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

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ISSN 0974-3618 www.rjptonline.org

REVIEW ARTICLE

Protein and Peptide Drugs: A Brief Review

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

1Institute of technology and Management, Gorakhpur (UP) India2Department of Pharmaceutical Sciences, Dibrugarh University, Assam, India.

*Corresponding Author E-mail:[email protected]

ABSTRACTPeptides and proteins continue to grow in popularity for their potential use in current drug therapy and protein drugmarket will reach $ 55.7 billion by the end of 2011. Protein and Peptide products for therapeutic use include a verydiverse range of product as Hormones, Growth factors, cytokines, vaccines and Monoclonal antibodies. Biotechnologyhas given many ways for the production of protein based drugs as Hybridoma cell technology, Recombinant DNAtechnology and transgenic animals. Different Technologies are developed for the improved delivery of proteintherapeutics 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 .

INTRODUCTION:The living cells produce an impressive diversity ofmacromolecules that serve as structural components,biocatalysts, hormones, receptors or repositories ofgenetic information. These macromolecules arebiopolymers constructed of monomer units or buildingblocks and for proteins the monomer units are - aminoacids.1

In todays medicine peptides and proteins continue togrow in popularity for their potential use in drugtherapy. Protein based drugs are any substance thatuses the different proteins that occur naturally in anyliving organism to diagnose, prevent and treat diseasesand conditions to restore or maintain normal bodyfunctions. Hormones, serum proteins and enzymes havebeen used as protein drug ever since the commercialIntroduction of Insulin, thyroid hormone, and factor viiifrom 1920 through 1940.2 Molecular biology has nowgiven us the tools to expand the range of peptide andprotein 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 aresult of advances in genomics and proteomics. Thesehave led to the discovery of numerous protein andpeptide drugs of therapeutic potential, a number ofwhich are already applied clinically.3

Received on 25.11.2008 Modified on 23.01.2009Accepted on 16.02.2009 RJPT All right reservedResearch J. Pharm. and Tech. 2(2): April.-June.2009,;Page 228-233

BIOCHEMICAL APPROACH -STRUCTURE OF PEPTIDES AND PROTEINS:Two amino- acid molecules can be covalently joinedthrough a substituted amide linkage, termed a peptidebond to yield a dipeptide , when many amino-acids arejoined , the product is called polypeptide. Proteins arepolymers of aminoacids , with each amino acid residuejoined to its neighbor by a specific type of covalent bond.

Four levels of protein structure are commonly defined. Adescription of all covalent-bonds linking amino acidresidues in a polypeptide chain is its Primary structure.The most important element of primary structure is thesequence of amino acid residues. Secondary structurerefers to particularly stable arrangements of amino-acidsresidues giving rise to recurring structural patterns.Tertiary structure describes all aspects of the threedimensional folding of polypeptide. When a protein hastwo or more polypeptide subunits, their arrangement inspace is referred to as quaternary structure.4

BIO TECHNOLOGY APPROACH-

PRODUCTION OF PROTEIN AND PEPTIDEDRUGS:Prior to advances in protein and peptide biotechnologysuch as r.DNA technology and transgenic animals, thefew proteins drugs available were taken directly fromhuman and animal corpses and insulin required to treatdiabetes was collected from slaughtered pigs.

Biotechnology has given many ways for the productionof protein based drugs.

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Table: 1 Hormone therapeutics in current use:

HORMONE TRADE NAME ( PRODUCTS ) GENERAL USE OR ACTION

Thyrotropin ReleasingHormone. ( TRH)

PROTIRELIN THYPINONE,ZOLADES

USED IN PRIMARY BY HYPOTHYROYDISM

Gonadotropin-ReleasingHormone. (GnRH)

LUTEPULAC,FACTRAL INDUCE OVULATION AND SPERMATOGENESIS

Somatostatin SANDOSTATIN INHIBITION OF THE RELEASE OF GROWTH HORMONE FROM THEANTERIOR PITUITARY

Growth Hormone

Releasing factor

USED IN GROWTH HORMONE DEFICIENCY

AdrenocorticotropinHormone. ( ACTH )

CORTICOTROPIN,ACDH,ACTHAR

USED IN DIAGNOSIS OF PRIMARY ADRENO CORTICALINSUFFICIENCY OR ADRENO CORTICAL FUNCTION.

Oxytocin PITOCIN, SYNTOCINON TO INDUCE LABOR DURING CHILD BIRTH

Vasopressin ADH,PITTRESSIN USED IN REDUCING EPISODE OF SPONTANEOUS OR TRAUMA-INDUCED BLEEDING.

Human CHORIONICGonadotropin.

PRAFASE HP,PREGNYL FOR EARLY PREDICTION OF PREGNANCY

Corticotropin- ReleasingHormone

CORTICORELIN,CORTICORELIN OVINE

A DIAGNOSTIC AGENT FOR PATIENTS WITH ACTH-DEPENDEND CUSHING SYNDROME

Somatotropin SOMATROPIN, NUTROPIN, SEROSTIM USED FOR DWARFISM

Gonadotropins PERGONAL, REPRONEX,FERTINEX, GONAL-F, FOLLISTIM

FOR INDUCING OVULATION IN FEMALE AND SPERMATOGENESISIN MALES.

Parathyroid Hormone PRATHAR A DIAGNOSTIC AGENT FOR PATIENTS WITH HYPOCALCEMIA

DUE TO HYPOPARAPHYROIDISMHuman Calcitonin CIBACALCIN USED FOR TREATING PAGETS DISEASE OF BONE

Insulin IN DIABETES

GlucagonGLUCAGEN EMERGENCYKIT AND DIAGNOSTIC KIT

USED IN THE TREATMENT OF SEVERE HYPOGLYCEMICREACTIONS IN DIABETIC PATIENTS

CHEMICAL SYNTHESIS:Powerful Techniques now make direct chemicalsynthesis an attractive option in many cases for smallpeptides and proteins.The complexity of proteins makesthe traditional synthetic approaches of OrganicChemistry for peptides with more than four or fiveamino acid residues. One problem is the difficulty ofpurifying the product after each step. The major break

through in this technology was provided by R.Brucemarrifield in 1962.5 He developed a alternative methodof peptide synthesis, on solid phase, has certainly grownin popularity and eliminated some of these problems, butnot without its own limitations .

HYBRIDOMA CELL TECHNOLOGY FOR THE

MONOCLONAL ANTIBODIES:In hybridoma technology, a mouse or other small animalis sensitized with an antigen. When an enough antibodyagainst the selected antigen has been attained animal issacrificed and its spleen cells are collected. Spleen cellscontain large no. of B- lymphocytes.6

To produce MAbs , B cells are fused with immortalmyeloma cells in the presence of fusogens they replicateand lessly and can be used to produce specific proteinbased drugs called Monoclonal antibodies.7,8

The MAbs produced contains various mouse proteinsand other contaminants that might require purification.9

RECOMBINANT DNA TECHNOLOGY:-The Introduction of r.DNA technology or geneticengineering has given a new way and Cost efficientsource of proteinbased drugs. These technologies arecapable of producing large amount of drugs.

The basic technique in r.DNA technology involveisolating or copying a gene; inserting the precise gene in

to transmissible vector that can be transcribed, amplifiedand propagated by host cells ; biochemical machinery ;transferring it to that host cell ; and facilitating thetranscription into m.RNA and translation in to proteins.10

TRANSGENIC ANIMALS FOR PROTEIN DRUG

PRODUCTION:Transgenic animal has been gaining application forprotein therapeutics. Since the development oftransgenic supermice in 1982 and the development ofthe 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 proteindrug. The DNA gene for the desired protein is coupledwith a DNA signal directing its production in themammary glands. The new gene functions in themammary glands so that the protein drug is made only inthe milk.11

Because of the long time periods involved and lowsuccess rates, developing transgenic animals is currentlyvery expensive.

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FIGURE:-1 Production of mono clonal antibodies:

PHARMACEUTICAL APPROACH-

THE CURRENT STATUS OF PROTEIN AND

PEPTIDE DRUGS:The total global market for proteins drugs was $ 47.4billion in 2006. The market will reach $ 55.7 billion bythe end of 2011, an average annual growth rate of3.3%.

Modified and engineered antibodies present the largestshare of the total global market. In 2006 , this sector heldalmost 56% of the global market. Hormone proteindrugs have the largest AAGR. It will reach $ 11.4 billionby 2011.12

Protein and peptide products for therapeutic use includea very diverse range of products as Hormones , Growthfactors , Cytokines , Vaccines and Monoclonal-antibodies. The important milestones in peptide hormonediscovery as the isolation ,structure determination andsynthesis of oxytocin by du Vigneaud and co workers

and the determination of the structure of insulin by thesanger.13,14

HORMONES:There are wide variety of peptide and protein hormones,natural as well as their analogs , that are commerciallyavailable for the treatment of various diseases or areused for diagnostic purposes. Several of these hormonesare obtained synthetically some from natural sourceswhile others are obtained via genetic engineering (Table-1) .

MONOCLONAL ANTIBODIES:Antibodies usage has now extended to treatment; hencethese are promoted as potential candidates for themanagement of various diseases.15 The Abs synthesizedagainst a particular antigen ( Ag ) are known as amonoclonal antibodies (MAbs). By definition, MAbs are aclass of highly specific Abs produced by the clones ofsingle hybrid. Cell formed in the laboratory by the fusion of

B. lymphocytes with a tumor cell . The process ofproducing MAbs was invented by Kohler and Milstein in1975.16

Types of Mabs that are used in treatment currently-Naked Mabs- these are the antibodies without any drugor radioactive material.1. Rituximab-It is an Mab directed against the CD20antigen expressed on the surfaces of normal andmalignant B lymphocytes.2. Alemtuzumab- For B cell chronic lymphocyticleukemia.3. Bevacizumab-For metastatic colorectal cancer.

Conjugated Mabs- They are used to deliverradionucleides toxins , or cytotoxic drugs to a specifictissue or malignant cell population.1. Gemtuzumab- Mab derived from the CD33 antigen Itis the only immunotoxin to receive FDA approval.2. Ibritumonab tiuxetan-Radiolabeled Mabs ti treat Bcellnon-Hodgkins lymphoma.

OTHER SIGNIFICANT PEPTIDES AND

PROTEIN DRUGS:Except Hormone and Monoclonal antibodies many otherclass of peptide and protein therapeutics are available ina market as Cytokines, Clotting factors and differentVaccines (Table-2).

PEPTIDE ANTIBIOTICS:The development of novel antibiotics is a burningnecessity now a day. Among many proposed strategies ,studies on natural and artificial amphipathic peptidesacting on membranes of microorganism have yieldedpromising result . Currently more than 800 suchpeptideantibiotics have been described and some of themhave already entered clinical trials.17

A growing number of reports, support a role ofhemoglobin as a precursor of peptides able to kill

microorganism.18

PATENT EXPIRY OF SOME BIOTECH PROTEINS:The patents of the first generation of biopharmaceuticals derived from recombinant DNA such asInterferon , growth hormones and epoietins are expiring, opening up the possibility for competitors to introducebiosimilar 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 RegulationAtrial natriuretic peptide 28- peptide, vasodilator, increases glomerular filtration and diuresisBradykinin 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 Band 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- cellcarcinoma 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 PowderThymopentin Timunox 5- peptide used as immunomodulating investigation drug in HIV positivepatients.

KEY TECHNOLOGIES AND DELIVERY OF

PROTEIN DRUGS:Till now, injections are the most common means foradministering these protein and peptide drugs but due to

the poor patient compliance with injections manyalternative routes are also tried by researchers withvarying 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 ishampered by their rapid elimination from the circulationbecause 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 improvedpharmacokinetic properties and enhanced exposure ofthe protein therapeutics, which for the patient yields, lessfrequent administration, greater convenience, andimproved efficacy.29

PROTEIN FUSION AND CONJUGATES:Protein fusion technologies exploit the modular nature ofprotein functional domains and through molecularengineering, create a new protein ability that combines thelong 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 lifethrough the utilization of the FcRn receptor recyclingsystem .30

The other approach of protein fusion technologies forincreased time action of protein therapeutics are humanserum albumin ( HSA) or Transferrin ( Tf ) fusionproteins .31,32

PEGYLATION:In PEGYlation technology , a large polyethylene glycol (PEG ) moiety is covalently or reversibly attached to theprotein through a reactive group , for example amine ,thiol or Carboxylic Acid.

Pegylation increases molecular stability , changes thevolume of distribution and reduces Immune reactionsand polymer attached to the protein globule createssteric hindrance for the interaction of protected polypeptides with active sites of proteases , opsoninsand antigen processing cells .33,34

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TABLE:3:PATENT EXPIRY DATES FOR SOME PROTEIN AND PEPTIDE DRUGS :

PATENT/MARKETPRODUCT PATENT/MARKET INNOVATOR

EXCLUSIVITY EXPIRY US EXCLUSIVITY EXPIRY EUROPE

SOMATROPIN GENETECH EXPIRED EXPIRED

EUDERASE ABBOTT EXPIRED EXPIRED

INSULIN ELI LILLY EXPIRED EXPIRED

G-CSF AMGEN 2015 2006

IL-2 CHIRON 2006-2012 2005

TPA GENENTECH 2005-2010 2005

IFN- INTER MUNE 2005,2006,2012 2002,2004

ERYTHROPOIETIN- AMGEN 2013 2004

IFN-2 BIOGEN/ROCHE 2002 2003,2007

ABBREVIATIONS: G- CSF, GRANULOCYTE COLONY- STIMULATING FACTOR; IFN, INTERFERON; IL, INTERLEUKIN; TPA, TISSUEPLASMINOGEN ACTIVATOR

FIGURE:-3 Production of transgenic animals.

OTHER TECHNOLOGIES:Proteolytic enzymes present at most routes ofadministration are able to quickly metabolize mostpeptides. Many molecules are absorbed through deep

lungs in to the blood with high bioavailability.35 Fortransmembrane delivery of protein therapeutics theenhenced endosomal escape is achieved through use oflytic peptides,PH sensitive polymer or swellable dendriticpolymers. Cargo proteins having ability to penetratemembranes are also used for transmembrane delivery.36

Controlled-release or depot technologies are also used forthe protein and peptide therapeutics as polymer baseddepot formulations. Different devices such as externalpumps, microchips and implantable pumps are the

potential option for increasing systemic exposure ofproteins.

TOXICITY AND SAFETY OF PROTEIN

THERAPEUTICS:The quality, safety and efficacy of biotechnology productsfor therapeutic use are intricately linked, far more so than

for conventional medicinal products, leading to the needfor increased communication between those responsiblefor ensuring product quality and those responsible fornon-clinical safety testing. Safety issues includemicrobiological safety (due to the use of biologicalmaterials either during the manufacturing process or as anintegral part of the products), pharmacological/ biologicaltoxicity (due to excessive primary pharmacology orundesirable secondary pharmacology), immunogenicityand potential tumourigenicity (for example, for growthfactors, immunosuppressive monoclonal antibodies andcell therapy products). Genotoxicity and intrinsicchemical toxicity are less of a problem for biotechnologyproducts.37

IMMUNOGENICITY:Immunogenicity is one of the major Concerns in thedevelopment and application of biotherapeutics. Thepatients can mount sustained Immune responses toprotein therapeutics with the production of neutralizineantibodies that can compromise efficacy or safety of thisdrugs.38The Immunogenicity of protein drugs can be ascribed toa few Immunodominant helper T lymphocyte epitopes ,and that reducing the MHC binding affinity of theseHTL epitopes contained with in these proteins cangenerate drugs with lower Immunogenicity.39

Recently method for Investigation of theImmunogenicity of a protein drugs has been developedwith the help of Equilibrium dialysis and liquidchromatography/ tandin mass spectroscopy.40

CONCLUSION:However Protein and peptide therapeutics have beenavailable for centuries. For instance, vaccinations withscabs that contained the small pox virus had beenpracticed for centuries. Also estrogen, insulin and other

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

This article has tried to present an overview of theprotein and peptide therapeutics. The field is advancingrapidly, and every doctor or pharmacist must stay

current with the literature on Protein therapeutics.

Different drug delivery systems, technologies, still haveto be developed for enhancing exposure of proteintherapeutics. The new and relevant approaches toimprove human risk assessment for novel proteintherapeutics is also needed but the Introduction ofsecond entry biopharmaceuticals or protein drugs isdesirable . They will play important role in the life cycleof drugs and reduce health care cost; they will also forcethe innovator Companies to improve their products.

ACKNOWLEDGEMENTS:

The authors express their thanks to Dr.Kamal kishore ,Professor, AIIMS, New Delhi for their valuablesuggestions , and to Mr. Shriram Mishra Dept. ofPharmacy, I.T.M, Gida, Gorakhpur for their kindcooperation.

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