Alan R. Fritzberg

Melanoma Therapy via Peptide-Targeted α-Radiation

Purpose: The therapeutic efficacy of a unique melanoma-targeting peptide conjugated with an in vivo generated α-particle-emitting radionuclide was evaluated in the B16/F1 mouse melanoma animal model. α-Radiation is densely ionizing, resulting in high concentrations of destructive radicals and irreparable DNA double-strand breaks. This high linear energy transfer overcomes radiation-resistant tumor cells and oxygen effects resulting in potentially high therapeutic indices in tumors such as melanoma. Experimental Design: The melanoma targeting peptide, 1,4,7,10-tetraazacyclodecane-1,4,7,10-tetraacetic acid (DOTA)-Re(Arg11)CCMSH, was radiolabeled with 212Pb, the parent of 212Bi, which decays via α and β decay. Biodistribution and therapy studies were done in the B16/F1 melanoma-bearing C57 mouse flank tumor model. Results:212Pb[DOTA]-Re(Arg11)CCMSH exhibited rapid tumor uptake and extended retention coupled with rapid whole body disappearance. Radiation dose delivered to the tumor was estimated to be 61 cGy/μCi 212Pb administered. Treatment of melanoma-bearing mice with 50, 100, and 200 μCi of 212Pb[DOTA]-Re(Arg11)CCMSH extended their mean survival to 22, 28, and 49.8 days, respectively, compared with the 14.6-day mean survival of the placebo control group. Forty-five percent of the mice receiving 200 μCi doses survived the study disease-free. Conclusions: Treatment of B16/F1 murine melanoma–bearing mice with 212Pb[DOTA]-Re(Arg11)CCMSH significantly decreased tumor growth rates resulting in extended mean survival times, and in many cases, complete remission of disease. 212Pb-DOTA-Re(Arg11)CCMSH seems to be a very promising radiopharmaceutical for targeted radionuclide therapy of melanoma.

The development of technetium-99m-labelled interleukin-2: A new radiopharmaceutical for the In vivo detection of mononuclear cell infiltrates in immune-mediated diseases

We describe here a new method for labelling interleukin-2 (IL-2) in high specific activity with 99mTc for in vivo studies in man. Labelling was performed via a two-step reaction using an N3S bifunctional chelating agent. To optimise the reaction, factors affecting the incorporation of 99mTc into the N3S ligand were studied. The conjugation of the preformed N3S chelate ligand to IL-2 was then similarly optimised. Various strategies for purifying the 99mTc-IL-2 were explored including size-exclusion, ion-exchange, and several modes of reversed-phase chromatography. The radiochemical purity of the purified protein was determined by HPLC, ITLC, TCA precipitation, and SDS-PAGE. The receptor binding capacity of 99mTc-IL-2 was studied. Biodistribution studies in normal mice were performed with 99mTc-IL-2 purified using different techniques or labelled after prolonged storage and compared to 125I-IL-2.

Pretargeted α Emitting Radioimmunotherapy Using 213Bi 1,4,7,10-Tetraazacyclododecane-N,N′,N″,N‴-Tetraacetic Acid-Biotin

Purpose: The use of an α emitter for radioimmunotherapy has potential advantages compared with β emitters. When administered systemically optimal targeting of intact antibodies requires >24 h, therefore limiting the use of short-lived α emitters. This study investigated the biodistribution of bismuth-labeled biotin in A431 tumor-bearing mice pretargeted with antibody B3-streptavidin (B3-SA) and examined the therapeutic efficacy of the α emitter, 213Bi-labeled biotin. Experimental Design: Biotinidase-resistant 7,10-tetraazacyclododecane-N,N′,N″,N‴-tetraacetic acid (DOTA)-biotin was radiolabeled with 205,206Bi or 213Bi. Treatment of tumor-bearing mice began by administration of B3-SA (400 μg) to target the tumor sites for 24 h. Then, an agent containing biotin and galactose groups was used to clear the conjugate from the circulation. Four h later, bismuth-radiolabeled DOTA-biotin was given, and biodistribution or therapy was evaluated. Dose escalation treatment from 3.7–74 MBq was performed, and the effects on tumors of different sizes were investigated. Tumor growth, complete blood cell counts, toxicity, and survival were monitored. Results: Radiolabeled biotin cleared rapidly. Rapid tumor uptake resulted in much higher tumor:nontumor targeting ratios than achieved with the directly labeled monoclonal antibody. Dose escalation revealed that 74 MBq caused acute death of mice, whereas 0.37–37 MBq doses inhibited tumor growth and prolonged survival significantly. Evidence of mild hematological toxicity was noted. At therapeutically effective doses renal toxicity was observed. Conclusions:213Bi-DOTA-biotin, directed by the Pretarget method to tumor-targeted B3-SA, showed a therapeutic effect, although the therapeutic index was low. The source of the toxicity was most likely related to the renal toxicity.

Radiohalogenated small molecules for protein labeling

Haloaryl compounds are lithiated and thereafter metalated with one of the following organometallic groups: Sn(n--Bu)3 or SnMe3. The resulting aryltin compound can be transmetalated in site-specific reaction with one of the following organometallic groups: HgX, Hg(OAc)2, BX3, or BZ2, wherein X is Cl, Br, or I, and Z is alkyl or alkoxy. The metalated compounds are subsequently radiohalogenated via a demetalation reaction. A functional group suitable for conjugation to protein can be added subsequent or preferably prior to the radiohalogenation. Also compounds of the formula: R1 --Ar--R2, wherein R1 is either a radiohalogen or any one of the organometallic groups stated above, Ar is aromatic or heteroaromatic ring, and R2 is a short-chain substituent that does not activate the aromatic ring and that bears a functional group, or a precursor thereof, suitable for conjugation to protein under conditions that preserve the biological activity of the protein. The radiohalogenated small molecules are conjugated to proteins such as monoclonal antibodies for use in diagnosis and therapy.

Synthesis of a new class of Tc chelating agents: N2S2 monoaminemonoamide (mama) ligands

Abstract A novel N 2 S 2 -monoamine amide (MAMA) bifunctional chelate was designed and synthesized for labeling of antibodies with Tc-99m. The chelate forms Tc complexes at a range of pHs and shows faster kinetics of complexation than N 2 S 2 -diamidedithiol (DADS) ligands. The MAMA chelate contains a carboxyl group for covalent attachment to amino groups on proteins.

Radiohalogenated for proteins

Haloaryl compounds bearing a defined functional group or precursor are metalated with either Sn(n-Bu)3 or SnMe3. The resulting aryltin compound may be transmetalated in site-specific reaction with one of the following organometallic groups: HgX2, Hg(OAc)2, BX3, or BZ3, wherein X is Cl, Br, or I, and Z is alkyl or alkoxy. The stannylated or otherwise metalated compounds are subsequently radiohalogenated via a demetalation reaction. The Stannylated or otherwise metalated compounds are subsequently radiohalogenated via a demetalation reaction. The functional group is suitable for conjugation to protein and can be present or be added subsequent, but most preferably prior, to the radiohalogenation. Also compounds of the formula: R1 --Ar--R2, wherein R1 is either a radiohalogen or any one of the organometallic groups stated above, Ar is aromatic or heteroaromatic ring, and R2 is a short-chain substituent that does not highly activate the aromatic ring an that bears a functional group suitable for conjugation to protein under conditions that preserve the biological activity of the protein. The radiohalogenated small nolecules are conjugated to proteins such as monoclonal antibodies for use in diagnosis and therapy.

Approaches to improved antibody- and peptide-mediated targeting for imaging and therapy of cancer

Antibodies with their ability to selectivity bind antigens have been of great interest in targeting radiation, drugs and toxins to tumors. Limited success in delivery of radioactivity has been enjoyed with conventional attachment to antibodies. This is due to slow tumor targeting processes and slow disappearance from blood and variable uptake and disappearance from the excretory organs, liver and kidney. Preliminary studies in animal models and patients have shown promise in increasing the tumor to blood exposure ratio by pretargeting antibody followed by small molecule delivery of radioactivity using a molecular capture system. Efficient capture of the small molecule radioactivity carrier by tumor localized antibody and rapid clearance and excretion of the untargeted radioactivity decreases the background problem for imaging and lowers marrow toxicity for radioimmunotherapy. Small peptide ligands that bind to receptor bearing tumors offer similar advantages.

Relative role of Tc-99m-diethyl-IDA and Tc-99m-sulfur colloid in the evaluation of liver function.

Imaging of diffuse liver disease is traditionally done with Tc-99m-sulfur colloid. The advent of Tc-99m-labeled hepatobiliary agents led the authors to reevaluate the role of Tc-99m-sulfur colloid. A total of 95 paired Tc-99m-diethyl-IDA and Tc-99m-sulfur colloid studies were performed in 61 patients (60 paired studies in liver transplant patients and 35 paired studies patients who had not received liver transplants). The following parameters were visually graded on a five-point scale: Tc-99m-diethyl-IDA clearance and time of appearance of intestinal activity; and Tc-99msulfur colloid liver size, bone marrow activity, splenic size, splenic activity, and lung activity. Total serum bilirubin levels were used as the standard measure of liver function. The Tc-99m-diethyl-IDA parameters correlated more highly with total serum bilirubin than did the Tc-99m-sulfur colloid parameters in both the transplant and nontransplant groups. In conclusion, Tc- 99m-diethyl-IDA appears to be the preferred radiopharmaceutical for evaluation of hepatocellular function, although both agents may be needed in certain clinical situations.

A Clinical Comparison of 99mTc-Diethyl-lminodiacetic Acid, 99mTc-Pyridoxylideneglutamate, and 131l-Rose Bengal in Liver Transplant Patients1

The relative merits of three radiopharmaceuticals for evaluating liver transplant patients were determined in paired studies. In 86% of studies both 131I-RB and 99mTc-PG gave similar information for differentiation of hepatocellular disease and billary tract obstruction. 99mTc-PG probably demonstrates the biliary tract and small intestine better early after injection (8%); 131I-RB is probably better in showing the colon at 24 hours when intestinal activity is not seen by 1 hour (6%). 99mTc-diethyl-IDA is superior in all respects when compared to 99mTc-PG. The blood retention method (20 min./5 min.) showed that none of the radiopharmaceuticals was a reliable indicator of hepatocyte function when compared to total serum bilirubin.

Communication of intrahepatic cavities with bile ducts: demonstration with Tc-99m-diethyl-IDA imaging.

Tc-99m-diethyl-IDA imaging demonstrated intrahepatic bile leaks in two patients. The first patient had an intrahepatic abscess associated with a common bile duct stone; he was successfully treated with common duct lithotomy and antibiotics, but without surgical drainage of the intrahepatic abscess. The second patient had bile leakage into an intrahepatic cavitary hematoma after abdominal trauma.