J. Joseph Marr

Purine and Pyrimidine Metabolism

Publisher Summary This chapter discusses the metabolism of purine and pyrimidine nucleotides in parasitic protozoa and helminths. They are the monomeric units of both DNA and RNA; ATP serves as the universal cellular energy source, adenine nucleotides are components of three key coenzymes (NAD+, FAD and CoA), they are used to form activated intermediates, such as uridine-diphosphate glucose, and they serve as metabolic regulators. The results of studies by investigators show that purine metabolism in these pathogens differs from that of their mammalian hosts. Whereas mammalian cells synthesize the purine ring de novo from simple precursors, all the parasitic protozoa and helminths that have been investigated lack de novo purine synthesis and utilize preformed purines obtained from their host environment. These pathogens use various purine salvage pathways to supply their purine nucleotide requirements. The chapter explains that the difference in purine metabolism between host and parasite is an area of potential chemotherapeutic intervention that is actively being explored. Unlike purines, pyrimidine metabolism,with the exception of the amitochondrial protists Giardia lamblia, Trichomonas vaginalis and Tritrichomonas foetus, is similar to mammalian cells, all pathogenic protozoa and helminths are able to synthesize pyrimidines de novo. Like mammalian cells, their ability to salvage pyrimidines is limited when compared to purine salvage. The chapter summarizes that with the exception of G.lamblia and T.vaginallis, all the parasitic organisms discussed in the chapter possess ribonucleotide reductase and can synthesize purine deoxynucleotides based on one or more of the following criteria: organisms multiply in the absence of deoxynucleosides, reductase activity can be detected in cell extracts, bases or ribonucleosides are incorporated into DNA or organisms are sensitive to growth inhibition by hyroxyurea (a ribonuleotide reductase inhibitor).

Allopurinol in the Treatment of American Cutaneous Leishmaniasis

BACKGROUND Pentavalent antimony, the generally accepted treatment for leishmaniasis, is given parenterally, and it is expensive and not readily available in developing countries. An inexpensive, orally administered compound would be a substantial advance in treatment. Previous studies in vitro have shown synergism between allopurinol and pentavalent antimony in tissue-culture systems. We designed this clinical study to determine whether synergism could be demonstrated in patients. METHODS We performed a randomized, controlled study of the efficacy of allopurinol plus meglumine antimoniate (Glucantime), as compared with meglumine antimoniate alone, in patients with cutaneous leishmaniasis, who were recruited from a village in southeastern Colombia. In addition, those who declined injections were treated with allopurinol alone, and those who declined any treatment were considered controls. All the patients were followed for one year after the completion of treatment. Lesions that healed completely at three months and remained healed during follow-up were considered to be cured. RESULTS The cure rate for patients treated with meglumine antimoniate was 36 percent; the addition of allopurinol increased the rate to 74 percent (P less than 0.001). Treatment with allopurinol alone yielded a cure rate of 80 percent (P less than 0.001). There were no cures among the untreated patients. There was no significant difference between the cure rate with allopurinol plus meglumine antimoniate and that with allopurinol alone. No major toxic effects were observed. CONCLUSIONS For the treatment of American cutaneous leishmaniasis, the combination of allopurinol and meglumine antimoniate is significantly more effective than meglumine antimoniate alone, probably because of the efficacy of allopurinol alone, which appears to be as good as the combination.

Pyrazolopyrimidine metabolism in the pathogenic trypanosomatidae

Pyrazolopyrimidines are purine analogues. These compounds are metabolized by the pathogenic hemoflagellates and other members of the family Trypanosomatidae as though they were purines. This metabolic sequence does not exist in man or other mammals. In the hemoflagellates, the pyrazolopyrimidine base, of which allopurinol is the paradigm, undergoes ribosylphosphorylation to the ribonucleotide. This ribonucleotide may remain as such or be aminated to the amino analogue and further converted to the aminopyrazolopyrimidine ribonucleoside triphosphate. The latter is incorporated into RNA. This metabolic sequence has been demonstrated in the genera Leishmania and Trypanosoma.

Purine metabolism in Leishmania donovani amastigotes and promastigotes

Purine metabolism in Leishmania donovani amastigotes was found to be similar to that of promastigotes with the exception of adenosine metabolism. Adenosine kinase activity in amastigotes is approximately 50-fold greater than in promastigotes. Amastigotes deaminate adenosine to inosine through adenosine deaminase, an enzyme not present in promastigotes. Inosine is cleaved to hypoxanthine and phosphoribosylated by hypoxanthine-guanine phosphoribosyltransferase. Promastigotes cleave adenosine to adenine and deaminate adenine to hypoxanthine via adenase, an enzyme not present in amastigotes. Hypoxanthine is phosphoribosylated by hypoxanthine-guanine phosphoribosyltransferase.

Inhibition of growth of Toxoplasma gondii by qinghaosu and derivatives.

The antimalarial compound qinghaosu (artemisinin) was tested in vitro for the ability to inhibit plaque formation by Toxoplasma gondii in fibroblasts. Qinghaosu at 0.4 microgram/ml for 5 days eliminated all plaques and microscopic foci of T. gondii. At 1.3 micrograms/ml for 14 days, qinghaosu completely eliminated T. gondii. Pretreatment of host cells or T. gondii with qinghaosu had no effect on T. gondii growth. There was no apparent toxicity to human fibroblasts in long-term studies. Of the six qinghaosu derivatives tested, dihydroqinghaosu, 1-propyl-ether-qinghaosu, and 1-butyl-ether-qinghaosu were comparable to qinghaosu. Ethyl-ether-qinghaosu (arteether) and sec-butyl-ether-qinghaosu were more effective. Methyl-ether-qinghaosu (artemether) was the most effective, with a potency approximately 10-fold greater than that of qinghaosu.

The activity of ketoconazole and other azoles against Trypanosoma cruzi: biochemistry and chemotherapeutic action in vitro

Trypanosoma cruzi epimastigotes in culture medium, and amastigotes and trypomastigotes in cultured human diploid lung cells were exposed to the antimycotic agent ketoconazole and their growth and/or sterol biosynthesis observed. Propagation of epimastigotes and amastigotes was impaired by concentrations of ketoconazole achievable in human serum, and amastigotes were more sensitive than were epimastigotes. Epimastigotes and trypomastigotes (non-dividing stage) displayed changes in their membrane sterol content such that the amounts of normal, end-product sterols (ergosterol, ergosta-5,7-dien-3 beta-ol, 24-ethylcholesta-5,7,22-trien-3 beta-ol, 24-ethylcholesta-5,7-dien-3 beta-ol) were notably decreased and the amounts of 14 alpha-methyl sterol precursors of these sterols (24-methylenedihydrolanosterol, obtusifoliol, lanosterol) were increased. Other azole drugs, itraconazole and fluconazole, when tested on epimastigotes, evoked the same qualitative pattern of changes in free sterols. Itraconazole was nearly as potent as ketoconazole, but fluconazole was significantly less potent. The nature of the sterols found in T. cruzi and the actions of azole drugs on their biosynthesis were similar in many respects to those observed in fungi and in Leishmania species. By analogy, it would seem that the primary mechanism of action of azole drugs on T. cruzi life-cycle stages is the impairment of the cytochrome P-450 sterol 14 alpha-demethylase. The consequent loss of normal sterols and accumulation of 14 alpha-methyl sterols may be responsible for the coincident retardation or cessation of growth.

Biological action of inosine analogs in Leishmania and Trypanosoma spp.

Previous investigations have suggested that inosine analogs would be good models for the development of chemotherapeutic agents active against pathogenic hemoflagellates. We have systematically modified the five-membered heterocyclic ring of six inosine analogs and tested them for their antiprotozoal activities and toxicity to a mammalian cell line. All six analogs were very active against the three protozoan pathogens Leishmania donovani, Trypanosoma cruzi, and Trypanosoma gambiense. Two of the six, 9-deazainosine and allopurinol ribonucleoside, had very little toxicity for mouse L cells and offer promise as potential chemotherapeutic agents.

Inosine analogs as chemotherapeutic agents for African trypanosomes: metabolism in trypanosomes and efficacy in tissue culture.

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.

Sterilization of Ommaya reservoir by instillation of vancomycin

We describe the management of a patient with a Staphylococcus epidermidis infection of an Ommaya reservoir that was being used for the treatment of carcinomatous meningitis. Intravenous vancomycin failed to eradicate the organism. We treated our patient successfully with parenteral antibiotics and local instillation of vancomycin (25 micrograms/ml) without removing the reservoir. The patient tolerated the intraventricular vancomycin well. We recommend this approach in the treatment of infected Ommaya reservoirs in patients who have diseases with extremely poor prognoses and who wish to be discharged from the hospital early.

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.