PET Course KFSH&RC PET - Pebbtides-based PET Radiopharmaceuticals: Potential Tools against Cancer Dr. subhani okarvi peptides-based pet radiopharmaceuticals

1. Peptides-based PET
Radiopharmaceuticals:
Potential Tools against Cancer
Dr. Subhani M Okarvi, PhD
Scientist, Cyclotron & Radiopharmaceuticals Department
Research Center

2. Introduction
• Radio-pharmaceutical: (Radioactive Drug)
Radio:
Pharmaceutical:
• RPs are drugs containing a radionuclide and are
designed for:
 diagnostic or therapeutic use
 to deliver radionuclide to the disease sites (tumors)
RPs: mostly small organic molecules: i.e., peptides
• Peptides are compounds that contain AAs linked by peptide
bonds.

3. Peptide-based Radiopharmaceuticals:
“The Wave of the Future”
• new and useful class of radiopharmaceuticals
• small in size
• can be synthesized chemically
• easy to radiolabel
• rapid blood clearance
• rapid pharmacokinetics
• rapid uptake by target tissues
• rapid excretions
• not toxic
• high receptor affinity
• chemical/molecular modifications
• ability to attach BFCA at the C- or N-terminus
• high tumor penetration
• specific in vivo distribution
• many clinically relevant molecular and cellular targets

4. Peptide-based Radiopharmaceuticals
Many human tumors overexpress various types of
peptide receptors
Radiolabeled peptides designed for binding these
receptors can be used for the detection and treatment
of cancer
Also, radiolabeled peptides can be used as carriers to
deliver therapeutic radionuclide to tumor sites for
peptide receptor radiotherapy

5. Small Peptides
• Due to their small size, peptide molecules exhibit
favorable pharmacokinetics:
- rapid uptake by target tissue
- rapid blood clearance
- images to be acquired earlier after injection
• The rapid pharmacokinetics are ideal for labeling
peptides with a radioisotope that has a short T1/2 (i.e.,
68Ga, T1/2 = 68 min)
• The challenge is to label bioactive peptides with 68Ga,
without altering biological properties of the peptides

6. Peptides as
diagnostic imaging agents
• Tumor imaging (SST, VIP, NT, α-MSH
CCK/gastrin, Substance-P, α-M2, Bombesin)
• Infection/inflammation imaging
• (i.e., chemotactic)
• Thrombus imaging (i.e., RGD)
• Tumor-antigens derived peptides (HER2 & MUC1)
New class of peptides:
- Cardioactive peptides

7. BN-like peptides
• Bombesin (BN), a small and linear 14 amino acid
peptide initially isolated from the frog skin
- pGlu-Gln-Arg-Leu-Gly-Asn-Gln-Trp-Ala-Val-Gly-His-Leu-Met-
NH2
- Many human tumors (breast, prostate, gastric, renal,
etc. ) overexpress receptors for BN peptide
- Low expression in normal tissues

8. Why Bombesin-like peptides?
• The C-terminal 7-14 AA (i.e., Gln-Trp-Ala-Val-Gly-
His-Leu-Met-NH2) is necessary for retaining
receptor binding affinity & preserving the
biological activity of BN-like peptides
• A Ga-68 chelating agent can be introduced
at the N-terminal (which is not involved in
receptor binding) of the BN-like peptides
• A number of available human tumor cell lines
that overexpressed receptors specific for BN

9. Tumor antigens derived peptides
• Peptide-based radiopharmaceuticals derived from the
tumor antigens, such as HER2/neu and epithelial
mucin (MUC1), can be useful for tumor imaging
because of their overexpression on several human
cancers, including breast and ovarian cancer.
• The low-expression of these antigens on normal
tissues makes them promising targets for the diagnosis
and therapy of cancer.

10. HER-2/neu derived-peptides
(for diagnosis/treatment of breast and other cancer)
The receptors for HER-2/neu
are overexpressed in a variety
of cancers (breast & ovarian)
Peptides derived from the breast cancer- associated
antigens can be useful for imaging and therapy of
breast cancer
The low expression of this tumor antigen on normal
tissues makes it attractive molecular target for tumor
imaging and therapy.

11. MUC-1 (a tumor-associated antigen)
The human epithelial mucin MUC-1 is an example of a
tumor-associated antigen
MUC-1 is elevated in many breast cancer patients
and overexpressed on almost all human epithelial
cancers, including:
>90% of breast, ovarian, pancreatic, colorectal, lung,
prostate and gastric cancers
The low expression of this tumor antigen on normal
tissues makes MUC1 an attractive target for tumor
imaging and therapy.

13. Peptide as a targeting molecule

14. Cytotoxic Peptide Conjugates
• Conjugation of cytotoxic drugs (i.e. MTX, DOX) to
receptor binding peptides may be useful for targeted
delivery of cytotoxic peptide conjugate to tumor cells
for radiotherapy
• In this study, a BN peptide is conjugated to a well-
known and widely-used anticancer agent, methotrexate
(MTX), which is chemically suitable for conjugation to
peptides.

15. Theranostics ⇒ (Diagnosis +
Therapy)
• Theranostic Radiopharmaceuticals in molecular imaging:
• molecular receptor targeting agents (e.g., peptides)
• first radiolabeled with diagnostic radionuclide, and then
• same peptide radiolabeled with therapeutic radionuclide
• Thus same peptide can be used for both diagnosis and
therapy of a particular disease (cancer)
• diagnosis, for example; using 99mTc/68Ga-labeled BN
analogs, is followed by
• personalized therapy using 177Lu/90Y-labeled BN analogs

16. • Synthesis of peptides by SPPS
• Radiolabeling of peptides with 68Ga
(Sn-tart exchange method)
• Radio-HPLC analysis/purification
• In vitro studies (chemical stability, plasma stability, cell-
binding, etc.)
• In vivo biodistribution in normal mice
• In vivo tumor targeting in tumor-bearing mice
Experimental
Methods

17. Methods of peptide Synthesis
Solution-phase peptide
synthesis
- complex and time-consuming,
suitable for large scale synthesis
- Low-yield synthesis
Solid-phase peptide synthesis
(SPPS)
- Fast, efficient, suitable for small-
scale synthesis, high-yield
synthesis

18. Radiolabeling Methods

19. Quality Control of Radiolabeled Peptides
• Radio-TLC

20. Radio-HPLC analysis

21. Choice of a Radionuclide
• The choice of the radiometal is dependent on:
• the medical treatment objective (for diagnosis, or
therapy),
• the availability
• the required radiation dose
• the physical half-life
• the cost
• other relevant factors

22. Ga-68 Properties
• Physical properties
• Halflife T1/2 = 68 min
• Positron branching 89% (PET nuclide)
• Available via a 68Ge/68Ga generator
• Mother 68Ge cyclotron produced (T1/2 = 271 d)
• Chemical properties of Ga-68
• Trivalent metal
• Chelation chemistry
• Applicability
• Short half-life useful for molecules with fast biokinetics
(Peptides, Ab-fragments, small complexes,…)

23. Ga-68
• It is true that the 511 keV photons from Ga-68 are
10-times more penetrating than 99mTc and may
require automation and remote handling to produce
the number of patient doses per day needed.
• The 68-min half-life of Ga-68 may limit the number of
doses prepared at one time.
• However, the rapid in-growth of Ga-68 on the
generator will allow for multiple elutions, approx.
every 2 h.

24. Ga-68

25. Ga-68 chemistry

26. Labeling of Peptides with Ga-68
• 25–50 µg of DOTA-coupled peptide
• 200–300 µL of 2.5M Sodium acetate
• 68GaCl3 (~2 mCi, ~500 µL in 0.6 M HCl)
• heating at 90°C for 20-30 min

27. Complexation with Ga-68
Donor set: N4O2

28. 111In-Octreotide (OctreoScan)
4 h post-injection of
111In-octreotide
images in the anterior
and posterior views
revealed abnormal
radiotracer
localization in the left
adrenal gland (arrows),
the known site of the
neuroendocrine tumor
(paraganglioma).

29. PET
Imaging
Modality
Radiation
spectrum
Resolution Main
advantages
Limitations
Positron
Emission
Tomography
(PET)
High-
energy
gamma
rays
1‒2 mm - high
sensitivity
-isotopes of
biological
important
atoms for
substitution
- quantitative
- translational
to clinic
-Cyclotron
needed for the
production of
short-lived
isotopes
- Radiation dose

30. 68Ga application – Production of 68Ga-DOTATOC
-Production of chelator (DOTA) and peptide (TOC)
-Elution of 68Ga
-Chelation reaction of 68Ga with DOTATOC
-Administration to the patient (receptor binding to
cell receptor) and PET scan
Ga +

31. 68Ga-SST

32. 111In vs. 68Ga-SST

33. 111In-Octreoscan vs. 68Ga-DOTATOC

34. Labeling Peptides with Lu-177
Macrocyclic chelator, e.g. DOTA
Metal-free conditions for radiolabeling:
Metal-free water, pure reagents (acid, buffers)
Metal impurities decrease labeling efficiency dramatically

35. Copper-64
• Copper-64 [t1/2=12.7 h; positron energy, 0.656 MeV, 17.9%)
• Eβ−max=0.57 MeV (39%); 43.1% electron capture]
• a promising isotope for site-directed PET imaging and radiotherapy
• The half-life of 64Cu is long enough for:
• drug preparation,
• quality control,
• drug incorporation, circulation and patient imaging/therapy
• But, widespread usage of 64Cu radiometal as a diagnostic tool is
limited because of
• transmetallation reactions in vivo into serum proteins, namely,
superoxide dismutase, found in blood and liver tissue.

36. Cu-64
•Production of no-carrier-added Cu-64 by the
reaction of 64Ni(p,n)64Cu on a biomedical cyclotron
• its availability now increased
•It shows some in vivo demetallation of radiometal
from complexing ligand, resulting in
•accumulation and retention of tracer in non-target
tissues

37. Cu-64
• An effective a radiometal labeled molecular imaging
should have:
• High degree of kinetic inertness onto the complex,
• preventing the dissociation of radiometal from
bioconjugate under in vivo conditions
• should rapidly clear from non-target tissues
• produce high-quality, high-contrast diagnostic PET
images

38. Cu-64
• For example, DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-
tetraacetic acid) or TETA (1,4,8,11-tetraazacyclotetradecane-1,4,8,11-tetraacetic
acid), when used to complex Cu-64,
• result in high in vivo accumulation and retention of
radiometal in liver tissue, presumably caused by
dissociation of Cu-64 radionuclide from ligand and
subsequent complexation of metal to superoxide
dismutase in hepatic tissue

39. 64Cu-labeled Bombesin peptide analog

40. Cu-64

41. Clinical studies with 64Cu-DOTATATE

42. Zr-89
• Interest in 89Zr as it exhibits a long half-life, making it
• ideally suited for imaging studies with slowly-accumulating bioactive
molecules,
• allowing for imaging of biological processes at late time-points after
tracer injection
• produces positrons with about 22.3% and mean energy 0.389 MeV
• which is between the positron energies of 18F (mean β+ energy of
0.250 MeV) and 68Ga (mean β+ energy of 0.836 MeV)
• thus allows high resolution PET images.
• 89Zr is produced via cyclotron using 89Y (which has a natural
abundance of 100%) as target material in the 89Y(p,n)89Zr
• nuclear reaction and can be obtained in high isolated yields of 99.5%
and high radionuclidic purity of 99.99%

43. Radiolabeling of 89Zr via DFO

44. Cu-64 vs. Zr-89
• a longer half-life of
12.7 h together with
• main positron energy
of 0.653 MeV and
• low spatial resolution
loss of 0.7 mm,
allowing for an
• extended imaging of
biological processes.
• 89Zr is even more interesting for
the radiolabeling of slowly-
accumulating
radiopharmaceuticals as it
exhibits
• a longer half-life of 3.27 days,
being particularly
• suited for in vivo imaging of
antibodies, nanoparticles and
other large biomolecules
• However, this results in higher
absorbed organ doses than
• in case of 18F-labeled radiotracers

45. Labeling peptides via 18F-SFB

47. [18F]Galacto-RGD PET images in a patient with invasive ductal breast cancer of left
breast,
Maximum-intensity projection of [18F]Galacto-RGD PET and planar images show
primary tumor, lymph-node (LN) metastases, and osseous metastasis with good
tumor/background contrast.

48. Nuclear Medical Imaging

49. Biodistribution in mice
In vivo biodistribution of the
radiopeptides were performed on
healthy mice at different time points
• For tumor implantation, ~ 5 x 106 breast
cancer cells were injected s.c. into nude mice.

50. Implantation of human tumors in nude mice

51. Intravenous tail vein and Intratumor
injection

52. PET/CT nanoscan for
small animal imaging
- High resolution
- High sensitivity
- allow both CT (anatomical) &
- PET (functional) images
- laborious

53. In vivo Tumor targeting of 68Ga-BN in nude mice with MDA-MB-231

54. PreClinical evaluation in animals

55. UNIQUE CHARACTERISTICS OF
SMALL-ANIMAL PET
• Small-animal PET and human-PET both use similar
image formation techniques and share some
common image quality issues.
• Rats and mice are not as cooperative as humans.
• Rodents do not remain still through an imaging
session that usually lasts several minutes.
• Anesthesia must be used for most imaging
procedures.

56. Molecular Imaging in Cancer
Subhani M. Okarvi, PhD, BCNP, DABSNM
King Faisal Specialist Hospital & Research Centre,
Riyadh, KSA

57. MI procedures
• MI system typically consists of:
• an imaging agent, or probe
• an imaging device and
• a molecular target
• A variety of imaging agents are used to visualize
cellular activity, such as the chemical processes
involved in metabolism, oxygen use or blood flow.
- In nuclear medicine, which is a branch of MI, the
imaging agent is a radiotracer, a compound that
contain a radioactive atom or an isotope.
- Interaction of the target with a “labeled agent” can be
detected externally by one or more imaging
modalities

59. Modalities of Molecular Imaging

61. A General Approach for Tumor Targeting

63. Cellular internalization of
radiolabeled peptides

64. In vivo peptide receptor targeting of cancer
After the radiopeptide bound to receptor on
cancer cells, the receptor-peptide complex is
internalized

65. Imaging and Therapy

66. Peptide receptor tumor targeting

67. Radiolabeled peptide binds to receptor on cancer cell surface and enters
into the cell, followed by emission of radiation that destroys

68. Chemical structures of FA & MTX
N
N
N
N
NH2
H2N
N
CH3
N
O COOH
COOH
H
N
N
N
NH2N
N
O N
O COOH
COOH
H
H
Folic Aacid
MTX

69. DOTA-TOC - Cancer targeting agent

70. DOTA-MTX conjugate