Pharmacy 361

1. Pharmacy 361 Protein Formulation & Delivery David Wishart david.wishart@ualberta.ca 3-41 Athabasca Hall

2. Today’s lecture notes are available at: http://www.wishartlab.com/

4. The Human Genome Project • First Draft completed on June 26, 2000 • “Finished” on May 18, 2006 • 3,260,000,000 bp on 24 chromosomes • 3,201,762,515 bases sequenced (92.3%) • 20,251 genes (so far) • All FDA approved drugs target just 1768 different proteins

5. Proteins • Polypeptides composed of covalently linked amino acids • Polypeptides with <40 amino acids are called peptides • Polypeptides with >40 amino acids are called proteins • Function of a protein determined by its non-covalent 3D structure

6. Amino Acids

7. Polypeptides

8. Lactate Dehydrogenase: Mixed a / b Immunoglobulin Fold: b Hemoglobin B Chain: a Protein Structure

9. Protein Pharmaceuticals • 150 FDA approved protein drugs (http://www.drugbank.ca) • >60% are recombinant (rDNA) proteins • Protein pharmaceutical market is currently approaching $130 billion/yr • By 2015 it is expected to reach $158 billion/yr

10. Finding More About Protein Drugs http://www.drugbank.ca or just google “drugbank”

11. DrugBank DrugCards

14. Searching DrugBank

15. Searching DrugBank

16. Searching By Text

17. DrugBank Statistics

18. Part Time Jobs Available • Up to $25/hour (depends on quals.) • Work from home, flexible hours • Learn more about drugs, drug actions, pharmacology • Annotation of DrugBank4 • Chance to co-author a research paper • Contact me by email or Dr. AnChi Guo (anchiguo@gmail.com)

19. Classes of Protein Pharmacueticals • Vaccines (peptides, parts of proteins, killed bacteria) • Peptides (oxytocin, pitocin) • Blood products (Factor X, Factor VIII, gamma globulin, serum albumin) • Recombinant therapeutic proteins (herceptin, humulin, alferon, etc.)

20. Vaccines • Diptheria(Corynebacterium diphtheriae) - diptheria toxin • Tetanus(Clostridium tetani) - tetanus toxin • Whooping cough(Bordetella pertussis) - acullelar extract Tetanus Toxin HC Fragment

21. Therapeutic Proteins • Insulin (diabetes) • Interferon b (relapsing MS) • Interferon g (granulomatous) • TPA (heart attack)

22. Actimmune (If g) Activase (TPA) BeneFix (F IX) Betaseron (If b) Humulin Novolin Pegademase (AD) Epogen Regranex (PDGF) Novoseven (F VIIa) Intron-A Neupogen Pulmozyme Infergen Therapeutic Proteins

23. The Problem with Proteins • Very large and unstable molecules • Structure is held together by weak noncovalent forces • Easily destroyed by relatively mild storage conditions • Easily destroyed/eliminated by the body • Hard to obtain in large quantities

24. The Problem with Proteins(in vivo - in the body) • Elimination by B and T cells • Proteolysis by endo/exo peptidases • Small proteins (<30 kD) filtered out by the kidneys very quickly • Unwanted allergic reactions may develop (even toxicity) • Loss due to insolubility/adsorption

26. Denaturation Aggregation Precipitation Adsorption Deamidation Oxidation Disulfide exchange Proteolysis The Problem with Proteins(in vitro - in the bottle) Noncovalent Covalent

27. Noncovalent Processes Denaturation Adsorption

28. Noncovalent Processes Aggregation Precipitation

29. Covalent Processes • Deamidation - conversion of Asn-Gly sequences to a-Asp-Gly or b-Asp-Gly • Oxidation - conversion RSR’ to RSOR’, RSO2R’ or RSO3R’ (Met & Cys) • Disulfide exchange - RS- + R’S-SR’’ goes to RS-SR’’ + R’S- (Cys) • Proteolysis - Asp-Pro, Trypsin (at Lys) or Chymotrypsin (at Phe/Tyr)

30. Deamidation

31. How to Deal with These Problems? Storage Formulation Delivery Pharmaceutics

32. Storage - Refrigeration • Low temperature reduces microbial growth and metabolism • Low temperature reduces thermal or spontaneous denaturation • Low temperature reduces adsorption • Freezing is best for long-term storage • Freeze/Thaw can denature proteins

33. Storage - Packaging • Smooth glass walls best to reduce adsorption or precipitation • Avoid polystyrene or containers with silanyl or plasticizer coatings • Dark, opaque walls reduce hn oxidation • Air-tight containers or argon atmosphere reduces air oxidation

34. Storage - Additives • Addition of stabilizing salts or ions (Zn2+ for insulin) • Addition of polyols (glycerol and/or polyethylene glycol) to solubilize • Addition of sugars or dextran to displace water or reduce microbe growth • Use of surfactants (CHAPS) to reduce adsorption and aggregation

35. Storage - Freeze Drying • Only cost-effective means to prepare solid, chemically active protein • Best for long term storage • Removes a considerable amount of water from protein lattice, so much so, that some proteins are actually deactivated

36. Freeze Drying • Freeze liquid sample in container • Place under strong vacuum • Solvent sublimates leaving only solid or nonvolatile compounds • Reduces moisture content to <0.1%

37. Sublimation vs. Melting

38. Protein Pharmaceutics Storage Formulation Delivery

39. The Problem with Proteins(in vivo) • Elimination by B and T cells • Proteolysis by endo/exo peptidases • Small proteins (<30 kD) filtered out by the kidneys very quickly • Unwanted allergic reactions may develop (even toxicity) • Loss due to insolubility/adsorption

40. Protein Formulation • Protein sequence modification (site directed mutagenisis) • PEGylation • Proteinylation • Microsphere/Nanosphere encapsulation • Formulating with permeabilizers

41. Site Directed Mutagenesis E343H

42. 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 • Strategic placement of cysteines to produce disulfides to increase Tm • Protein engineering (size, shape, etc.)

43. PEGylation O O O O +

44. PEGylation • PEG is a non-toxic, hydrophilic, FDA approved, uncharged polymer • Increases in vivo half life (4-400X) • Decreases immunogenicity • Increases protease resistance • Increases solubility & stability • Reduces depot loss at injection sites

45. Proteinylation + Protein Drug ScFv (antibody)

46. Proteinylation • Attachment of additional or secondary (nonimmunogenic) proteins for in vivo protection • Increases in vivo half life (10X) • Cross-linking with Serum Albumin • Cross-linking or connecting by protein engineering with antibody fragments

47. Microsphere Encapsulation 100 mm

48. Encapsulation • Process involves encapsulating protein or peptide drugs in small porous particles for protection from “insults” and for sustained release • Two types of microspheres • nonbiodegradable • biodegradable

49. Types of Microspheres • Nonbiodegradable • ceramic particles • polyethylene co-vinyl acetate • polymethacrylic acid/PEG • Biodegradable (preferred) • gelatin • polylactic-co-glycolic acid (PLGA)

50. PLGA - Structure