Pharmacokinetics and Pharmacodynamics of ( (cid:2) )-Primaquine and ( (cid:3) )-Primaquine Enantiomers in Rhesus Macaques ( Macaca mulatta )

Primaquine (PQ) remains the sole available drug to prevent relapse of Plasmodium vivax malaria more than 60 years after licen-sure. While this drug was administered as a racemic mixture, prior studies suggested a pharmacodynamic advantage based on differential antirelapse activity and/or toxicities of its enantiomers. Oral primaquine enantiomers prepared using a novel, easily scalable method were given for 7 days to healthy rhesus macaques in a dose-rising fashion to evaluate their effects on the blood, liver, and kidneys. The enantiomers were then administered to Plasmodium cynomolgi -infected rhesus macaques at doses of 1.3 and 0.6 mg/kg of body weight/day in combination with chloroquine. The ( (cid:3) )-PQ enantiomer had higher clearance and apparent volume of distribution than did ( (cid:2) )-PQ and was more extensively converted to the carboxy metabolite. There is evidence for differential oxidative stress with a concentration-dependent rise in methemoglobin (MetHgb) with increasing doses of ( (cid:2) )-PQ than seen for ( (cid:3) )-PQ. There was a marked, reversible hepatotoxicity in 2 of 3 animals dosed with ( (cid:3) )-PQ at 4.5 ( (cid:3) )-PQ in combination with chloroquine was successful in preventing P. cynomolgi disease relapse at doses of 0.6 and 1.3 mg/kg/day, of 2 animals receiving ( (cid:2) )-PQ at 0.6 mg/kg/day relapsed. ( (cid:3) )-PQ was also associated with hepatotoxicity at higher as seen previously, not been identiﬁed as a clinical concern in humans during > 60 years of use. Limited evidence for increased MetHgb generation with the ( (cid:2) ) form in the rhesus macaque model suggests that it may be possible to improve the therapeutic window for hematologic toxicity in the clinic by separating primaquine into its enantiomers. Pharmacokinetic-pharmacodynamic study. In the healthy-animal study, pharmacokinetic blood sampling was done at 0, 0.5, 1, 2, 4, 8, 24, 72, 120, 168, and 216 h on the last day of the 3- and 4.5-mg/kg dose with 2 ml collected at each time point in heparinized sodium/lithium Vacu-tainer tubes. Samples were centrifuged at 2,500 rpm for 20 min, and then thesupernatant(plasma)wastransferredandkeptat (cid:3) 80°Cuntilanalysis was performed. Plasma was separated into two tubes with 200 to 400 (cid:7) l of plasma collected in each, and a third tube was used for erythrocytes. LC-MSanalysis. Theliquidchromatography-massspectrometry(LC-MS) analysis methods of levels in plasma for each PQ enantiomer and their respective carboxyprimaquine (cPQ) metabolites in rhesus macaques were modiﬁed from our previous studies (16). Chromatography was performed on a Chiracel OD-H instrument (250 by 4.6-mm [inner diameter],5- (cid:7) mparticlesize,andprecolumnofthesamematerial,910by 4.0 mm [inner diameter]; Chiral Technologies Inc.). The mobile phase used for analysis was 20 mM ammonium formate, pH 3.56 (A), and 0.1% formic acid in ACN (B). The starting gradient had an initial composition of 81% A and 19% B for 10 min, increasing to 60% ACN at 2 min, and then was held for 16 min at a ﬂow rate of 0.5 ml/min. Total analysis 30 min. max ),areaundertheconcentration-timecurvefromtimezeroto inﬁnity (AUC 0- (cid:8) ), and mean residence time (MRT). Because the fraction of dose absorbed cannot be estimated for extravascular models, apparent volume of distribution (V/F) and apparent clearance (CL/F) were substi-tuted for V and CL. Pharmacodynamic (PD) endpoints were also monitored for the two doses, including percentages of decrease in hemoglobin (%Hgb), increase in reticulocytes (%Retic), decrease in methemoglobin (%MetHgb), and increase in alanine aminotransferase (%ALT) compared to the baseline values for each animal, with a two-tailed Student’s t test comparison for difference in means between the dosing groups. The PK parameters for both PQ and cPQ were compared with PD variables of interest, including mean %Hgb, %Retic, %MetHgb, and %ALT. Data analysis and graphical representation were completed using SPSS, v.12. 0, and GraphPad Prism, v5.0, respectively.