Anthony D. Theoharides

Analysis of artesunic acid and dihydroqinghaosu in blood by high-performance liquid chromatography with reductive electrochemical detection.

A new high-performance liquid chromatography (HPLC) method using reductive electrochemical detection has been developed for the analysis of the antimalarial drugs artesunic acid (ARTS) and dihydroqinghaosu (DQHS) in blood. Presently, this method has been validated to 4 micrograms/ml for ARTS and 200 ng/ml for DQHS. Pharmacokinetic studies in the rabbit show that after intravenous administration (100 mg/kg) ARTS is metabolized rapidly to DQHS and has a t1/2 of 1.7 min in blood. DQHS data were fit to non-linear regression models consisting of the sum of two exponential terms. For phases 1 and 2, t1/2 values of 3.0 +/- 0.4 and 29 +/- 2 min were calculated, respectively. In vitro studies in which ARTS was incubated with blood from various species show that rabbit blood hydrolyzes ARTS at a much greater rate than rat or human blood. Incubation of ARTS with rabbit blood in the presence or absence of diisopropylfluorophosphate suggested that this hydrolysis reaction is catalyzed by plasma and red blood cell esterases. These results suggest that future pharmacokinetic studies in both animals and man should focus on the measurement of DQHS rather than ARTS.

Hepatic metabolism of artemisinin drugs—I. Drug metabolism in rat liver microsomes

1. In this communication, metabolism of the semisynthetic antimalarial drugs of the artemisinin class (beta-arteether, beta-artelinic acid and dihydroartemisinin) in rat liver microsomes, is reported. 2. Dihydroartemisinin was the major early metabolite of arteether (57%) and artelinic acid (80%); in addition, arteether was hydroxylated in the positions 9 alpha- and 2 alpha- of the molecule. 3. Dihydroartemisinin was further metabolized by extensive hydroxylation of its molecule; we were able to identify four hydroxylated derivatives of DQHS, but not the exact positions of the hydroxyl groups. 4. The rates of NADPH-supported metabolism of arteether, artelinic acid and dihydroartemisinin in rat liver microsomes were: 4.0, 2.5 and 1.3 nmol/min/mg of microsomal protein, respectively. 5. The apparent affinity constants of arteether and artelinic acid for the microsomal metabolizing system, calculated from the rates of product formation, were 0.54 mM and 0.33 mM (for arteether) and 0.11 mM (for artelinic acid), respectively. The appearance of two affinity constants indicated that arteether was metabolized by two different isoenzymes of cytochrome P-450 in rat liver microsomes.

Thermal decomposition products of dihydroahtemisinin (dihydroqinghaous)

Abstract Dihydroartemisinin (2), a sodium borohydride reduction produot of artemisinin (1), undergoes themolysis at 190 °C to give desoxyartemisinin (3) and a preponderant decomposition product (4) consisting of 2 epimers 4a, (2S, 3R, 6S)-2-(3-oxobutyl)-3-methyl-6-[(R)2-propanal]-cyclohexanone, and 4b, (2S, 3R, 6R)-2-(3-oxobutyl)-3-methyl-6-[(R)2-propanal]-cyclohexanone.

Structure-activity relationships of putative primaquine metabolites causing methemoglobin formation in canine hemolysates☆☆☆

A rapid and reproducible in vitro test system was developed to measure the methemoglobin (MHb)-forming properties of various 8-aminoquinoline derivatives. Initial rates and extents of reaction were measured spectrophotometrically with either canine hemolysates from which ferrihemoglobin reductase was removed, or with purified human oxyhemoglobin (Hb). The results demonstrate that primaquine derivatives that can be oxidized to quinones or iminoquinones (5-hydroxy,6-desmethyl primaquine; 5-hydroxyprimaquine; 5,6-dihydroxy-8-aminoquinoline; and 5-hydroxy, 6-methoxy-8-aminoquinoline) are potent MHb-forming compounds. Studies on the extent of reaction in hemolysates and purified oxyhemoglobin suggest that the extent of MHb formation may be limited by the rate at which the corresponding iminoquinones or quinones arylate nucleophiles. The effects of glutathione, mannitol, ascorbate, and superoxide dismutase on the rate and extent of hemoglobin oxidation by 5,6-dihydroxy-8-aminoquinoline suggest that these compounds oxidize Hb similar to the mechanism known for dimethylaminophenol (DMAP), in which Hb oxidizes the quinoline to semiquinone radical and quinone species which are the oxidizing and arylating agents.

Hepatic metabolism of artemisinin drugs. II : Metabolism of arteether in rat liver cytosol

1. In this communication, in vitro metabolism of a semisynthetic antimalarial drug arteether in rat liver cytosol is reported. 2. Whenever 14C-labeled arteether was mixed with rat liver cytosol, a crude postmicrosomal fraction of liver cell homogenates, an appearance of three major 14C-labeled metabolites was always attested: deoxy-dihydroartemisinin, AEM-1 (Baker et al., 1988) and metabolite MW286. 3. Transformation of arteether into deoxyDQHS was catalyzed by an enzyme present in the rat liver cytosol, whose activity depended on the presence of NAD+/NADH and a low molecular, dialyzable factor present in the cytosol. The maximal activity of this enzyme was 0.31 nmol of deoxyDQHS formed/min/mg of cytosolic protein. 4. AEM-1 and metabolite mol. wt 286 have been formed directly from arteether by a chemical interaction of the drug with the cytosolic fraction, probably in a non-enzymatic reaction. 5. Taking together the in vitro data of arteether metabolism in rat liver cytosol, presented in this communication, and in vitro data in rat liver microsomes, presented in the preceding communication (Leskovac and Theoharides, 1991), we were able to postulate an integral pathway of Phase I metabolism of arteether in a whole rat liver cell.

Regioselective hydroxylation of prostaglandins by constitutive forms of cytochrome P-450 from rat liver: formation of a novel metabolite by a female-specific P-450

Previous studies demonstrated that liver microsomes from untreated rats catalyze the omega, omega-1, and omega-2 hydroxylation of prostaglandins [K. A. Holm, R. J. Engell, and D. Kupfer (1985) Arch. Biochem. Biophys. 237, 477-489]. The current study examined the regioselectivity of hydroxylation of PGE1 and PGE2 by purified forms of P-450 from untreated male and female rat liver microsomes. PGE1 was incubated with a reconstituted system containing cytochrome P-450 RLM 2, 3, 5, 5a, 5b, 6, or f4, NADPH-P-450 reductase, and dilauroylphosphatidylcholine in the presence or absence of cytochrome b5. Among the P-450 forms examined, only RLM 5 (male specific), 5a (present in both sexes), and f4 (female specific) yielded high levels of PGE hydroxylation. With PGE1, RLM 5 catalyzed solely the omega-1 hydroxylation and 5a catalyzed primarily the omega-1 and little omega and omega-2 hydroxylation. By contrast, f4 effectively hydroxylated PGE1 and PGE2 at the omega-1 and at a novel site. Based on retention on HPLC and on limited mass fragmentation, we speculate that this site is omega-3 (i.e., 17-hydroxylation). Kinetic analysis of PGE1 hydroxylation demonstrated that the affinity of f4 for PGE1 is approximately 100-fold higher than that of RLM 5; the Km values for f4, monitoring 19- and 17-hydroxylation of PGE1, were about 10 microM. Surprisingly, cytochrome b5 stimulated the activity of RLM 5a and f4, but not that of RLM 5. Hydroxylation of PGE2 by RLM 5 was at the omega, omega-1, and omega-2 sites, demonstrating a lesser regioselectivity than with PGE1. These findings show that the constitutive P-450s differ dramatically in their ability to hydroxylate PGs, in their regioselectivity of hydroxylation, and in their cytochrome b5 requirement.

Metabolism of a potential 8-aminoquinoline antileishmanial drug in rat liver microsomes

The metabolism of the 8-aminoquinoline, 8-(6-diethylaminohexylamino)-6-methoxy-lepidine dihydrochloride (WR 6026 X 2HCl), was studied in a rat hepatic microsomal system. The results show that WR 6026 X 2HCl was metabolized into two more polar compounds. The structures of these metabolites as proven by gas chromatography-mass spectrometry, ultraviolet absorption, and high performance liquid chromatography were: 8-(6-ethylaminohexylamino)-6-methoxy-lepidine (metabolite 1) and 8-(6-diethylaminohexylamino)-6-methoxy-4-hydroxymethyl quinoline (metabolite 2). The formation of both metabolites was NADPH dependent and also linearly dependent on incubation time and microsomal protein concentration at 0.24 mM WR 6026 X 2 HCl. Studies on the effects of pretreatment of animals with either phenobarbital or Aroclor 1254 suggest that cytochrome P-450 isozymes catalyzed both N-deethylation and hydroxylation reactions. N-deethylase activity was induced by either pretreatment: however, hydroxylase activity was unaffected by phenobarbital pretreatment and significantly elevated by Aroclor 1254 pretreatment. These results suggest that these two reactions are catalyzed by different cytochrome P-450 isozymes. The formation of these two metabolites in vivo may play an important role in the antileishmanial activity of WR 6026 X 2HCl.

ω-1 and ω-2 hydroxylation of prostaglandins by rabbit hepatic microsomal cytochrome P-450 isozyme 6☆

Incubation of prostaglandin El (PGE1) with liver microsomes from control rabbits and from rabbits treated with ethanol or imidazole yielded 18-, 19-, and 20-hydroxy metabolites, representing hydroxylation at w-2, w-l, and w carbons, respectively. The current investigation demonstrates that rabbit liver P-450 isozyme 6 effectively catalyzes the w-l and w-2 hydroxylation of PGEl and PGE2. Additionally, a small amount of product with chromatographic characteristics of the corresponding 20-hydroxy metabolite has been detected. The incorporation of cytochrome & into the reconstituted system did not enhance the rate of PGEl hydroxylation and had no effect on the ratio of products formed. The Km value for the w-l and w-2 hydroxylation of PGEl with P-450 isozyme 6 from imidazole-treated rabbits was approximately 140 PM; the I’,,,,,, (nmol product mine1 nmol P-450-l) were 2.1 and 1.1 for the o-l and o-2 hydroxylations, respectively. These rates represent the highest activities by hepatic P-450 isozymes for hydroxylation of PGs, and suggest that isozyme 6 is responsible for the w-2 hydroxylation of PGEs observed in rabbit liver microsomes. o 19% Academic press, I,,~.

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.

High Performance Liquid Chromatographic Method for Detection of Physostigmine and Eseroline in Plasma Using a Silica Gel Column and a Perchloric Acid Mobile Phase

Abstract A high-performance liquid chromatographic (HPLC) procedure that employs a silica gel column, a perchloric acid in methanol mobile phase and fluorescence detection has been developed for the determination of the concentrations of physostigmine and its metabolite, eseroline, in plasma. The method requires plasma samples to contain an ascorbic acid anti-oxidation solution. Plasma samples are extracted with 5 ml of methyl-t-butyl ether under alkaline conditions. The organic phase is evaporated to dryness and reconstituted with mobile phase. The quantitation range is 0.1 to 5.0 ng/ml (base) for both compounds. Mean ± S.D. recoveries for 4 concentrations within this range were 80.7 ± 4.3% for physostigmine and 84.1 ± 3.6% for eseroline. Interday and intraday (n = 6) coefficients of variation, respectively, for 4 concentrations in the 0.2 to 3.0 ng/ml range were 2.82 – 6.99% and 1.59 – 6.48% for physostigmine and 3.59 – 6.82% and 4.05 – 8.78% for eseroline. Bias for blind samples at 4 concentrations fro...