Effect Of Primaquine Research Articles

Safety of single-dose primaquine as a Plasmodium falciparum gametocytocide: a systematic review and meta-analysis of individual patient data

BackgroundIn 2012, the World Health Organization (WHO) recommended single low-dose (SLD, 0.25 mg/kg) primaquine to be added as a Plasmodium (P.) falciparum gametocytocide to artemisinin-based combination therapy (ACT) without glucose-6-phosphate dehydrogenase (G6PD) testing, to accelerate malaria elimination efforts and avoid the spread of artemisinin resistance. Uptake of this recommendation has been relatively slow primarily due to safety concerns.MethodsA systematic review and individual patient data (IPD) meta-analysis of single-dose (SD) primaquine studies for P. falciparum malaria were performed. Absolute and fractional changes in haemoglobin concentration within a week and adverse effects within 28 days of treatment initiation were characterised and compared between primaquine and no primaquine arms using random intercept models.ResultsData comprised 20 studies that enrolled 6406 participants, of whom 5129 (80.1%) had received a single target dose of primaquine ranging between 0.0625 and 0.75 mg/kg. There was no effect of primaquine in G6PD-normal participants on haemoglobin concentrations. However, among 194 G6PD-deficient African participants, a 0.25 mg/kg primaquine target dose resulted in an additional 0.53 g/dL (95% CI 0.17–0.89) reduction in haemoglobin concentration by day 7, with a 0.27 (95% CI 0.19–0.34) g/dL haemoglobin drop estimated for every 0.1 mg/kg increase in primaquine dose. Baseline haemoglobin, young age, and hyperparasitaemia were the main determinants of becoming anaemic (Hb < 10 g/dL), with the nadir observed on ACT day 2 or 3, regardless of G6PD status and exposure to primaquine. Time to recovery from anaemia took longer in young children and those with baseline anaemia or hyperparasitaemia. Serious adverse haematological events after primaquine were few (9/3, 113, 0.3%) and transitory. One blood transfusion was reported in the primaquine arms, and there were no primaquine-related deaths. In controlled studies, the proportions with either haematological or any serious adverse event were similar between primaquine and no primaquine arms.ConclusionsOur results support the WHO recommendation to use 0.25 mg/kg of primaquine as a P. falciparum gametocytocide, including in G6PD-deficient individuals. Although primaquine is associated with a transient reduction in haemoglobin levels in G6PD-deficient individuals, haemoglobin levels at clinical presentation are the major determinants of anaemia in these patients.Trial registrationPROSPERO, CRD42019128185

Global economic costs due to vivax malaria and the potential impact of its radical cure: A modelling study.

In 2017, an estimated 14 million cases of Plasmodium vivax malaria were reported from Asia, Central and South America, and the Horn of Africa. The clinical burden of vivax malaria is largely driven by its ability to form dormant liver stages (hypnozoites) that can reactivate to cause recurrent episodes of malaria. Elimination of both the blood and liver stages of the parasites ("radical cure") is required to achieve a sustained clinical response and prevent ongoing transmission of the parasite. Novel treatment options and point-of-care diagnostics are now available to ensure that radical cure can be administered safely and effectively. We quantified the global economic cost of vivax malaria and estimated the potential cost benefit of a policy of radical cure after testing patients for glucose-6-phosphate dehydrogenase (G6PD) deficiency. Estimates of the healthcare provider and household costs due to vivax malaria were collated and combined with national case estimates for 44 endemic countries in 2017. These provider and household costs were compared with those that would be incurred under 2 scenarios for radical cure following G6PD screening: (1) complete adherence following daily supervised primaquine therapy and (2) unsupervised treatment with an assumed 40% effectiveness. A probabilistic sensitivity analysis generated credible intervals (CrIs) for the estimates. Globally, the annual cost of vivax malaria was US$359 million (95% CrI: US$222 to 563 million), attributable to 14.2 million cases of vivax malaria in 2017. From a societal perspective, adopting a policy of G6PD deficiency screening and supervision of primaquine to all eligible patients would prevent 6.1 million cases and reduce the global cost of vivax malaria to US$266 million (95% CrI: US$161 to 415 million), although healthcare provider costs would increase by US$39 million. If perfect adherence could be achieved with a single visit, then the global cost would fall further to US$225 million, equivalent to $135 million in cost savings from the baseline global costs. A policy of unsupervised primaquine reduced the cost to US$342 million (95% CrI: US$209 to 532 million) while preventing 2.1 million cases. Limitations of the study include partial availability of country-level cost data and parameter uncertainty for the proportion of patients prescribed primaquine, patient adherence to a full course of primaquine, and effectiveness of primaquine when unsupervised. Our modelling study highlights a substantial global economic burden of vivax malaria that could be reduced through investment in safe and effective radical cure achieved by routine screening for G6PD deficiency and supervision of treatment. Novel, low-cost interventions for improving adherence to primaquine to ensure effective radical cure and widespread access to screening for G6PD deficiency will be critical to achieving the timely global elimination of P. vivax.

The association of CYP2D6 gene polymorphisms in the full-length coding region with higher recurrence rate of vivax malaria in Yunnan Province, China

BackgroundAccumulating evidence suggest that compromised CYP2D6 enzyme activity caused by gene mutation could contribute to primaquine failure for the radical cure of vivax malaria. The current study aims to preliminarily reveal the association between the recurrence of vivax malaria in Yunnan Province and CYP2D6 gene mutation by analysing polymorphisms in the entire coding region of human CYP2D6 gene.MethodsBlood samples were collected from patients with vivax malaria, who received "chloroquine and 8-day course of primaquine therapy" in Yunnan Province. The suspected relapsed cases were determined by epidemiological approaches and gene sequence alignment. PCR was conducted to amplify the CYP2D6 gene in the human genome, and the amplified products were then sequenced to compare with the non-mutation “reference” sequence, so as to ensure correct sequencing results and to determine 9 exon regions. Subsequently, the DNA sequences of 9 exons were spliced into the coding DNA sequence (CDS), which, by default, is known as maternal CDS. The paternal CDS was obtained by adjusting the bases according to the sequencing peaks. The mutation loci, haplotypes (star alleles), genotypes and odds ratios (OR) of all the CDSs were analysed.ResultsOf the119 maternal CDS chains in total with 1491 bp in length, 12 mutation sites in the 238 maternal and paternal CDS chains were detected. The c.408G > C mutation was most frequently detected in both suspected relapsed group (SR) and non-relapsed group (NR), reaching 85.2% (75/88) and 76.0% (114/150), respectively. The c.886C > T mutation was most closely related to the recurrence of vivax malaria (OR = 2.167, 95% CI 1.104–4.252, P < 0.05). Among the 23 haplotypes (Hap_1 ~ Hap_23), Hap_3 was non-mutant, and the sequence structure of Hap_9 was the most complicated one. Five star alleles, including *1, *2, *4, *10 and *39, were confirmed by comparison, and CYP2D6*10 allele accounted for the largest percentage (45.4%, 108/238). The frequency of CYP2D6*2 allele in the SR group was significantly higher than that in the NR group (Χ2 = 16.177, P < 0.05). Of the defined 24 genotypes, 8 genotypes, including *4/*4, *4/*o, *2/*39, *39/*m, *39/*x, *1/*r, *1/*n, and *v/*10, were detected only in the SR group.ConclusionMutation of CYP2D6*10 allele accounts for the highest proportion of vivax malaria cases in Yunnan Province. The mutations of c. 886C > T and CYP2D6*2 allele, which correspond to impaired PQ metabolizer phenotype, are most closely related to the relapse of vivax malaria. In addition, the genotype *4/*4 with null CYP2D6 enzyme function was only detected in the SR group. These results reveal the risk of defected CYP2D6 enzyme activity that diminishes the therapeutic effect of primaquine on vivax malaria.

Effect of Primaquine on Gametocyte Carriage in the Case-Management of Uncomplicated Falciparum Malaria with Acts: Nigerian Perspective

Background: Drugs that kill or inhibit sexual stages of Plasmodium such as Primaqiune (PQ) could potentially amplify or synergize the impact of first line antimalarials by blocking transmission to mosquitoes. This study examined the effect of Primaquine on gametocyte carriage in the case management of uncomplicated falciparum malaria with artemisinin-based combination therapy (ACT) with the overall purpose of possibly recommending it as an adjunct drug for malaria control. Methods: A total of 181 patients with uncomplicated falciparum malaria, normal glucose-6-phosphate dehydrogenase (G6PD) enzyme levels, and haemoglobin levels ≥ 8 g/dL completed this two-arm randomized blinded clinical trial to test the efficacy of a single dose PQ (0.75 mg/kg) on falciparum gametocytaemia. 88 subjects were assigned to a standard 3-day course of Dihydroartemisinin-Piperaquine (DHP) alone (n = 88) while 93 others had DHP combined with a single dose of PQ on day 3 (n = 93). A 28-day follow-up schedule carried out in the outpatient clinic of a Primary health facility in Vom, Plateau State Nigeria where study participants were seen on days 1, 3, 7 and then weekly to assess the presence of asexual parasites and gametocytes by microscopy. A Kaplan-Meier analysis was employed to determine the survival function of gametocytes on day 3. The data was analyzed using Epi info version 7.1.5. Results: With a gametocyte prevalence of 27.1%, gametocyte carriage rate was lower in the PQ group due to higher probability of clearing gametocytes (Breslow test χ2 = 8.306, df = 1, p = 0.004) and significantly less likely to harbor gametocytes by day 7 when compared to the DHP-alone group (χ2 = 6.218, df = 1, p = 0.013). Conclusion: Addition of single-dose 0.75 mg/kg PQ was associated with reduced gametocyte carriage as a result of faster gametocyte clearance and lower incidence of gametocyte development in DHP-treated patients. PQ as gametocytocidal drug may be useful in combination with artemisinin-based combination therapy (ACT) regimen to clear gametocytes and thereby interrupt malaria transmission to mosquito vector more effectively than ACT alone.

Factors Affecting Primaquine Combination Treatment in Malaria Patients in Selangor, Malaysia.

Introduction:Primaquine is vital for the management of liver-stage Plasmodium vivax and Plasmodium ovale malaria. However, primaquine effectiveness is dependent on various factors and differs between populations. Therefore, this study was conducted to identify factors that affect the length of stay and relapse during primaquine combination treatment in malaria-infected patients in the local setting.Materials and Methods:A retrospective study on the use of primaquine combination among P. vivax and P. ovale infected patients in Selangor, Malaysia within a 5-year period from 2011 to 2015 was obtained from the National Malaria Case Registry, Malaysia. Data collected were patient characteristics (age, gender, nationality, glucose-6-phosphate dehydrogenase, pregnancy); disease characteristics (survival, past malaria infection, parasite type, presence of gametocyte, parasite count, week onset, severity, transmission type); and treatment characteristics (type of antimalarial, treatment completion). Outcome measures were length of stay and relapse during a 1-year follow-up.Results:A total of 635 patients were included in the study. Based on a multivariate logistic regression analysis, the significant predictors for length of stay were gender (P = 0.009) and indigenous transmission (P < 0.001). Male patients had a shorter length of stay than females by 0.868 days (P = 0.009), and indigenous transmission took 1.82 days more compared to nonindigenous transmission (P < 0.001). Predictors for relapse were indigenous transmission of malaria (P = 0.019), which was 15.83 times more likely to relapse than nonindigenous transmission (P < 0.01).Conclusions:This study reveals that the effectiveness of primaquine was clinically associated with gender and indigenous transmission. To that end, vigilant monitoring of primaquine use is required to reduce relapse and future transmission.

Attenuation of Colchicine Toxicity in Drug-resistant Cancer Cells by Co-treatment with Anti-malarial Drugs

Colchicine (COL) is a well-known and potent microtubule targeting anticancer agent. The purpose of our study was to identify conditions that increase sensitization of COL-resistant cancer cells that overexpress P-glycoprotein (P-gp). The anti-malarial drugs chloroquine (CHL), mefloquine (MEF) and primaquine (PRI) have been shown to increase sensitization in drug-resistant KBV20C cells via P-gp inhibition. Therefore, we tested whether co-treatment of COL with PRI, CHL or MEF increases sensitivity in COL-resistant KBV20C cells over that of cells treated with COL alone and whether these effects are attributable to P-gp activity. Interestingly, we found that both CHL and PRI, but not MEF, reduced cytotoxicity in KBV20C cells receiving high concentrations of COL, suggesting that the effects of CHL and PRI have specific mechanisms among the anti-malarial drugs. The effects of CHL and PRI were specific to COL-resistant cells, since we did not detect a reduction in cytotoxicity in drug-sensitive parent KB cells. These data suggest that CHL and PRI inhibit the signaling pathways of COL-treated-resistant cells without P-gp inhibition. Furthermore, we studied the molecular mechanisms underlying the effects of COL-CHL co-treatment in KBV20C cells. FACS analysis, annexin V staining and western blot analysis revealed that G2 arrest and apoptosis were lower in cells co-treated with COL and CHL than in cells treated with COL alone. We also found that pH2AX, pHistone H3 and pRb expression was highly reduced in COL-CHL co-treated cells but not in COL-VIB co-treated cells. In addition, expression of the p21 protein, which correlates with drug-resistant phenotypes, increased in cells receiving COL-CHL co-treatment over that of COL-treated cells. These results suggest that reduced G2 arrest and apoptosis resulting from COL-CHL co-treatment was attributable to DNA damage and reduced cell cycle progression. These findings provide important information regarding the prevention of COL toxicity in COL-resistant cells and indicate that CHL, PRI and MEF may contribute to sensitization in COL-resistant cells.

Editorial Commentary: Single-Dose Primaquine as Gametocytocidal Treatment in Patients With Uncomplicated Falciparum Malaria

Editorial Commentary: Single-Dose Primaquine as Gametocytocidal Treatment in Patients With Uncomplicated Falciparum Malaria

The Effect of Primaquine on Gametocyte Development and Clearance in the Treatment of Uncomplicated Falciparum Malaria With Dihydroartemisinin-Piperaquine in South Sumatra, Western Indonesia: An Open-Label, Randomized, Controlled Trial

Artemisinin-based combination therapy is very effective in clearing asexual stages of malaria and reduces gametocytemia, but may not affect mature gametocytes. Primaquine is the only commercially available drug that eliminates mature gametocytes. We conducted a 2-arm, open-label, randomized, controlled trial to evaluate the efficacy of single-dose primaquine (0.75 mg/kg) following treatment with dihydroartemisinin-piperaquine (DHP) on Plasmodium falciparum gametocytemia, in Indonesia. Patients aged ≥5 years with uncomplicated falciparum malaria, normal glucose-6-phosphate dehydrogenase enzyme levels, and hemoglobin levels ≥8 g/dL were assigned by computerized-generating sequence to a standard 3-day course of DHP alone (n = 178) or DHP combined with a single dose of primaquine on day 3 (n = 171). Patients were seen on days 1, 2, 3, and 7 and then weekly for 42 days to assess the presence of gametocytes and asexual parasites by microscopy. Survival analysis was stratified by the presence of gametocytes on day 3. DHP prevented development of gametocytes in 277 patients without gametocytes on day 3. In the gametocytemic patients (n = 72), primaquine was associated with faster gametocyte clearance (hazard ratio = 2.42 [95% confidence interval, 1.39-4.19], P = .002) and reduced gametocyte densities (P = .018). The day 42 cure rate of asexual stages in the DHP + primaquine and DHP-only arms were: polymerase chain reaction (PCR) unadjusted, 98.7% vs 99.4%, respectively; PCR adjusted, 100% for both. Primaquine was well tolerated. Addition of single-dose 0.75 mg/kg primaquine shortens the infectivity period of DHP-treated patients and should be considered in low-transmission regions that aim to control and ultimately eliminate falciparum malaria. Clinical Trials Registration. NCT01392014.

The Technology for Preparation of Generic (Monoenantiomeric) Antimalarial Drug Primaquine by Using Supercritical Fluid Chromatography. Separation of Primaquine from Quinocide: Simultaneous Resolution of the Enantiomers of Primaquine and Their Separation from Quinocide in One Run

Malaria is one of the most harmful diseases on the globe. According to the World Health Organization (WHO), several million people die every year from malaria, and most of them are children. Hundreds of millions of fresh cases of ma- laria are registered by the WHO every year, and more than one-third of the earth’s population lives in malaria-endemic areas. Primaquine is an important antimalarial drug because it has gametocytocidal properties and prevents relapse in most cases. However, primaquine is a highly toxic substance, especially to the Negroid race (in Africa, Australia and North, Latin and South America) and some others. Negroid male children are most vulnerable to the toxic effects of primaquine. The toxicity of primaquine can be enhanced in mixtures with other antimalarial drugs. In the present study, unprocessed primaquine and primaquine tablets highly contaminated with quinocide (I. Brondz, Historical Overview of Chromatography and Related Techniques in Analysis of Antimalarial Drug Primaquine (Editor I. Brondz), Nova Sci- ence Publishers, Inc. (2011) ISSN 978-1-61761-944-1) are discussed versus monoenantiomeric primaquine as a drug. The contamination of primaquine with quinocide enhances the toxicity of primaquine by additive or synergistic action. The use of contaminated primaquine can be harmful. Development of a useful antimalarial vaccine can take a decade or longer. This paper describes the possibility of preparing antimalarial generic monoenantiomeric primaquine, free of both quinocide contamination and the ineffective enantiomer of primaquine, using fractionation by supercritical fluid chromatography equipped with a new experimental High Resolution Isomer Column (HRIC). By this approach, it is pos- sible to produce a significant amount of pharmacologically active enantiomer of primaquine at relatively low cost for a broad range of patients sensitive to contaminated primaquine. Leading pharmacopoeias should no longer deny the pre- sence of the toxic contaminant quinocide in relatively high concentrations in unprocessed primaquine and in prima- quine tablets. New standards for antimalarial primaquine diphosphate tablets must be adopted in pharmacopoeias and by the pharmaceutical industry.

Plasmodium vivax treatments

For over 50 years, the treatment of Plasmodium vivax has relied on a combination of chloroquine and primaquine, but this strategy is under threat. Chloroquine efficacy is now compromised across much of the vivax endemic world and there are significant operational difficulties in deploying primaquine. We review the recent advances in P. vivax chemotherapy that may influence the future management of this neglected pathogen. New-generation artemisinin combination therapies (ACTs) have shown potent efficacy against the erythrocytic stages of both drug-resistant P. vivax and Plasmodium falciparum. Antimalarial regimens containing slowly eliminated drugs provide a measure of protection against the first, and possibly second, relapse of tropical strains of P. vivax, but reliable radical cure is needed to prevent future relapses. Primaquine is currently the only licensed hypnozoitocidal treatment, but requires long treatment courses and its effectiveness in different endemic settings remains largely unknown. In regions coendemic for P. vivax and P. falciparum, a unified treatment policy for malaria of any parasitological cause is likely to confer the greatest individual and public health benefit. Optimizing the safety and effectiveness of primaquine through the development of rapid diagnostic tests for glucose-6-phosphate dehydrogenase deficiency and improving drug adherence will be crucial endeavors in the fight against vivax malaria.

Optimally timing primaquine treatment to reduce Plasmodium falciparum transmission in low endemicity Thai-Myanmar border populations

BackgroundEffective malaria control has successfully reduced the malaria burden in many countries, but to eliminate malaria, these countries will need to further improve their control efforts. Here, a malaria control programme was critically evaluated in a very low-endemicity Thai-Myanmar border population, where early detection and prompt treatment have substantially reduced, though not ended, Plasmodium falciparum transmission, in part due to carriage of late-maturing gametocytes that remain post-treatment. To counter this effect, the WHO recommends the use of a single oral dose of primaquine along with an effective blood schizonticide. However, while the effectiveness of primaquine as a gametocidal agent is widely documented, the mismatch between primaquine's short half-life, the long-delay for gametocyte maturation and the proper timing of primaquine administration have not been studied.MethodsMathematical models were constructed to simulate 8-year surveillance data, between 1999 and 2006, of seven villages along the Thai-Myanmar border. A simple model was developed to consider primaquine pharmacokinetics and pharmacodynamics, gametocyte carriage, and infectivity.ResultsIn these populations, transmission intensity is very low, so the P. falciparum parasite rate is strongly linked to imported malaria and to the fraction of cases not treated. Given a 3.6-day half-life of gametocyte, the estimated duration of infectiousness would be reduced by 10 days for every 10-fold reduction in initial gametocyte densities. Infectiousness from mature gametocytes would last two to four weeks and sustain some transmission, depending on the initial parasite densities, but the residual mature gametocytes could be eliminated by primaquine. Because of the short half-life of primaquine (approximately eight hours), it was immediately obvious that with early administration (within three days after an acute attack), primaquine would not be present when mature gametocytes emerged eight days after the appearance of asexual blood-stage parasites. A model of optimal timing suggests that primaquine follow-up approximately eight days after a clinical episode could further reduce the duration of infectiousness from two to four weeks down to a few days. The prospects of malaria elimination would be substantially improved by changing the timing of primaquine administration and combining this with effective detection and management of imported malaria cases. The value of using primaquine to reduce residual gametocyte densities and to reduce malaria transmission was considered in the context of a malaria transmission model; the added benefit of the primaquine follow-up treatment would be relatively large only if a high fraction of patients (>95%) are initially treated with schizonticidal agents.ConclusionMathematical models have previously identified the long duration of P. falciparum asexual blood-stage infections as a critical point in maintaining malaria transmission, but infectiousness can persist for two to four weeks because of residual populations of mature gametocytes. Simulations from new models suggest that, in areas where a large fraction of malaria cases are treated, curing the asexual parasitaemia in a primary infection, and curing mature gametocyte infections with an eight-day follow-up treatment with primaquine have approximately the same proportional effects on reducing the infectious period. Changing the timing of primaquine administration would, in all likelihood, interrupt transmission in this area with very good health systems and with very low endemicity.

The Effect of Primaquine on Lysosomal Protein in Cultured Rat Hepatocytes

We previously reported that chloroquine disrupted lysosomes, but not the shift to low-density lysosomes. In the present study, the effects of primaquine on lysosomal integrity in cultured rat hepatocytes were studied by measuring lysosomal enzyme β-glucuronidase (β-G) or lysosomal-associated membrane glycoprotein (lamp-1) in the cytosolic fraction obtained from cells permeabilized by digitonin, and in the cytosolic fraction obtained by conventional cell fractionation or in Percoll density gradient fractions. The percentage disruption of lysosomes in living cells by 50 μM or 100 μM of primaquine was 1% or 4%, respectively, and lysosomes disrupted by homogenization or centrifugation during cell fractionation by 50 μM or 100 μM of primaquine were 2% or 7%, respectively. The decrease of β-G and lamp-1 in lysosome fractions (fractions 16 to 18) on a Percoll density gradient (1 to 18 fractions) in 50 μM or 100 μM primaquine-treated cells was 9% or 19% for β-G, and 16% or 24% for lamp-1, respectively. The decrease of β-G and lamp-1 in the lysosome fraction was higher than the disruption of lysosomes in living cells or by homogenization or centrifugation during cell fractionation by 50 μM or 100 μM of primaquine. Also, the peak fraction numbers of the subcellular distribution of β-G and lamp-1 on a Percoll density gradient by 50 μM primaquine-treated cells were fraction 17 (high density) and 4 (low density), while those by 100 μM primaquine-treated cells were fraction 6 (low density) or 4 (low density). From these data, we infer that the main effect of primaquine is to cause a shift of lysosomal protein to low density, and then to cause differences in the proportion of membrane and luminal proteins of lysosomes in low-density fractions, although the main effect of chloroquine was the disruption of lysosomes.

Is primaquine useful and safe as true exo-erythrocytic merontocidal, hypnozoitocidal and gametocidal antimalarial drug?

The main objective of this paper is to make available in a single document, a sequence of events that have been published on the biology of malaria parasites and their interaction with the human host, looking for arguments for effective and save treatment: what we know and what we would like to know about the effects of primaquine in order to justify its use in clinical and public health practice. The practicioner should be aware that the antimalarial activity, hemolytic and methemoglobinemic side effects, and detoxification of primaquine are all thought to depend on various biotransformation products of the drug. In spite of the universal use during over six decades, their site and mechanism of formation and degradation and their specific biologic effects remain very poorly understood in human beings. The mature gametocytes of P. falciparum are naturally resistant to chloroquine and other blood merontocides, but they are usually eliminated with a single dose of 1.315 mg/kg per os (p.o.) of primaquine phosphate (equivalent to 0.75 mg-base). Rather than empirically, related with relapses frequency, dosage schedules should only be determined through consideration of the kinetics and dynamics of the drug and its effect on sporozoites, pre and exo-erythrocytic merontes, hypnozoites and gametocytes of P. vivax. Where medical care services are not available or not capable to detect glucose-6-phosphate dehydrogenese- (G-6-PD) deficiencies and deleterious effects of the drug, we recommend not to use primaquine. Both, P. vivax primary clinical attack and P. vivax relapses, as and when they occur should be treated with a course of 10 mg/kg chloroquine-base p.o. Prevention of relapses is probably related to strain characteristics of P. vivax hypnozoites populations involved. If well informed and qualified medical care workers decide to use primaquine in the absence of enzime deficiencies and are able to follow-up the clinical, toxicological and parasitic results, a daily dose of 0.25 mg/kg primaquine-base during 14 days could be administered safety for possible prevention of P. vivax relapses.

Primaquine Alters Antioxidant Enzyme Profiles in Rat Liver and Kidney

The effects of primaquine treatment on antioxidant enzyme activities were investigated in rat liver and kidney. Male Sprague-Dawley rats were treated with 0.21 mg/kg daily for two weeks (chronic treatment) or a single dose at 0.21 or 0.63 mg/kg. Antioxidant enzyme activities were determined in liver and kidney cytosolic fractions whereas glutathione (GSH) and malondialdehyde (MDA) levels were determined in tissue samples. Results for the liver showed increases in cytosolic superoxide dismutase (SOD) and glutathione peroxidase (GPX) enzymatic activities after chronic primaquine treatment. Levels of MDA, a marker for lipid peroxidation, were also increased by more than 50% indicating enhanced oxidative damage in the liver. In the single dose study, 0.63 mg/kg primaquine caused a more than 100% increase in liver SOD and a 36% increase in NAD (P) H: quinone oxidoreductase (NQOR) activities. Results for the kidney, however, showed fewer primaquine-induced changes in antioxidant enzyme activities when compared to the liver in both the chronic and single dose studies. Overall, our results indicate that primaquine treatment causes an oxidative stress in the two rat organs. These results are consistent with the known pro-oxidant effects of primaquine in vivo, and supplement current knowledge on the effects of antimalarial drugs on various enzyme systems.

Store-activated Ca2+ inflow in Xenopus laevis oocytes: inhibition by primaquine and evaluation of the role of membrane fusion.

The role of membrane fusion in the activation of store-activated Ca2+ channels (SACCs) in the plasma membrane of Xenopus laevis oocytes was investigated with primaquine, an inhibitor of vesicle trafficking, reagents that disrupt the cytoskeleton, and reagents that activate or inhibit the functions of monomeric and trimeric GTP-binding regulatory proteins. Ca2+ inflow was assessed by measuring the rate of increase in the fluorescence of the intracellular Ca2+ chelator fluo-3 after the addition of extracellular Ca2+ to oocytes previously incubated in the absence of added Ca2+. Primaquine inhibited the 3-deoxy-3-fluoro Ins(1,4,5)P3 (Ins(1,4,5)P3F)-stimulated increase in Ca2+O,-induced fluo-3 fluorescence with no detectable effect on the release of Ca2+ from intracellular stores. The effect of primaquine was observed within 1.5 min, showed similarity to the inhibition induced by Gd3+, was reversible, and was observed when primaquine was added either before or after activation of the SACCs. The degree of inhibition of Ca2+ inflow by primaquine was halved when the extracellular concentration of Ca2+ was increased from 3.1 to 12.5 mM. Primaquine also inhibited Ca2+ inflow through cholera toxin-activated divalent cation channels and Drosophila Trpl channels (expressed in oocytes after injection of trp1 cRNA). These results indicate that primaquine inhibits open SACCs, possibly by directly inhibiting Ca2+ flow through the channel pore. Colchicine plus cytochalasin B, Brefeldin A, the peptide Arf-1 (2-17) (introduced by microinjection), lovastatin or pertussis toxin did not inhibit the Ins(1,4,5)P3F stimulated increase in fluo-3 fluorescence. In contrast, guanosine 5'-[gamma-thio]triphosphate (GTP[S]), guanosine 5'-[beta, gamma-imido]triphosphate (p[NH]ppG) and A1F4-, but not guanosine 5'-[beta-thio]diphosphate, inhibited the Ins(1,4,5)P3F-stimulated increase in fluo-3 fluorescence. Co-administration of GTP did not prevent the inhibition by GTP[S] of FA1F4-. Staurosporine largely prevented the inhibition of store-activated Ca2+ inflow by GTP[S]. It is concluded that membrane fusion processes are unlikely to be involved in the link between the release of Ca2+ from the endoplasmic reticulum and activation of SACCs. The idea that this link is achieved by direct interaction of a protein(s) in the endoplasmic reticulum membrane with the SACC protein is briefly discussed.

Susceptibility of glucose-6-phosphate dehydrogenase deficient red cells to primaquine, primaquine enantiomers, and its two putative metabolites: II. Effect on red blood cell membrane, lipid peroxidation, MC-540 staining, and scanning electron microscopic studies

The effects of primaquine (PQ), its enantiomers [(+)PQ,(−)PQ] and hydroxy metabolites [5-hydroxyprimaquine (5HPQ) and 6-desmethyl-5-hydroxyprimaquine (6D5HPQ)] on cell membranes of glucose-6-phosphate dehydrogenase (G-6-PD) deficient red cells were studied in vitro. There was no significant effect of PQ on the malonyldialdehyde (MDA) content of normal and heterozygous red cells, but it caused a significant increase in MDA in G-6-PD deficient red cells (P < 0.05). There was no noticeable difference between the effects of the two enantiomers on this variable (P > 0.05). Compared to PQ, the hydroxy metabolites produced a significantly greater increase in MDA in all the groups studied (P < 0.001). Of the two hydroxy metabolites, 6D5HPQ was more toxic than 5HPQ. Staining with MC 540 showed that exposure to PQ, its enantiomers and two putative metabolites produced significant fluorescence, indicating that the drug produces marked alterations in membrane fluidity. Although the fluorescence was seen both in normal and heterozygous cells, the effect was marked in hemizygous deficient red cells (P < 0.001). Scanning electron microscopic (SEM) studies revealed that PQ enantiomers had a stomatocytic effect on red cells of normal, heterozygous and hemizygous G-6-PD deficient red cells, whereas the putative metabolites had an echinocytic effect. The effects were most pronounced in G-6-PD deficient red cells.

Primaquine Is Lethal for Intracellular but Not Extracellular Trypanosoma cruzi

Primaquine has been used to treat Chagas' disease in humans and has been reported to be active against extracellular Trypanosoma cruzi. Experiments were designed to evaluate the relative activity of primaquine against extra- and intracellular T. cruzi and to determine if primaquine might be combined advantageously with ketoconazole. Primaquine at 0.5 micrograms/ml significantly inhibited T. cruzi replication in infected mouse peritoneal macrophages and also effectively treated infected L929 cells. To examine the effect of primaquine on extracellular organisms, tissue culture T. cruzi were incubated with primaquine for different periods of time and then used to infect macrophages. Incubation with 10 micrograms/ml for 14 hr but not 8 hr significantly inhibited but did not eradicate replication. Incubation of spleen amastigotes or blood trypomastigotes for 2 hr with 10 micrograms/ml did not inhibit replication. Incubation of extracellular tissue culture T. cruzi with primaquine for 2 hr did not potentiate the activity of ketoconazole against intracellular organisms. The combination of primaquine and ketoconazole administered to acutely infected mice significantly decreased parasitemias in comparison to treatment with primaquine or ketoconazole alone. Thus primaquine acts primarily on intracellular rather than extracellular T. cruzi. Primaquine and ketoconazole appear to have additive activity in vivo.

Oxidant stress in malaria as probed by stable nitroxide radicals in erythrocytes infected with Plasmodium berghei. The effects of primaquine and chloroquine

Erythrocytes from normal mice and mice infected with the malarial parasite Plasmodium berghei reduce the water-soluble spin probes 2,2,6,6-tetramethylpiperidine-4-hydroxy- N-oxyl (TEMPOL), 2,2,6,6-tetramethylpiperidine- N-oxyl (TEMPO) and 2,2,6,6-tetramethylpiperidine-4-keto- N-oxyl (TEMPONE) at similar rates under both air and N 2 atmospheres. The ESR signal of the lipid-soluble spin probe 5-doxyl-stearate is stable on incorporation into erythrocytes from normal mice. In contrast, parasitized red cells reduce this nitroxide probe, at a rate which increases with the level of parasitemia. Inhibitors of electron transport such as KCN and NaN 3, increase the rate of reduction. We propose that nitroxide reduction occurs via the electron transport chain in the parasite. The antimalarial drug primaquine causes reduction of both water-soluble and lipid-soluble spin probes. This action of primaquine is independent of its ability to release H 2O 2 from oxyhemoglobin, and is ascribed to the ability of primaquine to accelerate flux through the hexose monophosphate shunt. The increased production of NADPH results in increased rates of reduction of the nitroxide radicals. Methylene blue, which also increases flux through the shunt, is even more effective than primaquine at reducing the nitroxides. Chloroquine has no such effect. Parasitized mice treated with chloroquine six hours prior to ESR measurements show less nitroxide reducing capacity than do untreated mice. Chloroquine is known to decrease flux through the hexose monophosphate shunt. The metabolic influences of the two antimalarial drugs are, thus, quite different.

Effect of lysosomotropic amines on the secretory pathway and on the recycling of the asialoglycoprotein receptor in human hepatoma cells.

We studied the intracellular transport of secretory and membrane proteins in the human hepatoma cell line HepG-2 infected with vesicular stomatitis virus. Cells were pulse-labeled in the presence of [35S]methionine and chased in the presence of the lysosomotropic agent primaquine. At a concentration of 0.3 mM primaquine effectively inhibited the secretion of albumin and, to a lesser extent, that of orosomucoid and transferrin. The drug also prevented the budding of virus particles at the cell surface. The intracellular transport to the Golgi complex of the membrane protein VSV-G was not affected by primaquine as it acquires resistance to endo-beta-N-acetylglucosaminidase H at the same rate as in control cells. Addition of primaquine at various times after the initiation of the chase period indicates that the effect of primaquine occurs just before secretion. In confirmation of the biochemical data, immunocytochemical localization of albumin in cells treated with NH4Cl demonstrated that albumin accumulated in vesicles at the trans side of the Golgi complex. The effect of primaquine on secretion was also compared with its effect on receptor recycling. The dose-response characteristics of the effect of primaquine on receptor recycling are identical to those of the effects on protein secretion and virus budding. These results indicate that both processes involve the same transport mechanism, and/or that they occur via at least one identical intracellular compartment.

Primaquine and lysosomotropic amines inhibit malaria sporozoite entry into human liver cells

The binding and entry of Plasmodium berghei sporozoites to human hepatoma HepG2 cells is inhibited in a dose-dependent manner by primaquine, chloroquine and other lysosomotropic amines. The site of action of these agents appears to be the hepatoma cell itself, not the sporozoite. While this inhibitory effect of primaquine is rapidly reversible, the precise mechanism responsible for this effect is not presently known.