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PROTEIN AND PEPTIDE DRUG DELIVERY: PERSPECTIVES FROM

PHARMACEUTICS

Dr. YAMINI D. SHAH

L.M. COLLEGE OF PHARMACY

AHMEDABAD

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More than 7000 naturally occurring peptides • Hormones, • Neurotransmitters, • Growth factors, • Ion channel ligands

peptides are selective and efficacious signaling molecules that bind to specific cell surface receptors• Ex. G protein-coupled receptors (GPCRs) or ion channels,

where they trigger intracellular effects. peptides represent an excellent starting point for the design of

novel therapeutics and their specificity has been seen to translate into • Excellent safety• Tolerability• Efficacy profiles in humans.

The main disease areas: Metabolic diseases and Oncology.

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Most peptide therapeutics are injectables cyclosporine (NeoralTM) and desmopressin (MinirinTM)

Only a few oral peptide drugs in existence. Challenges relating to development of oral peptides include

acidic and enzymatic degradation of the molecules in both the gastrointestinal tract and when crossing the intestinal mucosa via either active transport or passive diffusion.

Chemical strategies in the design of peptides for oral administration include the previously mentioned stabilizing of the secondary structures, such as stapled peptides, building hydrophobic faces, cyclization, N-methylation, and establishment of intramolecular hydrogen bonds.

Various biotechnology companies are active in this field of peptide drug research, including Ra Pharma, Peptidream, and Cyclogenix (cyclic peptides), and Bicycle Therapeutics.

Formulary aspects and permeability enhancers are also important.

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Injectable peptide medicines will remain the preferred solution over orally administered peptides.

Ex. Patients with diabetes treated with insulin. Technologies have been invented for the insertion of membrane

permeability elements, ‘cell penetrating peptides’, such as penetratin or the transcription trans-activating (TAT) sequences, to reach intracellular targets with peptides to some degree.

In antibody peptide conjugates: the antibody part might take the role as the targeting

entity, the peptide is the effecter part.

Biotechnology companies working with peptide conjugates include Angiochem and 3B Pharmaceuticals, among others.

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Current multifunctional peptides in development include antimicrobial peptide drug candidates that have additional biological functions, such as immune stimulation and wound healing. Also, the trend towards multifunctional peptides can be seen in the GLP-1 agonist field.

Which represents an established drug class with several products, includin ByettaTM (exenatide), BydureonTM (exenatide), VictozaTM (liraglutide), LyxumiaTM (lixisenatide), and most recently TanzeumTM (albiglutide), g being introduced to the market with great commercial success.

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Proteins have the most dynamic and diverse role of any macromolecule in the body.

Catalysing biochemical reactions, forming receptors and channels in membranes, providing intracellular and extracellular scaffolding support, and transporting molecules within a cell or from one organ to another.

It is currently estimated that there are 25,000–40,000 different genes in the human genome, and with alternative splicing of genes and post translational modification of proteins (for example, by cleavage, phosphorylation, acylation and glycosylation), the number of functionally distinct proteins is likely to be much higher.

From the perspective of disease mechanisms, as disease may result when any one of these proteins contains mutations or other abnormalities, or is present in an abnormally high or low concentration.

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Advantages of Protein Therapeutics: Proteins often serve a highly specific and complex set of

functions that cannot be mimicked by simple chemical compounds.

The action of proteins is highly specific, there is often less potential for protein therapeutics to interfere with normal biological processes and cause adverse effects.

The body naturally produces many of the proteins that are used as therapeutics, these agents are often well tolerated and are less likely to elicit immune responses.

For diseases in which a gene is mutated or deleted, protein therapeutics can provide effective replacement treatment without the need for gene therapy, which is not currently available for most genetic disorders.

the clinical development and FDA approval time of protein therapeutics may be faster than that of small molecule drugs

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Most are now produced by recombinant DNA technology. Production systems for recombinant proteins include:

Bacteria, Yeast, Insect cells, Mammalian cells, Transgenic animals and plants.

The system of choice can be dictated by the cost of production or the modifications of the protein

Ex. Glycosylation, Phosphorylation proteolytic cleavage

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Protein glycosylation patterns can have a dramatic effect on the activity, half life and immunogenicity of the recombinant protein in the body.

Ex. The half-life of native erythropoietin, a growth factor

important in erythrocyte production, can be lengthened by increasing the glycosylation of the protein.

Darbepoetin a is an erythropoietin analogue that is engineered to contain two additional amino acids that are substrates for N‑linked glycosylation reactions. When expressed in Chinese hamster ovary cells, the analogue is synthesized with five rather than three N‑linked carbohydrate chains; this modification causes the half-life of darbepoetin to be threefold longer than that of erythropoietin

Peptide

20- 50 amino acids

Primary sequence

Secondary not found in chain length having 20 aas

No tertiaryNo quaternary

Function of a small peptide will be dependent only on the functional groups of various amino acids

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Structure of Protein and Peptide:

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Biologics may include: Vaccines, Recombinant proteins, Genes, Synthetic tissues and Viruses,

As a result of extensive research in molecular biology and biotechnology and all cell biology

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• The cyclic peptide cyclosporine A may be only 1.2 kda but a typical mab will be 144 kda;

Molecular weight

• leading to chemical and physical instability, in turn effecting the shelf life and also question arisen whether to make liquid or solid dosage formulations

pka values

• High water solubility but increase in viscosity at concentrations above 50 mg/ml, how to maintain viscosity in case of subcutaneous injection

Water solubility

• Silicone-coated glass syringesInteractions with packaging

• The product must be compatible with aseptic processing and manufacture, use of freeze/ spray drying of protein drugs.

Stability

Considerations in Pre Formulation Studies For Peptide and Protein:

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Chromatography and light scattering, to determine purity and aggregation

Microscopy, to observe particulates 2 to10 micrometer

IR spectroscopy, for conformational changes;

Isoelectric focusing, for determination of the isoelectric point

Mass spectrometry, for chemical degradation

Calorimetry and fluorescence, for determination of unfolding/stabilization

Analytical Method Employed With Peptides and Proteins:

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General Problems Associated With Biomacromolecules:

Get destroyed at stomach pH Some get destroyed at intestinal pH or due to the presence of

proteases Presence of cell-cell tight junctions in the epithelial cells of

intestine do not readily transit Semi permeable cell membrane

Presence of efflux proteins such as P- glycoprotein and cell glycocalyze

Size larger bio macro molecules Short half lives of bio macro molecules Receptor mediated endocytosis Susceptibility to chemical and physical degradation

(aggregation) Very Potent

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Monoclonal antibodies (mabs) : Monoclonal antibodies (mabs) are most commonly administered

by subcutaneous routes and are shown patient compliance Depends on frequency of administration depot inj: once every

month or even less patient compliance is not problem but need for daily injections of insulin has in itself driven many researchers to develop non invasive protein delivery mainly oral and pulmonary

Inhalable insulin marketed by Pfizer inc. under the name of Exubera was launched in 2006.But after around a year was removed from the market reason due to lack of acceptance uncertainty over patient compliance and safety, cost.

Neverthless research on going in the following area as under Nasal, Dermal, CNS.

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Examples of Therapeutic Monoclonal Antibodies:1

181. A.K. Pavlou, M.J. Belsey, The therapeutic antibodies market to 2008, Eur. J. Pharm. Biopharm. 59 (2005) 389-396.

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Formulation parameters to be closely monitored to avoid Degradation, Aggregation Changes in ph Ionic strength Metal impurities Temperature Irradiation Gases (particularly oxygen and moisture) Shear and surface adsorption.

Excipients to stabilize proteins Polyethylene glycol (PEG); Non ionic surfactants, Amino acids.

Excipient compatibility must be screened for in the liquid and/or solid state for solid state if formulated in,solid state optimization of the lyophilization cycle.

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Case Study of Insulin Insulin is usually administered by subcutaneous injection or a

pump and cannula. The subcutaneous route is more convenient, and is inevitably

used for control of type I diabetes. An added challenge to insulin delivery is the need for a pulsatile

release system which would follow the normal post prandial response of a sudden rise in blood glucose levels.

Oral insulin delivery is perhaps the primary target, which must overcome enzymatic degradation and poor permeability in the GIT.

Beyond current commercialization activities, there lies a large volume of research employing micro emulsions and nano particulates and microspheres of polysaccharides/polyesters/ eudragit to achieve oral insulin delivery; oral bioavailability remains around 10% or less .

Nasal and buccal delivery have generated the most attention.

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The nasal cavity is highly vascularized and bypasses the hepatic metabolism but suffers from a low surface area (compared to the lung)

Chitosan derivatives are commonly used materials for these routes because chitosan acts as a permeation enhancer by transiently loosening the epithelial tight junctions.

Sublingual delivery has the advantage of ease of administration, but the challenge includes the multilayered structure of the buccal mucosa and the constant flow of saliva.

Ex. Following a regular insulin regime in type I diabetics, it was

reported that oral insulin spray caused similar changes in blood glucose compared to subcutaneous insulin injection.

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Controlled Delivery of Peptide and Protein: The use of biodegradable polyester microspheres for controlled

delivery is not limited to a specific route of administration, which may be either invasive (e.g. subcutaneous/depot injection) or non invasive (e.g. oral, inhaled).

Conversely, polymers used in non invasive buccal delivery may be required for mucoadhesion but not controlled delivery.

There is a well established track record in the use of PLGA microspheres for the controlled delivery of peptide hormones (Table 1)

Optimization of an encapsulation protocol is therefore dependent on the physicochemical characteristics of the peptide/protein and the polymer itself

Monomer composition Molecular weight Glass transition.

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Commercially Available Biodegradable Drug Delivery System:2

2. F. Mohamed, C.F. van der Walle, Engineering biodegradable polyester particles with specific drug targeting and drug release properties, J. Pharm. Sci. 97 (2008) 71-87.

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Encapsulation Technique Must Ensure Main Criteria:

The structure of the peptide/protein remains intact, particularly with respect to aggregation and neutralizing antibodies.

The size of the spheres has to support the administration route aimed for from microns for injectable and inhalable particles, to nanoparticles for oral administration.

The drug release profile must be appropriate to the intended medical application Ex.vaccine, long-term release, delayed release

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Toxicity Profiles: The complex nature of proteins, peptides derived through

biotechnological processes present many challenges when it comes to understanding their physicochemical and therapeutic behavior.

Their specific primary, secondary, tertiary and quaternary structures, play key roles in defining the integrity and biological activity of bio macromolecules.

This is the case whether they are directly incorporated into simple, formulated systems, or use more sophisticated delivery platforms.

Safety and toxicity must be evaluated through the use of scientifically sound methods as a matter of paramount importance and necessity.

Through appropriate species selection, relevant models, appropriate use of biomass, no effect or reduced efficacy to allergic reactions, thrombocytopenia and anemia.

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Factors To Be Considered Before Commercialization of A Peptide Protein Drug Delivery: For Final fill finish product manufacturing Selection of the methods and experiments Selection of raw material and manufacture to make

therapeutic protein delivery Development of the final formulation. Carrier platform selection of the delivery system vial/ syringe

or other applicator system. Emphasis on determining essential processing parameters

which affect the quality of product identification, of quality attribution of the product.

Establish specifications which define product quality. Conduct appropriate tests to justify these specification Conduct DOE to plan on testing critical quality attributes.

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Implement A Disciplined Approach By Performing: Quality by design (QBD), Process analytical technology (PAT), Failure mode effects and criticality analysis (FMECA), Risk assesment practices When all this done carry out successful trials Run batches Carry out formal validation Carry out manufacture of a predetermined batches with

protocols and standard operating procedures Establish that process can reproducibly meet Established critical quality attributes Failure

REFERENCES A.K. Pavlou, M.J. Belsey, The therapeutic antibodies market to

2008, Eur. J. Pharm. Bio- pharm. 59 (2005) 389-396. J. Jones, Amino Acid and Peptide Synthesis, The Bath Press,

Bath, 2002. K. Sun-Chang, L.J. Hyun, K.M. Hyung, K.J. Hyun, H. Seung-

Suh, L. Hyun-Soo, Method for mass production of antimicrobial peptide. Patent applicant: Samyang Genex Co. Ltd., Korea Advanced Inst Sci & Tech, Korea. Publication: WO9854336 (1998).

T.E. Creighton, Proteins: Structures and Molecular Properties, W.H. Freeman, New York, 1993.

C.F. van der Walle, G. Sharma, M. Ravi Kumar, Current approaches to stabilising and analysing proteins during microencapsulation in PLGA, Expert Opin. Drug Deliv. 6 (2009) 177-186.