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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.

An investigation was conducted in which the stabilities of four structurally different biotin derivatives were assessed with regard to biotinamide bond hydrolysis by the enzyme biotinidase. The biotin derivatives studied contained an extra methylene in the valeric acid chain of biotin (i.e., homobiotin), or contained conjugated amino acids having hydroxymethylene, carboxylate, or acetate functionalities on a methylene alpha to the biotinamide bond. The biotinidase hydrolysis assay was conducted on biotin derivatives that were radioiodinated at high specific activity, and then subjected to diluted human serum at 37 degrees C for 2 h. After incubation, assessment of biotinamide bond hydrolysis by biotinidase was readily achieved by measuring the percentage of radioactivity that did not bind with avidin. As controls, an unsubstituted biotin derivative which is rapidly cleaved by biotinidase and an N-methyl-substituted biotin derivative which is stable to biotinidase cleavage were included in the study. The results indicate that increasing the distance from the biotin ring structure to the biotinamide bond by one methylene only decreases the rate of biotinidase cleavage, but does not block it. The data obtained also indicate that placing a hydroxymethylene, carboxylate, or acetate alpha to the biotinamide bond is effective in blocking the biotinamide hydrolysis reaction. These data, in combination with data previously obtained, which indicate that biotin derivatives containing hydroxymethylene or carboxylate moieties retain the slow dissociation rate of biotin from avidin and streptavidin [Wilbur, D. S., et al. (2000) Bioconjugate Chem. 11, 569-583], strongly support incorporation of these structural features into biotin derivatives being used for in vivo targeting applications.

An investigation was conducted to determine the affect of structural variation of biotin conjugates on their dissociation rates from Av and SAv. This information was sought to help identify optimal biotin derivatives for in vivo applications. Fifteen biotin derivatives were conjugated with a cyanocobalamin (CN-Cbl) derivative for evaluation of their "relative" dissociation rates by size exclusion HPLC analysis. Two biotin-CN-Cbl conjugates, one containing unaltered biotin and the other containing iminobiotin, were prepared as reference compounds for comparison purposes. The first structural variations studied involved modification of the biotinamide bond with a N-methyl moiety (i.e., sarcosine conjugate), lengthening the valeric acid side chain by a methylene unit (i.e., homobiotin), and replacing the biotinamide bond with thiourea bonds in two conjugates. The rate of dissociation of the biotin-CN-Cbl derivative from Av and SAv was significantly increased for biotin derivatives containing those structural features. Nine additional biotin conjugates were obtained by coupling amino acids or functional group protected amino acids to the biotin moiety. In the conjugates, the biotin moiety and biotinamide bond were not altered, but substituents of various sizes were introduced alpha to the biotinamide bond. The results obtained from HPLC analyses indicated that the rate of dissociation from Av or SAv was not affected by small substituents alpha to the biotinamide (e.g., methyl, hydroxymethyl, and carboxylate groups), but was significantly increased when larger functional groups were present. On the basis of the results obtained, it appears that biotin conjugates which retain an unmodified biotin moiety and have a linker molecule conjugated to it that has a small functional group (e.g., hydroxymethylene or carboxylate) alpha to the biotinamide bond are excellent candidates for in vivo applications. These structural features are obtained in the biotin amino acid conjugates: biotin-serine, biotin-aspartate, biotin-lysine, and biotin-cysteine. Importantly, these biotin derivatives can be readily conjugated with other molecules for specific in vivo applications. In our studies, these derivatives will be used in the design of new biotin conjugates to carry radionuclides for cancer therapy using the pretargeting approach.

In this investigation, studies were conducted to determine if size exclusion HPLC could be used to assess relative association rates (on-rates) and dissociation rates (off-rates) of biotin derivatives from avidin (Av) and streptavidin (SAv). For easy detection and quantification of biotin derivatives, molecules that can be detected by UV absorbance were conjugated to biotin. Concern that conjugation of the chromophoric moieties (dyes) might affect biotin binding with Av and SAv or might interact with the HPLC column led to evaluation of 10 biotin-dye conjugates. The dyes conjugated with biotin included dansyl, cyanocobalamin (CN-Cbl), coumarin 343, Lissamine-rhodamine, fluorescein, Cascade Blue, Lucifer Yellow, Oregon Green, tetramethylrhodamine, and Alexa Fluor 594. The biotin-dye conjugates were initially evaluated to determine their peak characteristics on two different size exclusion HPLC columns. Measurement of the percent of biotin-dye conjugate bound with Av in the presence of an equal quantity of biotin provided an association rate relative to biotin. All of the biotin-dyes tested had association rates within a factor of 3x (slower) that of biotin. The relative dissociation rate of biotin-dye conjugates was assessed by challenging the biotin conjugate bound to Av or SAv with a large excess of biotin. All of the initial biotin-dye conjugates tested bound Av and SAv tightly resulting in very slow dissociation rates. From the biotin-dye conjugates studied, biotin-CN-Cbl, 6b, was selected as the best conjugate for the HPLC assay. To test the HPLC assay, an iminobiotin-CN-Cbl conjugate, 13a, and a biotin-sarcosine-CN-Cbl conjugate, 13b, were synthesized. The fact that the iminobiotin does not bind with Av at physiological pH was easily detected in the size exclusion HPLC assay. The biotin-sarcosine-CN-Cbl conjugate was expected to have a more rapid dissociation rate than the other biotin-dye conjugates. This was confirmed in that HPLC assay. Although 13b bound tightly with Av in the absence of added biotin, it was completely released within 1 h when challenged by an excess of biotin. A slower dissociation of 13b was noted with SAv. The results obtained indicate that CN-Cbl conjugates of biotin derivatives can be used to determine relative on-rates and off-rates of biotin derivatives with Av and SAv. The studies also demonstrated that the biotin-CN-Cbl conjugate, 6b, can be used as a reference compound to compare on-rates and off-rates of nonchromophoric biotin derivatives.

The high affinity of biotin for streptavidin has made this pair of molecules very useful for in vivo applications. To optimize reagents for one potential in vivo application, antibody-based pretargeting of cancer, we have prepared a number of new biotin and streptavidin derivatives. The derivatives developed include new radiolabeled biotin reagents, new protein biotinylation reagents, and new biotin multimers for cross-linking and/or polymerization of streptavidin. We have also modified streptavidin by site-directed mutation and chemical modification to improve its in vivo characteristics, and have developed new reagents for cross-linking antibody fragments with streptavidin. A brief overview of these new reagents is provided.

This report describes an investigation aimed at preparation of radioiodinated cyanocobalamin (CN-Cbl) monomers and dimers with improved water solubility and decreased nonspecific binding. In the investigation, synthesis and radioiodination reactions of one monomeric and two dimeric CN-Cbl derivatives were conducted. The initial step in the synthesis of the CN-Cbl derivatives was mild acid hydrolysis of CN-Cbl, 1, followed by separation of the resultant corrin ring b-, d-, and e-monocarboxylate isomers. The investigation was limited to preparation of conjugates of CN-Cbl-e-carboxylate, 2, as earlier studies had shown binding of that isomer with recombinant human transcobalamin II (rhTCII) was similar to CN-Cbl. In a second synthetic step, the hydrophilic linker moiety, 4,7,10-trioxa-1,13-tridecandiamine, 3, was conjugated with 2 to form the adduct, 4. The synthesis of a monomeric CN-Cbl derivative, 6a, which can be used for radioiodination, was accomplished by reaction of 4 with p-tri-n-butylstannylbenzoate tetrafluorophenyl (TFP) ester, 5a. Two CN-Cbl dimers containing the arylstannane radioiodination moiety were also synthesized. The first dimer, 8a, was synthesized by cross-linking 4 with a stannylbenzoyl-aminoisophthalate di-TFP ester, 7a. The second dimer, 11a, was synthesized by reacting benzene tricarboxylate tri-TFP ester, 10, in a stepwise manner with 1 equiv of the adduct of 5a and 3 (forming 9a), followed by 2 equiv of 4. Iodobenzoate HPLC standards, 6b, 8b, and 11b, used in the radioiodination studies, were prepared in a manner similar to that of the stannylbenzoate derivatives. Radioiodinations were performed by reacting 6a, 8a, or 11a with N-chlorosuccinimide and Na[(125)I]I in methanol under neutral conditions. Radiochemical yields of 17-42% were obtained. Evaluation of the binding properties of radiolabeled CN-Cbl conjugates with rhTCII showed that the dimer of CN-Cbl, 11b, bound more avidly than the monomer, 6b, and that the binding affinity of the dimer is essentially equivalent to that of unmodified CN-Cbl. Incubation of radioiodinated monomer, [(125)I]6b, and dimer, [(125)I]11b, with rhTCII followed by size-exclusion chromatographic analysis provided data that the monomer bound one rhTCII molecule whereas two rhTCII molecules were bound to approximately 30% of the dimer.

We are investigating the hypothesis that biotin multimers can be used with streptavidin and monoclonal antibody conjugates in cancer pretargeting protocols to provide a method of increasing the amount of radioactivity bound on cancer cells in patients. As part of that investigation, a series of biotinylated Starburst dendrimers (BSBDs) have been prepared and evaluated in vitro and in vivo. In this study, a new biotinidase-stabilized, water-solubilizing biotinylation reagent was prepared and reacted with Starburst (PAMAM) dendrimers, generations 0, 1, 2, 3, and 4. The reaction conditions employed resulted in perbiotinylation of generation 0 (four biotin moieties conjugated), generation 1 (eight biotin moieties conjugated), generation 2 (16 biotin moieties conjugated), and generation 3 (32 biotin moieties conjugated). With generation 4, incomplete biotinylation was achieved resulting in the largest portion of that BSBD having 51 biotin moieties (of 64 possible) conjugated. The ability of each BSBD to cross-link streptavidin (SAv) was examined in an in vitro assay. In that assay, an assessment was made of the quantity of [125I]SAv bound with polystyrene-bound SAv after treatment with the synthesized BSBDs. All BSBDs cross-linked the polystyrene-bound SAv with [125I]SAv; however, the amount of [125I]SAv bound varied with the different BSBDs. Roughly 1 equiv of [125I]SAv was bound when Starburst dendrimers containing three or four biotin moieties (generation 0) were used. Two equivalents were bound with BSBD generation 1, and 4 equiv were bound with BSBDs generations 2, 3, and 4. To assess the distribution of BSBDs generations 0, 1, and 2 in mice (at 4 h postinjection), a method was developed for radioiodinating them using the NHS ester of p-[125I]iodobenzoate ([125I]PIB). It was found that the radioiodinated BSBDs had low blood concentrations (i.e., 0.13-0.20% ID/g) at the 4 h time point. In fact, most tissues examined had low concentrations of biotinylated dendrimers, except kidney and liver. Kidney had the highest concentration of [125I]-labeled BSBDs, and its concentration increased with increasing size and charge of dendrimer (e.g., 8-48% ID/g). On the basis of the increased radioactivity observed in the in vitro assay and the rapid clearance from blood in mice, additional in vivo studies with perbiotinylated Starburst dendrimer, generation 2, are planned.

An investigation has been conducted to determine if the kidney localization of recombinant streptavidin can be decreased to improve its characteristics in pretargeting protocols. Three different methods of accomplishing this were evaluated. The first method, blocking kidney uptake with a preadministration of recombinant streptavidin in which biotin occupied all of the binding sites, was unsuccessful. In a second method, l-lysine administration was used to block kidney localization. This method worked well, decreasing the concentration to 29% of the unmodified amount at 8 h postinjection. However, this method suffered from a requirement for constant infusion of lysine during the period of observation. A third method, use of succinylated recombinant streptavidin, was found to be the best approach. Succinylation of streptavidin was readily accomplished with very good protein recovery. With the succinylated streptavidin, the kidney concentration was only 14% of that of nonmodified streptavidin at 4 h postinjection. While these results demonstrated that the concentration of streptavidin could be decreased in the kidney, it was important to assess whether the tumor colocalization of streptavidin with biotinylated antibody was affected under those conditions. As part of our continuing investigation of pretargeting, a new water-solubilized biotinidase-stabilized biotinylation reagent was prepared. Using that reagent in a pretargeting experiment, an equivalent quantity of succinylated recombinant streptavidin as biotinylated antibody Fab' was localized in a tumor xenograft model. In that experiment, the kidney concentration was decreased to less than 10% of that obtained with unmodified recombinant streptavidin at 24 h postinjection. The results of our investigation have demonstrated that succinylation of streptavidin improves its distribution characteristics for pretargeting applications. The fact that succinylated streptavidin has no specific tissue localization should allow its use as a carrier of radioactivity in "two-step" pretargeting protocols.

Polymerization and/or cross-linking of recombinant streptavidin (r-SAv) with biotin derivatives containing two biotin moieties (biotin dimers) or three biotin moieties (biotin trimers) has been investigated as a model for reagents to be used to increase the amount of radioactivity on cancer cells in tumor pretargeting protocols. In the investigation, six biotin dimers and three biotin trimers were synthesized. Most biotin derivatives synthesized had ether containing linker molecules incorporated to improve their aqueous solubility. The synthesized biotin dimers contained linker moieties which provided distances (when fully extended) of 13-49 A between biotin carboxylate carbon atoms, and the biotin trimers contained linker moieties which provided distances of 31-53 A between any two biotin carboxylate atoms. All of the biotin derivatives were evaluated for their ability to polymerize r-SAv in solution. When the biotin derivatives were mixed with r-SAv, none of the biotin dimers caused polymerization, but all of the biotin trimers resulted in complete polymerization. Some of the biotin dimers did cross-link r-SAv (to form r-SAv dimers, trimers, etc.), but the percentage of cross-linking was low (< or = 40%). The length of the linker molecule was important in cross-linking of biotin dimers. While linkers which provided distances of 13 and 19 A between biotin carboxylate carbon atoms did not result in cross-linking, a linker which provided a 17 A distance resulted in a small (< or = 10%) amount of cross-linking. Also cross-linking was increased in biotin dimers with linkers which provided distances between biotin carboxylate carbon atoms of > or = 23 A. Cross-linking of streptavidin bound in polystyrene wells with biotin dimers and trimers was also examined. In those experiments, an excess of each biotin derivative was incubated at 37 degrees C for 10-30 min in polystyrene wells containing bound SAv. After the excess biotin derivative was rinsed from the wells, an excess of r-[125I]SAv was incubated for another 10-30 min. The amount of r-[125I]SAv bound after rinsing the excess from the wells was an indicator of the extent of cross-linking that occurred. The process of alternating additions of reagents was repeated four times to demonstrate that bound radioactivity could be increased with each addition of [125I]SAv. The results of cross-linking r-SAv in polystyrene wells paralleled results from cross-linking in solution.

Analogues of cyanocobalamin (CN-Cbl), with functional groups attached to either the various propionamide groups of the corrin ring or to the ribose-nucleotide linker arm, have been evaluated in a cobalamin (Cbl)-dependent in vitro cell growth assay. In this bioassay, CN-Cbl supported, in a dose-dependent manner, the growth of the murine lymphoma BW5147 and the Cbl carrier protein, human apo-transcobalamin II, reduced the required concentration of Cbl by 100-1000-fold. Any chemical modification of Cbl decreased its ability to support cellular viability and proliferation, with several of the modifications abrogating activity completely. All of the Cbl analogues that promoted growth required the presence of apo-transcobalamin II for the optimal support of cell growth. Generally, Cbl analogues modified at the d-position of the corrin ring and, to a lesser degree, analogues modified at the b- position supported cell growth, whereas analogues with modifications at the e-position did not support cell growth. Mixing experiments demonstrated an inverse order of potency of Cbl analogues to inhibit cell growth. Thus, Cbl analogues with modifications at the e-position were potent inhibitors, whereas b-analogues exhibited only partial inhibitory activity at high molar excess, and d-analogues had no inhibitory activity at all. These results indicate that modifications at the e-position of Cbl abolish the ability of Cbl to support cell growth and generate potent inhibitors of Cbl-dependent cell growth.

As part of our development of antibody pretargeting for cancer therapy, an investigation has been conducted to examine the stability of water solubilized, radioiodinated biotin derivatives toward biotinidase degradation in mouse and human serum. Eight new biotin derivatives were synthesized to conduct the study. The biotin derivatives synthesized contained (1) the biotin moiety, (2) a water solubilizing linker moiety, (3) p-iodobenzoate or p-tri-n-butylstannylbenzoate moieties, and (4) in some of the compounds, N-methyl or alpha-methyl containing moieties were added to block biotinidase activity. The linker moiety, 4,7,10-trioxa-1,13-tridecanediamine, 5, was included in the biotin derivatives to improve their water solubility, and it also functioned as a 17 A spacer between the biotin and the benzoyl moieties. Four of the new biotin derivatives (12, 14, 22, and 23) contained a p-tri-n-butylstannylbenzoyl moiety as precursors which could be radioiodinated in the last synthetic step. The other four biotin derivatives (13, 15, 24, and 25) contained p-iodobenzoyl moieties and were used as HPLC reference standards. Initial studies involved radioiodination of 12 to yield [125I]13. Radioiodinated 13, which did not contain a moiety for blocking biotinidase activity, was found to be rapidly degraded in both mouse and human serum at 37 degrees C. Derivatives which were designed to be stable to biotinidase incorporated N-methyl and alpha-methyl moieties adjacent to the biotin carboxylate group. In one set of biotin derivatives (14 and 15), the N-methyl moiety was obtained by incorporating N,N-dimethyl-4,7,10-trioxa-1,13-tridecanediamine, 9, as a linker in the place of 5. In the second set of biotin derivatives (22 and 24), the N-methyl moiety was introduced by incorporating a sarcosine (N-methylglycine) moiety between biotin and 5. The radioiodinated N-methyl containing biotin derivatives [125I]15 and [125I]24 were found to be very stable to biotinidase degradation. An alpha-methyl group was obtained in a pair of biotin derivatives (23 and 25) by incorporating a 3-aminobutyric acid moiety between biotin and 5. The radioiodinated alpha-methyl containing derivative, [125I]25, was found to have an intermediate stability with regards to biotinidase degradation.

Several cobalamin (Cbl) dimers have been prepared for evaluation as potential antiproliferative agents in the treatment of AIDS-related lymphoma. The Cbl dimers were synthesized by cross-linking Cbl carboxylates, produced by acid hydrolysis of the b-, d-, and e-propionamide side chains of cyanocobalamin (CN-Cbl), through an isophthalate molecule. Linking molecules were used between the Cbl carboxylates and the isophthalate moiety. The linkers were incorporated to provide a distance between the two Cbl molecules such that the dimeric Cbls might bind two molecules of transcobalamin II (TCII), the Cbl transport protein in plasma. Initially, the linking moiety used was 1,12-diaminododecane, but the resulting dimers had low aqueous solubility. To improve the solubility of the dimers, 4,7,10-trioxa-1,13-tridecanediamine was employed as the linking moiety. This improved the water solubility of the dimers considerably, while retaining the distance between the Cbl molecules at 41-42 A (fully extended). To introduce additional substitution on Cbl dimers, 5-aminoisophthalic acid was used as the cross-linking reagent. p-Iodobenzoyl and p-(tri-n-butylstannyl)benzoyl conjugates of 5-aminoisophthalate were synthesized and used to prepare Cbl dimers. The stannylbenzoyl-conjugated Cbl dimers were prepared as precursors to be used in radioiodination reactions, and the iodobenzoyl-conjugated Cbl dimers were prepared as HPLC standards for the radioiodinated product. Attempts to iodinate/radioiodinate the stannylbenzoyl Cbl dimers were unsuccessful. Although an explanation for this is not readily apparent, the failure to react may be due to the lipophilicity of the linker used and the steric environment of the two Cbl moieties. A biotinylated derivative of 5-aminoisophthalate was also synthesized and used to prepare biotinylated-Cbl dimers. In a competitive rhTCII binding assay with [57Co]CN-Cbl, Cbl dimers containing the lipophilic diaminododecane linking moiety had decreased binding avidities compared to those of Cbl monomers substituted at the same corrin ring carboxylate. However, Cbl dimers containing the water-solubilizing trioxadiamine linker appeared to have avidities similar to those of the Cbl monomers.

An evaluation of the use of a biotinylated monoclonal antibody Fab' fragment in tumor pretargeting was conducted. As a model system, tumor colocalization of avidin or recombinant streptavidin (r-streptavidin) and the biotinylated Fab' fragment (Fab'-S-biotin) of A6H, an antirenal cell carcinoma antibody, was evaluated in athymic mice bearing human renal cell carcinoma xenografts. A new water soluble sulfhydryl reactive biotinylation reagent, N-(13-N-maleimdo-4, 7,10-trioxatridecanyl)-biotinamide, was synthesized and used for biotinylation of Fab'. A biodistribution of ChT-labeled A6H Fab'-S-biotin was conducted. Data from that distribution indicated that the Fab'-S-biotin localized well (i.e. 28% ID/g at 24 h) to human tumor xenografts in athymic mice. Subsequently, a biodistribution study involving pretargeting radioiodinated A6H Fab'-S-biotin to tumor xenografts, followed by administration of r-streptavidin at 4 or 20 h, was conducted. Specific colocalization of r-streptavidin to tumors containing the A6H Fab'-S-biotin was evident from the data obtained. In a similar biodistribution study, specific colocalization of avidin to tumors pretargeted with A6H Fab'-S-biotin was also observed. The avidin used in the study was radioiodinated with the N-hydroxysuccinimidyl ester of p-[125I]iodobenzoate ([125I]PIB-NHS). Very low concentrations (e.g. 0.35% ID/g) of avidin colocalized at the tumor. To further show that specific colocalization within the tumor xenografts had occurred with biotinylated A6H Fab', radioiodinated avidin and r-streptavidin were co-injected into athymic mice bearing tumor xenografts to obtain their distributions without having biotinylated Fab' present. At 20 h postinjection, only small differences in the blood and tumor concentrations of either protein were observed, indicating that the specific tumor colocalization seen in the previous two biodistributions must have been due to the presence of Fab'-S-biotin. Calculations were conducted to estimate how much r-streptavidin (as a molar ratio) was colocalized. From the data obtained it was estimated that 36-61% of the tumor-localized Fab'-S-biotin molecules were bound with r-streptavidin and 4-23% bound with avidin, under the conditions studied.

A new method of preparing radiolabeled cobalamin derivatives has been developed. The method involves the use of cobalamin-tri-n-butylstannyl hippurate conjugates as intermediates to obtain radioiodinated cobalamin-iodohippurate conjugates. The arylstannyl functionality was used as an exchangeable group to obtain high specific activity radioiodinations and to circumvent some deleterious side reactions common to cobalamins under electrophilic iodination conditions. The first step in the synthesis of tri-n--butylstannyl hippurate conjugates was to obtain free carboxylate groups on the cobalamin moiety. This was accomplished by mild acid hydrolysis of the b-, d-, or e-propionamide side chains on the corrin ring, followed by careful separation of the isomeric products. The second step was to couple a linking molecule (diaminododecane) to the carboxylate. The final step was to conjugate p-tri-n-butylstannyl hippurate to the cobalamin-diaminododecane adduct. All three isomeric cobalamin-p-tri-n-butylstannyl hippurate conjugates were prepared, as were the corresponding cobalamin-p-iodohippurate conjugates (HPLC standards). Radioiodination reactions were conducted with N-chlorosuccinimide and Na[*I]I in Me OH using conditions previously developed for arylstannylations. However, unlike the previous reactions, a key factor in obtaining the desired radioiodinated cobalamins was that the reaction be conducted under neutral conditions. Isolated yields of 40-65% were obtained for all three cobalamin isomers. Specific activities of 10-33% theoretical were obtained for the radioiodinated cobalamins. Evaluation of competitive binding of (nonradioactive) cobalamin-iodohippurate conjugates with recombinant human transcobalamin II showed that the e-isomer bound nearly as well as [57Co]cyanocobalamin (50%), whereas the b-isomer had decreased binding (6%) and the d-isomer was significantly decreased in its binding (0.7%). Two biodistributions of the radioiodinated e-isomer were conducted in athymic mice. One biodistribution investigated tissue localization in mice bearing a renal cell carcinoma xenograft, and the other biodistribution investigated tissue localization when the radioiodinated cyanocobalamin was mixed with 1% BSA prior to injection. A comparison of the results of the two biodistributions and a discussion of how they relate to previous [57/60Co]cyanocobalamin biodistributions are provided.

Six cobalamin-biotin conjugates have been prepared. The cobalamin-biotin conjugates were prepared to evaluate the effect that the location of attachment had on the binding with transcobalamin II (TCII), the cobalamin binding protein in plasma, and to evaluate their potential use for in vitro and in vivo applications. This study focused only on the effect of binding with TCII. To decrease the possibility of steric problems in binding of the cobalamin conjugates with TCII, and biotin's binding with streptavidin or avidin, moieties of 11-18 atoms in length were used as linkers. Four biotin conjugates were prepared which were attached to the corrin ring of the cobalamin molecule (on b-, c-, d-, and e-side chains). One conjugate was attached to the 5'-OH of the ribose moiety, and another conjugate was attached at the cobalt metal (in place of the cyanide moiety of cyanocobalamin). Competitive binding studies were conducted where various amounts of the cobalamin-biotin conjugates and their precursor cobalamin derivatives competed with [57Co]cyanocobalamin for binding of recombinant human TCII (rhTCII). Evaluation of cobalamin derivatives which were conjugated at the 5'-OH of ribose or the cobalt metal center indicated that conjugation at either of these positions had little effect on binding with rhTCII. However, conjugates where the attachment was made on the corrin ring substituents had a large variation in binding with rhTCII. Conjugates on the e-propionamide side chain had little effect (relative affinity was equal to or decreased less than a factor of 3) on binding with rhTCII, conjugates of the b-isomer had decreased binding (relative affinity decreased less than a factor of 10), conjugates of the d-propionamide had further decreased binding (relative affinity decreased between 44 and 69 times), and conjugates on the c-acetamide group had poor binding to rhTCII (relative affinity decreased between 295 and 1160 times). The significance of the side chains on the corrin ring in providing specificity and high-affinity binding with rhTCII is discussed.

One hundred eighty-six breast cancer patients with either T1 or T2 primary tumors and negative axillary lymph nodes (N-) underwent only local therapy at Norwalk Hospital from 1978-83. No adjuvant chemotherapy or hormone therapy was given for this favorable group of patients. The actuarial five-year disease free (NED) survival rate for these (N-) patients was 91% (+/- 3%, 95% confidence limits) and the ten-year NED survival rate was 79% (+/- 8%). These NED survival rates are vastly superior to the NED survival rates quoted by the National Cancer Institute (NCI) Clinical Alert for (N-) breast cancer patients who received no adjuvant therapy (67%-77% at three to four years median follow-up time). The NCI Clinical Alert is based on three clinical trials which included (N-) patients with larger tumors than T1 or T2 and excluded patients with small primary tumors. Based on these three clinical trials which demonstrated improved NED survival for (N-) patients treated with adjuvant therapy compared to nontreated patients, the NCI Clinical Alert recommended that adjuvant therapy be considered for all (N-) patients. We conclude that it is premature to recommend that all (N-) breast cancer patients should routinely receive adjuvant therapy until more information is available.