1. FACULTY OF SCIENCE Prof Geoff Currie, BPharm, MMedRadSc, MAppMngt, MBA, PhD Faculty of Science, Charles Sturt University Faculty of Medicine and Health Sciences, Macquarie University Rural Clinical School, University of NSW Peptides Imaging and Therapy May 28 – 30, 2015, Montréal, Québec

2. FACULTY OF SCIENCE Educational Objectives Upon completion of this activity, the participant will be able to: 1. Discuss amino acids, peptides, and proteins in medicine. 2. Describe the appropriate radiolabeling principles, including radionuclide selection (SPECT, PET, and therapy). 3. Describe the radiolabeling methods. 4. Apply knowledge to prototype model.

3. FACULTY OF SCIENCE Disclosure Statement: No Conflict of Interest May 28 – 30, 2015, Montréal, Québec I do not have an affiliation, financial or otherwise, with a pharmaceutical company, medical device or communications organization. I have no conflicts of interest to disclose ( i.e. no industry funding received or other commercial relationships). I have no financial relationship or advisory role with pharmaceutical or device-making companies, or CME provider. I will not discuss or describe in my presentation at the meeting the investigational or unlabeled ("off-label") use of a medical device, product, or pharmaceutical that is classified by Health Canada as investigational for the intended use.

4. FACULTY OF SCIENCE.1. Discuss amino acids, peptides, and proteins in medicine.

5. FACULTY OF SCIENCE Amino Acids About 300 amino acids present in various animals, plants, and microbial systems Only 20 amino acids are coded by DNA to appear in proteins. Cells produce proteins with different properties and activities by joining the 20 amino acids in many different combinations and sequences. The properties of proteins are determined by the physical and chemical properties of their monomer units, the amino acids.

6. FACULTY OF SCIENCE Basic Structure of Amino Acids Amino acids are the basic structural units of proteins consisting of an amino group, (-NH2) A carboxyl group (-COOH) a hydrogen atom (-H) and a (variable) distinctive (R) group. All of the substituents in amino acid are attached (bonded) to a central α carbon atom. This carbon atom is called α because it is bonded to the carboxyl (acidic) group.

7. FACULTY OF SCIENCE Basic Amino Acid Structure

8. FACULTY OF SCIENCE Basic Amino Acid Structure D (+) amino acid L (-) amino acid

9. FACULTY OF SCIENCE Peptide Bonds Peptide bonds are formed via a condensation reaction forming peptides and proteins out of chains of amino acids; polymerisation. Peptides tend to be small comprising just a few amino acids (eg. some hormones and neurotransmitters). Proteins are polypeptides and vary in the number of peptides and their configuration.

10. FACULTY OF SCIENCE Peptide Bonds Peptide bonds are covalent bonds. An amide linkage between carboxyl group of one amino acid and the amino group of another. Peptide bonds are resistant to conditions that normal denature proteins. Peptide bonds can be broken in high acidic or basic conditions at elevated temperatures.

11. FACULTY OF SCIENCE Peptide Bonds

12. FACULTY OF SCIENCE Peptide Bonds

13. FACULTY OF SCIENCE Peptide Bonds

14. FACULTY OF SCIENCE Peptide Bonds

15. FACULTY OF SCIENCE Proteins Proteins can be large molecules ranging from less than 50 amino acids to more than 10000 with complex shape and structure. While peptide bonds tend to suggest a linear structure, proteins are folded into a variety of 3 dimensional shapes that determine functionality. Protein structure can be thought of in terms of primary, secondary, tertiary and quaternary structures.

16. FACULTY OF SCIENCE Proteins Primary structure relates to the protein configuration arising from the amino acid sequence in the polypeptide chain. Secondary structure relates to the manner in which the polypeptide chain is folded (hydrogen bonds). Tertiary structure relates to the interactions associated with amino acid side chains. Quaternary structure relates to interactions between different polypeptide chains within the same protein.

17. FACULTY OF SCIENCE Primary Structure of Proteins

18. FACULTY OF SCIENCE Secondary Structure of Proteins

19. FACULTY OF SCIENCE Tertiary Structure of Proteins

20. FACULTY OF SCIENCE Quaternary Structure of Proteins

21. FACULTY OF SCIENCE Cell Surface Receptors Cell surface receptors are specialised proteins that take part in communication between the cell and the outside world. Extracellular signalling molecules (usually hormones, neurotransmitters, cytokines, growth factors or cell recognition molecules) attach to the receptor, triggering changes in the function of the cell. Cell surface receptors can be over-expressed in certain diseases and thus become a target for diagnosis and therapy.

22. FACULTY OF SCIENCE Receptor Concept

23. FACULTY OF SCIENCE Receptor Concept

24. FACULTY OF SCIENCE.2. Describe the appropriate radiolabeling principles, including radionuclide selection (SPECT, PET, and therapy).

25. FACULTY OF SCIENCE Anatomy of a Bioconjugate Content Zeglis, et al. Dalton Transactions 2011, 40: 6168-6195.

26. FACULTY OF SCIENCE Anatomy of a Bioconjugate Biomolecule Type (peptide) Target (receptor) Nuclide (ligand) PET SPECT Therapy Radiometal Radiohalogen Zeglis, et al. Dalton Transactions 2011, 40: 6168-6195. Radiohalogen Direct Labeling Prosthetic Radiometal Chelator Conjugation

27. FACULTY OF SCIENCE Zeglis, et al. Dalton Transactions 2011, 40: 6168-6195. Peptide Chelator Linker Radionuclide

28. FACULTY OF SCIENCE Limitations of Non Metals in PET Short half-lives only allow evaluation of short biological processes (minutes to a few hours) using rapid pharmacokinetic profiles. Short half-lives and necessity of incorporating the radioisotopes into the core structure of the tracer (rather than in an appended chelator or prosthetic group) necessitate complex syntheses. Often requires a on-site cyclotron facility.

29. FACULTY OF SCIENCE Biomolecules Development in the production, purification, and radiochemistry of PET tracers of the metals 64 Cu (copper), 68 Ga (gallium), 86 Y (yttrium), 89 Zr (zirconium), Half-lives consistent with the biological half-lives. All 4 form stable chelate complexes suitable for the radiolabeling of biomacromolecules. Most important application of PET radiometals is the development of tracers based on peptides

30. FACULTY OF SCIENCE Biomolecule Labelling Principles Radiometals. Radiometal is almost never directly attached to the biomolecule itself. Radionuclide is bound to a chelating moiety (e.g. DOTA). Chelating moiety is covalently attached to the biomolecule (linker). The intent is to alter the biochemical properties as little as possible.

31. FACULTY OF SCIENCE Biomolecules Key (choice) is matching the radioactive half-life to the biological half-life of the biomolecule

32. FACULTY OF SCIENCE Therapeutic Radionuclides 177Lu 6.7 d Beta/gamma (208KeV 11%) 1 mm

33. FACULTY OF SCIENCE Pre Targeting Challenge to balance maximizing absolute amount of radionuclide that can be delivered to the tumor and meeting the requirement that the tumor-to- normal organ dose ratios be as high as possible. The problem is that large molecules such as antibodies provide the highest tumor accumulation, while smaller molecules such as peptides provide the highest tumor-to-normal organ dose ratios.

34. FACULTY OF SCIENCE Pre Targeting 68 Ga is an inappropriate choice for labeling fully intact IgG molecules, because it will decay through a number of half-lives before the antibody reaches its fully optimal biodistribution within the body. One solution is to use longer lived radiometals 64 Cu, 86 Y, and 89 Zr especially with fully intact mAbs. Increases radiation dose! But pre targeting may be the best approach.

35. FACULTY OF SCIENCE Pre Targeting Break the treatment strategy into two steps. The first involving an unlabelled macromolecule that can biologically take as long as necessary to localise in the target in high concentration. Followed later by administration of a radiolabeled small molecule that binds specifically to the protein or peptide.

36. FACULTY OF SCIENCE Pre Targeting

37. FACULTY OF SCIENCE Pre Targeting 111 In: Rossin, et al. Angew. Chem. Int. Ed. 2010; 49: 3375-8. 64 Cu: Zeglis, et al. J. Nucl. Med. 2013; 54: 1-8. 18 F: Devaraj, et al. PNAS. 2013; 109: 4762-4767.

38. FACULTY OF SCIENCE.3. Describe the radiolabeling methods.

39. FACULTY OF SCIENCE Radiolabelling Methods Two processes need to be considered. The first is the labelling of the ligand (radionuclide) to the chelate. The second is attaching that ligand-chelate complex to the biomolecule. This can be direct labelling, use of prosthetic groups, or click chemistry in particular

40. FACULTY OF SCIENCE PET Direct Labelling Prosthetic groups Al-F NOTA Click chemistry

41. FACULTY OF SCIENCE Direct Labelling

42. FACULTY OF SCIENCE Direct Labelling

43. FACULTY OF SCIENCE Direct Labelling

44. FACULTY OF SCIENCE Direct Labelling

45. FACULTY OF SCIENCE Prosthetic Group Activation

46. FACULTY OF SCIENCE Prosthetic Group Activation

47. FACULTY OF SCIENCE Prosthetic Group Activation

48. FACULTY OF SCIENCE Issues with Prosthetic Groups Multiple steps Long synthesis time Require HPLC purification Hard to automate

49. FACULTY OF SCIENCE Advantages of Metal Chelates Single step Rapid Reaction in aqueous solution Little substrate required Minimal purification Kit formulation

50. FACULTY OF SCIENCE Click Chemistry

51. FACULTY OF SCIENCE PET Chelators NOTA

52. FACULTY OF SCIENCE PET Chelators DOTA

53. FACULTY OF SCIENCE PET Chelators TETA

54. FACULTY OF SCIENCE Click Chemistry A desirable click chemistry reaction would: be modular be wide in scope give very high chemical yields generate only benign by-products be stereospecific be physiologically stable have simple reaction conditions use readily available materials and reagents use no or a benign solvent Provide simple product isolation.

55. FACULTY OF SCIENCE Direct Labelling

56. FACULTY OF SCIENCE Click Chemistry

57. FACULTY OF SCIENCE Click Chemistry 4-[18F] fluorobenzoic acid (FBA)

58. FACULTY OF SCIENCE Click Chemistry - Selectivity

59. FACULTY OF SCIENCE Click Chemistry - Modularity

60. FACULTY OF SCIENCE.4. Apply knowledge to prototype model. Somatostatin Receptor Imaging and Therapy Theronostic Pairs

61. FACULTY OF SCIENCE Somatostatin Dota Chelator

62. FACULTY OF SCIENCE Affinities 1234512345 SSR Lanreotide Octreotide (NOC) Octreotate Courtesy John Buscombe formerly Royal Free Hosp London

63. FACULTY OF SCIENCE Title Content Courtesy John Buscombe formerly Royal Free Hosp London

64. FACULTY OF SCIENCE Ga-68 Courtesy John Buscombe formerly Royal Free Hosp London Positive PET and negative 111In

65. FACULTY OF SCIENCE In-111 Oct Ga-68 PET Similar Lesions Courtesy John Buscombe formerly Royal Free Hosp London

66. FACULTY OF SCIENCE Ga-68 PET/CT more lesions than In-111 Oct Ga-68 PET In-111 Oct Courtesy John Buscombe formerly Royal Free Hosp London

67. FACULTY OF SCIENCE Title Content Baum et al 2012 Theronostics

68. FACULTY OF SCIENCE Title Content Baum et al 2012 Theronostics

69. FACULTY OF SCIENCE 90Y octreotate therapy Content Baum et al 2012 Theronostics

70. FACULTY OF SCIENCE Title Content

71. FACULTY OF SCIENCE.5. Questions.