D. C. Warhurst

In vitro and In vivo evaluation of betulinic acid as an antimalarial

The lupane‐type triterpene betulinic acid was isolated from an ethanol extract of the root bark of the Tanzanian tree Uapaca nitida Müll‐Arg. (Euphorbiaceæ). The in vitro antiplasmodial IC50 values of betulinic acid against chloroquine resistant (K1) and sensitive (T9‐96) Plasmodium falciparum were found to be 19.6 µg/mL and 25.9 µg/mL, respectively. The in vitro activities of several related triterpenes were also evaluated. Betulin was found to be inactive at 500 µg/mL for both K1 and T9‐96. Ursolic acid exhibited IC50 values of 36.5 µg/mL and 28 µg/mL, and oleanolic acid exhibited IC50 values of 88.8 µg/mL and 70.6 µg/mL against K1 and T9‐96, respectively. When betulinic acid was tested for in vivo activity in a murine malaria model (P. berghei) the top dosage employed of 250 mg/kg/day was ineffective at reducing parasitaemia and exhibited some toxicity. Betulinic acid has not previously been evaluated for in vivo activity. This is believed to be the first compound to be isolated from U. nitida. Copyright

Comparison of the in vitro activities of quassinoids with activity against Plasmodium falciparum, anisomycin and some other inhibitors of eukaryotic protein synthesis.

Using the inhibition of incorporation of [3H]hypoxanthine as an index of viability of malaria parasites, it was shown that a chloroquine-sensitive strain of Plasmodium falciparum (T9-96) and a chloroquine-resistant strain (K1) did not differ in their sensitivities to the quassinoids ailanthinone, bruceantin and chaparrin. Similarly, there were no differences between the strains in their sensitivities to the protein synthesis inhibitors anisomycin, deacetylanisomycin, cephalotaxine, homoharringtonine, cycloheximide, puromycin and puromycin aminonucleoside. The IC50 values derived for ailanthinone and bruceantin, cycloheximide, homoharringtonine and puromycin were in the nanomolar range, whereas those for the anisomycins, cephalotaxine and the aminonucleoside of puromycin were micromolar or greater. Those drugs tested which contain an ester moiety (ailanthinone, bruceantin, anisomycin, homoharringtonine) were more active than the related drugs (chaparrin, deacetylanisomycin, cephalotaxine) that do not. Cross-resistance to inhibitors of protein synthesis appeared not to accompany resistance to chloroquine.

Plasmodium falciparum: effects of phaeanthine, a naturally-occurring bisbenzylisoquinoline alkaloid, on chloroquine-resistant and -sensitive parasites in vitro, and its influence on chloroquine activity.

Phaeanthine, a bisbenzylisoquinoline alkaloid which occurs naturally in Triclisia species, was extracted from Triclisia patens (Menispermaceae) obtained from Sierra Leone (West Africa). In vitro, phaeanthine was found to be twice as potent against a chloroquine-resistant Plasmodium falciparum strain (K1), as against a chloroquine-sensitive clone (T9-96), with 50% inhibitory concentrations of 365.85 (+/- 11.41) nM and 704.87 (+/- 81.48) nM respectively. At a sub-inhibitory concentration of 80.35 nM, chloroquine resistance was not reversed by phaeanthine. Isobolograms constructed from experiments with chloroquine/phaeanthine combinations showed antagonism in T9-96 and an additive effect in K1. In a 48-hour microtest, phaeanthine at antimalarial concentrations showed no cytotoxicity to mammalian (KB) cells in vitro.

FAILURE OF CHLOROQUINE-ERYTHROMYCIN AND CHLOROQUINE-TETRACYCLINE COMBINATIONS IN TREATMENT OF CHLOROQUINE-RESISTANT FALCIPARUM MALARIA IN EASTERN THAILAND

In Eastern Thailand the increasing resistance of Plasmodium falciparum to all available antimalarial drugs prompted a reassessment of chloroquine in combination with either erythromycin or tetracycline. Both combinations had produced encouraging results in vitro, in experimental animal models, or in preliminary clinical trials. In patients with uncomplicated falciparum malaria presenting with moderate parasitaemia the trial of chloroquine and tetracycline was abandoned after RIII resistance with clinical deterioration was encountered in 2 out of the first 5 patients studied. Of 16 patients treated with chloroquine+erythromycin, 11 showed resistance (RI-RIII). These regimens appear ineffective and potentially dangerous in Eastern Thailand.

The chemotherapy of rodent malaria, XXIV. The blood schizontocidal action of erythromycin upon Plasmodium berghei.

Erythromycin inhibits chloroquine-induced pigment clumping in Plasmodium berghei in vitro. The drug was therefore tested against infections of P. berghei in mice and was found to be active at non-toxic doses. Given orally, the stearate salt was more effective than the base, but subcutaneously the base was more effective than the stearate. Erythromycin potentiated the action of chloroquine against two chloroquine-resistant strains of rodent malaria, the mildly resistant NS, and the highly resistant RC strains of P. berghei, but not against the drug-sensitive N strain.

Field chloroquine-resistance determinants

Although sequence changes in the malarial multidrug-resistance gene, Pfmdr1, such as asn86tyr, have been linked with chloroquine-resistance in a collection of routinely cultured, laboratory strains of Plasmodium falciparum, an experimental cross indicates that another gene may be involved. Mammalian cells transfected with wild-type Pfmdr1 are more susceptible to chloroquine but 3′ mutations abolish the effect by increasing the lysosomal pH, probably due to defective anion-channel activity. In malarial parasites, overexpression of wild-type Pfmdr1 leads to increased chloroquine sensitivity. Use of PCR amplification on uncultivated material from Zaria confirms that a highly significant, though incomplete, association exists between asn86tyr and resistance to chloroquine, supporting the involvement of this gene. If a Pfmdr1gene with a resistance-related mutation were overexpressed, this might have a different effect from overexpression of a wild-type gene. A functional or non-functional anion channel associated with the carboxy-terminal end of the Pfmdr1 product could lead to conflicting effects of overexpression, depending on the presence or absence of mutations associated with either end. It is possible that the additional gene involved in resistance may regulate baseline lysosomal pH, which could have a primary influence on resistance, irrespective of mutations in Pfmdr1.

Unusual Plasmodium related to P simiovale

SIR, Plasmodium ovale is rare outside Africa, but is occasionally found in SE Asia and Oceania (5 cases have been reported to the PHLS Malaria Reference Laboratory as imported into the UK in the past 10 years). On Aug 24, 1992, blood samples from a patient suspected of having malaria were received by the Central Laboratory, St Mary’s Hospital, Portsmouth. The patient, a male of 35, had returned 2 months earlier from a 6-month trip to Papua New Guinea where he had not been taking proguanil prophylaxis regularly, and discontinued it on return. He had been treated for malaria with intravenous quinine 1 month before leaving Papua New Guinea.