RESUMO
Intact Eimeria tenella sporozoites and merozoites did not incorporate radiolabeled formate or glycine into their purine nucleotides suggesting a lack of de novo purine synthesis. However, [U-14C]glucose was incorporated into the cellular purine and pyrimidine nucleotide pools of both forms probably via conversion to radiolabeled ribose-1-phosphate and/or 5'-phosphoribosyl-1-alpha-pyrophosphate and the resulting action of various purine and pyrimidine salvage enzymes. Both forms of the parasite salvaged radiolabeled purine bases and nucleosides in a similar fashion. These purines were incorporated into ribonucleotides and into RNA and DNA. Adenine and inosine were transformed to hypoxanthine. Adenosine was converted to both inosine and hypoxanthine. Hypoxanthine and xanthine were not oxidized to uric acid but were metabolized to nucleotides. Guanosine was cleaved to guanine; guanine was deaminated to xanthine. The results demonstrate the presence of several purine salvage pathways. Purine phosphoribosylating and nucleoside phosphorylating activities as well as purine nucleoside cleaving and adenosine, adenine and guanine deaminating activities were evident. The metabolic evidence suggests the enzymes required to convert the newly formed nucleoside monophosphates to ATP and GTP were present also.
Assuntos
Eimeria/metabolismo , Purinas/metabolismo , Animais , Radioisótopos de Carbono , Glucose/metabolismo , Nucleosídeos de Purina/metabolismo , Nucleotídeos de Purina/metabolismo , RNA/biossínteseRESUMO
Culture forms of Trypanosoma cruzi are incapable of synthesizing purines de novo from formate, glycine, or serine and require an exogenous purine for growth. Adenine, hypoxanthine, guanine, xanthine and their respective ribonucleosides are equal in their abilities to support growth. Radiolabeled purine bases, with the exception of guanine, are stable and are converted to their respective ribonucleotides directly by phosphoribosyltransferase activity. Guanine is both converted to its ribonucleotide and deaminated to xanthine. Purine nucleosides are not hydrolysed to any extent but are converted to their respective ribonucleotides. This conversion may involve a rete-limiting ribonucleoside cleaving activity or a purine nucleoside kinase or phosphotransferase activity. The apparent order of salvage efficiency for the bases and their respective ribonucleosides is adenine greater than hypoxanthine greater than guanine greater than xanthine.
Assuntos
Purinas/metabolismo , Trypanosoma cruzi/metabolismo , Adenina/metabolismo , Animais , DNA/biossíntese , Guanina/metabolismo , Hipoxantinas/metabolismo , Nucleosídeos de Purina/metabolismo , Nucleotídeos de Purina/metabolismo , RNA/biossíntese , Xantinas/metabolismoRESUMO
Guanine arabinoside (araG) inhibited the in vitro growth of Giardia lamblia WB with an ED50 value of 4 microM. The inhibition was prevented completely by 2'-deoxyguanosine, prevented partially by guanine and guanosine, and not prevented by adenine, adenosine or 2'-deoxyadenosine. Extracts of G. lamblia grown in the presence of [8-3H]araG contained radiolabeled araGMP, araGDP and araGTP. The formation of araGTP during the exponential phase of cell growth increased with time and was dependent upon the araG concentration. AraG was incorporated into G. lamblia DNA in a time-dependent manner at a ratio of 1 araG for each 27 2'-deoxyguanosine residues. Short-term exposure of growing cultures to araG was inhibitory to DNA synthesis but not to RNA or protein synthesis. Over an extended period, synthesis of all three macromolecules was depressed. Attempts to measure araG phosphorylation by cell-free extracts of G. lamblia under a variety of nucleoside kinase and nucleoside phosphotransferase assay conditions were unsuccessful. In an attempt to understand further the action of araG, the metabolic pathways of guanine, guanosine and 2'-deoxyguanosine were delineated in detail. The presence of araG did not appear to cause any major alterations in the metabolism of these compounds; however, it was accompanied by a 3- to 4-fold increase in the endogenous pools of ATP and GTP.
Assuntos
Arabinonucleotídeos/farmacologia , Giardia/efeitos dos fármacos , Guanosina Trifosfato/análogos & derivados , Adenina/farmacologia , Adenosina/farmacologia , Animais , Replicação do DNA/efeitos dos fármacos , Desoxiadenosinas/farmacologia , Desoxiguanosina/farmacologia , Giardia/crescimento & desenvolvimento , Guanina/farmacologia , Guanosina/farmacologia , Guanosina Trifosfato/farmacologia , Biossíntese de Proteínas , RNA/biossínteseRESUMO
Leishmania donovani, grown in culture, salvaged radiolabeled purine bases which were distributed into adenine and guanine ribonucleotides and into the RNA of these cells. De novo synthesis of purines in L. donovani does not occur [J. J. Marr, R. L. Berens and D. J. Nelson, Biochim. biophys. Acta 544, 360 (1978)]. [8-14C]Adenine was rapidly deaminated to hypoxanthine via the action of an adenine aminohydrolase (EC 3.5.4.2). [8-14C]Guanine was also rapidly deaminated by guanase (EC 3.5.4.3) to form zanthine in these cells. Therefore, the formation of nucleotides of hypoxanthine and xanthine are the first committed steps of purine salvage in L. donovani. While purines are efficiently conserved by this parasite, the salvage of pyrimidines is not so dramatic. [2-14C]Orotic acid was converted to OMP and then incorporated into the pyrimidine nucleotides and into RNA, indicating the existence of the later steps of de novo pyrimidine synthesis. [6-14C]Thymidine was salvaged by L. donovani, being incorporated into the thymine deoxyribonucleotides and into DNA. The major pathway of thymidine metabolism in this parasite, however, was cleavage of the deoxyriboside linkage to form thymine, probably via the action of a thymidine phosphorylase (EC 2.4.2.4).
Assuntos
Leishmania/metabolismo , Purinas/metabolismo , Pirimidinas/metabolismo , Animais , BiotransformaçãoRESUMO
Two nucleosides related to the known antiprotozoal agent 1-(beta-D-ribofuranosyl)-1,5-dihydro-4H-pyrazolo-[3,4-d]pyrimidine-4-one (allopurinol riboside, 1) were prepared and evaluated against Leishmania donovani, Trypanosoma cruzi, and Trypanosoma gambiense. 3'-Deoxyinosine (2) exhibited potent antiprotozoal activity against the three protozoal pathogens with minimal toxicity for host cells. It was found to be especially effective against the Columbia strain of T. cruzi reported to be resistant to 1. The antiprotozoal activity of 2 appeared to be inversely related to the rate of cleavage of the glycosidic bond, as shown by metabolic profiles of 2 in the various pathogenic hemoflagellates and host cells. Combining the key structural elements of 1 and 2 led to the synthesis of 1-(3-deoxy-beta-D-erythro-pentofuranosyl)-1,5-dihydro-4H-pyrazolo[3,4-d] pyrimidin-4-one (3'-deoxy-allopurinol riboside, 3). which was found to be inactive as an antiprotozoal agent.
Assuntos
Antiprotozoários/síntese química , Inosina/análogos & derivados , Animais , Relação Dose-Resposta a Droga , Inosina/síntese química , Inosina/farmacologia , Leishmania donovani/efeitos dos fármacos , Macrófagos/efeitos dos fármacos , Testes de Sensibilidade Microbiana , Relação Estrutura-Atividade , Trypanosoma cruzi/efeitos dos fármacosRESUMO
Thiopurinol [4-thiopyrazolo(3.4-dyprimidine, TPP] and its ribonucleoside (TPPR) were effective in vitro against the intracellular and extracellular forms of L. braziliensis and L. mexicana. They also inhibited the transformation of the amastigote of L. donovani to the promastigote. These thio-analogues had about the same activity as allopurinol [4-hydroxypyrazolo(3.4-d)pyrimidine, HPP] and its ribonucleoside (HPPR). the thiopyrazolopyrimidines were converted primarily to the ribonucleoside-5' -phosphate (TPPR-MP) and to an unidentified metabolite, but not to any of the adenine ribonucleoside analogues previously shown to be formed from allopurinol and its ribonucleoside. There was an antagonism between the growth-inhibitory effects of allopurinol and thiopurinol. This is consistent with the findings that the intracellular concentrations of TPP and TPPR-MP are sufficient to inhibit the conversion of allopurinol to allopurinol ribonucleotide (HPPR-MP) by the hypoxanthine-guanine phosphoribosyltransferase by 30 per cent and the amination of HPPR-MP by adenylosuccinate synthetase by 50 per cent respectively. Consequently, the incorporation of the aminated product (aminopyrazolopyrimidine) into RNA was substantially decreased. The difference in metabolism between the thio- and hydroxypyrazolopyrimidines suggests a difference in their mechanisms of action against the pathogenic leishmania.
Assuntos
Alopurinol/análogos & derivados , Antiprotozoários , Leishmania/efeitos dos fármacos , Ribonucleosídeos/farmacologia , Tionucleosídeos/farmacologia , Alopurinol/metabolismo , Alopurinol/farmacologia , Animais , Cinética , Ribonucleosídeos/metabolismo , Tionucleosídeos/metabolismoRESUMO
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.
Assuntos
Adenilossuccinato Sintase , Alopurinol/análogos & derivados , Antibióticos Antineoplásicos/metabolismo , Antiprotozoários/metabolismo , Formicinas/metabolismo , Leishmania/enzimologia , Ligases , NADH NADPH Oxirredutases/antagonistas & inibidores , Ribonucleosídeos/metabolismo , Ribonucleotídeos/metabolismo , Alopurinol/metabolismo , Aminação , Animais , Antiprotozoários/farmacologia , Formicinas/toxicidade , GMP Redutase , Humanos , Inosina Monofosfato/metabolismo , Cinética , Células L/metabolismoRESUMO
STUDY OBJECTIVE: To evaluate the pharmacokinetics and safety of atovaquone suspension in volunteers infected with the human immunodeficiency virus ((HIV). DESIGN: Open-label, nonrandomized study. SETTING: Two clinical research centers. PATIENTS: Twenty-two HIV-infected volunteers with a median CD4 cell count of 37 cells/mm3. INTERVENTIONS: Patients received atovaquone suspension fasting or fed for 2-week periods with crossover at dosages of 500 mg/day, and randomization to fasting or fed at dosages of 750 and 1000 mg/day. A subset of patients also received 750 mg twice/day with food, and a subset of those who received 1000 mg/day fasting also received 1000 mg with food. During a long-term dosing phase, a subset of subjects were evaluated for an interaction between atovaquone and trimethoprim-sulfamethoxazole (TMP-SMX). MEASUREMENTS AND MAIN RESULTS: Average steady-state atovaquone concentrations at 500 mg were 6.7 +/- 3.2 microg/ml fasted and 11.3 +/- 5.0 microg/ml with food; at 750 mg, 9.9 +/- 7.1 microg/ml fasted and 12.5 +/- 5.9 microg/ml with food; at 1000 mg, 9.7 +/- 4.3 microg/ml fasted and 13.6 +/- 5.0 microg/ml with food; and at 1500 mg, 21.1 +/- 5.0 microg/ml with food. Thus, plasma concentrations were not proportional to dose. Concomitant food ingestion resulted in a 1.3- to 1.7-fold increase in values. Average steady-state concentrations were less than 10 microg/ml in 21% and more than 15 microg/ml in 36% of patients at 1000 mg/day with food; at 750 mg twice/day, all five patients had levels above 15 microg/ml. Atovaquone suspension was well tolerated; diarrhea, nausea, fatigue, and rash were the most common adverse events. Concomitant administration of TMP-SMX did not change atovaquone concentrations and resulted in small decreases in concentrations of TMP (16%) and SMX (10%). CONCLUSION: Plasma concentrations are significantly higher when atovaquone suspension is administered with food compared with fasting. Total doses of 1500 mg/day are likely to achieve concentrations effective for prophylaxis of Pneumocystis carinii pneumonia.
Assuntos
Antifúngicos/efeitos adversos , Antifúngicos/farmacocinética , Naftoquinonas/efeitos adversos , Naftoquinonas/farmacocinética , Infecções Oportunistas Relacionadas com a AIDS/metabolismo , Infecções Oportunistas Relacionadas com a AIDS/prevenção & controle , Adulto , Antifúngicos/administração & dosagem , Atovaquona , Jejum , Feminino , Interações Alimento-Droga , Humanos , Masculino , Pessoa de Meia-Idade , Naftoquinonas/administração & dosagem , Pneumonia por Pneumocystis/prevenção & controle , Combinação Trimetoprima e Sulfametoxazol/farmacocinéticaRESUMO
HPP-Rib is a potent antileishmanial agent, which has been useful in defining new and unusual purine metabolizing pathways in leishmaniae, in comparison with those in the host. The ribosyl linkage both in the parasite and in the host is resistant to cleavage. In the parasite there is a selective and marked conversion of HPP-Rib to HPP-Rib-5'-P and 4-APP ribonucleotides as well as incorporation into RNA, which does not occur in the host. These findings with HPP-Rib suggest a new chemotherapeutic approach which may be exploited in the treatment of leishmaniasis.
Assuntos
Alopurinol/análogos & derivados , Antiprotozoários , Leishmania/metabolismo , Ribonucleosídeos/metabolismo , Alopurinol/metabolismo , Alopurinol/farmacologia , Animais , Leishmania/efeitos dos fármacos , Leishmania/crescimento & desenvolvimento , Ribonucleosídeos/farmacologia , Ribonucleotídeos/biossíntese , Especificidade da EspécieAssuntos
Leishmania donovani/efeitos dos fármacos , Ribonucleosídeos/toxicidade , Tionucleosídeos/toxicidade , Animais , Divisão Celular/efeitos dos fármacos , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Leishmania donovani/metabolismo , Camundongos , Nucleotídeos de Purina/metabolismo , Ribonucleosídeos/metabolismo , Relação Estrutura-Atividade , Tionucleosídeos/metabolismoRESUMO
The growth of Leishmania donovani promastigotes and mouse L cells is differentially inhibited by several inosine analogs with modifications in the imidazole ring. The protozoal and mammalian cells also demonstrate differential metabolism of these analogs. 7-Deazainosine, 7-thia-7,9-dideazainosine, and formycin B were converted to their respective ATP analogs by both cell types. 8-Azainosine was converted to a GTP analog by mouse L cells; L. donovani did not metabolize this nucleoside. 9-Deazainosine and allopurinol riboside were metabolized only to their respective IMP analogs by L cells. L. donovani metabolized 9-deazainosine and allopurinol riboside to their ATP analogs and also metabolized 9-deazainosine to its GTP analog. All nucleosides studied were resistant to cleavage by either organism. From metabolism studies in the presence of a specific enzyme inhibitor, it was deduced that allopurinol riboside, formycin B, and 9-deazainosine were phosphorylated by at least two different routes in the mouse L cells. The metabolism of formycin B was inhibited 65% by the adenosine kinase inhibitor, 5-iodotubercidin, whereas the metabolism of allopurinol riboside (14% inhibition) and 9-deazainosine (0% inhibition) was only slightly affected by this inhibitor. The metabolism of allopurinol riboside and 9-deazainosine by L. donovani was not affected by 5-iodotubercidin. In contrast to the results of L cells, the metabolism of formycin B by L. donovani was also not affected by 5-iodotubercidin. The abilities of mouse L cells and L. donovani to metabolize these inosine analogs to the corresponding nucleotide analogs of ATP or GTP may be considered to be an activating step and correlates well with the respective cytotoxic effects of these compounds.
Assuntos
Fibroblastos/metabolismo , Inosina/análogos & derivados , Leishmania/metabolismo , Adenosina Quinase/antagonistas & inibidores , Trifosfato de Adenosina/metabolismo , Animais , Linhagem Celular , Cromatografia Líquida de Alta Pressão , Inibidores Enzimáticos/farmacologia , Guanosina Trifosfato/metabolismo , Inosina/metabolismo , Inosina Trifosfato/metabolismo , Camundongos , Tubercidina/análogos & derivados , Tubercidina/farmacologiaRESUMO
Atovaquone was compared to trimethoprim-sulfamethoxazole (TMP-SMZ) for the relationship of time receiving therapy, plasma drug concentrations, and incidence of adverse reactions in patients with AIDS-associated Pneumocystis carinii pneumonia. Treatment-limiting adverse events occurred in 9% of atovaquone-treated patients and 24% of TMP-SMZ-treated patients. Adverse events usually did not occur before day 7 for either treatment. Only the incidence of rash increased with increasing plasma concentrations of atovaquone. The incidence of anemia, neutropenia, and azotemia increased with increasing trimethoprim plasma concentration, while other adverse events (gastrointestinal disorders, rash, fever, and liver function abnormalities) were independent of plasma drug concentration.
Assuntos
Infecções Oportunistas Relacionadas com a AIDS/tratamento farmacológico , Antifúngicos/efeitos adversos , Naftoquinonas/efeitos adversos , Pneumonia por Pneumocystis/tratamento farmacológico , Combinação Trimetoprima e Sulfametoxazol/efeitos adversos , Adulto , Antifúngicos/sangue , Antifúngicos/farmacocinética , Atovaquona , Método Duplo-Cego , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Naftoquinonas/sangue , Naftoquinonas/farmacocinética , Combinação Trimetoprima e Sulfametoxazol/sangue , Combinação Trimetoprima e Sulfametoxazol/farmacocinéticaRESUMO
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.
Assuntos
Alopurinol/análogos & derivados , Antiprotozoários/farmacologia , Ribonucleosídeos/farmacologia , Trypanosoma cruzi/efeitos dos fármacos , Adenina/análogos & derivados , Adenina/farmacologia , Alopurinol/farmacologia , Animais , Antiprotozoários/uso terapêutico , Doença de Chagas/tratamento farmacológico , Resistência a Medicamentos , Formicinas/metabolismo , Formicinas/farmacologia , Inosina/metabolismo , Camundongos , Camundongos Endogâmicos DBA , Tionucleosídeos/farmacologia , Trypanosoma cruzi/metabolismoRESUMO
Certain purine analogs, the pyrazolopyrimidines, are effective chemotherapeutic agents against Leishmania spp. and Trypanosoma cruzi both in vitro and in some clinical models. Heretofore they have not been effective against the African trypanosomes; this suggested that these organisms were not comparable to the other pathogens with respect to their purine metabolism. We have studied the efficacy and metabolism of the pyrazolopyrimidine bases allopurinol and thiopurinol, their respective ribonucleosides, and the C-nucleosides formycin B and 9-deazainosine in Trypanosoma brucei subsp. gambiense and Trypanosoma brucei subsp. rhodesiense. The efficacy of these compounds was dependent on the purine content of the culture medium. The C-nucleosides were the most effective, with 90% effective doses for formycin B and 9-deazainosine of 0.01 and 2 micrograms/ml, respectively. Metabolism was the same in both the bloodstream and culture forms and identical to that reported for Leishmania spp. and T. cruzi. Both agents were phosphorylated to the ribonucleotide and then aminated to produce adenine nucleotide analogs. Growth inhibition studies were performed with three inosine analogs (allopurinol riboside, formycin B, and 9-deazainosine) on trypomastigotes grown in bone marrow tissue culture. Both C-nucleosides eradicated the infection at a concentration of 0.25 micrograms/ml. Unlike formycin B, 9-deazainosine is not known to be aminated by mammalian cells and appears to be relatively nontoxic in three different mammalian tissue culture systems. This nucleoside was very active against all pathogenic leishmaniae and trypanosomes investigated and is worthy of further study.
Assuntos
Inosina/análogos & derivados , Tripanossomicidas/metabolismo , Trypanosoma/metabolismo , Animais , Medula Óssea/parasitologia , Técnicas de Cultura , Inosina/metabolismo , Inosina/farmacologia , Camundongos , Camundongos Endogâmicos BALB C , Trypanosoma brucei brucei/metabolismo , Trypanosoma brucei gambiense/metabolismoRESUMO
Pneumocystis carinii pneumonia continues to be a cause of morbidity and mortality in AIDS patients. Current therapies have a high rate of toxicity and failure. Compound 566C80 is a 1-4,hydroxynaphthoquinone with potent antiprotozoal activity which shows good efficacy and safety in 21-day treatment trials of P. carinii pneumonia (PCP) in AIDS patients. Because there is a generally high recurrence rate after treatment of PCP and there may be a possible advantage in decreasing the P. carinii burden in the lung with extended anti-Pneumocystis therapy, we performed an open label-trial of the safety and efficacy of 42-day therapy with 566C80 for PCP in AIDS patients. Ten patients were enrolled and one was lost to follow-up. Eight of the remaining nine patients successfully completed 42 days of therapy with minimal toxicity. This trial suggests that 566C80 for 42 days can be an effective, safe, and well-tolerated oral therapy for PCP in AIDS patients.