A clinical-parasitological monotherapy cure in the treatment of experimental infection by a highly virulent strain ofToxoplasma gondii

Toxoplasmic encephalitis in patients with the acquired immunodeficiency syndrome (AIDS) is treated classically with pyrimethamine plus sulfadiazine. Unfortunately, up to 40% of these patients are unable to complete, the course of therapy because of adverse reactions to sulfonamides. This study considers the possible usefulness of monotherapies in the treatment of acute toxoplasmosis, producing parasitological cures 2–3 months after the date of infection. With this therapy, the main adverse effects are suppressed. Groups of mice infected with the RH strain ofToxoplasma gondii were treated with pyrimethamine alone, sulfadiazine alone, and pyrimethamine plus sulfadiazine for 7 d. Treatment with pyrimethamine plus sulfadiazine produced clinical cures in 100% of the infected mice 1 month after infection. Treatment with pyrimethamine gave a 60% survival rate (clinical cure), at 1 month postinfection. Finally, treatment with sulfadiazine produced a 60% survival rate at 1 month postinfection. Although the antitoxoplasmic regimen with pyrimethamine plus sulfadiazine has proven to be effective in intensive treatment of toxoplasmic encephalitis, relapses occur in more than 80% of cases after cessation of antitoxoplasmic therapy, making secondary prophylaxis mandatory. In this study the efficacy of treatment was also evaluated in terms of parasitological cure. None of the three therapies showed parasitological cure after 1 month of treatment. When the intervals were extended to a 3-month observation, monotherapy with pyrimethamine and sulfadiazine alone produced a parasitological cure.     

Treatment of pregnant BALB/c mice with sulphadoxine pyrimethamine or chloroquine abrogates Plasmodium berghei induced placental pathology

Malaria infection during pregnancy is a risk factor for foetus survival and is associated with abortion, premature delivery and low birth weight of infants in malaria endemic regions. In these regions, prophylactic measures and treatment mainly rely on chloroquine and sulphadoxine pyrimethamine, but their efficacy in reducing the placental pathology has not been studied. Therefore, the present study was designed to assess the effectiveness of chloroquine and sulphadoxine pyrimethamine treatment in reducing the placental pathology of Plasmodium berghei infected BALB/c mice. It was observed that pregnant-infected mice, treated either with chloroquine or sulphadoxine pyrimethamine had significantly lower percent parasitaemia, 100% survival and delivered normally compared with untreated pregnant-infected mice. Interestingly, antimalarial treatment significantly reduced malondialdehyde (MDA) levels, measure of lipid peroxidation and number of apoptotic cells in the placentae of pregnant-infected treated mice. Histologically also no morphological and cellular alterations were observed in the placentae of pregnant-infected treated mice. Taken together, the study shows the effectiveness of chloroquine and sulphadoxine pyrimethamine treatment, when administered in second trimester in abrogating malaria induced oxidative stress, apoptosis and histopathological alterations in the placenta, leading to normal foetal development.     

The Clastogenic effect of Pyrimethamine (Daraprim) on human chromosomes in lymphocyte cultures

In this study the clastogenic effect of pyrimethamine (Daraprim), a folic acid antagonist used for the treatment of toxoplasmosis and malaria on human chromosomes, was investigated. Pyrimethamine was added to in vitro lymphocyte cultures at six different concentrations: 0.05 (normal therapeutic dose), 0.1, 0.2, 0.4, 0.8, and 1.6 mg/ml. No proliferation was observed in any of the cultures containing 1.6 mg/ml pyrimethamine. The results of the cytogenetic evaluations show that the frequency of breaks and gaps increase significantly in dose-dependent manner. Thus, pyrimethamine has a clastogenic effect on human chromosomes.Abbreviations PHAM Phytohemagglutinin M - M Metaphase - G Gap - B break - R Rearrangement - NCA Number Chromosome Abnormalities - FA Folic acid - SCE Sister Chromatid Exchange     

Plasmodium falciparum: gene mutations and amplification of dihydrofolate reductase genes in parasites grown in vitro in presence of pyrimethamine

Samples of three pyrimethamine-sensitive clones of Plasmodium falciparum were grown for periods of 22-46 weeks in media containing stepwise increases in pyrimethamine concentrations and were seen to develop up to 1000-fold increases in resistance to the drug. With clone T9/94RC17, the dihydrofolate reductase (DHFR) gene was sequenced from 10 uncloned populations and 29 pure clones, all having increased resistance to pyrimethamine, and these sequences were compared with the sequence of the original pyrimethamine-sensitive clone. No changes in amino acid sequence were found to have occurred. Some resistant clones obtained by this method were then examined by pulsed-field gel electrophoresis, and the results indicated that there had been an increase in the size of chromosome 4. This was confirmed by hybridization of Southern blots with a chromosome 4-specific probe, the vacuolar ATPase subunit B gene, and a probe to DHFR. Dot-blotting with an oligonucleotide probe to DHFR confirmed that there had been increases up to 44-fold in copy number of the DHFR gene in the resistant strains. Resistant clones obtained by this procedure were then grown in medium lacking pyrimethamine for a period of nearly 2 years, and reversion nearly to the level of pyrimethamine sensitivity of the original clone T9/94RC17 was found to occur after about 16 months. Correspondingly, the chromosome 4 of the reverted population reverted to a size like that of the original sensitive clone T9/94RC17. The procedure of growing parasites in stepwise increases of pyrimethamine concentration was repeated with two other pyrimethamine-sensitive clones: TM4CB8-2.2.3 and G112CB1.1. (The DHFR gene of these clones encodes serine at position 108, in place of threonine as in clone T9/94RC17, and it was thought that this difference might conceivably affect the rate of mutation to asparagine at this position). Clones TM4CB8-2.2.3 and G112CB1.1 also responded by developing gradually increased resistance to pyrimethamine. However, in clone TM4CB8-2.2.3 a single mutation from Ile to Met at position 164 in the DHFR gene sequence was identified, and in clone G112CB1.1 there was a single mutation from Ala to Ser at position 16, but no mutations at position 108 were obtained in any of the clones studied here. In addition, chromosome 4 of clone TM4CB8-2.2.3 increased in size, presumably due to amplification of the DHFR gene. No increase in size was seen in clone G112CB1.1. We conclude that whereas some mutations producing changes in the amino acid sequence of the DHFR molecule may occur occasionally in clones or populations of P. falciparum grown in vitro in the presence of pyrimethamine, amplification of the DHFR gene following adaptation to growth in medium containing pyrimethamine occurs as a regular feature. The bearing of these findings on the development of pyrimethamine-resistant forms of malaria parasites in endemic areas is discussed.     

Antiplasmodial effects of the aqueous extract of Phyllantus amarus Schumach and Thonn against Plasmodium berghei in Swiss albino mice.

Phyllantus amarus Schumach and Thonn is a medicinal plant used commonly for the treatment of malaria-related symptoms by the general public in southeastern Nigeria. The present study determines the possible antiplasmodial effects of the aqueous extract of the leaves and stem of the plant against Plasmodium berghei infection using Swiss albino mice as models. The blood schizonticidal activity of the aqueous extract in early infection and in established Plasmodium berghei infection was assessed and compared to the activities of chloroquine and sulfadoxine/pyrimethamine. The repository activity of the extract was also assessed and compared to the activity of pyrimethamine. The LD50 of the aqueous extract of the leaves and stem of the plant was also determined using albino Wistar rats. The results show that the LD50 of the aqueous extract of Phyllantus amarus Schumach and Thonn was 650 mg/kg. In early infection, the extract at doses of 108.33 mg/kg, 165 mg/kg and 325 mg/kg was found to cause a significant dose-dependent suppression of P berghei parasites [P < P0.05] sulfadoxine/pyrimethamine caused a similar significant suppression of P berghei parasites [P < 0.05] while chloroquine at a dose of 5 mg/kg did not cause a significant effect on P berghei parasites. Similarly, the extract was found at all doses to cause a statistically significant [P < 0.05] suppression of P berghei parasites via a repository action. This effect was comparable to the effects of pyrimethamine a standard repository agent. In established infection, the extract at all doses administered, was found to significantly suppress P berghei parasites at 24 and 72-hour periods [P < 0.05]. Comparatively, sulfadoxine/pyrimethamine caused a similar statistical [P < 0.05] suppression of the parasites of P. berghei. However, the effects of sulfadoxine/pyrimethamine were more sustained over the 72-hour period. The present study therefore validates the local use of the extracts of Phyllantus amarus Schumach and Thonn as an antimalarial agent. Further studies are however recommended to identify and possibly characterize the potential antiplasmodial agents in the aqueous extract of the plant.     

Genome comparison of progressively drug resistant Plasmodium falciparum lines derived from drug sensitive clone

Chloroquine has been the mainstay of malaria chemotherapy for the past five decades, but resistance is now widespread. Pyrimethamine or proguanil form an important component of some alternate drug combinations being used for treatment of uncomplicated Plasmodium falciparum infections in areas of chloroquine resistance. Both pyrimethamine and proguanil are dihydrofolate reductase (DHFR) inhibitors, the proguanil acting primarily through its major metabolite cycloguanil. Resistance to these drugs arises due to specific point mutations in the dhfr gene. Cross resistance between cycloguanil and pyrimethamine is not absolute. It is, therefore, important to investigate mutation rates in P. falciparum for pyrimethamine and proguanil so that DHFR inhibitor with less mutation rate is favored in drug combinations. Hence, we have compared mutation rates in P. falciparum genome for pyrimethamine and cycloguanil. Using erythrocytic stages of P. falciparum cultures, progressively drug resistant lines were selected in vitro and comparing their RFLP profile with a repeat sequence. Our finding suggests that pyrimethamine has higher mutation rate compared to cycloguanil. It enhances the degree of genomic polymorphism leading to diversity of natural parasite population which in turn is predisposes the parasites for faster selection of resistance to some other antimalarial drugs.     

Pancytopenia as a complication of the treatment of toxoplasmosis]

Therapeutical doses of pyrimethamine are very close to toxic ones. Pyrimethamine is widely used in toxoplasmosis therapy. Hematological complications following pyrimethamine administration were seen in 4 patients treated for toxoplasmosis at our hospital in the last year. Pancytopenic syndrome was diagnosed in all four cases. Three patients recovered completely whereas ITP is persisting in one patient.     

Gametocytocidal and sporontocidal effects of antimalarial drugs on malaria parasites. II. Action of the folic reductase inhibitors, chlorguanide, and pyrimethamine against Plasmodium cynomolgi

The antimalarial activity of the dihydrofolate reductase inhibitors, chlorguanide and pyrimethamine, against gametocytes and sporogony of the simian parasite, Plasmodium cynomolgi was tested. Plasmodium cynomolgi infected rhesus monkeys (Macaca mulatta) were treated orally with multiple doses of chlorguanide (5.6 or 11.3 mg base/kg/Day X5) or a single dose of pyrimethamine (3 mg base/kg). Batches of mosquitoes (Anopheles maculatus) were allowed to feed immediately prior to and at appropriate intervals after drug administration. The effects of the drugs on the developmental stages of the parasite were assessed within the mosquito host. The results indicated that sporogony was interrupted in mosquitoes fed 1 hr after initial dosing with chlorguanide. With pyrimethamine, a clear-cut sporontocidal action was shown as early as 15 min after the drug was given. Both compounds inhibited further development of young oocysts and successive batches of mosquitoes fed 1 hr or more after medication ware consistently negative for sporozoites. Administration of either compound in the indicated doses did not prevent exflagellation, zygote or ookinete formation. However, the behavior of the latter forms was apparently altered by the action of the drug. There was a marked retarding effect in the formation of ookinetes in mosquitoes fed 24 hr after initial medication and thereafter. The schizontocidal and gametocytocidal action of chlorguanide was more evident than it was in pyrimethamine.     

Determination of plasma concentrations of dapsone,monoacetyl dapsone and pyrimethamine in human subjects dosed with maloprim

A high-performance liquid chromatographic method was developed to enable dapsone, monoacetyl dapsone and pyrimethamine to be measured simultaneously in plasma samples from volunteers in England and Malaysia who had been dosed with Maloprim. Mean half-lives of 25 and 80 h were calculated for dapsone and pyrimethamine, respectively, but there was wide individual variation. All subjects were found to be classifiable as “slow acetylators”.     

A model for testing compounds influencing porphyrin synthesis

The yeast Saccharomyces cerevisiae cultivated semi-anaerobically in a synthetic medium was used as a model to establish (a) total porphyrin synthesis, (b) ratio of intracellular to extracellular porphyrin concentrations. The antimalarials used for the therapy of porphyria cutanea tarda, chloroquine and pyrimethamine, reduced the total synthesis of porphyrins, pyrimethamine being more effective than chloroquine, like in porphyric patients. Both drugs exerted an antagonistic influence on the release of porphyrins from cells. Chloroquine reduced the concentration ratio of porphyrins while pyrimethamine increased it, apparently through inhibition of permeation of porphyrins. Combined treatment with the two compounds may hold promise for the therapy of porphyria cutanea tarda.     

Plasmodium falciparum: drug-resistant malaria complicating leukemias and lymphomas in children.

The present communication deals with drug-resistant Plasmodium falciparum malaria complicating hematologic malignancies (leukemias, n = 24, and lymphomas, n = 7) in children. Of 50 cases of hematologic malignancies, 31 patients were microscopically diagnosed as having P. falciparum infection (MP +). Initially, all the patients were treated with chloroquine. The results of primary treatment showed chloroquine resistance in 16 (51. 62%) cases. Of these 16 chloroquine-resistant cases, 13 were secondarily treated with a combination of pyrimethamine plus sulfamethopyrazine. The results of secondary treatment also revealed resistance to pyrimethamine plus sulfamethopyrazine in 6 of 13 (46. 10%) cases. The 6 pyrimethamine plus sulfamethopyrazine-resistant P. falciparum cases were finally cured by quinine therapy, against which no resistance was encountered. Conversely, in the control group comprising 38 cases of P. falciparum without malignancy, the incidence of chloroquine resistance was found in only 9 cases, which is rather low (23.70%). Of these 7 chloroquine-resistant cases, 5 were found to be sensitive to pyrimethamine plus sulfamethopyrazine treatment, while the 2 nonresponders were finally cured with quinine. The overall results of this study show a high prevalence of chloroquine resistance among clinical cases of falciparum malaria (25/69; 30.6%). Among the nonresponders (n = 20) 40% of cases were also resistant to the pyrimethamine plus sulfamethopyrazine combination. There was no resistance to quinine.     

Heterologous expression of active thymidylate synthase-dihydrofolate reductase from Plasmodium falciparum

The coding sequence of the bifunctional thymidylate synthase-dihydrofolate reductase (TS-DHFR) from a moderately pyrimethamine-resistant strain (HB3) of Plasmodium falciparum was assembled in a pUC expression vector. The coding sequence possesses unique Nco1 and Xba1 sites which flank 243 bp of the DHFR gene that include all point mutations thus far linked to pyrimethamine resistance. Wild-type (3D7) and highly pyrimethamine-resistant (7G8) TS-DHFRs were made from this vector by cassette mutagenesis using Nco1-Xba1 fragments from the corresponding cloned TS-DHFR genes. Catalytically active recombinant TS-DHFRs were expressed in Escherichia coli, albeit at low levels. Both TS and DHFR coeluted upon gel filtration and copurified upon affinity and anion exchange chromatography. Gel filtration and SDS-PAGE indicated that the enzyme was a dimer with identical 67-kDa subunits, characteristic of protozoan TS-DHFRs. Amino-terminal sequencing gave 10 amino acids which perfectly matched the sequence predicted from the nucleotide sequence. The recombinant TS-DHFR was purified to homogeneity by 10-formylfolate affinity chromatography followed by Mono Q FPLC. The inhibition properties of pyrimethamine toward the purified recombinant enzymes show that the point mutations are the molecular basis of pyrimethamine resistance in P. falciparum.     

Inhibition of Pneumocystis dihydrofolate reductase by analogs of pyrimethamine, methotrexate and trimetrexate.

Dihydrofolate reductase was obtained from Pneumocystis carinii isolated from heavily infected lungs of female Sprague-Dawley rats infected by transtracheal inoculation. The enzyme differed significantly from other forms of dihydrofolate reductase in response to KCl and to antifolate drugs. Dihydrofolate reductase from P. carinii was used to assess activity of analogs of pyrimethamine, methotrexate, and trimetrexate. One pyrimethamine analog was selective for P. carinii dihydrofolate reductase; potency was in the micromolar range. In contrast, 21 methotrexate analogs and 2 trimetrexate analogs were selective for P. carinii dihydrofolate reductase; potencies for these were in the nanomolar range.     

Trimethoprim-resistant bacteria: cross-resistance patterns

One hundred and eighty one strains (clinically isolated) of trimethoprim-resistant bacteria were tested for sensitivity to proguanil, pyrimethamine, methotrexate and methasquin. All these agents had less intrinsic antibacterial activity than trimethoprim, and there was marked cross-resistance between trimethoprim on the one hand and pyrimethamine and methasquin on the other. Methotrexate was of very low activity against Gram-negative bacilli, but was highly inhibitory for streptococci. Proguanil had approximately the same activity for all the organisms tested, and appears not to act as an anti-folate agent.     

Plasmodium berghei: diet and drug dosage regimens influencing selection of drug-resistant parasites in mice

Two different diets for the host and three drug dosage regimens were used to select lines resistant to sulfadoxine and pyrimethamine from the parent strain of the rodent malaria parasite Plasmodium berghei [the N (K173) strain]. A higher yield of resistance was obtained when a high parasitemia was present at the beginning of the drug pressure schedule. The development of resistance to the association of sulfadoxine plus pyrimethamine was accelerated by a relatively high para-aminobenzoic acid (PABA) content diet. Reproducibility was satisfactory when one of the dosage regimens was applied independently by two different technicians at different times.     

Inclusion complexes of pyrimethamine in 2-hydroxypropyl-beta-cyclodextrin: characterization, phase solubility and molecular modelling

The inclusion complexation of pyrimethamine in 2-hydroxypropyl-beta-cyclodextrin has been investigated by 2D (1)H NMR, FTIR and UV/visible spectroscopy and also by molecular modelling methods (AM1, PM3, MM3). From the phase-solubility diagram a linear increase was observed in pyrimethamine aqueous solubility in the presence of 2-hydroxypropyl-beta-cyclodextrin, evidencing the formation of a soluble inclusion complex. According to the continuous variation method (Job's plot) applied to fluorescence measurements, a 1:1 stoichiometry has been proposed for the complex. Concerning the structure of the complex, a Cl-in orientation of pyrimethamine in the 2-hydroxypropyl-beta-cyclodextrin cavity has been proposed from the theoretical calculations, being confirmed by two-dimensional (1)H NMR spectroscopy (ROESY). The thermal behaviour has also been studied, providing complementary evidences of complex formation.     

Comparative studies on dihydrofolate reductases from Plasmodium falciparum and Aotus trivirgatus.

Dihydrofolate reductase (E.C. 1.5.1.3) from Plasmodium falciparum and from its host, the owl monkey (Aotus trivirgatus), were partially purified and characterized. The molecular weight of the parasite enzyme was estimated to be over 10 times as high as that of the host enzyme. The host enzyme had 2 pH optima whereas the parasite enzyme only one. The activity of the host enzyme was greatly stimulated by KCl and urea, while that of the parasite enzyme was inhibited at high concentrations of such chaotropic agents. Km of the parasite enzyme was significantly higher than that of the host enzyme. The parasite enzyme had much lower Ki for pyrimethamine than the host enzyme. Dihydrofolate reductases isolated from pyrimethamine-resistant and pyrimethamine sensitive strains of P. falciparum were found to be similar.     

Dihydrofolate Reductase from Eimeria tenella: Rationalization of Chemotherapeutic Efficacy of Pyrimethamine

SYNOPSIS. Dihydrofolate reductase activity of 0.2 nmole of dihydrofolate reduced/min/mg protein was detected in crude extracts of unsporulated oocysts of Eimeria tenella. The enzyme was purified by a combination of affinity and ion-exchange chromatography. Its molecular weight was estimated as 240,000 daltons. An anticoccidial drug pyrimethamine is a potent inhibitor of the activity of E. tenella dihydrofolate reductase ( K _t = 3 nM), but it is less effective an inhibitor of dihydrofolate reductase from chicken liver. This difference may explain the in vivo therapeutic action of pyrimethamine against Coccidia.     

Comparison of chloroquine,pyrimethamine and sulfadoxine,and chlorproguanil and dapsone as treatment for falciparum malaria in pregnant and non-pregnant women,Kakamega District,Kenya.

OBJECTIVE--To compare treatment and protection against falciparum malaria in pregnant and non-pregnant women with three drug regimens. DESIGN--Prospective intervention study with six weeks'' follow up. Patients received one of three drug regimens in order of entry. SETTING--Primary care hospital and secondary girls'' school in rural western Kenya. PATIENTS--158 of 988 pregnant women (89 primigravid and 69 multigravid) in the third trimester and 105 of 1488 non-pregnant schoolgirls of reproductive age were parasitaemic (more than 500 asexual forms/microliter. These women were divided into three treatment groups by gravid state. INTERVENTIONS--Women were treated with chloroquine base 25 mg/kg over three days or pyrimethamine 75 mg and sulfadoxine 1500 mg as a single dose or chlorproguanil 1.2 mg/kg and dapsone 2.4 mg/kg as a single dose. MAIN OUTCOME MEASURES--Parasitaemia and haemoglobin concentrations measured at seven day intervals for six weeks. RESULTS--Primigravid women were more likely to be parasitaemic on follow up than multigravidas or nulligravidas, whose response was about the same. Parasites did not clear by day 7 in primigravidas in six (20%) of 30 who received chloroquine, three (8%) of 35 treated with pyrimethamine and sulfadoxine, and none of 23 treated with chlorproguanil and dapsone. At day 28, 83%, 19%, and 67% of primigravidas in these treatment groups were parasitaemic. Haemoglobin concentrations rose in all women, but improvement was sustained only in women who remained free of parasites. CONCLUSIONS--Clearance of parasites was better with either pyrimethamine and sulfadoxine or chlorproguanil and dapsone than with chloroquine. Longest protection was obtained with pyrimethamine and sulfadoxine.     

Plasmodium yoelii: activity of azithromycin in combination with pyrimethamine or sulfadoxine against blood and sporozoite induced infections in Swiss mice

The efficacy of pyrimethamine or sulfadoxine administered in combination with azithromycin was examined in a rodent malaria model. Outbred Swiss mice infected with blood stage parasites were treated from day 0 to day 3 and efficacy of different regimens was monitored in terms of the curative response and the delay time to reach 2% parasitaemia (2% DT). Administration of azithromycin alone at 60 mg/kg/day produced curative response while lower doses showed marginally delayed 2% DT. A marked potentiation in activities of pyrimethamine (100-fold) or sulfadoxine (10-fold) was observed when administered at non-curative doses of 0.1 mg/kg/day in combination with azithromycin (30 mg/kg/day) against blood stage parasites. A combination of 10 mg/kg/day azithromycin with 0.3 mg/kg/day sulfadoxine was also curative. Likewise in the causal prophylactic test, a combination regimen comprising 1/16th and 1/3rd the individual curative doses of pyrimethamine and azithromycin, respectively, prevented the development of patent infection after Plasmodium yoelii sporozoite challenge. Our results suggest that a combination of azithromycin with the second line treatment regimen of fansidar may enhance the therapeutic efficacy of the latter and also provide better prophylaxis against Plasmodium falciparum malaria.