protein and-peptide-drug-delivery-systems

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


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


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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barriers to oral absorption Preformulation and Formulation

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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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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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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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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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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protein and-peptide-drug-delivery-systems

Education

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p p absorption

oral absorption preformulation

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peptides drugs

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