Cancer-targeting biomolecules labeled with 211At must be stable to in vivo deastatination, as control of the 211At distribution is critical due to the highly toxic nature of alpha-particle emission. Unfortunately, no astatinated aryl conjugates have shown in vivo stability toward deastatination when (relatively) rapidly metabolized proteins, such as monoclonal antibody Fab' fragments, are labeled. As a means of increasing the in vivo stability of 211At-labeled proteins, we have been investigating antibody conjugates of boron cage moieties. In this investigation, protein-reactive derivatives containing a nido-carborane (2), a bis-nido-carborane derivative (Venus Flytrap Complex, 3), and four 2-nonahydro-closo-decaborate(2-) derivatives (4-7) were prepared and conjugated with an antibody Fab' fragment such that subsequent astatination and in vivo tissue distributions could be obtained. To aid in determination of stability toward in vivo deastatination, the Fab'-borane conjugates were also labeled with 125I, and that material was coinjected with the 211At-labeled Fab'. For comparison, direct labeling of the Fab' with 125I and 211At was conducted. Direct labeling with Na[125I]I and Chloramine-T gave an 89% radiochemical yield. However, direct labeling of the Fab' with Na[211At]At and Chloramine-T resulted in a yield of <1% after quenching with NaS2O5. As another comparison, the same Fab' was conjugated with p-[211At]astatobenzoate NHS ester, [211At]1c-Fab', and (separately) with p-[125I]iodobenzoate NHS ester, [125I]1b-Fab'. An evaluation in athymic mice demonstrated that [211At]1c-Fab' underwent deastatination. In contrast, the high in vivo stability of [125I]1b-Fab' allowed it to be used as a tracer control for the natural distribution of Fab'. Although found to be much more stable in vivo than [211At]1c-Fab', the biodistributions of nido-carborane conjugated Fab' ([125I]2-Fab'/ [211At]2-Fab') and the bis-nido-carborane (VFC) ([125I]3-Fab'/[211At]3-Fab') had very different in vivo distributions than the control [125I]1b-Fab'. Biodistributions of closo-decaborate(2-) conjugates ([125I]4-Fab'/[211At]4-Fab', [125I]6-Fab'/[211At]6-Fab', and [125I]7-Fab'/[211At]7-Fab') demonstrated that they were stable to in vivo deastatination and had distributions similar to that of the control [125I]1b-Fab'. In contrast, a benzyl-modified closo-decaborate(2-) derivative evaluated in vivo ([125I]5-Fab'/[211At]5-Fab') had a very different tissue distribution from the control. This study has shown that astatinated protein conjugates of closo-decaborate(2-) are quite stable to in vivo deastatination and that some derivatives have little effect on the distribution of Fab'. Additionally, direct 211At labeling of Fab' conjugated with closo-decaborate(2-) derivatives provide very high (e.g., 58-75%) radiochemical yields. However, in vivo data also indicate that the closo-decaborate(2-) may cause some retention of radioactivity in the liver. Studies to optimize the closo-decaborate(2-) conjugates for protein labeling are underway.

An investigation has been conducted to assess the in vivo stability of a series of astatinated benzamides and astatinated nido-carborane compounds in mice. It was hypothesized that the higher bond strength of boron-astatine bonds in the nido-carboranes might provide increased stability toward in vivo deastatination. Four tri-n-butylstannylbenzamides were prepared for radiohalogenation and evaluation in vivo. Those compounds were N-propyl-4-(tri-n-butylstannyl)benzamide 1a, N-propyl-3-(tri-n-butylstannyl)benzamide 2a, ethyl 4-tri-n-butylstannylhippurate 3a, and 4-tri-n-butylstannyl-hippuric acid 4a. Seven mono-nido-carboranyl derivatives were prepared for radiohalogenation and in vivo evaluation. Four of the seven mono-carboranyl derivatives (5a, 6a, 7a, 13a) contained a 3-(nido-carboranyl)propionamide functionality, and the remaining compounds (8a, 8g, 10a) contained a 4-(nido-carboranyl)aniline functionality. Two additional derivatives (11a, 12a) were prepared that contained bis-(nido-carboranylmethyl)benzene moieties (also referred to as Venus flytrap complexes (VFCs). All benzamide and nido-carborane compounds underwent facile iodination and radiohalogenation, except a 4-(nido-carboranyl)aniline derivative, 8a. Iodination of 8a resulted in a mixture, of which the desired iodinated product was a minor component. Therefore, radiohalogenation was not attempted. It is believed that the mixture of products is due to the presence of a thiourea bond. Previous studies have shown that thiourea bonds can interfere with halogenation reactions. In vivo comparisons of the compounds were conducted by co-injection of dual labeled (125/131I and 211At) compounds. Tissue distribution data were obtained at 1 and 4 h postinjection of the radiolabeled compounds, as that was sufficient to determine if astatine was being released. Stability of the astatinated compound was assessed by the difference in concentration of radioiodine and astatine in lung and spleen. All of the benzamides were found to undergo rapid deastatination in vivo. The nido-carborane derivatives appeared to be slightly more stable to in vivo deastatination; however, they had long blood residence times. The surprising finding was that the VFC derivatives did not release 211At in vivo, even though they rapidly localized to liver. This finding provides encouragement that stable conjugates of 211At may be attained if appropriate modifications of the VFC can be made to redirect their excretion through the renal system.

Pretargeted radioimmunotherapy specifically targets radiation to tumors using antibody-streptavidin conjugates followed by radiolabeled biotin. A potential barrier to this cancer therapy is the presence of endogenous biotin in serum, which can block the biotin-binding sites of the antibody-streptavidin conjugate before the administration of radiolabeled biotin. Serum-derived biotin can also be problematic in clinical diagnostic applications. Due to the extremely slow dissociation of the biotin-streptavidin complex, this endogenous biotin can irreversibly block the biotin-binding sites of streptavidin and reduce therapeutic efficacy, as well as reduce sensitivity in diagnostic assays. We tested a streptavidin mutant (SAv-Y43A), which has a 67-fold lower affinity for biotin than wild type streptavidin, and three bivalent bis-biotin constructs as replacements for wild-type streptavidin and biotin used in pretargeting and clinical diagnostics. Biotin dimers were engineered with certain parameters including water solubility, biotinidase resistance, and linker lengths long enough to span the distance between two biotin-binding sites of streptavidin. The bivalent biotins were compared to biotin in exchange, retention, and off-rate assays. The faster off-rate of SAv-Y43A allowed efficient exchange of prebound biotin by the biotin dimers. In fluorescent competition experiments, the biotin dimer ligands displayed high avidity binding and essentially irreversible retention with SAv-Y43A. The off-rate of a biotinidase-stabilized biotin dimer from SAv-Y43A was 4.36 x 10(-)(6) s(-)(1), over 640 times slower compared to biotin. These findings strongly suggest that employing a mutant streptavidin in concert with a bivalent biotin can mitigate the deleterious impact of endogenous biotin, by allowing exchange of bound biotin and retention of the biotin dimer carriers.

We are investigating the use of an 211At-labeled anti-CD45 monoclonal antibody (mAb) as a replacement of total body irradiation in conditioning regimens designed to decrease the toxicity of hematopoietic cell transplantation (HCT). As part of that investigation, dose-escalation studies were conducted in dogs using 211At-labeled anti-canine CD45 mAb, CA12.10C12, conjugated with a maleimido-closo-decaborate(2-) derivative, 4. Unacceptable renal toxicity was noted in the dogs receiving doses in the 0.27 – 0.62 mCi/kg range. This result was not anticipated, as no toxicity had been noted in prior biodistribution and toxicity studies conducted in mice. Studies were conducted to understand the cause of the renal toxicity and to find a way to circumvent it. A dog biodistribution study was conducted with 123Ilabeled CA12.10C12 that had been conjugated with 4. The biodistribution data showed that 10-fold higher kidney concentrations were obtained with the maleimido-conjugate than had been obtained in a previous biodistribution study with 123I-labeled CA12.10C12 conjugated with an amine-reactive phenylisothiocyanato-CHX-A” derivative. The difference in kidney concentrations observed in dogs for the two conjugation approaches led to an investigation of the reagents. SE-HPLC analyses showed that the purity of the CA12.10C12 conjugated via reduced disulfides was lower than that obtained with amine-reactive conjugation reagents, and non-reducing SDS-PAGE analyses indicated protein fragments were present in the disulfide reduced conjugate. Although we had previously prepared closo-decaborate(2-) derivatives with amine-reactive functional groups (e.g. 6 & 8), a new easily synthesized, amine-reactive (phenylisothiocyanate) derivative, 10, was prepared for use in the current studies. A biodistribution was conducted with co-administered 125I- and 211At-labeled CA12.10C10 conjugated with 10. In that study, lower kidney concentrations were obtained for both radionuclides than had been obtained in the earlier study of the same antibody conjugated with 4 after reduction of disulfide bonds.

A "wet chemistry" approach for isolation of 211 At from an irradiated bismuth target is described. The approach involves five steps: (1) dissolution of bismuth target in conc. HNO 3 ; (2) removal of the HNO 3 by distillation; (3) dissolution of residue in 8 M HCl; (4) extraction of

In vivo deastatination has been a major problem in the development of reagents for therapeutic applications of the α-particle emitting radionuclide 211 At. Our prior studies demonstrated that the use of a closo-decaborate(2-) ([closo-B 10 H 9 R] 2− ) moiety for 211 At labeling of biomolecules provides conjugates that are stable to in vivo deastatination. In this investigation, the closo-decaborate(2-) moiety was compared with the structurally similar closo-dodecaborate(2-) ([closo-B 12 H 11 R] 2− ) to determine if one has more favorable properties than the other for use in pendant groups as 211 At labeling molecules. To determine the differences, two sets of structurally identical molecules, with the exception that they contained either a closo-decaborate(2-) or a closo-dodecaborate(2-) moiety, were compared with regards to their synthesis, radiohalogenation, stability to in vivo deastatination and tissue distribution. Quite different rates of reaction were noted in the synthetic steps for the two closo-borate(2-) moieties, but ultimately the yields were similar, making these differences of little importance. Differences in radiohalogenation rates were also noted between the two closo-borate(2-) moieties, with the more electrophilic closo-decaborate(2-) reacting more rapidly. This resulted in somewhat higher yields of astatinated closo-decaborate(2-) derivatives (84% vs 53%), but both cage moieties gave good radioiodination yields (e.g. 79-96%). Importantly, both closo-borate(2-) cage moieties were shown to have high stability to in vivo deastatination. The largest differences between pairs of compounds containing the structurally similar boron cage moieties were in their in vivo tissue distributions. I]1b, was evaluated, the route of excretion appeared to be hepatobiliary rather than renal. Identical biotin derivatives containing the two closo-borate(2-) cage moieties had similar tissue distributions, except the closo-decaborate(2-) derivative had lower concentrations in kidney (1h, 19.9%ID/g; 4h, 24.4%ID/g vs. 1h, 38.9%ID/g; 4h, 40.6%ID/g). In summary, the higher reactivity, faster tissue clearance, and lower kidney concentrations make the closo-decaborate(2-) more favorable for further studies using them in reactive groups for 211 At labeling of biomolecules.

545The glyoxalase-enzyme system, which has been known since 1913 [l, 21, is widely distributed in nature and catalyses the conversion of methylglyoxal (or other a-ketoaldehydes) to i)-lactate (or other a-hydroxy acids) with reduced glutathione serving as the cofactor [3-71. The system consists of two enzymes, glyoxalase I and glyoxalase I1 [8]. Glyoxalase I (EC 4.4.1.5) acts upon the equilibrium adduct of methylglyoxal and glutathione, a hemimercaptal, to form the thioester, S-i Aactoylglutathione (SLG). Glyoxalase I1 (EC 3.1.2.6) hydrolyses the thioester to regenerate glutathione and to liberate free iAactic acid. Many suggestions for the function(s) of the glyoxalase system have been made and include protection against a-ketoaldehyde toxicity [8], regulation of cell growth [9, 101 and a glycolytic bypass from dihydroxyacetone phosphate to I )-lactate, via methylglyoxal synthase and the glyoxalases [ 111. Several years ago the glyoxalase system was reported to affect cell-free microtubule assembly [ 121. Synthesis of competitive inhibitorsOne of the approaches employed in exploring possible functions of the glyoxalases has been with the use of competitive inhibitors of glyoxalase I and glyoxalase 11. From 1957 onwards, a number of glyoxalase I inhibitors, derived from Flutathione, have been synthesized [ 13-17]. More recently competitive inhibitors of glyoxalase 11, also derived from glutathione, have been prepared and studied [ 18-21 1 ; these inhibitors are typically thiocarbonate or thiocarbonate-carbamate derivatives of glutathione. Those derivatives which have been found to be effective glyoxalase I1 inhibitors are summarized in the generalized structure: OOCHCI lICHLCONHCHCONHCH,COO -I I NI I S I I K' R Abbreviations used: CH-G, S-carboxybenzoxylglutathione; DiFMOC-G, N,S-fluorenylmethoxycarbonylglutathione; FMOC-G, S-fluorenylmethoxycarbonylglutathione; 0-, m-, p-NCH-G, S-(0, m-, p-nitrocarbobenzoxyglutathione); SLG, S-1)-lactoylglutathione. This report is concerned mainly with the synthesis of new glyoxalase I1 inhibitors (in particular N,S-bisfluorenylmethoxycarbonylglutathione [DiFMOC-GI and some of its diesters) and their effects on purified mammalian glyoxalase I1 and on mammalian cells in culture. We will touch on the subject of the inhibition of purified preparations of glyoxalase I1 by a variety of purine nucleotides as well. W e have prepared S-fluorenylmethoxycarbonylglutathione (FMOC-G) and N,S-biscarbobenzoxy glutathione (DiCB-G) previously and have found them to be quite potent and specific competitive inhibitors of glyoxalase I1 purified from mammals and plants 1201. Recently we have prepared and studied N,S-bisfluorenylmethoxycarbonyl glutathione (DiFMOC-G). Table 1 lists a number of glyoxalase I1 inhibitors that have been reported, as well as the newly synthesized DiFMOC-G. The latter compound is the most potent competitive inhibitor of glyoxalase I1 known to us that has been synthesized ( K , = 0.75 pM, calfliver glyoxalase 11). The thiocarbonate derivatives of glutathione, which bind ve...

Abstract. The size of affibody molecules makes them suitable as targeting agents for targeted radiotherapy with the ·-emitter 211 At, since their biokinetic properties match the short physical half-live of 211 At. In this study, the potential for this approach was investigated in vivo. Two different HER-2 binding affibody molecules were radiolabeled with 211 At using both the linker PAB (N-succinimidyl-para-astatobenzoate) and a decaborate-based linker, and the biodistribution in tumorbearing nude mice was investigated. The influence of L-lysine and Na-thiocyanate on the 211 At uptake in normal tissues was also studied. Based on the biokinetic information obtained, the absorbed dose was calculated for different organs. Compared with a previous biodistribution with 125 I, the 211 At biodistribution using the PAB linker showed higher uptake in lungs, stomach, thyroid and salivary glands, indicating release of free 211 At. When the decaborate-based linker was used, the uptake in those organs was decreased, but instead, high uptake in kidneys and liver was found. The uptake, when using the PAB linker, could be significantly reduced in some organs by the use of L-lysine and/or Na-thiocyanate. In conclusion, affibody molecules have suitable blood-kinetics for targeted radionuclide therapy with 211 At. However, the labeling chemistry affects the distribution in normal organs to a high degree and needs to be improved to allow clinical use.