Protein and-peptide-drug-delivery-systems

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Protein and-peptide-drug-delivery-systems

  1. 1. PROTEIN AND PEPTIDE DRUGDELIVERY SYSTEMS
  2. 2. 2 INTRODUCTION  Proteins are the most abundant macromolecules in the living cells, occurring in all cells and all parts of cells.  Cells can produce proteins that have strikingly different properties and activities, by joining same 20 amino acids in many different combinations and sequences.  The term protein is used for molecules composed of over 50 amino acids, and peptide for molecules composed of less than 50 amino acids. BY VISHAL SHARMA
  3. 3. 3  Scientific advances in molecular and cell biology have resulted in the development of two new biotechnologies. The first utilizes RECOMBINANT DNA to produce protein products.  The second technology is HYBRIDOMA TECHNOLOGY. Various proteins and peptides drugs are epidermal growth factor, tissue plasminogen activator. BY VISHAL SHARMA
  4. 4. 4 PROTEIN AND PEPTIDE DRUGS  Management of illness through medication is entering a new era in which a growing number of biotechnology produced peptide and protein drugs are available for therapeutic use.  Ailments that can be treated effectively by this new class of therapeutic agents include cancers, memory impairment, mental disorders, hypertension. BY VISHAL SHARMA
  5. 5. 5 MARKETED PROTEINS IN FREEZE DRIED FORMULATIONS Product Formulation Route IndicationMetrodin FSH 75 IU i.m. Induction of ovulationPergonal FSH and LH i.m. infertility Profasi HCG i.m. Infertility Elspar Asparginase i.m. i.v. LeukemiaGlucagon Glucagon i.m. i.v. s.c. Hypoglycemia Acthar Corticotropin i.m. i.v. s.c. Hormone Deficiency BY VISHAL SHARMA
  6. 6. 6 MARKETED PEPTIDES IN READY TO USE FORMULATIONS Product Formulation Route Indication Pitressin 8-Arginine i.m. s.c. Post operative Vasopressin abdominal distension Lupron Leuprolide s.c. Prostatic cancer Syntocinon Oxytocin i.m. i.v. Labour induction Sandostatin Octreotide s.c. Intestinal tumour Calcimar Salmon s.c. hypercalcemia BY VISHAL calcitonin SHARMA
  7. 7. 7 SUSTAINED RELEASE DOSAGE FORMS Product Formulation Route Indication Lupron Leuprolide i.m. Prostatic cancer H.P.Acthar gel ACTH i.m. s.c. Antidiureti c Pitrressin tannate Vasopressin i.m. Endocrine in oil tannate cancer BY VISHAL SHARMA
  8. 8. 8 PROTEIN AND PEPTIDE DRUGS  They are therapeutically effective only by parenteral route.  Repeated injections are required.  Therapeutic applications of these drugs rely on successful development of viable delivery systems to improve their stability and bioavailability. BY VISHAL SHARMA
  9. 9. 9 APPROACHES BY VISHAL SHARMA
  10. 10. 10 NON PARENTERAL SYSTEMIC DELIVERY / NON INVASIVE  These routes are useful for long term therapy.  Higher patience compliance (oral)  Reduction in administration cost  Without permeation enhancers lower bioavailability is achieved when these routes are used.  Lower bioavailability is due to poor mucosal permeability. BY VISHAL SHARMA
  11. 11. 11 IT INVOLVES  Oral route  Transdermal route  Nasal  Pulmonary  Rectal  Vaginal BY VISHAL SHARMA
  12. 12. 12 CHALLENGES  Large molecular size  Susceptibility to enz. Degradation  Short plasma half life  Ion permeability  Immunogenicity  Aggregation  Denaturation etc BY VISHAL SHARMA
  13. 13. 13 ABSORPTION OF PROTIENS FOLLOW BY VISHAL SHARMA
  14. 14. 14 ABSORPTION MECHANISM  90% of nutrient absorb in small intestine.  P & P absorption is limited by acidic environment , action of enz. ,non absorptive nature of epithelial.  Through paracellular and transcellular mech. They absorbed into blood or lymph (in villi) BY VISHAL SHARMA
  15. 15. DEVELOPMENT OF DELIVERY SYSTEMS FOR PEPTIDE AND15 PROTEIN BASED PHARMACEUTICALS Considerations are to be given for following aspects :  barriers to oral absorption  Preformulation and Formulation considerations  Pharmacokinetic considerations  Analytical considerations  Regulatory considerations BY VISHAL SHARMA
  16. 16. DEVELOPMENT OF DELIVERY16 SYSTEMS FOR PEPTIDE AND PROTEIN BASED PHARMACEUTICALS Considerations are to be given for following aspects :  barriers to oral absorption  Preformulation and Formulation considerations  Pharmacokinetic considerations  Analytical considerations  Regulatory considerations BY VISHAL SHARMA
  17. 17. 17 BARRIERS TO ORAL ABSORPTION  Age related development of macromolecule permeability barrier  Physical barrier - Size , charge ,solubility  Chemical barriers- pH solubility profile  Enz. Barriers  Interplay b/w P-glycoprotien & CYP3A4 BY VISHAL SHARMA
  18. 18. AGE RELATED DEVELOPMENT OF MACROMOLECULE PERMEABILITY18 BARRIER  It was found out that permeability of the neonates intestine is good for the macromolecules and as the age increases the permeability was reduce for macro. Mol. & inc for small molecules. BY VISHAL SHARMA
  19. 19. 19 PHYSICAL BARRIER  Size ,charge and solubility is in our hand to change by formulation and chemistry change.  For ex. Sustained release human insulin by attaching with lipophilic molecule. BY VISHAL SHARMA
  20. 20. 20 Surface adsorption :  Glass and plastic surfaces adsorbs proteins and peptides.  To avoid surface adsorption albumin, gelatin, sodium chloride can be used. Aggregation behaviour :  To prevent aggregation additives are used such as : urea, glycerol, EDTA, lysine, poloxamer 188. BY VISHAL SHARMA
  21. 21. 21 CHEMICAL BARRIERS pH :  Solution pH is important for stability purpose. For simple peptides pH of minimum degradation should be identified. Peptides are usually formulated at slightly acidic pH (3-5). For proteins pH is set away from isoelectric pH to avoid aggregation.  Insulin is more stable at pH 5.4. However for solubility reasons insulin injection pH are 2.5-3.5 or 7-7.8. BY VISHAL SHARMA
  22. 22. 22 ENZ. BARRIERS/PROTEIN INSTABILITIES  The degradation of proteins and peptides can be divided into two main categories : 1. Those that involve a covalent bond. 2. Those involving a conformational change. This process is often referred to as denaturation. BY VISHAL SHARMA
  23. 23. 23 PEPTIDE FRAGMENTATION  The peptide bond (RNH-CO-R) is succeptible to hydrolysis.  Peptide bonds are considered stable unless hydrolysis is assisted by neighbouring group. Hydrolysis rate is affected by solution pH. DEAMIDATION  It means removal of ammonia from amide moiety. Deamidation is the major factor for instability of insulin, ACTH, Human Growth Hormone. In acidic media peptides deamidate by direct hydrolysis. BY VISHAL SHARMA
  24. 24. 24 OXIDATION Sulphur containing amino acids are prone to oxidation. MAILLARD REACTION In the maillard reaction the carbonyl group (RCH=O) from glucose can react with the free amino group in a pepide to form a Schiff base. This reaction is acid catalysed. DIMERISATION AND POLYMERIZATION Insulin forms a small amount (about 1%) of covalent dimer and polymer during two years cold storage. Production of these species increases as temperature increases. BY VISHAL SHARMA
  25. 25. 25 ENZYMES BY VISHAL SHARMA
  26. 26. 26 PROTEASE INHIBITORS  Coadministration of protease inhibitors provides a viable means to circumvent the enzymatic barrier in achieving the delivery of peptide and protein drugs.  Th e choice of protease inhibitors will depend on the structure of these therapeutic drugs, and the information on the specifi city of proteases is essential to guarantee the stability of the drugs in the GI tract.  A number of inhibitors including aprotinin (trypsin /chymotrypsin inhibitor), amastatin, bestatin, boroleucine, and puromycin (aminopeptidase inhibitors) have been reported for this purpose BY VISHAL SHARMA
  27. 27. 27 INTERPLAY B/W P- GLYCOPROTIEN AND CYP3A4  P-gp is ABC transporter associated with MDR  CYP3A4 are enz. BY VISHAL SHARMA
  28. 28. 28 BY VISHAL SHARMA
  29. 29. 29 PERMEATION ENHANCER  Without permeation enhancers lower bioavailability is achieved when these routes are used.  Lower bioavailability is due to poor mucosal permeability.  Sodium tauroglycocholate is commonly used penetration enhancer. BY VISHAL SHARMA
  30. 30. DEVELOPMENT OF DELIVERY30 SYSTEMS FOR PEPTIDE AND PROTEIN BASED PHARMACEUTICALS Considerations are to be given for following aspects :  barriers to oral absorption  Preformulation and Formulation considerations  Pharmacokinetic considerations  Analytical considerations  Regulatory considerations BY VISHAL SHARMA
  31. 31. PREFORMULATION AND31 FORMULATION CONSIDERATIONS  Denaturation stabilizers  Maximising oral protein and peptide absorption  Chemical Modifications  Amino acid Modification  Hydrophobization  Conjugation with polymers BY VISHAL SHARMA
  32. 32. 32 DENATURATION o Specific confirmation is required for proteins to exert pharmacological and physiological activities. Denaturation is a process of altering protein confirmation. Heat, organic solvents, high salt concentration, lyophilization can denature proteins.  Protein confirmation refers to the specific tertiary structure, which is determined by the primary and secondary structures and the disulfide bonds and is held together by three forces : hydrogen bonding, salt bridges, and hydrophobic interactions. BY VISHAL SHARMA
  33. 33. 33 COMMON STABILIZERS SERUM ALBUMIN :  It can withstand heating to 60o C for 10 hours.  At pH 2 albumin molecule expands and elongates but can return to native confirmation reversibly. Also, it shows good solubility. BY VISHAL SHARMA
  34. 34. 34 AMINO ACIDS Glycine is most commonly used stabilizer.  Mechanism of action of amino acids as stabilizers may be one of the following :  Reduce surface adsorption.  Inhibit aggregate formation.  Stabilize proteins against heat denaturation. BY VISHAL SHARMA
  35. 35. 35 SURFACTANTS  They cause denaturation of proteins by hydrophobic disruption. However judicious use of surfactants can protect proteins from other denaturants. Proteins have tendency to concentrate at liquid/liquid or liquid/air interface. Due to this proteins may adopt non native confirmation and such confirmation is having less solubility.  Optimal concentration of surfactants for stabilization should be greater than cmc. Ionic surfactants are more effective stabilizers than non ionic surfactants.  Various surfactants used are : poloxamer 188, polysorbate. BY VISHAL SHARMA
  36. 36. 36 POLYHYDRIC ALCOHOLS AND CARBOHYDRATES :  They contain –CHOH-CHOH- groups which are responsible for stabilizing proteins. They stabilize proteins against denaturation caused by elevated temperature or by freeze drying or by freeze thaw cycles.  Many important therapeutic proteins and peptides are derived from blood such as immune globulin, coagulation factors. For viral destruction pasteurization at 60o C for 10 hours is needed. Hence thermal stability is needed. Long chain polyhydric alcohols are more effective as stabilizers. e.g. sorbitol, xylitol. BY VISHAL SHARMA
  37. 37. 37  Mechanism of action as stabilizers for polyhydric alcohols is that they have effect on structure of surrounding water molecules which strengthens hydrophobic interactions in protein molecules.  Mechanism of action as stabilizers for carbohydrates is that they provide dry network that provides significant support for protection.  Polyhydric alcohols used are sorbitol, mannitol, glycerol, PEG.  Carbohydrates used are glucose, mannose, sucrose, ribose. BY VISHAL SHARMA
  38. 38. 38 ANTI-OXIDANTS Thiol compounds such as thioacetic acid, triethanolamine, reduced glutathione and metal chelants such as EDTA are used as antioxidants. MISCELLANEOUS  Certain enzymes can be stabilized by using compounds having similar structures of enzymes. e.g. Glucose stabilizes glucoamylase while aspargine stabilizes asparginase.  Compounds forming stable complex through ionic interaction with proteins can stabilize proteins.  Calcium is essential for thermal stability of certain amylases or proteases. SHARMA BY VISHAL
  39. 39. MAXIMISING ORAL PROTEIN39 AND PEPTIDE ABSORPTION  1. Amino acid modifications Metkephamid, an analog of methionine enkephalinwith substitution of glycine₂ by l- alanine and modified methionine, readily penetrated across the nasal mucosa with 54% bioavailability relative to subcutaneous administration but was orally inactive. BY VISHAL SHARMA
  40. 40. 40 2. Hydrophobization  Hydrophobization of peptides may be attempted by two approaches. The first ispeptide backbone modification to include more of hydrophobic amino acids; the second would be covalent conjugation of a hydrophobic moiety—for example, a lipid orpolymeric tail.  Increasing the hydrophobicity of a peptide or protein by surface modification using lipophilic moieties may be of particular benefit to transcellular passive or active absorption by membrane penetration or attachment, respectively; or it may simply aid in the increased stability of the protein. BY VISHAL SHARMA
  41. 41. 41  EXAMPLE  lipophilic modificationof TRH by covalent conjugation of lauric acid to this tripeptide (Lau- TRH). The derivative was more stable in rat plasma and was rapidly converted to TRH in the intestinal mucosal homogenate. BY VISHAL SHARMA
  42. 42. 42 CONJUGATION WITH POLYMER  One of the most commenly used technique is (PEG)-ylation technology.  Enlarges the active molecule by attaching a web like shield of hydrated PEG polymer chain around the molecule. BY VISHAL SHARMA
  43. 43. 43 BENEFITS  increase clearance half life  Provide possibility of drug to stay more in the circulation.  Increase molecular stability  Change the vol. of distribution  Reduce immune response BY VISHAL SHARMA
  44. 44. DEVELOPMENT OF DELIVERY44 SYSTEMS FOR PEPTIDE AND PROTEIN BASED PHARMACEUTICALS Considerations are to be given for following aspects :  barriers to oral absorption  Preformulation and Formulation considerations  Pharmacokinetic considerations  Analytical considerations  Regulatory considerations BY VISHAL SHARMA
  45. 45. 45 PHARMACOKINETIC CONSIDERATIONS  Basal insulin secretion in healthy subjects shows circadian rhythm with peak time at 15:00 hrs.  It has been suggested that larger amount of insulin is needed in afternoon and night.  Hence delivery systems could be designed by considering such aspects. BY VISHAL SHARMA
  46. 46. DEVELOPMENT OF DELIVERY46 SYSTEMS FOR PEPTIDE AND PROTEIN BASED PHARMACEUTICALS Considerations are to be given for following aspects :  barriers to oral absorption  Preformulation and Formulation considerations  Pharmacokinetic considerations  Analytical considerations  Regulatory considerations BY VISHAL SHARMA
  47. 47. 47 ANALYTICAL CONSIDERATIONS  Many tests are required for stability of protein products to assure identity, purity, potency and stability of formulation.  Due to complexity of proteins bioassay are required to assess potency of the formulation. Bioassay are of two types : in vitro and in vivo.  In case of in vitro bioassays response of cells to hormones and growth factors is monitored. In case of in vivo bioassay pharmacological response of animals to proteins is monitored : e.g., post injection blood sugar in rabbits is monitored for bioassay of insulin. BY VISHAL SHARMA
  48. 48. 48 U.V. SPECTROSCOPY  Proteins containing aromatic amino acid residues such as phenyl alanine, tyrosine, tryptophan can be detected by u.v. spectroscopy.  Ultraviolet spectroscopy can be used for in process quality control.  Protein aggregates scatter u.v. light and absorbance increases. Hence u.v. spectroscopy can be used to monitor protein aggregation. BY VISHAL SHARMA
  49. 49. 49 BRADFORD ASSAY :  This assay employs the principle that in the presence of proteins absorption maximum of coomassie brilliant blue dye changes from 465nm to 595nm. BIURET TEST :  Structure of biuret and proteins are similar. Biuret in presence of proteins or peptides reduces copper to cuprous ions in alkaline solutions and colour complex is developed. BY VISHAL SHARMA
  50. 50. 50 THERMAL ANALYSIS  Differential scanning calorimetry (DSC) is gaining widespread use as a tool for investigating transitions of confirmation as a function of temperature and, more importantly, the effect of potential stabilizing excipients in a protein solution. The apex of the endothermic peak is the transition temperature between native and partially unfolded confirmations. BY VISHAL SHARMA
  51. 51. 51 ELECTROPHORESIS  Most often used technique for protein products is sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE).  Proteins are denatured by boiling in the SDS solution. All charges of protein are masked by negative charge of dodecyl sulphate.  Thus protein moves on polyacrylamide gel strictly on basis of size of protein molecule.  This technique is useful for determining molecular weight of proteins.  For visualization of proteins on the gel reagents used areBYsilver nitrate, coomassie brilliant blue dye. VISHAL SHARMA
  52. 52. 52 LIQUID CHROMATOGRAPHY To study stability of proteins and peptides HPLC is useful technique. Various modes used are  Normal Phase HPLC  Reverse Phase HPLC  Ion Exchange  Chromatofocusing BY VISHAL SHARMA
  53. 53. DEVELOPMENT OF DELIVERY53 SYSTEMS FOR PEPTIDE AND PROTEIN BASED PHARMACEUTICALS Considerations are to be given for following aspects :  barriers to oral absorption  Preformulation and Formulation considerations  Pharmacokinetic considerations  Analytical considerations  Regulatory considerations BY VISHAL SHARMA
  54. 54. 54 REGULATORY CONSIDERATIONS Four federal agencies regulates biotechnology products : 1. US Food and drugs administration (USFDA) 2. Environmental protection agency (EPA) 3. Occupational safety and health administration (OSHA) 4. US Department of agriculture (USDA) BY VISHAL SHARMA
  55. 55. 55  Nasal route : Poor permeability is common problem. Proteolytic enzymes in nasal mucosa degrades the administered drugs.  Pulmonary route : Monodisperse aerosol with a mass median aerodynamic diameter of 3 µm was reported to achieve alveolar deposition of 50% or more drug. BY VISHAL SHARMA
  56. 56. 56  Ocular route : Ocular absorption can be enhanced by use of nanoparticles, liposomes, gels, ocular inserts.  Buccal route : Mucoadhesive dosage forms can be used.  Rectal route : solid dispersion of insulin with mannitol can produce rapid release of insulin from suppositories. BY VISHAL SHARMA
  57. 57. 57  Transdermal route : Skin has very low proteolytic activity. Two types of iontophoresis are used : DIRECT CURRENT MODE PULSE CURRENT MODE  Vaginal route : Especially useful to deliver hormones. Not much accepted in developing countries. BY VISHAL SHARMA
  58. 58. 58 PARENTERAL ROUTE  Most efficient route.  Extremely short duration of action.  Hence, viable drug delivery techniques are to be developed such as controlled drug delivery systems for prolongation of biological activity.  Complications arising from this route are : Thrombophlebitis Tissue necrosis immunogenicity BY VISHAL SHARMA
  59. 59. 59 PARENTERAL ROUTE BIO DEGRADABLE POLYMERS BASED DRUG DELIVERY SYSTEMS :  Microspheres are used as drug carriers which are made of natural or synthetic polymers.  Natural polymers have advantage that they are biocompatible and inexpensive. But they are lacking purity. Synthetic polymers are PLA, PGA, PLGA.  Mechanism of degradation are : firstly random chain scission occurs. Then soluble oligomeric products are formed which then gets converted to soluble monomers. BY VISHAL SHARMA
  60. 60. 60 Cont……………  PLGA biodegrades into lactic and glycolic acids. These acids enter into TCA cycle and then eliminated as carbon dioxide and water. Injectable controlled release formulations of certain drugs are formulated using lactide/glycolide copolymers. Such drugs are LHRH, calcitonin, insulin.  Nanoparticles made of PLGA, albumin polystyrene have potential for targeted drug delivery. BY VISHAL SHARMA
  61. 61. 61 LIPOSOMES BASED DRUG DELIVERY SYSTEMS  Liposomes are microscopic vesicles composed of one or more lipid layers that enclose aqueous compartments. Liposome membranes are semi permeable and can thus be used as controlled release systems. Liver is natural target for liposomes.  Disadvantage is low stability of liposomes. BY VISHAL SHARMA
  62. 62. 62 HYDROGEL BASED DRUG DELIVERY SYSTEMS  Hydrogels have advantage of biocompatibility. Insulin has been incorporated into hydrogels and widely investigated.  Emulsions , multiple emulsions, micro emulsions, resealed erythrocytes can also be used to deliver protein and peptide drugs. BY VISHAL SHARMA
  63. 63. 63 APPLICATIONS  Oral peptides today  Nasal delivery of proteins  Pulmonary delivery of proteins  Polymeric protein delivery to increaser half life  Sustained release peptide systems  Chemical altered protiens BY VISHAL SHARMA
  64. 64. 64 ORAL PEPTIDES TODAY  Desmopressin acetate (DDAVP) is a synthetic analogue of 8 arginine vasopressin: ant diuretic hormone. Marketd by aventis pharmaceutical and is approved for diabetes insipidus.0.16 % bioavilable  Novartis and roche pharmaceutical market cyclosporin (small lyophilic mol. For graft rej.) 30% bioavailibilty BY VISHAL SHARMA
  65. 65. 65 NASAL DELIVERY OF PROTIENBrand company drug Used for bioavailibiname lityMiacalcin® Novartis Calcitonin osteoporosi 3% analogue sSynarel® Hoffman la- LHRH endometros 2.8% roche agonist is naferlinDDAVP Vasopressi Diabetes 3% n analogue insipidus BY VISHAL SHARMA
  66. 66. 66 PULMONARY DELIVERY OF PEPTIDE  Various companies like Nektar , Alkermes , Aradigm have developed arosolised insulin showing about 10% bio available as compared to SC.  Particle size in important in transfer of molecule from pulmonary.  Size of insulin should be 0.5-3 micron.  See figure back side BY VISHAL SHARMA
  67. 67. 67 BY VISHAL SHARMA
  68. 68. 68 POLYMERIC PROTEINS  Sustained release protien  LHRH agonist goserelin with PLGA marketd by AstraZeneca : administer SC 14-16 gauge needle.  Octreotide LAR (long acting release) by novartis for gastroentopancreatic endocrine tumors.  Neutropin Depot® by Alkermes and Genetech ; human growth hormones. BY VISHAL SHARMA
  69. 69. 69 CHEMICALLY ALTERED PROTEINS  Prepared by PEGylation  First PGA product FDA approved was Enzon’s Adagen® (bovine enx. Adenosine deaminase) For ADA def. severe combined disease.  Another its product was Oncasper® (l-as.paragenase)  AMINO ACID SUBSTITUTION  Rapid insulin Eli-Lilly ; lys pro insulin is an ex. BY VISHAL SHARMA
  70. 70. 70 CONCLUSION  Protein and peptide based pharmaceuticals are rapidly becoming a very important class of therapeutic agents and are likely to replace many existing organic based pharmaceuticals in the very near future.  Peptide and protein drugs will be produced on a large scale by biotechnology processes and will become commercially available for therapeutic use. BY VISHAL SHARMA
  71. 71. 71 REFERENCES 1) Agrawal S, Udupa N, Protein and peptide drug delivery : recent advances. In : Jain NK, editor. Progress in controlled and novel drug delivery systems. 1 st ed. Delhi : CBS Publishers; 2004.p.184-204. 2) Chien YW : Novel drug delivery systems. 2nd ed. New York : Marcel Dekker Inc; 2005.p.631-745. 3) Yu Chang John Wang : Parenteral products of proteins and peptides. In : Lieberman HA, Avis KE, editors. Pharmaceutical dosage forms : Parenteral medications, volume 1. 2nd ed. New York Marcel Dekker Inc; 2005.p.283-320. 4) Block JH, Beale JM. Wilson and Gisvoldˈs textbook of organic medicinal and pharmaceutical chemistry. 11 th ed. Philadelphia : Lippincott Williams and wilkins; 2005.p.851-852. BY VISHAL SHARMA
  72. 72. 72 5) Patel NK, Pharmaceutical suspensios. In : Lachman l, Lieberman HA, Kanig JL, editor. The theory and practice of pharmacy. 3 rd ed. Mumbai : Varghese Publishing House; 1987.P.488-489. 6) Aulton ME : Pharmaceutics : The science of dosage form design. 2 nd ed. Toronto : Churchill livingstone; 2006.p.544-553. 7) Poon CY : Clinical Analysis. In : Troy DB, editor. Remington : The Science of Dosage form Design. 21st ed. Volume 1. Philadelphia : Lippincott Williams and wilkins; 22005.p.577-578. 8) www.ida.lib (accessed on 15/4/2010.) 9) Rang HP, Dale MM : Pharmacology. 5th ed. Toronto : Churchill livingstone; 2003.p.386-388. BY VISHAL SHARMA
  73. 73. 73 10) Massey FH, Sheliga TA : Development of aggregation resistant insulin formulations. Pharm Res; 3 : 26S (1986). 11) www.wikipedia.org (accessed on 08/4/2012.) 12) Agharkar SN, Motola S. Preformulation research of parenteral medications.In : Lieberman HA, Avis, KE, editors. Pharmaceutical dosage forms : parenteral medications; volume 1. 2nd ed. New York : Marcel Dekker Inc; 2005.p.150-155. BY VISHAL SHARMA
  74. 74. 74 BY VISHAL SHARMA

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