Henry C. Nathan

Potentiation by inhibition of drug degradation : 6-substituted purines and xanthine oxidase

Abstract The administration of the xanthine oxidase inhibitor. 4-hydroxypyrazolo (3, 4-d)pyrimidine, concurrently with 6-mercaptopurine, results in a marked decrease in the metabolic oxidation of the latter to 6-thiouric acid in both the mouse and man. The inhibition of metabolic degradation by this means results in several-fold potentiations of 6-mercaptopurine, 6-methylthiopurine, 6-propylthiopurine, and 6-chloropurine in trials against adenocarcinoma 755 and of 6-mercaptopurine and 6-chloropurine as inhibitors of the immune response of mice to sheep erythrocytes.

Detection of Agents which Interfere with the Immune Response

Summary A screening test is described for materials which suppress the immune response during its inductive phase. This test is based on the formation of hemagglutinins to sheep red blood cells in mice. 6-Mercap-topurine, 6-thioguanine and their S-imidazolyl derivatives, B.W. 57-322 and B.W. 57-323, show high activity. The authors wish to acknowledge their indebtedness to Dr. Robert Schwartz for the suggestion which stimulated this investigation, and to thank Dr. R. Y. Calne and Dr. A. W. Frisch for the opportunities to read their papers in advance of publication.

Differential sensitivity of Trypanosoma brucei rhodesiense isolates to in vitro lysis by arsenicals

Clinical isolates of Trypanosoma brucei rhodesiense, which were resistant to arsenical drugs in murine infections, were examined for resistance in vitro. A rapid lysis assay was developed which was able to predict in vivo sensitivity to melarsoprol (Mel B, Arsobal) and melarsen oxide. The assay was based on the finding that long slender bloodforms of drug-sensitive isolates would lyse in the presence of arsenicals upon incubation in heat-inactivated fetal bovine serum. On the basis of plots of decrease in the absorbance of trypanosome suspensions vs time of incubation with drug, L50 values, reflecting the drug concentration necessary for lysis of 50% of the cells within 30 min. were calculated for five strains. These values ranged from less than 30 microM for arsenical-sensitive strains to greater than 75 microM in proven arsenic refractory isolates. Calcium was essential for lysis, and the presence of the Ca2+ chelator EGTA (10 mM) in serum delayed lysis of sensitive strains. Ca2+ channel antagonists (Verapamil, Diltiazem), however, did not enhance lysis of refractory isolates when used at 20 to 30 microM. Intracellular concentrations of reduced trypanothione, the apparent target of arsenicals, were similar for all isolates, approximately 1.02 +/- 0.28 nmol/10(8) cells, as detected by monobromobimane derivitization and HPLC analysis. Uptake of melarsen oxide was found to be reduced in arsenical refractory strains. Uptake was judged by reduction of free reduced trypanothione as a result of formation of the trypanothione-arsenic complex Mel T. Little change was found in arsenical-resistant strains, but sensitive strains had 50 to 70% reductions in trypanothione levels after incubation with a low (1 microM) level of melarsen oxide.

Combination chemotherapy of drug-resistant Trypanosoma brucei rhodesiense infections in mice using DL-alpha-difluoromethylornithine and standard trypanocides.

Combinations of DL-alpha-difluoromethylornithine (DFMO; eflornithine; Ornidyl) with either suramin or melarsen oxide were found to be effective against acute laboratory model infections with Trypanosoma brucei rhodesiense. We used clinical isolates known to be resistant to these drugs when used singly. An infection with a melarsen oxide-refractory isolate was cured by a combination of low-dose DFMO (0.5% in the drinking water) plus low-dose suramin (1 mg/kg of body weight given intraperitoneally). Another strain, moderately resistant to arsenical drugs, was cured with combinations of 4% DFMO with 5 mg of melarsen oxide per kg. Furthermore, a combination of DFMO (2% in the drinking water) and suramin (20 mg/kg) provided a 100% cure rate in a central nervous system model, although the same doses of these drugs used singly were completely ineffective. The synergism of DFMO and suramin against an acute infection was improved when suramin was given at the end of the DFMO administration. No adverse interactions were observed when high doses of DFMO combined with high doses of suramin were administered to uninfected mice. These results suggest that combinations of DFMO and suramin should be examined clinically for activity in arsenical-drug-refractory cases of East African sleeping sickness.

Synergism between 9-deazainosine and DL-alpha-difluoromethylornithine in treatment of experimental African trypanosomiasis.

Kinetoplastid hemoflagellates are sensitive to growth inhibition by various purine analogs. In this study the activities of 9-deazainosine (9-DINO), formycin B, and sinefungin were compared in experimental murine Trypanosoma brucei subsp. brucei infections, both singly and in combination with the ornithine decarboxylase inhibitor DL-alpha-difluoromethylornithine (DFMO, eflornithine). Used singly, all of the purine analogs were able to suppress an acute T. brucei subsp. brucei infection. 9-DINO and formycin B were the most active. None of the purine analogs was curative when used singly against a strain causing chronic central nervous system infection. 9-DINO was highly effective when used in combination with DFMO in curing this central nervous system infection and another more stringent experimental infection. Neither sinefungin nor formycin B was active in combination with DFMO in curing the central nervous system experimental infection. 9-DINO was metabolized to phosphorylated derivatives of 9-deazaadenosine and 9-deazaguanosine by bloodstream trypomastigotes, but not by murine erythrocyte suspensions or kidney or liver homogenates--a potential rationale for the selectivity of the analog. These studies indicate that 9-DINO is a potent, nontoxic purine analog which, in combination with DFMO, is capable of late-stage cures of African trypanosomiasis.

The effects of 6-mercaptopurine on Lactobacillus casei

Abstract The inhibitory action of 6-mercaptopurine has been shown to be manifested by an interference with purine interconversion. The incorporation of adenine and of adenylic acid are affected in different ways by 6-mercaptopurine. These facts have been fitted to a schematic representation of a purine interconversion cycle compatible with the known metabolic behavior of L. casei.

Suppression of the immune response by drugs in combination.

Summary Combinations of purine with pyrimidine analogs were tested for their effects on formation in mice of hemagglutinins to red blood cells. Greater than additive effects were obtained with 6-mercaptopurine and thioguanine in combination with 5-bromodeoxyuridine, 4-thiodeoxyuridine, 5-acetoxyuracil and 6-azauracil plus urethan. 5-Bromodeoxyuridine is notable for the magnitude of the ratio of maximum tolerated dose to minimum effective dose.

Polyamines antagonize both the antileukemic activity and the reverse transcriptase stimulatory activity of 4,4′-diacetyldiphenylurea bis(guanylhydrazone) (DDUG)

(Diacetyldiphenylurea)bis(guanylhydrazone) (DDUG) functions as a cationic trypanocide antagonized in vivo by exogenous concomitant addition of the biologically active polyamine, spermine. It also inhibits the DNA polymerases of L1210 murine leukemia cells. We have found that DDUG stimulates Rauscher murine leukemia virus DNA polymerase activity in a manner similar to polyamines. Such stimulation does not occur if DNA synthesis is carried out on spermine + activated DNA complexes. We also show that the in vivo antileukemic activity of DDUG in the L1210 ascites mouse model is antagonized by biologically active polyamines. These studies suggest a new intracellular target for the antileukemic activity of DDUG: interference with polyamine function.