Radiosensitizer conjugation to the carcinoma 19-9 monoclonal antibody.

Misonidazole was covalently conjugated (3-68 mol drug/mol antibody) to 19-9 monoclonal antibody directed against a colorectal carcinoma tumor-associated antigen as a method for targeting radiosensitizing agents. This attachment was accomplished by the mixed anhydride method using the hemisuccinate derivative of misonidazole. Evaluation of conjugates in vitro shows a loss of antibody binding activity with increasing loading levels; however, significant binding activity is retained even at relatively high sensitizer/antibody ratios. This observation was consistent in three binding assays: a competitive radioimmunoassay; an enzyme immunoassay; and an affinity column assay. From these studies, it was concluded that the optimal loading factor for misonidazole-antibody conjugates, when it is used for immunochemotherapy lies between 8 and 15. In vitro release studies indicated that conjugates are hydrolytically stable (t1/2 = 4 days) under physiological conditions.

Selective inactivation of lymphocytes after psoralen/ultraviolet light (PUVA) treatment without affecting systemic immune responses.

Psoralens, together with ultraviolet light A (PUVA), are used for the treatment of several proliferative diseases. It has been suggested that the effectiveness of this treatment is due to induction of a specific immune response by PUVA-treated lymphocytes that down-regulates untreated cells of the same specificity. The present studies show that the clinically used psoralen analogue 8-methoxypsoralen (8-MOP), as well as 4,8-dimethyl-5'-(pyridiniummethyl)psoralen bromide (4NQ), a derivative that does not cross the cell membrane, when activated by UVA light, render lymphocytes unresponsive to mitogenic, antigenic, or phorbol myristate acetate + ionomycin-induced stimulation. This state of unresponsiveness was not reversed by exogenous recombinant interleukin-2. Treatment of lymphocytes with 4NQ and UVA light had no effect on the viability or the homing pattern of the cells to spleen or liver, whereas 8-MOP induced toxicity in about 50% of cells after 3 days in culture. Using an adoptive transfer mouse model, we found that antigen-specific lymphocytes treated with PUVA (8-MOP or 4NQ) had no effect on the ability of these mice to respond to a subsequent challenge with the same or an irrelevant antigen. This was tested by measuring the T cell proliferative responses of lymph node cells from mice primed with keyhole limpet hemocyanin (KLH) or fowl gamma globulin (FGG) before or after adoptive transfer of large numbers of KLH- or FGG-specific PUVA-treated lymph node cells. No effects of antigen-primed PUVA-treated cells on antigen-primed untreated cells were observed in vitro in mixed lymphocyte cultures responding to the relevant antigen. These results suggest that, in our model system, PUVA induces cell inactivation but does not affect systemic T cell responses.

Biodistribution and pharmacokinetics of S-35-labeled 5-thio-D-glucose in hamsters bearing pancreatic tumors.

5-thio-D-glucose (5-TDG) exerts profound effects on rapidly metabolizing tissues. We have used liquid scintillation counting to study the tissue distribution and pharmacokinetics of S-35-labeled 5-TDG in hamster models of pancreatic tumors. In the normal hamster, initial uptake of S-35 activity into kidney, liver, and blood was high, but rapidly decreased with time. The pancreatic uptake (% dose/g) never exceeded 0.75%. This level occurred only at the earliest times after administration. Uptake in all three tumor models exceeded that in pancreatic tissue within 15 min of injection. The highest tumor-to-pancreas ratio was seen in the duct-tumor model, which also exhibited the most favorable tumor-to-tissue ratio when compared with kidney, liver and muscle. Favorable ratios were most pronounced at 6 and 24 hr after injection. These studies provide impetus for the use of 5-TDG as a model compound for the synthesis of potentially useful agents for clinical detection of pancreatic tumors.

Radiosensitization of human colorectal tumor cells by 4-nitro-5-sulfonatoimidazoles.

Misonidazole, a clinically-effective 2-nitroimidazole hypoxic cell radiation sensitizer, and 12 4-nitro-5-sulfonatoimidazoles were tested in cultured human SW1116 colorectal adenocarcinoma cells for radiosensitizing efficiency. Octanol-water partition coefficients and HPLC capacity factors were determined for all agents as measurements of lipophilicity, and an excellent correlation was found between the two measurements. Cytotoxicity, in vitro glutathione reactivity, and one-electron reduction potential were also determined for each compound to evaluate potential utility as macromolecularly transported radiosensitizers. Ten members of the set were found to be 40 to 300 times more radiotoxic than misonidazole, but no correlation was found between their radiosensitizing efficiencies and the chemical and physical parameters.

Synthesis and antibacterial and anticancer evaluations of alpha-methylene-gamma-butyrolactones.

Nine new alpha-methylene-gamma-butyrolactones were synthesized by the Reformatsky condensation of ethyl alpha-bromomethylacrylate with ketones, aldehydes, and an epoxide. A unique spirobutyrolactone class was prepared by reaction of the zinc alkyl derivative and N-methylisatins. The compounds were evaluated against L-1210 and P-388 leukemia and the 9KB carcinoma of the nasopharynx. They also were screened in a microbiocidal and an antifungal assay. The spiro methylene lactone of 5-iodo-N-methylisatin displayed activity in the P-388, 9KB, and antifungal screens.

Cyanogen condensations as a route to 3-amino-2-imino-1,3-benzothiazin-4-ones with CNS depressant potential.

Seven new 3-amino-2-imino-1,3-benzothiazin-4-ones, prepared from the condensation of cyanogen and 2-mercaptobenzhydrazides, were evaluated in a neuropharmacological mouse profile. CNS depression was observed in several members of the class.

Alloxan analogues as potential pancreatic-imaging radiopharmaceuticals.

Three families of alloxan derivatives, 5-arylthiobarbituric, 5-aryliminobarbituric, and 5-aryldialuric acids, were prepared as prospective radioiodine-transporting radiopharmaceuticals for the delineation of pancreatic insulinomas. Members of each class were screened for effects on blood sugar levels in a rat glucose tolerance assay. Transient hyperglycemia was observed with 5-(2,4-dichlorophenyl)iminobarbituric acid. No agent evaluated induced permanent diabetes at the doses tested.

Effect of liposome and cyclodextrin entrapment on retardation of glutathione decomposition of nitroimidazolyl sulfones.

A new class of radiosensitizing pharmaceuticals derived from 4-nitro-5-imidazolyl sulfones has its clinical potential compromised by a metabolic reaction with plasma glutathione which leads to a much less active metabolite. Entrapment of two members of the class in three different liposomes, one polymerized liposome, and a beta-cyclodextrin system has shown that this glutathione condensation can be suppressed by a rate factor of nearly 50-fold. Stabilizations of these metabolically labile radiosensitizers appear to relate to their lipid-buffer partition coefficients and to the fluidity of the liposome membrane in which they are entrapped.

New synthesis of 1,4,2-dioxazoles and their pharmacological properties.

A series of 3-substituted 5-methoxycarbonyl-5-methoxycarbonylmethyl-1,4,2-dioxazoles was prepared by addition of hydroxamic acids to acetylene esters. Eleven of these previously unknown compounds were submitted to general pharmacological screening, and several displayed modest CNS depressant activity.

Synthesis and evaluation of an 111In-labeled porphyrin for lymph node imaging.

111In-labeled tetra(4-N-methylpyridyl)porphyrin was investigated as a possible lymph node imaging agent. A clinically feasible method for the preparation of this radioactive pharmaceutical was developed from experiments with the synthesis and characterization of the unlabeled complex. The in vivo distribution of the compound in Wistar rats was determined as a function of time. Favorable lymph node-blood, lymph node-muscle, and specific lymph node-surrounding tissue ratios were obtained.

Macromolecular attachment as a metabolic stabilizer for a labile radiosensitizer.

4-Nitro-5-sulfonylimidazoles represent a class of hypoxic cell radiation sensitizers whose in vitro activity greatly exceeds that of the current clinical standard, misonidazole. However, in vivo studies with these 4,5-disubstituted imidazoles have shown that a rapid reaction with circulating thiols decomposes the agent and compromises its clinical utility. Drug-macromolecule conjugates prepared in this study from poly-L-glutamate, succinylated poly-L-lysine, dextran, and a succinylated polylysine-antibody, all demonstrated protection of the drug from glutathione displacement. 1-Methyl-4-nitro-5-imidazolyl 4-aminophenyl sulfone conjugated to a succinylated poly-L-lysine-antibody was greater than 7 times less reactive than the unbound drug. Polymer transport may offer a useful tumor-delivery mechanism for these highly reactive radiation sensitizers.

Syntheses and tissue distribution of 99m-Tc-sulfonylureas.

Four new tolbutamide analogs were prepared and evaluated as hypoglycemics. Hypoglycemic action was observed in two members of the class, and 99m-Tc-chelates were tested as potential radiopharmaceutical imaging agents.

Syntheses of psoralen analogues and evaluation of their inhibition of epidermal growth factor binding.

An exchange hydrogenation reaction on trioxsalen yields two isomeric dihydro analogues for which a combined HPLC-supercritical fluid chromatography method supplies purified materials. These reduced compounds and a related benzodipyranone are biologically active and inhibit the binding of epidermal growth factor on HeLa cells to an even greater extent than trioxsalen. This observation suggests a non-DNA target may play a role in the overall effects of psoralens on cells.