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.     

Inhibition of tubulin assembly by antileprosy drug dapsone

The effect of dapsone on assembly-disassembly process of bovine brain tubulin was examined. The drug was found to readily bind tubulin dimer and that in its presence colchicine binding to tubulin was enhanced. Although dapsone associated with tubulin at a site other than the colchicine binding site, distinct inhibition of microtubule assembly was detected.     

Genetic diversity of Plasmodium falciparum parasites from Kenya is not affected by antifolate drug selection

The genotypes of merozoite surface protein-1, merozoite surface protein-2 and glutamine rich protein are frequently used to distinguish recrudescence from reinfection when parasitaemia reappears after antimalarial drug treatment. However, none of the previous reports has clearly assessed the change of genetic diversity following drug treatment. In the present study, we have assessed the impact of pyrimethamine/sulfadoxine and chlorproguanil/dapsone on the genetic diversity of isolates and the multiplicity of infection in patient isolates from Kilifi, Kenya. We have analysed the length polymorphism of merozoite surface protein-1, merozoite surface protein-2 and glutamine rich protein and the data clearly show that treatment with pyrimethamine/sulfadoxine and chlorproguanil/dapsone did not change the multiplicity of infection found in patients, in contrast to the selection that these drugs exert on the genes encoded by the target enzymes. In addition, we report that children of less than 2 years tend to have fewer numbers of clones per isolate when compared with older children. Overall, this study shows that the selection for genes that confer drug resistance is not a factor in reducing the genetic diversity of parasite clones in a patient.     

Substrate proton to heme distances in CYP2C9 allelic variants and alterations by the heterotropic activator, dapsone

CYP2C9 polymorphisms result in reduced enzyme catalytic activity and greater activation by effector molecules as compared to wild-type protein, with the mechanism(s) for these changes in activity not fully elucidated. Through T₁ NMR and spectral binding analyses, mechanism(s) for these differences in behavior of the variant proteins (CYP2C9.2, CYP2C9.3, and CYP2C9.5) as compared to CYP2C9.1 were assessed. Neither altered binding affinity nor substrate (flurbiprofen) proton to heme-iron distances differed substantially among the four enzymes. Co-incubation with dapsone resulted in reduced substrate proton to heme-iron distances for all enzymes, providing at least a partial mechanism for the activation of CYP2C9 variants by dapsone. In summary, neither altered binding affinity nor substrate orientation appear to be major factors in the reduced catalytic activity noted in the CYP2C9 variants, but dapsone co-incubation caused similar changes in substrate proton to heme-iron distances suggesting at least partial common mechanisms in the activation of the CYP2C9 forms.     

Plasmodium falciparum: in vitro activity of sulfadoxine and dapsone in field isolates from Kenya: point mutations in dihydropteroate synthase may not be the only determinants in sulfa resistance

We have determined the relationship between point mutations in the gene that encodes the sulfa target, dihydropteroate synthase (DHPS) and the chemosensitivity profile to sulfadoxine and dapsone in 67 isolates from Kilifi, Kenya. We assessed the presence of mutations at codons 436, 437, 540, 581, and 613 of dhps. The results showed that the dhps genotype had a strong influence on the sensitivity to sulfadoxine and dapsone, but that the correlation was far from perfect. Eleven isolates carried a wild-type dhps allele, but were resistant to sulfadoxine (IC(50) values >10 microg/ml), and 4/28 isolates were classed as sensitive to sulfadoxine (IC(50) values <10 microg/ml), but carried a triple mutant (436/437/613) allele of dhps. These data show that in low folate medium in vitro, the dhps genotype alone did not account completely for sulfadoxine or dapsone resistance; other factors such as the utilisation of exogenous folate must also be considered.     

Comparison of in vivo and in vitro inactivation of endospores of <Emphasis Type="Italic">Rhinosporidium seeberi</Emphasis> following dapsone treatment

Rhinosporidiosis in humans and animals, caused by Rhinosporidium seeberi, can now be termed an emerging infective disease worldwide. The pharmacokinetics of dapsone when used as an antimicrobial agent in patients with rhinosporidiosis was compared with its pharmacodynamics in the inactivation of purified rhinosporidial endospores in vitro. A marked discrepancy was noted between the in vitro inactivation, which commenced on day 1 and was a maximum at day 4, whereas earlier reports on the response of endospores in patients under dapsone therapy indicated that the degeneration and disappearance of the endospores was not observed for 18–36 weeks after therapy began. Reasons for this discrepancy that operate in vivo are postulated: (a) impermeability of the barriers of down-growths of squamous epithelium in which the endospore-containing rhinosporidial sporangia are embedded; (b) presence of a mucoid matrix in which the endospores exist within the sporangia. These explanations contribute to resolving the controversial problem of general correlations between in vitro and in vivo results from studies on the action of antimicrobial drugs.     

Plasmodium berghei: Efficacy of 5-fluoroorotate in combination with commonly used antimalarial drugs in a mouse model

Resistance to antimalarial antifolates necessitates a search for new antimetabolites targeting other enzymes of the folate metabolic pathway. In this study, 5-fluoroorotate (FOA), reported to be an inhibitor of thymidylate synthase, was assayed against Plasmodium berghei NK 65 in mice, with(out) an oral uridine supplement. FOA (2.5 and 5.0 mg/kg bw.) was tested alone, or in a double and triple combination with a fixed oral dose of 1.25 and 2.5 mg/kg of pyrimethamine (PYR); 1.0 and 2.0 mg/kg of dapsone (DAP); 1.0 and 2.0 mg/kg of artesunate (ART). FOA achieved high suppression which ranged from 95.7% to aparasitaemic, activity that was dose-dependent. At the highest dosages used, FOA-PYR and FOA-DAP-ART combinations were synergistic with 100% cure rate, while FOA-PYR-ART was antagonistic. Drugs in a synergistic combination may exert less resistance selection pressure, thus FOA-PYR and FOA-DAP-ART warrant further evaluation with an ultimate object of possible clinical use against drug-resistant malaria.     

Measurement of physiological concentrations of dapsone and its monoacetyl metabolite: a miniaturised assay for liquid or filter paper-absorbed samples

A modification of existing HPLC assay methods is described for the measurement of dapsone and monoacetyldapsone in 50-μl samples of plasma and whole blood. This method, in particular the use of small sample volumes dried onto filter paper strips, is applicable to multi-sample clinical and pharmacokinetic studies in children with malaria, who are often anaemic, and where sample volume must be kept to a minimum. Basified samples were extracted into 5 ml of ethyl acetate-tert.-butylmethyl ether (1:1, v/v), chromatographed on a μBondapaK C₁₈, 10 μm column with water-acetonitrile-glacial acetic acid (81:17.5:5, v/v) containing 2 g/l 1-octanesulphonic acid as the mobile phase and detected at 274 nm.     

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.     

Selective inhibition of human neutrophil chemotaxis to N-formyl-methionyl-leucyl-phenylalanine by sulfones

The therapeutic efficacy of the sulfones, dapsone, and sulfoxone in neutrophilic dermatoses may be related to the effects of these drugs on neutrophil function. Therefore we determined whether neutrophil chemotactic migration to various chemoattractants could be inhibited by sulfones in vitro. The chemotactic responses of human neutrophils from healthy donors were tested by using N-formyl-methionyl-leucyl-phenylalanine (F-met-leu-phe), purified human C5a, and leukocyte-derived chemotactic factor (LDCF). Therapeutic concentrations of sulfones selectively inhibited neutrophil chemotaxis to F-met-leu-phe, but did not affect neutrophil chemotaxis to LDCF or C5a. Inhibition of neutrophil chemotaxis to F-met-leu-phe was induced by both dapsone and sulfoxone at a concentration of 10 micrograms/ml without affecting random migration, and the inhibition was reversed by washing the neutrophils. When dapsone- and sulfoxone-treated neutrophils (100 micrograms/ml) were stimulated with F-met-leu-phe, neutrophil superoxide generation was not inhibited. Sulfapyridine (10 micrograms/ml) also selectively inhibited neutrophil chemotaxis to F-met-leu-phe; however, sulfamethoxazole and sulfisoxazole did not affect chemotaxis. The inhibitory effects of dapsone, sulfoxone, and sulfapyridine could not be demonstrated with granulocytes from rabbits or guinea pigs nor with human monocytes. Experiments with radiolabeled dapsone showed rapid, nonspecific, and reversible binding of dapsone to human neutrophils. These data suggest that a mechanism of action of sulfones in neutrophilic dermatoses may be a selective inhibition of neutrophil migration to as yet undefined chemoattractants in the skin.     

Effector-mediated alteration of substrate orientation in cytochrome P450 2C9

Cytochrome P450 2C9 (CYP2C9)-mediated flurbiprofen 4'-hydroxylation is activated by the presence of dapsone resulting in reduction of the K(m) for flurbiprofen hydroxylation and an increase in V(m). Previous spectral binding studies have demonstrated that the binding of flurbiprofen with CYP2C9 is increased (decrease in K(S)) by the presence of dapsone. We hypothesized that the two compounds are simultaneously in the active site with the presence of dapsone causing flurbiprofen to be oriented more closely to the heme. T(1) relaxation rates determined by NMR were used to estimate the distances of protons on these compounds from the paramagnetic heme-iron center. Samples contained 0.014 microM CYP2C9 and 145 microM flurbiprofen in the presence and absence of 100 microM dapsone. Estimated distances of various flurbiprofen protons from the heme ranged from 4.2 to 4.5 A in the absence of dapsone and from 3.2 to 3.8 A in the presence of dapsone. The 4' proton of flurbiprofen, the site of metabolism, showed one of the greatest differences in distance from the heme in the presence of dapsone, 3.50 A, as compared to the absence of dapsone, 4.41 A. Dapsone protons were less affected, being 4.40 A from the heme in the absence of flurbiprofen and 4.00-4.01 A from the heme in the presence of flurbiprofen. Molecular modeling studies were also performed to corroborate the relative orientations of flurbiprofen and dapsone in the active site of CYP2C9. Shift of the 4' proton of flurbiprofen closer to the heme iron of CYP2C9 in the presence of dapsone may play a role in activation.     

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.     

Experimental and Clinical Studies on Rifampicin in Treatment of Leprosy

Rifampicin showed high activity against experimental leprosy, inhibiting the multiplication of dapsone-sensitive and dapsone-resistant strains of Mycobacterium leprae in mice fed 5 mg./kg. body weight. In a formal pilot-type trial on six previously untreated patients with active lepromatous leprosy, rifampicin (600 mg. daily by mouth) was as effective as standard treatment with dapsone. Myco. leprae, however, appeared to be killed more rapidly by rifampicin than by dapsone or other antileprosy drugs so far studied. This was confirmed on a further 10 patients, including two with dapsone resistance, and from the infectivity in mice of bacilli recovered from patients during treatment with rifampicin or dapsone. These results are consistent with the bactericidal activity of rifampicin against other micro-organisms, which could be important to the chemotherapy of leprosy, since all antileprosy drugs in current use are bacteriostatic.     

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.     

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.     

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.     

Activation of cytochrome P450 2C9-mediated metabolism: mechanistic evidence in support of kinetic observations

Studies were designed to investigate the possible mechanisms associated with the kinetic observation of CYP2C9 activation by dapsone and its phase I metabolite, N-hydroxydapsone. Kinetic studies suggested that dapsone activated CYP2C9-mediated flurbiprofen 4(')-hydroxylation by decreasing the K(m) (alpha=0.2) and increasing the V(max) (beta=1.9). Interestingly, N-hydroxydapsone also activated flurbiprofen 4(')-hydroxylation by increasing V(max) (beta=1.5) but had no effect on K(m) (alpha=0.98). To study the effects of these modulators on the binding affinity of flurbiprofen, spectral binding studies were performed. In the presence of dapsone, the spectral binding constant (K(s)) for flurbiprofen was reduced from 14.1 to 2.1 microM, while in the presence of N-hydroxydapsone, the K(s) remained unchanged (14.0 microM), which suggests that dapsone causes an increase in the affinity of flurbiprofen for CYP2C9, whereas N-hydroxydapsone does not. Additionally, stoichiometry measurements under activation conditions in the presence of dapsone resulted in a doubling of both NADPH and oxygen consumption for flurbiprofen 4(')-hydroxylation, with an overall increase in metabolite formation and a decrease in formation of peroxide and excess water. Interestingly, the presence of N-hydroxydapsone generally caused the same effects on stoichiometry as those of flurbiprofen 4(')-hydroxylation but failed to reduce excess water formation, which suggests that, while N-hydroxydapsone activates CYP2C9, it does so less efficiently and possibly through a mechanism different from that of dapsone.     

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.     

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.