Research J. Pharm. and Tech.2 (2): April.-June. 2009.

ISSN 0974-3618 REVIEW ARTICLE

1*

www.rjptonline.org

Protein and Peptide Drugs: A Brief Review

1

Pandey Rishabh, 2Singh AV, 1Pandey Awanish, 1Tripathi Poonam, 1SK Majumdar and 2Nath LK
2

Institute of technology and Management, Gorakhpur (UP) India Department of Pharmaceutical Sciences, Dibrugarh University, Assam, India. *Corresponding Author E-mail: pandey.rishabh@rediffmail.com

ABSTRACT

Peptides and proteins continue to grow in popularity for their potential use in current drug therapy and protein drug market will reach $ 55.7 billion by the end of 2011. Protein and Peptide products for therapeutic use include a very diverse range of product as Hormones, Growth factors, cytokines, vaccines and Monoclonal antibodies. Biotechnology has given many ways for the production of protein based drugs as Hybridoma cell technology, Recombinant DNA technology and transgenic animals. Different Technologies are developed for the improved delivery of protein therapeutics but there are number of safety issues, relating to protein products that differ from those raised by low molecular weight products.

KEYWORDS: Protein, Hormone, Monoclonal Antibodies, Cytokines, Immunogenicity .

The living cells produce an impressive diversity of macromolecules that serve as structural components, biocatalysts, hormones, receptors or repositories of genetic information. These macromolecules are biopolymers constructed of monomer units or building blocks and for proteins the monomer units are - amino acids.1 In todays medicine peptides and proteins continue to grow in popularity for their potential use in drug therapy. Protein based drugs are any substance that uses the different proteins that occur naturally in any living organism to diagnose, prevent and treat diseases and conditions to restore or maintain normal body functions. Hormones, serum proteins and enzymes have been used as protein drug ever since the commercial Introduction of Insulin, thyroid hormone, and factor viii from 1920 through 1940.2 Molecular biology has now given us the tools to expand the range of peptide and protein based drug to combat poorly controlled diseases. However the potentially huge impact of this class of drug in therapy has become apparent only recently ,as a result of advances in genomics and proteomics. These have led to the discovery of numerous protein and peptide drugs of therapeutic potential, a number of which are already applied clinically.3
Received on 25.11.2008 Accepted on 16.02.2009 Modified on 23.01.2009 RJPT All right reserved

INTRODUCTION:

STRUCTURE OF PEPTIDES AND PROTEINS: Two amino- acid molecules can be covalently joined through a substituted amide linkage, termed a peptide bond to yield a dipeptide , when many amino-acids are joined , the product is called polypeptide. Proteins are polymers of amino- acids , with each amino acid residue joined to its neighbor by a specific type of covalent bond . Four levels of protein structure are commonly defined. A description of all covalent-bonds linking amino acid residues in a polypeptide chain is its Primary structure. The most important element of primary structure is the sequence of amino acid residues. Secondary structure refers to particularly stable arrangements of amino-acids residues giving rise to recurring structural patterns. Tertiary structure describes all aspects of the three dimensional folding of polypeptide. When a protein has two or more polypeptide subunits, their arrangement in space is referred to as quaternary structure.4 BIO TECHNOLOGY APPROACHPRODUCTION OF PROTEIN AND PEPTIDE DRUGS: Prior to advances in protein and peptide biotechnology such as r.DNA technology and transgenic animals, the few proteins drugs available were taken directly from human and animal corpses and insulin required to treat diabetes was collected from slaughtered pigs. Biotechnology has given many ways for the production of protein based drugs.

BIOCHEMICAL APPROACH -

Research J. Pharm. and Tech. 2(2): April.-June.2009,;Page 228-233

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Table: 1 Hormone therapeutics in current use: HORMONE TRADE NAME ( PRODUCTS ) Thyrotropin Releasing PROTIRELIN THYPINONE, Hormone. ( TRH) ZOLADES Gonadotropin-Releasing LUTEPULAC, FACTRAL Hormone. (GnRH) Somatostatin SANDOSTATIN Growth Hormone Releasing factor Adrenocorticotropin Hormone. ( ACTH ) Oxytocin Vasopressin Human CHORIONIC Gonadotropin. Corticotropin- Releasing Hormone Somatotropin Gonadotropins Parathyroid Hormone Human Calcitonin Insulin Glucagon

Research J. Pharm. and Tech.2 (2): April.-June. 2009. ,

GENERAL USE OR ACTION USED IN PRIMARY BY HYPOTHYROYDISM INDUCE OVULATION AND SPERMATOGENESIS INHIBITION OF THE RELEASE OF GROWTH HORMONE FROM THE
ANTERIOR PITUITARY

USED IN GROWTH HORMONE DEFICIENCY CORTICOTROPIN, ACDH,

ACTHAR

PITOCIN, SYNTOCINON ADH,PITTRESSIN PRAFASE HP, PREGNYL CORTICORELIN, CORTICORELIN OVINE SOMATROPIN, NUTROPIN, SEROSTIM PERGONAL, REPRONEX, FERTINEX, GONAL- F, FOLLISTIM PRATHAR CIBACALCIN GLUCAGEN EMERGENCY
KIT AND DIAGNOSTIC KIT

USED IN DIAGNOSIS OF PRIMARY ADRENO CORTICAL INSUFFICIENCY OR ADRENO CORTICAL FUNCTION. TO INDUCE LABOR DURING CHILD BIRTH USED IN REDUCING EPISODE OF SPONTANEOUS OR TRAUMAINDUCED BLEEDING. FOR EARLY PREDICTION OF PREGNANCY A DIAGNOSTIC AGENT FOR PATIENTS WITH ACTHDEPENDEND CUSHING SYNDROME

USED FOR DWARFISM FOR INDUCING OVULATION IN FEMALE AND SPERMATOGENESIS IN MALES. A DIAGNOSTIC AGENT FOR PATIENTS WITH HYPOCALCEMIA
DUE TO HYPOPARAPHYROIDISM

USED FOR TREATING PAGETS DISEASE OF BONE IN DIABETES USED IN THE TREATMENT OF SEVERE HYPOGLYCEMIC
REACTIONS IN DIABETIC PATIENTS

CHEMICAL SYNTHESIS: Powerful Techniques now make direct chemical synthesis an attractive option in many cases for small peptides and proteins.The complexity of proteins makes the traditional synthetic approaches of Organic Chemistry for peptides with more than four or five amino acid residues. One problem is the difficulty of purifying the product after each step. The major break through in this technology was provided by R.Bruce marrifield in 1962.5 He developed a alternative method of peptide synthesis, on solid phase, has certainly grown in popularity and eliminated some of these problems, but not without its own limitations . HYBRIDOMA CELL TECHNOLOGY FOR THE MONOCLONAL ANTIBODIES: In hybridoma technology, a mouse or other small animal is sensitized with an antigen. When an enough antibody against the selected antigen has been attained animal is sacrificed and its spleen cells are collected. Spleen cells contain large no. of B- lymphocytes.6 To produce MAbs , B cells are fused with immortal myeloma cells in the presence of fusogens they replicate and lessly and can be used to produce specific protein based drugs called Monoclonal antibodies.7,8 The MAbs produced contains various mouse proteins and other contaminants that might require purification.9

RECOMBINANT DNA TECHNOLOGY:The Introduction of r.DNA technology or genetic engineering has given a new way and Cost efficient source of proteinbased drugs. These technologies are capable of producing large amount of drugs. The basic technique in r.DNA technology involve isolating or copying a gene; inserting the precise gene in to transmissible vector that can be transcribed, amplified and propagated by host cells ; biochemical machinery ; transferring it to that host cell ; and facilitating the transcription into m.RNA and translation in to proteins.10 TRANSGENIC ANIMALS FOR PROTEIN DRUG PRODUCTION: Transgenic animal has been gaining application for protein therapeutics. Since the development of transgenic supermice in 1982 and the development of the first mice to produce a human drug , tPA , in 1987 . Transgenic animal in health care are animals that have been genetically modified to produce a particular protein drug. The DNA gene for the desired protein is coupled with a DNA signal directing its production in the mammary glands. The new gene functions in the mammary glands so that the protein drug is made only in the milk.11 Because of the long time periods involved and low success rates, developing transgenic animals is currently very expensive.

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FIGURE:-1 Production of mono clonal antibodies:

Research J. Pharm. and Tech.2 (2): April.-June. 2009. ,

MONOCLONAL ANTIBODIES: Antibodies usage has now extended to treatment; hence these are promoted as potential candidates for the management of various diseases.15 The Abs synthesized against a particular antigen ( Ag ) are known as a monoclonal antibodies (MAbs). By definition, MAbs are a class of highly specific Abs produced by the clones of single hybrid. Cell formed in the laboratory by the fusion of B. lymphocytes with a tumor cell . The process of producing MAbs was invented by Kohler and Milstein in 1975.16 Types of Mabs that are used in treatment currentlyNaked Mabs- these are the antibodies without any drug or radioactive material. 1. Rituximab-It is an Mab directed against the CD20 antigen expressed on the surfaces of normal and malignant B lymphocytes. 2. Alemtuzumab- For B cell chronic lymphocytic leukemia. 3. Bevacizumab-For metastatic colorectal cancer. Conjugated Mabs- They are used to deliver radionucleides toxins , or cytotoxic drugs to a specific tissue or malignant cell population. 1. Gemtuzumab- Mab derived from the CD33 antigen It is the only immunotoxin to receive FDA approval. 2. Ibritumonab tiuxetan-Radiolabeled Mabs ti treat Bcell non-Hodgkins lymphoma.

PHARMACEUTICAL APPROACHTHE CURRENT STATUS OF PROTEIN AND PEPTIDE DRUGS: The total global market for proteins drugs was $ 47.4 billion in 2006. The market will reach $ 55.7 billion by the end of 2011, an average annual growth rate of 3.3%. Modified and engineered antibodies present the largest share of the total global market. In 2006 , this sector held almost 56% of the global market. Hormone protein drugs have the largest AAGR. It will reach $ 11.4 billion by 2011.12 Protein and peptide products for therapeutic use include a very diverse range of products as Hormones , Growth factors , Cytokines , Vaccines and Monoclonalantibodies. The important milestones in peptide hormone discovery as the isolation ,structure determination and synthesis of oxytocin by du Vigneaud and co workers and the determination of the structure of insulin by the sanger.13,14 HORMONES: There are wide variety of peptide and protein hormones, natural as well as their analogs , that are commercially available for the treatment of various diseases or are used for diagnostic purposes. Several of these hormones are obtained synthetically some from natural sources while others are obtained via genetic engineering (Table1) .

OTHER SIGNIFICANT PEPTIDES AND PROTEIN DRUGS: Except Hormone and Monoclonal antibodies many other class of peptide and protein therapeutics are available in a market as Cytokines, Clotting factors and different Vaccines (Table-2). PEPTIDE ANTIBIOTICS: The development of novel antibiotics is a burning necessity now a day. Among many proposed strategies , studies on natural and artificial amphipathic peptides acting on membranes of microorganism have yielded promising result . Currently more than 800 such peptideantibiotics have been described and some of them have already entered clinical trials.17 A growing number of reports, support a role of hemoglobin as a precursor of peptides able to kill microorganism.18 PATENT EXPIRY OF SOME BIOTECH PROTEINS: The patents of the first generation of bio pharmaceuticals derived from recombinant DNA such as Interferon , growth hormones and epoietins are expiring , opening up the possibility for competitors to introduce biosimilar products (Table-3).19,20,21

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TABLE- 2: COMMERCIALLY AVAILABLE SIGNIFICANT PEPTIDES AND PROTEINS: Name Trade Name General Use or Action Angiotensin II Vasoconstricting 8 peptide involved in blood Pressure Regulation Atrial natriuretic peptide 28- peptide, vasodilator, increases glomerular filtration and diuresis Bradykinin 9- peptide produced in response to tissue damage, inflammation, viral infections. Denileukin diftitox Ontak rDNA- derived fusion protein consisting of diphtheria toxin fragments A and B and Interleukin to. Used in treating cutaneous T-cell lymphoma. Deslorelin Somagard GnRH super agonist Used in treating central precocious puberty. Interferon alfa- 2 a referon A Protein of r DNA origin that has orphan DRUG and is Used for a number of malignancies: leukemia, Renal- cell carcinoma, AIDS related Kaposis sarcoma. Interleukin- 2 Tecaleukin Protein of r DNA origin that has orphan DRUG and is Used for renal- cell carcinoma and metastatic malignant melanoma. Pentagastrin Peptavion 5- peptide analog of the c-terminal portion of gastrin. Used in diagnosing gastric secretory problems. Secretin Secretin Ferring 27- peptide Used in diagnosing zollinger- Ellison syndrome Powder Thymopentin Timunox 5- peptide used as immunomodulating investigation drug in HIV positive patients.

Research J. Pharm. and Tech.2 (2): April.-June. 2009. ,

KEY TECHNOLOGIES AND DELIVERY OF PROTEIN DRUGS: Till now, injections are the most common means for administering these protein and peptide drugs but due to the poor patient compliance with injections many alternative routes are also tried by researchers with varying degrees of success like oral , buccal, intranasal, pulmonary, transdermal, ocular and rectal.22-27
FIGURE :-2: r.DNA technology :

The use of many polypeptides as therapeutic agents is hampered by their rapid elimination from the circulation because of enzymatic degradation, renal filtration , uptake by the reticulo endothelial system and accumulation in non targeted organs and tissues.28 Different technologies are developed for the improved pharmacokinetic properties and enhanced exposure of the protein therapeutics, which for the patient yields, less frequent administration, greater convenience, and improved efficacy.29 PROTEIN FUSION AND CONJUGATES: Protein fusion technologies exploit the modular nature of protein functional domains and through molecular engineering, create a new protein ability that combines the long serum persistence of an endogenous protein with the biological activity of the protein of therapeutic interest. Fc fusion technology generates the molecular entities ( Fc domain of antibodies ) with increased half life through the utilization of the FcRn receptor recycling system .30 The other approach of protein fusion technologies for increased time action of protein therapeutics are human serum albumin ( HSA) or Transferrin ( Tf ) fusion proteins .31,32 PEGYLATION: In PEGYlation technology , a large polyethylene glycol ( PEG ) moiety is covalently or reversibly attached to the protein through a reactive group , for example amine , thiol or Carboxylic Acid. Pegylation increases molecular stability , changes the volume of distribution and reduces Immune reactions and polymer attached to the protein globule creates steric hindrance for the interaction of protected polypeptides with active sites of proteases , opsonins and antigen processing cells .33,34

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TABLE: 3: PATENT EXPIRY DATES FOR SOME PROTEIN AND PEPTIDE DRUGS : PRODUCT PATENT/MARKET INNOVATOR SOMATROPIN EUDERASE INSULIN G- CSF IL-2 TPA IFNERYTHROPOIETINIFN- 2 ABBREVIATIONS: G- CSF,
PLASMINOGEN ACTIVATOR

Research J. Pharm. and Tech.2 (2): April.-June. 2009. ,

GRANULOCYTE COLONY- STIMULATING

GENETECH ABBOTT ELI LILLY AMGEN CHIRON GENENTECH INTER MUNE AMGEN BIOGEN/ROCHE

EXPIRED EXPIRED EXPIRED 2015 2006-2012 2005-2010 2005, 2006, 2012 2013 2002 FACTOR; IFN, INTERFERON;

EXCLUSIVITY EXPIRY US

PATENT/MARKET

EXPIRED EXPIRED EXPIRED 2006 2005 2005 2002,2004 2004 2003, 2007 IL, INTERLEUKIN;

EXCLUSIVITY EXPIRY EUROPE

TPA, TISSUE

FIGURE:-3 Production of transgenic animals.

potential option for increasing systemic exposure of proteins. TOXICITY AND SAFETY OF PROTEIN THERAPEUTICS: The quality, safety and efficacy of biotechnology products for therapeutic use are intricately linked, far more so than for conventional medicinal products, leading to the need for increased communication between those responsible for ensuring product quality and those responsible for non-clinical safety testing. Safety issues include microbiological safety (due to the use of biological materials either during the manufacturing process or as an integral part of the products), pharmacological/ biological toxicity (due to excessive primary pharmacology or undesirable secondary pharmacology), immunogenicity and potential tumourigenicity (for example, for growth factors, immunosuppressive monoclonal antibodies and cell therapy products). Genotoxicity and intrinsic chemical toxicity are less of a problem for biotechnology products.37 IMMUNOGENICITY: Immunogenicity is one of the major Concerns in the development and application of biotherapeutics. The patients can mount sustained Immune responses to protein therapeutics with the production of neutralizine antibodies that can compromise efficacy or safety of this drugs.38 The Immunogenicity of protein drugs can be ascribed to a few Immunodominant helper T lymphocyte epitopes , and that reducing the MHC binding affinity of these HTL epitopes contained with in these proteins can generate drugs with lower Immunogenicity.39 Recently method for Investigation of the Immunogenicity of a protein drugs has been developed with the help of Equilibrium dialysis and liquid chromatography/ tandin mass spectroscopy.40 However Protein and peptide therapeutics have been available for centuries. For instance, vaccinations with scabs that contained the small pox virus had been practiced for centuries. Also estrogen, insulin and other

OTHER TECHNOLOGIES: Proteolytic enzymes present at most routes of administration are able to quickly metabolize most peptides. Many molecules are absorbed through deep lungs in to the blood with high bioavailability.35 For transmembrane delivery of protein therapeutics the enhenced endosomal escape is achieved through use of lytic peptides,PH sensitive polymer or swellable dendritic polymers. Cargo proteins having ability to penetrate membranes are also used for transmembrane delivery.36 Controlled-release or depot technologies are also used for the protein and peptide therapeutics as polymer based depot formulations. Different devices such as external pumps, microchips and implantable pumps are the

CONCLUSION:

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hormone replacement therapy were in use for decades. The development of Hybridoma technology, transgenic animals and r.DNA technology led to an explosion of protein based drugs.

Research J. Pharm. and Tech.2 (2): April.-June. 2009. ,

This article has tried to present an overview of the protein and peptide therapeutics. The field is advancing rapidly, and every doctor or pharmacist must stay current with the literature on Protein therapeutics. Different drug delivery systems, technologies, still have to be developed for enhancing exposure of protein therapeutics. The new and relevant approaches to improve human risk assessment for novel protein therapeutics is also needed but the Introduction of second entry biopharmaceuticals or protein drugs is desirable . They will play important role in the life cycle of drugs and reduce health care cost; they will also force the innovator Companies to improve their products. The authors express their thanks to Dr.Kamal kishore , Professor, AIIMS, New Delhi for their valuable suggestions , and to Mr. Shriram Mishra Dept. of Pharmacy, I.T.M, Gida, Gorakhpur for their kind cooperation.

ACKNOWLEDGEMENTS:

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