32129428 protein and peptide drug delivery systems

8/11/2019 32129428 Protein and Peptide Drug Delivery Systems

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PROTEIN AND PEPTIDE DRUG

DELIVERY SYSTEMS Guided by :

Mrs. M.R.P. RAOPharmaceutics department)

Presented by :Mr.Dhanesh H. Sali.

AISSMS COLLEGE OF PHARMACY, PUNE


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CONTENTS

IntroductionProtein and peptide drugsParenteral drug delivery systems

Non Parenteral drug delivery systemsDevelopment of drug delivery systemsInsulinConclusionReferences

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Scientific advances in molecular and cell biology

have resulted in the development of two newbiotechnologies. The first utilizes recombinant DNAto produce protein products. The second technology is hybridoma technology.

Various proteins and peptides drugs are epidermalgrowth factor, tissue plasminogen activator.


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MARKETED PROTEINS IN FREEZE DRIEDFORMULATIONS

Product Formulation Route Indication

Metrodin FSH 75 IU i.m. Induction ofovulation

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. HormoneDeficiency 6


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MARKETED PEPTIDES IN READY TO USEFORMULATIONSProduct Formulation Route Indication

Pitressin 8-ArginineVasopressin

i.m. s.c. Post operativeabdominaldistension

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


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SUSTAINED RELEASE DOSAGE FORMS

Product Formulation Route Indication

Lupron Leuprolide i.m. Prostaticcancer

H.P.Acthargel

ACTH i.m. s.c. Antidiuretic

Pitrressintannate inoil

Vasopressin tannate

i.m. Endocrinecancer

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P ROTEIN AND PEPTIDE DRUGS

They are therapeutically effective only by parenteralroute.Repeated injections are required.

Therapeutic applications of these drugs rely onsuccessful development of viable delivery systemsto improve their stability and bioavailability.

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PLGA biodegrades into lactic and glycolicacids. These acids enter into TCA cycle andthen eliminated as carbon dioxide andwater. Injectable controlled release

formulations of certain drugs are formulatedusing lactide/glycolide copolymers. Suchdrugs are LHRH, calcitonin, insulin.Nanoparticles made of PLGA, albuminpolystyrene have potential for targeted drugdelivery.

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BASIC PRINCIPLES OF SELFREGULATED DEVICES

COMPETITIVE DESORPTION :

Insulin molecules with covalently attached sugarmolecules are used that is also known asglycosylated insulin. These are complementary to major binding site ofconA. It is carbohydrate binding protein.

Glycosylated insulin can be bound to conA andreversibly displaced from conA by glucose in directproportion of glucose concentration.

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MEMBRANE CONTROLLEDDEVICES

Glucose oxidase is immobilized in cross linkedpolymers. In absence of external glucose aminegroups are unprotonated and membrane porosity issuch that insulin molecules are unable to diffuse

out.Glucose when diffused into membranes getsoxidized by glucose oxidase to gluconic acid whichprotonates amino groups due to which membraneporosity increases and now insulin can diffuse out.

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EROSION CONTROLLEDDEVICES

Reaction between glucose and glucose oxidasegenerates gluconic acid. Poly(ortho esters)polymers erodes as pH decreases. Release ofinsulin 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.


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Transdermal route :Skin has very low proteolytic activity.

Two types of iontophoresis are used :DIRECT CURRENT MODEPULSE 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 considerationsPharmacokinetic considerations Analytical considerationsRegulatory considerations

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P REFORMULATION AND F ORMULATION CONSIDERATIONS

Preformulation data is to be generated for followingaspects :

Isoelectric pointpH solubility profilepH stability profileExcipient compatibilities

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P HARMACOKINETIC CONSIDERATIONS

Basal insulin secretion in healthy subjects showscircadian rhythm with peak time at 15:00 hrs. It has been suggested that larger amount of insulinis needed in afternoon and night.Hence delivery systems could be designed byconsidering such aspects.

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U.V. SPECTROSCOPY Proteins containing aromatic amino acid residuessuch as phenyl alanine, tyrosine, tryptophan can bedetected by u.v. spectroscopy. Ultraviolet spectroscopy can be used for in processquality control. Protein aggregates scatter u.v. light andabsorbance increases. Hence u.v. spectroscopycan 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 bluedye changes from 465nm to 595nm.

BIURET TEST :

Structure of biuret and proteins are similar. Biuret inpresence of proteins or peptides reduces copper tocuprous ions in alkaline solutions and colour complex isdeveloped.


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THERMAL ANALYSISDifferential scanning calorimetry (DSC) is gainingwidespread use as a tool for investigatingtransitions of confirmation as a function oftemperature and, more importantly, the effect ofpotential stabilizing excipients in a protein solution.The apex of the endothermic peak is the transitiontemperature between native and partially unfoldedconfirmations.

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ELECTROPHORESISMost often used technique for protein products issodium dodecyl sulphate polyacrylamide gelelectrophoresis (SDS-PAGE).Proteins are denatured by boiling in the SDS

solution. All charges of protein are masked bynegative charge of dodecyl sulphate.Thus protein moves on polyacrylamide gel strictlyon basis of size of protein molecule. This technique is useful for determining molecularweight of proteins.For visualization of proteins on the gel reagentsused are silver nitrate, coomassie brilliant blue dye.

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PROTEIN INSTABILITIES

The degradation of proteins and peptides can bedivided 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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OXIDATIONSulphur containing amino acids are prone to oxidation.

MAILLARD REACTIONIn the maillard reaction the carbonyl group (RCH=O)

from glucose can react with the free amino group in apepide to form a Schiff base. This reaction is acidcatalysed.

DIMERISATION AND POLYMERIZATIONInsulin forms a small amount (about 1%) of covalentdimer and polymer during two years cold storage.Production of these species increases as temperatureincreases.


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DENATURATION

o Specific confirmation is required for proteins to exertpharmacological 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 tertiarystructure, which is determined by the primary andsecondary 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 60 o C for 10 hours. At pH 2 albumin molecule expands and elongates butcan return to native confirmation reversibly. Also, it shows

good solubility.Mechanism for such behaviour may be one of thefollowing :

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 maybe one of the following :Reduce surface adsorption.

Inhibit aggregate formation.Stabilize proteins against heat denaturation.


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POLYHYDRIC ALCOHOLS ANDCARBOHYDRATES :

They contain CHOH-CHOH- groups which areresponsible for stabilizing proteins. They stabilizeproteins against denaturation caused by elevatedtemperature or by freeze drying or by freeze thaw

cycles.Many important therapeutic proteins and peptidesare 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 chainpolyhydric alcohols are more effective asstabilizers. e.g. sorbitol, xylitol. 42


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Mechanism of action as stabilizers for polyhydric alcoholsis that they have effect on structure of surrounding watermolecules which strengthens hydrophobic interactions inprotein molecules. Mechanism of action as stabilizers for carbohydrates is

that they provide dry network that provides significantsupport for protection.Polyhydric alcohols used are sorbitol, mannitol, glycerol,PEG.

Carbohydrates used are glucose, mannose, sucrose,ribose.

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ANTI-OXIDANTSThiol compounds such as thioacetic acid, triethanolamine,

reduced glutathione and metal chelants such as EDTA areused as antioxidants.

MISCELLANEOUS

Certain enzymes can be stabilized by using compoundshaving similar structures of enzymes. e.g. Glucose stabilizesglucoamylase while aspargine stabilizes asparginase.Compounds forming stable complex through ionic interactionwith proteins can stabilize proteins.Calcium is essential for thermal stability of certain amylases orproteases.


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Protamine zinc insulin (PZI) contain an excess quantity ofzinc to retard absorption. According to BRITISH

PHARMACOPOEIA of 1958, a phosphate buffer is addedto each individual vial containing acidified solution ofinsulin, protamine and zinc so that pH is between 6.9 to7.3. The preparation is compounded in final container by

mixing PZI and buffer in filling operations.Main problem in using insulin is to avoid plasmaconcentration fluctuations. To solve this problem variousformulations are available varying in onset and durationof action.


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USP INSULIN TYPE STRENGTHS


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USP INSULIN TYPE STRENGTHS

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

Isophane insulin suspension (NPHInsulin)

U-40 mixed,U-400 mixed

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

U-100

Insulin zinc suspension (Lenteinsulin)

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


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REFERENCES

1) Agrawal S, Udupa N, Protein and peptide drug delivery :recent advances. In : Jain NK, editor. Progress in controlledand novel drug delivery systems. 1 st ed. Delhi : CBSPublishers; 2004.p.184-204.

2) Chien YW : Novel drug delivery systems. 2 nd ed. New York :Marcel Dekker Inc; 2005.p.631-745.

3) Yu Chang John Wang : Parenteral products of proteins andpeptides. In : Lieberman HA, Avis KE, editors. Pharmaceuticaldosage forms : Parenteral medications, volume 1. 2 nd ed. NewYork Marcel Dekker Inc; 2005.p.283-320.

4) Block JH, Beale JM. Wilson and Gisvold s textbook of organicmedicinal 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. Thetheory and practice of pharmacy. 3 rd ed. Mumbai :Varghese Publishing House; 1987.P.488-489.

6) Aulton ME : Pharmaceutics : The science ofdosage form design. 2 nd ed. Toronto : Churchilllivingstone; 2006.p.544-553.

7) Poon CY : Clinical Analysis. In : Troy DB, editor.Remington : The Science of Dosage form Design.21 st 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. 5 th ed. Toronto

: Churchill livingstone; 2003.p.386-388.


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32129428 protein and peptide drug delivery systems

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