STUDY OF POLYMORPHISM IN DOXEPIN

HYDROCHLORIDE

By
RAKESH PANDA

Dissertation submitted to the

Rajiv Gandhi University of Health Sciences,
Karnataka, Bangalore

In partial fulfillment

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of the requirements for the degree of

MASTER OF PHARMACY
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PHARMACEUTICAL CHEMISTRY
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Under the guidance of

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Dr. Gopal Krishna Rao

Department of Pharmaceutical Chemistry

Al-Ameen College of Pharmacy
Bangalore- 560027

MARCH - 2011
 AL-AMEEN COLLEGE OF PHARMACY
Hosur Road, Bangalore – 560 027

Rajiv Gandhi University of Health Sciences,

Karnataka

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DECLARATION BY THE CANDIDATE
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I hereby declare that this dissertation entitled “Study of
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polymorphism in Doxepin Hydrochloride” is a bonafide and genuine
research work carried out by me under the guidance of
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Dr. Gopal Krishna Rao, Professor and Head, Dept. of

Pharmaceutical Chemistry.

Date:-

Place: Bangalore RAKESH PANDA

 AL-AMEEN COLLEGE OF PHARMACY
Hosur Road, Bangalore – 560 027

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CERTIFICATE BY THE GUIDE
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This is to certify that the dissertation entitled “Study of
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polymorphism in Doxepin Hydrochloride” is a bonafide research
work done by Mr. RAKESH PANDA in partial fulfillment of the
requirement for the Degree of Master of Pharmacy in
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Pharmaceutical Chemistry, under my personal supervision and

guidance in the laboratories of our institution and R. L. Fine
chemicals, Yelahanka, Bangalore.

Date: - Dr. Gopal Krishna Rao

Professor and Head,
Place: Dept. of Pharmaceutical Chemistry,
Al-Ameen college of Pharmacy,
Hosur Road, Bangalore-560027
 AL-AMEEN COLLEGE OF PHARMACY
Hosur Road, Bangalore – 560 027

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CERTIFICATE BY THE CO-GUIDE
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This is to certify that the dissertation entitled “Study of
EV
polymorphism in Doxepin Hydrochloride” is a bonafide research
work done by Mr. RAKESH PANDA in partial fulfillment of the
requirement for the Degree of Master of Pharmacy in
PR

Pharmaceutical Chemistry, under my personal supervision and

guidance.

Date:- Dr. RAMESHA A. R.

Vice President (Technical),
Place: [Link] Chemicals Limited,
Yelahanka, Bangalore
 AL-AMEEN COLLEGE OF PHARMACY
Hosur Road, Bangalore – 560 027

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CERTIFICATE BY THE CO-GUIDE
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This is to certify that the dissertation entitled “Study of
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polymorphism in Doxepin Hydrochloride” is a bonafide research
work done by Mr. RAKESH PANDA in partial fulfillment of the
requirement for the Degree of Master of Pharmacy in
PR

Pharmaceutical Chemistry, under my personal supervision and

guidance.

Date:- Dr. REEMA RAZDAN

Professor,
Place: Dept. of Pharmacology,
Al-Ameen College of Pharmacy,
Hosur Road, Bangalore 560027
 AL-AMEEN COLLEGE OF PHARMACY
Hosur Road, Bangalore – 560 027

ENDORSEMENT BY THE HOD, PRINCIPAL/HEAD OF THE

INSTITUTION

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This is to certify that the dissertation entitled “Study of
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polymorphism in Doxepin Hydrochloride” is a bonafide research
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work done by Mr. RAKESH PANDA under the guidance of
Dr. Gopal Krishna Rao, Professor and Head, Dept. of
Pharmaceutical Chemistry.
PR

Dr. Gopal Krishna Rao Prof. B.G. Shivananda

Professor and Head, Principal
Dept. of Pharmaceutical Chemistry
Date: Date:
Place: Bangalore Place: Bangalore
 COPYRIGHT

DECLARATION BY THE CANDIDATE

I hereby declare that the Rajiv Gandhi University of Health

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Sciences, Karnataka shall have the rights to preserve, use and
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disseminate this dissertation / thesis in print or electronic format
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for academic / research purpose.
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Date: RAKESH PANDA

Place: Bangalore

© Rajiv Gandhi University of Health Sciences, Karnataka

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 ACKNOWLEDGEMENT

I would like to take privilege to thank the selfless people

from the core of my heart who with their constant support,

affection, inspiration and encouragement made me to feel

comfortable to successfully complete this venture.

It is my privilege to express my gratitude and heartfelt

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thanks to my esteemed guide Dr. GOPAL KRISHNA RAO, Professor and
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Head, Dept. of Pharmaceutical Chemistry, for his dedicated

support and encouragement to excel in what I do and have

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instilled confidence to reach greater heights in life. All in

all, it s my fortune to have him as my guide.

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Words are not enough to thank my esteemed research [Link]

Dr. RAMESHA A.R., Vice President (Technical), R. L. Fine Chem,

Bangalore for his dedicated support, and affection to begin the

project and for the encouragement to reach the final

destination, his kind personal consideration, inspiration,

motivation and a guiding attitude during the course of my

study has been truly comfortable.

 I wish to express my profound gratitude to Prof. B G

Shivananda, Principal, Al-Ameen College of Pharmacy, Bangalore,

for providing the support, encouragement and necessary

facilities during my course of study.

I am also thankful to [Link] K. Roy, Managing Director, R.

L. Fine Chem, Bangalore, for providing me the necessary

drugs,equipments and facilities at his esteemed research

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laboratory during my project work .
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A special word of thanks to Dr. Naseeruddin Inamdar, Professor

& Head, Dept. of Pharmacology and Dr. Reema Razdan, Professor,

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Dept. of Pharmacology for their constant support, valuable

suggestions and guidance during my antidepressant activity

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study of the compounds.

Dr. Sanjay Pai. PN, Professor and Head, Department of

Quality Assurance, deserves a special mention for the advice

and guidance shown in my project, interpretation of analytical

data and the freedom given to me that I enjoyed each moment

working with the analytical instruments.

 I consider it an honor and privilege in thanking

Mr. Rajasekaran, Lecturer and Dr Monica Kachroo, Assistant

Professor Dept. of Pharmaceutical Chemistry, for their

unrelenting support given to me to perform the research work.

I take this opportunity to acknowledge and thank my crew of

dedicated teachers for their constant support and

encouragement, Mrs. Kalpana Devi, Dept. of Pharmaceutical

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Chemistry, for her support given to me.
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I would like to convey my sincere thanks to Indian

Institute of Science, Bangalore for providing the necessary XRD

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and DSC spectras of my synthesized polymorphs for my project

work.
PR

To office staff, library staff and especially my

Department non-teaching staff Mr. Shaiq, Mr. Ravi, Mr. Ayub, Mr.

Vajid, Mrs. Sabiha, and others who have always extended their

full hearted support.

I shall forever remain indebted to my dear friends Radha,

Shadab, Abhishek, Nidhi, Niranjan, Guru, Divya, prity, hanif and

others, whose loving gestures made world a better place for me,

without whose immense help and support my project would not have

been in the form it is.

A special thanks to my juniors, Datendra, Rasul, Jahirul,

Jasim, Susmita, Soujanya and Neelam for lending a helping hand

whenever I needed.

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A special thanks to Mansur and Surekha for lending a

helping hand during antidepressant activity study.

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My cordial thanks goes to my friends Sravan, Maaz,
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Abhijith, Payam and my other batch mate Vanitha, whose love

boosted my confidence, whose criticism helped me improvise

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myself as person, whose supporting and caring hands held mine

in the darkest of hours made me a person what I am today.

Above all, Thank You to the Almighty, who has given me this

opportunity to extend my gratitude to all those people who have

helped me and guided me throughout my life. I bow my head in

complete submission before Him for blessings poured on me.

 There is no friendship, no love, like that of the parent for

the child.

-- Henry Ward Beecher

My father Prof. R. C. Panda who not only provided me with the

wings to fly and chase my dreams but also stood beneath to hold

me each time I fell and imbibe in me the energy and confidence

to fly better again and again. A deep respect my mother Mrs. Geeta

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Rani Panda for her love and full faith in me. I would also thank
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my sister Rachita Panda for her love and affection.
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Date:
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Bangalore: Rakesh Panda

 LIST OF ABBREVIATIONS USED

°
C : Degree centigrade
mg : Milligram
g : Gram
hr : Hours

cts : Centimeters

IR : Infra red spectra

ml : Milli liter

L : Litre

Mol : Mole

Mol. Form. :

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Molecular formula
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Mol. Wt. : Molecular weight

% : Percent
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KBr : Potassium bromide

XRD : X-ray diffraction

DSC : Differential Scanning Calorimetry

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BA : Bioavailability

SEM : Standard error of mean

 ABSTRACT

Polymorphism is the crystallisation of the same compound in more than one distinct

crystal architecture and is associated with different crystal packing arrangements; this

phenomenon is very common in pharmaceuticals. More recently, the polymorphism

of drugs has been the subject of intense interest in the pharmaceutical industry. In

particular, the variation in solubility between different polymorphs is important for

pharmaceuticals, as it can affect drug efficacy, bioavailability and safety. Because

they have different crystal structures, polymorphs have different chemical and

physical properties; they have different melting points, different chemical reactivity,

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different dissolution rates and different bioavailability.

Attempts for crystallization of 3-(dibenzo [b,e]oxepin-11(6H)-ylidene)-N,N-

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dimethylpropane-1-amine hydrochloride (doxepin hydrochloride), an antidepressant

compound, were made and doxepin hydrochloride was found to produce one distinct
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polymorph. This polymorph was characterized by FTIR, DSC and XRD studies. Shift

in the peak position as well as absence of some peaks in IR spectra showed the
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presence of polymorph and differences in the enthalpy in DSC indicated the

differences between polymorphs. Since, XRD is the predominant tool for the study of

polycrystalline materials, comparison of relative intensities with interplanar spacing

of XRD pattern, confirmed the existence of one polymorph.

In current study, this polymorph was evaluated for their in vivo antidepressant activity

using forced swim test and tail suspension test models. Out of the 3 different doses

investigated, at 15 mg/kg i.p and 30 mg/kg i.p doses, GKRP-7 was found to have

enhanced activity when compared with same doses of parent compound, doxepin

hydrochloride.

Keywords: Doxepin HCl, polymorph, FTIR, DSC, XRD, antidepressant activity

 TABLE OF CONTENTS

CONTENTS PAGE NO.

Sl. No.

1 INTRODUCTION 1 – 32

2 OBJECTIVES 33

3 REVIEW OF LITERATURE 34-43

4 METHODOLOGY 44-54

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5 CHARACTERISATION OF PLYMORPHS 55-70
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6 RESULTS 71-83
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7 PHARMACOLOGICAL ACTIVITY 84-92

8 DISCUSSION 93-96
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9 CONCLUSION 97-98

11 SUMMARY 99

12 BIBLIOGRAPHY 100-107
 LIST OF TABLES
TABLE PARTICULARS PAGE
NO. NO.
1. Classical crystallization methods 11-12
2. Classification of antidepressant drugs 27
3. Pharmacokinetics of Doxepin hydrochloride 31
4. List of chemical and reagents 44
5. Solvents used for crystallisation 71
6. Comparison of IR values of Doxepin HCl and its polymorphs 72
7. XRD data of Doxepin HCl 73
8. XRD data of GKRP-1 74
9. XRD data of GKRP-2 75

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10. XRD data of GKRP-3 76
11. XRD data of GKRP-4 77
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12. XRD data of GKRP-5 78
13. XRD data of GKRP-6 79
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14. XRD data of GKRP-7 80

15. XRD data of GKRP-8 81
16. Thermal data for Doxepin HCl and its polymorphs 82
17. Bulk density of Doxepin HCl and its polymorphs 83
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18. Grouping of animals for antidepressant study 85

19. Observation for forced swim test 86
20. Observation for tail suspension test 89
 LIST OF FIGURES
FIGURE PARTICULARS PAGE
NO. NO.
1. General relationship between polymorphs, solvates and 7
amorphous.
2. Mechanism of action of Tricyclic antidepressants 30

3. IR spectrum of Doxepin hydrochloride 55

4. IR spectrum of GKRP-1 56
5. IR spectrum of GKRP-2 57
6. IR spectrum of GKRP-3 58
7. IR spectrum of GKRP-4 59

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8. IR spectrum of GKRP-5 60
9. IR spectrum of GKRP-6 61
10. IR spectrum of GKRP-7 62
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11. IR spectrum of GKRP-8 63
12. XRD pattern of Doxepin hydrochloride 64
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13. XRD pattern of GKRP-1 65

14. XRD pattern of GKRP-2 65
15. XRD pattern of GKRP-3 66
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16. XRD pattern of GKRP-4 66

17. XRD pattern of GKRP-5 67
18. XRD pattern of GKRP-6 67
19. XRD pattern of GKRP-7 68
20. XRD pattern of GKRP-8 68
21. XRD pattern of Doxepin HCl and its polymorphs 69
22. DSC of Doxepin HCl 70
23. DSC of GKRP-7 70
24. Effect of doxepin and its polymorph GKRP-7 on immobility 87
time in forced swim test in mice.
25. Effect of doxepin and its polymorph GKRP-7 on immobility 90
time in tail suspension test in mice.
 Study of polymorphism in Doxepin hydrochloride INTRODUCTION

1. INTRODUCTION

The physicochemical properties of the active pharmaceutical ingredients are the key

factors to the development of appropriate dosage forms. Most of the organic

substances exists in solid state as polymorphs, pseudopolymorphs, of amorphous

forms. Since all physicochemical properties in solid state are affected mainly in terms

of solubility, dissolution, bioavailability, processability and stability, it is mandatory

to investigate the polymorphic behavior of active ingredients.

The performance of many drug substances depends on the ability to manufacture

reproducibly a particular crystal form, i.e. a certain crystal habit, a solvate or a

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polymorph. One crystalline modification may show 10 times the solubility of other

forms of the same compound and this will affect the bioavailability.
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Solvates and hydrates are formed by many drug substances, for example, oestradiol,

forms solvate with 30 solvents. Adduct have different solubilities and different
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dissolution rate from the anhydrous compounds and this affects the bioavailability.

The amorphous form of the antibiotic novobiocin is at least 10 times more soluble
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than crystalline form. The amorphous novobiocin is rapidly absorbed and is

therapeutically active, while the crystalline form is not absorbed and is therapeutically

inactive.

Anhydrous forms generally have significantly greater dissolution rates and

consequent bioavailability than the hydrous form. The anhydrous form of caffeine,

theophylline and glutethimide dissolve more rapidly in water than corresponding

hydrous forms of these drugs1.

Department of Pharmaceutical Chemistry, AACP 1

 Study of polymorphism in Doxepin hydrochloride INTRODUCTION

1.1 DEFINITIONS
Polymorphism

‘Polymorphism’ comes from the Greek word, Polus = many and morph = shape. Thus

it is defined as the ability of a substance to exist as two or more crystalline phases that

have different arrangements or conformations of the molecules in the crystal lattice. It

essentially means that in different polymorphs, the same molecule exists in different

ways. If this difference is because of packing, it is termed as packing polymorphism

and if it is due to difference in conformation, it is called conformational

polymorphism. As a result of polymorphism, molecules have different arrangements

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in the unit cell of its crystal and thus display different physical properties. These

include different packing properties, thermodynamic properties such as solubility, free

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energy, melting point, etc.,

spectroscopic properties, kinetic properties such as dissolution rate, stability, and

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mechanical properties such as hardness, compatibility, tableting, tensile strength, etc.

Polymorphism is very important in those areas of chemical research where full

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characterization of a material has a pivotal role in determining its ultimate use, e.g., in

pharmaceutical, pigment, agrochemical, explosive and fine chemical industries2.

About 40% of all organic compounds can exist in various polymorphic forms.

Nearly 70% of the barbiturates and 65% of sulfonamides exhibit polymorphism3.

Department of Pharmaceutical Chemistry, AACP 2

 Study of polymorphism in Doxepin hydrochloride INTRODUCTION

For example,

Drug Drug polymorph

Propylphenazone Form I, Form II

Oxybuprocaine hydrochoride Form A, Form B4

Pitofenone hydrochloride 6 Forms5

Famotidine Form A, Form B

Chloramphenicol palmitate 3 Forms

Carbamazepine 3 Forms

More recently, the polymorphism of drugs has been the subject of intense interest in

the pharmaceutical industry. In particular, the variation in solubility between different

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polymorphs is important for pharmaceuticals, as it can affect drug efficacy,
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bioavailability and safety.

Polymorphs can convert spontaneously from less stable to more stable forms, and the
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most stable polymorph will be the least soluble. New forms may appear as a result of

a change in the manufacturing process, even if the change is just in the equipment
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used to dry the final drug substance. A change may even occur whilst materials are

being stored, with no recorded change in the storage conditions. The appearance of a

new polymorphic form of an API is a major concern for pharmaceutical companies6.

Therefore, it is essential to understand various polymorphic forms of a given drug and

their physicochemical properties. In this study, we have investigated different

polymorphic forms of Doxepin Hydrochloride and their physicochemical properties.

Department of Pharmaceutical Chemistry, AACP 3

 Study of polymorphism in Doxepin hydrochloride INTRODUCTION

Pseudo polymorphism

This term refers to crystalline materials with solvent molecules as an integral part of

the structure. It includes solvates and hydrates4.

In pharmaceuticals, polymorphs and pseudo polymorphs can affect bioavailability and

effective clinical use.

Pseudo polymorphs are not strictly polymorphs because they differ from each other in

solid crystalline phase, such as through the incorporation of solvents or water.

Psuedopolymorphs may also be suitable for development and should be identified and

evaluated during solid form screening activities.

e. g.:

Drug

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Ampicillin Ampicillin monohydrate

Ampicillin trihydrate
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1.2 CRYSTALLIZATION

A crystal is, by definition, a solid that has a regularly repeating internal structure
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(arrangement of atoms). This internal periodicity was surmised in the 17th century

from the regularities of the shapes of crystals, and was proved in 1992 when it was

shown that a crystal could act as a three dimensional diffraction grating for X rays,

since X rays have wavelengths comparable to the distances between atoms in

crystals7.

In comparison to crystalline polymorphs, the amorphous form of a drug is generally

expected to be less chemically stable due to the lack of a three dimensional crystalline

lattice, higher free volume and greater molecular mobility8.

The performance of many drug substances depends on the ability to manufacture

reproducibly a particular crystal form, i.e. a certain solvate or a polymorph. One

Department of Pharmaceutical Chemistry, AACP 4

 Study of polymorphism in Doxepin hydrochloride INTRODUCTION

crystalline modification may show 10 times the solubility of another form of the same

compound and this will affect the bioavailability. Since it is now recognized that most

organic compounds, when studied carefully, exist in more than one crystal form, not

only final drug substance but also intermediates may be polymorphic and this may

affect reactivity, solubility and particularly solids-handling characteristics. The latter

will be important in product isolation, when the ability to remove impurities in the

mother liquors will depend to some extent on how well the solid retains the liquors:

this is dependent on crystal form. Development chemists should therefore be aware of

likely problems during scale-up9.

1.3 SOME COMMON TERMINOLOGIES

Crystalline solids

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It implies to an ideal crystal where the structural units, termed unit cells, are repeated

regularly and indefinitely in three dimensions in space. The unit cell has a definite
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orientation and shape defined by the translational vectors, a, b, and c and as a result

has a definite volume V, which contains the atoms and molecules necessary for
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generating the crystal e.g. Ice, sodium chloride, diamond.

Amorphous solids

A material without a real or apparent crystalline nature. It contains no long range

ordering of atoms. Many substances that appear superficially to be amorphous may, in

fact, be composed of many tiny crystals7.

Hydrated forms, Solvate forms and co-crystals

When a solid crystallizes, entrapping a water molecule in a lattice, it is termed as

hydrate. The water of crystallization may evaporate on heating or even upon standing

resulting in an anhydrous form, which may be amorphous.

Department of Pharmaceutical Chemistry, AACP 5

 Study of polymorphism in Doxepin hydrochloride INTRODUCTION

When a solid crystallizes, entrapping solvent molecules in a specific lattice position

and in a fixed stoichiometry, results in a solvate. If the solvent of crystallization,

entrapped randomly in a crystal lattice (in channels, layers or cage), the solid is

termed as nonstoichiometric solvate. Solvates may loose solvent by heating, or even

simply upon standing, thereby transforming into amorphous forms. If incorporated

solvent is water then it is called as hydrate.

Co-crystals are multicomponent crystals based on hydrogen bonding interactions

without the transfer of hydrogen ions to form salts. This is an important feature, since

Bronsted acid-base chemistry is not a requirement for the formation of co crystals.

Primary difference between solvates and co-crystals is the physical state of the

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individual components. If one component is liquid at room temperature then the
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crystals are designated as solvates, if both the components are solid at room

temperature then the crystals are designated as co-crystals. Solvates have potential to
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enhance drug dissolution rate, as shown as solvates of spiranolactone. Solvated

crystals however are often unstable, leading to disolvation during storage and this
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solvent loss may lead to amorphous phase crystallizing into less soluble forms.

Co-crystals, however, tend to be product of more rational design and are stable,

particularly as a co-crystallizing agent at room temperature. As with other crystalline

system, polymorphic crystals are not uncommon. At least 20 have been reported to

date, including caffeine and glutaric acid polymorphic co-crystals. Examples of some

co-crystals are10:

a) Co-crystals of aceclofenac.

b) Co-crystals of 5-nitrouracil.

c) Co-crystals of indomethacin.

d) Co-crystals of fluoxetine hydrochloride (Prozac).

Department of Pharmaceutical Chemistry, AACP 6

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