Alan R. Ketring

Development of an in vitro model for assessing the in vivo stability of lanthanide chelates

An in vitro model was developed to evaluate the in vivo stability of lanthanide polyaminocarboxylate complexes. The ligand-to-metal ratios for the chelates EDTA, CDTA, DTPA, MA-DTPA (monoamide-DTPA) and DOTA with the lanthanides lanthanum, samarium, and lutetium were optimized to achieve > or = 98% complexation yield for the resultant radiolanthanide complexes. The exchange of the radiolanthanides from their EDTA, CDTA, DTPA, MA-DTPA and DOTA complexes with Ca(2+) was determined by in vitro adsorption and in vitro column studies using hydroxyapatite (HA), an in vitro bone model. In vitro serum stability of these radiolanthanide complexes was used as an additional indicator of in vivo stability, although the mechanism of instability in serum will be different than with bone. The in vitro studies were consistent with the expected findings that the smallest lanthanide (Lu) formed the most stable complexes. In vivo studies were done to validate the in vitro model. Biodistribution studies in normal CF-1 mice showed that in vivo stability of the complex (i.e., the more lanthanide remaining in complex form) could be assessed by a combination of the urinary, bone and liver uptake. For example, biodistribution studies demonstrate that high urinary excretion correlated with complex stability, while high liver plus bone uptake correlated with complex instability. The urinary excretion of the EDTA complexes decreased from (177)Lu to (140)La indicating a loss in stability in the direction of (140)La, consistent with the in vitro studies. The more stable a lanthanide complex is, the lower its exchange with HA in vitro will be, and the lower its combined bone plus liver uptake and higher its urinary excretion will be in vivo. This investigation indicates that the in vivo stability can be determined by a screening method that measures the degree of exchange from the lanthanide chelate with hydroxyapatite (HA) and its serum stability.

153Sm-EDTMP and 186Re-HEDP as bone therapeutic radiopharmaceuticals

Two new radiopharmaceuticals, 186Re-1-hydroxyethylidenediphosphonate (186Re-HEDP) and 153Sm-ethylenediaminetetramethylenephosphonate (153Sm-EDTMP), have been proposed as palliative treatments for metastatic bone cancer. Biolocalization properties of these chelates in animals as well as the physical decay and production properties of the respective radionuclides are consistent with those of a therapeutic agent. Subtherapeutic doses of both agents have been administered to human cancer patients to determine their biokinetics and skeletal localization. The 186Re-HEDP studies were conducted at the University of Cincinnati while the 153Sm-EDTMP studies were conducted at the University of Missouri-Columbia. The pharmacokinetics of these agents in humans were consistent with those found in animals. Imaging studies show that the retained activity localizes primarily in the skeleton with high selective uptake in skeletal lesions; there is no visualization of other organs or soft tissue. This paper will review the development and preparation procedures for these radiopharmaceuticals and briefly summarize the animal and patient data.

Reactor-produced radionuclides at the University of Missouri Research Reactor

A revolution in radiotherapy has been developing in recent years, based on more sophisticated targeting methods including radioactive intra-arterial microspheres, chemically-guided bone agents, labeled monoclonal antibodies, and isotopically-tagged polypeptide receptor-binding agents. The isotopes of choice for these applications are reactor-produced beta emitters such as Sm-153, Re-186, Re-188, Ho-166, Lu-177, and Rh-105. The University of Missouri Research Reactor (MURR) has been in the forefront of research into means of preparing, handling, and supplying these high specific activity isotopes in quantities appropriate not only for research, but also for patient trials in the U.S. and around the world. Considerable effort has been expended to develop techniques for irradiation, handling, and shipping isotopes worldwide. The MURR has also served as a highly reliable production source for isotopes, with one of the best operating histories of any isotope production reactor in the world.

Comparison of Systemic Toxicities of 177Lu-DOTMP and 153Sm-EDTMP Administered Intravenously at Equivalent Skeletal Doses to Normal Dogs

Bone-seeking radiopharmaceuticals have been used to effectively treat cancer arising from and metastasizing to bone in humans and dogs. The rate of complete tumor control is low, and the geographic distribution of available compounds is limited by their half-lives. This experiment was done to evaluate in normal dogs the toxicity of 177Lu-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetramethylene phosphonate (177Lu-DOTMP) used as a potential therapeutic radiopharmaceutical. Methods: Four normal purpose-bred dogs were administered 177Lu-DOTMP at a dose of 8.14 MBq/kg and monitored for 84 d for evidence of toxicity in the bone marrow and vital organs. Results: No statistically significant alterations in the biochemical profile, white blood cell count, or platelet count were observed in any dog. Very mild decreases in the red cell count were seen on day 84. No microscopic evidence of toxicity was present at necropsy. Conclusion: The dogs receiving 177Lu-DOTMP tolerated the administration and the effects of the compound without apparent clinical toxicity. The results of this experiment support the further evaluation in tumor-bearing dogs of 177Lu-DOTMP as a potential therapy for metastatic bone cancer and primary bone tumors in humans and dogs.

198Au-labeled hydroxymethyl phosphines as models for potential therapeutic pharmaceuticals

The development of novel gold-198 complexes with water-soluble phosphines is reported. A series of cationic and hydrophilic 198Au complexes containing the ligands tris(hydroxymethyl)phosphine (THP, 1) 1,2-bis[bis(hydroxymethyl)phosphino]benzene (HMPB, 2), and 1,2-bis[bis(hydroxymethyl)phosphino]ethane (HMPE, 3) were prepared and evaluated as models for potential gold-199 radiopharmaceuticals. The 198Au complexes were formed in high radiochemical purity by simply mixing H198AuCl4 with the respective ligand. The complexes were shown to exhibit high in vitro stability over wide pH ranges and temperatures. However, only the 198Au(HMPB)2+ complex was found to exhibit good in vivo stability. HPLC analyses indicated that the 198Au complexes with these three phosphine ligands produced singular species with similar retention times as compared to their known macroscopic complexes.

Proton irradiation parameters and chemical separation procedure for the bulk production of high-specific-activity 186gRe using WO3 targets

Abstract Rhenium-186g (T1/2= 89.2 h) is a β− emitter suitable for therapeutic applications. Current production methods rely on reactor production via 185Re(n,γ) which results in low specific activities, thereby limiting its use. Production by p,d activation of enriched 186W results in a 186gRe product with a higher specific activity, allowing it to be used for targeted therapy with limited receptors. A test target consisting of pressed, sintered natWO3 was proton irradiated at Los Alamos (LANL-IPF) to evaluate product yield and impurities, irradiation parameters and wet chemical Re recovery for proof-of-concept for bulk production of 186gRe. We demonstrated isolation of 186gRe in 97% yield from irradiated natWO3 targets within 12 h of end of bombardment (EOB) via an alkaline dissolution followed by anion exchange. The recovery process has potential for automation, and WO3 can be easily recycled for recurrent irradiations. A 186gRe batch yield of 42.7 ± 2.2 μCi/μAh or 439 ± 23 MBq/C was obtained after 24 h in an 18.5 μA proton beam. The target entrance energy was determined to be 15.6 MeV. The specific activity of 186gRe at EOB was measured to be 1.9 kCi (70.3 TBq) mmol−1, which agrees well with the result of a previous 185,186mRe co-production EMPIRE and TALYS modeling study assuming similar conditions. Utilizing enriched 186WO3, we anticipate that a proton beam of 250 μA for 24 h will provide batch yields of 256 mCi (9.5 GBq) of 186gRe at EOB with specific activities even higher than 1.9 kCi (70.3 TBq) mmol−1, suitable for therapy applications.

An Rh-105 complex of tetrathiacyclohexadecane diol with potential for formulating bifunctional chelates☆

1,5,9,13-Tetrathiacyclohexane-3,11-diol (16S4-diol), a sulfur crown ether analog, was studied as a potential chelating agent to complex no-carrier-added (NCA) grade 105Rh(III) in high yield at low ligand concentrations. trans-[RhCl2(16S4-diol)]chi (chi = Cl, PF6) was prepared using nonradioactive RhCl3.3H2O and characterized by UV-Vis, nuclear magnetic resonance (NMR) and X-ray crystallography. It was shown to have a +1 charge with the Rh(III) metal center coordinated to the four S atoms equatorially and two Cl atoms in trans axial positions. The 105Rh-16S4-diol complex prepared with NCA 105Rh(III)-chloride reagent was found to exhibit identical chromatographic properties as trans-[Rh(III)Cl2(16S4-diol)]+ (including silica and C-18 thin-layer chromatography [TLC] and electrophoresis). The preparation of 105Rh-16S4-diol complex formation optimized for conditions of pH, temperature, time, % ethanol and quantity of 16S4-diol resulted in yields > 90%. Very low quantities of 16S4-diol (3 nmol) complex NCA 105Rh(III) under relatively mild reaction conditions (heating at 64 degrees C for 90 min) in the presence of ethanol (10%), yielded the high specific activity 105Rh-16S4-diol complex as a single cationic species. The 105Rh-16S4-diol complex was shown to be stable for > or = 4 days in physiological buffers at room temperature and in human serum at 37 degrees C.

Rhodium-105 complexes as potential radiotherapeutic agents

Rhodium-105 complexes have been investigated for their suitability as the basis of potential bifunctional chelating agents for therapeutic radiopharmaceuticals. Rhodium-105 is a reactor-produced therapeutic radionuclide that is available in high specific activity. The chemistry and biology of several six-coordinate Rh(III) complexes of the general form [RhCl2L]+, where L is a tetradentate ligand containing at least three thioethers donor atoms, is discussed. The backbone chain length of the acyclic or macrocyclic ligand determines the geometry about the Rh(III) centre (cis vs. trans), with the larger ligands preferentially forming trans-dichloro complexes. The stability of all of the 105Rh complexes is very high (>5 days) and the biological clearance properties of the complexes are consistent with their relative lipophilicities.

Comparison of pretargeted and conventional CC49 radioimmunotherapy using 149Pm, 166Ho, and 177Lu.

The therapeutic efficacies of radiolabeled biotin, pretargeted by monoclonal antibody (mAb)-streptavidin fusion protein CC49 scFvSA, were compared to those of radiolabeled mAb CC49, using the three radiolanthanides in an animal model of human colon cancer. The purpose of the present study was to compare antibody pretargeting to conventional radioimmunotherapy using (149)Pm, (166)Ho, or (177)Lu. Nude mice bearing LS174T colon tumors were injected sequentially with CC49 scFvSA, the blood clearing agent biotin-GalNAc(16), and (149)Pm-, (166)Ho-, or (177)Lu-DOTA-biotin. Tumor-bearing mice were alternatively administered (149)Pm-, (166)Ho-, or (177)Lu-MeO-DOTA-CC49. Therapy with pretargeted (149)Pm-,(166)Ho-, and (177)Lu-DOTA-biotin increased the median time of progression to a 1 g tumor to 50, 41, and 50 days post-treatment, respectively. Therapy with (149)Pm-,(166)Ho-, and (177)Lu-MeO-DOTA-CC49 increased the median time to progression to 53, 24, and 67 days post-treatment, respectively. In contrast, saline controls showed a median time to progression of 13 days postinjection. Treatment with pretargeted (149)Pm-, (166)Ho-, and (177)Lu-biotin or (149)Pm-, (166)Ho-, and (177)Lu-CC49 increased tumor doubling time to 18-36 days, compared to 3 days for saline controls. Among treated mice, 23% survived >84 days post-therapy, and 11% survived 6 months, but controls survived <29 days. Long-term survivors showed tumor growth inhibition or partial regression, extensive necrosis in residual masses, and no evidence of nontarget tissue toxicity at necropsy. Both pretargeted and conventional RIT demonstrated considerable efficacy in an extremely aggressive animal model of cancer. Our results identified (177)Lu as an optimal radiolanthanide for future evaluation of these agents in toxicity and multiple-dose therapy studies.

Biodistribution of model 105Rh-labeled tetradentate thiamacrocycles in rats

105Rh(III)Cl2 complexes with a limited series of [14]ane- and [16]ane- thia macrocycles were prepared and their biodistributions in Sprague-Dawley rats studied. These studies demonstrate that modifications in the structure and composition of the 105Rh-thia macrocycle complexes produce significant differences in their uptake and retention in both the liver and kidneys. The results indicate that the cis-Rh(III)Cl2-[14]ane thiamacrocycles exhibit less kidney retention than the corresponding trans-Rh(III)Cl2-[16]ane thiamacrocycles. In addition, the presence of a side chain containing a carboxylate group will produce decreased retention of activity in the kidneys. HPLC analysis of urine from these animals indicates no observable in vivo metabolism or dissociation of these chelates in the blood stream.