Although the antimalarial activity, hemolytic and methemoglobinemic side effects, and detoxification of primaquine are all thought to depend on various biotransformation products of the drug, their site and mechanism of formation and degradation are unknown and their specific biologic effects remain very poorly understood, particularly in humans. We have therefore explored the feasibility of studying primaquine metabolism in cultured human cells. We found that the biotransformation of primaquine can be investigated in vitro in serum-supplemented liquid cultures of partially synchronized and exponentially growing human erythroleukemic K562 cells. Further, these cells can be replaced by cells present in normal bone marrow. Primaquine is rapidly and predominantly converted in vitro into carboxyprimaquine (CPQ) in a quantitative manner and without further modification. In addition to CPQ, a compound Xc that is not 6-methoxy-8-aminoquinoline, and is not derived from CPQ, appears in minor amounts in a delayed fashion. With the K562 as well as with the bone marrow cells the formation of CPQ from primaquine can be totally blocked by large concentrations of the nitrosourea, 1,3-bis-(2-chloroethyl)-nitrosourea (BCNU). With bone marrow, increasing blockade of CPQ formation by BCNU leads invariably to a progressive and striking accumulation of Xc. The availability of reproducible, quantitative, and practical new tools for the study of primaquine metabolism in vitro raises a number of challenging questions and may improve understanding of the mode of action, toxicology, and pharmacogenetics of 8-aminoquinolines.

The role of the 8-aminoquinolines against malaria is currently focused on their activity as tissue schizontocides in the treatment and prophylaxis of vivax and ovale malaria. In addition they are gametocytocidal and sporontocidal and can prevent transmission of falciparum malaria. Of the two 8-aminoquinolines presently in use primaquine is the most generally available and quinocide, a structural isomer of primaquine, is used in the USSR. Although primaquine is the safest available 8-aminoquinoline it does have significant toxic effects, the best known being the induction of haemolytic anaemia in individuals with glucose-6-phosphate dehydrogenase (G6PD) deficiency. Furthermore, the radical cure of vivax and ovale malaria requires the administration of primaquine even in partially immune individuals over a period of at least several days. Nevertheless, because no other available tissue schizontocide is more effective or less toxic than primaquine there is a recognised need for investigations to differentiate the efficacy and the toxicity of prima-quine as well as to discover new, less toxic, tissue schizontocides. Thus the Third Meeting of the Scientific Working Group on the Chemotherapy of Malaria of the World Health Organization held in Geneva, Switzerland in October 1980 was entirely devoted to the field of tissue schizontocidal drugs.

A high-performance liquid chromatographic method was developed for the simultaneous determination of primaquine and its metabolites from plasma and urine samples obtained after oral administration of primaquine diphosphate. Following partial deproteinization with acetonitrile, samples were chromatographed by direct injection onto a cyano column with UV detection at 254 nm. Levels as low as 100 ng/mL per 20-μL injection were quantitated. Preliminary pharmacokinetic analysis is reported for two human volunteers after oral doses of 60 mg and 90 mg. Two apparent plasma metabolites and two possible urinary metabolites of primaquine are also reported.

With the objective of reducing analysis time and maintaining good efficiency, there has been substantial focus on high-speed chromatographic separations. Recently, commercially available ultra-performance liquid chromatography (UPLC) has proven to be one of the most promising developments in the area of fast chromatographic separations. In this work, a new isocratic reverse phase chromatographic method was developed using UPLC for primaquine phosphate bulk drug. The newly developed method is applicable for assay and related substance determination of the active pharmaceutical ingredient. The chromatographic separation of primaquine and impurities was achieved on a Waters Acquity BEH C18, 50 × 2.1 mm, 1.7 μm column within a short runtime of 5 min. The method was validated according to the regulatory guidelines with respect to specificity, precision, accuracy, linearity and robustness. Forced degradation studies were also performed for primaquine phosphate bulk drug samples to demonstrate the stability indicating power of the UPLC method. Comparison of system performance with conventional HPLC was made with respect to analysis time, efficiency and sensitivity.

In vitro studies on primaquine have been carried out to examine its ability to stimulate the oxidative pathway of glucose metabolism in human erythrocytes and in vivo studies were carried out after ingestion of the drug to determine plasma levels and to investigate the formation of metabolites and the effects of the drug on human erythrocytes. These investigations showed that:1) Two mechanisms are involved in the stimulation of the oxidative pathway. This was demonstrated by comparing the effects of methylene blue, ascorbic acid, primaquine, and other drugs on normal, glutathione-reductase-deficient, and G6PD-deficient erythrocytes. A start was made towards classifying drugs according to the mechanism by which they stimulate CO(2) production.2) Following oral ingestion of primaquine, three as yet unidentified metabolites were present, two in the plasma and one in the urine. The rapid disappearance of primaquine from the plasma (within 24 hours) was confirmed.3) Two factors that stimulate glucose oxidation in human erythrocytes were found in plasma; one occurred only in fresh plasma, when EDTA was present, and the other occurred in all plasma and serum samples studied.4) The erythrocytes of blood drawn 24 hours after the ingestion of primaquine (after primaquine had disappeared from the plasma) showed increased ability to oxidize glucose.It is not yet known whether serum or plasma prepared from blood drawn 24 hours after ingestion of primaquine has the ability to increase the oxidation of glucose.

Although a number of reports has recently suggested naloxone (NX) might affect dopamine (DA) agonist-elicited behavior, the effect of NX on apomorphine-induced sterotyped behavior (AISB) has not yet been systematically investigated. Doses of NX varying from 0.20 to 20.0 mg/kg were administered prior to the injection of apomorphine (0.5 mg/kg), a directly acting DA agonist, and the effect of NX on AISB was subsequently assessed. A sequential doubling of the dose of NX administered from 0.20 to 3.20 mg/kg generated a partial antagonist curve for AISB which fell in a linear manner from 0.20 to 0.40 mg/kg NX and thereafter became essentially asymptotic with 20 mg/kg producing the same effect on AISB as 0.4 mg/kg. Pretreatment with 0.4 mg/kg NX produced a sigmoidal inhibition of the stereotypy induced by successive doses of apomorphine (0.10–0.25 mg/kg). In a subsequent study, a low dose of NX (0.05 mg/kg) was combined with the specific DA receptor antagonist haloperidol (0.01 or 0.05 mg/kg) to determine if extremely low doses of the two antagonists could act synergistically. This was subsequently confirmed. Moreover, while a low dose of haloperidol (0.20 mg/kg) completely abolished AISB, 100 × this dose of NX (20.0 mg/kg) was only partially effective in this respect, indicating that NX, unlike haloperidol, exerts a partial antagonist effect relative to AISB. Furthermore, the marked synergistic effect of the very low doses of NX and haloperidol on blocking AISB was greater than their additive postsynaptic effects, suggesting that the effect of NX on AISB may in part be presynaptic.

The disposition of sulfalene was studied in eight individuals before and during an infection with a chloroquine-resistant strain of Plasmodium falciparum. Isoniazid acetylator phenotype was determined in each individual prior to the administration of sulfalene. Following the administration of sulfalene before infection with malaria, a significant difference in half-life of non-acetylated sulfalene and percent acetylation of sulfalene in plasma was observed between rapid and slow acetylators. When sulfalene was administered during malaria, this difference was no longer apparent. Individuals who did not respond to the therapeutic administration of sulfalene alone were treated with a combination of sulfalene and pyrimethamine. Three individuals were cured by sulfalene without pyrimethamine and one was cured by the drug combination. Three of the four individuals who were not cured by any dose of sulfalene or the drug combination were slow acetylators. There was no distinct correlation between clinical response and maximum levels or half-life of nonacetylated sulfalene. These findings suggest that acetylator phenotype does not influence the therapeutic response of individuals infected with falciparum malaria to sulfalene or to the combination of sulfalene and pyrimethamine. Further information is presented, however, to confirm the importance of an as yet unidentified host factor(s) in determining therapeutic response to these agents.