Properties of drugs

TYPES OF TABLETS
AND THEIR
CHARECTERISTICS.

By
Dr. Paul Wanjala Muyoma

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 CONTENTS

 Introduction
 Classification of tablets
 Characteristics of tablets
 TableT’s dissoluTion/disinTegraTion TesTs, friabiliTy
tests & hardness
 Culture and sensitivity
 sterility tests of fluids
 References

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definition
 Tablet is defined as a compressed solid dosage form containing
medicaments with or without excipients.

 Tablets are solid, flat or biconvex dishes, unit dosage form, prepared
by compressing a drugs or a mixture of drugs, with or without
diluents.

 Tablets vary in shape and differ greatly in size and weight, depending
on amount of medicinal substances and the intended mode of
administration.

 It is the most popular dosage form and 70% of the total medicines
are dispensed in the form of Tablet.

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General properties

 Accurate dosage of medicament, uniform in weight, appearance and

diameter.

 Have the strength to withstand the rigors of mechanical shocks

encountered in its production, packaging, shipping and dispensing.

 Release the medicinal agents in the body in a predictable and

reproducible manner.

 Elegant product, acceptable size and shape.

 Chemical and physical stabilities.

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advantages
 Production aspect
 Large scale production at lowest cost
 Easiest and cheapest to package and ship
 Greatest chemical and microbial
stability over all oral dosage form.
 User aspect (doctor, pharmacist, patient)
 Easy to handling
 Lightest and most compact
 Greatest dose precision & least content
variability
 Coating can mark unpleasant tastes &
improve patient acceptability
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disadvantages
 Some drugs resist compression into
dense compacts.
 Drugs with poor wetting, slow dissolution,
intermediate to large dosages may be
difficult or impossible to formulate and
manufacture as a tablet that provide
adequate or full drug bioavailability.
 Bitter taste drugs, drugs with an
objectionable odor, or sensitive to oxygen
or moisture may require encapsulation or
entrapment prior to compression or the
tablets may require coating.

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classification of tablets

Use wise
Tablet for oral ingestion
In oral cavity
By other routes
to prepare solution

Structure wise
Divisible tablet
Classification of Aperture tablet
Concave-convex tablet
Tablets Core tablet
Layered and inlay tablet

Drug Action
Modified Release tablet Timed release/
sustained release/ prolonged release tablet
Delayed action tablet e.g., Enteric coated
Bisacodyl tablet
Rapidly dissolving/ disintegrating tablet

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ORAL TABLETS FOR INGESTION

 Standard compressed tablets e.g., Paracetamol tablet

 Multiple compressed tablets
I. Compression coated tablet
*Sugar coated tablet
*Film coated tablet
*Gelatin coated tablet
*Enteric coated tablet
II. Layered tablet
III. Inlay tablet
 Targeted tablet
I. Floating tablet
II. Colon targeting tablet
 Chewable tablet e.g., Antacid tablet
 Dispersible tablet
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TABLETS USED IN THE ORAL CAVITY

I. Lozenges and troches

II. Sublingual tablet e.g., Vitamin-c tablet
III. Buccal tablet e.g., Vitamin-c tablet
IV. Dental cones
V. Mouth dissolved tablet/ rapidly dissolving tablets

TABLETS ADMINISTERED BY OTHER

ROUTES
I. Vaginal tablet e.g., Clotrimazole tablet
II. Rectal tablet
III. Hypodermic tablet
IV. Implants

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TABLETS USED TO PREPARE SOLUTION

 Effervescent tablet e.g., Dispirin tablet

(Aspirin)
 Molded tablets
I. Hypodermic tablet
II. Dispensing /soluble tablet e.g.,
Enzyme tablet (Digiplex)
 Tablet triturates e.g., Enzyme tablet
(Digiplex)

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I. Standard compressed tablet
 These are the standard uncoated tablets made by
either
➢ direct compression
➢ wet granulation
➢ dry granulation
 They may be used for local action in GIT/
systemic action.
 In addition to medicinal agents
 They usually contain several pharmaceutical
adjuvants.

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Here are the steps for a
successful wet granulation tablet
manufacturing process:
 Weigh, mill, and mix your active pharmaceutical
ingredients APIs with powdered excipients.
 Prepare the binder solution.
 Mix your binder solution with powders to create a damp
mass.
 Wet screen the dampened powder into pellets or granules
using a mesh screen.
 Dry the moist granules.
 Use dry screening to size granulation.
 Mix the dried granules with lubricant and disintegrants.
 Compress the granules into tablets.

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Here are the steps for a
successful dry granulation tablet
manufacturing process:
 Weigh and mill formulation ingredients like drug
substances and excipients.
 Mix the milled powders.
 Compress the mixed powders into slugs.
 Mill and sieve the slugs.
 Compress them into tablets.

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Here are the steps for a
successful direct compression
tablet manufacturing process:
 Mill therapeutic agents and excipients.
 Mix the milled powders, disintegrants, and lubricants.
 Compress the tablets.

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15
 Multiple compressed tablet
II. compression coated tablet
 Function like sugar-coated or film-coated tablets or gelatin-
coated, enteric coated.
 Coating of a tablet may
a. mask a bitter taste, odor, color of the substance
b. conceal an unpleasant or mottled appearance
c. provide a barrier for a substance irritating to the stomach
d. Provide physical and chemical protection for one inactivated by
gastric juice.
e. Control the release of drug from the tablet.
o There are 3 principle designs in compression-coating machines
i. Colton model
ii. Stokes model
iii. Manesty dry- cota model

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 film coated pills sugar coated pills
 Disadvantages:
i. Improper centration of core either vertically/ horizontally produces
weak edges and coating will not hold together.
ii. More expensive because of multiple granulation.

Unequal coating cocking off centre

Faults in compression coating

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II. Layered tablet
 Multilayer tablets (2 or 3) are prepared by
repeated compression of powders and are
made primarily to separate incompatible drugs from each other.
o It makes possible SR release preparation with the IR quantity in
one-layer and slow-release portion in the second, a third layer with
an intermediate release might be added.
For example,
1. A mixture containing Phenylephedrin HCL
and Ascorbic Acid with Paracetamol.
Paracetamol + phenylephedrine Hydrochloride → one layer
Paracetamol + ascorbic acid → another layer.
2. Analgesic-antipyretic decongestant containing aspirin and phenyl
propanolamine. A thin layer of placebo is placed between them
to negate the chemical incompatibility of active ingredient.

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 Equipment- Versa press
 Advantages-
i. Coloring the separate layers provides many possibilities for
unique tablet identity.
ii. Pre compression lengthens dwell time and aid in bonding.
iii. Layer presses find employment in manufacture of chewable
antacid tablets. (MgO heavy USP) first layer, (Al(OH)2 dried gel
USP) as second layer.

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III. INLAY TABLET
o DOT OR BULL’S EYE TABLET
o Instead of completely surrounded by the coating, its top surface is
completely exposed.

 It has some advantages over compression coated tablets:

i). Less coating material is required.
ii). Core is visible, so coreless tablets can be easily detected.
iii). Reduction in coating forms a thinner tablet and thus
freedom from capping of top coating.
 Used in sustained release preparations to reduce the size and weight
of tablet.

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 Equipment: Stokes, Colton or kilian machines
 Example-
i. 25 mg of hydrochlorothiazide in the bull’s eye, 600 mg of
potassium chloride in outside portion.
 Disadvantages- Poor- centration is a much more obvious defect in
inlay tablet.
 Also, color reaction due to incompatibility between the core and
coating are obvious.

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Targeted tablets
 Under this category we have two types of tablets:
I. GASTRO- RETENTIVE TABLET
 Opted when active pharmaceutical ingredients (API) release is
desired in stomach (antacids, APIs used against H. pylori infection)
 Floating tablet
 To retain the drug for longer time period in stomach following
approaches can be used:
❖ Low density tablet
❖ Tablet that can expand in gastric environment (swelling or unfolding).
❖ Using muco - adhesive polymer
Supine position is to be avoided.
o Drugs like diazepam, levodopa, benserazide and ciprofloxacin are
successfully marketed.

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II. COLONIC TABLETS
 For the drugs having poor absorption in stomach or small intestine,
colonic drug delivery is an answer of choice.
 The pH in this region varies from 6.4-7 and presence of microbial
flora plays an important role in drug release.
 Various mechanisms adopted for drug release in this area are:

❖Coating with pH sensitive polymer e.g., Eudragit

S100 and L100
❖Biodegradable polymer which are sensitive to
colonic bacteria.
❖Bio- adhesive polymer e.g., poly carbophils/poly
ethanes.
❖Redox sensitive polymers.

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Chewable tablets
 Chewable tablets are to be chewed and thus mechanically
disintegrated in the mouth, so that NO DISINTEGRANT IS
INCLUDED.

 Flavoring, sweetening and coloring agents are important.

 Sorbitol and mannitol are common examples of fillers in chewable

tablets, (mannitol has negative heat of solution which results in
cooling effect and also has sweetening action).

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Advantages of chewable
tablets
 Provide quick and complete disintegration of the tablet and thus
obtain a rapid drug effect after swallowing and dissolution.

 Easy administration, especially for infants and elderly people.

 Patient convenience through elimination of need of water for

swallowing.

 Examples

i. Chewable Aspirin tablets (for children in the treatment of

rheumatoid and to prevent clot formations in adults).

ii. Antacid tablets

iii. Chewable multi vitamin tablet 25

Dispersible tablets
 Disintegrate either rapidly in water to form stabilized suspension or
disperse instantaneously in the mouth to be swallowed without the
aid of water.
 The properties such as porosity, hardness, DT, increase in viscosity
after dispersion are necessary to investigate during manufacturing.
 ADVANTAGES:
i. For pediatric patients who cannot swallow.
ii. For API’s unstable if formulated in liquid formulation.
iii. Faster onset of action compared to standard compressed tablet.
iv. Patients having prolonged illness who are prone to nauseatic
sensation.
 Examples: Analgesics (aspirin, ibuprofen etc.)

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Lozenges and trouches
 Lozenges are flavored medicated dosage forms intended to be sucked
and held in mouth or pharynx.

 Two lozenge forms include hard (or boiled) candy lozenges and
compressed tablet lozenges (TROUCHES).

Lozenges may be used for;

 Local medications in the mouth or throat,
e.g., local anesthetics, anti-histamines,
decongestants, analgesics, demulcents, antiseptics &
antibiotics
 Systemic drug uptake.

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 A soft variety of lozenge, called a pastille, consists
of medicament in a gelatin or glycero-gelatin or in a
base of acacia, sucrose and water.

 No disintegrant is included in compressed

lozenges composition

 Other additives (binder and filler) must have

pleasant taste or feeling during dissolution.

 Common binder used in compressed lozenges is

gelatin, common fillers are (Sorbitol,
mannitol and glucose).

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sublingual tablets
 Requirements of sublingual tablets are speed of absorption and a
correspondingly rapid physiological response.
 Intended to be placed beneath
the tongue and held there until
absorption has taken place.
 Absorption through oral cavity
avoids first pass metabolism.
1. Molded sublingual tablets- Sublingual tablet is
Prepared from soluble ingredients so that placed under the
tongue (rich in
the tablets are completely and rapidly soluble. blood supply)
Examples- codeine phosphate tablets, scopolamine HBr tablets,
nitroglycerine tablets.

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2. Compressed sublingual tablets-
 This type has less weight variation, better content uniformity, also
harder and less fragile.
 Examples-
a. glyceryl trinitrate
b. isoprinosine sulphate
c. nitroglycerine tablet
d. erythrityl tetra nitrate
e. isosorbide dinitrate

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Buccal tablets
 Intended to be dissolved in buccal pouch.
 Tablets are designed not to disintegrate.
 It is placed near the opening of parotid duct to provide the
medium to dissolve the tablet.

 Buccal tablets are most often used when replacement

hormonal therapy is the goal, e.g., Methyl testosterone,
testosterone propionate.

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Long- acting
buccal tablets-
 Use of viscous natural or synthetic gums
or mixtures of gums can be compressed
to form a hydrated surface layer from which
the medicament slowly diffuses and is
available for absorption through buccal
mucosa.
 Using Polyacrylic co-polymer (carbopol
934) blended with HPC or sodium
cascinate.
 Mucoadhesive polymers like PANA and
carbopol 934 are used.
 Examples- nitroglycerin buccal tablets
prochlorperazine maleate buccal tablet

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Dental cones

 These tables are designed to be loosely packed in the empty socket

remaining following a tooth extraction.

 Main purpose behind the use of this tablet is either to prevent

multiplication of bacteria in the socket by employing a slow
releasing antibacterial compound or to reduce bleeding by an
astringent or coagulant containing tablet.

 It’s formulated to dissolve or erode slowly in presence of a small

volume of serum or fluid over 20-40 minutes period.

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Mouth Dissolved tablets/
rapidly Dissolving tablets
 Also known as orally disintegrating tablets.
 Preferred when fast action or relief is
desired.
 Several techniques are used to prepare these
tablets, including lyophilization, soft direct
compression.
 Taste masking poses numerous challenges
since the drug product dissolves in mouth,
either by flavoring technique or by micro
encapsulation or nano -encapsulation.
 Most commonly used drugs under this
formulation are the agents active against
Migraine. 34
Vaginal tablets
 Designed for vaginal administration in treatment of local vaginal
infections, for systemic absorption and absorption into vaginal
tissue.

 Can be inserted with aid of an appliantor.

 In the treatment of localized vaginal infections such as, Candida

albicans, yeast and Haemophilus vaginalis.

 Examples:
- Cyclodextran formulations of hydrophilic drugs such as amino-
glycosides, β- lactum antibiotics and peptides.
- Propanolol

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Rectal tablets
 Old and acceptable means of treatment.
 The volume and nature of rectal fluid, its buffer
capacity, pH and surface tension play a large part in
this but are subject to wide variation, even within
single subject, resulting in variability of absorption
by this route.
 Advantages:
 Tablets have distinct advantages over suppositories
is
i. Not requiring refrigeration
ii. Better product stability even at room temperature
iii. Suppositories containing such compounds as
Aspirin and Penicillin G sodium have limited
product stability even under refrigeration.
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Rectal tablets

 Disadvantages:

i. Possibility of premature expulsion of the dosage

form before sufficient absorption takes place.
ii. Unfortunately, little is known about the rectal
delivery from tablet.
iii. Additional studies would be required to
demonstrate the extent of bioavailability.
 Examples:

 Rectal tablet Prochlorperazine.

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implants
 Designed for subcutaneous implantation by surgical
procedure where they are slowly absorbed over a period of
months or a year.
 Special injector with a hollow needle and plunger is
used to administer the rod shaped tablet.
 For other shapes surgery is used.
 They are sterile formulations without excipients.
 Mainly these tablets are prepared to deliver growth hormones
to food producing animals.
 Ear is preferred site for administration of drug.
 Safety problems

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Effervescent tablets
 Effervescent tablets are dropped into a glass of
water before administration during which CO2 is
liberated. This facilitates tablet disintegration and
drug dissolution; the tablet disintegration should
complete within few minutes.

 (Effervescence is a special mechanism for

disintegration) CO2 is created by the reaction
between carbonate or bicarbonate and a
weak acid such as citric acid or tartaric acid.

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Advantages of
effervescent
tablets
i. Rapid drug action e.g., analgesics and antacids.
ii. Significant differences in absorption kinetics
(gastric emptying rate, rapid tablet dissolution)
iii. Facilitate drug intake, for example vitamins.
iv. Special conditions
Low RH (25%) and moderate to cool temperature (250c) in
processing areas is essential.
Effervescent tablets should be protected from moisture, may
be packed in blister packs.

 Examples
 wide range of effervescent tablets include antibiotics,
ergotamines, digoxin, methadone, L- dopa.
Preparation for veterinary use have also been
developed.

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Molded tablets
I. Hypodermic tablets
 They are intended to be added in Water
For Injection (WFI) of sterile water to
form a clear solution which is to be
injected parenterally.
o Widely used by rural physician due to its
portability.
o Can be used for medicaments whose
stability in water is very poor.
o Their use in this manner should be
discouraged, since the resulting
solutions are not sterile.

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II. Dispensing/ soluble tablet
 They are to be added to water or other solvents to make a solution
containing a fixed concentration of API.
 Should contain no insoluble materials (including glidants, binders
etc.), since they will be made into clear solution.

 Examples
 Mercuric chloride dispensing tablet (antiseptic), tablet for
ophthalmic drops of neomycin sulfate.
 Others include topical local anesthetics (cocaine),
antibiotics (bacitracin).

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Tablet triturates
 They are small, usually cylindrical, molded
or compressed tablets containing small
amounts of usually potent drugs.
 Only a minimal pressure is applied during
their manufacturing, since they must be
readily and completely soluble in water.

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Structure wise
 Divisible tablet
It is sometimes necessary to administer
one-half or one-fourth of a tablet and
under such circumstances tablets are
generally scored once in the middle or
twice with lines perpendicular to one another.
o V-shaped double layer tablets with scoring in the centre have been
designed.
o Apertured tablets
Designed with a view to achieve constancy in the surface area during
disintegration & dissolution.

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Structure wise

 Concave-convex tablets
These tablets have been designed with a view to keep surface area of the
structure relatively constant during the dissolution process.
Area is lost on the convex surfaces and gained at the concavities.

 Core tablets
These tablets have a central core over which another layer of material is
compressed and are generally made by two successive compressions.
Separate incompatible ingredients.

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ENTERIC COATED TABLET
 Protect the drug from being destroyed by gastric contents,
either enzymes or highly acidic gastric fluids. E.g., low pH destroys
some drugs (erythromycin).

 Prevent or reduce nausea and vomiting associated with a drug’s

irritation of gastric mucosa. E.g., aspirin, strong electrolytes such as
NH4Cl, KCl.

 Deliver the drug to its absorption site in the intestine.

 Deliver the drugs intended for local action in the intestine. E.g.,
intestinal antibacterial or antiseptic agents.

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47
Coating composition
❖ Enteric polymers e.g., shellac, CAP, HPMC phthalate, methacrylic
acid, polyvinyl acetate phthalate, co-polymers like Eudragit L100,
Eudragit S100.
❖ Plasticizers e.g., glycerin, PEG, propylene glycol, castor oil,
phthalate esters.
❖ Solvents
 Enteric coating materials-
 Action of enteric coating results from difference in the composition
of gastric and intestinal environments with respect to both pH and
enzymes.
1. Materials that rely on erosion
2. Materials that rely on change in pH/ pH sensitive materials

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Modified release tablets
 Release the medicament slowly for long time duration after
administration of a single tablet.
 Used to target the site-specific releases.

Comparison of blood concentration vs. time

 Any adjuvant that can alter water uptake rate, swelling, and gelling
characteristics can alter the release rate of API. E.g., electrolytes in
HPMC matrix tablet.

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Modified release tablets

 The drug release can be modified by providing suitable

microenvironment pH in the tablet. E.g., acidic polymer, succinic
acid etc.
 Inclusion of alkaline polymers results in desirable drug release of
acidic drugs.
Advantages-
o Patient compliance
Disadvantages-
 Increases the cost of manufacturing.
 Chances of burst of drug release.
 In case of accidental poisoning, the doctor has to deal with special
treatment problems.
 Due to large size, patient may feel difficulties in swallowing as the
matrixing agent to drug ratio is high.
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Tablets for miscellaneous use
Reagent tablets-
 For qualitative and quantitative tests
 In diabetics to estimate urinary sugar levels,
presence of acetone, aldehydes or ketones in
urine.
 To determine presence of albumin in urine for
the detection of occult blood.
 Tablets fulfill countless other needs- for water
purification, artificial sweeteners,
nutritional ingredients for diet control,
cleaners for dentures, general cleaners and
disinfectants, fertilizers for house plants and
even Easter egg colors.
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Summary
 With advancement in technology and increase in
awareness towards modification in standard tablet to
achieve better acceptability as well as bioavailability,
newer and more efficient tablet dosage forms are
being developed.
 The main reasons behind formulation of different
types of tablets are to create a delivery system that is
relatively simple and inexpensive to manufacture.
 Provide the dosage form that is convenient from
patient’s perspective and utilize an approach that is
unlikely to add complexity during regulatory
approval process.
 To understand each dosage form, tablets here are
classified by their route of administration and by the
type of drug delivery system they represent within
that route. 52
 TableT’s
dissolution/disintegration
tests, friability tests, hardness
SNO CONTENTS
1 QUALITY CONTROL TEST DEFINITION
2 INPROCESS QC TESTS FOR TABLETS
FOR UNCOATED TABLET
FOR COATED TABLET
3 OFFICIAL TESTS FOR TABLETS
WEIGHT VARIATION TEST
FRIABILITY
DISINTEGRATION TEST
DISSOLUTION
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QUALITY CONTROL TEST
 QC refers to produce (or) a set of steps taken during
manufacturing of a product to ensure that it meets
requirements
 QC is the monitoring process through which
manufacturer measures actual quality performance
compares it with standards & finds out the causes of
deviation from standard to ensure quality product not
once but every time .
 The most commonly required ISO standards that are
applicable for all kinds of Pharmaceutical Industry are
as listed below:
1. ISO 9001 Standard: Quality Management System.
2. ISO 14001 Standard: Environmental Management
System.
3. ISO 27701 Standard: Privacy Information Management
System.

54
Tests conducted on Tablets

UNOFFICIAL TESTS OFFICIAL TESTS

 1. Appearance/  1. Identification Tests
Description  2. Friability Test
 2. Thickness and  3. Disintegration Test
Diameter  4. Weight Variation Test
 3. Hardness  5. Uniformity of Dosage
Unit Test
 4. Organoleptic
properties  6. Dissolution Test
 7. Assay Test
 8. Impurities Test

55
INPROCESS QC TESTS FOR
TABLETS

For coated tablet For uncoated tablet

 Appearance  Appearance

 Dimensions  Dimensions

 Adhesion test

 Resistance to abrasion

56
Specific Pharmacopoeial Tests
of Tablets

 1. Microbiological
Examination of Tablets
 2. Acid-Neutralizing
Capacity
 3. Quality test of
Splitting Tablets with
Functional Scoring
 4. Water content

57
COATED TABLET
 These are the tests conducted when the tableting is under process
Appearance :
 The tablet should be free from cracks, depression ,pinholes etc.
Dimensions :
 The dimensions of the tablets, thickness and diameter
 Measured by using digital vernier calipers or screw gauge
Adhesion test :
 This measured by using tensile strength testers
 The force required to peel the film from the tablet surface is measured
Resistance to abrasion :
 The ability of the coating to remain stuck to the tablet surface is tested
 Any defects in the formulation of coating solution can be tested

58
UNCOATED TABLETS

 Appearance :

 It should be free from cracks, depression, pinholes

etc.

 Colour & polish of the tablet should be uniform on

whole surface

 There should be no signs of coating

 Surface should be smooth

59
UNCOATED TABLETS
 Dimensions :
 The dimensions of the tablets, thickness & diameter are
measured by using digital vernier calipers and screw gauge

60
UNCOATED TABLETS: HARDNESS
 It is defined as the crushing strength of
the tablet or force required to break a
tablet across the diameter
 Hardness of the tablet is the indication
of strength
 Why do we do hardness test for tablets ?
 Tablet should be stable to mechanical
stress & transportation
 Degree of hardness varies with different
manufacturers & different tablets

61
HARDNESS
 It is a valuable test ,which influence the tablet
dissolution & disintegration

 Hardness varies: Depending upon the type &

concentration of the binding agent.

 Binding agents (e.g.); acacia mucilage, starch paste,

sugar syrup, methyl cellulose dispersion etc.

 The force is measured in kilograms & the hardness of

about 4kg is considered to be satisfactory for
uncoated tablet

62
Various types of hardness
testers used are
 Various types of hardness testers used are :

Monsanto tester
The tablet is placed across the spindle & anvil. Knob is
adjusted to hold the tablet in position. The reading of the
pointer is adjusted to zero. Pressure is slightly increased to
63
break the tablet
Various types of hardness
testers used are

Pfizer tester

It works on the mechanical principle as a pair of pliers

Tablet is compressed between holding anvil & a piston

connected to the direct force reading gauge.

64
Various types of hardness
testers used are
 Erweka tester

Tablet is placed on the lower anvil & the anvil is

adjusted so that the tablet just touches the upper
test anvil
65
Various types of hardness
testers used are
 Schleuniger tester

Operates in horizontal position

An anvil is driven by an electric motor presses the tablet at

a constant load rate against a stationary anvil until the
tablet breaks

66
 Other tablet hardness testers
Strong-Cobb Tester for Tablets
 The Strong-Cobb tester pushes an anvil against an unmoving platform.
 The tester has a hydraulic meter from which you can see the results, which are the same as
that of Monsanto tester.

Dr. Schleuniger Pharmatron Tester for Tablets

 The Dr. Schleuniger Pharmatron tester functions in a horizontal posture.
 An electric motor propels an anvil to press a tablet at a consistent rate.
 The force pushes the tablet against a static anvil until it breaks.
 A scale indicator records the reading which is the hardness value of the tablet.

Kraemer Elektronik Tablet Testing

 The Kraemer Elektronik tablet hardness testing machine was the first automated tablet
hardness tester system for self-activation at tablet presses.
 The machine was developed by Mr. Norbert Kraemer, a German mechanical engineer in
Darmstadt, Germany.
 A patented feeder chute separates the tablets moving them on a horizontal star wheel via
different testing points.
 The Kraemer Elektronik tablet hardness tester machine measures weight, width,
length/diameter, thickness, and hardness of capsules and tablets.
67
Why test for hardness?
 · To determine the tablet disintegration
An exceedingly hard tablet could designate excessive bonding possibilities between excipients and active
ingredients, which can hinder proper disintegration of the tablet required for a correct dosage.
Conversely, a softer tablet could indicate weak bonding and may result in premature dissolution when
consumed by the patient.

 · To determine tablet handling

A soft tablet may not sustain the handling and could crack or chip during the successive processing phases
of production,
These many include coating and packaging and during transportation.

 · To determine the material ingredients

Knowing the mechanical features of a solid-dose tablet can give important information necessary for
optimizing material ingredients.
The nature of active constituent(s), the kind of binder used, and the composition of the constituent(s) in
the tablet will impact on the hardness of the tablet.

 · To determine the production process

Knowing the right tablet press speed, granulation flow, and air in the powder is important in formulating
the appropriate table.
For that matter, you need to control these parameters during production to manufacture a tablet with
the correct hardness.

68
 These testers Do not produce same results for
the same tablet: Weight variation test for tablets

USP – OFFICIAL LIMITS BP - OFFICIAL LIMITS

Tablet weight limit Tablet weight limit
130mg or less ±10 % 80mg or less ±10 %
130 – 324 mg ±7.5 % 80mg – 250 mg ±7.5 %
> 324 mg ±5 % > 250mg ±5%
IP – OFFICIAL LIMITS
Tablet weight limit
80mg or less ±10 %
80mg – 250 mg ±7.5 %

> 250mg ±5% 69

 Test procedure :

 30 tablets are randomly selected for the test .every tablet in each batch should have uniform weight .

 20 tablets are weighed individually. Average weight is calculated from individual weight of all tablets.

 Individual weight is compared with average weight.

 The percentage difference in the weight variation should be with in the permissible limits.

 The percentage deviation is calculated by using the formula:

% weight variation = individual weight – average weight

---------------------------------------------×100
individual weight

70
 Result :

 Out of 20 tablets , if 2 tablets deviate the limit =

perform test for other 10 tablets

 Out of 30 tablets , if 2 tablets deviate the limit = the

batch passes the test

71
Friability test:
 It is used to measure the strength of the tablet.

 It is used to measure tablet to withstand mechanical shock

& abrasion without crumbling during the handling of
manufacturing, packaging, shipping, and consumer use.

 The friability of tablets is indicated by chipping, capping

(or) breaking

 Friability is strictly adhered to coated tablets.

 Friability problem is encountered with thin tablets ,large

diameter tablets, granules (excessively dried or excessive
fine granules).

72
Friability test :
 The extent of friability is measured by using Roche
Friabilator
 It rotates at a rate of 25 rpm.
 10 tablets are weighed collectively & placed in the
chamber of friabilator.
 In the friabilator the tablets are exposed to rolling
resulting from free fall of the tablets within the chamber
of friabilator.

73
Friability test:
 After 100 revolutions (4 min ) the tablets are taken out from the
friabilator and intake tablets are again weighed collectively.

 % friability is calculated by using the formula :

 friability = W1 – W2
--------------- × 100
W1

 W1 = Weight of the tablet before test

 W2 = Weight of the tablet after test

74
 Name of some brand of Friability Test Apparatus
1. ERWEKA Tablet Friability Tester
2. Copley Tablet Friability Tester
3. PTF Tablet Friability Tester
4. HMK-1601 Tablet Friability Tester
5. CS-1/2/3 Tablet Friability Tester
6. Electrolab Dual Drum Friability Tester
 Causes of High friability of tablets
1. Inadequate binder.
2. Over-drying of granules.
3. The use of some excipients such as microcrystalline cellulose,
silicified microcrystalline cellulose, magnesium silicate,
polysorbate, and sodium stearyl fumarate, gives low friability at
lower compression pressures.
4. Too much or too little compression pressure.
5. Over-lubrication.
6. Improper tablet design.
75
Content uniformity test :
 This test is applied to assure uniform potency for tablets of
low dose drugs
 The test is applicable to tablets that contain 10mg / < 10mg
(or) < 10%w/w of active ingredients
procedure:
 Select 30 tablets randomly from the batch.
 At least 10 of them are assayed individually.
 Out of 10 tablets 9 tablets must contain not less than 85% not
more than 115% of labelled drug content.
 10th tablet may not contain < 75% or > 125 % of labelled drug
content.

76
Content uniformity test :
 Result :

 Batch passes the test

 If there is any deviation then perform the assays individually

for 20 tablets

 Out of 30 tablets 3 tablets can be within 75 – 125 % & the

others should be within 85 – 115 %

77
Disintegration test (DT):

 Disintegration is the process of breakdown of tablet into

smaller particles or granules .

 There is no correlation between dissolution & disintegration .

 Disintegration is a pre-requisite for the dissolution .

78
Disintegration test (DT):

DISINTEGRATION TEST APPARATUS

79
 Disintegration test (DT):

 It has a basket rack assembly .

 Basket has 6 cylinders (77.5 mm long, 21.5 mm internal diameter , 2mm thick )

 Tubes are held vertically by 2 super imposed plastic plates

(90 mm in diameter 6.75 mm thick ) and have perforations of size of cylinders.

 Lower plate has woven stainless steel (ss) wire attachment .

 Upper plate is covered by ss disc perforated by 6 holes .

 Plates are held rigidly in position & 77.5mm part by vertical metal rod at the
periphery

80
Disintegration test (DT):
 Metal rod is fixed at the centre (or) upper plate to enable the
assembly to be attached to the device for raising & lowering it
smoothly at constant frequency of between 28 – 32 cycles per
minute through a distance of 50 – 60 mm

 DISCS :

 Discs are used to prevent the floating of tablet & to impart abrasive
action to the tablet.

 Dimensions : 20.7 mm in diameter, 9.5 mm thick.

 They are made up of transparent plastic & pierced with 5holes

each 2 mm in diameter 1 in centre & 4 are equally spaced on the
circle.

81
DISINTEGRATION TIME
TABLET TYPE DISINTEGRATION TIME

Uncoated 15 minutes
Plain coated tablet 60 minutes
Enteric coated tablet 3 hours

Dispersible tablet 3 minutes

Effervescent tablet ˂ 3 minutes
Sublingual tablet 4 hours
Buccal tablet 4 hours
Vaginal tablet 60 minutes
chewable tablet not required

82
Types of disintegration
testers
 1) Fully Automated Tablet Disintegration Tester Machine
This kind of Disintegration Tester automatically detects
the individual disintegration times for a tablet or/ and
other solid dosage forms. As the word automatic states,
they operate independently. ...
 2) Semi-automated Tablet Disintegration Testing
Machine ...
 3) Manual Tablet Disintegration Tester Machines ...
 4) Single Disintegration Tester Machines ...

83
DISSOLUTION TEST:

 Dissolution is a process in which a solid substance solubilizes

in a given solvent ( mass transfer from the solid surface to the
liquid phase.

or

 It is a process by which drug released from solid dosage form

& immediately goes into molecular solution.

 Rate of drug absorption for drugs is often determined by rate of

drug dissolution from the tablet.

84
DISSOLUTION TEST:

85
DISSOLUTION TEST:
 Based on sink (or) non sink conditions
 dissolution apparatus are classified as :

i. Closed compartment apparatus

ii. Open compartment apparatus

 Types of dissolution apparatus (official)

 According to IP :
 TYPE I : PADDLE
 TYPE II : BASKET

 According to BP :
 TYPE I : BASKET
 TYPE II : PADDLE
 TYPE III : FLOW THROUGH CELL

86
DISSOLUTION TEST:
According to USP:
 TYPE I: ROTATING BASKET

 TYPE II: PADDLE

 TYPE III: RECIPROCATING CYLINDER

 TYPE IV: FLOW THROUGH CELL

 TYPE V: PADDLE OVER DISC

 TYPE VI: ROTATING CYLINDER

 TYPE VII: RECIPROCATING DISC

87
TYPE
: I: BASKET TYPE:
Design :

Vessel Made up of borosilicate glass

Hemispherical bottom
Inner diameter 98 to 106 mm
Capacity 1000 ml
Height 160 to 210 mm

Shaft Stainless steel 316

Rotates smoothly without significance .

Wooble positioned in such a way that its axis is

not more than 2 mm from vertical axis of the
88

vessel 88
BASKET TYPE 89
 Basket Type

Basket stainless steel 316

made of # 22mesh
gold coatings upto 0.0001 inch
placed at a distance of 2cm from bottom

Water bath maintained at 37 ± 0.5 ˚c

Agitation 100 RPM

Use capsules, tablets, delayed release, suppositories,

floating dosage forms.
90
TYPE II : PADDLE TYPE

DESIGN :

Shaft The blade passes through shaft so that bottom of the

blade fuses with bottom of the shaft

Stirring elements made up of tefflon

for laboratory purposes
stainless steel 316
Water bath maintain at 37± 0.5 ˚c

Sinkers platinum wire is used to prevent capsule / tablet from

floating

Agitation 50 to 75 RPM
Basket mesh size ranges from 10 to 80 can be used

91
92
TYPE II PADDLE APPARATUS
93
TYPE III : RECIPROCATING CYLINDER:

DESIGN :

Vessel cylindrical flat bottom glass vessel.

Agitation type reciprocating generally 5 to 35 RPM

Volume of dissolution fluids 200 – 250 ml

water bath maintained at 37± 0.5 ˚c

Use extended release eg : chloramphenicol

chewable tablets eg : carbamazepine

94
RECIPROCATING CYLINDER
95
Type IV : FLOW THROUGH CELL:

DESIGN :

Reservoir For dissolution medium

Pump forces dissolution medium through cell

holds the sample
flow rate 10 – 100 ml / min
laminar flow is maintained
peristalic / centrifugal pumps are not recommended

Water bath 37± 0.5 ˚c

96
97
TYPE V : PADDLE OVER DISC:

DESIGN :

Sample holder disc assembly that hold the product in

such a way that release surface is parallel with paddle

Paddle is directly attached over disc assembly

Samples are drawn away between the surface of medium &

top of the paddle blade

Volume 900ml

Water bath 32 ˚c
98
TYPE V : PADDLE OVER DISC:

99
TYPE VI : ROTATING CYLINDER :

DESIGN :

Vessel In place of basket ,cylinder is used

Cylinder stainless steel 316

Sample mounted to cuprophan ( inner porous cellulosic

material ) an entire system is adhere to cylinder

Dosage unit is placed in cylinder & released from

outside

water bath maintained at 37± 0.5 ˚c

100

Use transdermal patches 100

TYPE VII :RECIPROCATING DISC :

Vessel flat bottom cylindrical vessel

volume of dissolution medium 50 – 200ml

Sample is placed on disc shaped holders

Agitation reciprocation
reciprocating frequency 30 cycles / min

Water bath maintained at 37± 0.5 ˚c

Use transdermal patches

101
Limits :

Dissolution testing & interpretation can be done by 3 stages

(S1,S2,S3)

In stage 1 : 6 tablets were tested & are acceptable if all of the tablets
are not less than Q + 5%

In stage 2 : additional 6 tablets were tested .if all average of 12 tablets

is greater than or equal to Q & no unit is less than Q – 15%

If the tablets fails the test

In stage 3 : all the average of 24 tablets is greater than or equal to

Q & if not more than tablets are less than Q - 15 %
102
Summary

103
Summary

104
THANK you

105
 Culture and
sensitivity; sterility
tests of
PHARMACEUTICAL
MATERIALS

By
Dr. Paul Wanjala Muyoma

1
Sterilization

 Sterilization – An introduction
 Importance of sterilization
 Methods
▪ Advantages & Disadvantages
 Effects of Sterilization

2
STERILIZATION:-

❖ The process of killing or

removing bacteria and all other
forms of living micro-organisms
and there spares from preparation.
❖ Essential concept in the
preparation of sterile
pharmaceutical products
❖ Its aim
▪ is to provide a product that is safe and eliminates the
possibility of introducing
3
❖ Medical Sterilization
❖ Prevents the Growth of Diseases
In any medical tool/device used, bacteria comes onto it. If left unchecked or
not disinfected properly, it is highly likely that bacteria will grow.

❖ Prevents the Spread of Diseases

If surgical equipment is not properly sterilized, patients treated are
exposed to a disease the previous patient had.

❖ Prevents Double Surgeries

If unsterilized equipment is used, it can cause an infection leading to another
surgery later on in order to remove it.
This is costly and can cause many life-threatening complications.
4
4
METHOD OF STERILIZATION
 THREE METHOD :-
1. Physical method
a) Dry heat sterilization
b) Moist heat sterilization
c) Sterilization by radiation (gamma radiation)

2. Chemical method
a) Gaseous sterilization
b) Sterilization by disinfectant

3. Mechanical method
Pass through bacteria-proof filter

5
5
Sl. Physical Method of Instruments used
No. Sterilization
1 Dry Heat Oven

2 Moist Heat Autoclave

3 Radiation Gamma-ray Chamber

6
6
DRY HEAT STERILIZATION
 Instrument- ‘OVEN’
OVEN : -
specially designed instrument - electrically heated and
thermostatically controlled.
Expose at 160 ºC for 1 hour.

Advantage-
it is suitable method for sterilization of
substances destroyed by moisture.

Disadvantage-
long heating time, high temperature.
oven
7
7
MOIST HEAT STERLIZATION

 Instrument- ‘AUTOCLAVE’
Heating process in autoclave - saturated steam under pressure is allowed to
penetrated through materials for 15 minutes and temperature 121º c.

Advantage-
Microorganism are killed most efficiency in lesser time
due to high pressured saturated steam

Disadvantage-
Unsuitable for materials not withstanding
temperature of 115ºC or more during heating

AUTOCLAVE
8
8
STERELIZATION BY RADIATION
Two techniques involved:
 Alteration of chemicals lead to form new compound in cells destroying the
micro-organism itself
 Vital structure like nuclear protein are destroyed killing the micro-organism.
▪ e.g., Co-60 - used for gamma ray sterilization process.
Gamma rays –
 generally obtained from radio isotope(Co-60) during disintegration of unstable atoms
 kill micro-organisms by isolating atoms of essential substance of cells present in them.

9
9
ADVANTAGE
1. No significant rise in temperature
2. Continuous process due to short exposure time.

DISADVANTAGE
1. May lead to color change.
2. Solubility of preparation leading to decomposition of certain materials.

10
10
CHEMICAL METHOD

 Gaseous sterilization-

 Ethylene oxide used.

 Special type of chemical sterilization using gases and vapors
 The gas used is safe & non-inflammable.
 Now-a-days, ethylene oxide most widely used gaseous sterilization agent in
medical science.

11
11
Advantage:
1. It has penetration power quite useful for sterilizing surgical instruments
(such as catheter, needles, plastics, disposables)

Disadvantages:
1. Very slow sterilization process
2. Very costly equipment

12
12
DISINFECTION
 Decontamination - removal of microorganisms
contaminating an object
 Preservation - preventing methods of microbes-
caused spoilage of susceptible products
(pharmaceuticals, foods)
 Sanitization - removal of microbes that pose a
threat to the public health, food industry, water
conditioning
❑ sanitizer-an agent, usually a detergent, that reduces the
numbers of bacteria to a safe level

13
13
DISINFECTANTS

Chemical agents
 Alcohols, aldehydes, halogen, phenols, surfactants, heavy metals
▪ e.g., ethylene oxide – most commonly used for sterilization
 Advantages:
1. Widely used in hospitals for materials that cannot withstand steam sterilization
 Disadvantages
1. 40-60% humidity in sterilizing chamber

14
14
MECHANICAL METHOD
 The solution to be sterilized is passed through depth-filter or screen-filter
which includes
 Particulate filters
 Microbial filters
 Final filter

 Pharmaceutical solutions are sterilized by this method.

 The micro-organism are physically removed by adsorption on the filter
medium or by mechanism.
 Filtration filling and sealing processes are under a septic condition.
 Sterilization test must be done.

15
15
SOME EFFECTS OF STERILIZATION
There are 3 effects:
1. Gamma radiation sterilization of medical device is
common but irradiation effect at 2.5 mega rad on a bone
replacement material when started and modified
property when investigated by Creep test.
2. Irradiation increase Creep resistance of material
with the formation of crosslink and then increase in
crystality respectively.
3. Biodegradable materials sterilized by gamma
radiation may be associated with some adverse effect.
NB: Creep deformation generally occurs when a
material is stressed at a temperature near its melting
point. 16
16
FUTURE PROSPECTS
❖ In the near future the main challenge in
biomedical engineering is to make the
sterilization process
1. More reliable
2. Reusable
3. Non-polluting
4. Cost effective
5. Less time consuming and
6. Better availability
at hospitals, pharmaceutical company, drug industry and food
processing plants.

17
17
Difference between
sterilization & disinfection :
 Sterilization is defined as the process where all the
living microorganisms, including bacterial spores are
killed.
 Disinfection is the process of elimination of most
pathogenic microorganisms (excluding bacterial
spores) on inanimate (nonliving) objects.
 Sterilization is an absolute condition while disinfection
is not.

 The two are not synonymous.

18
Methods of sterilization:

 Sterilization can be achieved by physical, chemical and

physiochemical means.
 Physical methods include – heat (dry or moist heat)-
ultraviolet light- ionising radiations- filtration through
abacteria proof filter
 Chemical methods involves the use of- liquid and-
gaseous sterilizing agents.

19
Methods of sterilization:
Methods widely applied to
Pharmaceutical Preparations:
 Heat Filtration Combined physical and chemical method
involving heat in the presence of a bactericide
 Methods mainly used for Surgical materials and
Equipment :
1. Ionizing radiations
2. Gaseous sterilization
3. Liquid sterilizing agents
4. Antiseptics and disinfectants.

20
Methods for pharmaceutical
preparations:
The British Pharmacopoeia has five methods for ensuring
that injections are sterile:
 Dry heat
 Moist heat
 Moist heat in the presence of a bactericide
 Filtration through a bacteria-proof filter
 Aseptic technique during preparation

21
Pharmaceutical Importance
of Sterilization :
 Moist heat sterilization is the most efficient biocidal agent. In the pharmaceutical
industry it is used for: Surgical dressings, Sheets, Surgical and diagnostic equipment,
Containers, Closures, Aqueous injections, Ophthalmic preparations and Irrigation fluids
etc.
 Dry heat sterilization can only be used for thermo stable, moisture sensitive or moisture
impermeable pharmaceutical and medicinal. These include products like; Dry powdered
drugs, Suspensions of drug in non aqueous solvents, Oils, fats waxes, soft hard paraffin
silicone, Oily injections, implants, ophthalmic ointments and ointment bases etc.
 Gaseous sterilization is used for sterilizing thermolabile substances like; hormones,
proteins, various heat sensitive drugs etc.
 U.V light is perhaps the most lethal component in ordinary sunlight used in sanitation of
garments or utensils.
 Gamma-rays from Cobalt 60 are used to sterilize antibiotic, hormones, sutures, plastics
and catheters etc.
 Filtration sterilizations are used in the treatment of heat sensitive injections and
ophthalmic solutions, biological products, air and other gases for supply to aseptic areas.
They are also used in industry as part of the venting systems on fermentors, centrifuges,
autoclaves and freeze driers. Membrane filters are used for sterility testing.

22
Physical method of
sterilization: HEAT
 Heat is considered to be most reliable method of
sterilization of articles that can withstand heat. Those
articles that cannot withstand high temperatures can
still be sterilized at lower temperature by prolonging
the duration of exposure.
 There are two types of heat – dry heat and moist heat.
Moist heat is heat along with moisture.
 Dry heat kills microorganisms by oxidation.
 Moist heat kills microorganisms by coagulation of
proteins that leads to denaturation of proteins.

23
Sterilization by Heat Factors
affecting sterilization by heat
are:
 Nature of heat: Moist heat is more effective than dry heat
 Temperature and time: temperature and time are inversely proportional. As
temperature increases the time taken decreases.
 Number of microorganisms: More the number of microorganisms, higher the
temperature or longer the duration required.
 Nature of microorganism: Depends on species and strain of microorganism,
sensitivity to heat may vary. Spores are highly resistant to heat.
 Type of material: Articles that are heavily contaminated require higher
temperature or prolonged exposure. Certain heat sensitive articles must be
sterilized at lower temperature.
 Presence of organic material: Organic materials such as protein, sugars, oils and
fats on poorly cleaned equipment increase the time required.
 Other factors: whether or not the devices /articles were properly loaded into the
sterilizer , whether or not the sterilizing agent is properly delivered into the
system , the sterilizer’s condition and maintenance protocol and whether or not
the correct sterilization method and cycle were used.

24
Sterilization by Heat Factors
affecting sterilization by heat
are:
 Sterilization by Heat Susceptibility of microorganisms to heat
can be expressed by:
1. Thermal death point (TDP) is the lowest temperature at
which all the microorganisms in a particular liquid
suspension will be killed in 10 minutes.
2. Thermal death time (TDT) is the minimum length of time
for all bacteria in a particular liquid culture to be killed at
a given temperature.
3. Decimal reduction time (DRT or D-value) is the time in
minutes in which 90% of a population of bacteria at a given
temperature will be killed. It is related to bacterial heat
resistance.
25
Sterilization by DRY HEAT
 Examples of Dry heat sterilization are:
 1. Incineration -This is a method of destroying
contaminated material by burning them in incinerator.
Articles such as soiled dressings; animal carcasses,
pathological material etc. should be subjected to
incineration. This technique results in the loss of the
article, hence is suitable only for those articles that
have to be disposed.
 2. Red heat- Articles such as bacteriological loops,
straight wires, tips of forceps and searing spatulas are
sterilized by holding them in Bunsen flame till they
become red hot.
 3. Flaming - This is a method of passing the article over
a Bunsen flame, but not heating it to redness. Articles
such as scalpels, mouth of test tubes, flasks, glass slides
and cover slips are passed through the flame a few
times.4. Hot air oven
26
Sterilization by DRY HEAT
 Sterilization by dry heat is usually carried out in an
apparatus known as hot-air oven in which heat is transferred
from its source to the load by radiation, convection and to a
lesser extent by conduction.
 The first stage in the design of a heat sterilization process is
choice of suitable temperatures and times.
 This depends on the need to obtain a sterile product which is
influenced by the stability of the material/preparation.
 Using dry heat for 1½ hours at 100°C will destroy all
vegetative bacteria, 3 hrs at 140°C for most resistant spores
and 1½ hours at 115°C for mould spores.

27
Sterilization by DRY HEAT
 Pharmacists must consider the stability of their
products and should not expose them to conditions
greatly in excess of those needed to produce sterility
e.g., the B.P. recommends 150°C for 1 hr. for oily
solutions.
 There is no objection to the use of high temperatures
where harmful effects cannot result e.g., for glass
vessels and containers and for these the B.P. specify not
less than 1 hr. at not lower than 160°C.

28
HOT AIR OVEN (Sterilization
by dry heat)
 The design of the oven must satisfy the following
requirements:
 Every article inside must receive the correct exposure,
wherever it is placed.
 The sterilizing temperature must be reached quickly and
maintained with little variation.
 Hot air oven: This method was introduced by Louis Pasteur.
Articles to be sterilized are exposed to high temperature
(160° C -180 ° C) for duration of 1 -2 hours in an
electrically heated oven. Since air is poor conductor of
heat, even distribution of heat throughout the chamber is
achieved by a fan. The heat is transferred to the article by
radiation, conduction and convection.
29
HOT AIR OVEN (Sterilization
by dry heat)
 Dry-heat sterilization is accomplished by thermal
(heat) conduction, convection and radiation.
 Initially, heat is absorbed by the exterior surface
of an item and then passed to the next layer.
 Eventually, the entire object reaches the
temperature needed for sterilization.
 Death of microorganisms occurs with dry heat by
oxidation that leads to slow destruction of
protein.

30
HOT AIR OVEN (Sterilization
by dry heat)
 Parts of a hot air oven:
 i) An insulated chamber made of aluminium or stainless steel
surrounded by an outer case containing glass-fibre insulation and
electric heaters.
 ii) A fan (to allow circulation of hot air)
 iii) Perforated Shelves ( perforated to allow circulation of hot
air)
 iv) Thermocouples
 v) Temperature sensor
 vi) Door locking controls with asbestos gasket that provides a
tight seal.
 vii) vents (on top of the oven).

31
HOT AIR OVEN (Sterilization
by dry heat)
 Operation of a hot air oven:
 i) Articles to be sterilized are first wrapped or enclosed in containers of
cardboard, paper or aluminum. Mouths of flasks, test tubes and both
ends of pipettes must be plugged with cotton wool. Articles such as
petri dishes and pipettes may be arranged inside metal canisters and
then placed.
 ii) Then, the articles must be placed at sufficient distance so as to
allow free circulation of air in between them and to ensure
uninterrupted air flow.
 iii) Oven may be pre-heated for materials with poor heat conductivity.
 iv) The door can then be shut and the heater and the fan is switched
on.
 v) When the thermometer shoes that the oven air has reached
sterilizing temperature , heating is continued for the required period of
time.
 vi) The temperature is allowed to fall to 40°C, prior to removal of
sterilized material ; this prevents breakage of glassware.
32
Pharmaceutical Applications
of sterilization by DRY HEAT
 1. Glassware –
Glassware that are regularly sterilized by dry heat includes flasks,
beakers, tubes, containers (e.g., ampoules), pipettes, petri dishes and
all glass syringes.
At first, they must be thoroughly degreased by washing in hot water
and detergent and rinsing well, followed by a final three rinses in a
pyrogenic distilled water.
New or very dirty articles should be soaked first in chromic cleaning
solution overnight.
Then they are dried in a drying oven at about 65°C.

33
Pharmaceutical Applications
of sterilization by DRY HEAT
2. Other equipment include:
 some articles of porcelain (such as mortars, pestles,
evaporating basins and tiles) and Metals (such as beakers,
dishes of stainless steel, scissors, scalpels and ointment
tubes).
3. Oils and similar anhydrous materials:
 Dry heat sterilization is of particular importance when
contact with moisture must be avoided e.g. Powders
 Vehicles used for oily injections (e.g. fixed oils, ethyl oleate
and other fatty acid esters)
 Ingredients of ointment bases (e.g. liquid, soft and hard
paraffins ,wool fat, wool alcohols and beeswax
 Medical lubricants (e.g. glycerol).
34
Pharmaceutical Applications
of sterilization by DRY HEAT
4. Powders
 Dusting powders fall into two groups:
 medical and surgical powders.
 Medical powder is used to treat superficial skin
conditions and so sterility is not essential. They must be
free from dangerous pathogens and so must be
sterilized by maintaining the powder at not less than
160°C for at least an hour.
 Surgical powders must be sterile because they are used
in body cavities and major wounds or on burns.

35
Pharmaceutical Applications
of sterilization by DRY HEAT
 The following substances present special problems that complicate
their sterilization by heat :
 Starch – Starch does not flow easily because its particles tend to stick
together and this is made worse if the moisture content is high.
However, if it is dried at 100°C for about an hour and then powdered
(with other ingredients) before sterilization (at 150 °C for 1 hr) its
flow properties are enhanced.
 Sulphonamides – The main problem with sulphonamide which is used
as a diluent in penicillin is to produce a free-flowing powder without
discoloration. A number of factors are involved such as particle size,
moisture content, envelope paper and other added substances (e.g.
Kaolin and zinc oxide gave a more free-flowing powder). The usual
method is to use crystals of suitable fineness, to dry these in a thin
layer at 100 °C, to pack preferably in double paper envelopes and
then to sterilize by maintaining at 150 °C for 1 hr.

36
Pharmaceutical Applications
of sterilization by DRY HEAT
 The following substances present special problems that
complicate their sterilization by heat :
 Lactose – Lactose has been used occasionally as a
diluent for penicillin. Since penicillin must be kept dry
to avoid decomposition, the lactose should be dried in
an oven at 105 °C, then sterilized and finally mixed
with the antibiotic. Paper envelopes do not give
sufficient protection against moisture and a well-closed
sifter vial should be used.

37
Advantages of dry-heat
sterilization :
 It is an effective method of sterilization of heat stable articles.
 It is the only method of sterilizing oils and powders.
 Provided sufficient time for penetration is allowed, it is suitable
for assembled equipment, e.g., all glass-syringes.
 In moist-heat sterilization, steam or water must be in contact
with every surface and this is not always possible for e.g., on the
closely fitted adjacent surfaces of the barrel and plunger of an
assembled syringe.
 It is less damaging to glass and metal equipment than moist heat.
Repeated exposure of glass to moisture at high temperatures can
produce clouding and alkali extraction and rusting is a serious risk
when instruments are sterilized by wet methods.

38
Disadvantages of dry-heat
sterilization:
 Due to high temperatures, long exposure and very long
heating up times, most of the medicaments, rubbers
and plastics are too much thermolabile for sterilization
by dry heat.
 It is unsuitable for surgical dressings.
 Dry-heat sterilization takes longer than steam
sterilization, because the moisture in the steam
sterilization process significantly speeds up the
penetration of heat and shortens the time needed to kill
microorganisms.

39
Sterilization by moist heat:

 Microorganisms can be exposed to moist heat by using

Hot water, Boiling water, Steam at atmospheric
pressure (steaming), Steam under pressure
(autoclaving)

 A long exposure at a relatively low temperature may

cause more damage to a pharmaceutical preparation
than a shorter treatment at a higher temperature.

 This explains why boiling and steaming are not used for
sterilization of parenteral solutions.

40
Principles of sterilization by
steam under pressure
(autoclaving)
 Pressure itself has no sterilizing power. Steam under pressure can be
used as a method of sterilization as it can provide temperatures high
enough to destroy microorganisms quickly. Steam for sterilization are
of two types:
i. Wet saturated steam
ii. Dry saturated steam
 Wet saturated steam is produced in a portable boiler from water
present inside it and since this steam is in constant contact with water,
it will always contain water droplets and thus known as wet saturated
steam.
 Dry saturated steam is produced in a separate boiler and then with the
help of a pipe is transferred to another boiler or tank where it can be
used for sterilization purpose. This steam practically contains no water
droplets and is highly efficient for sterilization of intra-venous fluids
and surgical dressings.

41
Principles of sterilization by
steam under pressure
(autoclaving)
 Steam is described as saturated when it is at a
temperature corresponding to liquid boiling point
appropriate to its pressure.
 Example: if appropriate equivalent temperature is
115°C and the corresponding steam pressure is 1.7 bars,
that steam is saturated.
 Steam pressure Approximate equivalent (bars)
temperature (°C)

42
Saturated Steam: an efficient
sterilizing agent
 Saturated steam is an efficient sterilizing agent
because-
 1. A large percentage of its Heat energy is in the form of
Latent heat :
 The heat energy in steam is in two forms which are
sensible heat and latent heat:
 i) Sensible heat – The heat required to raise the
temperature of water from its freezing point (0° C) to
boiling point (100 ° C).
 ii) Latent heat – Latent heat is the additional heat
needed to convert water from its boiling point (100 °
C) to steaming point at the same temperature (100 °
C).
43
Saturated Steam: an efficient
sterilizing agent
 Whenever the saturated steam touches the cool
surface of an article inside, the saturated steam
condenses and liberates all its latent heat immediately.
 The large amount of latent heat is given to the article
and makes a major contribution in raising it to
sterilization temperature.
 Since all the sensible heat is retained by the condensate
there is no fall of temperature in the surroundings.
 So, saturated steam is a much better heating agent than
hot air because the heat content of the hot air is small
and the heat transfer is slow and accompanied by a
drop in air temperature.
44
Saturated Steam: an efficient
sterilizing agent
 It Condenses on Cooling: The protein coagulation by
which moist heat kills microorganisms occurs at lower
temperatures if plenty of moisture is available.
 A possible explanation is that the coagulation
temperature of egg albumen depends on the amount of
water present –Water (%) Coagulation Temperature (°C)
If the level of water is low, high temperature is needed
as suggested from the above data.
 Therefore, the lethal agent in steam sterilization is
very hot water and so the readiness of saturated steam
to condense is a tremendous advantage.

45
Saturated Steam: an efficient
sterilizing agent
 When it Condenses it Contracts to an Extremely Small
Volume. At 121°C only 1 ml of water of is produced from the
condensation of 865ml of steam. As a result, a region of low
pressure is created into which more steam rapidly flows. This,
in turn, condenses, gives up its latent heat and contracts, and
the cycle is repeated until the article has been raised to steam
temperature.
 This property of saturated steam ensures quick penetration
throughout bulky porous materials such as surgical dressings.
 The inferiority of hot air in this respect can be explained which
compares the heating up times for a roll of flannel. Hot air
Saturated Steam Air temperature °C Steam temperature °C
Temperature inside roll °C Temperature inside roll °C after 3
hours after 10 minutes
46
Saturated Steam: an efficient
sterilizing agent
To summarize, the advantages of saturated steam as
sterilizing agent are –
i. It flows quickly to and if required into every article in
the load (volume contraction).
ii. It rapidly heats the load to sterilization temperature
(liberation of latent heat).
iii. It provides, at high temperatures, the moisture
essential for killing microorganisms (production of
condensate).

47
Types of Steam
sterilizers/autoclaves
 The apparatus for sterilization by steam under pressure is
called an autoclave or steam sterilizer.
 They are of two types:
i. pressure-controlled
ii. temperature controlled
 In a pressure-controlled type the pressure gauge is the sole
indicator of the internal conditions and therefore, all the
air must be removed before the sterilizing exposure begins
and it is made of aluminium alloy.
 In the temperature-controlled type a thermometer or
thermostat is used to indicate or ensure respectively that
the exposure temperature has been reached and it is not
essential to expel the air. It is made of stainless steel.

48
Autoclave/ Steam Sterilizers

Parts of an autoclave: Autoclaves, or steam sterilizers

essentially consist of following parts:
 i) A cylindrical or rectangular chamber, with capacities
ranging from 400 to 800 liters.
 ii) Water heating system or steam generating system
 iii) Steam outlet and inlet valves
 iv) Single or double doors with locking mechanism.
 v) Thermometer or temperature gauge
 vi) Pressure gauges

49
Operation of an autoclave:
 STEP 1: Decontaminate, clean and dry all instruments and other items
to be sterilized.
 STEP 2: All jointed instruments should be in the opened or unlocked
position, while instruments composed of more than one part or sliding
parts should be disassembled.
 STEP 3: Instruments should not be held tightly together by rubber
bands or any other means that will prevent steam contact with all
surfaces.
 STEP 4: Arrange packs in the chamber to allow free circulation and
penetration of steam to all surfaces.
 STEP 5: When using a steam sterilizer, it is best to wrap clean
instruments or other clean items in a double thickness of muslin.
(Unwrapped instruments must be used immediately after removal from
the sterilizer, unless kept in a covered, sterile container.)
 STEP 6: Sterilize at 121°C for 30 minutes for wrapped items, 20
minutes for unwrapped items; time with a clock.

50
Operation of an autoclave:
 STEP 7: Wait 20 to 30 minutes (or until the pressure gauge reads
zero) to permit the sterilizer to cool sufficiently. Then open the lid
or door to allow steam to escape. Allow instrument packs to dry
completely before removal, which may take up to 30 minutes. (Wet
packs act like a wick drawing in bacteria, viruses and fungi from
the environment.) Wrapped instrument packs are considered
unacceptable if there are water droplets or visible moisture on the
package exterior when they are removed from the steam sterilizer
chamber. If using rigid containers (e.g., drums), close the gaskets.
 STEP 8: To prevent condensation, when removing the packs from
the chamber, place sterile trays and packs on a surface padded with
paper or fabric.
 STEP 9: After sterilizing, items wrapped in cloth or paper are
considered sterile as long as the pack remains clean, dry (including
no water stains) and intact. Unwrapped items must be used
immediately or stored in covered, sterile containers.

51
Advantages of pressure-
controlled autoclaves:
 The venting and constant escape of steam during
exposure ensures the absence of air.
 The lid is easier to fit and the method of fitting doesn’t
reduce the effective depth of the autoclave.
 The pressure regulator is simple and requires less
attention than a thermostat.

52
Advantages of temperature-
controlled autoclaves:
 The internal temperature is controlled and shown.
 The chief material of construction is stainless steel.
Alkaline solution will attack the aluminium alloy of
pressure-controlled autoclave.
 The method of closure is safer because the lid cannot
be removed while steam is at pressure inside. Additional
safety is provided by downward discharge of the vent.
 The wire basket allows easy air drainage and is more
satisfactory than a solid chamber in which there is the
possibility of air layering or pocketing.

53
Large Sterilizers
There are two types of large
sterilizers:
i. Surgical-dressings sterilizer

ii. Sterilizer for bottled fluids

54
1. Surgical-dressings sterilizer
The following stages are involved in the sterilization of surgical dressings:
 1. Suitably packed dressings are correctly loaded into the chamber.
 2. The door is closed, and steam admitted to the jacket.
 3. Air is partially or almost completely removed by the vacuum.
 4. Dry saturated steam is admitted and if necessary, may be used to
displace the rest of the air.
 5. Heating-up and exposure are carried out; air (drained from the
dressings) and condensate are automatically discharged meanwhile.
 6. The supply steam is then cut off and the chamber vented.
 7. The dressings are dried either by drawing a high vacuum or by using a
partial vacuum to suck warm sterile air through them.
 8. When high vacuum drying has been used the vacuum is broken by
admitting sterile air.

55
2. Sterilizer for bottled fluids
The following stages are involved in the sterilization of surgical
dressings:
 1. The bottles are loaded correctly.
 2. The door is closed.
 3. In some modern equipment, air is removed by high vacuum.
 4. Dry saturated steam is admitted to displace the air.
 5. Heating-up and exposure are carried out, air and condensate being
discharged meanwhile.
 6. The supply steam is cut off.
 7. Either (a) The chamber steam is allowed to vent slowly to reduce
the internal pressure to atmospheric OR (b) A fine mist of cold water
is sprayed over the bottles to cool them to safely below 100 °C when
they can be removed immediately.
56
Applications of autoclaving:
 Some of the glass equipment used in aseptic
technique has rubber parts (e.g., rubber closures)
and this must be autoclaved.
 Suitable exposures for glassware and closures are
115°C for 30 minutes (British Pharmacopoeia) 0r
121°C for 15 minutes.
 There are more than 100 official injections, and
the majority is sterilized by autoclaving (B.P
mentions 115 to 116°C for 30 minutes but also
allows a shorter time at a higher temperature
(e.g., 15 min at 121°C if the medicament is
sufficiently thermostable such as sodium chloride).
57
Advantages of autoclaving:
 Autoclaving destroys microorganisms more efficiently than
dry heat and therefore a shorter exposure at a lower
temperature is possible.
 It can be used for a large proportion of the official
injections.
 In a sterilizer supplied with dry saturated steam porous
materials can be sterilized without damage.
 Equipment or components of rubber and certain plastics
such as nylon and P.V.C will withstand the conditions.
 Disadvantages of autoclaving: It is unsuitable for anhydrous
materials such as powders and oils.
 It cannot be used for injections and articles such as some
plastics that deteriorate at 115°C.

58
Testing the efficiency of
sterilizers
 To know whether the equipment is
performing efficiently , certain tests are
needed such as
 Direct test (sterility testing)– Each article
of the load should be tested which is
highly expensive and time consuming.
Moreover very skilled people are required
to prevent contamination.
 Indirect test – are of 3 types. They are:
Instrumental, Cultural, Chemical.
59
Testing the efficiency of
sterilizers
 Indirect tests:
 1. Instrumental test– Temperature, Pressure and Time are the
factors that affect instruments. Thermostat or thermometer are
used to check whether optimum temperature (high temperature
120°C) is maintained. High pressure (1.5 atm) is checked using a
pressure gauge. Whether a fixed time (20 mins) is maintained is
checked using a stopwatch.
 2. Cultural test– The article is put into autoclave with
bacteriospore Bacillus subtilis. The limitation is that Bacillus
subtilis is mesophilic so when it reaches or comes into contact with
boiling water at 100°C it dies. Therefore either soil samples or
thermophilic bacterial spore such as Bacillus stearothermophyllus
is used.

60
Testing the efficiency of
sterilizers
Indirect tests:
3. Chemical test-
 i) Witness tube
 ii) Klintex paper
 iii) Test tablet
i) Witness tube:
 It is a sealed tube either containing acetanilid (melts at 115°C) or
benzoic acid (melts at 121°C). These chemicals melts when sterilizing
temperature is reached. Depending upon the appearance of these
chemical, it is known whether sterilization is complete or not.
 Apart from these chemicals, dye such as methylene blue can be used.
The article + benzoic acid will show one colour before melting and
article + benzoic acid + the dye will show another colour after
melting. The function of the dye is recognition whether melting is
complete.

61
Testing the efficiency of
sterilizers
ii) Klintex paper:
 When the Klintex paper is placed in autoclave, the word autoclave is
displayed in black on the paper against a pale backgound which
confirms sterlization is complete.

iii) Test tablet:

 The tablet contains 75% starch, 24% lactose and 1% magnesium
trisilicate. Before autoclaving, the appearance is white and hard.
After autoclaving, the appearance is brown and gelatinous. This
change occurs at 115°C after 24 minutes.

62
Sterility Testing

A sterility test may be defined as — ‘a test that critically

assesses whether a sterilized pharmaceutical product is
free from contaminating microorganisms’.
Sterility testing are intended for detecting the presence of
viable forms of microorganisms in or on the pharmacopeial
preparations.
All the parenteral products of the B.P must comply with the
sterility testing required for that sample.

63
Preparations for which
sterility test is required:
 Ready-made injections – it includes both solution and
suspension, both aqueous and oily.
 Solids for injection – it includes materials from
biological sources e.g., heparin, hyaluronidase and the
antibiotics.
 Ophthalmic products – eye drops, eye ointments, eye
lotions.
 Water for injections, Human blood and human blood
products, Immunological products – vaccines, antiserum
plants, Surgical sutures

64
 Information given by a
sterility test:
 Sterility means free from living micro-organisms and therefore it
is not possible to claim that a batch of products is sterile unless –
The entire content of every container in the batch has been
tested and The test provides optimum conditions for the growth
and multiplication of every organism, vegetative or spore,
healthy or injured, that might be a contaminant.
 Unfortunately, neither of these conditions can be satisfied
because –In sterility testing the article or preparation under test
is either destroyed (e.g., an injection solution) or made
unusable (e.g., a syringe); therefore, only part of the batch is
sampled.
 Even great care is taken to provide media and incubation
conditions satisfactory for most organisms it is impossible to
supply all the variations necessary to ensure that every type and
condition of contaminant will grow.

65
Information given by a
sterility test:
 Therefore, Sterility testing should not be used as
the sole means of controlling sterile processing.
Heat sterilization methods can be checked
instrumentally and bacteriologically and
procedures involving asepsis may be controlled by
careful supervision of operatives, regular air
sampling (within and outside the screen) and full–
scale runs using nutrient broth.
 To obtain suitable samples from sterility tests it is
necessary to take sufficient samples, to use
sensitive culture media and during testing, to
reduce accidental contamination to a minimum.
66
Precautions against
accidental contamination:
 Ventilated aseptic room supplied with bacteriologically
cleaned air, Highly trained staff.
 Adequate control test should be performed at the
same time.
 In case of a negative result: sterility of the sample is
confirmed.
 In case of a positive result: contamination of the sample.

67
CONTROL TESTS Negative control
– in these no growth is expected.

 A container of medium from each batch used for the test is

incubated at the same time as the test containers. This control serves
three purposes :
i. - It confirms that the medium is sterile.
ii. - It shows that the oxidation-reduction qualities of the indicator-containing
anaerobic media are satisfactory. If they are not, the colour quickly spreads
down from the surface of the medium.
iii. - It serves as a standard with which the corresponding test container can be
compared during and after incubation.
 A faint turbidity is more easily detected if the suspect tube is examined
at the side of the control. Any substance, other than the sample, added
to the test tube should be proved sterile by incubating suitable
amounts in appropriate media. An example is penicillinase, a solution
of which has been used in testing certain penicillins.

68
CONTROL TESTS: Positive Controls –
in these growth is expected.
 The sensitivity of the media must be confirmed. Each type of medium is
inoculated with an appropriate organism (i.e., an exacting aerobe,
anaerobe or yeast) and after incubation under suitable conditions is
examined for growth. The European Pharmacopoeia suggests that
Staphylococcus aureus as the aerobe, Clostridium sphenoides as the
anaerobe and Candida albicans as the yeast.
 When a medium capable of detecting aerobes and anaerobes and fungi is
used for the test the different organisms should be added to a separate
control containers because if they are inoculated into the same one it
may not be possible to decide whether both have grown or not.
 The medium must be shown capable of supporting the growth of small
numbers of bacteria in the presence of the sample.
 The bacteria are incubated at 30 to 32°C for 7 days and yeast 22 to 25
°C for 2 days.
69
CONTROL TESTS
Controls to check working conditions and operator’s
technique –
General air sampling: This shows that the high quality of
the air supply to the room is being maintained.
Air sampling at each working space: Settling plates under
and near the screen help to detect poor technique and
particularly excess movement.
‘Dummy runs’: Tests are performed with materials known
to be sterile e.g., ampoules or bottles of Water for
Injections or sodium chloride that have been sterilized for
longer times and/ or higher temperatures than normal.

70
Microbial Count Air:
 Air itself contain microorganism but it cannot
produce microorganism. Since air does not contain
any nutrient material and it should not allow
microorganism to grow in it. However, only those
microorganisms that can tolerate desiccation and
that can tolerate continuing dry state are present
in air. Microorganisms that are present in air are:
 1. Spore forming bacteria such as Bacillus and
Clostridium
 2. Non-spore forming bacteria such as
corynebacterium, staphylococcus, streptococcus
 3. Certain moulds
71
Methods to determine the
condition of air :
 Exposure of a petri-dish containing nutrient agar to
air and then from the observation of the result
(number and shape and size of the colonies),
conclusion can be drawn whether air is contaminated
or not.
 By air sampling machine – The air collected by air
sampling machine is taken on a Petri dish containing
nutrient agar or on plastic strip or in membrane
filter. In case of using plastic strip and membrane
filter, these should be inoculated in a media that
contains nutrient. Highly dense colonies represent
more contamination.
72
Principle of sterility testing:

 Sterility tests are exclusively based upon

the principle that in case the bacteria
are strategically placed in a specific
medium that caters for the requisite
nutritive material and water and
maintained at a favorable temperature
(37 ± 2°C), the microbes tend to grow,
and their legitimate presence may be
clearly indicated by the appearance of a
turbidity in the originally clear medium.

73
Principle of sterility testing:

 Tests for sterility are adequately designed to reveal the

presence of microorganisms in the ‘samples’ used in the
tests.
 However, the interpretation of results is solely based
upon the assumption that the contents of each and
every container in the batch, had they been tested
actually, would have complied with the tests.
 As it is not practically possible to test every container, a
sufficient number of containers must be examined to
give a suitable degree of confidence in the ultimate
results obtained of the tests.

74
Aseptic Processing:

 Aseptic processing is concerned with the preparation of

those sterile products that cannot be subjected to a
terminal heating process because the medicaments they
contain are thermolabile.
 The most important example is Sterilization by
Filtration.
 Other examples include: the packaging of thermolabile
solids for injection the preparation of injections from
such solids the preparation of sterile dusting powders
containing thermolabile medicaments the preparation
of eye ointments.

75
Sterilization by Filtration:
 Filtration process does not destroy but removes the
microorganisms.
 It is used for both the clarification and sterilization of liquids
and gases as it can prevent the passage of both viable and
non-viable particles.
 The major mechanisms of filtration are sieving, adsorption
and trapping within the matrix of the filter material.
 Sterilizing grade filters are used in the treatment of heat
sensitive injections and ophthalmic solutions, biological
products and air and other gases for supply to aseptic areas.
 They are also used in industry as part of the venting systems
on fermenters, centrifuges, autoclaves and freeze driers.
Membrane filters are used for sterility testing.

76
 Sterilization by Filtration:
 Application of filtration for sterilization of gases: HEPA (High efficiency
particulate air) filters can remove up to 99.97% of particles >0.3
micrometer in diameter.
 Air is first passed through prefilters to remove larger particles and then
passed through HEPA filters.
 The performance of HEPA filter is monitored by pressure differential
and airflow rate measurements.
 Application of filtration for sterilization of liquids: Membrane filters of
0.22 micrometer nominal pore diameter are generally used, but
sintered filters are used for corrosive liquids, viscous fluids and organic
solvents.
 The factors which affects the performance of filter is the titre
reduction value, which is the ratio of the number of organism
challenging the filter under defined conditions to the number of
organism penetrating it. The other factors are the depth of the
membrane, its charge and the tortuosity of the channels.
77
Sterilization by Filtration:
 Filtration through a bacteria-proof filter is a
suitable method for the sterilization of injections
containing thermolabile medicaments.
 However, the solid or medicament must be stable in
solution or compatible with water.
 The process involves four stages :
1. Filtration of the solution through a bacteria-proof
filter.
2. Aseptic distribution of the filtered solution into
previously sterilized containers.
3. Aseptic closure of the containers.
4. Testing of samples for sterility.

78
Classification of bacteria-
proof filter:
There are four classes:
 Sintered ceramics – It is made from finely
ground porcelain. (may be used several times)
 Fibrous pads – containing asbestos and wood
cellulose. (one time use)
 Sintered glass – made from borosilicate glass
(may be used several times).
 Microporous plastics – prepared from cellulose
esters, particularly the acetate or nitrate (one
time use).
79
Filtration techniques in
sterility testing:
 Contaminants are removed from the sample by filtration through a
sterile bacteria-proof filter pad.
 Then bactericides and inhibitory medicaments are removed from
the organisms and filter by washing with a sterile solvent and
 finally the whole of the pad incubated in a suitable culture
medium.
 Previously asbestos-cellulose filter pad was used which has been
replaced by a membrane filter because the membranes are so thin
that the retention of inhibitory substances is very small.
 Asbestos-cellulose pads are relatively thick and fibrous and
therefore may absorb and retain sufficient inhibitor to cause
bacteriostasis in the culture medium.
 Quick filtration. Oil pass through easily and quite quickly and
there is no need to dissolve them in an organic solvent first.

80
Advantages of sterilization by
filtration:
 Wide application. They can be used for Solutions with or without
inhibitory properties. Soluble solids with or without inhibitory
properties. Insoluble solids without inhibitory properties. Oils
Ointments, provided a non-inhibitory solvent or dispersing medium can
be found.
 Articles, such as syringes that can be rinsed with a sterile fluid. A very
large volume can be tested with one pad. Therefore, the method is
applicable to the testing of poorly soluble solids. A much smaller
volume of broth is required than for testing by direct inoculation
into the culture media. They are applicable to substances for which no
satisfactory inactivators are known e.g., many antibiotics.
 Some strongly adsorbed antibacterial agents such as the mercurial
and the quaternary ammonium compounds can be inactivated on the
filter by treatment with the appropriate neutralizing solution.
Subculturing is often eliminated e.g., for oils and oily preparations.
81
Disadvantages of sterilization
by filtration:
 Even with membrane filters the possibility of
adsorption of sufficient medicament cannot be
disregarded.
 Highly skilled staff and
 exceptionally good aseptic techniques are necessary.

82
Tests for Sterility:
 Tests for sterility are carried out by two methods:
(a) Membrane Filtration Method
(b) Direct Transfer / Inoculation Method.
 The Membrane Filtration Method is used as the method
of choice wherever feasible.
 Media used in Sterility Testing:
 Fluid Thioglycollate Medium (Medium 1) and
 Soybean-Casein Digest Medium (Medium 2) are the two
media generally used for tests for sterility.

83
 Tests for Sterility: 1. Method of
Membrane
Procedure
Filtration
 The filter should be a membrane filter disc of cellulose esters or other
suitable plastics, having a nominal average pore diameter not exceeding
0.45 μm.
 The membrane should be held firmly in a filtration unit which consists of a
supporting base for the membrane, a receptacle for the fluid to be tested, a
collecting reservoir for the filtered fluid, and the necessary tubes or
connections.
 The apparatus is so designed that the solution to be filtered can be
introduced and filtered under aseptic conditions.
 It permits the aseptic removal of the membrane for transfer to medium
or it is suitable for carrying out the incubation after adding the medium to
the apparatus itself.
 Cellulose nitrate filters are recommended for aqueous, oily and weakly
alcoholic solutions and cellulose acetate filters for strongly alcoholic
solutions.
 The entire unit should be sterilized by appropriate means with the
membrane filter and sterile airways in place.
 The method of sterilization should not be deleterious to the membrane,
e.g., weaken it or change the nominal average pore diameter.84
 Tests for Sterility: 2. Method
of Direct Transfer Procedure:
Liquids and soluble or dispersible solids:
 Appropriate quantities of the preparation to be examined are added
directly into Medium 1 and Medium 2.
 Approximately equal quantities of the preparation should be added to each
vessel of medium.
 The test vessels of Medium 1 is incubated at -°C and the vessels of Medium 2
is incubated at -°C.
 The volume of Medium 1 should be such that the air space above the medium
in the container is minimized.
 The volume of Medium 2 should be such that sufficient air space is left above
the medium to provide conditions that permit the growth of obligate aerobes.
 Unless otherwise prescribed, in no case should the volume of material under
test be greater than 10% of the volume of the medium alone, i.e., 90%
medium and 10% product.
 If a large volume of product is to be tested it may be preferable to use
concentrated media, prepared so as to take the subsequent dilution into
account. 85
 Tests for Sterility:
 Where appropriate the concentrated medium may be added directly
to the product in its container.
 Wherever possible solid articles such as devices should be tested by
immersion in or filling with culture media.
 Immerse all parts of each article in sufficient medium contained in
one vessel to completely cover all parts.
 The volume of Medium 1 should be such that the air space above the
medium in the container is minimized.
 The volume of Medium 2 should be such that sufficient air space is left
above the medium to provide conditions that permit the growth of
obligate aerobes.
 Place half the articles into Medium 1 and the remaining half into
Medium 2.
 Incubate the test vessels of Medium 1 at °C and the vessels of Medium
2 at °C.
 Ointments and oily preparations: Ointments and oily preparations may
be tested by the method of Direct Transfer if testing by the method of
Membrane Filtration is not feasible, i.e., when a suitable 86solvent is not
available .
 Tests for Sterility: Incubation and
examination of sterility tests:
 All test vessels of Medium 1 are incubated at °C.
 The vessels of Medium 2 are incubated at °C.
 All test and control vessels, other than the subculture vessels referred to
below, must be incubated for at least 14 days unless microbial
contamination is detected at an earlier time.
 If turbidity, precipitate, or other evidence of microbial growth during
incubation is seen: the suspected growth is examined microscopically by
Gram stain; colonies of each type of micro-organism present are examined
for colonial morphology and cellular morphology by Gram stain.
 Interpretation of the test results: If microbial growth is not evident in any
of the vessels inoculated with the product, the sample tested complies
with the test for sterility, if microbial growth is evident the product does
not comply with the test for sterility unless it can be clearly demonstrated
that the test was invalid for causes unrelated to the product being
examined.
87
Sterilization by Gas:
 Sterilization by gas involves sterilization with a chemical in the
gaseous state.
 The chemically reactive gases such as formaldehyde, (methanol,
H.CHO) and ethylene oxide (CH2) 2O possess biocidal activity.
 Ethylene oxide is a colorless, odorless, and flammable gas.
 The mechanism of antimicrobial action of the two gases is assumed to
be through alkylations of sulphydryl, amino, hydroxyl and carboxyl
groups on proteins and amino groups of nucleic acids.
 The concentration ranges (weight of gas per unit chamber volume) are
usually in range of mg/L for ethylene oxide and mg/L for formaldehyde
with operating temperatures of 45-63°C and 70-75°C respectively.
 Both of these gases being alkylating agents are potentially mutagenic
and carcinogenic.
 They also produce acute toxicity including irritation of the skin,
conjunctiva and nasal mucosa.

88
 Sterilization by Gas: Ethylene
oxide sterilizer:
 An ethylene oxide sterilizer consists of a chamber of Litre capacity and
surrounded by a water jacket.
 Air is removed from sterilizer by evacuation, humidification and
conditioning of the load is done by passing sub-atmospheric pressure
steam, then evacuation is done again and preheated vaporized ethylene
oxide is passed.
 After treatment, the gases are evacuated either directly to the outside
atmosphere or through a special exhaust system.
 Ethylene oxide gas has been used widely to process heat-sensitive
devices, but the aeration times needed at the end of the cycle to
eliminate the gas made this method slow.
 Low temperature steam formaldehyde (LTSF) sterilizer: An LTSF
sterilizer operates with sub atmospheric pressure steam. At first, air is
removed by evacuation and steam is admitted to the chamber.
89
Plasma Sterilization Hydrogen
Peroxide Sterilization:
 This method disperses a hydrogen peroxide solution in a vacuum chamber,
creating a plasma cloud.
 This agent sterilizes by oxidizing key cellular components, which inactivates the
microorganisms.
 The plasma cloud exists only while the energy source is turned on. When the
energy source is turned off, water vapor and oxygen are formed, resulting in no toxic
residues and harmful emissions.
 The temperature of this sterilization method is maintained in the 40-50°C range,
which makes it particularly well-suited for use with heat-sensitive and moisture-
sensitive medical devices. The instruments are wrapped prior to sterilization and can
either be stored or used immediately.
 An advantage of the plasma method is the possibility, under appropriate
conditions, of achieving such a process at relatively low temperatures (≤50 °C),
preserving the integrity of polymer-based instruments, which cannot be subjected to
autoclaves and ovens.
 Furthermore, plasma sterilization is safe, both for the operator and the patient, in
contrast to EtOH.
90
Sterilization by Radiation:
 Two types of radiation are used:

1. Ionizing and
2. Non-ionizing.
 Non-ionizing rays are low energy rays with poor penetrative
power while
 Ionizing rays are high-energy rays with good penetrative power.
 Since radiation does not generate heat, it is termed "cold
sterilization".

91
 Sterilization by Radiation:
Non-ionizing rays:
 Rays of wavelength longer than the visible light are non-ionizing.
Microbicidal wavelength of UV rays lie in the range of nm, with 260 nm being
most effective.
 UV rays are generated using a high-pressure mercury vapor lamp. It is at
this wavelength that the absorption by the microorganisms is at its
maximum, which results in the germicidal effect.
 UV rays induce formation of thymine-thymine dimers, which ultimately
inhibits DNA replication.
 UV readily induces mutations in cells irradiated with a non-lethal dose.
 Microorganisms such as bacteria, viruses, yeast, etc. that are exposed to
the effective UV radiation are inactivated within seconds.
 Since UV rays don’t kill spores, they are considered to be of use in surface
disinfection.
 Disadvantages of using UV rays include low penetrative power, limited life
of the UV bulb, some bacteria have DNA repair enzymes that can
overcome damage caused by UV rays, organic matter and dust prevents its
reach, rays are harmful to skin and eyes. It doesn't penetrate glass, paper
or plastic.
92
Sterilization by Radiation:
Ionizing rays:
 Ionizing rays are of two types, particulate and electromagnetic rays.
 Electron beams are particulate in nature while gamma rays are
electromagnetic in nature. High-speed electrons are produced by a linear
accelerator from a heated cathode. Electron beams are employed to sterilize
articles like syringes, gloves, dressing packs, foods and pharmaceuticals.
Sterilization is accomplished in few seconds. Unlike electromagnetic rays, the
instruments can be switched off. Disadvantage includes poor penetrative
power and requirement of sophisticated equipment.
 Electromagnetic rays such as gamma rays emanate from nuclear disintegration
of certain radioactive isotopes (Co 60, Cs 137). They have more penetrative
power than electron beam but require longer time of exposure. These high-
energy radiations damage the nucleic acid of the microorganism. A dosage of
2.5 megarads kills all bacteria, fungi, viruses and spores. It is used
commercially to sterilize disposable petri dishes, plastic syringes,
antibiotics, vitamins, hormones, glasswares and fabrics. Disadvantages
include; unlike electron beams, they can’t be switched off, glasswares tend to
become brownish, loss of tensile strength in fabric. Bacillus pumilus E601 is
used to evaluate sterilization process.

93
THANK you

94
Pharmaceutical analysis

Dr. Paul Wanjala Muyoma

 Introduction
 All pharmaceutical finished products undergo
rigorous QC testing in order to confirm their
conformance to predetermined specifications.
 Potency testing is of obvious importance, ensuring
that the drug will be efficacious when administered
to the patient.
 A prominent aspect of safety testing entails analysis
of product for the presence of various potential
contaminants (Table 1).
 An overview of the range of finished-product tests of
recombinant protein biopharmaceuticals is outlined
below.
Table 1 The range and medical significance of potential impurities present
in biopharmaceutical products destined for parenteral administration

Impurity Medical consequence

Microorganisms Potential establishment of a
severe microbial infection
Viral particles Potential establishment of a
severe viral infection
Pyrogenic substances Fever response that, in serious
cases, culminates to death
DNA Significance is unclear- could
bring about an immunological
response
Contaminating proteins Immunological reactions.
Potential adverse effects if the
contaminant exhibits an
unwanted biological activity
 Protein-based contaminants
 Most of the chromatographic steps undertaken during
downstream processing are specifically included to
separate the protein of interest from additional
contaminant proteins.
 Proteins may be introduced during upstream or
downstream processing.
 For example, animal cell culture media are typically
supplemented with bovine serum/fetal calf serum (2–
25 per cent), or with a defined cocktail of various
regulatory proteins required to maintain and stimulate
growth of these cells.
 Protein-based contaminants
 Downstream processing of intracellular microbial
proteins often requires the addition of endonucleases to
the cell homogenate to degrade the large quantity of DNA
liberated upon cellular disruption.
 The clinical significance of protein-based impurities
relates to
(a) their potential biological activities and
(b) their antigenicity.
 Whereas some contaminants may display no undesirable
biological activity, others may exhibit activities
deleterious to either the product itself (e.g., proteases that
could modify/degrade the product) or the recipient patient
(e.g., the presence of contaminating toxins)
Protein-based contaminants
 Their inherent immunogenicity also renders likely and
immunological reaction against protein-based
impurities upon product administration to the recipient
patient.
 Although the product itself is likely to be non-
immunogenic (usually being coded for by a human
gene), contaminant proteins will be endogenous to the
host cell, and hence foreign to the human body.
 This is particularly likely if a requirement exists for
ongoing, repeat product administration (e.g.,
administration of recombinant insulin)
 Immunological activation of this type could also
potentially (and more seriously) have a sensitizing
effect on the recipient against the actual protein
product.
Protein-based contaminants
 In addition to distinct gene products, modified forms of
the protein of interest are also considered impurities,
rendering desirable their removal from the product
stream.
 Modified product “impurities” may compromise the
product in a number of ways, e.g.:-
1. biologically inactive forms of the product will reduce
overall product potency;
2. some modified product forms remain biologically active,
but exhibit modified pharmacokinetic characteristics
(i.e., timing and duration of drug action);
3. modified product forms may be immunogenic.
 Removal of altered forms of the
protein of interest from the
product stream
 Modification of any protein will generally alter
some aspect of its physicochemical
characteristics.
 This facilitates removal of the modified form by
standard chromatographic techniques during
downstream processing.
 Most downstream procedures for protein-based
biopharmaceuticals include both gel-filtration
and ion-exchange steps.
Removal of altered forms of the
protein of interest from the
product stream
 Aggregated forms of the product will be effectively removed
by gel filtration (because they now exhibit a molecular mass
greater by several orders of magnitude than the native
product).
 This technique will also remove extensively proteolysed forms
or glycoprotein variants of the product.
 Disulfide bond formation, partial denaturation and limited
proteolysis can also alter the shape and surface charge of
proteins, facilitating their removal from the product by ion
exchange or other techniques, such as hydrophobic
interaction chromatography.
Table 2: Methods used to characterize (protein-based) finished product
biopharmaceuticals. An overview of most of these methods is presented over the
next several sections
S/N
1. Non-denaturing gel electrophoresis

2. Denaturating (SDS) gel electrophoresis

3. Two dimensional electrophoresis

4. Capillary electrophoresis
5. Peptide mapping
6. HPLC (mainly RP-HPLC)
7. Isoelectric focusing
8. Mass spectrometry
9. Amino acid analysis
10. N-terminal sequencing
11. Circular dichroism studies
12. Bioassays and immunological assays
 Product potency
 Any biopharmaceutical must obviously conform
to final product potency specifications.
 Such specifications are usually expressed in
terms of ‘units of activity’ per vial of product
(or per therapeutic dose, or per milligram of
product).
 Bioassays represent the most relevant potency-
determining assay, as they directly assess the
biological activity of the biopharmaceutical.
 Bioassay involves applying a known quantity of
the substance to be assayed to a biological
system that responds in some way to this
applied stimulus.
 Product potency
 The response is measured quantitatively, allowing an
activity value to be assigned to the substance being
assayed.
 An example of a straightforward bioassay is the
traditional assay method for antibiotics (disc or well
diffusion methods).
 The biological system used can be whole animals,
specific organs or tissue types, or individual
mammalian cells in culture.
 Bioassays of related substances can be quite similar in
design.
 Specific growth factors, for example, stimulate the
accelerated growth of specific animal cell lines.
Product potency
 Relevant bioassays can be undertaken by incubation of
the growth-factor-containing sample with a culture of
the relevant sensitive cells and radiolabeled
nucleotide precursors.
 After an appropriate time period, the level of
radioactivity incorporated into the DNA of the cells is
measured.
 This is a measure of the bioactivity of the growth
factor.
 The most popular bioassay of EPO involves a mouse-
based bioassay (EPO stimulates red blood cell
production, making it useful in the treatment of
certain forms of anemias).
 Basically, the EPO-containing sample is administered
to mice along with radioactive iron (57Fe).
 Product potency
 Subsequent measurement of the rate of incorporation
of radioactivity into proliferating red blood cells is
undertaken.
 Although bioassays directly assess product potency
(i.e., activity), they suffer from several drawbacks,
including:
1. Lack of precision. The complex nature of any
biological system, be it an entire animal or individual
cell, often results in the responses observed being
influenced by factors such as metabolic status of
individual cells, or (in the case of whole animals)
subclinical infections, stress levels induced by human
handling, etc.
 Product potency
2. Time. Most bioassays take days, and in some cases
week, to run.
3. Cost. Most bioassay systems, in particular those
involving whole animals, are extremely expensive to
undertake.
 Because of such difficulties alternative assays have been
investigated, and sometimes are used in conjunction
with, or instead of, bioassays.
 The most popular alternative assay system is the
immunoassay.
 Immunoassays employ monoclonal or polyclonal
antibody preparations to detect and quantify the
product.
 The specificity of antibody–antigen interaction ensures
good assay precision.
Product potency
 The use of conjugated radiolabels (RIA) or enzymes
(EIA) to allow detection of antigen–antibody binding
renders such assays very sensitive.
 Furthermore, when compared with a bioassay,
immunoassays are rapid (undertaken in minutes to
hours), inexpensive, and straightforward to
undertake.
 In most such systems, the antibody is immobilized on
the internal walls of the wells in a multi-well microtitre
plate, which therefore serves as collection of reaction
mini-test tubes.
 One of the most popular EIA systems currently in use is
that of the ELISA (Figure 1)
 In this form it is also often referred to as the double
antibody sandwich technique.
Figure 1: Basic principle of ELISA system
 Determination of protein
concentration
 Quantification of total protein in the final product
represents another standard analysis undertaken by QC.
 Several different protein assays may be potentially
employed (Table 2).
 Detection and quantification of protein by measuring
absorbency at 280 nm is perhaps the simplest such
method.
 This approach is since the side chains of the amino
acids tyrosine and tryptophan absorb at this
wavelength.
 Determination of protein
concentration
 The method is popular, as it is fast, easy to perform
and is non-destructive to the sample.
 However, it is a relatively insensitive technique, and
identical concentrations of different proteins will
yield different absorbance values if their content of
tyrosine and tryptophan vary to any significant
extent.
 Hence, this method is rarely used to determine the
protein concentration of the final product, but it is
routinely used during downstream processing to
detect protein elution off chromatographic columns,
and hence track the purification process.
Table 2
 Detection of protein-based product
impurities:-
 SDS polyacrylamide gel electrophoresis (SDS-PAGE)
represents the most used analytical technique in the assessment of
final product purity.
 It provides high-resolution separation of polypeptides on the
basis of their molecular mass.
 Bands containing as little as 100 ng of protein can be
visualized by staining the gel with dyes such as Coomassie
blue.
 Subsequent gel analysis by scanning laser densitometry
allows quantitative determination of the protein content of each
band.
 Detection of protein-based product
impurities:-
 The use of silver-based stains increases the
detection sensitivity up to 100-fold, with
individual bands containing as little as 1ng of
protein usually staining well.
 However, because silver binds to protein non-
stoichiometrically, quantitative studies using
densitometry cannot be undertaken.
 SDS-PAGE is normally run under reducing
conditions.
 Addition of a reducing agent such as β-
mercaptoethanol or dithiothreitol (DTT) disrupts
interchain (and intrachain) disulfide linkages.
 Individual polypeptides held together via disulfide
linkages in oligomeric proteins will thus separate
from each other on the basis of their molecular
mass.
 Detection of protein-based product
impurities:-
 The presence of bands additional to those
equating to the protein product generally
represent protein contaminants.
 Such contaminants may be unrelated to the
product or may be variants of the product itself
(e.g., differentially glycosylated variants,
proteolytic fragment, etc.).
 Further characterization may include western
blot analysis.
 This involves eluting the protein bands from the
electrophoretic gel onto a nitrocellulose filter.
 The filter can then be probed using antibodies
raised against the product.
 Binding of the antibody to the ‘contaminant’
bands suggests that they are variants of the
product.
 Detection of protein-based product
impurities:-

 One concern relating to SDS-PAGE-based purity

analysis is that contaminants of the same molecular
mass as the product will go undetected as they will
comigrate with it.
 Two-dimensional electrophoretic analysis would
overcome this eventuality in most instances.
 The most commonly utilized method entails
separation of proteins by isoelectric focusing (see
below) in the first dimension, with separation in the
second dimension being undertaken in the presence of
SDS, thus promoting band separation on the basis of
protein size.
 Detection of protein-based product
impurities:- Isoelectric focusing

 Isoelectric focusing entails setting up a pH gradient

along the length of an electrophoretic gel.
 Applied proteins will migrate under the influence
of an electric field until they reach a point in the
gel at which the pH equals the protein’s isoelectric
point pI (the pH at which the protein exhibits no
overall net charge; only species with a net charge
will move under the influence of an electric field).
 Isoelectric focusing thus separates proteins on the
basis of charge characteristics.
 Detection of protein-based product
impurities:- Isoelectric focusing
 This technique is also utilized in the
biopharmaceutical industry to determine
product homogeneity.
 Homogeneity is best indicated by the
appearance in the gel of a single protein
band, exhibiting the predicted pI value.
 Isoelectric focusing also finds application in
analysing the stability of biopharmaceuticals
over the course of their shelf life.
Capillary electrophoresis:-
 Separation is based upon different rates of
protein migration upon application of an electric
field.
 This separation occurs within a capillary tube
with a diameter of 20–50 μm and be up to a 1 m
long.
 This, in turn, allows operation at a higher
current density, thus speeding up the rate of
migration through the capillary.
 Sample analysis can be undertaken in 15–30 min,
and on-line detection at the end of the column
allows automatic detection and quantification of
eluting bands.
 The speed, sensitivity, high degree of automation
and ability to quantitate protein bands directly
render this system ideal for biopharmaceutical
analysis.
 High-performance liquid
chromatography:-
 Most of the chromatographic strategies used to separate
proteins under ‘low pressure’ (e.g., gel filtration, ion
exchange, etc.) can be adapted to operate under high
pressure.
 Reverse-phase-, size-exclusion- and, to a lesser extent,
ion-exchange-based HPLC chromatography systems are
now used in the analysis of a range of biopharmaceutical
preparations.
 On-line detectors (usually a UV monitor set at 220 or 280
nm) allows automated detection and quantification of
eluting bands.
HPLC is characterized by a number of features that
render it an attractive analytical tool. These
include:

 excellent fractionation speeds (often just

minutes per sample);
 superior peak resolution;
 high degree of automation (including data
analysis);
 ready commercial availability of various
sophisticated systems
 Mass spectrometry:-

 It is now possible to determine the molecular

mass of many proteins to within an accuracy
of +/-0.01 per cent.
 A protein variant missing a single amino acid
residue can easily be distinguished from the
native protein in many instances.
 Immunological approaches to
detection of contaminants:-

 Most recombinant biopharmaceuticals are produced in

microbial or mammalian cell lines.
 Thus, although the product is derived from a human
gene, all product-unrelated contaminants will be
derived from the producer organism.
 These non-self proteins are likely to be highly
immunogenic in humans, rendering their removal
from the product stream especially important.
 Immunological approaches to
detection of contaminants:-

 Immunoassays have found widespread

application in detecting and quantifying
product impurities.
 These assays are extremely specific and very
sensitive, often detecting target antigen down
to parts per million levels.
 Many immunoassays are available
commercially, and companies exist that will
rapidly develop tailor-made immunoassay
systems for biopharmaceutical analysis.
 Additional tests to detection of
contaminants:-
 Application of the analytical techniques
discussed thus far focuses upon detection of
proteinaceous impurities.
 A variety of additional tests are undertaken
that focus upon the active substance itself.
 Tests performed to verify the product
identity include;
i. amino acid analysis,
ii. peptide mapping,
iii. N-terminal sequencing and
iv. spectrophotometric analyses.
 Amino acid analysis:-
 Amino acid analysis remains a characterization
technique undertaken in many laboratories, in
particular if the product is a peptide or small
polypeptide (molecular mass ≤10 kDa.).
 The strategy is simple:
 Determine the range and quantity of amino
acids present in the product and compare the
results obtained with the expected (theoretical)
values.
 The results should be comparable.
 Although this technique is relatively
straightforward and automated amino
acid analyzers are commercially
available.
 It is subject to several disadvantages that
limits its usefulness in biopharmaceutical
analysis. These include:
1. Hydrolysis conditions can destroy/modify
certain amino acid residues,
2. The method is semi-quantitative rather
than quantitative;
3. Sensitivity is at best moderate; low-level
contaminants may go undetected.
 Peptide mapping:-
 A major concern relating to biopharmaceuticals
produced in high-expression recombinant
systems is the potential occurrence of point
mutations in the product’s gene, leading to an
altered primary structure (i.e., amino acid
sequence).
 The approach most used to detect alterations
in amino acid sequence is peptide (fingerprint)
mapping.
 Peptide mapping:-
 Peptide mapping entails exposure of the protein
product to a reagent that promotes hydrolysis of
peptide bonds at specific points along the protein
backbone.
 This generates a series of peptide fragments.
 These fragments can be separated from each other
by a variety of techniques, including one- or two-
dimensional electrophoresis, and RP-HPLC in
particular.
 A standardized sample of the protein product when
subjected to this procedure will yield a characteristic
peptide fingerprint, or map,
 Two-dimensional separation of the peptides is
far more likely to resolve each peptide
completely from the others.
 In the case above, for example,
chromatography (in the vertical dimension)
alone would not have been sufficient to resolve
peptides 1 and 3 fully.
 During biopharmaceutical production, each
batch of the recombinant protein produced
should yield identical peptide maps.
 N-terminal sequencing:-
 N-terminal sequencing of the first 20–30
amino acid residues of the protein product
has become a popular quality control test for
finished biopharmaceutical products. The
technique is useful, as it:
1. Positively identifies the protein;
2. Confirms (or otherwise) the accuracy of the
amino acid sequence of at least the N-
terminus of the protein;
3. Readily identifies the presence of modified
forms of the product in which one or more
amino acids are missing from the N-terminus.
 N-terminal sequencing:-
 N-terminal sequencing is normally
undertaken by Edman degradation.
 Facilitate fast and automated
determination of up to the first 100
amino acids from the N-terminus of most
proteins, and usually requires a sample
size of less than 1 μmol to do so.
 Analysis of secondary and
tertiary structure:-
Although a protein’s three-dimensional
conformation may be studied in great detail
by X-ray crystallography or NMR spectroscopy,
routine application of such techniques to
biopharmaceutical manufacture is
impractical, both from a technical and an
economic standpoint.
 More recently proton-NMR has also been
applied to studying higher orders of protein
structure.
 Endotoxin and other
pyrogenic contaminants:-

 Pyrogens are substances that, when they enter the

blood stream, influence hypothalamic regulation of
body temperature, usually resulting in fever and in
severe cases results in patient death.

 Pyrogens represent a diverse group of substances,

including various chemicals, particulate matter and
endotoxin (LPS), a molecule derived from the outer
membrane of Gram-negative bacteria.
 Endotoxin and other
pyrogenic contaminants:-
 In many instances the influence of pyrogens on body
temperature is indirect.
 For example, entry of endotoxin into the bloodstream
stimulates the production of IL-1 by macrophages.
 It is the IL-1 that directly initiates the fever response
(hence its alternative name, ‘endogenous pyrogen’).
 Effective implementation of GMP (good manufacturing
practice) minimizes the likelihood of product
contamination by pyrogens.
 For example, GMP dictates that chemical reagents
used in the manufacture of process buffers be
extremely pure.
 Endotoxin and other
pyrogenic contaminants:-
 Such raw materials, therefore, are unlikely to contain
chemical contaminants displaying pyrogenic activity.
 Furthermore, GMP encourages filtration of virtually all
parenteral products through a 0.45 or 0.22 μm filter
at points during processing and prior to filling in final
product containers (even if the product can
subsequently be sterilized by autoclaving).
 As an additional safeguard, the final product will
usually be subject to a particulate matter test by QC
before final product release.
Contamination of the final product
with endotoxin is more difficult to
control because:-
1. Many recombinant biopharmaceuticals are
produced in Gram-negative bacterial systems;
thus, the product source is also a source of
endotoxin.
2. Most biopharmaceutical preparations will be
contaminated with low levels of Gram-negative
bacteria at some stage of manufacture.
 NB: This is one of many reasons why GMP
dictates that the level of bioburden in the
product stream should be minimized at all
stages of manufacture.
Contamination of the final product
with endotoxin is more difficult to
control because:-

3. The heat stability exhibited by endotoxin

means that autoclaving of process
equipment will not destroy endotoxin
present on such equipment.
4. Adverse medical reactions caused by
endotoxin are witnessed in humans at
dosage rates as low as 0.5 ng per kilogram
body weight.
Endotoxin, the molecule:-
 The structural detail of a generalized
endotoxin (LPS) molecule is presented in
Figure 7.7.
 As its name suggests, LPS consists of a
complex polysaccharide component linked
to a lipid (lipid A) moiety.
 Most of the LPS biological activity
(pyrogenicity) is associated with its lipid A
moiety.
 This usually consists of six or more fatty
acids attached directly to sugars such as
glucosamine.
Pyrogen detection:-
 Pyrogens may be detected in parenteral
preparations (or other substances) by several
methods.
 Two such methods are widely employed in the
pharmaceutical industry.
1. The rabbit pyrogen test:
 This entails parenteral administration of the
product to a group of healthy rabbits, with
subsequent monitoring of rabbit temperature using
rectal probes.
 Increased rabbit temperature above a certain point
suggests the presence of pyrogenic substances.
 Pyrogen detection:-
 The product is considered to have passed the test if
the total (summed) increase of the temperature of all
three animals (rabbits) is less than 1.15 °C.
 If the total increase recorded is greater than 2.65 °C
then the product has failed.
 However, it is also subject to a few disadvantages,
including:
a. it is expensive (there is a requirement for animals, animal facilities
and animal technicians);
b. excitation/poor handling of the rabbits can affect the results
obtained, usually prompting a false positive result;
c. subclinical infection/poor overall animal health can also lead to false
positive results.
d. Use of different rabbit colonies/breeds can yield variable results.
e. Another issue of relevance is that certain biopharmaceuticals (e.g.,
cytokines such as 1L-1 and TNF) themselves induce a natural
pyrogenic response.
 Pyrogen detection:-

2. In vitro assay; the Limulus ameobocyte lysate

(LAL) test.
 This is based upon endotoxin-stimulated
coagulation of amoebocyte lysate obtained
from horseshoe crabs.
 This test is now the most widely used assay for
the detection of endotoxins in
biopharmaceutical and other pharmaceutical
preparations.
Pyrogen detection:-
 Development of the LAL assay was based upon the
observation that the presence of Gram-negative
bacteria in the vascular system of the American
horseshoe crab, Limulus polyphemus, resulted in the
clotting of its blood.
 Tests on fractionated blood showed that the factor
responsible for coagulation resided within the crab’s
circulating blood cells, i.e., the amoebocytes.
 Further research revealed that the bacterial agent
responsible for initiation of clot formation was
endotoxin.
 Pyrogen detection:-
 The endotoxin molecule activates a coagulation cascade
quite similar in design to the mammalian blood
coagulation cascade (Figure 7.8).
 Activation of the cascade also requires the presence of
divalent cations such as calcium or magnesium.
 The LAL reagent is prepared by extraction of blood
from the horseshoe crab, followed by isolation of its
amoebocytes by centrifugation.
 After a washing step, the amoebocytes are lysed and the
lysate dispensed into pyrogen-free vials.
 The assay is normally performed by making a series of
1:2 dilutions of the test sample using (pyrogen-free)
WFI-water for injections-(and pyrogen-free test tubes).
Pyrogen detection:-

 A reference standard endotoxin preparation is treated

similarly.
 LAL reagent is added to all tubes, incubated for 1 h,
and these tubes are then inverted to test for gel (i.e.,
clot) formation, which would indicate presence of
endotoxin.
 More recently, a colorimetric-based LAL procedure
has been devised.
 This allows spectrophotometric analysis of the test
sample, facilitating more accurate end-point
determination.
 The LAL system displays several advantages
when compared with the rabbit test, most
notably:-

1. Sensitivity – endotoxin levels as low as a few

picograms per millilitre of sample assayed will
be detected.
2. Cost – the assay is far less expensive than the
rabbit assay.
3. Speed – depending upon the format used, the
LAL assay may be conducted within 15–60 min.
 Its major disadvantage is its selectivity: it only
detects endotoxin-based pyrogens.
 Removing of Endotoxin:-

 Endotoxin present in the earlier stages of production is

often effectively removed from the product during
chromatographic fractionation.
 The endotoxin molecule’s highly negative charge often
facilitates its effective removal from the product stream
by ion-exchange chromatography.
 Gel-filtration chromatography also serves to remove
endotoxin from the product.
 Although individual LPS molecules exhibit an average
molecular mass of less than 20 kDa, these molecules
aggregate in aqueous environments and generate
supramolecular structures of molecular mass 100–1000
kDa.
 Removing of Endotoxin:-

 The molecular mass of most biopharmaceuticals is

considerably less than 100 kDa (Table 7.4).
 The proteins would thus elute from gel-filtration
columns much later than contaminating endotoxin
aggregates.
 Should the biopharmaceutical exhibit a molecular
mass approaching or exceeding 100 kDa, then
effective separation can still be achieved by
inclusion of a chelating agent such as EDTA in the
running buffer.
 This promotes depolymerization of the endotoxin
aggregates into monomeric (20 kDa) form.
DNA as a contaminant:
 The clinical significance of DNA-based
contaminants in biopharmaceutical products
remains unclear.
 The concerns relating to the presence of DNA in
modern biopharmaceuticals focus primarily
upon the presence of active oncogenes in the
genome of several producer cell types (e.g.,
monoclonal antibody production in hybridoma
cell lines).
 Parenteral administration of DNA contaminants
containing active oncogenes to patients is
considered undesirable.
DNA as a contaminant:

 The concern is that uptake and

expression of such DNA in human cells
could occur.
 There is some evidence to suggest that
naked DNA can be assimilated by some
cells at least, under certain conditions.
 Guidelines to date state that an
acceptable level of residual DNA in
recombinant products is of the order of
10 pg per therapeutic dose.
 DNA Detection:-
 DNA hybridization studies (e.g., the ‘dot blot’ assay)
utilizing radiolabeled DNA probes allows detection of
DNA contaminants in the product, to levels in the
nanogram range.
 The process begins with isolation of the contaminating
DNA from the product.
 This can be achieved, for example, by phenol and
chloroform extraction and ethanol precipitation.
 The isolated DNA is then applied as a spot (i.e., a
‘dot’) onto nitrocellulose filter paper, with
subsequent baking of the filter at 80°C under
vacuum.
 This promotes (a) DNA denaturation, yielding single
strands, and (b) binding of the DNA to the filter.
 DNA Detection:-
 A sample of total DNA derived from the cells in which the product
is produced is then radiolabeled with 32P using the process of nick
translation.
 It is heated to 90°C (promotes denaturation, forming single
strands) and incubated with the baked filter for several hours at
40°C.
 Lowering the temperature allows reannealing of single strands via
complementary base-pairing to occur.
 Labelled DNA will reanneal with any complementary DNA strands
immobilized on the filter.
 After the filter is washed (to remove non-specifically bound
radiolabeled probe)
 It is subjected to autoradiography, which allows detection of any
bound probe.
 Quantification of the DNA:-
 Quantification of the DNA isolated from the product
involves concurrent inclusion in the dot blot assay of
a set of spots, containing known quantities of DNA, and
being derived from the producer cell.
 After autoradiography, the intensity of the test spot is
compared with the standards.
DNA Removal:
 In many instances there is little need to incorporate
specific DNA removal steps during downstream
processing.
 Endogenous nucleases liberated upon cellular
homogenization come into direct contact with cellular
DNA, resulting in its degradation.
Quantification of the DNA:-

 Commercial DNase’s are sometimes

added to crude homogenate to reduce
DNA-associated product viscosity.
 Most chromatographic steps are also
effective in separating DNA from the
product stream.
 Ion-exchange chromatography is
particularly effective, as DNA exhibits a
large overall negative charge.
Microbial and viral contaminants :-
 Finished-product biopharmaceuticals, along
with other pharmaceuticals intended for
parenteral administration, must be sterile
(the one exception being live bacterial
vaccines).
 The presence of microorganisms in the final
product is unacceptable for a number of
reasons:
1. Parenteral administration of contaminated
product would likely lead to the
establishment of a severe infection in the
recipient patient.
Microbial and viral contaminants :-

2. Microorganisms may be capable of

metabolizing the product itself, thus
reducing its potency.
3. Microbial-derived substances secreted
into the product could adversely affect the
recipient’s health. Examples include
endotoxin secreted from Gram-negative
bacteria.
Microbial and viral contaminants :-
 Sterilization of biopharmaceuticals by filtration,
followed by aseptic filling into a sterile final-
product container, inherently carries a greater risk
of product contamination.
 Biopharmaceutical products are also subjected to
screening for the presence of viral particles prior to
final product release.
 Although viruses could be introduced, for example,
via infected personnel during downstream
processing, proper implementation of GMP
minimizes such risk.
 Any viral particles found in the finished product are
most likely derived from raw material sources.
 Microbial and viral contaminants :-
 Examples could include HIV or hepatitis viruses
present in blood used in the manufacture of blood
products.
 Such raw materials must be screened before
processing for the presence of likely viral
contaminants.
 Producer cell lines are screened during product
development studies to ensure freedom from a
variety of pathogenic advantageous agents, including
various species of bacteria, fungi, yeast,
mycoplasma, protozoa, parasites, viruses and prions.
 Suitable microbiological precautions must
subsequently be undertaken to prevent producer cell
banks from becoming contaminated with such
pathogens.
Removal of Viruses :-

1. Gel-filtration chromatography, for example,

effectively separates viral particles from most
proteins on the basis of differences in size.
2. Filtration through a 0.22 μm filter effectively removes
microbial agents from the product stream but fails to
remove most viral types.
 Repeat filtration through a 0.1 μm filter is more
effective in this regard.
3. Alternatively, incorporation of an ultrafiltration step
(preferably at the terminal stages of downstream
processing) also proves effective.
Removal of Viruses :-
4. Heating the product to between 40 and 60°C for
several hours inactivates a broad range of
viruses.
 Many biopharmaceuticals can be heated to such
temperatures without being denatured
themselves.
 Such an approach has been used extensively to
inactivate blood-borne viruses in blood products.
5. Exposure of product to controlled levels of UV
radiation can also be quite effective, while
having no adverse effect on the product itself.
Removal of Viruses :-
Viral assays:
 Viral assays currently available will detect
only a specific virus, or at best a family of
closely related viruses.
 Current viral assays fall into one of three
categories:
1. immunoassays.
2. assays based on viral DNA probes.
3. bioassays.
Removal of Viruses :-
1. Immunoassays capable of detecting a wide
range of viruses are available commercially.
 The sensitivity, ease, speed and relative
inexpensiveness of these assays render them
particularly attractive.
2. An alternative assay format entails the use of
virus-specific DNA probes.
 These can be used to screen the
biopharmaceutical product for the presence of
viral DNA.
 The assay strategy is similar to the dot blot
assays used to detect host-cell-derived DNA
contaminants, as discussed earlier.
 Removal of Viruses :-
3. Viral bioassays: different formats have also been
developed.
 One format entails incubation of the final product
with cell lines sensitive to a range of viruses.
 The cells are subsequently monitored for cytopathic
effects or other obvious signs of viral infection.
 A range of mouse-, rabbit or hamster-antibody
production tests may also be undertaken.
 These bioassays entail administration of the product
to a test animal.
 Any viral agents present will elicit production of
antiviral antibodies in that animal.
 Removal of Viruses :-
 Serum samples (withdrawn from the animal
approximately 4 weeks after product
administration) are screened for the presence of
antibodies recognizing a range of viral antigens.
 This can be achieved by enzyme immunoassay, in
which immobilized antigen is used to screen for
the virus-specific antibodies.
 These assay systems are extremely sensitive, as
minute quantities of viral antigen will elicit strong
antibody production.
 A single serum sample can also be screened for
antibodies specific to a wide range of viral
particles.
 Time and expense factors, however, militate
against this particular assay format.
Miscellaneous contaminants:-
 Could include buffer components,
precipitants (ethanol or other solvents, salts,
etc.), proteolytic inhibitors, glycerol, anti-
foam agents, etc.
 In addition to these, other contaminants
may enter the product during downstream
processing in a less controlled way.
 Examples could include metal ions leached
from product-holding tanks/pipework, or
breakdown products leaking from
chromatographic media.
 For this reason, high-quality glass vials are
often used.
 Miscellaneous contaminants:-
 In some instances, it may be necessary to
demonstrate that all traces of specific contaminants
have been removed prior to final product filling.
 This would be true, for example, of many proteolytic
inhibitors added during the initial stages of
downstream processing to prevent proteolysis by
endogenous proteases.
 Some such inhibitors may be inherently toxic, and
many could (inappropriately) inhibit endogenous
proteases of the recipient patient.
 Various chemical-coupling methods may be used to
attach affinity ligands to the chromatographic support
material.
Miscellaneous contaminants:-

 Some such procedures entail the use of toxic

reagents, which, if not entirely removed after
coupling, could leach into the product.
 Improvements in the chemical stability of
modern chromatographic media, however, have
reduced such difficulties, and most
manufacturers have carried out extensive
validation studies regarding the stability of their
product.
 The possibility exists, however, that
uncharacterized contaminants may persist,
remaining undetected in the final product.
 As an additional safety measure, finished
products are often subjected to ‘abnormal
toxicity’ or ‘general safety’ tests.
 Miscellaneous contaminants:-

 Standardized protocols for such tests are outlined

in various international pharmacopoeias.
 These normally entail parenteral administration of
the product to at least five healthy mice.
 The animals are placed under observation for 48
h and should exhibit no ill effects (other than
expected symptoms).
 The death or illness of one or more animals
signals a requirement for further investigation,
usually using a larger number of animals.
THANK YOU
References
www.tufts.edu/med/neurosurgery/
 Wonderful image on first phenytoin slide

http://www-
clinpharm.medschl.cam.ac.uk/pages/teaching/i
mages/
 Excellent clinical pharmacology resource
http://www.bnf.org/BNF/bnf/current/3617.htm
 BNF guide to management of status epilepticus
DRUG DISPOSITION

Dr. Paul Wanjala Muyoma

Introduction

 Pharmacology-the study of the effects of

chemical substances on the function of living
systems. Has two broad divisions;-
 Pharmacodynamics and Pharmacokinetics
 Pharmacodynamics-is what the drug does to the
body (drug action) i.e., the biological and
therapeutic effects of drugs.
 Revision:
 [You studied about the relationship between drug concentration
and tissue response and the nature of that response.]

85
Introduction…
 Pharmacokinetics- is what the body does
to the drug (drug disposition).
 Several processes collectively determine
the concentration of drug at its site of
action and how the concentration alters
with time.
 These dispositional processes are
studied quantitatively in the science of
pharmacokinetics.

86

86
Introduction:…
 Disposition is a comprehensive term that includes
absorption, distribution & elimination (Metabolism &
Excretion):-i.e.

 Absorption
 Distribution ADME
 Metabolism
 Excretion

87
Introduction:…
 Absorption-the entry of drug molecules
into the systemic blood via the mucous
membrane (of, for example, the
alimentary or respiratory tracts), via the
skin or from the site of an injection.
 Distribution-the movement of drug
molecules between the water, lipid &
protein constituents of the body.
 Elimination-the removal of the original
drug molecule from the body by
excretion or by metabolism ( alteration
of the structure of the molecule)

88
Introduction…
 Designing dosage regimens to optimize
therapy and to minimize toxicity is
greatly assisted by an understanding of the
pharmacokinetic properties of drugs.
 Also, variability in responses to drugs and
selectivity of drug actions between
tissues can have dispositional as well as
pharmacodynamic causes.
 Many unwanted effects of single drugs or
interactions between drugs have a
dispositional basis.
89
DRUG ABSOPTION AND
DISTRIBUTION

90
Introduction
 In order to work, drugs need to achieve
an adequate concentration in their
target tissues.
 The two fundamental processes that
determine the concentration of a drug at
any moment and in any region of the body
are:
1. Translocation of drug molecules and
2. Chemical transformation

91
Introduction (cont.)
 Discussion on:
1. Drug translocation
2. Factors that determine absorption and
3. Factors that determine distribution
 These are critically important for
choosing appropriate routes of
administration.
 Chemical transformation by drug
metabolism, and other processes involved
in drug elimination.

92
Drug Absorption &
Distribution:
Learning Objectives;-
 At the end of the session a learner
should be able to understand:

1. The movement of drugs across

cellular barriers
2. Binding of drugs to plasma proteins
3. Drug absorption and bioavailability
4. Drug distribution

93
Definitions
 Drug: broadly speaking is any
substance that when absorbed into
the body of a living organism, alters
normal bodily function.
 There is no single precise
definition, as there are different
meanings in:
i. drug control law,
ii. government regulations,
iii. medicine and
iv. colloquial usage 94
Definitions (cont.)
In pharmacology a ‘Drug’ can be defined as:
Any chemical substance used in treatment,
cure, prevention or diagnosis of a
disease or used to otherwise enhance
physical or mental well being.
A habit-forming narcotic; any substance
that causes physiological or emotional
dependence
OR
An illegal substance that some people
smoke or inject etc. to give them
pleasant or exciting feelings
95
Definitions (cont.)
Absorption:
 The process of a liquid, gas or other
substance being taken in e.g., Vit D is
necessary to aid the absorption of
calcium from food.
OR
 The passage of a drug from its site of
administration into the plasma.
OR
 The movement of drug molecules
through membranes to reach the blood

96
Definitions(cont)

 Plasma:
The colorless liquid part of blood in
which the blood cells, etc. are
suspended

97
Translocation of Drug
Molecules
 Drug molecules move around the body in two
ways:
1. Bulk flow transfer (i.e., in the blood
stream)
2. Diffusional transfer ( i.e., molecule
by molecule, over short distances)

98
Movement of drug molecules
across cell barriers
 Cell membranes form the barriers between
aqueous compartments in the body.
 A single layer of membrane separates the
intracellular from the extracellular
compartments
 An epithelial barrier such as the g.i.t. mucosa
or renal tubule, consists of a layer of cells
tightly connected to each other so that
molecules must traverse at least two cell
membranes (inner and outer) to pass from
one side to another
 further reading (Revise)

99
Movement of drug molecules
across cell barriers…
 There are 4 main ways by which small
molecules cross cell membranes:
1. by diffusing directly through the lipid (lipid
diffusion)
2. by diffusing through aqueous pores formed
by special proteins (‘aquaporins’) that
traverse the lipid (aqueous diffusion)
3. by combination with a transmembrane
carrier protein that binds a molecule on one
side of the membrane then changes
conformation and releases it on the other
(specific carrier systems)
4. by pinocytosis
100
 Movement of drug molecules
across cell barriers…
1. Lipid Diffusion
 To traverse cellular barriers (e.g., GIT
mucosa, renal tubule, blood-brain-barrier,
placenta), drugs have to cross lipid
membranes
 Drugs cross lipid membranes mainly by
a) passive diffusional transfer and
b) carrier mediated transfer
 The main factor that determines the rate of
passive diffusional transfer across membranes
is a drug’s lipid solubility. Molecular weight
is less important factor 101
Movement of drug molecules
across cell barriers
Lipid Diffusion (cont.)
 The driving force is the concentration gradient
i.e., the rate of transport increases directly in
proportion to the concentration gradient.
 The permeability constant is closely related to
the lipid/water partition coefficient of the drug.
 The lipid/water partition coefficient is a
physicochemical property that expresses the
relative solubility of the drug in lipid compared
with water.
 To measure the lipid/water partition coefficient, a
small amount of drug is added to a mixture of an
oily solvent (usually n-octanol, the physicochemical
properties of which mimic those of cell
membranes) and water.
102
Lipid Diffussion…
 The mixture is shaken until equilibrium is reached
and the two layers are allowed to separate.
 The lipid/water partition coefficient is the
concentration of the drug in the oily phase divided
by its concentration in the aqueous phase.
 Drugs with high lipid/water partition coefficient
are often described in pharmacokinetic shorthand
as being lipid soluble.
 Drugs with low lipid/water partition coefficients
markedly less than 1.0 are described as water
soluble

103
Lipid Diffussion…
 Lipid soluble drug molecules or species ( i.e.,
those with high lipid/water partition
coefficient) penetrate membranes rapidly,
whereas water-soluble drugs or species (i.e.,
those with small lipid/water partition
coefficients) do not.
 For drugs that ionize in aqueous solution, only
the non-ionized species is lipid soluble.
 The proportions of ionized and non-ionized
species are determined by the pH of the
medium and the ionization constant of the drug.
 (the pKa=acid dissociation constant=the pH at
which one half of the drug molecules are
ionized)
104
Movement of drug molecules
across cell barriers….
2. Aqueous diffusion
 Water filled pores can be penetrated by
water-soluble drugs if the molecules are
small enough (MW less than 100 Daltons)
 This is a mechanism by which some highly
water-soluble molecules pass rapidly
through membranes ( e.g., Ethanol
(MW=46 Da.); Urea (MW=60 Da).;
Cycloserine (MW=99 Da)
 In general drugs are too large to pass
through these water-filled pores as most
drugs have MW between 100 and 400
daltons. 105
Movement of drug molecules
across cell barriers…
3. Specific carrier systems.
 Many cell membranes possess
specialized transport mechanisms that
regulate entry and exit of physiologically
important molecules (such as sugars,
amino acids, neurotransmitters).
 Such transport systems involve a carrier
molecule, i.e., a transmembrane protein
which binds one or more molecules or
ions, changes conformation and releases
them on the other side of the membrane.
106
Movement of drug molecules
across cell barriers…
Specific Carrier system (cont.)
 Such systems may operate as:
1. purely passively, ( without any energy
source; in this case they merely
facilitate the process of transmembrane
equilibration of the transported species
in the direction of the electrochemical
gradient a mechanism known as
facilitated diffusion
2. active transport-may be coupled to the
electrochemical gradient of Na+; in this
case transport can occur against an
electrochemical gradient
107
Movement of drug molecules
across cell barriers…
Specific Carrier System (cont)
 Some membranes possess active transport or passive
facilitated diffusion systems which exhibit substrate
specificity, stereospecificity, saturability and
competition between analogues; e.g., natural amino acids
and their analogues, (e.g. Levodopa, Methyldopa),
thyroxine sodium, antimetabolites (e.g., purines &
pyrimidine analogues)
 With carrier mediated transport the carrier sites become
saturated at high ligand concentrations and the rate of
transport does not increase beyond this point
 Few drugs meet the structural requirements for carrier
transport.
Note: Read more on diffusion through lipid & carrier mediated
transport
108
Movement of drug molecules
across cell barriers…
4. Pinocytosis involves invagination of part
of the cell membrane and the trapping
within the cell of a small vesicle
containing extracellular constituents. The
vesicle contents can then be released
within the cell or extruded from its other
side.
 Is important for the transport of some
macromolecules (e.g., insulin, which
crosses the blood-brain-barrier by this
process), but not for small molecules.
109
Binding of drugs to
plasma proteins
 At therapeutic concentrations in plasma,
many drugs exist mainly in bound form.
 The fraction of drug that is free in aqueous
solution can be as low as 1%, the reminder
being associated with plasma protein.
 Free drug: It is the unbound drug that is
pharmacologically active.
 The most important plasma protein in
relation to drug binding is albumin, which
binds many acidic drugs (e.g., warfarin,
NSAIDs, sulfonamides) and a smaller number
of basic drugs (e.g., tricyclic
antidepressants, chlorpromazine)

110
Binding of drugs to
plasma proteins…
 Other plasma proteins, including beta-
globulin and an acid glycoprotein that
increases in inflammatory disease, have also
been implicated in the binding of certain basic
drugs, such as quinin
 The amount of a drug that is bound to protein
depends on three factors:
i. The concentration of free drug
ii. Its affinity for the binding sites
iii. The concentration of proteins

111
Binding of drugs to
plasma proteins…
 At first approximation, the binding
reaction can be regarded as a simple
association of the drug molecules with a
finite population of binding sites, exactly
analogous to drug-receptor binding
Note: (revise on the binding of drug
molecules to cells)
D + S = DS
free binding complex
drug site
112
Binding of drugs to
plasma proteins…
 Binding sites on plasma albumin bind many
different drugs, so competition can occur
between them.
 Administration of drug B can thus reduce
the protein binding, and hence increase
the free plasma concentration, of drug A.
 To do this, drug B needs to occupy an
appreciable fraction of the binding sites.
 Few therapeutic drugs affect the binding
of other drugs because they occupy, at the
therapeutic plasma concentrations, only a
tiny fraction of the available sites.
113
Binding of drugs to
plasma proteins…
 Sulfonamides are an exception because
they occupy about 50% of the binding
sites at therapeutic concentrations and
so can cause unexpected effects by
displacing other drugs or, in premature
babies, bilirubin
 Competition between drugs for protein
binding can lead, rarely, to clinically
important drug interactions.

114
Drug Disposition: Summary
 Consider how the physical processes described
i.e. diffusion, penetration of membranes &
binding to plasma protein etc. influence the
overall disposition of drug molecules in the
body.
 Drug disposition is divided into 4 stages:
1. Absorption from site of administration
2. Distribution within the body
3. Metabolism
4. Excretion
115
DRUG ABSORPTION

116
Drug Absorption
 Drug absorption is the movement of drug
molecules through membranes to reach the
blood.
 It is therefore important for all routes of
administration, except the intravenous
injection
 There are instances, such as inhalation of a
bronchodilator aerosol to treat asthma,
where absorption is not required for the
drug to act,
 In most cases the drug must enter plasma
before reaching its site of action

117
Routes of Administration
 The main routes of administration are:
1. oral
2. sublingual
3. rectal
4. application to other epithelial surfaces
(e.g. skin, cornea, vagina and nasal
mucosa)
5. inhalation
6. injection: -subcutaneous, -intramuscular,
intravenous and - intrathecal

118
Formulations and routes of
administration
 Most drugs are administered as a
medicine, formulated along with other
materials known as excipients, which are
pharmacologically inactive.
 The formulations serves some or all of the
following:
1. To enable the administration of an
accurately measured dose
2. To improve drug stability
3. To present the drug in a convenient
form for administration
4. To regulate the rate of disintegration
and/or solution of the medicine
119
Formulations and routes of
administration
1.Oral administration
Most drugs are taken by mouth and swallowed. Little absorption
occurs
until the drug enters the small intestine.
1.1 Oral formulation
a. Tablets or capsules
Advantages are:
i. Precise control of dose and chemical.
ii. Stability of drugs as dry solids.
Two processes precede absorption: (disintegration & dissolution)
 Tablets often contain excipients that cause them to swell on
contact with water and disintegrate into fine particles.
 Dissolution then occurs from the surface of the particles.
 For many medicines the rate of dissolution is faster the smaller the
particles because of the larger surface area /mass ratio
120
of the
particles.
Formulations and routes
of administration…
Drug absorption from the intestine
 Above 75% of a drug given orally is
absorbed in 1-3hrs, but numerous
factors alter this, some physiological
and some to do with the formulation
of the drug.
 The main factors are:
1. g.i.t. motility
2. Splanchnic blood flow
3. Particle size and formulation
4. Physicochemical factors
121
Formulations and routes
of administration…
Rate of Absorption.
The rate of absorption from the gut can be influenced by many
factors.
1. Drug absorption can occur from the stomach but small
intestine is quantitatively much more important because of its
vastly greater area and blood flow.
2. If the time to gastric emptying is increased (e.g. by taking
a drug with food) then the rate of absorption is usually
decreased.
3. If dissolution from the formulation is slower than the rate
of drug absorption, then absorption is described as
dissolution rate limited.
4. However, if dissolution is rapid, the permeability
characteristics of the drug dictate the rate of absorption.
122
Formulations and routes of
administration
1.2. Sustained And Delayed Release Oral
Formulations.
 Rapid dissolution of drugs may cause local damage
to the gut mucosa (Aspirin, KCl, and Iron Salts) or
systemic adverse effects due to a brief, high peak
conc. of drug in the blood (Aminophylline producing
CNS stimulation).

 Alternatively the duration of drug response may

be too short for practical day to day treatment
(Quinidine).

 These problems are sometimes solved by producing

pharmaceuticals formulations that release drug
slowly or after a delay.
123
Formulations and routes
of administration
1.3. Enteric Coated Tablets
 The coating dissolves on reaching a non-acidic
medium.
 It is used to protect the drug from acid (e.g.
erythromycin) or protect the stomach from
the drug (e.g. Aspirin, Prednisolone).
 The thicker the coating the slower it
dissolves.

124
Formulations and routes
of administration
2. Sublingual administration
 Absorption directly from the oral cavity is
sometimes useful (provided the drug does not
have a displeasing taste) when a rapid response
is required, particularly when the drug is either
unstable at gastric pH or rapidly metabolized by
the liver.
 E.g. of sublingual drug is Glyceryl trinitrate
 Drugs absorbed from the mouth pass straight
into the systemic circulation without entering
the portal system and so escape first-pass
metabolism
125
Formulations and routes
of administration
3. Other Mucous Membranes (application to other
epithelial surfaces
 These routes can be used for drugs that are
required to provide a local effect or for
systemic absorption for drugs that are
susceptible to intestinal or liver metabolism.
 Since the venous drainage of these membranes
occurs into systemic circulation, gut and liver
enzymes are avoided.
 The rate of systemic absorption is dictated by
surface area and / or blood flow.

126
Formulations and routes of
administration

3.1 Buccal Mucosa

 It provides a convenient site for highly
lipid-soluble compounds (Glyceryl
Trinitrate in the prophylaxis of effort of
angina and treatment of heart failure).

127
Formulations and routes
of administration
3.2. Respiratory Mucosa.
 Bronchial mucosa and alveoli provide the
largest surface area of any mucous
membrane and are accessible to vapors and
ultra-fine droplets (aerosols).
 Particle size is of critical importance as only
small particles reach the smallest airway
 EXAMPLES USES
 1. Halothane, Nitrous Oxide- Anaesthesia
 2. Salbutamol- Relief of Asthma.

128
Formulations and routes
of administration
3.3. Nasal Mucosa
 It provides an inefficient (because a
high proportion of dose is wasted) but
convenient route of entry for
relatively low MW peptides. They can
be administered as a nasal spray.
 Examples
1. Lypressin- Diabetes Insipidus.
2. Sodium Cromoglycate-Allergic rhinitis
(local effect).
129
Formulations and routes
of administration
3.4. Rectal Mucosa (Rectal administration)
 It provides a useful absorptive surface when
the patient is un-conscious, asleep, vomiting or
suffering local gastric adverse effects.
Examples.
1. Rectal Diazepam soln.-Serial Seizure.
2. Sulphasalazine suppositories-Ulcerative
colitis.
3. Prednisolone foam-Ulcerative colitis.
4. Metronidazole supp.-Prophylaxis of surgical
wound infection.
130
Formulations and routes
of administration
3.5 Skin
 Healthy skin is a highly specialized protective envelope
that prevents un-controlled water loss.
 Skin is several mm thick and normally has a low blood
flow.
 Thus entry of water soluble compounds is limited.
 However, many lipid soluble drugs can enter and
produce therapeutic or unwanted effects.
 Examples Effects:
1. Glyceryl Trinitrate-Prophylaxis of effort Angina
2. Glucocorticoids-ACTH suppression.

Infants and toddlers are specially vulnerable to the undesired effects of substances
131
absorbed through the skin because of their large surface area/mass ratio
Formulations and routes
of administration
Diseased Skin.
 In diseased skin, due to extensive burns, wounds
or dermatitis, the water barrier is lost, even
water soluble drugs can enter and fatal accidents
can result.
 Examples Unwanted effects
 1. Sulphonamides Crystalluria
 2. Neomycin Deafness

132
Formulations and routes
of administration
4. PARENTERAL ROUTES
 The route is necessary when proportion of the
orally administered drug that is absorbed is low.
 Absorption rate is dictated by blood flow at the
site of injection unless sustained release
formulations are used.
a) INTRAVENOUS (I/V)
 Absorption is complete when the injection is
complete.
 I/V route is used when a rapid effect is required,
the drug is too irritant by other routes or when
abdominal surgery prevents the use of the oral
routes.

133
Formulations and routes
of administration
b) INTRAMUSCULAR
 Skeletal muscles have a rich capillary
plexus.
 The capillary endothelium has large water
filled pores that are freely permeable even
to water soluble drugs of high MW.
 Many emergency drugs (analgesics, anti-
emetics and oxytocics) are given by this
route.
 The rate of absorption is proportional to the
dose, the extent of dispersion through the
muscle and the rate of tissue blood flow

134
Formulations and routes
of administration
c) SUBCUTANEOUS.
 Subcutaneous tissues are poorly perfused
especially in low cardiac output states
(haemorrhagic shock, acute diabetic
ketoacidosis).
 It is the standard one for self injection
(diabetes)
 Absorption is relatively slow even in healthy
subjects and may be delayed by administration
of sustained release formulations (depot
insulin) or by co-administration of a
vasoconstrictor (adrenaline with lignocaine).
135
Formulations and routes
of administration…
d) Intrathecal:
 Injection of a drug into the sub-
arachnoid space via a lumbar
puncture needle is used for some
specialized purposes.
 E.g. methotrexate is administered in
this way in the treatment of certain
childhood leukaemias to prevent
relapse in the CNS.
 Regional anaesthesia can be produced
by intrathecal administration of a local
anaesthetic such as bupivacaine
136
Formulations and routes
of administration
SUSTAINED AND DELAYED RELEASE
PARENTERAL FORMULATIONS.
 An ester of drug and long chain fatty acid (
Fluphenazine decanoate) is dissolved in non-
toxic oil.
 Drug very slowly diffuse out from an
injection site over a period of weeks.
 The rate limiting step is hydrolysis to
Fluphenazine at the surface of the oil.
 This formulation is used for maintenance
dosage in psychotic patients who otherwise
fail to take prescribed treatment.

137
Formulations and routes
of administration
SUSPENSION OF INSOLUBLE COMPLEX
1. A complex between soluble drug and a
relatively inert molecule is almost
insoluble.
2. Soluble drug is slowly released from
suspension at the site of I/M or S/C
injection.
 This extends the duration of action of
benzyl penicillin (Procaine penicillin)
or Insulin (Isophane Insulin).
138
Formulations and routes
of administration
SUSPENSION OF CRYSTALS.
 Notably Insulin Zinc Susp. The smallest
particles (amorphous) give most rapid
absorption .
 The largest crystals (crystalline) gives
the slowest absorption and longest
duration of action.

139
Drug Absorption and
Bioavailability
SYSTEMIC BIOAVAILABILITY
 To get from the lumen of the small intestine into the
systemic circulation, a drug must not only penetrate the
intestinal mucosa , it must also run the gauntlet of
enzymes that may inactivate it in gut wall and liver.
 The term bioavailability is used to indicate the
proportion of drug that passes into the systemic
circulation after oral administration, taking into
account both absorption and local metabolic
degradation
 Can be defined as the fraction [F] of the dose of drug
administered that reaches the systemic circulation.
 For IV injection F equal 1.0 but by other routes,
particularly after oral administration, F may be less
than 1.0.
140
Drug Absorption and
Bioavailability…
SYSTEMIC BIOAVAILABILITY (cont)
REASONS FOR LOW SYSTEMIC BIOAVAILABILITY OF DRUGS
AFTER ORAL ADMINISTRATION

MECHANISM
1. Incomplete solution -Asprin in enteric coated Tablet
2. Breakdown in gut lumen - Benzylpenicillin
3. Binding in gut lumen -Tetracycline with divalent Cations
4. Negligible absorption -Gentamicin
5. Metabolism by gut wall -Isoprenaline, levodopa
6. Metabolism by liver - Glyceryl Trinitrate
 Oral route may still be suitable, despite extensive metabolism, if the
metabolites are pharmacologically active ( Aspirin is converted to
Salicylic Acid.)

141
DRUG DISTRIBUTION

142
DRUG DISTRIBUTION

 Distribution involves the movement of

drug molecules from the circulating
blood to other areas of the body,
including the sites of action of binding
and of elimination.

 Drug action at one site and not

another may be due to selective
access of drug [ pharmacokinetic
selectivity] rather than differences in
the sites of action
[ pharmacodynamic selectivity].
143
DRUG DISTRIBUTION
 For drugs applied to the site of action [
e.g. local anaesthetic agents injected
for nerve block, bronchodilator agents
inhaled as aerosols for local action in
airways ], absorption and distribution
reduce drug effects.
 Once a drug has entered the blood it
mixes rapidly [ circulation time= 20
seconds].

144
DRUG DISTRIBUTION
 The rate and extent of distribution depend
upon the relative arterial blood perfusion
rates of different organs and the
permeability characteristics of cell
membranes towards different drug
molecules.
 The initial driving force is the concentration
gradient between plasma and the site to
which distribution is occurring.
 There are two main factors that influence
drug distribution these are:
1. Hemodynamic (perfusion) and
2. Permeability factors
145
HAEMODYNAMIC (or
PERFUSION ) FACTORS
 The whole of the right heart output (and
therefore of the absorbed dose) is passed
through the lungs to the left heart.
 The bulk of the absorbed dose is then
carried rapidly to the vessel-rich group
of organs [ brain, myocardium, liver,
kidneys, adrenal glands and thyroid ],
which receive approximately 80% of the
cardiac output at rest.

146
HAEMODYNAMIC (or PERFUSION ) FACTORS…

 During late pregnancy this includes

placenta and the uterus which also
receive large volumes of blood
 A few minutes after a drug is injected as
an I/V bolus it is relatively concentrated
in the above organs.
 A drug is then more slowly distributed to
skeletal muscles, which have relatively
small blood flow at rest.
 A drug reaches the skin and adipose
tissue more slowly than muscles and
reaches avascular structures like tendons
and cartilage very slowly indeed.
147
PERMEABILITY FACTORS
 Arterial blood flow determines the rate
at which drug reaches the interstitial
fluid of a given organ or tissue.
 The capillary endothelia of most tissues
contain large pores ( 50-100 nm diameter
) and therefore present no barrier to
diffusion to even water-soluble
drugs/ionized species.
 Non-protein-bound drugs diffuse readily
into interstitial fluid.

148
PERMEABILITY FACTORS…
 Therefore, virtually all drugs can gain
access to the interstitial fluid/cell
surface.
 This may be their site of action ( e.g.
Penicillins)
 The fractional quantity of the drug that
is bound to plasma proteins remains
within the vascular system.

149
PERMEABILITY FACTORS…
 The rate of penetration into cells and
across special tissue barriers is,
however, dependent on the physico-
chemical properties of the drug
molecule, as the barrier is composed of
lipid membranes-
 Highly water soluble drugs ( e.g.
Gentamicin ) penetrate into cells more
slowly or not at all.
 Conversely, highly lipid soluble drugs (e.g.
Thiopentone and inhalation anaesthetics
agents) penetrate very rapidly.

150
RATE AND EXTENT OF
DISTRIBUTION
 For any drug the rate of distribution
to an organ can be perfusion limited
or permeability rate limited.
-Perfusion limits the rate of
distribution for highly lipid-soluble
drugs traversing most membranes
and for most drugs crossing
membranes with large pores.
-For example thiopentone is so lipid
soluble that its rate of entry into the
CNS is determined solely by cerebral
blood flow.
151
RATE AND EXTENT OF
DISTRIBUTION…
If a drug has a large affinity for tissue [ high
tissue/plasma partition coefficient ], then much drug
needs to be delivered before equilibrium is reached.
 Therefore, the time to equilibrium is long when
tissue perfusion is small and the drug has a high
tissue/blood partition coefficient
Permeability limits the rate of distribution for water-
soluble drugs crossing membranes with small pores.
-Here the time to equilibrium increases as the
permeability of the drug decreases and as the
proportion of the drug present as the ionized, non-
diffusible species increases.

152
BINDING OF DRUG BY PROTEIN [
OTHER MACROMOLECULES]:

 Virtually all drugs are adsorbed to

macro-molecules in tissues and in
plasma in a readily reversible manner
involving non-covalent bonds.
 These drug/macromolecule complexes
associate and dissociate with a half-time
measured in milliseconds.
 Rarely, therefore, is the dissociation of
the complex rate liming.

153
ASSOCIATION WITH SPECIFIC
BINDING SITES
 Many acidic drugs (e.g. Salicylic acid,
Sulphonamides, Warfarin) bind to one (
the same ) specific site on each albumin
molecule and some basic drugs ( e.g.
Diazepam, Propranolol ) bind to 1-acidic
glycoprotein and lipoprotein.
 Usually the number of binding sites
considerably exceeds the number of drug
molecules, therefore the fraction of bound
drug is constant over a wide range of total
drug concentrations
154
ASSOCIATION WITH SPECIFIC
BINDING SITES…
 A few drugs (e.g. Salicylic acid, Sodium
Valproate ) have such large affinities for
these specific binding sites that as the
total drug concentration in plasma
increases within the clinically
encountered range, the binding sites
become saturated.

155
BINDING OF DRUGS TO TISSUE MACRO-
MOLECULES:

 Most drugs equilibrate to larger concentration in

tissues than in plasma
-Such drug adsorption is reversible.
-Non-reversible binding can also occur.

(Drugs bound to other tissues other

than plasma proteins is shown in
the next table )

156
EXAMPLES
Drug Site Mechanism

Tetracycline Bones Binds when blood flow

Teeth is moderately
associated with
growth

Also chelation to Ca2+

Cyclophosphamide Nucleic acids Covalent bonding to

purine and pyrimidine
bases.

157
DISPOSITIONAL SIGNIFICANCE OF
PROTEIN BINDING:

The plasma protein/drug complex is a

pharmacologically inactive mass transit
system, carrying drugs to tissues.
 Plasma proteins act like reservoirs of
some drugs
 Protein binding often causes the total
plasma concentration of a drug to
exceed its aqueous solubility (e.g.
Phenytoin, Propranolol,
Benzylpenicillin).
 As a result, distribution is more rapid.

158
DISPOSITIONAL SIGNIFICANCE OF
PROTEIN BINDING:
The drug/ protein complex (plasma/ tissue) acts as
a reservoir, that both smoothens fluctuations in
the concentration of free drug in plasma water
and at the site of action, and prolongs the
action of the drug
 If the plasma albumin concentration is small (as
in nephrotic syndrome, liver failure, mal-
absorption, starvation), drug/protein complex is
reduced
 This may result in high concentration of
unbound or free drug if administered which
may give rise to toxicity

159
DISPOSITIONAL SIGNIFICANCE OF
PROTEIN BINDING:

 Two acidic drugs can compete for specific

binding sites on plasma albumin.
 As a result, the free plasma concentration of one
of these drugs may be much larger than when
there is no competing drug present, so that the
pharmacological effect is exaggerated
 At large plasma concentrations few drugs can
saturate the binding sites

160
SPECIAL BODY COMPARTMENTS
AND SPECIAL BARRIERS:
 The rate and extent of distribution of a drug to
and from these tissues can be limited either by
perfusion or permeability factors.
 For tissues with a lipid membrane between
plasma and the site of drug action and a large
blood flow ( e.g. brain, placenta ) drug
permeability across the membrane is usually
the limiting factor (BBB,BPB)
 Only for the most lipid-soluble drugs (e.g.
thiopentone) is blood flow the limiting factor
 Distribution into tissues with a small blood flow is
usually perfusion limited

161
BRAIN:
 Unlike the peripheral capillary
endothelium, capillary endothelia of
blood vessels of the CNS have
continuous tight junctions that provide
a gap-free lipid membrane between
blood and CSF
 Highly polar, water-soluble drugs (e.g.
Gentamicin) penetrates slowly, if at all.
 Non-polar, lipid-soluble drugs (e.g.
Thiopentone) penetrates rapidly, and
 drugs with intermediate solubility (e.g.
Tetracycline) penetrate at intermediate
rate.

162
The Blood Brain Barrier
(BBB)
 The brain is inaccessible to many drugs
 Inflammation can disrupt the integrity of
the BBB allowing entry of water soluble
drugs to enter the brain
 Drugs like penicillin readily enter the brain
during meningitis
 However in some parts of the brain like
the chemoreceptor trigger zone (CTZ)
the BBB is leaky and allows some drugs to
enter like domperidone a dopamine
receptor agonist
163
The Blood Brain Barrier
(BBB)
 Domperidone is useful in preventing nausea and
vomiting induced by dopamine receptor agonists like
apomorphine
 Fat insoluble drugs generally do not penetrate the BBB
 The stronger an acidic drug [the smaller the pka] the
smaller the concentration of unionized molecules at
pH 7.4 and the slower the rate of penetration into
brain-Salicylic acid (pKa 3) penetrates slowly.
 Similarly, the stronger a basic drug (the larger the
pKa) the smaller the concentration of unionized
molecules and the slower the rate of penetration into
brain- morphine (pKa 8) penetrates slowly.

164
The Blood Brain Barrier
(BBB)
 The penetration of acidic and basic drugs
depends also on the lipid solubility of
the un-ionized molecules
 Adrenaline has few CNS effects but less
polar amphetamine (no-OH group) has
marked CNS effects
 Several drugs are transferred from the
cerebrospinal fluid (CSF) to the plasma
across the choroids plexus against a
concentration gradient.

165
The Blood Brain Barrier
(BBB)
 Examples:-
Anions: Cations:
Benzylpenicillin tubocurarine
Probenecid
 This process reduces the concentration
of penicillins in the CSF and, therefore,
impairs their effectiveness against
bacterial infections within the CNS
 As a consequence, very large doses of
these antibiotics agents are required to
treat infection of the CNS
166
The Blood Brain Barrier
(BBB)
-Essential nutrients (amino acids, glucose, purines,
pyrimidines) are actively transported into the CSF
and brain.
-Levodopa (which is a naturally occurring amino acid ) and
its analogue methyldopa also enter by this means.

167
PLACENTA
 Lipid-soluble drugs cross the placenta
readily
 Hence general anaesthetic agents can
interfere with respiration in the newborn
child after administration to mother during
delivery
 Morphine and related analgesic agents
cause the same problem
 All drugs penetrate into the foetal
circulation at some rate.
 Even highly polar water-soluble drugs [
e.g. gentamicin ] penetrate to the foetus
slowly.

168
BREAST
 The breast is an example of a pharmacokinetic
deep compartment with a moderate blood
supply.
 Most drugs enter breast milk by passive
diffusion through lipid membranes.
 Compounds with a MW less than 100 Da
(Ethanol ) enter by passive diffusion through
water filled pores in the membrane.
 Iodine is actively transported and, therefore,
administration of radio-active iodide to mother
is an absolute contraindication to breast
feeding.

169
BREAST….
 For most drugs concentrations in milk are similar
to those in plasma at equilibrium.
 However, as the amount of drug in plasma is
usually small in relation to the total amount in
the body, so the total amount of drug delivered
to infant during breast feeding is small in
relation to doses recommended for therapeutic
purposes in infants.

170
BREAST….
 Hence feeding can be, continued
when the mother is taking digoxin,
tricyclic anti-depressant drugs
paracetamol, phenytoin, diuretic
agents and even warfarin.
 Breast feeding should be discouraged
where the mother is taking drugs for
prolonged periods and where the
drugs could have serious adverse
effects on the infant (radio-active
iodide, cytotoxic drugs, carbimazole,
theophylline, sulphonylurea oral
hypoglycaemic drugs).

171
EYE
 ANTERIOR COMPARTMENT:
-The conjunctiva, sclera, iris and ciliary
muscle receive a moderate blood supply
but cornea and lens are avascular.
-Drugs can penetrate to these structures and
the aqueous humour from the
conjuntival sac (e.g. Chloramphenicol).

172
EYE…
 As there is little perfusion of either side
of membranes, the rate of drug
movement is mainly proportional to the
lipid solubility of the drug and the
proportion that is non-ionized.
 As the stroma of the cornea has a large
water content, relatively water-soluble
drugs [pilocarpine] can penetrate into
aqueous humour.
 NOTE: benzylpenicillin is extruded from
aqueous humour as it is from the CSF
173
Posterior Compartment
 The sclera, choroids and retina are
moderately vascular but the vitreous
humour is avascular.
 Drugs cannot reach these structures by
diffusion from the conjuctival sac as the
diffusion distance is too great.
 Instead drugs reach these structures
from the systemic circulation.

174
SEROUS CAVITIES
 In general, all drugs enter and leave serous
cavities ( pleural, pericardial, peritoneal sacs,
joint spaces ) slowly.
 Water-soluble drugs penetrate slowly and lipid-
soluble drugs more rapidly.
 Acute inflammation facilitates the
penetration of drugs, as the movement of
water through the capillary epithelium
increases during inflammation.
 Chronic inflammation, however, leads to
fibrosis and this impedes penetration.
175
BONES AND TEETH
 Drug access is proportional to local
blood flow.
 Infection produces oedema, ischaemia
and avascular necrosis so that only
prompt treatment is effective.
 The growth region of bone is moderately
well perfused.
 Blood flow becomes very small when
growth ceases.
 Certain drugs and ions form complexes
with bone salt, especially in growing bone
(e.g. Lead, Fluoride, Tetracyclines).
176
SKIN AND NAILS
 These are avascular and thus penetration
of drugs from the systemic circulation is
very slow.
 Griseofulvin, an anti-fungal agent, has a
large affinity for keratin and so achieves
selectively large concentrations in skin
and nails.

177
ABSCESS CAVITIES
 Acute abscesses are thin walled and
have an increased local blood flow.
 Consequently antibiotics penetrate
readily.
 Chronic abscesses have thick avascular
walls and drugs do not penetrate.
 Similarly penetration into sputum is
slow.
 In acute ( but not chronic ) otitis media (
infection of middle ear ) the organisms
are accessible.

178
EXTENT OF DISTRIBUTION-
APPARENT VOLUME OF
DISTRIBUTION.
 After a dose of drug is administered distribution equilibrium will
eventually be reached
 Is an imaginary volume the drug would occupy if the concentration
of the drug in the body was the same as that which is found in
plasma.
 Vd = Amount of drug in body at equilibrium
_________________________________ =Vd/Cp ------in volume
Plasma drug concentration

Vd=Apparent Volume of distribution

Cp=Plasma concentration

179
 Examples of apparent volume of distribution (Vd)

Substance Volume (L) Equivalent

physiological
space

Evans blue, 3.5 Plasma water

Heparin
Inulin, Gentamicin 13.5 ECF

Tritiated water, 41.5 Total body water

ethanol
Digoxin 350 None

180
 Most drugs, however, have a
tissue/plasma partition coefficient much
greater than one, that is they exhibit
some tissue binding.
 These drugs therefore appear to have a
Vd greater than total body volume.

181
Summary:
Drug Distribution
 The major compartments are:
1. Plasma (5% of body weight)
2. Interstitial fluid (16%)
3. Intracellular fluid (35%)
4. Transcellular fluid (35%)
5. Fat ( 20%)
 Volume of distribution is defined as the
volume of plasma that would contain the
total body content of the drug at a
concentration equal to that in the plasma
182
Summary
Drug Distribution…
 Lipid-insoluble drugs are mainly
confined to plasma and interstitial
fluids; most do not enter the brain
following acute dosing
 Lipid soluble drugs reach all
compartments and may accumulate in
fat.
 For drugs that accumulate outside the
plasma compartment (e.g., in fat, or by
being bound to tissues) Vd may exceed
total body volume
183
THANK YOU
Therapeutic Drug
Monitoring

Dr. Paul Wanjala Muyoma

Introduction
This overview lecture is designed to
help with your learning around
common drugs which are prescribed
for therapeutic purpose.
As with many of the drugs prescribed
being potentially very dangerous, are
commonly mis-prescribed and there
remains a mythology around them
which lead to many of the errors
associated with their use.
 Aims and Objectives
By completing this lecture you should be able to
 Understand prescription of gentamicin, digoxin, warfarin,
heparin, Insulin, steroids and phenytoin
 Monitor drug therapy by taking appropriately timed drug
levels for gentamicin, phenytoin, digoxin and warfarin
 Write up an IV insulin sliding scale and insulin infusion
 Convert a patient from IV insulin to regular subcutaneous
dosing
 Appropriately prescribe anticoagulation intravenously,
subcutaneously and orally.
 Recognise the common side effects and toxicity of
gentamicin, warfarin, digoxin, phenytoin and steroids.
 Institute appropriate management to deal with toxic and
other side effects
Drug (1) – Gentamicin and the
aminoglycosides
Challenging practice
A 78 yo woman presents in A&E with severe urinary
sepsis.
She has hypertension but is otherwise fit and well.
She is on Bendrofluazide 2.5mg od.
Her U+Es are Na+131mmol/l, K 3.5+mmol/l Urea
13.8 mmol/l, Creatinine 127µmol/l.
(1) Prescribe a stat dose of gentamicin.
(2) When would you check the levels around the next
dose?
(3) How long will you continue the gentamicin?
Gentamicin Indications
 Principally used against clinically significant gram
negative sepsis – E. Coli, Proteus, Klebsiella,
Pseudomonas (Tobramycin may be preferred for
pseudomonas infection)
 Some anti-Staphylococcal effect (but is commonly
used in combination with other anti-staphylococcal
agents)
 Also used in eye and ear infections.

Why do we need to monitor?

 Like many of the drugs, gentamicin has a narrow
therapeutic window (NTW) which means toxicity,
particularly ototoxicity and nephrotoxicity, can be a
serious complication of treatment.
Gentamicin Dosing - I
 Previously (on ER) Gentamicin was given
(a) In relatively small, standard doses to
all patients regardless of weight, age
and renal function i.e. 80 mg TDS regime
(b) Often over inappropriately ‘long’
periods e.g. 7 days
 This led to toxicity; Particularly when
levels were unavailable / not done!
Gentamicin Dosing - II

 Now superseded by dosing with initial

‘Big Bolus’ dose (see next slide)
(Max 400mg total)
Measure trough LEVELS after approx 12 hrs

 Then depending on indications and patient

Further ONCE a day dosing
Generally not given for longer than 3 – 5 days unless
exceptional circumstances e.g. endocarditis

In endocarditis and pregnancy - The same dose (i.e. the once a

day dosing) is split into BD or TDS regime

NB – A wise person once pointed out to me that one should be

very wary of giving ototoxic drugs to blind people – It is not
an absolute contraindication but think about this!
Gentamicin Dosing – Rule of Thumb
Normal creatinine clearance - Gentamicin Dose
3 – 5 mg / kg
Reduced creatinine clearance – Gentamicin Dose
1 – 2 mg /kg

The following formula can be used to calculate creatinine clearance in order to

determine the doses and dosing interval when prescribing gentamicin.

Creatinine clearance (ml/min) = (N - age (years) ) x Wt (kg)

serum creatinine (µmol/l)

Where N = 150 for female patients; 160 for male patients >70 years,170 for male patients <70 years
This does rely on you knowing or guestimating patient’s weight correctly – always err on the side
of caution!
 Normal creatinine clearance is (male range 97 – 137ml/min) and (female range 88 –
128 ml/min) i.e. approximately 100ml/min
 It decreases with age (by approx 1ml/min/year from aged 20yo), reduced lean
body mass i.e. reduced muscle mass, gender (as above) and of course renal disease
 It is a useful guestimate of the Glomerular filtration rate (GFR).
Gentamicin Dosing - Infusion
Gentamicin comes in 80mg vials. It is important to try and
make your doses multiples of 40 to ease the nurses’ job in
making up the infusion.

Infusion
100ml 5% glucose or sodium chloride 0.9% over 60 minutes
(round to the nearest 40mg) to a maximum of 400mg.

Doses ≤ 240 mg – slow IV over 3 -5 minutes

≥ 240mg – over 30 minutes (as above)

Note it can be given IM with good effect BUT be wary of

DIC and raised INR in the sick or anticoagulated patient.
 Monitoring of Levels
After the initial BIG BOLUS
 Take the ‘trough’ level 6
– 14 hours POST dose
(conventionally 12 hours)
 This trough level should
be in the sub-therapeutic
range. As shown on the
graph <1 mg / l at 12hrs
 This applies to all
subsequent pre-dose
levels.
 If the trough or pre–dose
level is still in the
therapeutic window
(between the black lines),
the next dose should be
missed or delayed.
Monitoring of Levels - II
Pre level – (Trough) < 1mg/l
Post level (Peak) > 8 mg/l
 The post dose level is taken a minimum 30 minutes after dose –
usually measured 1 hour post dose.
 You don’t want it to be too high as this will mean that much of the
dose is above the therapeutic range and therefore will cause
toxicity.

Frequency of levels
 Ill patient – monitor daily or once / three days; Trough
(Pre) level is important as are massive peaks.
 Stable patient – STOP Treatment? Further levels once
every three days
 Multiple dosing (BD and TDS) – take levels after second
(bd) or third (tds) dose; then rules above apply.
Gentamicin challenge
You are the technician on acute medical on-call. The next patient
Mrs. Wafula is a previously fit and well woman who presents to A&E
with a 24 hour history of delirium and offensive urine. She is
haemodynamically stable.
Your Supervisor has asked you to give her a stat dose of gentamicin
and then write up her up for a course of IV cefuroxime.

U&Es: Na+ 142; K+4.9; Urea 11.6; Cr 120

Her weight is guestimated at 80Kg

1.Calculate her creatinine clearance

2.How does this affect your dosing of her gentamicin?
3.Using the drug chart provided write up the gentamicin and course of
IV cefuroxime.
4. What other therapeutic interventions will you write up?
Gentamicin challenge - Answers
(1) Creatinine clearance (ml/min) = (N - age (years) ) x Wt (kg)
serum creatinine (µmol/l)

= (150 – 60) x 80 = 60ml/min

120
Please note – her age will increase by 1 year for each year this
module is up and running! Change the calculation and answer
accordingly.
(2) This is a significantly reduced creatinine clearance
and thus this patient should receive a maximum of 1-2
mg /kg i.e., maximum dose 160mg
(3) See the chart on the next slide …
(4) She should also be written up for IV fluids, anti-
emetics and any regular medications.
So that’s what a slightly out of focus drug
chart looks like!
Anticoagulation

The verbs to Heparinise and to Warfarinise

Challenging Practice
 Remember neither Heparin nor Warfarin ‘thin’ the blood.
 They both stop the blood clotting!
 List three indications for heparin
 List three side effects of heparin
 Why may you use IV unfractionated heparin rather than SC low molecular
weight heparin?
 Which drug is used to reverse the anticoagulant effects of heparin?
Heparin
 Naturally occurring glycosaminoglycan
 Discovered in 1916 at John Hopkins University
but was not used clinically in humans until the
1930s.
 Derived from liver cells (‘Hepar’ is the Greek
for ‘Liver’)
 Two forms Unfractionated heparin (UH) and
fractionated, low molecular weight heparin
(LMWH)
 May be given subcutaneously or intravenously
 Should NEVER be given IM (think about it!)
Unfractionated Heparin (UH)
 Given intravenously
 Advantages
- Rapidly reversed by turning off the infusion (typically half life is 30minutes, so APTT
will return to normal within this period)
- This it is still used in patients where they may be at risk of bleeding but still need
anticoagulation; other indications include surgical patients, renal failure patients and
cardiac catheter patients
 Disadvantages
- Binds unpredictably and non-specifically to plasma proteins, macrophages and
vascular endothelium leading to an unpredictable response to dosing.
- Binding to plasma proteins can lead to heparin resistance, where very large doses
are required to achieve anticoagulation.
 Side effects
- Bleeding – if clinically significant needs to be reversed by (a) stopping the heparin
and (b) giving IV protamine infusion or FFP
- Heparin Induced Thrombocytopaenia [HIT] – heparin binds to platelet factor 4
forming a complex. UH may induce the production of an auto-antibody against this
complex, which in turn causes thrombocytopaenia. Paradoxically (despite low
platelets) there is an extension of existing thrombus and risk of further thrombosis.
- Osteopaenia – UH binds to osteoblasts, activating osteoclasts and thus leasds to
osteopaenia. This is only of concern in patients who need long term heparin e.g.
pregnant women who have had a DVT or PE early in their pregnancy (Warfarin is
contraindicated)
 Unfractionated Heparin (UH)
Dose (IV)
 Load with 4000 - 5000units stat (IV)
 Typically patients are then given between 20,000
and 50,000 units over 24hours (depending
principally on their lean and total body weight)
 Therapeutic level – aim to keep APTT at
(2 – 3 x control - approx 60 – 90 seconds)
 Monitor therapy by performing APTT at steady
state (6 hours after the infusion begins)
 Dose and infusion should then be adjusted
accordingly (see
http://www.hscj.ufl.edu/resman/manualpdfs/Heparin_Orders_Med_Surg_Crit_Care.pdf )

 APTT needs to be checked at least once every

24hours after therapeutic level is achieved.
A quick calculation …
http://www.hscj.ufl.edu/resman/manualpdfs/Heparin_Orders_Med_Surg_Crit_Care.pdf
Mr James Watt is a 43yo man who is to be admitted from
A&E with a suspected pulmonary embolism.
He is ‘guestimated’ to be 80Kg.
He has no known drug allergies.
Using the link (above) and the IV heparin protocol provided
(1)Write up the recommended heparin infusion on a fluid
chart.
(2)Please work out the rate of the infusion in units/hour and
ml/hour.
(3)At 6hours his APTT is 124seconds.
(4)Please recalculate the infusion rate in units/hr and
ml/hr.
Mr Watt – The answers
According to the protocol
 Write up an infusion of 25,000units of heparin in 250ml
5% dextrose (D5W) i.e. 100units/ml; Mr Watt is 80kg
 He should have had a stat dose of 5000u heparin IV
(which would have been written on the drug chart once
only section)
 The infusion rate for a patient with a PE should run at
15units/kg/hr = (15x80)u/hr = 1200u/hr
 If 100u/ml, this means the initial infusion rate is
12ml/hr.
 If the APTT is 124 seconds – the protocol recommends
that the infusion be turned off for 1 hour and then re-
started at a rate of 3u/kg/hr less than before i.e.
12u/kg/hr
 Thus new infusion rate = (12 x 80)u/hr = 960u/hr =
9.6ml/hr
Mr Watts - the answers
 Fractionated LMWH
 Derived by ‘fractionation’ or depolymerisation of the larger and longer
chained, naturally occurring unfractionated heparin
 Thus they are smaller molecules with shorter polysaccharide side chains
 Principally work by blocking coagulation factor Xa (unlike UH which also
blocks the action of thrombin)
 They have less affinity to plasma proteins, macrophages, endothelium and
osteoblasts
 These features mean they:
- cause less of the side effects of UH e.g. H.I.T and osteoporosis
 are more predictable in effect.
 have a longer half life and therefore need only be given once or twice / 24 hours
 can be administered subcutaneously
 Disadvantage – the long half life means they are less easily reversed so are
not used in ‘high risk’ patients or those with need for rapid, simple reversal of
their anticoagulation e.g. prior to operative intervention.
 Side effects – Bleeding; Other side effects are similar to UH but at a far lower
incidence .
 However they can NOT be used as a substitute for UH in patients with H.I.T; In
this case the heparinoid = Danaparoid is used. [Not to be confused with the
antipsychotic Danisparanoid]
LMWH – Indications and Doses
Acute Coronary Syndromes (ACS), Deep
Indications –
Vein Thrombosis (DVT) and Pulmonary Embolism
(PE) treatment and prophylaxis i.e. similar to those
of UH but LMWHs are now are used as the heparins of
choice.
 Two LMWH are commonly used include: Enoxaparin
(Clexane) and Dalteparin (Fragmin); Both are given
subcutaneously
 Both are dosed by units or mg/kg (thus you need to
know or guestimate the patient’s weight)
 Most hospitals will produce dose/Kg protocols for all
their main indications
LMWH
Indication Enoxaparin Dalteparin
(Clexane) (Fragmin)
Medical 40mg od 5000iu od
DVT prophylaxis
Surgical DVT / PE 40mg 12hours prior to 5000iu 12hours prior to
operation operation
prophylaxis 40mg for each day post- 5000iu for each day
operative that heparin is post-operative that
indicated heparin is indicated.

Treatment of 1.5mg/kg od 100iu/kg - bd

DVT or PE Or 1mg/kg bd
ACS 1mg/kg bd 120iu/kg bd
When to stop the Heparin
 If the patient is on prophylaxis for DVT/PE the heparin may be
stopped once they are mobilising well and clinically improving.
 In patients with ACS – the heparin is stopped when they are pain
free and mobilising well.
 In orthopaedic patients who have undergone total hip replacement
there is evidence to suggest that they should stay on heparin for
several months post-operatively (although this is still not common
practise).
 For medical patients you should think whether the patient would
benefit from long term anticoagulation with warfarin e.g. patients
with AF.
 In patients starting warfarin for DVT or PE, warfarin is started as
soon as the diagnosis is confirmed. The heparin is then continued
until the patient’s INR > 2.0.
 However there is evidence that patients should stay on the heparin
for several days from the time of their admission or diagnosis. This
varies from 5 to 10 days (and is additional to the initiation of
warfarin) – this is not common practise.
 DVT therapy is now commonly initiated as an outpatient (from the
A&E department) BUT all patients should be followed up to
consider possible underlying causes.
Heparin Links
 http://www.mja.com.au/public/issues/177_07
_071002/eik10205_fm.html
Nice summary of LMWH treatment

 http://www.rxkinetics.com/heparin.html
Nice summary of UH treatment (a little technified at the end!)

 http://www.bcshguidelines.com/pdf/heparin_2
20506.pdf
British Society for Haematology guidelines for heparin therapy – hot off
the press 2006

 http://www.hscj.ufl.edu/resman/manualpdfs/
Heparin_Orders_Med_Surg_Crit_Care.pdf
 coumA
+ RIN
anticoagulant

= WARFARIN
Challenging Practice
 List three indications for Warfarin therapy

 Write up a loading regime for a 41yo woman who

is on subcutaneous heparin and has just had a
left lower limb DVT confirmed

 List the essential steps before discharging a

patient on Warfarin

 A 71yo man who is on long term warfarin

treatment presents in A&E with a ‘torrential’
epistaxis. He is haemodynamically stable but his
INR is 9.9. What is your management?
 Warfarin
 The most famous coumarin anticoagulant (name
another!)
 Developed at The Wisconsin Alumni Research
Foundation ( http://www.warf.ws/ - for the
non-believers!)
 Hence its name WARFarin
 Indications
- Venous thrombo–embolic disease
- Pulmonary embolism
- Arterial thrombosis
- Atrial fibrillation/Stroke prophylaxis (primary and
secondary)
- Prosthetic heart valve
- Left ventricular aneurysm and large intra-cardiac
thrombus (primarily seen post MI) Warfarin induced skin necrosis

 Main side effect – Increased risk of bleeding

 Famously causes idiosyncratic skin necrosis at large
doses see …
(http://pathology.uc.edu/LABLINES/V7I6.pdf )
Monitor INR
 Therapeutic levels based on INR
 Target
INR 2 – 3: DVT, PE, AF, Arterial
thrombosis
INR 3 – 4: Metallic heart valve
 Warfarin levels will not reach steady state for several days so early INRs
may be misleading
Drug Interactions With Warfarin
Drugs that Increase INR – Drugs that Decrease INR –
Increase effect Reduce effect
[includes Enzyme inhibitors] [includes Enzyme inducers]
NSAIDs Phenytoin
Omeprazole / Cimetidine Carbamazepine
Macrolides Rifampicin
Ciprofloxacin Oral Contraceptives
Flu/Ketoconazole Griseofulvin

Isoniazid
Trimethoprim
Amiodarone / verapamil
aRetrovirals
Fennerty nomogram
This protocol was designed to
 achieve a target INR of 2 to 3 relatively quickly.
 reduce the risk of over anticoagulation which is more likely to occur
in patients who exhibit greater sensitivity to warfarin (e.g. elderly
patients, patients with liver disease, inadequate nutrition, or CHF).

 However: it does not eliminate INR overswings entirely,

and a lower loading dose of 5mg may be used in patients
thought to be especially at risk.
 It now appears in a modified form in the specific
anticoagulation part of many Trust drug charts (I.e. you
don’t have to memorise it!)
Warfarin Dosing
Warfarin and Haemorrhage
 Haemorrhage is the principal side effect of warfarin
therapy
 The risk is said to increase exponentially once INR goes
above 5.0 – particularly spontaneous intra-cranial
haemorrhage
 However 50% of patients who have bleeds whilst on
warfarin have INR< 4.0
 The risk of haemorrhage is increased by:
- Increasing age > 65yo (although this is multifactorial
and may be related more to increased co-morbidities
and polypharmacy)
- Co-morbidities – liver disease, hypertension, renal
failure, thrombocytopaenia and coagulopathy
- Drugs – enzyme inhibitors, alcohol excess, NSAIDs
- Previous GI or other significant haemorrhage.
Bleeding Hell! – What to do about it
 Major bleeding—stop warfarin; give vitamin K1 - 5 mg by slow intravenous
injection; give prothrombin complex concentrate PCC (factors II, VII, IX and X)
50 units/kg or (if no concentrate available) fresh frozen plasma 15 m/kg

 INR > 8.0, no bleeding or minor bleeding—stop warfarin, restart when INR < 5.0; if
there are other risk factors for bleeding give vitamin K1 0.5 mg by slow intravenous
injection or 5 mg by mouth (for partial reversal of anticoagulation give smaller oral
doses of vitamin K e.g. 0.5–2.5 mg using the intravenous preparation orally); repeat
dose of vitamin K if INR still too high after 24 hours

 INR 6.0–8.0, no bleeding or minor bleeding—stop warfarin, restart when INR < 5.0

 INR < 6.0 but more than 0.5 units above target value—reduce dose or stop
warfarin, restart when INR < 5.0

 Unexpected bleeding at therapeutic levels—always investigate possibility of

underlying cause e.g. unsuspected renal or gastro-intestinal tract pathology.
For the Warfarin addicted ….
http://www.acforum.org/doc_education_management.ppt#316
Another very nice overview of warfarin therapy – with really pretty
slides! A little out of date in parts but very good on the basic science.
http://www.bcshguidelines.com/pdf/oralanticoagulation.pdf
British Society for Haematology guidelines (2005) on oral anticoagulation
– everything you want to know and more!
http://www.mja.com.au/public/issues/181_09_011104/bak
10441_fm.html#CHDDCIFC
Another excellent overview from our friends down under – good on ya!
Be warned!
 All patients placed on warfarin
should be booked into a
specialist anticoagulation clinic
before being discharged.
 They should receive an
anticoagulation booklet once
established on warfarin
 Because it is a commonly
prescribed drug which has
potentially serious side effects,
multiple interactions and
‘complex’ follow up, warfarin
is the beloved drug of exams
and examiners
 Lots of OSCE potential there!
Digoxin
Challenging Practice
 List two indications for digoxin

 List three biochemical abnormalities which will potentiate digoxin

toxicity.

 List the common ECG changes seen with digoxin.

 What is the name of the drug used in severe digoxin toxicity?

Digoxin
Digoxin is a cardiac glycoside derived from the Foxglove plant –
‘Digitalis purpurea’
 Has been used by native tribesmen for centuries as a toxin for their
darts and arrows
 First used for cardiac conditions by the Romans
 Famously described by William Withering in 1785 as a treatment for
‘Dropsy’ (oedema)
 Indications
– Supraventricular tachyarrythmia (AF, Aflutter) – More recently its
use is being superseded by betablockers, rate limiting calcium
channel blockers, as well as amiodarone.
- Heart failure – likewise, its use has become restricted to end
stage disease
 Caution – Reduced Creatinine clearance (see gentamicin section);
Hypokalaemia
 Toxic effects potentiated by Hypokalaemia, hypomagnesaemia
(both commonly caused by diuretic therapy) and hypercalcaemia.
 Both hypomagnesaemia and hypercalcaemia interfere with its effect
on Na+/K+ ATPase.
Digoxin Dosing
 Loading (PO or IV)
- Patient needs ECG monitoring
- Loading dose 250 – 500 mcg (depending on
creatinine clearance)
- Further similar, repeat dose is given 8 hours
later

- Maintenance dose at 24 hours (from first

loading dose) = 62.5 – 125 mcg
- If rate is still poorly controlled dose can be
increased in 62.5mcg increments to a maximum
of 250 mcg / day
Side effects and interactions
Side effects
 Arrhythmia (classically bradycardia and various degrees of heart block.
Allegedly digoxin toxicity may cause any tachy or bradyarrhythmia!)
 Nausea and vomiting, diarrhoea.
‘Slow (bradycardia) and Sick’ – Digoxin toxicity
‘Fast (tachycardia) and Sick’ – Aminophylline toxicity
 Dizziness and confusion.
 Famously –Xanthopsia – Yellow (and green) colouring of the vision
 In practice – most patients (including the elderly!) have no problems on
digoxin (Despite its NTW and their co-morbidities)

Interactions
 Bradycardia inducing agents – betablockers, rate limiting calcium
channel blockers, amiodarone
 Quinidine
 Erythromycin
Digoxin Toxicity
 Toxicity often presents with non-specific signs and symptoms and as
with any patient on medications one needs a very high index of suspicion!

 Remember
- Drugs cause everything!
- ‘Slow and Sick’
– Digoxin toxicity
- The ‘reverse tick sign on an ECG is a sign of digoxin therapy
and not toxicity!
- Regular VEBs, Bradycardia, heart block and arrhythmia may
all be signs of digoxin toxicity

Correct hypokalaemia and other biochemical abnormalities

 Correct treatable causes of renal impairment
 For Severe Toxicity/Overdose one can give (Digibind ®) an IV
preparation made of Digoxin-specific antibody fragments.
Monitoring therapy
 Levels are taken on day (7 or after) of therapy
as this is when steady state is reached.
 Levels taken before this can often be
misleading.
 The level is taken at least 6 hours after the last
dose (this may also be a cause of false concern
when levels performed on admission fall well
within this 6 hour cut-off.)
 However very low or non-existent
concentrations on admission may confirm the
diagnosis of poor compliance.
 Normal range 1.0 – 2.6 nmol/l
 This may vary according to different labs
 Toxicity may occur within the normal range
when other factors e.g. hypokalaemia exist.
DIGOXIN Challenge
A 78yo man with Chronic obstructive pulmonary disease
(COPD) presents in A&E with a right lower lobe pneumonia
and fast AF.
U+Es: Na+ 141mmol/l, K+ 4.1 mmol/l Urea 32.8 mmol/l, Creatinine 140µmol/l.

He is guestimated to weigh 60kg. He has no known allergies.

The lovely 3rd year medical student has written up the
patient’s details for you.
1.Calculate his creatinine clearance
2.Write up the loading and maintenance doses of
digoxin on your charts. You should also write up the
other medications you would give him.
3.List three other investigations he may require.
Digoxin challenge - Answers
(1) Creatinine clearance (ml/min) = (N - age (years) ) x Wt (kg)
serum creatinine (µmol/l)
= (160 – 78) x 60 = 35.1ml/min
140

(2) See charts on the next 3 slides for answers

(3) Full blood count (FBC), Repeat U+Es, Calcium and

magnesium, Blood and sputum cultures
Chest radiograph
Electrocardiogram (ECG)
Arterial blood gas (ABGs)
When heart rate is improved - Echocardiogram
So just how many did you get?
…and there’s more!
References
 http://www.emedicine.com/med/topic568.htm

Recommended review on e.medicine

Insulin and Sliding Scales
Challenging practice
 What are the main differences between the previous generation of
insulins and human analogue insulins?

 Write up an insulin sliding scale, insulin infusion and IV fluids for a type 2
diabetic who has been admitted for a left total knee replacement
tomorrow morning.
Common insulin Regimes used in Diabetes
 Single Dose Intermediate or long acting insulin
(+/- Metformin)

 BD Regimen – Mixed Short / rapid and

Intermediate acting insulin

 QDS (‘Basal bolus’) Regimen – TDS Short / rapid

acting + Nocte Intermediate / long acting
insulin

 Sliding Scale – HONK; DKA; Peri-operatively;

Severely ill or patients who are NBM.
Human analogue insulins
 The previous generation of insulins are slowly being
phased out by the newer insulin analogues.
 In the 1980s most insulin was derived from bovine or
porcine sources.
 Then human insulin was genetically produced.
 Today a new generation of insulin analogues are being
produced by human recombinant DNA technology.
What’s the advantage of the rapid acting
analogues?
 The short or rapid acting analogues are said to be ‘more
physiological’ in their action I.e. they can be used to more
closely mimic the profile of endogenous insulin secretion.

 They are monomeric insulin molecules and this means they

are more rapidly absorbed.

 This in turn means they have a more rapid onset of

action and peak effect.

 For the patient this means they can be taken WITH or

just after a meal. [Rather than the 30minutes prior to a
meal as was required with the previous generation of
insulins]

 This has enabled diabetic patients to live far more

‘normal’ chaotic lives that the rest of us take for granted.
 What’s the advantage of the rapid
acting analogues?
 They have a shorter half life than previous insulins which means their
effects are eliminated more rapidly.
 This has led to reduced hypoglycaemic episodes particularly at night
(‘nocturnal hypoglycaemia’)
 However despite these positive differences these have so far not
translated into major differences in glycaemic control.
The advantage of long acting analogues
 The long acting analogue Glargine has full 24 hour
coverage.

 This means it can theoretically be given at any time

of the day or night which best suits the patient.This
needs to be the same time each day.

 Conventionally this is usually at breakfast or bedtime.

 Caution however must be used in patients with

significant renal impairment (DM is the commonest
cause of ESRF) because of its long half life.

 With reduced excretion there may be a ‘lag effect’

into the next day, increasing the risk of hypoglycaemia.
Human analogue insulins
Insulin Trade name Duration of Superseding
action
Insulin Lispro Humalog Rapid acting Actrapid insulin
Insulin aspart Novorapid Rapid acting Actrapid insulin

Lispro and Lispro Humalog mix25 Mix of

protamine (25% short acting: intermediate
suspension 75% intermediate) and short
acting
Insulin aspart and Novomix 30 Mix of Mixtard 30/70
aspart protmaine (30% short acting: intermediate Actrapid and
suspension 70% intermediate) and short isophane
acting
Glargine Lantus Long acting Monotard or
ultratard

Insulin zinc Humulin L (lente) Long acting

suspension
Insulin detemir Levemir Long acting
Sliding Scale Insulin
 Basic Indication – Hyperglycaemia and acute, severe illness
E.g. DKA, HONK coma, ACS, Stroke, Peri-operative patients, severe sepsis,
prolonged diarrhoea and vomiting, the unconscious diabetic patient.

 If possible it is always better to leave the patient on their regular insulin

regime

 Given IV – IV access is very important but may be quite difficult in the very
sick patient (Theoretically insulin can be given IM as hourly boluses– but in
practice this is never done; With IM injections it is very difficult to gain
glycaemic control and sick patients may have contra-indications such as DIC)

 Previously sliding scales often lead to ‘extreme’ swings in glycaemic control.

 This in turn led to rapid and harmful swings in potassium (hypokalaemia) and
osmotic equilibrium (cerebral oedema)

 Now we use sliding scales which (hopefully) lead to a more gentle reduction
in hyperglycaemia and stops the harmful swings in potassium levels and
osmotic changes.
 Sliding scale insulin
 It seems that every diabetologist in every hospital likes ‘their own’ sliding
scale.
 But some things should be true for all of them
 The Insulin infusion and fluids SHOULD run through the same
venflon. This means you don’t get one without the other!
 Potassium should be added to all bags of fluid unless the serum K+
is >5.0mmol/l
 Type 1 diabetics should NEVER be without insulin; even with low
blood sugars the infusion should not be turned off for prolonged
periods of time.
 The underlying problems must be treated.
 If the blood sugar does not come down – you need to give the
patient more insulin! This means re-thinking and re-writing the
sliding scale
 Dextrose should not be given to hyperglycaemic patients until their
blood glucose is ≤15mmol/l.
 Don’t stop the sliding scale until the patient is (a) eating and
drinking properly (b) clinically improved.
Example of Insulin sliding scale and infusion
Signature
Date Type of fluid Volume Added Rate of doctor

02.09.06 Normal saline 50ml 50 units of According to

Novarapid insulin sliding scale

02.09.06 Sliding scale Units per Hour

Blood glucose
0 – 4.0 Zero (Type2 DM)
0.5 (Type1)
4.1 – 6.0 1.0
6.1 – 7.0 2.0
7.1 – 8.0 3.0
8.1 – 10.0 4.0
10.1 – 12.0 5.0
12.1 – 15.0 6.0
>15.1 8.0

Remember: You would also need to write up regular fluids with potassium
Conversion from sliding scale to
regular insulin regime
 Patient should be clinically improving and eating and drinking adequate
amounts.

 Ideally if the patient is a known diabetic they should be converted back

to their regular insulin or oral hypoglycaemic agents.

 If this is not possible – e.g. new presentation of diabetes, unknown

regimen, insulin still required (wound healing and post MI) – then a
Basal bolus (QDS) regimen or less commonly a BD regimen should be
calculated (see next slides)

 Conversion from sliding scale should occur during the early part of the
working day to ensure no major problems occur. The sliding scale
should be stopped just prior to the meal and the regular insulin
administered with the meal (if a rapid acting analogue) or 30 minutes
prior to the meal if a human or other insulin is being administered.
Conversion from sliding scale to basal
bolus (QDS) insulin regimen
For QDS regimen
 Divide the total dose of insulin in previous 24 hours by two.
 Long acting (evening dose) = approximately 80% of one half.
 The 3 mealtime rapid acting doses = approximately 80% of the other half divided into 3
doses. Normally these would be equal doses in the morning and evening and a smaller
dose at lunchtime.
 But this may vary according to patient’s meals.
 Although this sounds very didactic in fact it is an educated guestimate of the patient’s
insulin requirements. You may find patient’s require very different doses once they are
better.
 These are only suggested regimens and you may find others have very different and even
more helpful views!

E.g. A 23yo newly diagnosed diabetic patient is being converted from his sliding scale to
regular QDS insulin. His total units of insulin over the past 24 hours has been 60 units.
Total 60 units
60/2 = 30 units
Thus evening dose of Glargine (80% of 30) = 24units
Doses of Novorapid AM 8units Lunch 6units PM 8units
 Conversion from sliding scale to
basal bolus (QDS) insulin regimen
 Divide total insulin dose received over 24 hours by 2.
 BD regimen typically total insulin – 2/3rd total in am; 1/3rd in pm
 However you would once again give approximately 80% of the total
insulin units that had been given over the past 24 hours.

E.g. A 63yo known type 2 diabetic man is recovering after major cardiac
surgery. He is eating and drinking well but the surgeon would like him
to remain on regular insulin to promote wound healing. He suggests a
BD regimen. The patient has received 60 units of insulin in the past 24
hours on his sliding scale.
Total = 60units
80% of 60u = 48u
Using Novomix 30
Morning dose = 2/3rds (48u) = 32u; Evening dose 1/3rd (48u) = 16u.
Insulin sliding scale - Example
Mrs Johnson, a 73yo type II diabetic, has been on
an insulin sliding scale for the past 72 hours after
being admitted with HONK pre-coma; She is now
eating and drinking normally and is currently on 3
units / hour of insulin. Her last blood glucose
performed an hour ago = 11.9 mmol/L.

Convert her to a QDS or BD regimen using

novorapid and glargine or novomix30. Please
prescribe your calculated doses on the drug chart
provided.
Sliding scale - Answer
QDS regimen – Glargine (nocte) and Novorapid (tds)
3u / hour = 72 units in 24 hours
≈ 80% of 72 = 58 units; 58/2 = 29units
Novorapid breakfast 10u; lunch 8u; evening 10u
Glargine 28units nocte

BD Regimen – Using Novomix 30

3u / hour = 72 units in 24 hours
≈ 80% of 72u = 58units
Morning dose = 2/3 of 58u ≈ 38units
Evening dose = 1/3 of 58u ≈ 18units

 All patients will require education re: Monitoring; Diet,

complications and hypoglycaemic episodes.
 They should be seen by the diabetes nurse specialist prior to their
discharge and follow up with the specialist diabetes services
confirmed.
Steroid Therapy

Professors Knight and Fowler realised that their ‘hobby’ was getting out of hand!
Challenging Practice
(1) List 5 different routes of administration for steroid therapy with an
indication for each.

(2) List 3 side effects of long term steroid therapy.

(3) List 3 contraindications to steroid therapy

Steroids - Indications
Indications
 Replacement for hypopituitarism and adrenal
insufficiency ( Hydrocortisone orally tds or bd)
 Anti-inflammatory
- Acute: Asthma, COPD, Vasculitis, Allergy, Cerebral
oedema (dexamethasone), Skin disease – eczema, Eye
disease – uveitis, iritis
- Chronic: Inflammatory Bowel Disease (IBD), Rheumatoid
arthritis, SLE, Myositis.
 Others – hypercalcaemia of malignancy, meningitis in
children (<15yo), Pemphigus, AIHA, ITP, Demyelination

NB: Anyone on steroid therapy long term (but not on

replacement therapy) – Don’t forget a Bone Sparing
Agent (BSA)!
‘Give them the ‘roids’ – The Fat man, The
House of God’
 ‘If patient is acutely unwell or ‘stressed’ (e.g.
peri-operative) – they need more, not less
steroids!’

• Routes of Administration – every possible way!

Topical (ointments, creams, drops (eyes))
Oral, IV, IM, PR (enema), Inhaler (nasal and
oral), Nebuliser
 Steroid dosing
Hydrocortisone replacement – (Hypopituitarism and Hypoadrenalism)
Oral: 10mg on waking; 5mg lunchtime; 5mg early
evening.
 Note Hydrocortisone 20mg/24 hours is the replacement
dose required in most patients. This is equivalent to a
patient on long term prednisolone 7.5mg/24hours.
 Thus if a patient is on a long term dose of prednisolone
>7.5mg/24 hours this will suppress their Hypothalamic-
Pituitary-Adrenal [HPA] axis.
 Therefore any patient who presents unwell on replacement
hydrocortisone or on long term prednisolone > 7.5mg/24
hours they need to be given EXTRA steroids either IM or IV
 Give Hydrocortisone 50 – 100mg 6 hourly or as an IV
infusion 1 -2mg/hour
Steroid dosing
Acute Asthma
Prednisolone 40mg od (preferably) or hydrocortisone 100mg 6 hourly
[Oral steroids are preferable over IV or IM therapy in any patient well enough to take
tablets. However in the severely unwell patient IV or IM therapy should be given.]

Acute arteritis, allergy, connective tissue disease

Prednisolone 40 – 60mg od or Hydrocortisone 100mg 6 hourly.

Intracerebral oedema
Dexamethasone Loading dose 10mg IV (if unwell)
Maintenance 2 – 4mg orally or IV /IM.

All Patients should be on the LOWEST possible maintenance dose to

keep them asymptomatic
Unwell Patients and steroids
 If a patient is on long term steroids or has known
Addison’s disease becomes acutely unwell, is NBM or
‘stressed’ (e.g. major surgery) they should be given
extra parenteral (IM or IV) steroid treatment.
 This also applies when the patient has suspected
Addison’s disease.
 IM is slow release and will give a smoother profile
whilst IV gives large, acute peaks and then rapidly
troughs because of its short half life.
 However IM dosing is contraindicated in patients with
Disseminated Intravascular Coagulation (DIC) or high
international normalized ratio (INR).
 If IV : 1 - 2mg/hour run in infusion over 24 hours i.e. 24
– 48mg over 24hours
 Seek an endocrinology opinion in any patient who you
are worried about.
Stopping Steroid Therapy
 Never stop long term steroid therapy abruptly.
 If the patient is acutely unwell they need more
NOT less!
 If they are clinically stable slowly reduce the
dose attempting to withdraw the steroids
completely.
 All patients should be maintained on the lowest
possible dose to keep them asymptomatic.
 Consider the use of other immunosuppressant
agents e.g. azothioprine or methotrexate which
may reduce the need for higher doses of
steroids
For each of the following scenarios
write out your steroid prescription.
 A 23yo with an acute exacerbation of their asthma
requiring admission. They are able to swallow tablets.

 A 79yo with possible temporal arteritis ( ESR = 112).

 A 24yo with Crohn’s disease now presenting with an

acute abdomen. He is on maintenance of prednisolone
10 mg od

 A 57yo on hydrocortisone replacement post pituitary

surgery is admitted with a severe community
pneumonia and drowsiness.

 An 83yo with a large left cerebral hemisphere tumour,

with surrounding oedema and mass effect to the right
Recommended doses
(1) Asthma – Stat prednisolone 40mg (po) followed by 5/7 (only)
course of prednisolone 40mg. You should have also included an
inhaled steroid e.g. beclomethasone, fluticasone or budesonide
2 puffs bd
http://www.brit-thoracic.org.uk/c2/uploads/BGMA.06_Manag_asthma.ppt

(2) Prednisolone 40mg PO od for 4 to 6 weeks, then slow reducing

dose. Success of treatment judged by reduced ESR and clinical
improvement.
http://turner-white.com/pdf/hp_feb03_giant.pdf
(3) And (4) To give hydrocortisone (IV) 24mg – 48mg over 24 hours
(you will need to write an infusion e.g. 24mg in 48mls of n.saline
to run IV at 2ml/hr. Or IM hydrocortisone 50 - 100mg qds).
(5) Patients with raised intracranial pressure and cerebral oedema
require dexamethasone PO, IM or IV 4mg qds. If acutely unwell -
Initially (IV) 10mg then 4mg (IM) 6hourly. If able to take tablets
4mg po QDS.
http://www.bnf.org/bnf/bnf/current/4271.htm?q=%22dexamethasone%22#_h
it
Phenytoin
Challenging Practice
 List three reasons why a patient on Phenytoin may present with a seizure.

 List some drugs which may interfere with the metabolism of Phenytoin.

 How long after a dose should you take the Phenytoin level?
Phenytoin
Indications
 Tonic -Clonic seizures
 Status Epilepticus
 Post-neurosurgical intervention
 Trigeminal neuralgia
 More recently it has become a second/third line
therapy in primary epilepsy because of its ‘nasty side
effect profile’ and the need to monitor its effects.

Therapy needs to be monitored because of:

 Narrow Therapeutic window (NTW)
 Zero order kinetics i.e. Non-linear relationship between
dose and plasma concentration. Thus, small increases
in the dose may lead to unpredictable (large) rises in
plasma concentration and precipitate toxicity.
Phenytoin – side effects
Acute (and chronic):
 Cerebellar syndrome – nystagmus, ataxia,
dysarthria.
 Paradoxical seizures – can be a sign of
toxicity and poor/non-adherence to
therapy
 Nausea and vomiting
 Sedation and confusion – thus it should be
given as single night time dose.

Chronic
 Aplastic anaemia and pancytopaenia
 Megaloblastosis (and anaemia) – Drug
effect and folate deficiency
 Hirsutism, coarse facies and acne
 Gingival hyperplasia
 Peripheral sensory neuropathy

Thus proving ‘DRUGS CAUSE EVERYTHING!’

Phenytoin Dosing and Monitoring
 Phenytoin is still used very effectively in status
epilepticus.
 It requires a loading dose with the patient in a
high dependency area, on an ECG monitor.
Loading dose

IV Infusion – 15mg/kg run at a rate NOT

exceeding > 50mg/minute.
Maintenance doses thereafter

IV infusion – 100mg 6 to 8 hourly

 When given IV it is very phlebitic and should be
followed by a saline flush whenever possible.
Phenytoin Dosing and Monitoring
 Steady state is reached after 7 to 10 days
 90% of the drug is protein bound in the serum thus significant
hypoalbuminaemia will effect the drug levels.
 Significant renal impairment (i.e. creatinine clearance <
20ml/min) will also effect the drug levels

Calculating phenytoin level correction

In hypoalbuminemia:
Corrected level= Measured phenytoin level
[(albumin x 0.2) + 0.1]

In renal failure: CrCL < 20 ml/min

Corrected level= Measured phenytoin level
[(albumin x 0.1) + 0.1]
 Levels
Post loading dose:
 2 to 4 hours (checks for significantly high levels i.e. potential toxicity)

 Trough level: Immediately prior to the regular daily dose.

- Taken on or after 10th day after starting regular therapy
- This gives information about the accumulating level of the drug
- Also checks patient adherence to drug

 Peak level: Phenytoin levels peak 3 to 9 hours post dose; Should be in the
therapeutic range (as below)
- Peak level is taken at 4 – 6 hours post dose; This is essential to check for potential
toxicity

Therapeutic Phenytoin levels

10 - 20 mg/l

(Note that reference ranges may vary between laboratories and local reference ranges should be consulted)
 Toxicity and overdose
 Because of its zero order kinetics (unpredictability of plasma
concentration with change of dose) - the dose should be increased
gradually by small 50mg increments.
 Once increased the dose should not be changed for several weeks.
 Its effect should be judged by monitored levels and clinical response
 Likewise, Phenytoin therapy should not be rapidly withdrawn unless
there is a life threatening complication (It may lead to status
epilepticus)

 Toxicity – see side effects; Usually cerebellar signs, reduced level of

consciousness, coma, hypotension and bradycardia.
 Toxicity is potentiated by enzyme inhibitors.
 Patients who present with severe toxicity may require intubation and
ventilation, cardiovascular support with inotropes and with very toxic
levels haemodialysis may be required to remove the drug from the
circulation.
Interactions – (see Warfarin section)
 Phenytoin is an enzyme inducer (interferes with metabolism OCP and
Warfarin, reducing their levels.)
 Plasma concentration increased by Enyme inhibitors - Increased TOXICITY
 Plasma concentration reduced by Enzyme inducers – Reduced Effect
 Some drugs e.g. ciprofloxacin have a unpredictable effect on phenytoin
levels.
Phenytoin Exercise
A 34yo man presents to A&E in
status epilepticus which has been
poorly responsive to IV and rectal
doses of diazepam. He is
estimated to be 75kg.
His WCC 15.9x109/l
RBG 7.1mmol/l
Na+ 123mmol/l
Otherwise his bloods are normal.

(1) Calculate and write up a

prescription for a phenytoin
infusion including the minimum
time for the infusion.

(2) His CT head scan is shown

opposite. Please write out your
further management.
Answers
(1) 15mg/kg (75Kg) = 15x75 = 1125mg total
(2) The infusion is made up of a solution of
phenytoin 50mg/ml – thus the infusion will be
1125/50 = 22.5ml
(3) Maximum rate = 50mg/min.
The solution is 50mg/ml i.e. the rate is 1ml/min.
Thus the infusion should run for a minimum of
1125/50 = 22.5 minutes.
If the infusion is 22.5ml it makes the calculation
easier to make it up to 30ml with normal saline,
not 30ml of 50mg/ml (1500mg infusion) and run it
over 30minutes.
Answers
The CT head scan shows a large right sided primary brain tumour with
surrounding oedema.
There is little midline shift but there is evidence of raised intracranial pressure.
His GCS will invariably be ≤7 as he is in ‘status’ therefore he will require
intubation and ventiltion (he would have require this for the CT head scan), IV
dexamethasone and referral to a neurosurgical ITU.
 Caco-2 [Caco2]

 Caco-2 [Caco2] are epithelial cells isolated from colon tissue

derived from a 72-year-old, White, male with colorectal
adenocarcinoma.
 This cell line is a suitable transfection host and has
applications in cancer and toxicology research.
 Transfection is a process by which foreign nucleic acids are
delivered into a eukaryotic cell to modify the host cell's
genetic makeup.
 Caco-2 [Caco2]
 Caco-2 cells, also known as Cancer coli-2, are a type of immortalized
cell line that were derived from a human colorectal adenocarcinoma.
 Today, Caco-2 cells are used to model the intestinal epithelial barrier.
 Jorgen Fogh developed them at Sloan-Kettering Cancer Research
Institute in 1977.
 One of the main reasons Caco-2 cells are used is because they can
spontaneously differentiate into a heterogeneous mixture of intestinal
epithelial cells when grown in culture.
 Additionally, when cultured under specific conditions, the cells
become differentiated and polarized, which makes them resemble the
enterocytes that line the small intestine.
 These cells have enterocyte-like tight junctions, microvilli, enzymes,
and transporters.
 One of the most common ways Caco-2 cells are used in research is
through a permeability assay, which is used to investigate intestinal
permeability.
 This assay predicts drug absorption in vivo by measuring compound flux
across polarized Caco-2 cell monolayers.
Caco-2 [Caco2]
 In general, a good Caco-2 permeability is considered to be above 5 x
10(-6) cm/s.
 For compounds with this level of permeability, human GI absorption
ranges from 50 to 100%.
 To differentiate Caco-2 cells, standard methods involve seeding
them on culture inserts fitted with polycarbonate filters, and
allowing spontaneous differentiation to proceed for two to three
weeks in culture medium containing 10% or 20% foetal bovine serum.
 It's important to note that Caco-2 cells are tumourigenic in nude mice
and forms moderately well differentiated adenocarcinoma consistent
with colonic primary (grade II) and it's also susceptible to Human
immunodeficiency virus 1.
 Caco-2 cells are mostly reported as non/low-mucus producing cells
and do not or hardly express mucin-related genes, such as MUC-2 and
MUC-6.
References
www.tufts.edu/med/neurosurgery/
 Wonderful image on first phenytoin slide

http://www-
clinpharm.medschl.cam.ac.uk/pages/teaching/i
mages/
 Excellent clinical pharmacology resource
http://www.bnf.org/BNF/bnf/current/3617.htm
 BNF guide to management of status epilepticus
Pharmacogenetics

MLP 421

Dr. Wanjala P. M.
 Background & Introduction

Contents Importance & Goals of Pharmacogenomics

Pharmacogenetic phenotypes

Pharmacogenomic tests Pharmacogenetics &

Drug development Pharmacogenetics in

clinical practice

Advantages and barriers of pharmacogenomics

What is pharmacogenomics
 Pharmacogenomics is an important example of
the field of precision medicine, which aims to
tailor medical treatment to each person or to
a group of people.
 Pharmacogenomics looks at how your DNA
affects the way you respond to drugs.
 In some cases, your DNA can affect whether
you have a bad reaction to a drug or whether
a drug helps you or has no effect.
 Pharmacogenomics can improve your health
by helping you know ahead of time whether a
drug is likely to benefit you and be safe for
you to take.
 Knowing this information can help your doctor
find medicine that will work best for you.
Background
Types of Genetic Variants
⚫A polymorphism is a variation in the
DNA sequence that is present at an
allele frequency of 1% or greater in a
population.

⚫ Two major types of sequence variation

are:
◦ single nucleotide polymorphisms (SNPs)
◦ insertions/deletions (indels).
⚫ Indels
are much less frequent in the
genome and are of low frequency.
 SNPs

A single nucleotide polymorphism (SNP), is a variation in

a single nucleotide that occurs at a specific position in the
genome, where each variation is present to some appreciable
degree within a population (e.g. >1%).
SNPs types

SNPs usually occur in non-coding regions

more frequently than in coding regions.
Non-coding SNPs in promoters/enhancers
or in 5′ and 3′ untranslated regions may
affect gene transcription or transcript
stability
cDNA Synthesis
cDNA Synthesis
 Application of cDNA
technique in pharmacology
 Genome sequencing, transcriptome, and proteome analysis are of
particular significance in pharmacogenomics.
 Transcriptome analysis can be done by methods of random cDNA
sequencing, mRNA display and, differential hybridization (i.e., cDNA
microarray and associated methods).
 Pharmacogenomic transcriptome analysis and pharmainformatics have
potential as strategies for defining novel drug targets in various diseases.
 Pharmacogenomics enhances the development, commercialization, and
clinical use of conventional pharmaceutical products for common
diseases, and it will eventually become a powerful tool for Evidence-Based
Medicine.
 It is also important to predict interindividual pharmacokinetic differences
by genetic polymorphisms of transporters or pharmacokinetic changes by
transporter-mediated drug interactions during drug development.
 Pharmacogenomics and pharmainformatics enable scientists to move
quickly and efficiently from targets to appropriate medicines.
 Definitions
PHARMACOGENETICS: The effect of
genetic variation on drug response,
including disposition (Pk), safety,
tolerability and efficacy (PD).

PHARMACOGENOMICS: It employs the

tools for surveying the entire genome to
assess multigenic determinants of drug
response.
Pharmacogenetics Pharmacogenomics

The study of genetic Use of genetic

basis for variability in information to guide the
drug response choice of drug and dose
on an individual basis
Importance of
Pharmacogenetics

“One-size-fits-all drugs”
only work for about 60 %
of the population at best
Goals of Pharmacogenetics
 Pharmacogenetic phenotypes
Genetic variations which affects the drug
response can be divided in 2 categories:

1. Variations affecting Pharmacokinetics

2. Variations affecting Drug receptor/target.

How does genetic variation
affect drug effect?

293
The fate of drug in the body
Variations affecting Pk
⚫ Phenotypic consequences of
the deficient CYP2D6
phenotype include
◦ increased risk of toxicity of
antidepressants or
antipsychotics (catabolized by
the enzyme)
◦ lack of analgesic effect of
codeine (anabolized by the
enzyme)
⚫ The ultra-rapid phenotype is
associated with extremely
rapid clearance and thus
decreased efficacy of
antidepressants.
CYP2D6
⚫ Debrisoquin-Sparteine oxidation type
of polymorphism:
CYP2D6 dependent oxidation of debrisoquin
and other drugs impaired
 CYP2C19
Aromatic hydroxylation of anticonvulsant mephenytoin

“Normal and (S) - mephenytoin is extensively hydroxylated by

extensive CYP2C19 before its glucuronidation and rapid
metabolizers” excretion in the urine, whereas
(R)-mephenytoin is slowly hydroxylated to N -
demethylated then to nirvanol, an active
metabolite
Poor metabolizers 1. Lack of stereospecific (S)-mephenytoin
hydroxylase activity, so both (S)- and (R)-
mephenytoin enantiomers are N -demethylated
to nirvanol, which accumulates in much higher
concentrations.
2. Increase the therapeutic efficacy of
omeprazole in gastric ulcer and
gastroesophageal reflux diseases.
CYP2C9
Relative contributions of
different phase II pathways

299
Relative contributions of
different phase II pathways

300
 GI toxicity in case of
Inactivation of MTHFR
Methotrexate

Serotonin receptor Responsiveness to

polymorphism Depression

Beta receptor Responsiveness to

polymorphism Asthma
Pharmacogenetics
and drug receptor
targets
Polymorphism in HMG-CoA Degree of lipid lowering
reductase following Statins

Polymorphism in Ion
Cardiac arrhythmias
channels

Polymorphism in ACE Renal Function Test

 Polymorphism-
modifying diseases
⚫ MTHFR polymorphism is linked to
homocysteinemia, which in turn affects thrombosis
risk. These polymorphisms do not directly affect the
Pk or PD of prothrombotic drugs, such as
glucocorticoids, estrogens, and asparaginase, but
may modify the risk of the phenotypic event
(thrombosis) in the presence of the drug.

⚫ Polymorphisms in ion channels (e.g., HERG,

KvLQT1, Mink, and MiRP1) increase the risk of
cardiac arrhythmias, which may be accentuated in
the presence of a drug that can prolong the QT
interval (e.g., macrolide antibiotics, antihistamines).
 Clinically
available
Pharmacogenomic
tests
A pharmacogenetic trait is any measurable or discernible trait
associated with a drug, including enzyme activity, drug or
metabolite levels in plasma or urine, effects on BP or lipid
levels, and drug-induced gene expression patterns
 deCode Genetics,
Navigenics, 23andMe
 Various type of test are
1. HLA gene tests
a) ABACAVIR & HLAB*5701
b) ANTICONVULSANTS & HLAB*1502
c) CLOZAPINE & HLA-DQ 1*0201

2. Drug metabolism related gene test

a) THIOPURINE & TPMT
b) 5-FLUOROURACIL (5-FU) & DPYD
c) TAMOXIFEN & CYP2D6
d) IRINOTECAN & UGT1A1*28
3) Drug target related gene test

a) Trastuzumab & HER 2

b) DASATINIB, IMATINIB & BCR-ABL 1

4) Combined (metabolism & target) gene test

a) WARFARIN & CYP2C9 + VKORC 1

GENOTYPING
Amplichip

Determine the genotype of

the patient in terms of two
CYP450 enzymes: 2D6 and
2C19

FDA approved the test on

Dec 24, 2004. The Amplichip
CYP450 test is the first FDA
approved pharmacogenetic
test.
Pharmacogenetics
& Drug
development
 Key players

31
Role of pharmacogenetics in drug development

1. Can indentify new targets. For eg.

a) Genome wide assessment could identify genes

whose expression differentiate inflammatory
process.

b) A compound could be identified that

can change expression of gene responsible for
inflammatory process.

c) That compound can serve as starting point for

anti-inflammatory drug development.
Role of pharmacogenetics in drug development
2) Pharmacogenetics may identify subsets of patients
who will have a very high or a very low likelihood of
responding to an agent.

a) So, drug can be tested on selected patients will

respond & low possibility of ADRs.
b) This will reduce the time & cost of drug
development.

3)Pharmacogenomics can identify the subset of

patient with higher risk of serious adverse effect. So,
these patients can be avoided in trials
Role of pharmacogenetics in drug development

• Pharmacogenetic data can be submitted to

responsible persons for their application.

• If pharmacogenetics studies on animals are available

then pharmacogenetic tests should be included in
clinical trials.

• During application sponsor should submit the

pharmacogenetic data voluntarily, intended to put on
label of the drug.
 Chemogenomics
• Chemogenomics, or chemical genomics, is the
systematic screening of targeted chemical libraries of
small molecules against individual drug target
families (e.g., GPCRs, nuclear receptors, kinases,
proteases, etc.) with the ultimate goal of identification
of novel drugs and drug targets.
Pharmacogenetics
in clinical practice
• Three major types of evidence that should
accumulate to implicate polymorphism in clinical
care.

1.Screens of tissues from individuals linking the

polymorphism to a trait.

2.Complementary preclinical studies.

3.Multiple supportive clinical phenotype/genotype

association studies.
• Despite considerable research activity,
pharmacogenetics are not yet widely utilized in
clinical practice.

• Dose adjustment on the basis of renal or hepatic

dysfunction can be accepted by clinician.

• But there is much more hesitation from clinician to

adjust the dose on pharmacogenetic ground.

• This can be due to resistance to accept or can be

due to unfamiliarity with the principles of genetics.
• Another hurdle in the path of Pharmacogenetics is
Genetic Discrimination.

• Genetic discrimination occurs if people are treated

unfairly because of differences in their DNA that
increase their chances of getting a certain
disease.

• For example, a health insurer might refuse to give

coverage to a woman who has a DNA difference
that raises her odds of getting breast cancer .

• Employers also could use DNA information to

decide whether to hire or fire workers.
 In America, Genetic Information
Non-discrimination Act (GINA)
2008
⚫ It is a federal law that protects Americans from
being treated unfairly because of differences in
their DNA that may affect their health.

⚫ The new law prevents discrimination from health

insurers and employers.
⚫ This has hardly been replicated in Africa but
indirectly “Equality and Freedom from
discrimination law exists in Kenya”.
Advantages of pharmacogenomics
1. To predict a patient’s response to drugs

2. To develop “customized” prescriptions

3. To minimize or eliminate adverse events

4. To improve efficacy and patient compliance

5. To improve rational drug development

Pharmacogenetic test need only be conducted once during

the life time.
Advantages of pharmacogenomics…

1. To improve the accuracy of determining

appropriate dosage of drugs
2. To screen and monitor certain diseases
3. To develop more powerful, safer vaccines
4. To allow improvements in drug discovery and
development
Barriers of Pharmacogenomics
1. Complexity of finding gene variations that affect drug
response.
⚫ Millions of SNPs must be identified and analyzed to
determine their involvement in drug response
2. Confidentiality, privacy and the use and storage of
genetic information
3. Educating healthcare providers and patients
⚫ Complicates the process of prescribing and
dispensing drugs
⚫ Physicians must execute an extra diagnostic step to
determine which drug is best suited to each patient
 Barriers of Pharmacogenomics..
4. Disincentives for drug companies to make
multiple pharmacogenomic products
⚫ Most pharmaceutical companies have been
successful with their “one size fits all” approach
to drug development
⚫ For small market- Pharmaceutical companies
hundreds of millions of dollars on
pharmacogenomic based drug development.
 Pharmacogenomics &
Personalized medicine
• Pharmacogenomics is still in early stages of
development.

• Much of the excitement regarding the promise of

human genomics hopes on the “PERSONALIZED
MEDICINE OR MAGIC BULLETS”.

• Reality of the added complexity of additional

testing & need for interpretation of results to
individualized dosing has been ignored.
Clinomics

As personalized medicine
Clinomics: is the study of advances, clinomics will
genomics data along be a bridge between
with its associated basic biological data and
clinical data. its effect on human
health.
 CONCLUSION
• Pharmacogenomics has great potential to optimize
drug therapy.

• Newer molecular diagnostic test will have to be

develop to detect polymorphisms.

• Pharmacotherapeutics decisions will soon become

fundamental for diagnosing the illness & guiding the
choice & dosage of medications.
 Scope of Pharmacogenomics

49
THANK YOU
References
1 Relling MV, Giacomini KM. Pharmacogenetics Brunton
Laurence, Chabner Bruce, Knollman Bjorn, editors.
Goodman and Gillman’s The Pharmacological Basis of
Theraputics.12ed. USA: McGraw Hills; 2011.p145-68.

2 Rang HP, Dale M M, Ritter JM, Flower RJ, Henderson

G. Pharmacogenetics, Pharmacogenomics &
Personalised medicine. Hyde Madelane, Mortimer
Alexandra, editors. Pharmacology. 7ed.Britain:
Elsevier Churchill Living stone; 2012.p132-8.
5 Semizarov D, Blomme D.Introduction genomics &
personalised medicine.Genomics in Drug
Discovery and Development .1ed. USA: Wiley;
2009.p1-24.

6 Dr. Hemant Banga’s Seminar on

Pharmacogenetics
 MLP 421
PHARMACOGNOSY
By: Dr. Paul Wanjala Muyoma
 Introduction
 Plants have been one of the important sources of
medicines since the beginning of human civilization.
 There is a growing demand for plant-based medicines,
health products, pharmaceuticals, food supplements
and cosmetics.
 According to the WHO survey 80% populations living in
the developing countries rely almost exclusively on
traditional medicine for their primary health care
needs.
 In addition, herbs have provided us some of the very
important life saving drugs used in the modern
medicine.
 However, among the estimated >350,000 plant species,
only 6% have been studied for biological activity and
about 15% have been investigated phytochemically.
 DEFINITION:

Pharmacy is the science of drug making/

deals with their procurement (bring
about), testing, storage and conversion
into suitable forms ( tablets, capsules,
emulsions etc.)
 CRUDE DRUG
It is the simple drug ,crude drugs are plant,
animal and their parts which after collection
are subjected only to drying or making them
into transverse or longitudinal pieces or
peeling (stripping off skin or bark).

They exist in natural forms.

 SOUCES OF DRUG
Drugs used in medicine may be organic and inorganic in
nature.
Organic drugs are essentially of 2 types:
1. Purely synthetic: The product of man ‘s creation of
new chemical entities ( structures) non
– existent before the era of

synthetic
chemistry.
2. Drugs of biological origin: Produced in the living cell,
 PHARMACOGNOSY

Recently it includes:
1Modern isolation techniques.
2 Pharmacological testing procedures to
prepare purified substances.
3Cultivation and propagation by tissue
culture
 PHARMACOGNOSY

 The word pharmacognosy was coined in 1815 by a

German Scientist SEYDLER.
Definition
 It is a branch of pharmacology dealing with
medicinal substances of biological origin and
especially medicinal substances obtained from
plants, animals and mineral.
 SCOPE OF PHARMACOGNOSY
Pharmacognosy has broad scope in the field of pharmacy such as :

1. ISOLATION and/or ANALYSIS OF PHYTOCHEMICAL :

• Eg; Strong acting substances such as glycosides from digitalis leaves,

• Alkaloids from the plants of Belladonna, Hyocyamus, Rauwlofia

• Morphine and other alkaloids from the plant opium are isolated and
clinical uses studied
 2. STRUCTURE ACTIVIT Y RELATIONSHIP:

Eg : Tubocurarine and Toxiferine from curare plant have muscle

relaxant properties because of quaternary ammonium
groups.

The hypotensive and tranquillizing actions of

reserpine are due to the trimethoxy benzoic acid
3. DRUGS OBTAINED BY PARTIAL SYNTHESIS OF NATURAL
PRODUCTS:

Eg : Preparation of Steroid hormones from diosgenin by acetolysis

and oxidation and further preparation of

cortisone by microbial reactions.

4. NATURAL PRODUCTS AS MODELS FOR SYNTHESIS OF NEW
DRUGS:

Eg: Morphine is the model of a large group of potent drugs .

Cocaine for local anaesthetics Atropine for certain spasmolytics

5. DRUGS OF DIRECT THERAPEUTIC USES :

 Among the natural constituents which even now cannot be replaced

are important group of antibiotics, steroids, ergot alkaloids, vincristine
etc

6. BIOSYNTHETIC PATHWAYS INVESTIGATION :

 Biosynthetic pathways are of primary and secondary metabolites.

 Some of the important pathways are Clavin ‘s cycle of photosynthesis,

 Shikimic acid pathway of aromatic compounds

 Acetate hypothesis for antharacene glycosides

 Isoprenoid hypothesis for terpens

7.CULTIVATION AND COLLECTION OF MEDICINAL PLANTS:
clove, cinchona , cinnamon, senna, opium, etc

8. PREPARATION OF HERBAL FORMULATIONS:

churnas, asvas, aristas, leha, etc

9. DEVELOPMENT OF TISSUE CULTURED PLANTS

Physical parameters
1. Moisture content
2. Ash values
3. Swelling factor
4. Extractive values
5. Melting point
6. Solubility
7. Optical rotation
8. Viscosity
 ASH VALUES

• The residue remaining after incineration is the ash

content of the drug. (inorganic salts of carbonates,
phosphates, silicates of sodium, potassium, calcium
and magnesium) is known as ash content.

• Ash value is a criterion to judge the identity OR

purity of the crude drug
TYPES OF ASH VALUES

1. Total ash value

2. Acid insolubleash value

3. Sulphated ash

value

4. Water soluble ash value

Total ash value:
Useful for detecting:

low grade products

exhausted products

excess of sand

earthy matter with drug

DETERMINATION
1. Weigh accurately about 3 g of the powdered drug
in a tared silica crucible

2. Incinerate the powdered drug by gradually

increasing the heat until free from carbon and cool.
Keep it in desiccators

3. Weigh the ash and calculate the % of the total ash

with reference to the air-dried sample
 Acid insoluble ash value:

1. Used for the determination of earthy matter

present on roots, rhizomes, and on the leaves.

2. Crude drugs contain calcium oxalate

crystals the amount may varies depending on
the environmental conditions.
 DETERMINATION

1. Boil the total ash obtained as above for 5 minutes with

25 ml of dilute HCL

2. Filter and collect the insoluble matter on the ashless

filter paper , wash the filter paper with hot water, ignite in
tared crucible, cool and kept in desiccators

3. Weigh the residue and calculate the acid insoluble ash

of the drug
Sulphated ash value:

Used for the detection of low grade products

Water soluble ash value:

Used to detect either material exhausted by water or

not (Tea leaves, Ginger rhizomes)

SWELLING FACTOR:

Significance:

• Useful in the evaluation of crude drugs containing

mucilage

• Useful for the detection of purity of the crude drug

 DETERMINATION

1. Transfer 1 gm of the seeds to a 25 ml

stoppered cylinder
2. Fill up to the 20 ml mark on the cylinder with
water.
3. Agitate gently and occasionally during 24 hours
and allowed to stand
4. Measure the volume occupied by the swollen
seeds
EXTRACTIVE VALUES:
Significance:
1. Useful for the evaluation especially when the
constituents of the drugs can not be readily estimated
by any other means

2. It also helps to indicate the nature of chemical

constituents present in the drug

3. Also helps in the identification of adulterants

 TYPES
1. Water soluble extractive values

2. Alcohol soluble extractive values

3. Ether soluble extractive values

1. Water soluble extractive value is applied for the drugs which contain
water soluble constituents such as tannins, sugars, plant acids and
mucilage.

2. Alcohol soluble extractive value is applied for the drugs which

contain alcohol soluble constituents such as tannins, resins and
alkaloids

Official method for the assay of myrrh & asafoetida

3.Ether soluble extractive value is applied for the
extraction of volatile oils, fixed oils and resins.

i. Volatile ether soluble extractive value

ii. Nonvolatile ether soluble extractive value

DETERMINATION

Water soluble extractive value:

1. Macerate about 5 gm of the accurately weighed
coarse powder with 100 ml of chloroform water in a
100 ml volumetric flask for 24 hours .

2.Shake frequently for first 6 hours

3.Filter rapidly through filter paper and evaporate 25
ml of water extract to dryness in a tared flat-bottomed
shallow dish.
4. Dry the residue at 105°C and weigh. Keep it in a
desiccators

5. Dry the extract to constant weight, finally, calculate

the % W/W of Water-soluble extractive value with
reference to the air-dried drug.
 Alcohol soluble extractive values

• Macerate about 5 g of the accurately weighed coarse

powder with 100 ml of 90 % alcohol in a 100ml
stoppered flask for 24 hours .

• Shake frequently for first 6 hours

• Filter rapidly through filter paper and collect the filtrate
evaporate 25 ml of alcohol extract to dryness in a tared
flat- bottomed shallow dish.
• Dry the residue at 105°C and weigh. Keep it in a desiccators

• Dry the extract to constant weight ,finally , calculate the % w/w of

alcohol soluble extractive value with reference to the air-dried drug.

• Separation of the alkaloid from unwanted material (Hardly any plant

which contains only one alkaloid exclusively). Separation of each
individual alkaloid from the mixture using chromatographic
techniques.

• Extraction depends mainly on:

The alkaline nature of most alkaloids

• The ability and ease of formation of alkaloidal salt with acids Relative
solubilities of resulting salts either in organic or aqueous media.
• Identification tests Mayer’s reagent, Dragenoorff's reagent, Wagner’s reagent,
Hager’s reagent are used.
• But proto and pseudo alkaloids do not respond to these tests and so they got
the names.
Dragenoorff's reagent
• Stock solution: mix bismuth subnitrate (oxynitrate; 1.7 g) with water (80 ml)
and glacial acetic acid (20 ml).
• Add potassium iodide solution (50% wlv, 100 ml). Shake or stir until dissolved.
Solution keeps indefinitely when stored in a dark bottle.
• Working solution: mix the stock solution ODD ml) with glacial acetic acid (200
ml) and make up to volume o litre) with distilled water.
• Keeps for 2-5 months when stored in a dark bottle.
 PRECIPITATION REACTION:
1. Mayer's or Valser's reagent (Potassium mercuric
iodide) gives white or yellow colour mostly amorphous
precipitates with alkaloids, except the purine bases,
ephedrine Colchicine and ricinine. Mercuric chloride
1.36 Potassium iodide 5.00 g Water to make 100 ml.
2. Wagner's reagent (Iodine- potassium iodide):-
It produces brown or reddish- brown precipitates with all
alkaloids. Iodine 1.3 g Potassium iodide 2.0 g Water to
make 100 ml 3.
2. Dragendorff's or Krauts reagent ( potassium iodide+
bithmus nitrate):-
It produces orange red precipitate which is usually
amorphous. Bismuth nitrate 8.0 g Nitric acid 20.5 g
Potassium iodide 27.2 g Water to 100 ml
 OTHEER PRECIPITATION REACTIONS :

1. Hager's reagent (picric acid):- Gives yellow crystalline precipitate.

2. Tannic acid solution: (5%w/v):- gives buff coloured ppt. which is soluble in
dil. acid or ammonia.
3. Ammonium reineckate solution: 2% solution, produces precipitates with
heterocyclic nitrogen alkaloids, with quaternary and some tertiary amines.
4. Van-Urks test:- (Para-dimethyl-amino-benzaldehyde + sulphuric acid)-- (for
Egot alkaloids).-gives Blue colour.
5. Vitali-Morin test:- test for solanceous alkaloids gives violet colour when
treated with conc. nitric acid and alcoholic KOH.
Mechanism:- These colour tests usually depend upon dehydration or
oxidation of the alkaloid with a resultant characteristic colour.
 Alkaloids
 The alkaloids represent a very extensive group of secondary
metabolites, with diverse structures, distribution in nature, and
important biological activities.

 ‘‘An alkaloid is a cyclic compound containing nitrogen in a

negative oxidation state which is of limited distribution in living
organisms’’.

 This definition includes both alkaloids with nitrogen as part of a

heterocyclic system and many exceptions with exocyclic nitrogen,
such as colchicines or capsaicin.

 Strong physiological effects and the selectivity of some alkaloids

present opportunities for utilizing the alkaloids in human
medicine.
 ALKALOID DESCRIPTION

 Contains nitrogen -usually derived from an amino

acid.
 Bitter tasting, generally white solids (exception -
nicotine is a brown liquid).
 They give a precipitate with heavy metal iodides.
 Caffeine, a purine derivative, does not precipitate
like most alkaloids.
 Alkaloids are basic -they form water soluble salts.
 Most alkaloids are well-defined crystalline
substances which unite with acids to form salts.
 In plants, they may exist In the free state.
 OCCURANCE and DISTRIBUTION
a). Ranunculaceae: Aconitine (aconite).
b). Legumioceae: Physostigmine (physostigma)
c). Loganiaceae: Strychnine and Brucine (Nux-vomica).
d). Papavaraceae: Morphine, Codeine, Thebaine
(Opium).
e). Solanaceae: Hyoscine (Belladona).
f). Berberidaceae: Berberine (Berberis).
g). Rubiaceae: Quinine and Quinidine (Cinchona).
h). Apocyanaceae: Reserpine (Rauwolfia) ,Vincristine
and Vinblastine (Vinca).
i). Liliaceae: Veriterine (Veratrum).
j). Clavicipitaceae: Ergotamine and Ergometrine (Ergot).
Any method will be a good technique, if it will accelerate:
1.Wetting of the surface of the herb particles.
2. Permeability of cell walls.
3. Rate of dissolution of cell contents in the solvent.
4. Outward diffusion of the solution.

EXTRACTION IDEAL PROPERTY OF SOLVENT

1. It should be non-toxic and selective.
2. It should not cause the extract to complex or dissociate.
3. It should be preservative in action.
4. It should promote rapid physiologic absorption of the extract.
5. It should be easily evaporated at low heat.
• NOTE:- Alcohol (Ethanol) will meet all above criteria.
 STAS-OTTO METHOD

The technique involve the distribution of alkaloidal bases between

acid or aqueous solution and immiscible organic solvent.

Method I: The powder is treated with alkalis to liberates the free

bases that can then be extracted with water immiscible organic
solvents.
A). stage1:- Powdered material is moistened with water and mixed
with alkali like sodium & potassium carbonate , ammonia, lime. Make
a paste with water, dry, re-powder.

Concept :- Lime (calcium hydroxide), combines with acid, tannins,

and other phenolic substances and sets free alkaloids.
 Method I:
B). Stage2:-- Extract the free alkaloids by hot continous percolation with
chloroform or any other organic solvents.(Concept :-The free alkaloids
dissolve together with other substances soluble in solvent.)

C). Stage3:--Agitate the chloroform soln. with successive portion of dil.

sulphuric acid separating the aqueous layer before adding the next portion of
acid.(Concept:-The alkaloids are converted into alkaloidal sulphates, which
being soluble in water, pass into aqeous layer)

D). Stage4:--Make the mixed aqueous liquid alkaline with ammonia, collect
the precipitate that forms, wash with water and dry. (Concept :- Ammonia
decomposes the alkaloidal sulphates forming ammonium sulphates, soluble
in water, and the free alkaloid which being practically insoluble in water is
precipitated)
 Method II:

1. The powder is extracted with water soluble organic solvents such as

MeOH or EtOH which are good solvents for both salts and free bases.
And the resultant extract is submitted to same process as that of
method1.

ADVANTAGE:-- This method requires no alkali, gives good penetration of

drug, and Economical.
 Method III

1. The powdered material is extracted with

water or aqueous alcohol containing dilute
acid.

Disadvantage:- Cheap but not used because it

also extract impurities like sugar, mucilage,
tannins, colouring matter.
 Extraction of liquid alkaloids

1. Plant powder is extracted directly with

acidified water.
2. Plant powder is extracted with acidified
alcoholic or a hydro alcoholic solution.
3. This is then followed by distillation under
vacuum (eliminates that alcohol, leaving
behind and acidic aqueous solution of
alkaloid salts).
 PURIFICATION OF ALKALOIDS
1. DIRECT CRYSTALLISATION FROM SOLVENT.
2. STEAM DISTILLATION.
3. CHROMATOGRAPHY TECHNIQUES.
4. GRADIENT pH TECHNIQUES
Though alkaloids are basic in nature, there are variations in the extent in the basicity
of various alkaloids of the same plant
• Depending on this character, the crude alkaloidal mixture is dissolved in 2% tartaric
acid solution and extracted with benzene so that the first fraction contains natural
and/or very weakly basic alkaloids.
• pH of the aqueous solution is increased gradually by 0.5 increment up to pH 9, and
extraction is carried out at each pH level with organic solvent, by this way alkaloids
with different basicity are extracted.
• Strongly basic alkaloids are extracted at the end.
 METHOD FOR STRUCTURE ELUCIDATION

1. U.V. Spectroscopy
2. IR Spectroscopy
3. Nuclear Magnetic resonance spectroscopy
4. Mass spectroscopy
5. X-Ray diffraction
 CLASSIFICATION OF ALKALOIDS BASED
ON THE RING STRUCTURE OR NUCLEUS

1. Pyridine-Piperidine --- Lobiline

2. Tropane--- Atropine
3. Quinoline --- Quinine
4. Isoquinoline --- Emetine
5. Indole--- Reserpine , Vincristine.
6. Imidazole --- Pilocarpine
7. Steroid --- Solanidine
8. Alkaloidal amines --- Ephedrine
9. Purine --- Caffeine , Theophylline
CLASSIFICATION OF ALKALOIDS BASED
ON PHARMACOLOGICAL ACTIVITY
1. Analgesics and narcotics: --- Morphine and codeine.
2. CNS stimulants: --- Caffeine and strychnine.
3. Anticancer: --- Vincristine, vinblastine and taxol.
4. Mydriatics: --- Atropine.
5. Anti-asthmatics: --- Ephedrine.
6. Anti- tussive: --- Codeine.
7. Expectorants: --- Lobeline.
8. Anti- hypertensive: --- Reserpine.
9. Smooth muscle relaxants: --- Atropine and papaverine
10. Skeletal muscle relaxants: --- d- tubocurarine.
11. Anthelmintics: --- Pelletierine and arecoline.
12. Antiparasitics: --- Quinine and emetine.
 CLASSIFICATION OF ALKALOIDS BASED ON
ROBINSON (BASED ON BIOSYNTHESIS)

1. Ornithine alkaloids ---- Pyrolline alkaloids (Nicotine)

2. Lysine alkaloids ---- Anabasine , Lupinine
3. Phenylalanine alkaloids ---- l-Hyoscyamine , Mescaline
4. Tryptophan alkaloids ---- Strychnine , Ergotamine ,
5. Tyrosine alkaloids ---- Morphine , Papaverine , Codeine
6. Terpenoid alkaloids ----- Squalene , Carotenoids
7. Purine alkaloids ---- Caffeine
Classification of Alkaloids based on sources

1. True (Typical) alkaloids that are derived from

amino acids and have nitrogen in a heterocyclic
ring. e.g. Atropine
2. Proto alkaloids that are derived from amino
acids and do not have nitrogen in a heterocyclic
ring. e.g. Ephedrine
3. Pseudo alkaloids that are not derived from
amino acids but have nitrogen in a heterocyclic
ring. e.g. Caffeine
4. False alkaloids are non alkaloids give false
positive reaction with alkaloidal reagents.
Biological Method of
Evaluation
( Bioassay)

The following methods are used:

1. Anti inflammatory activity

2. Analgesic activity

3. Antipyretic activity

4. Anti ulcer activity

5. Antidiabetic activity

6. Anthelmintic activity on earth worms

7.Cardiac activity- on frog and pigeon
8.Microbiological methods- living bacteria, yeast, molds are
used for the assaying vitamins and to determine the activity
of antibiotic drugs
SIGNIFICANCE:
1. The method is generally used when standardization
is not done satisfactory by chemical or physical
methods

2. When the quantity of the drug /sample are very less

then the drugs are evaluated by biological methods

These methods are performed on living animals,

isolating living organ and tissue, animal

preparation, and micro-organism

THANK YOU