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PROTEIN AND PEPTIDE DRUG DELIVERY
SYSTEMS
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.
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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.
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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.
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MARKETED PROTEINS IN FREEZE DRIED FORMULATIONS
Product Formulation Route Indication
Metrodin FSH 75 IU i.m. Induction of ovulation
Pergonal FSH and LH i.m. infertility
Profasi HCG i.m. Infertility
Elspar Asparginase i.m. i.v. Leukemia
Glucagon Glucagon i.m. i.v. s.c. Hypoglycemia
Acthar Corticotropin i.m. i.v. s.c. Hormone Deficiency
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MARKETED PEPTIDES IN READY TO USE FORMULATIONS
Product Formulation Route Indication
Pitressin 8-Arginine Vasopressin
i.m. s.c. Post operative abdominal distension
Lupron Leuprolide s.c. Prostatic cancer
Syntocinon Oxytocin i.m. i.v. Labour induction
Sandostatin Octreotide s.c. Intestinal tumour
Calcimar Salmon calcitonin
s.c. hypercalcemiaBY VISHAL SHARMA
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SUSTAINED RELEASE DOSAGE FORMS
Product Formulation Route Indication
Lupron Leuprolide i.m. Prostatic cancer
H.P.Acthar gel ACTH i.m. s.c. Antidiuretic
Pitrressin tannate in oil
Vasopressin tannate
i.m. Endocrine cancer
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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.
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APPROACHES
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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.
NON PARENTERAL SYSTEMIC DELIVERY / NON INVASIVE
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IT INVOLVES
Oral route
Transdermal route
Nasal
Pulmonary
Rectal
Vaginal
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CHALLENGES
Large molecular size
Susceptibility to enz. Degradation
Short plasma half life
Ion permeability
Immunogenicity
Aggregation
Denaturation etc
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ABSORPTION OF PROTIENS FOLLOW
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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)
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DEVELOPMENT OF DELIVERY 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
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DEVELOPMENT OF DELIVERY 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 considerationsBY VISHAL SHARMA
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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
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AGE RELATED DEVELOPMENT OF MACROMOLECULE PERMEABILITY
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.
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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.
PHYSICAL BARRIER
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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.
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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.
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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.
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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.
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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.
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ENZYMES
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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
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INTERPLAY B/W P-GLYCOPROTIEN AND CYP3A4
P-gp is ABC transporter associated with MDR
CYP3A4 are enz.
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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.
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30
DEVELOPMENT OF DELIVERY 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 considerationsBY VISHAL SHARMA
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PREFORMULATION AND FORMULATION
CONSIDERATIONS
Denaturation stabilizers
Maximising oral protein and peptide absorption
Chemical Modifications
Amino acid Modification
Hydrophobization
Conjugation with polymers
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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.
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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.
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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.
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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
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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.
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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.
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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.
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MAXIMISING ORAL PROTEIN AND PEPTIDE ABSORPTION
1. Amino acid modifications
Metkephamid, an analog of methionine enkephalinwith substitution of glycine by l-₂alanine and modified methionine, readily penetrated across the nasal mucosa with 54% bioavailability relative to subcutaneous administration but was orally inactive.
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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.
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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.
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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.
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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
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4444
DEVELOPMENT OF DELIVERY 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 considerationsBY VISHAL SHARMA
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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.
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464646
DEVELOPMENT OF DELIVERY 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 considerationsBY VISHAL SHARMA
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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.
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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.
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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.
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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.
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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 are silver nitrate, coomassie brilliant blue dye.
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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
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53535353
DEVELOPMENT OF DELIVERY 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 considerationsBY VISHAL SHARMA
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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)
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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.
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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
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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
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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
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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.
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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.
Cont……………
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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.
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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.
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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
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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
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NASAL DELIVERY OF PROTIEN
Brand name
company drug Used for bioavailibility
Miacalcin® Novartis Calcitonin analogue
osteoporosis
3%
Synarel® Hoffman la-roche
LHRH agonist naferlin
endometrosis
2.8%
DDAVP Vasopressin analogue
Diabetes insipidus
3%
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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
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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.
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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.
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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.
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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. 1st 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 Gisvold s textbook of organic medicinal and ˈpharmaceutical chemistry. 11 th ed. Philadelphia : Lippincott Williams and wilkins; 2005.p.851-852.
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5) Patel NK, Pharmaceutical suspensios. In : Lachman l, Lieberman HA, Kanig JL, editor. The theory and practice of pharmacy. 3rd ed. Mumbai : Varghese Publishing House; 1987.P.488-489.
6) Aulton ME : Pharmaceutics : The science of dosage form design. 2nd 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.
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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.
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