Relationship between plasma and red blood cell concentrations of quinine in Brazilian children with uncomplicated Plasmodium falciparum malaria on oral therapy / Relação entre as concentrações plasmáticas e eritrocitárias de quinina em crianças com malária por Plasmodium falciparum não complicada em terapia via oral

We determined the relationship between plasma and red blood cell concentrations of quinine in children with uncomplicated falciparum malaria from an endemic area of Amazonian region. Quinine was determined by high performance liquid chromatography with ultraviolet detection. In the steady state the ratio between plasma and red blood cell quinine concentration was 1.89 ± 1.25 ranging from 1.05 to 2.34. This result demonstrated that quinine do not concentrate in red blood cell of Brazilian children and characterize the absence of interracial difference in this relationship.

Neste estudo foi determinada a relação entre as concentrações plasmáticas e eritrocitárias de quinina em crianças com malária falciparum não complicada, oriundas de área endêmica da Região Amazônica. A quinina foi detrminada por cromatografia líquida de alta eficiência. No estado de equilíbrio, a relação foi 1,89 ± 1,25 variando de 1,05 a 2,34. Estes resultados demonstraram que a quinina não se concentra nos eritrócitos das crianças e caracterizaram a ausência de diferença racial nesta relação.

Quinine levels in patients with uncomplicated falciparum malaria in the Amazon region of Brazil

We examined the plasmatic concentrations of quinine in patients with uncomplicated falciparum malaria in an endemic area of the Amazon region in Brazil in a prospective clinical trial, in which a standard three-day course of oral quinine plus doxycycline was used. We measured the quinine in the plasma samples on days 0 and 3by high performance liquid chromatography. The mean concentration of quinine was 6.04 ±2.21 µg/mL in male patients and 5.98 ±1.95 µg/mL in female patients. No significant differences in quinine concentration were observed between these two groups. All samples collected before starting treatment were negative for quinine. This information could help in the development of strategies for the rational use of antimalarial drugs in Brazil.

Monoclonal antibodies to quinine.

Monoclonal antibodies (MAbs) to quinine conjugated to a carrier protein were produced. Quinine was converted into a hemisuccinate prior to covalently linked to bovine serum albumin (BSA) by reacting with N,N'-disuccinimidyl carbonate (DSC). Coupling ratio of quinine-BSA was 13:1 calculated by spectrophotometry and 14:1 by calculation from quinine standard curve. This immunogen was used for both monoclonal antibody production and for screening test, indirect ELISA. The specificity of quinine-BSA MAbs was examined by checking the cross reactivity with BSA and the structurally related antimalarial drug, mefloquine. Six MAbs belonging to IgG1 were obtained. These MAbs slightly reacted with mefloquine-BSA because of closely related structure of mefloquine to quinine and similar conjugate preparation procedure used for conjugation. One selected MAb against quinine-BSA, showed higher reactivity with blood samples from patients previously treated with quinine when compared to normal blood. This preliminary test indicated that MAbs obtained may be useful to be used as the probe for detection of quinine in biological fluids.

Plasma quinine levels in patients with falciparum malaria when given alone or in combination with tetracycline with or without primaquine.

Quinine has been an effective drug for severe chloroquine-resistant falciparum malaria. However, there has been a decline in the sensitivity of Plasmodium falciparum to quinine. In 1978-1979 the cure rate was 94% compared to 86% in 1979-1980 and 76% in 1980-1981. The combination of quinine and tetracycline has improved the cure rate to 95-100%. However, the mechanism responsible for this has not been identified. We have compared plasma quinine levels on day 2, day 5 and day 7 (before and at 2 hours after dosing) in twenty-one patients with acute falciparum malaria who were treated with quinine alone (8 patients) or quinine in combination with tetracycline (8 patients) or quinine with tetracycline and primaquine (5 patients). All patients who received combination of quinine and tetracycline with or without primaquine responded well to the treatment with no recrudescence. Two patients who were treated with quinine alone had RI responses. Plasma quinine concentrations from the quinine alone group were significantly lower than those obtained from combination groups on days 2, 5 and 7. The minimal plasma quinine levels from quinine alone group were all lower than MIC, ie below 10 micrograms/ml while those obtained from the combination group were higher than MIC for 7 days. The results from the present study suggest that tetracycline has influence on the maintenance of plasma quinine levels above MIC throughout the treatment period. Therefore, this must be one possible explanation for the better cure rate.

Determination of quinine and quinidine in biological fluids by high performance liquid chromatography.

A simple, specific, rapid and sensitive High Performance Liquid Chromatography (HPLC) method has been developed to measure plasma level of quinine and quinidine. The drugs were extracted successively from plasma at basic pH with chloroform and quantified on a revers-phase Z-module C18 HPLC column with fluorescence detector (excitation 340 nm, emission 425 nm). The isocratic mobile phase used was the mixture of 0.05 M ammonium formate and acetonitrile (93.5:6.5, v/v), adjusted pH to 2.0 with ortho-phosphoric acid. The limits of quantitation for these compounds were as low as 4 ng/ml of plasma, using a 0.25 ml specimen. Calibration curves were linear (R squared = 0.9994) in the range 0-7,000 ng/ml. An interassay reproducibility was 6.8%, 0.3% and 1.2% at the concentrations of 250 ng, 4,000 ng and 8,000 ng of quinine, respectively. Inter-assay coefficient of variation of quinidine was 1.8%, 2.7% and 3.7% at the concentrations of 250 ng, 1,500 ng and 3,000 ng, respectively. Recovery of quinine and quinidine were 76% and 81%, respectively. The method has been used in the analysis of quinine and quinidine in healthy volunteers receiving quinine or quinidine intravenously. The method is now being used to assay samples from field studies with satisfactory results.

Polarisation fluoroimmunoassay for quinine in serum and urine.

The development and validation of a polarisation fluoroimmunoassay for the antimalarial drug quinine is described. The assay is performed either by sequential addition of the reagents or by a single-reagent technique whereby the tracer and antibodies are premixed. Serum samples require pepsin digestion prior to assay while urine specimens are assayed directly. The reliability criteria of the assay are satisfactory and no cross-reaction is detected with quinidine (the optical isomer of quinine) or with common antimalarial drugs. The assay was applied to the measurement of quinine in urine specimens obtained from a single-dose pharmacokinetic study and the results correlated with those of the benzene extraction fluorescence method for quinine measurement.

In vivo and in vitro studies of quinine sensitivity of Plasmodium falciparum in Thailand.

Forty-four patients with falciparum malaria were studied. Nine patients were given quinine orally at a daily dose of 1.5 gm base for a period of 14 days. The mean parasite clearance in all 9 patients was 3.3 days, and none had recrudescence in follow-up examinations for 31 days. The in vivo study of these 9 patients showed sensitivity to quinine which correlated with the in vitro test, with concentration of quinine base 2.5-5.8 microgram/ml of blood that inhibited the maturation of Plasmodium falciparum parasites. The results of the in vitro test of 35 patients showed concentrations of quinine base 2.1-5.4 microgram/ml of blood were able to inhibit the maturation of P. falciparum parasites. Therefore, these studies indicate that Plasmodium falciparum are still sensitive to quinine and quinine remains to be the drug of choice for the treatment of falciparum malaria in Thailand.