Pathways of gallopamil metabolism. Regiochemistry and enantioselectivity of the O-demethylation processes

The oxidative O-demethylation of pseudoracemic gallopamil by rat and human liver microsomes was studied. By comparison of GC/MS retention times and fragmentation patterns with data from authentic standards, the four possible regioisomeric monophenolic metabolites, 2-(4-hydroxy-3,5-dimethoxyphenyl)-2-isopropyl-5-[(3,4- dimethoxyphenethyl)methylamino]-valeronitrile (2), 2-(5-hydroxy-3,4-dimethoxyphenyl)-2-isopropyl-5-[(3,4- dimethoxyphenethyl)methylamino]valeronitrile (3), 2-(3,4,5-trimethoxyphenyl)-2-isopropyl-5-[(4-hydroxy-3-methoxyphenethyl) -methylamino]valeronitrile (4), and 2-(3,4,5-trimethoxyphenyl)-2-isopropyl-5-[(3-hydroxy-4- methoxyphenethyl)methylamino]valeronitrile (5), were characterized. Rat liver microsomal oxidation produced all four regioisomeric monophenols which accounted for only 10% of the oxidative metabolism, the remaining 90% being N-dealkylation metabolites. Preference for metabolism of the O-methyl ethers at p-positions on each of the aromatic ring systems was noted, with more O-demethylation of the O-methyl ethers on the aromatic ring adjacent to the chiral center than on the aromatic ring in the short side chain. Significant enantio-selectivity was noted, the S/R ratios being 2.26, 1.97, 1.87 and 1.30 for formation of 2, 3, 4 and 5, respectively. Biliary excretion of the O-demethylated metabolites as conjugates, cleaved by beta-glucuronidase, was observed in rats after administration of pseudoracemic gallopamil. Significant stereoselectivity was noted, S/R ratios being 0.62, 1.61, 1.49 and 2.19 for 2, 3, 4 and 5, respectively. Human liver microsomal oxidation produced more p- than m-O-demethylation, with 4 less than 5, and 2 less than 3, but quantitatively the pathway is a minor one compared to N-dealkylation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Regiochemistry and enantioselectivity in the oxidative N-dealkylation of verapamil

The oxidative N-dealkylation of verapamil (1), a calcium channel antagonist, was examined in the presence of rat and human liver microsomes by using GC-MS methodology and synthesized regio-isomeric standards. All three possible secondary amine metabolites, N-methyl-4-(3,4-dimethoxyphenyl)-4-cyano-5-methylhexylamine (5), norverapamil (4), and N-methyl-2-(3,4-dimethoxyphenyl)ethylamine (3), were formed as microsomal metabolites. Compound 5 and norverapamil (4) were major products. Substrate stereoselectivity for the N-dealkylation process was determined when pseudoracemic verapamil[equimolar (S)-(-)-verapamil-d6 and (R)-(+):verapamil-d0] was used as substrate. In the presence of rat liver microsomes, a slight enantiomeric preference for the metabolism of (R)-verapamil to secondary amines 3 and 5 (S/R ratio = 0.88 and 0.78, respectively) was observed. In contrast, (S)-verapamil was preferentially metabolized to norverapamil (4) and primary amine 9 (S/R ratio = 1.20 for both). The enantioselectivity for the N-dealkylation process in the presence of human liver microsomes was slight and variable (six samples). Quantitatively, the major N-dealkylation routes in both microsomal systems yielded norverapamil (4) and secondary amine 5. Greater substrate enantioselectivity was observed for the N-dealkylation process in rat liver microsomes than in human liver microsomes. In rat liver microsomal studies, two aliphatic aldehydes (2 and 6) were successfully trapped as their O-methyloximes (7 and 11, respectively) by using methoxylamine. In addition, the alcohols formed from reduction of these aldehydes were observed, due in part to a direct reduction by NADPH.(ABSTRACT TRUNCATED AT 250 WORDS)     

N-dealkylation of arylpiperazine derivatives: disposition and metabolism of the 1-aryl-piperazines formed

In recent years several arylpiperazine derivatives have reached the stage of clinical application, mainly for the treatment of depression, psychosis or anxiety. Examples are the pyrimidinylpiperazine buspirone, the chlorophenylpiperazine derivatives nefazodone and trazodone, the dichlorophenylpiperazine aripiprazole and the benzisothiazolyl derivatives perospirone and ziprasidone. Most of them undergo extensive pre-systemic and systemic metabolism including CYP3A4-dependent N-dealkylation to 1-aryl-piperazines. These metabolites are best known for the variety of serotonin receptor-related effects they cause in man and animals, although some have affinity for other neurotransmitter receptors; others, however, are still largely unexplored despite uncontrolled use as amphetamine-like designer drugs. Once formed they distribute extensively in tissues, including brain which is the target site of most arylpiperazine derivatives, and are then primarily biotransformed by CYP2D6-dependent oxidation to hydroxylates which are excreted as conjugates; only 1-(2-benzisothiazolyl)-piperazine is more susceptible to sulfur oxidation than to aromatic hydroxylation. In studies analysing animal brain and human blood, 1-aryl-piperazine concentrations were either higher or lower than the parent compound(s), although information is available only for some derivatives. At steady state, the metabolite-to-parent drug ratios varied widely among individuals taking the same dosage of the same arylpiperazine derivative. This is consistent with the known individual variability in the expression and activity of CYP3A4 and CYP2D6. This review also surveys current published information on physiological and pathological factors affecting the 1-aryl-piperazine-to-parent drug ratios and examines the potential role of 1-aryl-piperazine formation in the pharmacological actions of the arylpiperazine derivatives that are already or will shortly be available in major markets.     

Microbial models of mammalian metabolism. N-dealkylation of furosemide to yield the mammalian metabolite CSA using Cunninghamella elegans.

Furosemide (Lasix), a widely used diuretic, is metabolized by the fungus Cunninghamella elegans (ATCC 36112) to 4-chloro-5-sulfamoyl anthranilic acid (CSA), a metabolite also present in mammalian systems. This metabolite was isolated following preparative-scale incubations of C. elegans, and was characterized by comparison with standard CSA using 13C-NMR, mass spectrometry (high-resolution mass spectra, electron impact mass spectra), UV, TLC, and HPLC with fluorescence detection. Because a known complication with furosemide studies is the spontaneous formation of CSA by decomposition of furosemide during incubation, extraction, and/or analysis, a time course study was conducted to determine the rate of CSA formation caused by metabolism vs. the relatively low rate of CSA formation caused by spontaneous decomposition.     

Metabolism of anethole. I. Pathways of metabolism in the rat and mouse

The metabolic fate of the naturally occurring food flavouring trans-anethole has been investigated in rats and mice. A single 50-mg/kg dose of trans-[methoxy-14C]anethole was given orally to female Wistar albino rats and by ip injection to male CD-1 mice. The major routes of elimination of 14C were the urine and expired air (as 14CO2). Excretion of 14C in the faeces and as volatile compounds in the expired air was very low (total less than 2% of the dose). Urinary metabolites were separated by solvent extraction, TLC and HPLC and were characterized by MS and GC-MS directly and following methylation or trimethylsilylation, the results being compared where possible with authentic standards. Eleven 14C-containing urinary metabolites were identified in the rat and ten in the mouse. These compounds arose from side-chain oxidation, side-chain cleavage and various conjugations. The major urinary metabolites were two isomers of 1-(4'-methoxyphenyl)propane-1,2-diol, 2-hydroxy-1-methylthio-1-(4'-methoxyphenyl)propane and 4-methoxyhippuric acid, the first three all being excreted as glucuronides. In addition to these 14C-labelled metabolites, 4-hydroxypropenylbenzene, the unlabelled product of oxidative O-demethylation of trans-[14C]anethole, was excreted extensively in urine as the glucuronide.     

Interaction between gallopamil and cardiac ryanodine receptors.

1. In a sarcoplasmic reticulum fraction obtained from rat hearts, the analysis of equilibrium [3H]-ryanodine binding showed high and low affinity sites (KD = 1.3 nM and 2.8 microM, Bmax = 2.2 pmol mg-1 and 27.8 pmol mg-1). The dissociation rate constant increased at 1 microM vs 4 nM [3H]-ryanodine concentration, and micromolar ryanodine slowed the dissociation of nanomolar ryanodine. 2. The binding of 4 nM [3H]-ryanodine was not affected by gallopamil, while the binding of 100 nM to 18 microM [3H]-ryanodine was partly displaced. Data analysis suggested that gallopamil inhibited low affinity [3H]-ryanodine binding, with IC50 in the micromolar range. 3. Gallopamil decreased the dissociation rate constant of 1 microM [3H]-ryanodine. While gallopamil alone did not affect the dissociation of 4 nM [3H]-ryanodine, gallopamil and micromolar ryanodine slowed it to a greater extent than micromolar ryanodine alone. 4. Our results are consistent with the hypothesis that the ryanodine receptor is a negatively cooperative oligomer, which undergoes a sequential alteration after ryanodine binding. Gallopamil has complex actions: it inhibits ryanodine binding to its low affinity site(s), and probably modulates the cooperativity of ryanodine binding and/or the transition to a receptor state characterized by slow ryanodine dissociation. These molecular actions could account for the previously reported effect of gallopamil on the sarcoplasmic reticulum calcium release channel.     

Effect of gallopamil on energy metabolism of the isolated perfused rat brain in the postischemic period

Summary The isolated perfused rat brain was used to demonstrate an effect of gallopamil on energy metabolism affected by ischemia. After a perfusion period of 30 min and 10 min of ischemia the isolated brain preparation was reperfused. From the onset of perfusion onwards, gallopamil (1 or 10 mol/l) was present in the medium. The higher concentration of gallopamil accelerated significantly the restoration of the high-energy phosphates in the recovery stage: after 2 min of recirculation the ATP and the creatine-P levels were higher and the AMP level was lower in cortical tissue of drug-treated brains than in untreated controls. These results suggest that gallopamil protected brain energy metabolism against ischemic damage.     

Red blood cell partitioning of gallopamil, verapamil and norverapamil.

In-vitro binding of calcium-antagonists gallopamil and verapamil (and its main metabolite norverapamil) to human red blood cells (RBCs) was investigated. The drugs are bound reversibly and dose dependent to RBCs in the same order of magnitude, with partition-coefficients of kRBC = 0.12-0.34 for gallopamil, kRBC = 0.10-0.30 for verapamil and kRBC = 0.10-0.27 for norverapamil. The data indicate that, although RBCs may act as subcompartments of the blood for this class of compounds, they may have no influence on therapeutic plasma concentrations, due to their low kRBC.     

Pathways involved in the metabolism and activation of polycyclic hydrocarbons

The metabolism and activation of the polycyclic aromatic hydrocarbons has been reviewed and the original contributions made to this area by Professor E. Boyland have been placed in context. The reactions involved in the formation, via epoxides, of hydroxylated derivatives have been outlined and conjugations with glucuronic and sulphuric acids and with glutathione have been discussed. Examples of secondary hydroxylation reactions have been given and the possible role that phenolic hydroxyl groups may play in activating epoxides considered. Mechanism by which polycyclic hydrocarbons are activated by metabolism to epoxides of various types have been included, mainly by reference to benzo[a]pyrene, benz[a]anthracene and chrysene. The tissue and species specific effects of polycyclic hydrocarbons have been referred to and the tissues that may act as targets in man for the initiation of malignancy by polycyclic hydrocarbons mentioned.     

Pathways involved in the metabolism and activation of polycyclic hydrocarbons

1. The metabolism and activation of the polycyclic aromatic hydrocarbons has been reviewed and the original contributions made to this area by Professor E. Boyland have been placed in context.

The reactions involved in the formation, via epoxides, of hydroxylated derivatives have been outlined and conjugations with glucuronic and sulphuric acids and with glutathione have been discussed.

3. Examples of secondary hydroxylation reactions have been given and the possible role that phenolic hydroxyl groups may play in activating epoxides considered.

4. Mechanism by which polycyclic hydrocarbons are activated by metabolism to epoxides of various types have been included, mainly by reference to benzo[a]pyrene, benz[a]anthracene and chrysene.

5. The tissue and species specific effects of polycyclic hydrocarbons have been referred to and the tissues that may act as targets in man for the initiation of malignancy by polycyclic hydrocarbons mentioned.     

Differential enantioselectivity and product-dependent activation and inhibition in metabolism of verapamil by human CYP3As.

In vitro studies of enantioselective metabolism of R-(+)- and S-(-)verapamil (VER) were conducted using human cDNA-expressed CYP3A isoforms, CYP3A4, CYP3A5, and CYP3A7. N-dealkylated products nor-VER [2,8-bis-(3,4-dimethoxyphenyl)-2-isopropyl-6-azaoctanitrile] and D617 [2-(3,4-dimethoxyphenyl)-5-methylamino-2-isopropylvaleronitrile] were the major metabolites for all CYP3A isoforms regardless of enantiomer. Enantioselectivity of CYP3A4 and CYP3A7 was most similar among the three isoforms. This coincides with the degree of homology of amino acids at the active sites and in the total amino acid sequences of the enzymes. Biphasic substrate inhibition was observed for the formation of nor-VER and D617, whereas simple biphasic kinetics were observed for the formation of O-demethylated products for both enantiomers with CYP3A4. The biphasic substrate inhibition was observed only for nor-VER, and simple biphasic kinetics were observed for D617 and O-demethylated products for both enantiomers with CYP3A5. However, with CYP3A7, D617 and O-demethylated products showed typical Michaelis-Menten kinetics, and only nor-VER displayed substrate (monophasic) inhibition. When metabolic rates of VER were determined in the presence of three different effectors, midazolam, testosterone, and nifedipine, activation, inhibition, or activation and inhibition of VER metabolism was observed depending on the enantiomers, metabolites, effectors, and cytochrome P450 isoforms. Addition of anti-CYP3A4 antibody inhibited formation of all metabolites for both CYP3A4 and CYP3A5. The atypical phenomena (biphasic substrate inhibition, activation, and inhibition depending on product formation) of VER kinetics could be adequately explained by introducing the concept of steric interaction into a two binding-site model.     

Liver sulphoxidative metabolism of albendazole in rat: enantioselectivity and effect of methimazole

The aim of the present work was to evaluate the effects of methimazole (MTZ) on the enantioselective sulphoxidation of albendazole (ABZ) by rat liver microsomes and tissue slices. Albendazole sulphoxide (ABZSO) was the metabolite recovered after the incubation with ABZ in both liver preparations. MTZ significantly reduced ABZSO production both in microsomes and slices. ABZSO production decreased as a function of MTZ concentration. The sulphoxidation reaction performed by rat liver explants in the presence of MTZ was 65% lower than that observed in controls. The reduction in the production of ABZSO in the presence of MTZ was mainly due to a lower production of (+) ABZSO. The results reported further contribute to the understanding of the enantioselective metabolism of ABZ. In addition, the work presented provides information on the comparison of two different liver tissue preparations for the evaluation of xenobiotic metabolism.     

Screening of Candida sp. for N-dealkylation of codeine

A number of Candida species were selected in order to test their ability to N- or O-demethylate codeine. Complex media and chemically defined media used in our experiments. Transformation products were analysed by reversed phase HPLC and TLC methods. N- and O-demethylation of codeine was performed by C. tropicalis NCYC 997, NCYC 405, NCYC 470 and C. albicans LSHTM 3153. Transformation by C. tropicalis NCYC 997 had better results. N- and O-demethylation occurred in the chemically defined medium. Change in the colour of the transformation mixture, in cultures containing more than 5 mmol.l-1 codeine, was attributed to codeine N-oxide, but its presence was not confirmed by HPLC. N- and O-demethylation of codeine by Candida sp. was compared to that occurred in mammals.     

Albendazole-praziquantel interaction in healthy volunteers: kinetic disposition, metabolism and enantioselectivity

AIM

This study investigated the kinetic disposition, metabolism and enantioselectivity of albendazole (ABZ) and praziquantel (PZQ) administered alone and in combination to healthy volunteers.

METHODS

A randomized crossover study was carried out in three phases (n = 9), in which some volunteers started in phase 1 (400 mg ABZ), others in phase 2 (1500 mg PZQ), and the remaining volunteers in phase 3 (400 mg ABZ + 1500 mg PZQ). Serial blood samples were collected from 0–48 h after drug administration. Pharmacokinetic parameters were calculated using a monocompartmental model with lag time and were analyzed using the Wilcoxon test; P≤ 0.05.

RESULTS

The administration of PZQ increased the plasma concentrations of (+)-ASOX (albendazole sulphoxide) by 264% (AUC 0.99 vs. 2.59 µg ml⁻¹ h), (−)-ASOX by 358% (0.14 vs. 0.50 µg ml⁻¹ h) and albendazole sulfone (ASON) by 187% (0.17 vs. 0.32 µg ml⁻¹ h). The administration of ABZ did not change the kinetic disposition of (+)-(S)-PZQ (–)-(R)-4-OHPZQ or (+)-(S)-4-OHPZQ, but increased the plasma concentration of (–)-(R)-PZQ by 64.77% (AUC 0.52 vs. 0.86 µg ml⁻¹ h).

CONCLUSIONS

The pharmacokinetic interaction between ABZ and PZQ in healthy volunteers was demonstrated by the observation of increased plasma concentrations of ASON, both ASOX enantiomers and (–)-(R)-PZQ. Clinically, the combination of ABZ and PZQ may improve the therapeutic efficacy as a consequence of higher concentration of both active drugs. On the other hand, the magnitude of this elevation may represent an increased risk of side effects, requiring, certainly, reduction of the dosage. However, further studies are necessary to evaluate the efficacy and safety of this combination.     

N-dealkylation of tertiary amides by cytochrome P-450.

1. N-Methyl-N-alkyl-p-chlorobenzamides (alkyl = Me, Et, nPr, nBu, PhCH2, isoPr and cylcoPr) underwent mono-N-dealkylation exclusively with phenobarbital-induced rat liver microsomes; with each compound both demethylation and dealkylation occurred. 2. The time-courses showed bilinear kinetics, but there was no evidence for general suicide-substrate activity with the cyclopropyl amide, and product ratios did not vary with time. 3. The demethylation/dealkylation ratio varied from 0.3 to 2.0 among the primary alkyl groups but was ca. 40 when the alkyl group was isoPr or cylcoPr. Dealkylation of the benzyl substituent was 2-3 times more favourable than for any other primary alkyl group. Despite wide variations in the demethylation/dealkylation ratios, at near-saturating concentrations of substrates the rates of total oxidation (demethylation plus dealkylation) varied little across the entire series. 4. The results of this study are consistent with a kinetic mechanism involving significant commitment to catalysis, substituent-induced metabolic switching at the product-determining step, a non-catalytic step which is partly rate-limiting in turnover, and a chemical mechanism involving H-atom abstraction as opposed to electron abstraction.     

Metabolism of the calcium antagonist gallopamil in man

The metabolism of gallopamil (5-[(3,4-dimethoxyphenyl)methylamino]-2-(3,4,5-trimethoxyphenyl) -2- isopropylvaleronitrile hydrochloride, Procorum, G) was studied after single administration (2 mg i.v., 50 mg p.o.) of unlabelled and labelled G (14G, 2H). TLC, HPLC, GLC, MS and RIA were used for assessment of G and its metabolites in plasma, urine and faeces. G clearance is almost completely metabolic, with only minimal excretion of unchanged drug. Metabolites represent most of the plasma radioactivity after p.o. administration. They are formed by N-dealkylation and O-demethylation with subsequent N-formylation, or glucuronidation, respectively. Compound A, derived by loss of the 3,4-dimethoxyphenethyl moiety of G is the main metabolite in plasma and urine (about 20% of the dose). This metabolite is accompanied by its N-formyl derivative (C), by the N-demethylated compound (H) and the acid (F), formed by oxidative deamination of A. Only 3 unconjugated monphenoles from several O-demthylated products showed distinct plasma levels which were nevertheless lower than metabolite A. These metabolites had no relevance to the pharmacodynamic action. Conjugated monophenolic and diphenolic products represented the major part in plasma and were excreted predominantly via the bile: they represented almost the whole faecal metabolite fraction. Less than 1% of the dose was recovered unchanged in the urine. About 50% of the dose is excreted by urine and 40% by faeces.