Carboxymethyldextran lactone: a preactivated polymer for amine conjugations.

The linking of amino haptens to carboxymethyldextran (CMD) requires carboxyl activation, for example, via carbodiimdes. We have discovered that substantial N-acylurea, derived from these carbodiimides, can be trapped on the CMD backbone. As an alternative, CMD can be conveniently lactonized by heating in inert solvents, and this carboxymethyldextran lactone (CLD) can be employed directly for amine conjugation.

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.

An enzymatic cycling procedure for beta-NADP+ generated by 3'-phosphodiesterase, 2':3'-cyclic nucleotide.

An enzymatic cycling procedure for beta-NADP+ generated by the enzyme 3'-phosphodiesterase, 2':3'-cyclic nucleotide (EC 3.1.4.37) from its substrate 2':3'-cyclic NADP+ is described. The enzymes glucose-6-phosphate dehydrogenase (EC 1.1.1.49) and diaphorase (EC 1.8.1.4) are used to cycle the cofactor between its oxidized and reduced forms in the presence of glucose-6-phosphate and p-iodonitrotetrazolium violet (INT) with the concomitant production of colored INT-formazan, monitored at 492 nm. The amplification is about 400-fold per hour and is sensitive enough to detect 6 x 10(-13) mol of NADP(H). A simple procedure for the optimization of this cycling assay is also described. Conjugates to 3'-phosphodiesterase, 2':3'-cyclic nucleotide may be used in heterogeneous enzyme immunoassays for the detection of small quantities of haptens or proteins in biological fluids.

A novel heterobifunctional linker for formyl to thiol coupling.

A maleimide hydrazide has been synthesized as a heterobifunctional cross-linking agent for thiol to formyl coupling. This linker has been applied to the coupling of the monoclonal antibody 17-1A, or an Fab' derived therefrom, to polyaldehyde dextran onto which the antineoplastic agent ellipticine has been attached. High binding avidities for the unshed antigen on the SW1116 colorectal tumor cell are retained in these drug-dextran-linker-antibody conjugates.

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.

Determination of degree of substitution of formyl groups in polyaldehyde dextran by the hydroxylamine hydrochloride method.

Colorimetric or potentiometric titration of the aldehyde residues in polyaldehyde dextran by the hydroxylamine hydrochloride/sodium hydroxide method has been found to be a convenient and accurate method to determine formyl content. Nitrogen combustion analyses on the isolated oximes confirmed the titrametric results.

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.

Misonidazole conjugates of the colorectal tumor associated monoclonal antibody 17-1A.

The radiation sensitizer misonidazole has been linked to the monoclonal antibody 17-1A which recognizes a nonshed antigen of a human gastrointestinal tumor. Linkage was accomplished through a hemisuccinate of misonidazole attached by a mixed anhydride coupling and gave a conjugate whose plasma half-life (for drug cleavage) was ca. 70 h. The degree of substitution on the antibody could be precisely regulated by varying the reactant ratios. The binding avidities of the resulting conjugates to the SW1116 colorectal tumor cells decrease logarithmically with increasing drug load. Four to six misonidazoles per antibody represented the optimum drug loading on this system. Enzymatic cleavage of the conjugate-drug union took place at both the ester and the amide linkages with the former scission predominating.

Hydrazide pharmaceuticals as conjugates to polyaldehyde dextran: syntheses, characterization, and stability.

The coupling of ellipticine and CI-921, two antineoplastic pharmaceuticals, to polyaldehyde dextran can be carried out under mild reaction conditions with novel acid hydrazides of the parent drugs. The resulting acyl hydrazones resist hydrolytic and enzymatic cleavage and offer a method for drug loading via dextran conjugates to monoclonal antibodies.

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.

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.

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.

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.

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.

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.