Inhibition of merozoite invasion and transient de-sequestration by sevuparin in humans with Plasmodium falciparum malaria.

SEVERE MALARIA: Even with the best available treatment, the mortality from severe Plasmodium falciparum malaria remains high. Typical features at death are high parasite loads and obstructed micro- vasculature. Infected erythrocytes (IE) containing mature parasites bind to the host receptor heparan sulfate, which is also an important receptor for merozoite invasion. To block merozoite invasion has not previously been proposed as an adjunctive therapeutic approach but it may preclude the early expansion of an infection that else leads to exacerbated sequestration and death. SEVUPARIN IN PHASE I STUDY: The drug sevuparin was developed from heparin because heparan sulfate and heparin are nearly identical, so the rationale was that sevuparin would act as a decoy receptor during malaria infection. A phase I study was performed in healthy male volunteers and sevuparin was found safe and well tolerated. SEVUPARIN IN PHASE I/II CLINICAL STUDY: A phase I/II clinical study was performed in which sevuparin was administered via short intravenous infusions to malaria patients with uncomplicated malaria who were also receiving atovaquone/proguanil treatment. This was a Phase I/II, randomized, open label, active control, parallel assignment study. Sevuparin was safe and well tolerated in the malaria patients. The mean relative numbers of ring-stage IEs decreased after a single sevuparin infusion and mature parasite IEs appeared transiently in the circulation. The effects observed on numbers of merozoites and throphozoites in the circulation, were detected already one hour after the first sevuparin injection. Here we report the development of a candidate drug named sevuparin that both blocks merozoite invasion and transiently de-sequesters IE in humans with P. falciparum malaria. TRIAL REGISTRATION: ClinicalTrials.gov NCT01442168.

Bioavailability enhancement of atovaquone using hot melt extrusion technology.

Emerging parasite resistance and poor oral bioavailability of anti-malarials are the two cardinal issues which hinder the clinical success of malaria chemotherapy. Atovaquone-Proguanil is a WHO approved fixed dose combination used to tackle the problem of emerging resistance. However, Atovaquone is a highly lipophilic drug having poor aqueous solubility (less than 0.2 µg/ml) thus reducing its oral bioavailability. The aim of the present investigation was to explore hot melt extrusion (HME) as a solvent-free technique to enhance solubility and oral bioavailability of Atovaquone and to develop an oral dosage form for Atovaquone-Proguanil combination. Solid dispersion of Atovaquone was successfully developed using HME. The solid dispersion was characterized for DSC, FTIR, XRD, SEM, and flow properties. It was filled in size 2 hard gelatin capsules. The formulation showed better release as compared to Malarone® tablets, and 3.2-fold and 4.6-fold higher bioavailability as compared to Malarone® tablets and Atovaquone respectively. The enhanced bioavailability also resulted in 100% anti-malarial activity in murine infection model at 1/8(th) therapeutic dose. Thus the developed methodology shows promising potential to solve the problems associated with Atovaquone therapy, namely its high cost and poor oral bioavailability, resulting in increased therapeutic efficacy of Atovaquone.

Failure of atovaquone-proguanil malaria chemoprophylaxis in a traveler to Ghana.

Clinical failure of Malarone™ chemoprophylaxis is extremely rare. We report a case of Plasmodium falciparum malaria in a returned traveler to Ghana who fully adhered to atovaquone-proguanil (Malarone™) chemoprophylaxis daily dosing, yet took the pills on an empty stomach. Screening of the P. falciparum isolate revealed triple codon mutation of Dhfr at positions 51, 59, and 108. Plasma drug levels of both atovaquone and proguanil revealed sub-therapeutic concentrations.

Emergence of resistance to atovaquone-proguanil in malaria parasites: insights from computational modeling and clinical case reports.

The usefulness of atovaquone-proguanil (AP) as an antimalarial treatment is compromised by the emergence of atovaquone resistance during therapy. However, the origin of the parasite mitochondrial DNA (mtDNA) mutation conferring atovaquone resistance remains elusive. Here, we report a patient-based stochastic model that tracks the intrahost emergence of mutations in the multicopy mtDNA during the first erythrocytic parasite cycles leading to the malaria febrile episode. The effect of mtDNA copy number, mutation rate, mutation cost, and total parasite load on the mutant parasite load per patient was evaluated. Computer simulations showed that almost any infected patient carried, after four to seven erythrocytic cycles, de novo mutant parasites at low frequency, with varied frequencies of parasites carrying varied numbers of mutant mtDNA copies. A large interpatient variability in the size of this mutant reservoir was found; this variability was due to the different parameters tested but also to the relaxed replication and partitioning of mtDNA copies during mitosis. We also report seven clinical cases in which AP-resistant infections were treated by AP. These provided evidence that parasiticidal drug concentrations against AP-resistant parasites were transiently obtained within days after treatment initiation. Altogether, these results suggest that each patient carries new mtDNA mutant parasites that emerge before treatment but are killed by high starting drug concentrations. However, because the size of this mutant reservoir is highly variable from patient to patient, we propose that some patients fail to eliminate all of the mutant parasites, repeatedly producing de novo AP treatment failures.

Lower atovaquone/proguanil concentrations in patients taking efavirenz, lopinavir/ritonavir or atazanavir/ritonavir.

HIV-infected travellers frequently use atovaquone/proguanil as malaria prophylaxis. We compared atovaquone/proguanil pharmacokinetics between healthy volunteers and HIV-infected patients taking efavirenz, lopinavir/ritonavir or atazanavir/ritonavir. The geometric mean ratio (95% confidence interval) area under the curve (AUC)0-->t for atovaquone relative to the healthy volunteers was 0.25 (0.16-0.38), 0.26 (0.17-0.41) and 0.54 (0.35-0.83) for patients on efavirenz, lopinavir/ritonavir and atazanavir/ritonavir, respectively. Proguanil plasma concentrations were also significantly lower (38-43%). Physicians should be alert for atovaquone/proguanil prophylaxis failures in patients taking efavirenz, lopinavir/ritonavir or atazanavir/ritonavir.

Alteration of pharmacokinetics of proguanil in healthy volunteers following concurrent administration of efavirenz.

Efavirenz and proguanil are likely to be administered concurrently for the treatment of patients with HIV and malaria. The metabolism of proguanil is mediated principally by CYP2C19 while efavirenz is known to inhibit this enzyme. This study therefore investigated the effect of efavirenz on proguanil disposition. Fifteen healthy volunteers were each given 300 mg single oral doses of proguanil alone or with the 9th dose of efavirenz (400mg daily for 11 days) in a crossover fashion. Blood samples were collected at pre-determined time intervals and analyzed for proguanil and its major metabolite, cycloguanil, using a validated HPLC method. Co-administration of proguanil and efavirenz resulted in significant increases (p < 0.05) in C(max), T(max), AUC(T) and elimination half-life (T(1/2beta)) of proguanil compared with values for proguanil alone [C(max): 2.55+/-0.24 mg/l vs 3.75+/-0.48 mg/l; T(max): 2.80+/-0.99 h vs 4.80+/-0.99 h; AUC(T): 45.58+/-12.75 mgh/l vs 97.00+/-23.33 mgh/l; T(1/2beta): 16.50+/-4.55 h vs 23.24+/-4.08 h]. Also, efavirenz caused a pronounced decrease in the AUC(metabolite)/AUC(unchanged drug) ratio of proguanil along with a significant decrease (p < 0.05) in C(max) and AUC of the metabolite. These results indicate that efavirenz significantly alters the pharmacokinetics of proguanil. These suggest that the protection against malaria by proguanil may be decreased when the drug is co-administered with efavirenz and the antimalarial efficacy is dependent on cycloguanil plasma levels.

Direct quantitative bioanalysis of drugs in dried blood spot samples using a thin-layer chromatography mass spectrometer interface.

The CAMAG thin-layer chromatography mass spectrometer (TLC-MS) interface has been assessed as a tool for the direct quantitative bioanalysis of drugs from dried blood spot (DBS) samples, using an MS detector, with or without high-performance liquid chromatography (HPLC) separation. The approach gave acceptable sensitivity, linearity, accuracy, and precision data for bioanalytical validations with and without the inclusion of HPLC separation. In addition, the direct elution technique was shown to increase assay sensitivity for a range of analytes representing a wide "chemical space" for pharmaceutical-type molecules over that obtained by conventional manual extraction of samples (punching of DBS and elution with solvent prior to HPLC-MS analysis). Investigations were performed to optimize extraction time, minimize sample-to-sample carry-over, and compare chromatographic performance. On the basis of this preliminary assessment, it has been demonstrated that the TLC-MS interface has the potential to be an effective tool for the direct analysis of drugs in DBS samples at physiologically relevant concentrations, an approach that could provide significant time and cost savings and greatly simplify bioanalytical procedures compared to current manual practices. Further, the increased sensitivity compared to that of manual extraction may enable the analysis of analytes not currently amenable to DBS sampling due to limitations in assay sensitivity.

Pharmacokinetics of chlorproguanil, dapsone, artesunate and their major metabolites in patients during treatment of acute uncomplicated Plasmodium falciparum malaria.

OBJECTIVE: Chlorproguanil (CPG)-dapsone (DDS)-artesunate was in development for the treatment of uncomplicated Plasmodium falciparum malaria. The pharmacokinetics of CPG, DDS, artesunate and their metabolites chlorcycloguanil (CCG), monoacetyl dapsone (MADDS) and dihydroartemisinin (DHA) were investigated in patients with P. falciparum given CPG-DDS alone or plus artesunate. METHODS: Adult patients from Malawi and The Gambia taking part in a phase II clinical trial were randomised to receive a 3-day treatment of CPG-DDS alone (2/2.5 mg/kg/day) or plus 1, 2 or 4 mg/kg/day artesunate. Blood samples for pharmacokinetic analysis were collected up to 24 h post-first dose. RESULTS: The pharmacokinetic analysis included 115 patients. For CPG, there was no significant effect of artesunate on C(max) or AUC(0-24), except the 90% confidence interval (CI) for AUC(0-24) for the 4 mg/kg artesunate dose was slightly below that for the standard bioequivalence range (90% CI 0.78, 1.11); this was not considered clinically relevant. Artesunate increased the CCG AUC(0-24) by 6-17% and C(max) by 0-16%. Artesunate had no significant effect on the rate or extent of absorption of DDS. For MADDS, artesunate increased the AUC(0-24) by 13-47% and C(max) by 8-45%. For 1, 2 and 4 mg/kg artesunate dosing, artesunate AUC(0-infinity) was 64.6, 151 and 400 ng.h/ml and C(max) 48.9, 106 and 224 ng/ml respectively; DHA AUC(0-infinity) was 538, 1,445 and 3,837 ng.h/ml and C(max) 228, 581 and 1,414 ng/ml respectively. Using a power model, the point estimates of slope were greater than 1 for artesunate AUC(0-t) by 16% and C(max) by 5% and for DHA by 39 and 21% respectively. CONCLUSION: Artesunate did not significantly affect CPG or DDS pharmacokinetics. For CCG and MADDS, small to moderate increases in exposure with artesunate dosing were observed. There was a greater than proportional increase in artesunate and DHA exposure with increasing artesunate dose. These effects are not considered to be clinically relevant. It should be noted that the CPG-DDS-artesunate programme has now been stopped following unacceptable haematological toxicity in patients with glucose-6-phosphate dehydrogenase deficiency during a phase III trial. In addition, the CPG-DDS combination has been withdrawn from clinical use.

Influence of the genetic polymorphisms in the 5' flanking and exonic regions of CYP2C19 on proguanil oxidation.

CYP2C19 is a polymorphic enzyme which metabolizes several clinically important drugs including proguanil. Variation in the 5' regulatory region may influence CYP2C19 activity. This study evaluates the relationship between proguanil metabolic ratio and genetic variations of CYP2C19 in a South Indian population. Fifty unrelated healthy subjects were genotyped for CYP2C19 (*)2 and (*)3 alleles and the 5' flanking region of CYP2C19 was sequenced. Plasma concentrations of proguanil and cycloguanil were estimated by reverse phase HPLC after single oral doses (200 mg) of proguanil. In silico docking analysis of transcription factors binding to its sites in CYP2C19 5' regulatory region was performed. The mean metabolic ratios (proguanil/cycloguanil) were highest in (*)1/(*)2 or (*)1/(*)3 subjects and in (*)2/(*)2 or (*)2/(*)3 as compared to (*)1/(*)1 subjects. Subjects with promoter region variation -98T>C showed decrease in the metabolic ratios irrespective of other variation, which may explain the deviation from the genotype-phenotype association of CYP2C19. In silico analysis predicted alteration in the interaction of transcription factors to their binding sites in the presence of variant alleles. The results of this study would be useful in predicting interindividual differences in the metabolism of substrates of CYP2C19.

Simultaneous determination of monodesethylchloroquine, chloroquine, cycloguanil and proguanil on dried blood spots by reverse-phase liquid chromatography.

A method for simultaneous analysis of chloroquine, proguanil and their metabolites from a whole blood sample (80 microL) dried on a filter paper was developed. Sample preparation included a liquid extraction from the filter paper, followed by a solid-phase extraction (C18 Bond Elut cartridge). Separation was obtained by reverse-phase liquid chromatography (HPLC) using a gradient elution on an X-Terra column; UV detection was made at 254 nm. This assay was linear between 150 and 2500 ng mL(-1) for chloroquine (and metabolite) and 300 and 2500 ng mL(-1) for proguanil and cycloguanil. The lower limit of quantification was close to 50 ng mL(-1) for chloroquine (and its metabolite) and 100 ng mL(-1) for proguanil (and its metabolite). No chromatographic interference from endogenous compounds or other tested anti-malarial drugs was evidenced. Chromatographic separation takes about 40 min with a coefficient of variation below 10.3% for within- and between-batch precision. The paper sampling method was validated in 10 healthy subjects treated by Savarine. The stability of compounds and metabolites on the filter paper was evaluated at four temperatures (-20, +4, 20 and 50 degrees C) and for 1, 5 and 20 days. Cycloguanil concentrations were not influenced by storage conditions, whereas, high temperatures and prolonged storage decreased chloroquine and proguanil levels. The proposed HPLC assay is accurate, precise and cost-effective; it can be used for pharmacokinetic and epidemiological studies on anti-malarial treatments.

Population pharmacokinetic and pharmacodynamic modelling of the antimalarial chemotherapy chlorproguanil/dapsone.

AIMS: To determine the population pharmacokinetics of chlorproguanil, dapsone and the active metabolite of chlorproguanil, chlorcycloguanil; and to estimate the duration of parasitocidal activity for chlorpoguanil/dapsone against Plasmodium falciparum isolates of varying sensitivity. METHODS: Rich and sparse pharmacokinetic data were collected prospectively from: healthy volunteers (n=48) and adults (n=65) and children (n=68) suffering from P. falciparum malaria. All subjects received 2.0 mg kg-1 of chlorproguanil and 2.5 mg kg-1 of dapsone. RESULTS: The population pharmacokinetic parameter estimates for chlorproguanil were ka=00.09 h-1 (intersubject variability was 44%), CL/F=51.53 l h-1 (57%), CLD/F=54.67 l h-1, V1/F=234.40 l (50%) and V2/F=1612.75 l; for dapsone were ka=00.93 h-1, CL/F=1.99 l h-1 (72%) and V/F=76.96 l (48%); and for chlorcycloguanil were CLm/Fm=3.72 l h-1 kg-1 (67%) and Vm/Fm=12.76 l kg-1 (64%). For dapsone, CL/F and V/F were both significantly positively correlated with body weight. For a 10-kg child, the mean duration of parasitocidal activity for chlorproguanil/dapsone against the three most susceptible P. falciparum strains was 4.5 days [5th and 95th percentiles 2.4, 7.3] for W282; 5.9 days (3.6, 9.7) for ItG2F6; and 6.1 days (3.7, 10.1) for K39. For an isolate with the ile-164-leu mutation, V1/S, activity ranged from 0.8 days (0.0, 3.3) for a 10-kg child to 1.8 days (0.0, 4.0) for a 60-kg adult. CONCLUSIONS: Plasmodium falciparum malaria has no effect on the pharmacokinetic parameters for chlorproguanil, dapsone or chlorcycloguanil. Chlorproguanil/dapsone will probably prove to be ineffective against parasite strains with the mutation ile-164-leu, were these to become prevalent in Africa.

Sensitive method for the quantitative determination of proguanil and its metabolites in rat blood and plasma by liquid chromatography-mass spectrometry.

A sensitive, simple and fast liquid chromatography tandem mass spectrometry (HPLC-MS/MS) method for the determination of proguanil (PG) and its metabolites, cycloguanil (CG) and 1-(4-chlorophenyl)biguanide (4CPB), was developed and validated over a concentration range of 1-2000 ng/mL using only 50 microL of blood or plasma. After a simple solvent precipitation procedure, the supernatant was analysed directly by HPLC-MS/MS. Separation was achieved using an ethyl-linked phenyl reverse phase column with polar endcapping with an acetonitrile-water-formic acid gradient. Mass spectrometry was performed using a triple quadrupole mass spectrometer operating in positive electrospray ionization mode. The elution of PG (254.07-->169.99), CG (252.12-->195.02) and 4CPB (212.06-->153.06) was monitored using selected reaction monitoring. The three compounds and the internal standard (chloroproguanil) were well separated by HPLC and no interfering peaks were detected at the usual concentrations found in blood and plasma. The limit of quantification of PG and CG was 1 ng/mL and 5 ng/mL for 4CPB in rat blood and plasma. The extraction efficiency of PG, CG and 4CPB from rat blood and plasma was higher than 73%. The intra- and inter-assay variability of PG, CG and 4CPB were within 12% and the accuracy within +/-5%. This new assay offers higher sensitivity and a much shorter run time over earlier methods.

Consumption of charcoal-broiled meat as an experimental tool for discerning CYP1A2-mediated drug metabolism in vivo.

Cytochrome P450 1A2 (CYP1A2) is a major drug-metabolising enzyme. Polycyclic aromatic hydrocarbons, present in high concentrations in tobacco smoke and charcoal-broiled meat, are known to induce CYP1A2. The purpose of the present study was to validate enzyme induction by consumption of charcoal-broiled meat as an experimental tool for discerning CYP1A2-mediated drug metabolism in vivo. Twenty-four healthy, non-smoking men, all extensive metabolisers of sparteine (CYP2D6), participated in the study. All participants were genotyped for a putative CYP1A2-inducibility genotype. In the study diet period charcoal-broiled meat was served for lunch and dinner for five consecutive days. All participants were tested with probe reactions for CYP1A2 (caffeine) and CYP2C19 (proguanil) before and after consuming the study diet. Further, in three subgroups, they were tested with either the CYP1A2-substrate tacrine or probe reactions for CYP3A4 (quinidine) or CYP2C9 (tolbutamide). Neither probe reactions for CYP1A2, CYP2C9, CYP2C19 or CYP3A4 were affected by consumption of charcoal-broiled meat as practised in this study. No modifying role of the CYP1A2-inducibility genotype was evident. A number of experimental limitations are discussed, among them the lack of standardisation of exposure, the timing of phenotyping, and the choice of probe reactions. In conclusion, consumption of charcoal-broiled meat as practised in the present study appears not to be a useful experimental tool for discerning CYP1A2-mediated metabolism in vivo.

Efficacy of antimalarial chemoprophylaxis among French residents travelling to Africa.

Controversy exists about which antimalarial chemoprophylaxis regimen should be used among travellers to Africa: the WHO and other experts recommend the use of mefloquine throughout sub-Saharan Africa, whereas French experts still support the combination of chloroquine and proguanil in most of West Africa (the so-called zone 2 countries). In this case-control study based at a travel clinic, we examined the compliance with antimalarial chemoprophylaxis and its efficacy among travellers to tropical areas. Cases were patients with Plasmodium falciparum malaria (n = 131). Controls were patients who had a negative malaria film (n = 158). Of all controls, only 36 (22.8%) were adequately protected (i.e. compliant with an adapted regimen of chemoprophylaxis). In zone 2 countries, the efficacy of the combined chloroquine and proguanil was 58% (95% CI 22-78%) for all users, but increased to 100% (95% CI 89-100%) for compliant users. In zone 3 countries, the efficacy of mefloquine was 90% (95% CI 51-98%) and 100% (95% CI 58-100%) for all users and compliant users, respectively.

Pregnancy and use of oral contraceptives reduces the biotransformation of proguanil to cycloguanil.

OBJECTIVE: To determine the effects of late pregnancy and also oestrogen supplementation on the CYP2C19-mediated biotransformation of proguanil (PG) to its active antifol triazine metabolite cycloguanil (CG). METHODS: Case control study conducted on the NW border of Thailand; a single dose of PG (4 mg/kg) was administered to Karen women in late pregnancy and a single blood and urine sample taken 6 h later. Women were studied in late pregnancy (>36 weeks) and restudied 2 months after delivery. A separate cohort of Karen women newly attending a birth-control clinic were studied before and 3 weeks into their first course of oral contraceptives (OCP: levonorgestrel 0.15 mg and ethinyloestradiol 0.03 mg). Forty-five pregnant women and forty-two healthy OCP users were studied. RESULTS: The results were similar in both groups; pregnancy and OCP use were both associated with reduced formation of cycloguanil (CG). Impaired PG biotransformation was seen in women with the "extensive metaboliser" phenotype (urine PG/CG ratio <10). CG levels, adjusted for dose, were a median (range) 73% (-59 to 420%) higher following the pregnancy than during the pregnancy in women characterised as extensive metabolisers ( P<0.001). CG levels in women characterised as extensive metabolisers were 34% (-54 to 323%) higher before than while taking the OCP ( P<0.01). CONCLUSION: Late pregnancy and OCP use impair biotransformation of the active antimalarial metabolite CG from the parent PG. This may be mediated by oestrogen inhibition of CYP2C19 activity. The dose of PG should be increased by 50% in these groups.

The pharmacokinetics of atovaquone and proguanil in pregnant women with acute falciparum malaria.

OBJECTIVE: To determine the pharmacokinetic properties of atovaquone, proguanil, and the triazine metabolite cycloguanil in women with recrudescent multi-drug resistant falciparum malaria during the second and third trimesters of pregnancy treated by artesunate-atovaquone-proguanil. METHODS: Serial plasma concentrations of atovaquone, proguanil and cycloguanil were measured in 24 women at baseline and after the final dose of the 3-day treatment with atovaquone (20 mg/kg/day) plus proguanil (8 mg/kg/day) plus artesunate (4 mg/kg/day) daily. RESULTS: The triple combination was well tolerated and highly effective. The outcomes of pregnancy were all normal. Population mean (+/- SEM) oral clearance (Cl/F) estimates were 313+/-33 ml/h/kg and 1109+/-43 ml/h/kg, total apparent volume of distribution (Vd/F) 13.0+/-1.3 l/kg and 22.9+/-1.4 l/kg, and terminal elimination half-life; 29.1 h and 14.3 h, for atovaquone and proguanil, respectively. Using conventional and population pharmacokinetic analyses, Cl/F and Vd/F estimates for both drugs were approximately twice, and plasma concentrations less than half those reported previously in healthy subjects and patients with acute malaria. CONCLUSION: Artesunate-atovaquone-proguanil is a promising treatment for multi-drug resistant falciparum malaria during pregnancy, but the dose of atovaquone-proguanil may need to be increased.

The impact of Malarone and primaquine on psychomotor performance.

INTRODUCTION: Recent evidence has established the effectiveness of Malarone and primaquine for chemoprophylaxis against Plasmodium falciparum malaria. Both have the advantage of providing causal prophylaxis and therefore require continued dosing for only 1 wk after departure from a malaria endemic area. Canadian Forces aircrews are often placed in situations that put them at risk for malaria infection but the safety of these drugs for use in aircrew has not been ascertained. This study was undertaken to determine whether or not Malarone or primaquine impact psychomotor performance. METHOD: Twenty-eight subjects (20 men and 8 women) ranging from 21 to 52 yr of age were assessed for psychomotor performance on 2 psychomotor test batteries at the end of a 7-d dosing protocol for each of placebo, Malarone, and primaquine treatment, in a double-blind crossover design with counterbalanced treatment order. All subjects were also assessed for psychomotor performance once per week during the 3-wk washout intervals. The daily Malarone dose was atovaquone 250 mg/proguanil 100 mg and the daily primaquine dose was 30 mg of base. In order to verify subject compliance with the medication dosing protocol, blood samples were drawn from all subjects at the end of each of the three 7-d loading protocols. All three medications were packaged in identical gelatin capsules for blinding purposes. At each psychomotor test session, all subjects completed a drug side-effect questionnaire, a mood questionnaire, and a sleepiness/fatigue questionnaire. RESULTS: There was no significant impact of Malarone or primaquine on serial reaction time, logical reasoning, serial subtraction, or multitask performance. With respect to drug adverse effects there were no significant main effects or interactions for the documented adverse effects of these medications (abdominal cramps, epigastric distress, nausea, vomiting, anorexia, headache, coughing and dizziness). CONCLUSIONS: There was no impact of either Malarone or primaquine on psychomotor performance, mood, sleepiness, or fatigue. The usual adverse effects of these medications were not significantly manifested in our subjects. These findings support the possible use of either Malarone or primaquine in aircrew for malaria chemoprophylaxis.

Persistence of atovaquone in human sera following treatment: inhibition of Plasmodium falciparum development in vivo and in vitro.

Published pharmacokinetic data indicate that after treatment of patients with therapeutic doses of atovaquone/proguanil hydrochloride (Malarone, GlaxoSmithKline Research Triangle Park, NC), the plasma half-lives of these drugs are 70h and 15h, respectively. However, using two biologic assays (mosquito transmission and in vitro asexual stage development), we demonstrate here that sera from volunteers treated with atovaquone/proguanil retained activity against Plasmodium falciparum up to 6 weeks after such treatment. This activity was due to atovaquone, as administration of this drug alone replicated the data obtained with the combination. Most notably, asexual stage development of an atovaquone-resistant strain (NGATV01) of P. falciparum was not inhibited by sera taken after atovaquone treatment. These data indicate that for atovaquone, biologic assays, though not quantitative, are more sensitive than the usual physicochemical assays. Also, persistence of atovaquone in plasma at low concentrations for long periods may increase the risk of resistant parasites arising.

Time-dependent pharmacokinetics and drug metabolism of atovaquone plus proguanil (Malarone) when taken as chemoprophylaxis.

OBJECTIVE: To determine the pharmacokinetic profiles of atovaquone (ATO), proguanil (PROG) and its active metabolite cycloguanil (CYCLO) with respect to possible accumulation and kinetic interaction upon repeated dosing with Malarone. METHODS: Thirteen healthy volunteers first received a single dose and then after 1 week, repetitive daily doses of Malarone (one tablet) for 13 days. For analysis of plasma drug concentrations, blood samples were collected at regular intervals over 8 days after a single dose and over 12 days after the last day of multiple dosing. Single-dose and steady-state pharmacokinetic parameters were determined for each individual. Genotyping of the gene coding for CYP2C19, a major enzyme catalyzing PROG metabolism, was performed using polymerase chain reaction, and in vitro enzyme kinetic experiments were carried out to study the possible effect of ATO on the catalytic activities of CYP2C19 and 3A4 using fluorometric assays. RESULTS: For ATO, the ratio of the area under the concentration-time curve (AUC) during the last dose interval to the AUC after the single dose (AUC(0- tau)/AUC(0- infinity)) was found to be 0.90 [95% confidence interval (CI) 0.56, 1.24] indicating absence of undue accumulation. AUC(0- tau), and peak plasma concentration at steady state (C(max,ss)) values were, however, threefold lower than those reported in human immunodeficiency virus-infected subjects after 12 multiple daily doses of 250 mg ATO alone. Four volunteers, with mean CYCLO/PROG AUC(0-tau) of 0.03 (-0.23, 0.09) were classified as poor metaboliser (PM) phenotypes. There was a significant increase in the AUC of PROG at steady state with a PROG AUC(0-tau)/AUC(0-infinity) ratio of 1.38 (1.07, 1.69) in extensive metaboliser (EM) phenotypes. CYCLO/PROG AUC ratios were significantly lower 0.67 (0.54, 0.81) at steady state than that after the first single dose in EM phenotypes. The in vitro kinetic experiments on recombinant enzymes (CYP2C19 and CYP3A4) suggested a possible inhibition of catalytic activity of CYP3A4 by ATO. CONCLUSIONS: There was no unexpected accumulation of ATO following repeated administrations of the combination. In EM phenotypes, PROG elimination was reduced at steady state. Also, at steady state, either the elimination of CYCLO was increased or its formation clearance decreased the latter possibly by inhibition of CYP3A4 by ATO.

Analysis of proguanil and its metabolites by application of the sweeping technique in micellar electrokinetic chromatography.

The method of applying large sample volumes in micellar electrokinetic chromatography termed sweeping is applied to determine the conservative limits of detection of some basic drugs in plasma and urine. The biguanides proguanil, 4-chlorophenylbiguanide and cycloguanil are used as models of basic drugs and the limits of detection obtained compared with those previously reported for capillary zone electrophoresis using field-amplified sample injection (FASI) and also by LC using off-line preconcentration. It is found that the sweeping method can be applied to extracts of such biological matrices. The limits of detection obtained by sweeping are improved over FASI for plasma but not for urine and the limits of detection are higher than those reported for LC, for these compounds.