formycin-b and allopurinol-riboside

We have developed a simple and reproducible system for infecting a human macrophage cell line (U937) with stationary-phase Leishmania donovani promastigotes. Four days after infection, greater than 90% of the promastigotes had transformed to amastigotes. The antileishmanial agents allopurinol riboside, formycin B, 9-deazainosine, and sodium stibogluconate effectively inhibited the growth of L. donovani amastigotes in this cell line. To study the capability of amastigotes in the U937 cell line to carry out biochemical reactions that could be monitored experimentally, we incubated the cells with radiolabeled 9-deazainosine. This purine analogue underwent metabolism in the amastigote phase similar to that occurring in the promastigote phase. This cell line should be useful for studies of parasite maturation and differentiation, parasite-human interactions, and antiparasitic drugs.

Topics: Allopurinol; Animals; Antimony Sodium Gluconate; Antiprotozoal Agents; Cell Line; Formycins; Humans; Inosine; Leishmania donovani; Macrophages; Ribonucleosides

Three analogs of inosine, formycin B, allopurinol ribonucleoside, and 9-deazainosine, were tested for their ability to suppress proliferation of Pneumocystis carinii in culture with WI-38 cells. The organism was inhibited by 9-deazainosine at 10 micrograms/ml, and there was some inhibition at 1 microgram/ml. Formycin B was effective only at 40 micrograms/ml. Allopurinol ribonucleoside had little effect.

Topics: Allopurinol; Animals; Antiprotozoal Agents; Cells, Cultured; Drug Combinations; Formycins; Inosine; Microbial Sensitivity Tests; Pneumocystis; Rats; Ribonucleosides; Sulfamethoxazole; Trimethoprim; Trimethoprim, Sulfamethoxazole Drug Combination

The anti-Trypanosoma cruzi effect of allopurinol ribonucleoside and formycin B was examined against infections of the sensitive Y and Peru strains in inbred mice, strain DBA/1. Allopurinol ribonucleoside given in the drinking water at doses calculated to be 239, 511 and 929 mg/kg/day for 28 days, prevented the death of the mice but did not eradicate the infection. Formycin B given orally at 100 and 10 mg/kg/day X 5 days, showed a similar effect.

Topics: Allopurinol; Animals; Antibiotics, Antineoplastic; Chagas Disease; Formycins; Mice; Mice, Inbred DBA; Ribonucleosides; Trypanocidal Agents

Allopurinol and allopurinol ribonucleoside tested in vitro and in vivo for activity against Leishmania donovani. Activity in vitro was low against the amastigote form of this parasite with ED50 values of the order of 54 and 96 microM and 86 and 213 microM respectively for the two compounds. In vivo inhibition of up to 47% was achieved with allopurinol ribonucleoside given in the drinking water. However, low blood levels were found in the mouse relative to those in man. Low in vivo activity was also seen with allopurinol ribonucleoside against L. major and other species of Leishmania causing cutaneous lesions. The metabolism of allopurinol ribonucleoside in aldehyde oxidase deficient mice (inbred strains DBA/1, DBA/2) resembled that of man, but the antileishmanial activity remained low. Other compounds, formycin B, sinefungin and the lepidine WR6026 were highly active against mice infected with L. donovani or L. major.

Topics: Adenosine; Allopurinol; Aminoquinolines; Animals; Antiprotozoal Agents; Cricetinae; Female; Formycins; Leishmania; Leishmaniasis; Leishmaniasis, Visceral; Macrophages; Mice; Mice, Inbred Strains; Ribonucleosides; Thionucleosides

Formycin B is cytotoxic toward Leishmania and is a potential chemotherapeutic agent for leishmaniasis. In order to determine the mechanism of action of formycin B, we have isolated and characterized clonal populations of formycin B-resistant Leishmania donovani. These formycin B-resistant clones are also cross-resistant to formycin A and allopurinol riboside-mediated growth inhibition. Incubation of the formycin B-resistant cells with [3H]formycin B indicates that, unlike wild type cells, the resistant populations cannot accumulate phosphorylated metabolites of exogenous [3H]formycin B. This is due to a defective transport system for formycin B in the resistant cells. However, wild type and mutant cells incorporate [3H]formycin A equally efficiently into [3H]formycin A-containing nucleotides and into RNA. These data suggest that formycin B cytotoxicity in Leishmania is not mediated by its incorporation as the adenosine analog into RNA. A plausible alternative hypothesis is proposed for the mechanism of action of the pyrazolo (4,3-d)pyrimidine C-nucleosides based upon depletion of an essential intracellular metabolite.

Topics: Allopurinol; Animals; Antibiotics, Antineoplastic; Cell Division; Chromatography, High Pressure Liquid; Drug Resistance; Formycins; Leishmania; Purines; Ribonucleosides; RNA

Strains of Trypanosoma cruzi differ in their susceptibilities to and metabolism of pyrazolopyrimidines. Allopurinol riboside can control but not eliminate infections with a sensitive strain in both tissue culture and mice. Formycin B, which proved to be greater than 10-fold more effective on a weight basis, showed a similar strain specificity but could eliminate an infection with a sensitive strain from tissue culture. However, this drug, unlike allopurinol riboside, was converted to toxic analogues of adenosine mono-, di-, and triphosphate by uninfected tissue culture cells. Thiopurinol and its riboside were effective against all strains unless culture was performed in purine-defined medium. Thus formycin B and allopurinol riboside appear to be good models for the design of antitrypanosomal agents. Suitable modification of the molecule may provide an effective chemotherapeutic agent.

Topics: Adenine; Allopurinol; Animals; Antiprotozoal Agents; Chagas Disease; Drug Resistance; Formycins; Inosine; Mice; Mice, Inbred DBA; Ribonucleosides; Thionucleosides; Trypanosoma cruzi

Formycin B 5'-monophosphate (Form B-MP) and allopurinol riboside 5'-monophosphate ( HPPR -MP) are isomers of IMP that are metabolically produced when Leishmania spp. are incubated with the antileishmanial agents formycin B and allopurinol or allopurinol riboside. The interactions of Form B-MP with succino -AMP synthetase and GMP reductase from both leishmanial and mammalian sources were compared with the data of earlier studies with HPPR -MP. Both analogs could substitute for IMP as a substrate for succino -AMP synthetase isolated from Leishmania donovani. The V'max values of Form B-MP and HPPR -MP were about 1% of the V'max of IMP. Only Form B-MP (and not HPPR -MP) could serve as an alternative substrate for mammalian succino -AMP synthetase. The V'max of Form B-MP was 40% that of IMP. The corresponding analogs of AMP, ADP and ATP were produced when Formycin B was incubated with mouse L cells. The Formycin A residue was incorporated into the cellular RNA. The amount of Formycin A-TP produced (relative to ATP) in mouse L cells was considerably less than that produced in Leishmania spp. Both Form B-MP and HPPR -MP were inhibitors of partially purified GMP reductase from L. donovani. The binding of Form B-MP and HPPR -MP to human GMP reductase was 40- and 100-fold weaker, respectively, than the binding to leishmanial GMP reductase. Pretreatment of promastigotes of L. donovani with either allopurinol or Formycin B resulted in greater than 95% reduction of the incorporation of the radiolabel from [14C]xanthine into ATP and greater than 80% reduction of the incorporation of the label into GTP. The HPPR -MP and Form B-MP present in these cells may have inhibited the leishmanial succino -AMP synthetase and GMP reductase. The analogs had little or no effect on the pool sizes of ATP and GTP of either mouse L cells or L. donovani.

Topics: Adenylosuccinate Synthase; Allopurinol; Amination; Animals; Antibiotics, Antineoplastic; Antiprotozoal Agents; Formycins; GMP Reductase; Humans; Inosine Monophosphate; Kinetics; L Cells; Leishmania; Ligases; NADH, NADPH Oxidoreductases; Ribonucleosides; Ribonucleotides

The pathogenic hemoflagellates of the genera Leishmania and Trypanosoma are major causes of human disease in the tropical and subtropical areas of the world. In general, the agents used to treat diseases caused by these organisms are toxic and not suitable for administration to the millions of people infected. Investigations over the past several years have shown that there are several major differences between man and these protozoans with respect to purine metabolism. The differences appear to offer promise for the development of effective chemotherapeutic compounds. These organisms do not synthesize purines de novo, as does man. They are able to concentrate pyrazolopyrimidines with the cell and metabolize them as purines through the salvage pathways, ultimately incorporating them into nucleic acids. This does not occur in mammals. The pyrazolopyrimidine base allopurinol, which has served as a prototype, is activated by a phosphoribosyltransferase to the ribonucleotide. The ribonucleotide is aminated to the 4-amino-pyrazolopyrimidine ribonucleotide and subsequently phosphorylated to the triphosphate form and incorporated into RNA. The pyrazolopyrimidine ribonucleosides formycin B and allopurinol ribonucleoside are activated through a nucleoside phosphotransferase. The resulting ribonucleotide is aminated and incorporated into RNA as described above. These metabolic peculiarities occur not only in the forms of these parasites which are found in the insect vectors but also in the intracellular forms which are pathogenic in man. The differences in the enzymology and metabolism of purines which exist in the genera Leishmania and Trypanosoma offer excellent opportunities for chemotherapeutic exploitation.

Topics: Adenine; Allopurinol; Animals; Formycins; Host-Parasite Interactions; Leishmania; Ribonucleosides; Thionucleosides; Trypanosoma cruzi