Thomas G. Brewer

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.

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.

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.

Plasmodium falciparum-Based Bioassay for Measurement of Artemisinin Derivatives in Plasma or Serum

ABSTRACT Artemisinin and its derivatives, artesunate and artemether, are rapidly acting antimalarials that are used for the treatment of severe and uncomplicated multidrug-resistant falciparum malaria. To optimize treatment regimens that use this new class of antimalarials, there is a need for readily available and reproducible assays to monitor drug levels closely in patients. A sensitive and reproducible bioassay for the measurement of the concentrations of artemisinin derivatives in plasma and serum is described. By modifying the in vitro drug susceptibility test, it was found that antimalarial activity in plasma or serum containing an unknown concentration of drug could be equated to the known concentrations of dihydroartemisinin (DHA) required to inhibit parasite growth. Dose-response curves for a Plasmodium falciparum clone (clone W2) and DHA were used as a standard for each assay. Assays with plasma or serum spiked with DHA proved to be reproducible (coefficient of variation, ≤10.9%), with a lower limit of quantitation equivalent to 2.5 ng of DHA per ml. For plasma spiked with artesunate or artemether, there was good agreement of the results obtained by the bioassay and the concentrations measured by high-performance liquid chromatography (HPLC) with electrochemical detection. The bioassay for measurement of the antimalarial activities of artemisinin derivatives in body fluids requires a smaller volume of plasma or serum and is more sensitive than the presently available HPLC methods, can provide pharmacodynamic parameters for determination of activity against the parasite, and should enhance the design of more appropriate dosage regimens for artemisinin drugs.

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.

Pharmacokinetics and kinetic-dynamic modelling of aminophenones as methaemoglobin formers

Methaemoglobin, the oxidized form of haemoglobin, can be formed by a variety of agents, most of which act to oxidize haemoglobin directly or indirectly. Cyanide has a higher affinity for methaemoglobin than for mitochondrial cytochromes, making methaemoglobin formation a basis for the treatment of cyanide poisoning. We used the beagle dog model to investigate the relationship between drug concentration and methaemoglobin levels for two candidate anti‐cyanide compounds. The compounds studied were the aminophenones p‐aminopropiophenone (PAPP) and p‐aminoheptylphenone (PAHP). Both PAPP and PAHP were given as intravenous boluses and as two different oral formulations.

The disposition of an antileishmanial 8-aminoquinoline drug in the isolated perfused rat liver: thermospray liquid chromatography-mass spectrometry identification of metabolites

1. The disposition of the candidate antileishmanial drug 8-(diethylaminohexylamino-6-methoxy-4-methyl quinoline dihydrochloride (I) has been investigated in the isolated perfused rat liver preparation after the administration of 5 mg/kg (25 microCi) of 14C-I. 2. The perfusate concentration of unchanged I declined biexponentially over the 4 h study period, with a distribution t1/2 of 3.3 +/- 0.3 min and a terminal t1/2 of 35.4 +/- 13.6 min. The area under the perfusate plasma concentration/time curve (AUC0-last time point) was 53.3 +/- 15.7 micrograms min/ml, representing 96% of the area under the curve extrapolated to infinity. the perfusate contained predominantly the carboxylic acid metabolite of I, as well as trace quantities of metabolites detected and identified in bile. 3. Biliary excretion of total 14C accounted for 18.2 +/- 5.0% of the dose, only 2.8 +/- 0.7% was identified by h.p.l.c. analysis as unchanged I. The remainder of the bile contained the desethyl metabolite of I as well as a minimum of 12 more polar metabolites. After 4 h, a total of 39.0 +/- 8.3% of dosed 14C was recovered from the liver tissue. Subcellular fractionation of the livers revealed 24.6 +/- 2.2% of 14C to be located in the 10,000 g pellet. 4. Thermospray liquid chromatography-mass spectrometry analysis of untreated bile and bile treated with beta-glucuronidase or aryl sulphatase permitted identification of some of these metabolites, revealing the presence of the parent drug, desethyl metabolite, 6-desmethyl glucuronide, the 6-desmethyl desethyl glucuronide and the side-chain cleaved 8-amino N-glucuronide metabolites of I, as well as the 6-desmethyl sulphate and the 6-desmethyl desethyl sulphate. Two dihydroxylated metabolites were also detected; however, further structure elucidation is required for unambiguous identification.

Rapid derivatization of alcohols with carboxylic-sulphonic mixed anhydrides for HPLC-UV/fluorescence analysis: Application to the detection of dihydroqinghaosu (DQHS) and its metabolites in biological samples

Abstract The formation of ester derivatives of alcohols for the purpose of HPLC-ultraviolet/fluorescence analysis is achieved rapidly when a mixture of the alcohol and the triethylamine salt of the required acid in chloroform or dichloromethane is treated with a solution of either 2,4,6-triisopropylbenzenesulphonyl chloride or 2,4,6-trimethylbenzenesulphonyl chloride (mesitylenesulphonyl chloride) followed by 4-dimethylaminopyridine in the same solvent. Aromatic carboxylic acids give a quantitative yield of the esters while the yield is reduced for arylacetic acids.

Comparative Efficacy of three Methemoglobin Formers in Delaying Effects of Infused Sodium Cyanide

A prophylactic treatment is needed to circumvent constraints of the current therapy for cyanide (CN-) intoxication: intravenous administration of sodium nitrite and sodium thiosulfate. Experiments were conducted to compare the anticyanide effects of three candidate pretreatment methemoglobin (MHb)-forming compounds in an anesthetized animal model. The experiment was conducted in 5 periods, with each of 9 animals receiving the vehicle control in period 1, 0.2 mg/kg p-aminopropiophenone (PA PP) in period 2, and 2.5 mg/kg WR242511 in period 3. Four of the animals received the vehicle control in period 4 followed by 7 mg/kg p-aminoheptanophenone (PA HP) in period 5, and 5 animals received 7 mg/kg PA HP in period 4 followed by the vehicle control in period 5. Sodium cyanide (Na CN) infusions for PAPP, PAHP, and WR242511 experiments were initiated when the predicted MHb level was approximately 5%. Infusions were stopped 10 s after no functional breaths (respiratory arrest) were observed. Blood samples were collected for hemoglobin (Hb), MHb, and total blood CN levels at scheduled time points. Time to respiratory arrest, percent MHb, and Na CN dose were the primary response parameters. A 11 pretreatment regimens effectively mitigated the effects of Na CN poisoning compared to the vehicle control (p <.05). No discernable differences in protection were provided by the three compounds. The results indicated that the protective effect is related to the MHb level rather than the specific pretreatment drug.

Mefloquine Effect on Disposition of Halofantrine in the Isolated Perfused Rat Liver

Halofantrine and mefloquine are antimalarial drugs used in the treatment of malaria, including that caused by chloroquine‐resistant Plasmodium falciparum. Reports of drug‐associated adverse reactions, including sudden death in one patient, have prompted concerns over the safety of halofantrine and the potential for drug‐drug interactions. We used the isolated perfused rat liver (IPRL) model to investigate a possible hepatic metabolic or pharmacokinetic drug‐drug interaction between halofantrine and mefloquine.