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Protein Therapeutics
(Pharmacodynamics, Pharmacokinetics & Immunogenicity of proteins & Peptides)

Peptide & Proteins

Proteins are complex, organic compounds composed of amino acids linked together through peptide bonds & cross linked between chains by sulfhydryl bonds, hydrogen bonds & Vander Waals forces. Two amino acid molecules can be covalently joined through a substituted amide linkage, termed a peptide bond, to yield a dipeptide. Such a linkage is formed by removal of the elements of water (dehydration) from the -carboxyl group of one amino acid and the -amino group of another.

Structure of Proteins
Primary structure- is the sequence of amino acid residues. Secondary structure- refers to particularly stable arrangements of amino acid residues giving rise to recurring structural patterns (-helix & -pleats) Tertiary structure- describes all aspects of the three-dimensional folding of a polypeptide. Quaternary structure -When a protein has two or more polypeptide subunits, their arrangement in space is referred to as Quaternary structure.

Sumanth Dept of Pharmacology

Proteins have the most dynamic and diverse role of any macromolecule in the body, catalysing biochemical reactions, constituting receptors and channels in membranes, providing intracellular and extracellular scaffolding support, and transporting molecules within a cell or from one organ to another. Protein therapeutics have several advantages over small-molecule drugs. Proteins often serve a highly specific and complex set of functions that cannot be mimicked by simple chemical compounds. Because the action of proteins is highly specific, there is often less potential for protein therapeutics to interfere with normal biological processes and cause adverse effects. Because the body naturally produces many of the proteins that are used as therapeutics, these agents are often well tolerated and are less likely to elicit immune responses. For diseases in which a gene is mutated or deleted, protein therapeutics can provide effective replacement treatment without the need for gene therapy, which is not currently available for most genetic disorders. The clinical development and FDA approval time of protein therapeutics may be faster than that of small-molecule drugs. Because proteins are unique in form and function, companies are able to obtain far-reaching patent protection for protein therapeutics. The last two advantages make proteins attractive from a financial perspective compared with small-molecule drugs.

Source
A relatively small number of protein therapeutics are purified from their native source, such as pancreatic enzymes from hog and pig pancreas and -1proteinase inhibitor from pooled human plasma. Instead, most therapeutic proteins are now produced by recombinant DNA technology and purified from a wide range of organisms. Production systems for recombinant proteins include bacteria, yeast, insect cells, mammalian cells, and transgenic animals and plants.

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Functional Classification of Protein Therapeutics

Group I: Enzymes and Regulatory Proteins Ia: Replacing a protein that is deficient or abnormal Ib: Augmenting an existing Pathway Ic: Providing a novel function or activity Group II: Targeted proteins IIa: Interfering with a molecule or organism IIb: Delivering other compounds or proteins Group III: Protein Vaccines IIIa: Protecting against a deleterious foreign agent IIIb: Treating an autoimmune disease IIIc: Treating Cancer Group IV Protein Diagnostics

Group I: Enzymes and Regulatory Proteins

Protein therapeutics in this group function by a classic paradigm in which a specific endogenous protein is deficient, and the deficient is then remedied by treatment with exogenous protein. Group Ia are used to replace a particular activity in cases of protein deficiency or abnormal protein production. Examples: 1. Endocrine disorders (Hormone deficiencies) Protein Insulin Insulin aspart Insulin glulisine Insulin lispro Isophane Insulin Function Regulates blood glucose Clinical Use Diabetes mellitus Diabetic ketoacidosis Diabetes mellitus

Insulin analogues with faster onset of action and shorter duration of action Insulin protamine crystalline formulation with somewhat slower onset of action and longer duration of action Insulin detemir Insulin analogues with slower onset Insulin glargine of action and longer duration of action Pramlintide Recombinant synthetic peptide analogue of human amylin 2. Hemostasis and Thrombosis

Diabetes mellitus

Diabetes mellitus

Diabetes mellitus in combination with Insulin

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Protein Factor VIII Factor IX Fibrinogen

Function Coagulation Factor Coagulation Factor Coagulation factor purified from pooled human plasma

Clinical Use Hemophilia A Hemophilia B Control of acute bleeding in patients with congenital fibrinogen deficiency

3. Metabolic Enzyme Deficiencies Protein -Glucocerebrosidase Alglucosidase Function Hydrolyses Glucocerebroside to glucose and ceramide Degrades glycogen by catalyzing the hydrolysis of 1,4 and -1,6 glycosidic linkages of lysosomal glycogen Clinical Use Gauchers Disease Pompe disease (Glycogen storage disease type II)

4. GIT disorders Protein Lactase Function Digests lactose; purified from fungus Aspergillus oryzae Clinical Use Gas, bloating, cramps, diarrhoea due to inability to digest lactose Cystic fibrosis, Chronic pancreatitis

Pancreatic enzymes Digests food (protein, fat & (lipase, amylase, carbohydrate) protease)

Group Ib enhance the magnitude or timing of a particular normal protein activity. Examples: 1. Hematopoiesis Protein Erythropoietin Epoetin Darbepoetin Function Stimulates erythropoiesis Clinical Use Anemia

Modified Erythropoietin with Anemia longer half-life; stimulates RBC production in the bone marrow Stimulates neutrophil proliferation, differentiation, and migration Neutropenia in AIDS or after chemotherapy or bone marrow transplantation

Granulocyte colonystimulating factor (GCSF), Filgrastim 2. Fertility

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Protein Human folliclestimulating hormone (FSH) Human Chorionic gonadotropin (HCG) Lutropin

Function Stimulates ovulation Stimulates ovarian follicle rupture and ovulation Recombinant human Luteinizing hormone

Clinical Use Assisted reproductive technology for infertility Assisted reproductive technology for infertility Infertility with LH deficiency

3. Hemostasis & Thrombosis Protein Tissue Plasminogen activator (tPA) Alteplase Reteplase Function Promotes fibrinolysis by binding fibrin and converting plasminogen to plasmin Contains the nonglycosylated kringle 2 and protease domains of human tPA; functions similar to tPA Nonrecombinant plasminogen activator derived from human neonatal kidney cells Clinical Use Pulmonary embolism MI Acute ischaemic stroke Management of MI

Urokinase

Pulmonary embolism

4. Immunoregulation Protein Interferon -2a Function Immunoregulator Clinical use Hairycell Leukaemia Kaposis Sarcoma Chronic Hepatitis C Hepatitis B Melanoma Follicular lymphoma

Interferon -2b

Immunoregulator

5. Growth Regulation
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Protein Octreotide

Lanreotide

Function Potent Somatostatin analogue; inhibits growth hormone, glucagon, and insulin Cyclical Somatostatin analog

Clinical Use Acromegaly Symptomatic relief of VIPsecreting adenoma Long-term therapy of acromegly

Group Ic Protein therapeutics include foreign proteins with novel functions and endogenous proteins that act at a novel time or place in the body. Examples: Protein Collagenase Function Obtained from fermentation by Clostridium histolyticum; digests collagen in necrotic base of wound Protease from the Carica papaya Recombinant Hirudin, a thrombin inhibitor from salivary gland of megicinal leech Hirido medicinalis Converts plasminogen to plasmin; produced by group c -hemolytic streptococci Inactivates Heparin by forming a stable 1:1 protamine:heparin complex Provides exogenous asparaginase activity Clinical Use Debridement of chronic dermal ulcers and severely burned areas Debridement of necrotic tissue Heparin-induced Trrombocytopenia Prophylaxis against deep vein thrombosis MI Pulmonary embolism Deep vein thrombosis Heparin overdose

Papain Lepirudin Desirudin

Streptokinase

Protamine

L-asparaginase

Acute lympholytic leukaemia (ALL), which requires exogenous asparagine for proliferation

Group II: Targeted Proteins

Group IIa: Interfering with a molecule or organism
Group IIa use antigen recognition sites of immunoglobulin (IgG) molecules or the receptor-binding domains of native protein ligands to guide the immune system to destroy specifically targeted molecules or cells.

Examples: 1. Immunoregulation
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Protein Abatacept Etanercept

Anakinra

Function Selective costimulation modulator Dimeric fusion protein between recombinant soluble TNF receptor and Fc portion of human IgG Recombinant interleukin-1 receptor antagonist

Clinical Use Rheumatoid arthritis Rheumatoid arthritis

Rheumatoid arthritis

2. Cancer Protein Bevacizumab Function Humanized mAb that binds all isoforms of vascular endothelial growth factor A(VEGF-A) MAB that binds Epidermal growth factor receptor (EGFR) Clinical use Colorectal cancer

Cetuximab

Colorectal cancer

Group IIb: Delivering other compounds or proteins

Protein-based, targeted delivery of molecules. Protein Denileukin diftitox Function Directs the cytocidal action of diphtheria toxin to cells expressing the IL-2 receptor Humanized anti-CD33 IgG4 kappa mAb conjugated to calicheamicin, a smallmolecule chemotherapeutic agent Clinical Use Persistent or recurrent cutaneous T-cell lymphoma expressing the CD25 component of the IL2 receptor Relapsed CD33-expressing acute myelogenous leukaemia

Gemtuzumab ozogamicin

Group III: Protein Vaccines

Protein Function Clinical Use Protecting against a deleterious foreign agent (Group IIIa)
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HBsAg

Non-infectious protein on surface of hepatitis B virus HPV vaccine Quadrivalent HPV recombinant vaccine; contains major capsid proteins from four HPV strains OspA Noninfectious lipoprotein on outer surface of Borrelia burgdorferi Treating an autoimmune disease (Group IIIb) Anti-Rh IgG Neutralizes Rh antigens that could otherwise elicit anti-Rh antibodies in an Rh-negative individual Treating Cancer (Group IIIc)

Hepatitis B vaccination Prevention of HPV infection

Lyme disease vaccination

HDNB

Group IV: Protein Diagnostics

Proteins in Group IV are not used to treat disease, but purified and recombinant proteins used for medical diagnostics (both in vivo and in vitro). Protein Capromab Function Imaging agent; indium-111labeled anti-PSA antibody; recognizes intracellular PSA Imaging agent; technetiumlabeled anti-CEA antibody Clinical Use Prostate cancer detection

Arcitumomab

Colon and breast cancer detection

Pharmacokinetics of Protein Therapeutics

Absorption
Enteral Administration
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Peptides and proteins, unlike conventional small molecule drugs, are generally not therapeutically active upon oral administration. The lack of systemic bioavailability is mainly caused by two factors: 1. High gastrointestinal enzyme activity 2. Low permeability through the Gastrointestinal mucosa Although various factors like permeability, stability, and GI transit time can affect the rate and extent of orally administered proteins the molecular size is considered the ultimate obstacle. Suggested approaches to increase the oral bioavailability of protein drugs include encapsulation into micro- or nanoparticles thereby protecting proteins from intestinal degradation. Other strategies are chemical modifications such as amino acid backbone modifications and chemical conjugations to improve the resistance to degradation and the permeability of the protein drug. Coadministration of protease inhibitors has also been suggested for the inhibition of enzymatic degradation.

Parenteral Administration
Most peptide and protein drugs are currently formulated as parenteral formulations because of their poor oral bioavailability. Major routes of administration include: Intravenous (IV) Subcutaneous (SC) Intramuscular (IM)

Other routes may include: Nasal Buccal Rectal Vaginal Transdermal Ocular Pulmonary drug delivery IV administration of peptides and proteins offers the advantage of circumventing presystemic degradation, thereby achieving the highest concentration in the biological system. Protein therapeutics given by the IV route include tissue plasminogen activator (t-PA) analogs Alteplase and Tenecteplase, the recombinant human erythropoietin epoetin-, and the granulocyte-colony-stimulating factor Filgrastim.

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Factors that limit bioavailability of proteins after SC or IM administration include variable local blood flow, injection trauma, and limitations of uptake into the systemic circulation related to effective capillary pore size and diffusion. Several peptide and protein therapeutics including Anakinra, Etanercept, Insulin and pegfilgrastim are administered as SC injections.

Distribution
The rate and extent of protein distribution is determined largely by their size and molecular weight physicochemical properties (charge, lipophilicity) protein binding dependency on active transport processes Since most of the therapeutic proteins have high molecular weights and are thus large in size, their apparent volume of distribution is usually small and limited to the volume of the extracellular space due to their limited mobility secondary to impaired passage through biomembranes. Besides the size-dependent sieving of macromolecules through the capillary walls, charge may also play an important role in the biodistribution of proteins. It has been suggested that the electrostatic attraction between positively charged proteins and negatively charged cell membranes (because of glycosaminoglycans) might increase the rate and extent of tissue distribution.

Protein Binding of Protein therapeutics

Physiologically active endogenous protein and peptides frequently interact with specific binding protein involved in their transport and regulation. Interaction with binding protein may enable or facilitate cellular uptake processes and thus affect the drugs pharmacodynamics. Similarly, therapeutically administered proteins may interact with endogenous binding proteins. It is a general pharmacokinetic principle, which is also applicable to proteins, that only the free Unbound fraction of drug substance is accessible to distribution and elimination process as well as interaction with its target structure at the site of action. Thus, protein binding may affect the pharmacodynamics, but also disposition properties of protein therapeutics. Specific binding proteins have been identified for numerous protein drugs, including recombinant human DNase for use as mucolytic in Cystic Fibrosis, growth hormone etc., Recombinant cytokines, for example, may after IV administration encounter various cytokine-binding proteins including soluble cytokine receptors and anticytokine antibodies.
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o In either case, the binding protein may either prolong the cytokine circulation time by acting as a storage depot or it may enhance the cytokine clearance. Apart from these specific bindings, peptides and proteins may also be nonspecifically bound to Plasma proteins. Metkephamid, a metenkephalin analog, was described to be 44% to 49% bound to albumin Octreotide, a Somatostatin analog, is up to 65% bound to lipoproteins

Elimination
Protein-based therapeutics are generally subject to the same catabolic pathways as endogenous or diabetic proteins. The end products of protein metabolism are thus amino acids that are reutilized in the endogenous amino acid pool for de novo biosynthesis of structural or functional proteins.

Proteolysis
The metabolic rate of protein degradation generally increases with decreasing molecular weight from large to small proteins to peptides, but it is also dependent on other factors such as size, charge, lipophilicity, functional groups and glycosylation pattern as well as secondary and tertiary structure.

Gastrointestinal Protein Metabolism

GIT is a major site of protein metabolism with high Proteolytic enzyme activity due to its primary function to digest proteins. Parenterally administered peptides and proteins may also be metabolised in the intestinal mucosa following intestinal secretion.

Renal Protein Metabolism and Excretion

The kidneys are a major site of protein metabolism for smaller sized proteins that undergo glomerular filtration.

Renal metabolism of peptides and small proteins is mediated through three highly effective processes: Glomerular filtration of larger, complex peptides and proteins followed by reabsorption into endocytic vesicles in the Proximal tubule and subsequent hydrolysis into small peptide fragments and amino acids.

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Glomerular filtration followed by intraluminal metabolism, predominantly by exopeptidases in the luminal brush border membrane of the proximal tubule. Peritubular extraction of proteins from post-glomerular capillaries with subsequent intracellular metabolism.

Hepatic protein Metabolism

Aside from renal and GI metabolism, liver may also play a major role in the metabolism of protein therapeutics. Exogenous as well as endogenous proteins undergo Proteolytic degradation to dipeptides and amino acids that are reused for endogenous protein synthesis. The rate of hepatic metabolism is largely dependent on the specific amino acid sequence of the protein. Proteolysis starts with endopeptidases that attacks in the middle part of the protein and the resulting oligopeptides are then further degraded by exopeptidases.

Chemical Modifications for Optimizing the Pharmacokinetics of Protein therapeutics:

Conjugation of high polymeric mass to protein drugs is generally aimed at preventing the protein from being recognized by the immune system as well as reducing its elimination via glomerular filtration or Proteolytic enzymes. Conjugation of protein drugs with PEG chains increases their molecular weight. PEGylation can also shield antigenic determinants on the protein drug. Eg: Pegfilgrastim is the PEGylated version of Granulocyte-colony-stimulating factor filgrastim.

Immunogenicity of Therapeutic Proteins

The therapeutic proteins currently available cover the whole spectrum, from completely foreign, like bacterial-derived asparaginase to completely-humanlike Interferon -2a. The foreign protein elicits antibodies by the classical pathway which includes ingestion and cleaving of the proteins into peptides by macrophages and
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dendritic cells, presentation of peptides by the MHC-II system and activation of B-cells and boosting, and affinity maturation and isotopic switching of the B-cells by helper T-cells. Furthermore, memory B-cells are induced. The general effects caused by an immune reaction to a therapeutic protein are Acute anaphylaxis, hypersensitivity, skin reactions, serum sickness etc.,

Factors Influencing Antibody Formation to Therapeutic proteins

Structural Factors Impurities Formulation Route of Administration Dose Patient Features

Structural Factors
The degree of nonself and presence of aggregates are the initial triggers of an antibody response to a therapeutic protein. Glycosylation is another important structural factor for the immunogenicity of therapeutic proteins.

Impurities
Impurities are apparently important factors in the immunogenicity of therapeutic proteins. Substances like host cell components, resins from chromatographic columns, or enzymes used to activate the product.

Route of administration
The subcutaneous route is the most immunogenic and the Intravenous the least immunogenic route.

Reducing Immunogenicity
Strategies involved are Changing the amino acid sequence of the protein. Linking proteins to polymers such as PEG and Low molecular weight Dextran. Reducing immunogenic response by Immunosuppressive treatments.

Challenges for Protein Therapeutics

Protein solubility, route of administration, distribution, and stability are all factors that can hinder the successful application of a protein therapy. Body may mount an immune response against the therapeutic protein. Protein to be physiologically active, post-translational modifications such as glycosylation, phosphorylation and Proteolytic cleavage are often required.
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Costs involved in developing protein therapies.

Conclusion
Medicine is entering a new era in which approaches to manage disease are being used at the level of the genetic and protein information that underlies all biology, and protein therapeutics are playing an increasingly important role.

References
David E.Golan, Armen H.Tashjian, Ehrin J. Armstrong, April W. Armstrong, Principles of Pharmacology, The Pathophysiologic Basis Of Drug Therapy,3rd Edition, Lippincott Williams & Wilkins, 2012 Pg no: 895-916 Daan J.A.Crommelin, Robert D.Sindelar, Bernd Meibohn, Pharmaceutical Biotechnology Fundamentals and Applications, Third Edition,published by Informa healthcare Pg No: 95-132

Sumanth Dept of Pharmacology