DISPOSITION AND ANTIMALARIAL ACTIVITIES OF THE DESETHYL METABOLITES OF CHLOROQUINE

Abstract

A fluorimetric method for the selective determination of chloroquine (chq) from biological samples following dichloromethane extraction is described. It involves the reaction of the dichloromethane extracted primary and secondary amine metabolites with ethylchloroformate. The CQ base present dichloromethane extract is a tertiary amine which does not react with chloroformates at room temperature. This is subsequently separated from the neutral carbonate derivatives of the metabolites by extraction with aqueous acid. The effectiveness of this method in separating cq from its metabolites in biological fluids is approximately a 100%. The percentage coefficient of variation at 25mg\ml level is 15.0% while this value is 13.5% at 150mg\ml level. The antimalarial activity of cq and its desethyl metabolites-desethyl-chloroquine (DCQ) and bisdesethylchloroquine (BDCQ) was studied in in-vitro and in rodant in-vivo models. The percentage suppression of p. berghei berghei asexual parasitaemia in mice was found to increase with increasing doses of CQ, DCQ and BDCQ in the 4-day test, indicating a dose-dependent susceptibility of the parasites to the 3 compounds. The ED95 was 3.9,3.7 and 3.9mg\kg for CQ, DCQ and BDCQ respectively. DCQ and BDCQ were found to possess antimalarial activity similar to that of CQ in the rane’s test which was used to assess the effect of the drugs in an established p. berghei berghei infection in mice. Results of the in-vitro microtechnique test with p. falciparum isolated from children showed that CQ,DCQ and BCDQ,at a concentration of 0.6 pm\ul blood caused total inhibition of maturation of the ring forms to schizonts. This indicates a high susceptibility of the p. falciparum isolates to these compounds. These results show that DCQ and BDCQ are approximately equiactive with CQ against p. berghei berghei in mice and drug sensitive p. falciparum in-vitro. The partitioning of CQ,DCQ and BDCQ between red blood cells (ABCs) and plasma was studied in-vitro using blood from healthy adults and children with proven p.falciparum parasitamia. Blood samples from healthy adult and those from children were incubated with varying concentrations of CQ,DCQ or BDCQ for 15 minutes and the ADC\plasma concentration ratio determined. DCQ and BDCQ were found to be concentrated in the ABCs of uninfected blood to a similar extent as CQ. The mean partition coefficient between normal ABCs and plasma are 3.67+-0.4, 4.08+-0.4 and 3.50+-0.3 for CQ, DCQ and BDCQ respectively. However, DCQ and BDCQ were concentrated to a significantly less extent than CQ in the red cells from malarial children. The mean partition coefficients for this group are 29.2+-2.0, 12.9+-1.2 and 12.0+-1.4 for CQ and BDCQ respectively. It is suggested that the reduced capacity of the infected ABCs to concentrate DCQ and BDCQ might have an important bearing on the development of resistance to CQ by p. falciparum. The tissue distribution of CQ,DCQ and BDCQ was studied 24 hours after intraperitoneal administration of 10mg\kg of either of the compounds to rats. The amount of each compound present in the ABCs, plasma, heart, lung, liver, kidney and spleen was determined . there was a similarity in the tissue distribution of DCQ, BDCQ and the parent compound(CQ). The highest concentration of each compound was found in the lung while the least concentration was found in the heart. The order of decreasing tissue concentration was found to be lung > spleen> kidney> liver> heart for CQ while this order was lung> spleen> kidney= liver> heart for both DCQ and BDCQ . The urinary excretion of these compounds was also studied following intravenous administration of 2.5, 5.0 and 10.0mg\kg of each of the compounds to rats. Urine samples were collected at 24 hour intervals for 10 days. The urinary excretion-time curves for the 3 compounds were similar. Maximum excretion was found to occur within the first 24 hour with a mean of 9%,6% and 15% of the administered doses of CQ, DCQ and BDCQ respectively. The mean urinary excretion half-life of 6.8 days obtained for DCQ is similar to a mean value of 6.3 days obtained for CQ in this study. Similarly, a mean urinary excretion half-life of 6.5 days was obtained for BDCQ. Results from the study also showed that much more BDCQ is excreted when compared to CQ or DCQ. The total recovery as percentage of administered doses of CQ, DCQ and BDCQ are 26.43, 25.23 and 63.70 respectively. This is consistent with the fact that BDCQ is more polar than CQ and DCQ. The fate of the of the unexcreted compounds is however unknown.