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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

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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.

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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.

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.1.Discuss amino acids, peptides, and proteins

in medicine.

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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.

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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.

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Basic Amino Acid Structure

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Basic Amino Acid Structure

D (+) amino acid L (-) amino acid

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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.

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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.

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Peptide Bonds

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Peptide Bonds

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Peptide Bonds

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Peptide Bonds

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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.

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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.

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Primary Structure of Proteins

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Secondary Structure of Proteins

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Tertiary Structure of Proteins

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Quaternary Structure of Proteins

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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.

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Receptor Concept

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Receptor Concept

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.2.Describe the appropriate radiolabeling

principles, including radionuclide selection (SPECT, PET, and therapy).

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Anatomy of a Bioconjugate

• Content

Zeglis, et al. Dalton Transactions 2011, 40: 6168-6195.

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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

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Zeglis, et al. Dalton Transactions 2011, 40: 6168-6195.

Peptide

Chelator

Linker

Radionuclide

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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.

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Biomolecules• Development in the production, purification, and

radiochemistry of PET tracers of the metals • 64Cu (copper),

• 68Ga (gallium),

• 86Y (yttrium),

• 89Zr (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

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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.

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Biomolecules

• Key (choice) is matching the radioactive half-life to the biological half-life of the biomolecule

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Therapeutic Radionuclides

177Lu 6.7 d Beta/gamma (208KeV 11%) 1 mm

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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.

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Pre Targeting

• 68Ga 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 64Cu, 86Y, and 89Zr especially with fully intact mAbs. Increases radiation dose!

• But pre targeting may be the best approach.

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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.

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Pre Targeting

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Pre Targeting

111In: Rossin, et al. Angew. Chem. Int. Ed. 2010; 49: 3375-8.64Cu: Zeglis, et al. J. Nucl. Med. 2013; 54: 1-8.18F: Devaraj, et al. PNAS. 2013; 109: 4762-4767.

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.3.Describe the radiolabeling methods.

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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

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PET

• Direct Labelling• Prosthetic groups• Al-F NOTA• Click chemistry

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Direct Labelling

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Direct Labelling

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Direct Labelling

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Direct Labelling

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Prosthetic Group Activation

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Prosthetic Group Activation

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Prosthetic Group Activation

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Issues with Prosthetic Groups

• Multiple steps • Long synthesis time • Require HPLC purification• Hard to automate

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Advantages of Metal Chelates

• Single step• Rapid• Reaction in aqueous solution• Little substrate required• Minimal purification• Kit formulation

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Click Chemistry

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PET Chelators

• NOTA

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PET Chelators

• DOTA

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PET Chelators

• TETA

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Click ChemistryA 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.

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Direct Labelling

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Click Chemistry

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Click Chemistry

• 4-[18F] fluorobenzoic acid (FBA)

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Click Chemistry - Selectivity

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Click Chemistry - Modularity

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.4.Apply knowledge to prototype model.

Somatostatin Receptor Imaging and Therapy

Theronostic Pairs

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Somatostatin Dota Chelator

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Affinities

1

2

3

4

5

SSR

Lanreotide Octreotide (NOC) Octreotate

Courtesy John Buscombe formerly Royal Free Hosp London

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Title

• Content

Courtesy John Buscombe formerly Royal Free Hosp London

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Ga-68

Courtesy John Buscombe formerly Royal Free Hosp London

Positive PET and negative 111In

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In-111 OctGa-68 PET

Similar Lesions

Courtesy John Buscombe formerly Royal Free Hosp London

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Ga-68 PET/CT more lesions than In-111 Oct

Ga-68 PET

In-111 Oct

Courtesy John Buscombe formerly Royal Free Hosp London

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Title

• Content

Baum et al 2012 Theronostics

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Title

• Content

Baum et al 2012 Theronostics

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90Y octreotate therapy

• Content

Baum et al 2012 Theronostics

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Title

• Content

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.5.Questions.