2. ) Department of Pharmaceutics.

3. CONTENTS  Protein & Peptides  Structure of protein  Classification of protein  Stability problems  Formulation Aspects  Barriers  Approaches  Parenteral delivery of protein and peptide  Ocular delivery of protein & peptide  Conclusion  References

4. Definitions  Protein: polypeptides which occur naturally and have a defined sequence of amino acids and a three- dimensional structure (e.g. insulin).  Peptide: a short chain of amino acid residues with a defined sequence (e.g. leuprolide).  Proteins - Chains of amino acids, each joined together by a specific type of covalent bond  Proteins formed by joining same 20 amino acids in many different combinations and sequences  Protein > 50 amino acids  peptide < 50 amino acids  Function of a protein determined by its non-covalent 3D structure

5. Structure of peptides and proteins  Proteins have in increasing order of complexity  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).

6. Protein Structure

8. Lactate Dehydrogenase: Mixed α /β Immunoglobulin Fold: β Hemoglobin B Chain: α

9. Functions  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

10. Applications of protein and peptide drug delivery system  Erythropoietin used for production of RBC.  Tissue plasminogen activator is used for Heart attack, Stroke.  Oxytocin maintain labor pain.  Bradykinin increases the peripheral circulation.  Somatostatin decrease bleeding in gastric ulcer.  Gonadotropin induce ovulation.  Insulin maintains blood sugar level.

11. Advantages  Improved patient compliance.  Reduced frequency of administration.  Reduced adverse effect profile.  Potential to reduce product development time and costs.  Possible new and broader therapeutic applications.

12. Disadvantages  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.

13. Problem with Proteins (in vivo – in the body)  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

14. 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  Defense proteins i.e. Immunoglobulin's and Antibodies  Regulatory proteins i.e. Insulin  Nutrient and storage proteins i.e. Ovalbumin

15. Stability Profile  Protein and peptide drugs have poor stability profile.  The degradation pathways of this class of drugs are due to chemical and physical instability  The high chemical and Physical instability presents peculiar difficulties in the purification, separation, formulation, storage and delivery of these compounds.  Physical instability involves transformations in the secondary, tertiary, or quaternary structure of the molecule.  These changes are manifested as denaturation, aggregation, precipitation and adsorption onto surfaces.  Chemical instability involves alteration in the molecular structure producing a new chemical entity, by bond formation or cleavage

16. Physical Instability  Denaturation  Aggregation  Precipitation  Adsorption onto surfaces

17. Denaturation  Peptides and proteins are comprised of amino acid residues and non-polar amino acid residues.  The hydrophobic, non-polar amino acid residues fold upon themselves in an aqueous environment to form globular molecules.  The hydrophilic, polar amino acid residues of these molecules are exposed to the aqueous environment.  On changing the aqueous environment to non-aqueous, they start unfolding and thereby exposing their hydrophobic residues to the hydrophobic environment.  This leads to the rearrangement and loss of quaternary and tertiary structure. On unfolding hydrophobic and hydrogen bonds are broken  The term denaturation is used to describe any nonproteolytic modification of the unique structure of a native protein that effects

18. Aggregation  Some proteins self-associate in aqueous solution to form oligomers.  Insulin, for example, exists in several states:  The zinc hexamer of insulin is a complex of insulin and zinc which slowly dissolves into dimers and eventually monomers following subcutaneous administration, conferring on it long acting properties

19. Surface adsorption and precipitation  Adsorption of proteins such as insulin on surfaces such as glass or plastic in giving sets:  can reduce the amount of agent reaching the patient  can lead to further denaturation, which can then cause  precipitation and the physical blocking of delivery ports in protein pumps.  Denaturation is facilitated by the presence of a large head space  allowing a greater interaction of proteins with the air–water interface.

20. Chemical Instability-Oxidation  Tryptophan, methionine, cysteine, histidine, and tyrosine amino acid side chains contain functionalities that are susceptible to oxidation. Methionine and cysteine can be oxidized by atmospheric oxygen and fluorescent light. Oxidation has been observed both in solution and in the solid state.  Oxidation of the methionine residues may cause a loss of bioactivity and, in the case of cysteine residues, the formation of nonnative disulphide bonds. Oxidation by atmospheric oxygen or auto-oxidation can be accelerated in the presence of certain metal ions such as copper and iron.  Methionine residues under acidic conditions are especially prone to oxidation by reagents such as hydrogen peroxide, producing methionine sulphoxide. Oxidation by peroxide may be a concern if the protein is processed in a manufacturing area that is sterilized using hydrogen peroxide vapor or using equipment that is so treated, In this case, experiments must be performed and procedures put in place to ensure that the protein is not oxidized during manufacturing.  Oxidation - conversion RSR’ to RSOR’, RSO2R’ or RSO3R’ (Met & Cys)

21. Deamidation  Deamidation is the hydrolysis of a side chain amide on glutamine and asparagine residues to yield a carboxylic acid. The deamidation reaction has been extensively studied and is widely observed in therapeutic proteins and peptides. Some protein delivery system processing and formulation conditions that result in an increase in temperature or pH have been shown to facilitate deamidation.  The deamidation process is important because of the potential loss in protein activity or function. Deamidation contributes to the reduction in catalytic activity of lysozyme and ribonuclease at high temperatures.  Deamidation - conversion of Asp-Glu sequences to a-Asp-Glu or b-Asp-Glu

22. Peptide Bond Hydrolysis  Aspartic acid residues have been implicated in the cleavage of peptide bonds, which have led to a decrease in biological activity. When lysozyme was heated to 90-100°C at pH 4, the loss in biological activity was attributed to hydrolysis of Asp-X bonds  Proteolysis - Asp-Pro, Trypsin (at Lys) or Chymotrypsin (at Phe/Tyr)

23. Disulphide Exchange  Many therapeutic proteins contain cysteine residues that form disulphide bonds. These bonds are important components of the structural integrity of proteins. Incorrect linkages of these disulphide bonds often lead to a change in the three-dimensional structure of the protein and therefore its biological activity. The aggregation of lyophilized formulations of bovine serum albumin, ovalbumin, p-lactoglobulin, and glucose oxidase was attributed to disulphide interchange. Racemisation and Beta Elimination  The reaction proceeds in both acidic and alkaline media, but the mechanisms are different. In neutral and alkaline media, the reaction is catalyzed by thiols. Thiols may be introduced during formulation (e.g., mercaptoethanol as an antioxidant) or by degradation of existing disulphide bonds via beta elimination of cysteine residues.

24. How to Deal with These Problems Storage Formulation Delivery

25. Protein Stabilization  Additives for Protein Stabilization  Protein Stabilization in the Solid State  Protein Stability within a Delivery Matrix  Interactions between the Delivery Matrix and the Protein  Internal Environment of the Delivery Matrix

26. Barriers for Protein and Peptide drug delivery  Enzymatic Barrier  Intestinal Epithelial Barrier  Capillary Endothelial Barrier  Blood Brain Barrier

30. Routes of drug transport across a mucous cell barrier

31. Classification

32. Protein Formulations 1 Protein sequence modification (site directed mutagenisis) PEGylation 2 Proteinylation Microspher encapsulation 3 Formulating with permeabilizers

33. Site Directed Mutagenesis  Allows amino acid substitutions at specific sites in a protein  i.e. substituting a Met to a Leu will reduce likelihood of oxidation E343H

34. PEGylation  PEG is a non-toxic, hydrophilic, FDA approved, uncharged polymer  Increases in vivo half life  Decreases immunogenicity  Increases protease resistance  Increases stability

35. Proteinylation  Attachment of additional or secondary (nonimmunogenic) proteins for in vivo protection  Cross-linking with Serum Albumin  Increases in vivo half life  Cross-linking or connecting by protein engineering with antibody fragments

36. Proteinylation + Protein drug scfc (antibody)

37. Parenteral Drug formulations  Intravenous  Intramuscular  Subcutaneous  Intradermal

38. Advantages & Disadvantages Route of delivery for 95% of proteins Allows rapid and complete action. Avoids first pass metabolism Advantages Problems with overdosing, necrosis Local tissue reactions/hypersensitivity Everyone hates getting a needle Disadvantages

39.  Polymers should be:  Biodegradable  Bio-compatible  Non-toxic  Examples:  Natural- Chitosan, Dextrin  Synthetic- Polylactides/ glycolide Polyanhydrides  Polyphosphoesters

40. Release Mechanism  Diffusion of drug out of the polymer  Drug Release by Polymer Degradation Hydrolysis Enzymatic (Phosphotases; Proteases etc.)

41. Microsphere Drug delivery Two types of microspheres Nonbiodegradable e.g, ceramic particles polyethylene co-vinyl acetate polymethacrylic acid/PEG Biodegradable (preferred) e.g, gelatin polylactic-co-glycolic acid (PLGA)

42. Magnetic Targeted Carriers(MTCs) Founder of FeRx and pioneer of magnetic targeted drug delivery is Dr. Kenneth Widder  Microparticles, composed of elemental iron and activated carbon  Drug is adsorbed into the MTCs and transported  The particles serve as delivery vehicles to the area of the tumor for site-specific targeting

43. Liposomal Drug delivery Spherical vesicles with a phospholipid bilayer  E.g, Bleomycin encapsulated in thermo sensitive liposome enhanced antitumor activity and reduced normal tissue toxicity  Liposome have recently been used successfully as vehicles for vaccines

44. Hydrogel Based Drug Delivery Hydrogels are three dimensional networks of hydrophilic polymers that are insoluble

45. Emulsion & Cellular carriers  Emulsion : Emulsion can be used for parenteral delivery of protein and peptides. Multiple emulsion further prolong the release of drug. e.g. subcutaneous administration of muramyl dipeptide in a w/o emulsion  Cellular carriers: Protein and peptides can be incorporated in erythrocytes to achieve the prolong release or targeting. Resealed erythrocytes as delivery system for c- reactive protein, and mainly used to target liver and spleen.

46. Emulsification

47. Coacervation

48. Extrusion And Spraying

49. Ocular Delivery of Peptide & Protein  Relevant anatomy and Physiology of the Human eye  Diameter of 23 mm  Three layers  Outermost coat : the clear, transparent cornea and the white, opaque sclera  Middle layer : the iris anteriorly, the choroid posteriorly, and the ciliary body at the intermediate part  Inner layer : retina

50. Peptides Useful in Ocular Pathology To treat infections and enzymes used to promote wound healing. a. Bacitracin  The drug is applied topically to the eye for a variety of conditions, including eyelid burns and corneal superficial punctate keratits.It is also used to treat optic neuritis.  Commercial bacitracin is a mixture of at least nine bacitracins, which is used for its antibacterial activity b. Chymotrypsin  Chymotrypsin is used clinically in the eye for enzymatic intracarpsular lens extraction. c. Cyclosporine  Cyclosporine are a group of biologically active metabolites produced by Fungi. When cyclosporine A is administered by nonocular route in rats, it produces significant blood levels, but it can not be detected in tissues.

51. IMPLANTS PROTEINS IN PUMPS : 1.Infusaid Model 400 Implantable Pump 2.Mechanical Insulin Pumps  Formulation is the beginning of successful drug delivery  Multiple potential interactions between the protein and the pump  Control of the material interface is most important  Device design and formulation need to work together and be regulated together.

52. ORAL PROTEIN DELIVERY :  Oral Insulin :  Buccal aerosol delivery system developed by Generex  Insulin is absorbed through thin tissue layers in mouth and throat  Insulin is formulated with a variety of additives and stabilizers to prevent denaturation on aerosolization and to stabilize aerosol particles  PH SENSITIVE MICROSPHERES :  Gel/Microsphere system with polymethacrylic acid + PEG  In stomach (pH 2) pores in the polymer shrink and prevent protein release  In neutral pH (found in small intestine) the pores swell and release protein  Process of shrinking and swelling is called complexation (smart materials)

53. NASAL DELIVERY OF PROTEINS :  Extensive microcirculation network underneath the nasal mucosa  Drug absorbed nasally can directly enter the systemic circulation before passing through the hepatic circulation  The nasal administration of peptides has attracted much interest now a days due to - Relatively rapid absorption of drug - Little metabolic degradation - Relative ease of administration - Selective to peptide structure and size  Enhancement of nasal absorption of insulin using polyacrylic acid as a vehicle  Enhancement in the nasal absorption of insulin entrapped in liposomes through the nasal mucosa of rabbits  Administration of insulin (1 IU/ kg) via the nasal route caused a significant decrease in the plasma glucose level  The nasal route appears to be a viable means of systemically delivering many small peptides

54. PULMONARY DELIVERY :  Deep lung, an attractive site of protein delivery due to - Relatively large surface area (100m 2 ) - Rapid absorption of drug into the blood stream through the alveoli  Dura and Inhale developed dry powder delivery systems for proteins  40% of the insulin administered in an aerosol, to the trachea of anaesthetized rabbit was absorbed  Albumin was largely absorbed within 48 hours of instillation into the lungs of guinea pigs and dogs

55. RECTAL DELIVERY  The rectal delivery offers many advantages  - Avoidance of drug dilution prior to reaching the systemic circulation - Reduction in first-pass metabolism - Rapid systemic absorption - Safe and convenient especially in case of neonates and infants - Greater dose may be administered - Withdrawal of drug is possible in case of adverse effects  Administration of insulin using the rectal route shows systemic absorption

56. TRANSDERMAL PATCHES :  Proteins embedded in a simple matrix with appropriate additives  Patch is coated with small needles that penetrate the dermal layer  Proteins diffuse directly into the blood stream via capillaries  Less painful form of parenteral drug delivery

57. REFERENCES :  Advances in controlled and Noval drug delivary by N.K. JAIN.  Oral protein and peptide drug delivery. In: Binghe W, Teruna S, Richard S, editors. Drug delivery: Principles and applications. New Jersey:Wiley Interscience:p.189.Rick.s.  Adessi C, Sotto C. Converting a peptide into a drug: Strategies to improve stability and bioavailability. Curr Med Chem. 2002;9:963–78.  Adessi C, Sotto C. Strategies to improve stability and bioavailability of peptide drugs. Frontiers Med Chem. 2004;1:513–27.  Sayani AP, Chien YW. Systemic delivery of peptides and proteins across absorptive mucosae. Crit Rev Ther Drug Carrier Syst. 1996;13:85–184.