Practical consideration on protein and peptide drug deliverySUCHANDRA BAGCHIM.S(PHARMACEUTICS)NIPERA1517PE10

Flow of seminar

Objective Introduction Structure of protein Classification of proteins Physicochemical parameters of proteins Protein degradation Rational for protein and peptide drug delivery Strategies to be used Advancement in protein delivery systems Toxicity and safety Commercialisation parameters and conclusions

Objective

The drug discovery of insulin in1922 marked the beginning of research and development to improve the means of delivering protein therapeutics to patients. From that period forward investigators have contemplated every possible route of delivery. Their research efforts have followed two basic pathways…..One pathway is focussed on non-invasive means of delivering proteins to the bodySecond pathway has been primarily aimed at increasing the biological half life of the therapeutic moleculesThe commercial success of protein delivery by nasal, oral and pulmonary routes have been more opportunistic rather than the application of the platform technologies application to every protein and peptides.

The major barriers of protein and peptide drug delivery

Poor intrinsic permeability Luminal and enzymatic degradation Rapid clearance Chemical and conformational stabilityThe success achieved by Sandoz with Cyclosporine formulations for proteins will remain highly compound specific.However recently novel oral delivery system for 5-CNAC, formulated with the peptide Salmon Calcitonin is in phase III clinical trial for the treatment of osteoporosis or osteoarthritis and could become the first marketed oral peptide. The present reviews focus on key findings and implications from studies undertaken till today for oral formulation of protein and peptides.

Introduction

The proteins are large molecules complex architecture. the peptides and proteins are seldom linear and adapt a variety of specific folded three dimensional patterns and conformations.The structure of proteins directly related to its functions so if shape gets altered functions also get loosened.There are generally four types of protein structures…1. Primary structure2. Secondary structure3. Tertiary structure4. Quaternary structure

Structure of proteins

Classifications of proteins

By functionEnzymes ,hormones, transport proteins, antibodies, structural proteins, major proteins, receptor, signalling proteins, storage proteins By location in living andMembrane proteins, external proteins, internal proteins, virus proteins By post translational modificationsNative proteins, glyco protein , cleaved protein, protein with disulphide bonds, protein complexes, chemically modified proteins, prions

Physicochemical properties of proteins and peptides

Solubility and partition coefficientAqueous solubility of protein and peptide is strongly dependent upon the p H,Presence of metallic ion, ionic strength and temperature. At isoelectric point the peptide solubility is minimal where the drug is having no charge.Peptides are very hydrophilic with very low octanol-water partition coefficient, so to improve the absorption of peptides by passive diffusion, their lipophilicity should be increased. Aggregation, self association and hydrogen bondingSelf aggregation tendency modifies their intrinsic properties. Human insulin was found to be more self aggregating than the bovine insulin. Addition of additives like non-ionic surfactants (Pluronic F68) stabilize the peptide formulation against self aggregation. Intermolecular hydrogen bonding with water decreases permeability of protein in lipid membrane.

Degradation pathways of protein and peptides

Sr. no. Physical instability

mechanisms

1 Denaturation Non-proteolytic modification of a unique structure of a native protein that affects definite change in chemical,Physical and biological properties. for eg. Urea, alcohol , acetic acid, SDS and PEG are denaturing agents.

2 Adsorption Amphiphilc nature of protein causes adsorption at various interfaces like air-water, air-solid etc.

3 Aggregation and precipitation

The denatured unfolded protein may rearrange themselves in such a manner that the hydrophobic parts aggregate and form agglomerates and hence give rise to precipitation of proteins

Protein degradation

sr,.no Chemical degradation mechanism1 Deamidation The hydrolysis of the amide containing terminal of protein

structure2 Oxidation and reduction Due to isolation, synthesis and storage of

proteins,temperature,ph ,metal ion accelerates the reaction . Glucagon is an exception after denaturation also it retains its biological activity after oxidation

3 Proteolysis Prolonged exposure to tempareture and ph and in presence of proteolytic enzymes

4 Disulfide exchange If any other disulphide linkage enter a structure and alters the protein structure

5 Racemization The alteration of L-amino acids to D-amino acids or vice versa.

6 B- elimination Formation of carbanion ion leads to beta elimination of amino acids under alkaline conditions

Why to go for protein drug delivery

Better medical treatments do not always require stronger medicines. The effectiveness depends upon the route of administration, optimised formulation in a proper drug delivery systems. The growth of biotechnology and human genome sequencing lead to

the production of protein and peptides drugs in reality. These macromolecules function as natural blueprints of original

proteins. Unfortunately stabilization of proteins is a challenge to pharmaceutical

delivery systems Their incredible selectivity. specificity and potent action

Strategies developed to deliver proteins

Modifying the physicochemical properties of proteins by prodrug approach and analogs of biological might protect the protein from proteolytic enzymatic degradation.Adding novel functionality to macromolecules such as peptide influx transporter and by adding inhibitors of efflux transportersUsing particulate carrier systems such as hydrogels, nanoparticles, microspheres, and lipid based drug delivery systems such as SLN,NLCs microemulsions ,liposomes etc

Various pharmaceutical approaches and their outcomes

Chemical modifications-improve enzyme stability Amino acid modification- improve membrane penetration Hydrophobization-resistance to degradation by enzymesFormulation vehicleEmulsion –protect drug from intestinal and luminal protease in GITMicrospheres- restrict release in favourite area of stomachNano particles-increase intestinal epithelial absorptionLiposomes-achieve site specific delivery

Advancement in drug delivery

Molecular conjugation – polypeptides conjugated with macromolecular carrier, such as polymer or protein. Advantage of this system it will increase absorption without creating any side effects like penetration enhancers which leads to transient openings leading to entry of many molecules along with drugs which is not desirable. NOBEX of GSK is based on this technology and in providing promising result in phase II of clinical trial.

Encapsulation-peptide encapsulation technology in particulate carriers has been developed extensively.as a result of their stability. Chitosan and hydrogels are the most extensively used in these days. Lipid microcarriers are most beneficial than liposomes for oral delivery.these particles are found to be more effective in vaccine delivery where it has been taken up by peyer’s patches in the GIT and subsequently targeted to immune systems.

Oral nanoscale carriers-in general nanoscale dimensions favour transport of particlesaxross the mucosa lepithelium. 100 nm poly(PLGA) particles diffused throughout the submucosal layers, whereas 10mm particleswere predominantly localised on epithelial lining of the tissue.

Colon specific drug delivery systems-interesting approach. Oral colon specific drug delivery system by using a coating of copolymer crosslinked with azoaromatic groups to form an impervious film which protects orally administered proteins from harsh GIT environment.

In the large intestine the microflora reduce the azo bonds, break the crosslinks and degrade the polymer film, thereby releasing drug in the colon for absorption.

Toxicity and safety regarding protein drug delivery system

This quality,safety, efficacy of a biotechnological products are interlinked.

Hence pharmacological safety, microbiological safety Immunogenicity/tumorigenicity Genetoxicity, monoclonal antibody etc could cause toxicity

Commercialisation factors

Key points to consider before commercial development of protein delivery methods Key point Considerations Manufacturing Costs of manufacturing process, scalability, yields Protein quality Effect of processing and delivery on protein Bioavailability Fraction of total drug delivered to circulation Safety/toxicity Impact of delivery method on clinical toxicology of the protein and

the site of administration

Conclusion

Peptide and protein drugs are currently used as parenteral therapies because of their poor bioavailability from different alternative routes of administration, including the p.o. route. Poor intestinal absorption of these drugs is due to their unfavorable physicochemical properties, such as high molecular weight, susceptibility to enzymatic hydrolysis, and high hydrophilicity. Moreover, there are several biological barriers to intestinal absorption of peptide and protein drugs across the GI tract. These include hydrolysis in the stomach, proteolytic degradation across the GI tract, and bacterial fermentation in the colon. Development of an effective oral delivery system, therefore, will require a thorough understanding of these barriers as well as the mechanisms involved in their absorption across the GI tract.

Thank you