James O. Peggins

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.

Mechanism of inhibition of cholinesterases by huperzine A.

Huperzine A, an alkaloid isolated from Huperzia serrata was found to reversibly inhibit acetylcholinesterases (EC 3.1.1.7) and butyrylcholinesterases (EC 3.1.1.8) with on- and off-rates that depend on both the type and the source of enzyme. Long-term incubation of high concentrations of purified cholinesterases (1-8 microM) with huperzine A did not show any chemical modification of huperzine A. A low dissociation constant KI was obtained for mammalian acetylcholinesterase-huperzine (20-40 nM) compared to mammalian butyrylcholinesterase-huperzine (20-40 microM). This indicates that the thermodynamic stability of huperzine-cholinesterase complex may depend on the number and type of aromatic amino acid residues in the catalytic pocket region of the cholinesterase molecule.

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.

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.

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.

Hepatic metabolism of artemisinin drugs—III. Induction of hydrogen peroxide production in rat liver microsomes by artemisinin drugs

1. In this communication, induction of hydrogen peroxide production by the semisynthetic antimalarial drugs of the artemisinin class (beta-arteether, beta-artelinic acid and dihydroartemisinin) in rat liver microsomes, is reported. 2. Endogenous, NADPH-dependent, production of hydrogen peroxide in rat liver microsomes was enhanced in the presence of arteether and artelinic acid, but not in the presence of dihydroartemisinin. 3. NADPH-dependent metabolism of arteether and artelinic acid was closely coupled to the drug-induced production of hydrogen peroxide. 4. The redox cycle of cytochrome P-450 was presented, which describes satisfactorily both the endogenous and the drug-assisted hydrogen peroxide production in rat liver microsomes; also, the rate-limiting step of the cycle was identified.

Biomimetic Metabolism of Artelinic Acid by Chemical Cytochrome P-450 Model Systems

AbstractPurpose. To study the reaction of artelinic acid with chemical model systems of cytochrome P-450 as a means of obtaining authentic samples of the putative metabolites necessary for identification of the mammalian metabolites of artelinic acid. Methods. Artelinic acid was reacted with different organic complexes of iron(II). The reaction products were isolated and characterized by NMR and thermospray mass spectroscopy. Results. Five compounds which are putative metabolites of artelinic acid were isolated from these reactions and unambiguously identified, while the identity of two other compounds await final confirmation. Conclusions. Standards of possible metabolites of artelinic acid can be produced by the reaction of the compound with ferrous complexes that may simulate cytochrome P-450 catalyzed metabolism of xenobiotics. This approach may provide a simple and versatile method for the formation of metabolites of artemisinin compounds which is more advantageous than previous approaches with fungal-based systems.

High-performance liquid chromatographic method for the determination of a candidate 8-aminoquinoline antimalarial drug (WR 242511) using oxidative electrochemical detection.

WR 242511 (or I) is a new compound of the 8-aminoquinoline class designed to replace primaquine for the treatment of malaria. In order to perform preclinical and clinical testing, an assay was needed to determine drug levels in plasma samples. A simple and reliable reversed-phase high-performance liquid chromatographic (HPLC) method for the measurement of I in plasma using oxidative electrochemical detection is described. A 250-microliters plasma sample containing WR 256408 (or II) as internal standard was extracted with tert.-butyl methyl ether-2-propanol. A 25-microliters aliquot of the extractant was used for HPLC analysis. The mobile phase was 50:50 acetonitrile-sodium acetate (50 mM, pH 6) with 1 mM EDTA. Compounds I and II were separated within 10 min. The limit of detection for I was 10 ng/ml (plasma) with a recovery around 72%. The method was validated in a dog experiment where levels were followed for 48 h. The method is sensitive and robust and can be used for routine drug analysis during pharmacokinetic studies.

The effects of age on the pharmacokinetics and biotransformation of theophylline in vivo and in vitro in the Mongolian gerbil (Meriones unguiculatus)

The effect of post maturational aging on the in vivo disposition of theophylline was examined in the Mongolian gerbils (Meriones unguiculatus) aged 30-39 (old), 12-18 (middle-aged) and 3 (young) months following a 20 mg/kg i.p. dose. Biotransformation of theophylline was also examined in liver microsomes from non-induced and 3-methylcholanthrene induced gerbils. Analysis of theophylline plasma kinetics showed decreased clearance, increased half-life and increased volume of distribution in old vs. young animals. Clearance to the 1,3-dimethyluric acid metabolite was similar for all age groups, while clearance to the 1-methyluric acid metabolite was significantly lower in the middle-aged group compared to that of young and old gerbils. Urinary recovery of 1-methylurate was increased in old vs. young and middle-aged animals while recovery of theophylline was decreased. 3-Methylcholanthrene induction resulted in decreased recovery of theophylline and increased recovery of 1,3-dimethylurate and 1-methylurate in young and middle-aged gerbils compared to non-induced controls. Decreased microsomal protein content was observed in old vs. young and middle-aged gerbils and an age-related decrease in cytochrome P-450 content (nmol P-450/g liver) was also observed. The rate of dimethylurate formation was decreased 37% in microsomes from old vs. young and middle-aged gerbils. 3-Methylcholanthrene administration resulted in a 2- and 1.5-fold increase in the rate of 1,3-dimethylurate formation in young and middle-aged gerbils, respectively. The results of these experiments indicate that the Mongolian gerbil may be useful for the study of the biochemical mechanisms underlying age-related changes in the biotransformation and kinetics of theophylline.

Primary toxic effects of anthraquinone-2-sulfonic acid in rat liver microsomes

(1) The endogenous, NADPH-supported production of H2O2 and of O2-.-radicals in rat liver microsomes was very strongly enhanced in the presence of anthraquinone-2-sulfonic acid (AQSA). (2) This induction of H2O2 and of O2-.-radicals was catalyzed by the microsomal NADPH:cytochrome P450 oxidoreductase (EC 1.6.2.4). (3) AQSA was reduced to AQSA radicals by reductase; AQSA radicals reduce molecular oxygen to O2-.-radicals, which are readily dismutated to H2O2 by the microsomal superoxide dismutase. (4) O2-.-radicals are the sole precursors of all AQSA-induced production of H2O2 in liver microsomes.