Presented by G.Ramya

Mpharm(phceutics)256212886041

MRCP

Under the guidance ofMrs.Yasmin Begum

Mpharm(PhD)

Proteins and Peptides drug delivery system

Contents

Protein & Peptides Structure of protein Classification of protein Stability problems Marketed formulations Conclusion References

Proteins: These are large organic compounds made of amino acids arranged in a linear chain and joined together by peptide bonds. Protein > 50 amino acids

Peptides: These are short polymers formed from the linking, in a defined order, of α-amino acids. peptide < 50 amino acids

Protein & Peptide

Each peptide or protein molecule is a polymer chain with α aminoacids linked together in a sequential manner by peptide bonds.

The peptide chains in a protein molecule are often folded into a specific three dimensional structure to fulfill a certain biological function.

The functional groups on each of the aminoacids and the sequence of the aminoacids in the peptide chains determine the conformation of the protein.

STRUCTURE OF PROTEIN

Based on their conformation proteins are classified as

Fibrous proteins Eg: collagen, keratin, elastin.

Globular proteins: insulin

STRUCTURE OF PROTEIN

There are four types.

Primary structure- The amino acid sequence.

Secondary structure- Regularly repeating local structures

stabilized by hydrogen bond.

Tertiary structure-Three dimensional structure of polypeptide

Quaternary structure-The structure formed by several protein

molecules (polypeptide chains).

STRUCTURE OF PROTEIN

Lactate Dehydrogenase: Mixed α /β

Immunoglobulin Fold: β

Hemoglobin B Chain: α

Protein Structure

Transport and storage of small molecules.

Coordinated motion via muscle contraction.

Mechanical support from fibrous protein.

Generation and transmission of nerve impulses.

Enzymatic catalysis.

Immune protection through antibodies.

Control of growth and differentiation via hormones.

FUNCTIONS

Classification of Proteins

According to their biological roles Enzymes – Catalyses virtually all chemical reactions i.e.

6GDH Transport proteins i.e. Haemoglobin of erythrocytes Contractile or Motile proteins i.e. Actin and Myosin Structural proteins i.e.Collagen in bones Defense proteins i.e. Immunoglobulins and Antibodies Regulatory proteins i.e. insulin Nutrient and storage proteins i.e. Ovalbumin

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.

Protein and Peptide drugs

Short biological half-life

Very large and unstable molecules.

Structure is held together by weak noncovalent forces.

Easily destroyed by relatively mild storage conditions and

gastric juices.

Hard to obtain in large quantities.

Disadvantages

Elimination by B and T cells

Proteolysis by endo/exo peptidases

Small proteins filtered out by the kidneys very quickly

Unwanted allergic reactions may develop (even toxicity)

Loss due to insolubility/adsorption

First-pass elimination in liver

Problem with Proteins (in vivo – in the body)

Physical instability Chemical instability Denaturation Deamidation Adsorption Oxidation and reduction Aggregation Proteolysis Precipitation Disulfide exchange Racemization β-elimination

Protein instabilities

Denaturation refers to any nonproteolytic modification of the unique structure of a native protein that affects definite changes in physical, chemical and biological properties.

Denaturation leads to rearrangement and loss of quarternary and tertiary structure and the hydrophobic and hydrogen bonds are broken.

Conditions include: Solvent changes pH change Alteration in ionic strength Temperature rise

Denaturation

Category Mechanism Examples

Polar and protic chemicals

Surfactants

Disrupt H-bonds

Hydrophobic disruption and charge group separation

Urea, Guanidine HCL, Alcohol, Acetic acid

Sodium dodecyl sulphate, Polyethylene glycol, Dodecyl ammonium chloride

Common Protein Denaturing agents

Proteins and peptides tend to absorb at interfaces such as air-water and air-solid.

This leads to conformational rearrangement which may cause loss or change in the biological activity.

It posses limitations with the drug delivery systems where protein content of formulation may remain adhered to the containers or drug delivery devices.

Overcome by addition of surfactants such as Pluronic F68 and Tween80.

Adsorption

Mechanism of protein aggregation involves protein denaturation and noncovalent association via hydrophobic residues.

Aggregation on a macroscopic scale leads to precipitation. Conditions favour include: Large air-water interface Agitation of the solution Presence of large headspace within the confines of

container. Increase in thermal motion of the molecules due to

agitation.

Aggregation and Precipitation

It involves the hydrolysis of the side chain amide linkage of an aminoacid residue leading to formation of free carboxylic acid.

Asparagine, glutamine, serine, glycine. Human growth harmone(hGH), bovine growth

harmone(bGH), prolactin, insulin, lysozyme and secretin. It leads to lowering of biological activity and alteration in

function.

Deamidation

Deamidation of L-Asparagyl residue

Methionine, cysteine, tryptophan and tyrosine. Commonly occurs during isolation, synthesis and storage of

proteins. Leads to loss in biological activity as observed in calcitonin,

corticotrophin, gastrin. Oxidising agents like hydrogen peroxide, dimethylsulphoxide

and iodine oxidise met to Met-sulphoxide.

Methionyl residue Methionyl sulfone residue

Oxidation

Hydrolysis of peptide bonds within the polypeptide or protein destroys or reduces the activity.

It may occur on prolonged exposure of proteins to extreme pH range, high temperature or proteolytic enzymes.

Bacterial contamination is the most common source of proteases.

Proteolysis

Disulfide bonds may break and reform with incorrect pairings which results in alteration of 3-dimensional structure followed by change in biological activity.

HSR’ + R-S-S-R ---- > R-S-S-R’

Peptide chain with more than one disulfide can enter into disulfide exchange reactions leading to scrambling of disulfide bridges and thereby change in alteration.

Disulfide Exchange

Alteration of L-aminoacids to D,L-mixtures. Except gly, all the mammalian aminoacids are chiral at the

carbon bearing chain and are susceptible to base catalysed racemization.

This reaction can be catalysed in neutral and alkaline media by thiols which may arise due to hydrolytic clevage of disulfides.

Racemization

The mechanism is similar to racemization which forms a carbonium intermediate.

Higher elimination rate under alkaline conditions leads to loss of biological activity.

Cys, Lys, Phe, Ser, and Thr

β-elimination

Photodegradation

Tryptophan, Tyrosine and Cysteine are susceptible to nonionizing radiation such as UV light.

Photoionisation can be either through direct interaction with aminoacid or indirectly via various sensitizing agents such as oxygen.

Photodegradation products include: S-S bond fission Conversion of tyrosine to DOPA Conversion of tryptophan to kynurenine and n- formyl-

Kynurenine.

Excipients Functions Examples

Surfactants Prevent denaturation and aggregation

Polysorbate 20, 80

Albumin Antiaggregatory Serum albumin

Sugars Stabilize protein against denaturation

Mannitol, propylene glycol, Sucrose, Lactose

Cryoprotectants Stabilize protein against very cold condition

Sugars, Amino acids, Amines, polyols, Salts

Commonly used excipients For solving physical and chemical stability problems

Osmolarity agent Stabilize pH Phosphate, Citrate Buffer

Preservatives Prevent from

microbes

Phenol, benzyl

alcohol

Antioxidants Prevent oxidation Ascorbic acid, sulphites, cysteine,

chelating agent

Solubility

enhancing agent

Increase solubility

of protein

Amino acids,

detergent,

cyclodextrin

Lyoprotectants Stabilize protein during freeze drying

Sugars

Applications

Protein/peptide drugs Biomedical applications

CVS activeAngiotensin 2 antagonistBradykininCaptopril

Lowering blood pressureImproving peripheral circulationHeart failure management

CNS activeCholecystokininΒ-endorphin

Suppressing appetiteRelieving pain

Applications

GI-activeGastrin antagonistPancreatic enzymes

Reducing secretion of gastric acidDigestive supplement

ImmunomodulatingBursinCyclosporinInterferon

Selective B-cell differentiatingharmoneInhibits functions of T-lymphocyteEnhancing activity of killer cells

Metabolism modulatingInsulinVassopressin

Treating diabetes mellitusTreating diabetes insipidus

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

MARKETED PROTEINS IN FREEZE DRIED 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

MARKETED PEPTIDES IN READY TO USE FORMULATIONS

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

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.

Conclusion

This poses an urgent challenge to 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.

Conclusion

REFERENCES

Controlled drug Delivery Concepts and Advances By Vyas, Roop Khar, Proteins and Peptides Delivery Considerations Page No: 503-511.

Advances in Controlled and Novel Drug Delivery By N.k.Jain, Oral delivery of protein and peptides Page no: 232-235.

Novel Drug Delivery System By Yie.W.Chein, Systemic Delivery of Peptide based pharmaceuticals Page no:631-637.

Drug Stability Principles and practices By J.Carstensen, Stability of Polypeptides and Proteins Page no:560-572