Regional Training Course on Therapeutic Radiopharmaceutical Production

1. 19-23 October 2009 The Manila Pavilion Hotel, Philippines C7-RAS-2.013-001- Regional Training Course on Therapeutic Radiopharmaceutical Production, QA/QC and implementation GMP Radiopharmaceuticals for Therapy and Concerns in Radiopharmaceutical Development Renata Mikolajczak, PhD Institute of Atomic Energy, Radioisotope Centre POLATOM, Poland

2. Radiopharmaceuticals Radiofarmaceutical is a substance formed in a chemical combination of two important components: • radioisotope, radioactive isotope of certain element – radiation emitted by this isotope is then registered and allows imaging of its distribution in the patients body or it can destroy the target tissue. • ligand, chemical compound, molecule or cell which is selectively taken up by the imaged organ or tissue.

3. Ligands Ligand is a carrier, which „delivers” radionuclide to the target tissue. Ligands are selected based on the knowledge on thier bahavior in this tissue – if their are taken up, metabolised or if they contribute to other physiologic processes Examples: • bones – phosphonates are build in the bone: 153Sm-EDTMP, 99mTc-MDP, 188Re-HEDP calcium analogue – 89SrCl2 • liver – colloids are taken up by rethiculo-endothelial liver cells – 99mTc-tin collloid • brain – lipophilic complexes can cross brain-blood barrier - 99mTc-HmPAO • infection – leukocytes or immunoglobulines – cells and molecules which naturally occur at the site of infection – 99mTc-HmPAO-leukocytes, 99mTc-IgG Frequently, the radionuclide in an ionic form has high affinity to certain tissue 131I, 123I for thyroid, 201Tl for the heart.

4. α β γ Design of radiopharmaceutical Selection of the ligand proper for the target Selection of the radioisotope with optimal radiation characterisitcs Courtesy A.Kolodziejczyk

5. Radionuclide therapy Over 50 years of experience with 131I in differentiated thyroid cancerDisseminated malignant disease requires a systemic approach and biological targeting

6. Radionuclides Progress due to increasing capacity for production in nuclear reactors and cyclotrons: • High specific activity or NCA radionuclides • Developments in generator techniques: 188W/188Re 90Sr/90Y 225Ac/213Bi 227Ac/223Ra Cost still remains a limiting factor and the availability is a must!

7. Isotope T1/2 (d) Mean range (mm) 199Au 3.1 410 169Er 9.3 510 177Lu 6.7 670 67Cu 2.6 710 105Rh 1.5 760 161Tb 6.9 770 47Sc 3.4 810 131I 8.0 910 -Emitters, short range (< 1000 mm)

8. Isotope T1/2 (d) Mean range (mm) 77As 1.6 1200 153Sm 1.9 1200 159Gd 0.8 1600 143Pr 13.6 1600 198Au 2.7 1600 111Ag 7.5 1800 109Pd 0.5 1800 186Re 3.8 1800 149Pm 2.2 1800 -Emiters, medium range (1000-2000 mm)

9. Isotope T1/2 (d) Mean range (mm) 165Dy 0.1 2200 89Sr 50 2500 166Ho 1.1 3200 32P 14 2900 188Re 0.7 3500 114mIn 50 3600 142Pr 0.8 3700 90Y 2.7 3900 76As 1.1 5000 -Emiters, large range (> 2000 mm)

10. Range of particulate radiations in tissue 10 a : cell nucleus b : single cell c : few cells d : 100’s of cells d 1 c Range (mm) 0.1 b 0.01 a Auger e- (few MeV)Low E -High E -(MeV)

11. Potential Advantage of Targeted Radiotherapy Due to the crossfire effect more complex (heterogeneous) tumors may benefit from targeted radionuclide therapy

12. Radionuclides, Selection criteria • -, -, Auger emitters • Half-life (1h-10d) • Energy • Low -emission • High specific activity • Labelling chemistry • Availability • Price

13. RADIOSYNOVECTOMY Injection of the radiocolloid Colloidal particles are rapidly phagcytosed by macrophages in the inflamed synovial memberane Cell damage and sclerosis of synovial mebrane Size of particles and radiation penetration range are a basis for selection of proper radiopharmaceutical

14. Characteristic of commercially available radiocolloids Combination of beta emitters and colloids/particles Options: Hydroxyapatites other particles with defined size other radionuclides: 166Ho, 188Re ?

15. Bone pain palliation Other options: 188Re-HEDP 117mSn-DTPA 177Lu-EDTMP (IAEA coordinated project) 223Ra (Alpharadin®, multicenter clinical study in Europe)

16. Therapeutic Radiopharmaceuticals Courtesy M. Venkatesh

17. Radiopharmaceuticals classification Radiopharmaceuticals, which biodistribution depends on their physico-chemical properties Radiofarmaceuticals, which biodistribution depends on their biological interaction, for example receptor binding S.Liu and D.S.Edwards, Chem.Rev.1999(99)2235-2268

18. CD20, CD22 3-integrins neovasculature important target for radiopeptides NIS transcription translation replication ↻ GPCRs m-RNA DNA protein extracellular matrix pO2↓ LAT GLUT-1 18FDG Norep-T [131I]MIBG soluble protein pH↓ EGFRs MMP Potential Targets for Molecular Imaging and Targeted Therapy in Cancer Courtesy H.Maecke, Basel

19. The goal of targeted radionuclide therapy Hence, requirements for the radiopharmaceutical: High labelling yield High radiochemical purity High specific activity High stability Deliver curative doses of radioactivity to the tumor cell Spare normal tissue

20. The ligands / vectors • G-protein coupled receptor ligands • Antibodies, fragments etc - CD20, 22, 33, 45 etc, - Her2new, PSMA, CEA, mucines etc. • Amino acids

21. Criteria of successful radionuclide therapy • Fast localisation  small molecules • Specific uptake  exquisite targets • High uptake  high target expression • Prolonged retention  internalization / KD • Rapid clearance from non target organs • Radiosensitivity / radiobiology

22. Schematic Representation of a Drug for Imaging and Targeted Therapy Chelator pharmacokinetic modifier Ligand Linker • Molecular Address • Antibodies, their fragments and modifications • Regulatory peptides and analogs thereof • Amino Acids • Target • Antigens (CD20, HER2) • GPCRs • Transporters • Reporting Unit • 99mTc, 111In, 67Ga • 64Cu, 68Ga • Gd3+ • Cytotoxic Unit • 90Y, 177Lu, 213Bi • 105Rh, 67Cu, 186,188Re Courtesy H. Maecke, Basel

23. Structure of DOTA-Tyr3-Octreotide our gold standard for the targeting of neuroendocrine tumors, a radiopeptide with high affinity to the somatostatin receptor 2 P. Powell, 1994 • DOTA-TOC cage 111In 90Y 67Ga 177Lu 68Ga213Bi Courtesy H. Maecke, Basel

24. 177Lu-DOTA,Tyr3octreotate Before first Tx 6 Mo after 4th Tx • M. de Jong, ESRR, Sopot, 2004

25. Advantages of using 177Lu in nuclear medicine • Physical properties - maximum energy of beta particles 500 keV (low bone marrow burden) - 10% of gamma radiation of modest energy, 208 keV - half-life of 6.65 days, no loss on decay • Flexibilityin specific activity to be obtained • Chemical properties– high aboundance of reactions with many compounds, useful for PRRT

26. The 177Lu isotope can be produced in a nuclear reactor in two ways: from 176Lu by (n,) reaction and from 176Yb by (n, ) and consecutive  decay By irradiation of enriched 176Lu in a high flux reactors the specific activities in the range of 20 -50 Ci/mg Lu can be reached. However, the nuclide of such properties is not easlily available due to the limited number of suitable reactors and logistic problems. For reactor production sites with lower flux, the irradiation of Yb followed by chemical separation of 177Lu could be a choice. Theoretical specific activity of 177Lu is 110 Ci/mg Lu

27. Limitations of radionuclide production mode Reactor produced radioisotopes by direct neutron irradiation: High concentrations of „cold” metal „Generator-like techniques” for nca radioisotopes: Separation methods are laborous and chemicals involved in the process are additional source of contamination

28. Lu isotopes with half-lifes > 1h

29. Irradiation conditions 177Lu was produced in the Maria reactor in Swierk, Poland, at the thermal and epithermal neutron flux of ~2,5 x 1014 and 1,2 x 1013 s-1 cm-2 respectively Target materials:176Lu enriched to 68,9%, Target material was dissolved in HCl

30. 177Lu produced by direct route Results of chemical impurities determination in selected batches of 177Lu Specific activities 17.8 – 26 Ci/mg Lu (EOB)

31. Results of 177mLu determination in selected batches of 177Lu Other radionuclidic impurities were below determination limits

32. Radiochemical aspects of preparation of therapeutic dose of 177Lu-DOTA-TATE • pH • Labelling kinetics (heating,contentrations) • Stability (free-radical scavengers) • Specific activity • Competing metals

33. DOTA-conjugated radioligands labeled with 90Y/177Lu for in vitro and in vivo use „Optimising conditions for radiolabelling of DOTA-peptides with 90Y, 111In and 177Lu at high specific activities” Breeman WAP et al. Eur J Nucl Med. Mol Imaging (2003) 30:917-920 Aim: Investigation of parameters influencing the reaction kinetics(pH, T, time, QC) of these radioligands

34. pH 0 pH 14 pH of the reaction? The rates of formation of the complexes of radiometal-DOTA were found to be inversely proportional to the [H+] Solubility of In3+, Ga3+, Y3+, and lanthanides  as pH • Szilagyi E et al.Inorganica Chimica Acta 2000Moerlein SM, Welch MJ. Int J Nucl Med Biol 1982

35. 100 Complex [%] formation of cation-[DOTA-tate] 50 0 2 4 6 pH Formation of [177Lu-DOTA0,Tyr3]octreotate complex, 80C, 10 • Breeman WAP et al. Eur J Nucl Med. Mol Imaging 2003

36. 100 C 80 C 100 C 111In Formation of [DOTA0,Tyr3]octreotate complex labeled with 90Y/177Lu or 111In 100 Complex [%] formation of cation-[DOTA-tate] 50 0 0 30 60 time (min) • Breeman WAP et al. Eur J Nucl Med. Mol Imaging 2003

37. Results of kinetic studies • Reactions with 177Lu and 90Y arecompleted after 20' at 80ºC, and with 111In after 30' at 100ºC • Conditions are optimal at pH 4 - 4.5 • Breeman WAP et al. Eur J Nucl Med. Mol Imaging 2003

38. Non-chelated metal Inert lanthanide complexes are essential for radiotherapy because in vivo dissociation to lanthanide from chelate results in bone and liver uptake • The lanthanides have a high affinity to bone because in vivo they mimic calcium • They are also taken up by the liver due to radiocolloid formation RCP>99.0% „Reduction of skeletal accumulation of radioactivity by co-injection of DTPA in [90Y-DOTA0,Tyr3]octreotide solutions containing free 90Y3+” WAP Breeman et al. Nucl Med Biol. 31 (2004) Additional advantage in QC by hPLC

39. IAEA CRP Nr 12131; „Comparative evaluation of therapeutic radiopharmaceuticals labelled with beta emitting isotopes” 2002-2005

40. Background The assessment of the relative effectiveness of different radiopharmaceuticals for cancer therapy is a difficult task owing to the large number of variables that must be considered: - related to the biological carrier - related to the radioisotope. Hence, the development of laboratory methods which can be used for reliable and efficient comparative evaluation of promising therapeutic radiopharmaceuticals is an important need.

41. Objectives of the CRP • Development of methods for labelling, purification and quality control of therapeutic radiopharmaceuticals for an appropriate disease model, based on relevant carrier molecules and radionuclides • Standardisation of in vitro methods for comparatively evaluating them for biological integrity, cell binding, serum stability, kinetics, internalisation and cytotoxicity • Establishment of in vivo models for comparatively evaluating biodistribution, in vivo stability and therapeutic efficacy

42. Summary – standardized protocols • Initial design – choice of target, ligand, radionuclide(s) and labelling strategies • Synthesis or purchase of cold precursors and availability of radionuclides • Radiolabelling development • Radiochemical purity analysis • Stability assessment • In-vitro receptor binding • Cellular processing and metabolism • In-vivo biodistribution and targeting • In-vitro efficacy • In-vivo efficacy

43. Laboratory set up for preparation of therapeutic doses Incubation block SepPak column assembled with filter and vaccum vial Peristaltic pump

45. In over 500 labelling procedures performed to the presented scheme we achieved reproducible results of RCP over 99.0% and the labelling yields over 95%. The radiolabelled preparations were stable when stored at room temperature, however the attention has to be paid that this stability was dependent on radioactive concentration of stored solution.

46. Stabilizers Radioharmaceuticals comprising -emitting radionuclides often undergo radiolysis during preparation and storage During radiolysis, emissions from radionuclide attack the chelate, targeting biomolecule, and other compounds in proximity, which results in decomposition or destruction of the metal chelator or/and biomolecule. „Ascorbic acid: useful as a buffer agent and Radiolytic Stabilizer for Metalloradiopharmaceuticals” Shuang Liu et al. Bioconjugate Chem. 2003, 14:1052-1056 Labelling conditions: Sodium acetate as buffer Ascorbic acid as stabilizer other stabilizers such as dihydroxybenzoic acidc

47. Stability of the labelled peptides Incubation time: 36 h Incubation at room temperature at various radioactive concentrations: 90Y-DOTATATE at 20 and 50 mCi/ml 177Lu-DOTATATE at 10 and 35 mCi/ml

48. Stability of 90Y-DOTATATE and 177Lu-DOTATATE depending on radioactive concentration, RT,radioactive concentration 20, 50 mCi/ml for 90Y-DOTATATE and 15, 35 mCi/ml for 177Lu-DOTATATE). IAEA CRP Comparative evaluation..

49. Specific ativity Succesful peptide receptor radionuclide therapy requires high specific activities of radiopharmaceutical • The amount of radioligand that can be administered is limited by affinity and the amount of receptors • Above the optimal dose an increase in ligand will increase the competition between unlabelled and labelled ligand for the same receptor and thus lower the uptake in receptor positive tissue • Pharmacological effects M. De Jong et al; Tumour uptake of the radiolabelled somatostatin analogue [DOTA0,Tyr3)octreotide is dependent on the peptide amount. Eur J Nucl Med (1999) 26:693-698

50. Effect of 177Lu specific activity on labelling yield www.mdsnordion.com