Protein and Peptide Drug Delivery Systems

PROTEIN AND PEPTIDE DRUG DELIVERY SYSTEMS

Guided by : Mrs. M.R.P. RAO(Pharmaceutics

department)

Presented by : Mr.Dhanesh H. Sali.

AISSMS COLLEGE OF PHARMACY, PUNE

CONTENTS

Introduction Protein and peptide drugs Parenteral drug delivery systems Non Parenteral drug delivery systems Development of drug delivery systems Insulin Conclusion References

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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.

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 6

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. hypercalcemia 7

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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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.

Judicious choice of route of administration should be done.

Complications arising from this route are : Thrombophlebitis Tissue necrosis immunogenicity 10

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.

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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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SELF REGULATED DEVICES:

These are capable of receiving the physiological feedback information and adjusting drug output from delivery systems in response to feedback information.

These devices are of two types : feedback signal modulates rate of drug

release.Feedback signal triggers drug release. 15

BASIC PRINCIPLES OF SELF REGULATED DEVICES

COMPETITIVE DESORPTION :

Insulin molecules with covalently attached sugar molecules are used that is also known as glycosylated insulin.

These are complementary to major binding site of conA. It is carbohydrate binding protein.

Glycosylated insulin can be bound to conA and reversibly displaced from conA by glucose in direct proportion of glucose concentration.

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MEMBRANE CONTROLLED DEVICES Glucose oxidase is immobilized in cross

linked polymers. In absence of external glucose amine groups are unprotonated and membrane porosity is such that insulin molecules are unable to diffuse out.

Glucose when diffused into membranes gets oxidized by glucose oxidase to gluconic acid which protonates amino groups due to which membrane porosity increases and now insulin can diffuse out.

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EROSION CONTROLLED DEVICES

Reaction between glucose and glucose oxidase generates gluconic acid. Poly(ortho esters) polymers erodes as pH decreases. Release of insulin can be modulated using this approach.

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Non parenteral systemic delivery

These routes are useful for long term therapy. 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.

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.

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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.

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DEVELOPMENT OF DELIVERY SYSTEMS FOR PEPTIDE AND PROTEIN BASED PHARMACEUTICALS

Considerations are to be given for following aspects :

Preformulation and Formulation considerations

Pharmacokinetic considerations Analytical considerations Regulatory considerations

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PREFORMULATION AND FORMULATION CONSIDERATIONS

Preformulation data is to be generated for

following aspects :

Isoelectric point pH solubility profile pH stability profile Excipient compatibilities

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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.

SALTS : Ammonium sulphate is a strong stabilizer.

Hence saturated solution of ammonium sulphate is used in protein purification process.

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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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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.

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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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.

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. 38

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.

Mechanism for such behaviour may be one of the following :

Inhibition of surface adsorption or cryoprotection.

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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.

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.

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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.

INSULIN Insulin was the first protein for which an amino

acid sequence was determined (by sangerʾs group in Cambridge in 1955)

It consists of two peptide chains (A and B of 21 and 30 amino acid residues respectively).

Insulin suspensions may be prepared by pH change method. Insulin has an isoelectric point at approximately pH 5. when it is mixxed with basic protein such as protamine, it is readily precipitated when pH is between isoelectric points of insulin and protamine i.e. pH 6.9 to 7.3.

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Protamine zinc insulin (PZI) contain an excess quantity of zinc to retard absorption. According to BRITISH PHARMACOPOEIA of 1958, a phosphate buffer is added to each individual vial containing acidified solution of insulin, protamine and zinc so that pH is between 6.9 to 7.3. The preparation is compounded in final container by mixing PZI and buffer in filling operations.

Main problem in using insulin is to avoid plasma concentration fluctuations. To solve this problem various formulations are available varying in onset and duration of action.

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Insulin Lispro : it is insulin analogue in which lysine and proline residues are switched. It is rapidly acting hence patient can inject immediately before start of meal.

Insulin Glargine : it is modified insulin analogue. Its intent is to provide constant basal insulin supply. It is clear solution, forms microprecipitate at physiological pH of subcutaneous tissue and absorption from subcutaneous site is prolonged. It avoids risk of night time hypoglycemia when used in conjunction with short acting insulin.

Name Particle size (µm)

Action Composition pH Duration (hours)

Insulin injection USP

prompt Insulin + zinc chloride

2.5-3.5 5-7

Prompt insulin zinc

suspension USP

2

rapid Insulin + zinc chloride +

buffer

7.2-7.5 12

Insulin zinc suspension

USP

10-40(70%)2(30%)

INTERMEDIATE Insulin + zinc chloride +

buffer

7.2-7.5 18-24

Extended insulin zinc suspension

10-40 Long-Acting Insulin + zinc chloride +

buffer

7.2-7.5 24-36

Globin zinc insulin

injection

INTERMEDIATE Globin +Insulin + zinc

chloride

3.4-3.8 12-18

Protamine zinc insulin

suspension

Long-Acting Protamine + insulin +zinc

7.1-7.4 24-36

Isophane insulin

suspension USP

30 INTERMEDIATE Protamine +zinc chloride

+insulin + buffer

7.1-7.4 18-24 48

USP INSULIN TYPE STRENGTHS

Insulin injection (regular insulin) U-40 mixed, U-100 mixed, U-500

Isophane insulin suspension (NPH Insulin)

U-40 mixed, U-400 mixed

Isophane insulin suspension (70%) and insulin injection (30%)

U-100

Insulin zinc suspension (Lente insulin)

U-40 mixed, U-100 mixed

Extended insulin zinc suspension (Ultra lente insulin)

U-40 mixed, U-100 mixed

Prompt insulin zinc suspension (semilente insulin)

U-40 mixed, U-100 mixed

Protamine zinc insulin suspension (PZI Insulin)

U-40 mixed, U-100 mixed 49

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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This poses an urgent challengeto the pharmaceutical industry to develop viable delivery systems for the efficient delivery of these complex therapeutic in biologically active form.

Much work needs to be done on the development of viable delivery systems for non parenteral administration to make peptide and protein pharmaceuticals commercially viable and therapeutically useful.

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 Gisvoldˈs textbook of organic medicinal and pharmaceutical 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 16/4/2010.)

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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