Population pharmacokinetics of the new antimalarial agent tafenoquine in Thai soldiers.

AIMS: To describe the population pharmacokinetics of tafenoquine in healthy volunteers after receiving tafenoquine for malaria prophylaxis. METHODS: The population consisted of 135 male Thai soldiers (mean age 28.9 years; weight 60.3 kg). All soldiers were presumptively treated with artesunate for 3 days plus doxycycline for 7 days to remove any pre-existing malaria infections. After the treatment regime, 104 soldiers (drug group) received a loading dose of 400 mg tafenoquine base daily for 3 days followed by 400 mg tafenoquine monthly for 5 consecutive months. In the placebo group, 31 soldiers were infected with malaria during the study period. They were re-treated with artesunate for 3 days plus doxycycline for 7 days followed by a loading dose of 400 mg tafenoquine daily for 3 days and then 400 mg tafenoquine weekly for prophylaxis. Blood samples were randomly collected from each soldier on monthly and weekly prophylaxis. Plasma tafenoquine concentrations were measured by h.p.l.c. Population pharmacokinetic modelling was performed using NONMEM. RESULTS: A one-compartment model was found best to describe the pharmacokinetics of tafenoquine after oral administration. Age and weight influenced volume of distribution (V/F), and subjects who contracted malaria had higher clearance (CL/F), but none of these factors was considered to have sufficient impact to warrant change in dosing. The population estimates of the first-order absorption rate constant (Ka), CL/F and V/F were 0.694 h(-1), 3.20 l h(-1) and 1820 l, respectively. The intersubject variability in these parameters (coefficient of variation, CV%) was 61.2%, 25.3% and 14.8%, respectively. The absorption and elimination half-lives were 1.0 h and 16.4 days, respectively. The residual (unexplained) variability was 17.9%. CONCLUSIONS: The population pharmacokinetics of orally administered tafenoquine have been determined in Thai soldiers under field conditions. This information, together with its known potent antimalarial activity, portends well for the application of tafenoquine as a useful prophylactic drug or for short-term radical treatment of vivax malaria.

Comparative pharmacokinetics and effect kinetics of orally administered artesunate in healthy volunteers and patients with uncomplicated falciparum malaria.

The single-dose pharmacokinetics of 100 mg of orally administered artesunate (AS) were studied in 6 patient volunteers with uncomplicated falciparum malaria and in 6 healthy volunteers. Plasma concentrations of both the parent drug, AS, and its major metabolite, dihydroartemisinin (DHA), were measured simultaneously by high-performance liquid chromatography (HPLC) with electrochemical detection (ECD). The antimalarial activity of each plasma sample measured by an in vitro bioassay (BA) was used to derive activity concentrations. Artesunate was absorbed rapidly and then almost completely hydrolyzed to DHA in patients, whereas hydrolysis was incomplete in healthy volunteers. The mean +/- standard deviation (SD) maximum concentration (Cmax) of AS was 296+/-110 nmol/L, the time to peak blood level (tmax was 0.71+/-0.66 hr, the half-life (t1/2,z) was 0.41+/-0.34 hr, and the bioavailability over 12 hr (area under the curve [AUC](0-12)) was 253+/-185 nmol hr/L. Measured by HPLC, the Cmax and AUC(0-12) values of DHA in patients with malaria were significantly greater than in volunteers (1,948+/-772 and 1,192+/-315 nmol/L; 4,024+/-1,585 and 1,763+/-607 nmol hr/L, respectively; P < or = 0.05). These differences were even greater when measured by BA. The Cmax for patients with malaria was 2,894+/-2,497 and 795+/-455 nmol/L for volunteers, and AUC(0-12) was 5,970+/-3,625 and 1,307+/-391 nmol hr/L, respectively (P < or = 0.05). In contrast, DHA parameter estimates for t1/2,z and tmax were similar between patients and healthy volunteers, with values of 0.80+/-0.30 versus 0.87+/-0.06 hr and 1.50+/-0.55 versus 1.13+/-0.52 hr, respectively (P > 0.5). Both drug metabolism and tissue protein binding could contribute to the differences between the antimalarial activity of artemisinin drugs in healthy volunteers and malaria infected patients.

Behavioral and neural toxicity of the artemisinin antimalarial, arteether, but not artesunate and artelinate, in rats.

Three artemisinin antimalarials, arteether (AE), artesunate (AS), and artelinate (AL) were evaluated in rats using an auditory discrimination task (ADT) and neurohistology. After rats were trained on the ADT, equimolar doses of AE (25 mg/kg, in sesame oil, n=6), AS (31 mg/kg, in sodium carbonate, n=6), and AL (36 mg/kg, in saline, n=6), or vehicle (sodium carbonate, n=6) were administered (IM) for 7 consecutive days. Behavioral performance was evaluated, during daily sessions, before, during, and after administration. Histological evaluation of the brains was performed using thionine staining, and damaged cells were counted in specific brainstem nuclei of all rats. Behavioral performance was not significantly affected in any rats treated with AS, AL, or vehicle. Furthermore, histological examination of the brains of rats treated with AS, AL, and vehicle did not show damage. In stark contrast, all rats treated with AE showed a progressive and severe decline in performance on the ADT. The deficit was characterized by decreases in accuracy, increases in response time and, eventually, response suppression. When performance on the ADT was suppressed, rats also showed gross behavioral signs of toxicity that included tremor, gait disturbances, and lethargy. Subsequent histological assessment of AE-treated rats revealed marked damage in the brainstem nuclei, ruber, superior olive, trapezoideus, and inferior vestibular. The damage included chromatolysis, necrosis, and gliosis. These results demonstrate distinct differences in the ability of artemisinins to produce neurotoxicity. Further research is needed to uncover pharmacokinetic and metabolic differences in artemisinins that may predict neurotoxic potential.

Arteether-induced brain injury in Macaca mulatta. I. The precerebellar nuclei: the lateral reticular nuclei, paramedian reticular nuclei, and perihypoglossal nuclei.

Malaria poses a threat across several continents: Eurasia (Asia and parts of Eastern Europe), Africa, Central and South America. Bradley (1991) estimates human exposure at 2,073,000,000 with infection rates at 270,000,000, illnesses at 110,000,000, and deaths at 1,000,000. Significant mortality rates are attributed to infection by the parasite Plasmodium falciparum, with an estimated 90% among African children. A worldwide effort is ongoing to chemically and pharmacologically characterize a class of artemisinin compounds that might be promising antimalarial drugs. The U.S. Army is studying the efficacy and toxicity of several artemisinin semi-synthetic compounds: arteether, artemether, artelinic acid, and artesunate. The World Health Organization and the U.S. Army selected arteether for drug development and possible use in the emergency therapy of acute, severe malaria. Male Rhesus monkeys (Macaca mulatta) were administered different daily doses of arteether, or the vehicle alone (sesame oil), for a period of either 14 days, or 7 days. Neuropathological lesions were found in 14-day arteether treated monkeys in the precerebellar nuclei of the medulla oblongata, namely: (1) the lateral reticular nuclei (subnuclei magnocellularis, parvicellularis, and subtrigeminalis), (2) the paramedian reticular nuclei (subnuclei accessorius, dorsalis, and ventralis), and the perihypoglossal nuclei (n. intercalatus of Staderini, n. of Roller, n. prepositus hypoglossi). The data demonstrate that the simina meduallry precerebellar nuclei have a high degree of vulnerability when arteether is given for 14 days at dose levels between 8mg/kg per day and 24 mg/kg per day. The neurological consequences of this treatment regimen could profoundly impair posture, gait, and autonomic regulation, while eye movement disorders might also be anticipated.

Acute high dose arteether toxicity in rats.

Acute high dose administration of the artemisinin antimalarial, beta-arteether (AE), was evaluated in rats using an auditory discrimination task (ADT) and histology. After rats were trained on the ADT, AE (25, 75, 125 mg/kg, i.m.) or vehicle (sesame oil) was administered and behavioral performance was evaluated for 11 consecutive days. Histological evaluation of the brains was performed using thionine and cupric-silver staining. Damaged cells were counted in specific brainstem nuclei of all rats and a qualitative analysis of the rostral-caudal extent of selected brains was performed. Behavioral performance was not significantly affected by any treatment although some evidence of disruption was observed, particularly after the largest dose. At 125 mg/kg, AE produced statistically significant neuropathology, including chromatolysis, in the nucleus trapezoideus and nucleus superior olive. AE at 75 mg/kg, produced significant neuropathology in the nucleus trapezoideus. Neither AE at 25 mg/kg, nor vehicle produced damage. Qualitative analysis revealed a pattern of neuropathology focused in the brainstem. The results show that, in rats, a single dose of AE can produce a pattern of brainstem neuropathology and that specific brainstem nuclei, including auditory nuclei, are particularly vulnerable. These results are consistent with, and extend, previous studies demonstrating brainstem neurotoxicity from repeated AE administration. Moreover, early detection of AE-induced neuropathology is problematic and may require selective examination of brainstem functions.

Comparison of a rapid field immunochromatographic test to expert microscopy for the detection of Plasmodium falciparum asexual parasitemia in Thailand.

We assessed a rapid, Plasmodium falciparum histidine rich protein 2 (PfHRP2)-based immunochromatographic test (ICT Malaria Pf Test), for detection of asexual P. falciparum parasitemia in 551 subjects in three groups: (1) symptomatic patients self-referring for diagnosis, (2) villagers in a screening survey, and (3) patients recently treated for P. falciparum malaria. Expert light microscopy was the reference standard. ICT test performance was similar for diagnostic and screening modes. Four findings emerged: (1) test sensitivity correlated directly with parasite density, (2) test band intensity correlated directly with parasite density, (3) persistent test positivity after parasite clearance precludes its use for monitoring early therapeutic responses, and (4) a false negative test at 18,000 parasites/microl is unexplained. We conclude that a strong positive ICT test is highly predictive of falciparum asexual parasitemia for the diagnosis of new cases of falciparum malaria in Thailand, but a negative test result is inadequate to exclude parasitemia < 300/microl, and in some instances, even a higher parasitemia.

Effects of Plasmodium berghei infection on cytochromes P-450 2E1 and 3A2.

Metabolism and disposition of most drugs used to treat malaria are substantially altered in malaria infection. Few data are available that specify effects of malaria infection on drug metabolism pathways in humans or animal model systems. In this report, studies were undertaken to determine the effect of Plasmodium berghei infection on cytochrome P-450 (CYP450) 2E1 and 3A2-mediated metabolism and enzyme expression in rat liver microsomes. Malaria infection (MAL) resulted in significant decreases in total cytochrome P-450 content (56%, P < 0.05) and NADPH cytochrome P-450 reductase activity (32%, P < 0.05) as compared to control (CON) rats. Chlorzoxazone 4-hydroxylase activity (CYP2E1-mediated) showed no significant difference between CON and MAL microsomes while testosterone 6-beta-hydroxylase activity (CYP3A2-mediated) was reduced by 41% (P < 0.05) in MAL. Enzyme kinetic studies and immunoblot analysis indicate that the loss of activity for CYP3A2 in malaria infection is due to significantly decreased CYP3A2 protein expression. The altered expression of CYP450s in malaria infection should be taken into account when treating patients with malaria in order to minimize drug-drug interactions or toxicity.

Randomized dose-ranging study of the safety and efficacy of WR 238605 (Tafenoquine) in the prevention of relapse of Plasmodium vivax malaria in Thailand.

WR 238605 is an 8-aminoquinoline developed for the radical cure of Plasmodium vivax. Forty-four P. vivax-infected patients were randomly assigned to 1 of 4 treatment regimens: 3 groups received a blood schizonticidal dose of chloroquine followed by WR 238605: group A (n=15) received 300 mg daily for 7 days; group B (n=11), 500 mg daily for 3 days, repeated 1 week after the initial dose; group C (n=9), 1 dose of 500 mg. A fourth group (D; n=9) received chloroquine only. Among patients who completed 2-6 months of follow-up (n=23), there was 1 relapse in group B (day 120) and 1 in group C (day 112). Among patients treated with chloroquine only, there were 4 relapses (days 40, 43, 49, and 84). WR 238605 was safe, well tolerated, and effective in preventing P. vivax relapse.

Arteether toxicokinetics and pharmacokinetics in rats after 25 mg/kg/day single and multiple doses.

Multiple doses of arteether (ARTE) at 25 mg/kg cause CNS and anorectic toxicities in rats. The same dose of ARTE was used to study the toxicokinetics (TK) after multiple injections and the pharmacokinetics (PK) following single administration. Animals were administered ARTE in sesame oil for 7 days, blood samples were collected using destructive sampling for up to 192 h after dosing and assayed by HPLC-ECD. Two other groups of rats were administered either a single 25 mg/kg i.v. or i.m. dose. In addition, the drug remaining in the i.m. injection site was measured. During the 7 day treatments, anorectic toxicity of ARTE was observed, and that caused significant reductions in food consumption and body weight after day 2. TK data on days 2-7 revealed marked changes compared to the PK parameters estimated on day 1. AUC (4367 ng x h/ml) on day 7 was 5-fold higher than AUC (905 ng x h/ml) on day 1. The volume of distribution at steady state (V(SS)) on day 7 (41.8 l) was 40% of the day 1 value of the V(SS) (104.3 l). Clearance (CL) was increased by 89% of the day 1 value, from 0.98 l/h to 1.85 l/h on day 7. The elimination t(1/2) of ARTE was also prolonged from 13.7 h (day 1) to 31.2 h (day 7). These data suggest that ARTE may have altered its distribution and elimination in rats as a result of the systemic toxicity. Analysis of the injection sites showed that 38% and 91% of the total amount of ARTE single dose remained in the muscles at 24 h (after first injection) and 168 h (at 24 h after 7 daily multiple doses), respectively. Fast and slow absorption phases from muscle were seen with t(1/2) of 0.97 h and 26.3 h, respectively. The apparent elimination t(1/2) of ARTE after i.m. injection (13.7 h) was much longer than that after i.v. dosing (0.67 h) due to the prolonged muscle absorption phase. Acute toxicity data of artemisinin drugs demonstrated that animals receiving a high single ARTE dose in sesame oil died between days 5-11, similar to artemether. When animals received dihydroartemisinin formulated in 50% DMAC/oil, or artesunic acid and artelinic acid in 0.9% saline vehicle, they died between days 1 and 2. This suggests that delayed onset toxicity and death in the ARTE rats may also be due to slow absorption and prolonged drug exposure. Therefore multiple i.m. administrations cause anorexia and drug accumulation, possibly affecting the toxicokinetics and efficacy of the drug.

Pharmacology and toxicology of artelinic acid: preclinical investigations on pharmacokinetics, metabolism, protein and red blood cell binding, and acute and anorectic toxicities.

The pharmacokinetics, metabolism, protein binding, red blood cell (RBC) binding, stability in vitro, and acute and anorectic toxicity of artelinic acid (ARTL) were investigated in various animal species and human blood samples. Absorption and distribution following 10 mg/kg intramuscular or oral administration in dogs and rats were very rapid with t1/2 0.12-0.54; there were also a high AUC (11,262 ng/h/mL) and Vss (9.5 L/kg), low CL (15 mL/min/kg) and long elimination time (t1/2 = 2.6 h), compared with rat data. Oral bioavailability of ARTL was 79.7% in dogs and 30.1% in rats. The conversion of ARTL to dihydroartemisinin (DART) in dogs (0.1-0.5% of total dose) after 3 routes of administration (intravenous, intramuscular and oral) was 10-fold lower than that in rats. In rats dosed with [14C]ARTL, unchanged ARTL accounted for less than 13% of the total radioactivity after all 3 administration routes, suggesting that ARTL was extensively biotransformed. The half-lives of total radioactivity (21-49 h) in urine were much longer than that of unchanged ARTL in plasma (1.4-3.7 h), indicating that some long-lasting metabolites of ARTL were formed in rats. The mass balance data showed that 77-83% of total radioactivity was recovered in urine and faeces. High binding capacity (79-95%) and low binding affinity (1.1-9.3 x 10-7 M) of ARTL were measured in rat, rabbit, dog, monkey and human plasma. The RBC/plasma ratios of [14C]ARTL were 0.35 and 0.44 for dog and human plasma, respectively. ARTL was much more stable than artesunic acid (ARTS) in rat and dog plasma, and both ARTL and ARTS were more stable in dog plasma than in rat plasma in vitro. The 50% lethal dose (LD50) of ARTL in rats was about 535 mg/kg. Multiple intramuscular dosing for 7 d of 50 mg/kg/d of ARTL caused mild anorectic toxicity compared to ARTS in rats. In contrast to 4 other artemisinin derivatives, ARTL seems to be a good antimalarial candidate as it has the highest plasma concentration, the highest binding capacities in RBC, the highest oral bioavailability, the longest elimination half-life, the lowest metabolism rate and the lowest toxicity at equivalent dose levels.

The in vitro metabolism of penclomedine in mouse, rat, and human systems.

Penclomedine is a multi-chlorinated alpha-picoline derivative that has demonstrated activity in several murine breast cancer models and is currently in clinical testing for use against solid tumors. This study evaluates the metabolism of penclomedine in several in vitro hepatic models, including microsomes, fresh liver slices, and the isolated perfused rat liver (IPRL). Both human and mouse liver slices as well as human and mouse liver microsomes under aerobic conditions resulted in limited metabolism of penclomedine to several oxidized metabolites, including penclomic acid, 4-demethylpenclomic acid, and 4-demethylpenclomedine. Microsomes under anaerobic conditions vigorously produced mainly reduced metabolites, primarily penclomedine dimers. This is in contrast to in vivo data, which showed rapid metabolism of penclomedine to primarily 4-demethylpenclomedine. The IPRL preparation, however, metabolized 50 microM penclomedine 90% within 90 min, producing primarily 4-demethylpenclomedine and penclomic acid. These were formed in roughly equimolar amounts and did not undergo significant further metabolism over 4 hr. Numerous highly polar biliary metabolites were also found. The IPRL preparation thus seems to most accurately reflect the in vivo situation.

Behavioral and neural toxicity of arteether in rats.

Repeated administration of the artemisinin antimalarial compound, 3-arteether (AE) (25 mg/kg, i.m.) was evaluated in rats using a two-choice, discrete trial, auditory discrimination task and subsequent neurohistology. Rats were trained to choose one of two response levers following presentation of white noise or a tone + white noise. Increasing and decreasing the intensity of the tone increased and decreased discriminability, respectively, and differential reinforcement density produced systematic changes in response bias. AE (n = 5) or vehicle (n = 5) was injected daily (9-12 days). Initial injections of AE did not affect behavioral performance. Continuing daily injections produced significant decreases in choice accuracy and significant increases in choice reaction time. When overt signs of severe toxicity were observed, rats were sacrificed and significant neural pathology was observed in the nucleus trapezoideus of AE-treated rats. In a subsequent experiment, AE was injected for 3 (n = 5), 5 (n = 5), or 7 (n = 5), consecutive days and performance was examined for an additional 7 days. Behavioral disruption was only observed in rats receiving AE for 7 days and the greatest degree of disruption occurred after AE injections were completed. Histopathological examination showed significant neural pathology in the nuclei trapezoideus, superior olive, and ruber of rats receiving 7- and 5-day AE regimens, and in the nucleus trapezoideus of rats receiving the 3-day regimen. Thus, behavioral disruption reflected, but did not predict, neuropathology. These results confirm and extend earlier results demonstrating neurotoxicity of AE in rats. Further, these results demonstrate that the auditory discrimination task provides an objective behavioral measure of AE neurotoxicity, and thus, can serve as a valuable tool for the safety development of AE and other artemisinin antimalarial compounds.

In vitro and in vivo reversal of chloroquine resistance in Plasmodium falciparum with promethazine.

The effect of combining promethazine with chloroquine was examined against Plasmodium falciparum in vitro in the Aotus-P. falciparum model and in bioassays from volunteers given promethazine. The combination of chloroquine plus promethazine (1 x 10(-6) M) reversed chloroquine resistance in standard P. falciparum clones and patient parasite isolates from Nigeria. The combination reduced the 50% inhibitory concentrations (IC50s) for chloroquine against resistant parasites by 32-92%. Coadministration of promethazine with chloroquine also demonstrated a dose-dependent effect in Aotus monkeys infected with chloroquine-resistant P. falciparum. Monkeys were given a chloroquine dose (20 mg/kg of body weight for seven days), which normally has no effect on parasitemia, plus 10, 20, 40, or 80 mg of promethazine/kg of body weight. In one monkey, parasitemia was suppressed at the lowest promethazine dose, but re-treatment with 20 mg/kg resulted in clearance of parasitemia. Initial treatment with chloroquine and 20 or 40 mg/kg of promethazine cleared parasitemia in some animals followed by recrudescence. Re-treatment at higher doses cured one monkey and resulted in initial clearance and delayed recrudescence 28 or 63 days after treatment in two monkeys. Recrudescent parasitemia in the two monkeys was low (10 parasites/microl of blood) and subsequently cleared without re-treatment. An in vitro bioassay model was developed to examine the effects of clinically achievable doses of promethazine on parasites susceptibilities in vitro. Plasma samples taken at hourly intervals from patients given a single oral dose of 25 mg of promethazine decreased the IC50 values for chloroquine by 20-58% with the most significant reductions occurring in plasma obtained from volunteers 3-4 hr after ingestion. Plasma obtained from two volunteers 6 hr after ingestion of the drug demonstrated no effect on chloroquine susceptibility, suggesting that study of the pharmacokinetic disposition and potential interaction is warranted to optimize the dose regimen in patients for antimalarial efficacy. Historic use of this drug combination for treatment or prevention of chloroquine-associated pruritus or as an antiemetic suggest that the combination is safe and effective when used at standard dosages. The results from this study demonstrate that promethazine is a potent modulator of chloroquine resistance. Clinical evaluation of therapeutic regimens is required to validate clinical efficacy of this promising combination for treatment of uncomplicated chloroquine-resistant malaria.

Metabolism of beta-arteether to dihydroqinghaosu by human liver microsomes and recombinant cytochrome P450.

beta-Arteether (AE) is an endoperoxide sesquiterpene lactone derivative currently being developed for the treatment of severe, complicated malaria caused by multidrug-resistant Plasmodium falciparum. Studies were undertaken to determine which form(s) of human cytochrome P-450 catalyze the conversion of beta-arteether to its deethylated metabolite, dihydroqinghaosu (DQHS), itself a potent antimalarial compound. In human liver microsomes, AE was metabolized to DQHS with a Km of 53.7 +/- 29.5 microM and a Vmax of 1.64 +/- 1. 78 nmol DQHS/min/mg protein. AE biotransformation to DQHS was inhibited by ketoconazole and troleandomycin. Ketoconazole was a competitive inhibitor, with an apparent Ki of 0.33 +/- 0.11 microM. Because AE is being developed for patients who fail primary treatment, it is possible that AE may be involved in life-threatening drug-drug interactions, such as the associated cardiotoxicity of mefloquine and quinidine. Coincubation of AE with other antimalarials showed mefloquine and quinidine to be competitive inhibitors with a mean Ki of 41 and 111 microM, respectively. Metabolism of AE using human recombinant P450s provided evidence that cytochrome P450s 2B6, 3A4, and 3A5 were the primary isozymes responsible for its deethylation. CYP3A4 metabolized AE to dihydroqinghaosu at a rate approximately 10 times that of CYP2B6 and approximately 4.5-fold greater than that of CYP3A5. These results demonstrate that CYP3A4 is the primary isozyme involved in the metabolism of AE to its active metabolite, DQHS, with secondary contributions by CYP2B6 and -3A5.

The pharmacokinetics and bioavailability of dihydroartemisinin, arteether, artemether, artesunic acid and artelinic acid in rats.

The pharmacokinetics and bioavailability of dihydroartemisinin (DQHS), artemether (AM), arteether (AE), artesunic acid (AS) and artelinic acid (AL) have been investigated in rats after single intravenous, intramuscular and intragastric doses of 10 mg kg(-1). Plasma was separated from blood samples collected at different times after dosing and analysed for parent drug. Plasma samples from rats dosed with AM, AE, AS and AL were also analysed for DQHS which is known to be an active metabolite of these compounds. Plasma levels of all parent compounds decreased biexponentially and were a reasonable fit to a two-compartment open model. The resulting pharmacokinetic parameter estimates were substantially different not only between drugs but also between routes of administration for the same drug. After intravenous injection the highest plasma level was obtained with AL, followed by DQHS, AM, AE and AS. This resulted in the lowest steady-state volume of distribution (0.39 L) for AL, increasing thereafter for DQHS (0.50 L), AM (0.67 L), AE (0.72 L) and AS (0.87 L). Clearance of AL (21-41 mL min(-1) kg(-1)) was slower than that of the other drugs for all three routes of administration (DQHS, 55-64 mL min(-1) kg(-1); AM, 91-92 mL min(-1) kg(-1); AS, 191-240 mL min(-1) kg(-1); AE, 200-323 mL min(-1) kg(-1)). In addition the terminal half-life after intravenous dosing was longest for AL (1.35 h), followed by DQHS (0.95 h), AM (0.53 h), AE (0.45 h) and AS (0.35 h). Bioavailability after intramuscular injection was highest for AS (105%), followed by AL (95%) and DQHS (85%). The low bioavailability of AM (54%) and AE (34%) is probably the result of slow, prolonged absorption of the sesame-oil formulation from the injection site. After oral administration, low bioavailability (19-35%) was observed for all five drugs. In-vivo AM, AE, AS and AL were converted to DQHS to different extents; the ranking order of percentage of total dose converted to DQHS was AS (25.3-72.7), then AE (3.4-15.9), AM (3.7-12.4) and AL (1.0-4.3). The same ranking order was obtained for all formulations and routes of administration. The drug with the highest percentage conversion to DQHS was artesunic acid. Because DQHS has significant antimalarial activity, relatively low DQHS production could still contribute significantly to the antimalarial efficacy of these drugs. This is the first time the pharmacokinetics, bioavailability and conversion to DQHS of these drugs have been directly compared after different routes of administration. The results show that of all the artemisinin drugs studied the plasma level was highest for artelinic acid; this reflects its lowest extent of conversion to DQHS and its slowest rate of elimination.

Comparative clinical trial of artesunate suppositories and oral artesunate in combination with mefloquine in the treatment of children with acute falciparum malaria.

A randomized pilot study to compare the safety and efficacy of artesunate suppositories (15 mg/kg/day for three days) versus oral artesunate (6 mg/kg/day for three days), both in combination with mefloquine (25 mg/kg), was conducted in 52 Thai children with uncomplicated multidrug-resistant falciparum malaria. Forty-five patients (87%) had a full 28-day follow-up in the hospital to assess efficacy and exclude reinfection. Mean [range] times to fever clearance of the two groups were similar (42 hr [15-104] versus 42 hr [6-119]). Artesunate suppositories resulted in significantly longer times to achieve 50% and 90% reductions of the initial parasite counts (17 and 26 hr versus 9 and 15 hr; P < 0.05 and P < 0.001). Time [range] to parasite clearance was longer in the artesunate suppositories group (42 hr [14-93] versus 35 hr [16-69]), but the difference was not significant. The cure rates by days 28 were not significantly different, 92% for artesunate suppository-treated patients and 100% for oral artesunate-treated patients. Both drug regimens are safe and effective. Further studies are needed to characterize the pharmacokinetic properties and the optimum regimen of artesunate suppositories for the treatment of severe malaria.

Dose-dependent brainstem neuropathology following repeated arteether administration in rats.

Histopathological effects of the artemisinin antimalarial, beta-arteether, were evaluated in rats. Arteether (3.125-12.5 mg/kg/day, IM, in sesame oil) was administered for 7 consecutive days. Seven days following the last injection, histological evaluation of the brainstem was performed. Rats treated with 12.5 mg/kg showed significant neuropathology, including chromatolysis, in the nucleus trapezoideus and nucleus superior olive. To a lesser extent, neuropathology was present in the nucleus ruber. Mild neuropathology was also detected in other brainstem regions examined. Although no statistically significant neuropathology was found for the groups treated with 6.25 mg/kg/day and 3.125 mg/kg/day, substantial neuropathology was observed in a single rat in each of these treatment conditions. These results confirm and extend previous studies demonstrating brainstem neurotoxicity from artemisinin antimalarials. Furthermore, these results suggest that, in rats, brainstem auditory pathways may be particularly vulnerable. Early detection of arteether neuropathology may, therefore, require examination of auditory functions.

Factors relating to neurotoxicity of artemisinin antimalarial drugs "listening to arteether".

The discovery of the occult brainstem neurotoxicity subsequent to widespread deployment of artemisinin derivatives has created particular problems. That is, the clinical setting for artemisinin use is problematic for accomplishing what ordinarily would be addressed in phase I-II clinical trials. Nevertheless, it is clear that an urgent and vital need exists for the deployment and widespread availability of artemisinins. The work done to date has already yielded a substantial body of evidence that, while incomplete, provides guidelines for artemisinin use to minimize the risk of these drugs while preserving their much-needed efficacy. The evidence thus far shows that route of administration, oil/water solubility and concentration-duration of drug level, are critical determinants of toxicity and can be given appropriate consideration in the clinical decisions regarding route, choice of drug used, and drug regimens. In this regard, an oral, water-soluble drug with moderately rapid clearance may be the most attractive choice in the absence of significant differences in efficacy. The same body of evidence clearly shows that toxicity can, and does, develop with no obvious or useful clinical marker. Therefore, the development and validation of a test that can reliably detect the onset of injury, at a reversible stage, is a critical path task for any future development in this class. More complete understanding of mechanisms, kinetics, and molecular targets of neurotoxicity, will certainly be forthcoming. A continuing, more generalized use of these drugs, however, cannot be fully endorsed without a useful, practical clinical test of toxicity. The requirement is especially critical in light of the reality that those patients receiving artemisinin derivatives live in high risk environments and are likely to receive repeated courses of therapy with little likelihood of close, post marketing surveillance.

Effects of Plasmodium berghei infection on arteether metabolism and disposition.

Arteether (AE) is primarily deethylated to dihydroqinghaosu (DQHS) in rats and humans. Conversion of AE to DQHS was impaired in microsomes from rats infected with Plasmodium berghei. The Km for AE was 175.1 +/- 49.1 and 124.4 +/- 115.1 mumol/l, and Vmax was 2.24 +/- 0.45 and 1.22 +/- 0.67 nmol AE formed/mg protein/min in control and infected microsomes (p < 0.05), respectively. Calculated intrinsic clearance (CLint = initial Vmax/Km) for AE was only 4% lower in infected microsomes. Apparent pharmacokinetic parameter estimates for AE using the isolated perfused rat liver demonstrated no differences (p > 0.05) in volume of distribution, clearance, and half-life between normal and infected animals. Malaria infection resulted in decreased biliary excretion of free AE and DQHS. The majority of AE is eliminated via biliary excretion of conjugated DQHS, which is approximately 500-fold higher than free DQHS and 75-fold higher than free AE on a molar basis.

Arteether: risks of two-week administration in Macaca mulatta.

Male rhesus monkeys (Macaca mulatta) were administered daily doses of the antimalarial drug arteether. The 14-day treated group received either 24 mg/kg/day, 16 mg/kg/day, or 8 mg/kg/day. The seven-day treatment group received either 24 mg/kg/day or 8 mg/kg/day. All control cases in each group received the sesame oil vehicle alone. Neurologic signs were absent for animals in the seven and 14-day treatment groups except for one monkey which showed diffuse piloerection on day 14, and another monkey receiving 24 mg/kg/day for seven days showed mild lethargy after the fourth day. Mild, sporadic anorexia was noted in all animals by day 14, and a single animal showed diffuse piloerection on day 14. Surgical anesthesia preceded killing by exsanguination and was accompanied by perfusion fixation of the central nervous system. Brain sections were cut and then stained for study by light microscopy. Evidence of neuronal pathology, both descriptive and numerical, was collected. The neuroanatomic and neuropathologic findings demonstrated that arteether produced extensive brainstem injury when administered for 14 days. The magnitude of brainstem neurotoxicity was dose-dependent, where injury was greatest at the 24 mg/kg/day dose level, less at the 16 mg/kg/day dose level, and least at the 8 mg/kg/day dose level. Arteether induced multiple systems injury to brainstem nuclei of 1) the reticular formation (cranial and caudal pontine nuclei, and medullary gigantocellular and paragigantocellular nuclei); 2) the vestibular system (medial, descending, superior, and lateral nuclei); and 3) the auditory system (superior olivary nuclear complex and trapezoid nuclear complex). The vestibular nuclei and the reticular formation were most severely injured, with the auditory system affected less. The cranial nerve nuclei (somatic and splanchnic) appeared to escape damage, with the exception of the abducens nerve nucleus. The same brainstem nuclear groups of seven-day treated monkeys appeared normal. The statistical data are concordant with the descriptive data in demonstrating neurotoxic effects. In summary, no neurologic deficits were detected in any of the vehicle control monkeys (14-day and seven-day cases). Monkeys in the 14-day treatment group were free of clinical neurologic signs throughout the first week. At day 14, fine horizontal nystagmus was seen in one monkey, and another monkey exhibited diffuse piloerection. Monkeys in the seven-day treatment group were free of clinical neurologic signs except for one case. This monkey was treated with 24/mg/kg/day of arteether and exhibited lethargy after the fourth day. These indications of dysfunction arose too late to be practical indicators of neurotoxicity.