A preliminary pharmacokinetic study of the antimalarial drugs, proguanil and chlorproguanil.

Pharmacokinetic parameters for cycloguanil and chlorcycloguanil, the active metabolites of proguanil (Paludrine] and chlorproguanil (Lapudrine) have been measured in a bioassay which assesses the in-vitro growth inhibition of a cycloguanil- and chlorcycloguanil-sensitive strain of Plasmodium falciparum produced by dilutions of plasma collected after oral administration of the pro-drugs. A single compartment model is applicable for cycloguanil with mean rate constants of elimination of 0.0624 h-1 and availability of 0.2398 h-1. The elimination profile for chlorcycloguanil indicates partition of drug into more than one compartment. In 2 of 10 subjects dosed with proguanil and 1 of 11 subjects dosed with chlorproguanil, the active metabolite levels were significantly lower than the mean for the other subjects. Abnormally low cycloguanil or chlorcycloguanil plasma levels may be of importance in relation to effective prophylaxis against malaria.

Response of Plasmodium falciparum to dihydrofolate reductase inhibitors in Malindi, Kenya.

The response of Plasmodium falciparum isolates to dihydrofolate reductase inhibitors (DHFRI) was examined in Malindi, Kenya. All 20 infected children treated with pyrimethamine/sulphadoxine responded. In contrast, after treatment with pyrimethamine, parasitaemia in 9 of 14 infections failed to clear or recrudesced during the seven-day follow-up. In a 48-hour in vitro test, five of six isolates resistant to pyrimethamine in vivo had a minimal inhibitory concentration (MIC) to pyrimethamine greater than or equal to 300 nmoles/1 compared with less than or equal to 100 nmoles/1 for the four sensitive isolates; four isolates did not grow. MIC to M-B 35769, an experimental DHFRI structurally similar to pyrimethamine were the same (six isolates) or 10-fold lower (three isolates). In the laboratory four of five isolates adapted to in vitro culture had the same MICs as in the field while one isolate became less responsive to both drugs. Cycloguanil (the active metabolite of proguanil) was more active in vitro in the laboratory than pyrimethamine or M-B 35769.

Antagonism of sulfadoxine and pyrimethamine antimalarial activity in vitro by p-aminobenzoic acid, p-aminobenzoylglutamic acid and folic acid.

The activity of pyrimethamine and sulfadoxine against two strains of Plasmodium falciparum has been studied in vitro by a radioisotopic technique. Low level antagonism of pyrimethamine resulted from the inclusion of p-aminobenzoic acid, p-aminobenzoylglutamic acid or folic acid in the test medium. Sulfadoxine activity was antagonised slightly by p-aminobenzoic but not by p-aminobenzoylglutamic acid, and antagonised markedly by folic acid at concentrations above 4 X 10(-8) M. At 10(-7) M folic acid, a concentration lower than that of normal RPMI medium 1640, sulfadoxine activity was reduced 7000 to 9000-fold in comparison with controls. These results are of importance in terms of the utilisation of folates by P. falciparum, the susceptibility of the parasite to antifolate drugs and the in vitro determination of parasite susceptibility.

In vitro antimalarial activity of tetrahydrofolate dehydrogenase inhibitors.

Three tetrahydrofolate dehydrogenase (dihydrofolate reductase = EC 1.5.1.3) inhibitors were tested for antimalarial activity against Plasmodium falciparum, using an in vitro radioisotopic technique. Activity of each drug was tested in both normal RPMI medium 1640 and in modified medium (containing no p-aminobenzoic acid and 2.27 X 10(-8) M folic acid) after a 24- or 48-hour exposure. Activity was increased 20- to 85-fold using the modified medium and the longer exposure time. Under all conditions, pyrimethamine and cycloguanil were of equal or greater potency than an experimental pyrimethamine analogue, M&B 35769, against pyrimethamine-sensitive strains, but M&B 35769 was more active than either pyrimethamine or cycloguanil against pyrimethamine-resistant strains.

The activity of proguanil and its metabolites, cycloguanil and p-chlorophenylbiguanide, against Plasmodium falciparum in vitro.

Using an in vitro radioisotopic method, the activity was measured of proguanil, its metabolites cycloguanil and p-chlorophenylbiguanide (PBG), pyrimethamine, and chloroquine against seven Kenyan and three South East Asian strains of Plasmodium falciparum. Five Kenyan isolates were sensitive to both pyrimethamine and cycloguanil in vitro, while the Smith and two Kenyan strains were resistant to both these drugs. Cross-resistance was incomplete: the Camp strain was resistant to pyrimethamine but not cycloguanil, and the FVO strain was resistant to cycloguanil but not pyrimethamine. Both proguanil and PBG exhibited weak antimalarial activity in vitro, but inhibitory blood levels of either compound are unlikely to occur after a normal human dose of proguanil. The results indicate that the activity of proguanil against P. falciparum is due entirely to the action of its active metabolite cycloguanil.

Synergistic antimalarial activity of pyrimethamine and sulfadoxine against Plasmodium falciparum in vitro.

Two strains of Plasmodium falciparum were tested in vitro for sensitivity to the dihydrofolate reductase inhibitor pyrimethamine, the p-aminobenzoic acid (PABA) analogue sulfadoxine, and combinations of both drugs. One strain was sensitive and one resistant to pyrimethamine in vitro. Parasites cultured in medium containing neither folic acid nor PABA were inhibited by 10(-6) M sulfadoxine, a concentration well below that achievable after therapeutic dosage. Folic acid added to this medium at a physiological concentration of 0.01 mg/liter caused a 1,000-fold reduction in sulfadoxine activity; a 100-fold higher concentration of folic acid caused a 10-fold reduction in pyrimethamine activity. Sulfadoxine in a concentration of 10(-7) M was able to potentiate pyrimethamine activity in PABA-free medium with no added folic acid or with 0.01 mg folic acid/liter. These data indicate that P. falciparum can utilize exogenous folic acid, and suggest that sulfadoxine may potentiate pyrimethamine activity by simultaneous inhibition of dihydrofolate reductase.

Effectiveness of amodiaquine as treatment for chloroquine-resistant Plasmodium falciparum infections in Kenya.

Studies were conducted in Malindi, Kenya, to assess the response of Plasmodium falciparum to chloroquine and amodiaquine in vivo (by an extended 14-day test) and in vitro (with the Rieckmann micro test). In-vivo resistance was demonstrated in 19 of 69 (28%) infections treated with chloroquine, but in only 2 of 60 (3.3%) of those treated with amodiaquine (p less than 0.001). In-vitro resistance to chloroquine was demonstrated in 15 of 23 (65%) tests. In contrast, 22 of the same 23 isolates were sensitive to amodiaquine in vitro. Effective concentrations by probit analysis for 50% and 99% (EC50 and EC99) inhibition, respectively, were 180.7 and 4319.6 nmol/l for chloroquine and 12.2 and 147.0 nmol/l for amodiaquine. The results suggest that amodiaquine is effective for the treatment of chloroquine-resistant falciparum malaria in Kenya.

A new in vitro test for pyrimethamine/sulfadoxine susceptibility of Plasmodium falciparum and its correlation with in vivo resistance in Kenya.

A useful in vitro method for field evaluation of Plasmodium falciparum sensitivity to pyrimethamine/sulfadoxine is described. Thirty-five Kenyan schoolchildren infected with P. falciparum were treated with this drug combination and followed up for 5 weeks. In vitro tests for sensitivity to these drugs and to chloroquine were performed before starting treatment. All infections cleared within 7 days of treatment, but 5 children had recurrent parasitaemia within 35 days. The original isolates from 4 of these 5 children had an in vitro response to pyrimethamine/sulfadoxine similar to a known strain that was resistant to these drugs; only 4 of the remaining 30 isolates from patients in whom recurrent parasitaemia did not occur had a resistant in vitro response (P = 0.006). In the patient with recurrent parasitaemia whose initial isolate appeared sensitive to pyrimethamine/sulfadoxine, the recurrent isolate had a resistant pattern in vitro, suggesting either reinfection or selection of a resistant subpopulation following treatment. The in vitro response to this drug combination was correlated with the in vitro response to either drug alone and with the in vitro response to chloroquine. Two of the 5 infections with recurrent parasitaemia after initial pyrimethamine/sulfadoxine treatment were resistant to chloroquine in vivo. The in vitro test for pyrimethamine/sulfadoxine should be useful for mapping the spread of multidrug-resistant P. falciparum.

The stability of adrenaline ophthalmic solutions on sterilization and storage.

The stability of adrenaline ophthalmic solutions, at pH 5.8 and 7.4, to sterilization and storage conditions has been studied. Solutions sterilized by filtration or heating at 98 degrees C for 30 min showed no detectable degradation at either pH value, whilst sterilization at higher temperatures resulted in losses of up to 30%. Total degradation increased with increasing sterilization temperature at both pH values.