Cytochrome P450 isoforms involved in metabolism of the enantiomers of verapamil and norverapamil.
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AIMS: The present study was conducted to evaluate metabolism of the enantiomers of verapamil and norverapamil using a broad range of cytochrome P450 isoforms and measure the kinetic parameters of these processes. METHODS: Cytochrome P450 cDNA-expressed cells and microsomes from a P450-expressed lymphoblastoid cell line were incubated with 40 microm concentrations of R- or S-verapamil and R- or S-norverapamil and metabolite formation measured by h.p.l.c. as an initial screening. Those isoforms exhibiting substantial activity were then studied over a range of substrate concentrations (2.5-450 microm ) to estimate the kinetic parameters for metabolite formation. RESULTS: P450s 3A4, 3A5, 2C8 and to a minor extent 2E1 were involved in the metabolism of the enantiomers of verapamil. Estimated Km values for the production of D-617 and norverapamil by P450 s 3A4 and 3A5 were similar (range=60-127 microm ) regardless of the enantiomer of verapamil studied while the Vmax estimates were also similar (range=4-8 pmol min-1 pmol-1 P450). Only nominal production of D-620 by these isoforms was noted. Interestingly, P450 2C8 readily metabolized both S- and R-verapamil to D-617, norverapamil and PR-22 with only slightly higher Km values than noted for P450s 3A4 and 3A5. However, the Vmax estimates for P450 2C8 metabolism of S- and R-verapamil were in general greater (range=8-15 pmol min-1 pmol-1 P450) than those noted for P450 s 3A4 and 3A5 with preference noted for metabolism of the S-enantiomer. Similarly, P450 s 3A4, 3A5 and 2C8 also mediated the metabolism of the enantiomers of norverapamil with minor contributions by P450 s 2D6 and 2E1. P450s 3A4 and 3A5 readily formed the D-620 metabolite with generally a lower Km and higher Vmax for S-norverapamil than for the R-enantiomer. In contrast, P450 2C8 produced both the D-620 and PR-22 metabolites from the enantiomers of norverapamil, again with stereoselective preference seen for the S-enantiomer. CONCLUSIONS: These results confirm that P450s 3A4, 3A5 and 2C8 play a major role in verapamil metabolism and demonstrate that norverapamil can also be further metabolized by the P450s. (+info)
The influence of CYP2D6 activity on the kinetics of propafenone enantiomers in Chinese subjects.
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AIMS: To determine role of CYP2D6 activity in the pharmacokinetics of propafenone (PPF) enantiomers in native Chinese subjects. METHODS: Sixteen extensive metabolizers (EMs) and one poor metabolizer (PM), whose phenotype had been previously assessed with dextromethorphan metabolic phenotyping, were enrolled. Blood samples (0 approximately 15 h) were taken after oral administration of a single dose (400 mg) of racemic-propafenone hydrochloride. A reverse-phase h.p.l.c. method with pre-column derivatization was employed to quantitate enantiomeric concentrations of propafenone in plasma. RESULTS: For the EM subjects, S-PPF was less rapidly metabolized and had higher peak plasma concentrations than R-PPF (413+/-143 vs 291+/-109 ng ml-1, P<0.001). The AUC was markedly higher for S-PPF than for R-PPF (2214+/-776 vs 1639+/-630 microg h l-1, P<0.001), whereas the clearance of S-PPF was significantly lower than that of R-PPF (96.0+/-39.0 vs 138+/-78 l h-1, P<0.01). There were no differences in t1/2, and Cmax between the two isomers (P >0.05). In the one PM subject, not only did S-PPF appear to undergo less rapid metabolism than R-PPF, but the subject also showed 2 approximately 3 fold differences in Cmax, CL and AUC compared with EMs. The correlation coefficients (rs ) between dextromethorphan metabolic ratio (lg MR) and pharmacokinetic parameters (Cmax, CL and AUC) were 0.63, -0.87, 0.87 for S-PPF and 0. 57, -0.73, 0.86 for R-PPF, respectively. CONCLUSIONS: Our results suggest that CYP2D6 activity contributes to the pharmacokinetic variability of propafenone enantiomers in Chinese subjects. (+info)
First total synthesis of N-4909 and its diastereomer; a stimulant of apolipoprotein E secretion in human hepatoma Hep G2 cells.
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Both (R)- and (S)-3-hydroxy-13-methyltetradecanoic acids were prepared via a lipase-catalyzed enantioselective acylation. The total synthesis of N-4909 and its diastereomer were achieved by a coupling of either (R)- or (S)-3-hydroxy-13-methyltetradecanoic acid moiety with a hexapeptide moiety and by a cyclization with HATU (O-(7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate) and HOAt (1-hydroxy-7-azabenzotriazole) in a high dilution condition. The R configuration of 3-hydroxy-13-methyltetradecanoic acid was found to be important for stimulating the activity of apolipoprotein E secretion in human hepatoma Hep G2 cells. (+info)
Site-directed mutagenesis of putative substrate-binding residues reveals a mechanism controlling the different stereospecificities of two tropinone reductases.
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Two tropinone reductases (TRs) constitute a key branch point in the biosynthetic pathway of tropane alkaloids, which are mainly produced in several solanaceous plants. The two TRs share 64% identical amino acid residues and reduce the 3-carbonyl group of a common substrate, tropinone, but they produce distinct alcohol products with different stereospecific configurations. Previous x-ray crystallographic analysis has revealed their highly conserved overall folding, and the modeling of tropinone within the putative substrate-binding sites has suggested that the different stereospecificities may be determined solely by the different binding orientations of tropinone to the enzymes. In this study, we have constructed various mutant TRs, in which putative substrate-binding residues from one TR were substituted with those found in the corresponding positions of the other TR. Substitution of five amino acid residues resulted in an almost complete reversal of stereospecificity, indicating that the different stereospecificities are indeed determined by the binding orientation of tropinone. Detailed kinetic analysis of the mutant enzymes has shown that TR stereospecificity is determined by varying the contributions from electrostatic and hydrophobic interactions and that the present TR structures represent highly evolved forms, in which strict stereospecificities and rapid turnover are accomplished together. (+info)
Stoichiometry of sulfonylurea-induced ATP-sensitive potassium channel closure.
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Hypoglycemic sulfonylureas (e.g., glibenclamide, glipizide, and tolbutamide) exert their stimulatory effect on excitatory cells by closure of ATP-sensitive potassium (KATP) channels. These channels are heteromultimers composed with a 4:4 stoichiometry of an inwardly rectifying K+ channel (KIR) subunit 6.x plus a sulfonylurea receptor (SUR). SUR1/KIR6.2 reconstitutes the neuronal/pancreatic beta-cell channel, whereas SUR2A/KIR6.2 and SUR2B/KIR6.1 (or KIR6.2) are proposed to reconstitute the cardiac and the vascular smooth muscle-type KATP channels, respectively. SUR2A and SUR2B are splice variants of a single gene differing only in their C-terminal 42 amino acids. Affinities of sulfonylureas for rat SUR2A, rat or human SUR2B, and a SUR2 chimera containing the C-terminal 42 amino acids of SUR1 did not differ significantly, implying that the C terminus does not form part of the binding pocket. Consistent with these findings, reconstituted SUR2A/KIR6.2 and SUR2B/KIR6.2 channels revealed similar sensitivities for glibenclamide and tolbutamide. Dissociation constants of sulfonylureas for SUR2A and SUR2B were 10- to 400-fold higher than for SUR1, however, amazingly the benzoic acid derivative meglitinide did not show lower affinity for SUR2 isoforms. Potencies of glibenclamide, glipizide, tolbutamide, and meglitinide to inhibit activity of SUR1/KIR6.2 and SUR2B/KIR6.2 channels were 3- to 6-fold higher than binding affinities of these drugs with concentration-inhibition relations being significantly steeper (Hill coefficients 1.23-1.32) than binding curves (Hill coefficients 0.93-1.06). The data establish that the C terminus of SURs does not affect sulfonylurea affinity and sensitivity. We conclude that occupation of one of the four SUR sites per channel complex is sufficient to induce KATP channel closure. (+info)
Comparative pharmacokinetics and tissue distribution of the d-enantiomers of para-substituted methylphenidate analogs.
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A comparative study of the plasma pharmacokinetics and tissue distribution of the d-threo enantiomers of methylphenidate (MPH), para-bromomethylphenidate (p-Br MPH), and para-methoxymethylphenidate (p-OCH3 MPH) was conducted in rats after i.p. administration of a 37 micromol/kg dose. The plasma kinetic data was fit to a two-compartment model with absorption and lag time as well as evaluated by noncompartmental methods. All three compounds attained maximal concentration within 10 min of injection. Calculated mean residence time and elimination half-life values for d-p-Br MPH were significantly longer than those for d-MPH and d-p-OCH3 MPH, and clearance of the bromo derivative was substantially lower than the latter two compounds. Tissue distribution studies of the three d-threo enantiomers revealed that para-substitution of d-MPH had a profound effect on the distribution pattern of these drugs. The highest concentration of drug was found in the kidney and lung for d-MPH, lung and liver for d-p-Br MPH, and lung and brain for d-p-OCH3 MPH. The bromo derivative was found in the highest concentration in the central nervous system at 30, 120, and 180 min whereas levels of d-MPH were twice as high as d-p-OCH3 MPH at 30 min but slightly lower than the latter at 120 min. Related studies on the lipophilicity, plasma protein binding, and resistance to plasma degradation of these compounds were also conducted. The combined data from these experiments along with the pharmacokinetics and central nervous system distribution of these drugs provide explanations for discrepancies between the in vivo and in vitro activity of these compounds described in previous work. (+info)
Presystemic metabolism of albendazole: experimental evidence of an efflux process of albendazole sulfoxide to intestinal lumen.
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Albendazole (ABZ) presystemic clearance was studied in rat by perfusion of a 25 microM ABZ solution in isolated intestinal loops. Significant secretion of the active metabolite, ABZSO, into the lumen was observed. The metabolite was also present in mesenteric blood. After 30 min of intestinal perfusion, 64% of the ABZ dose had disappeared from lumen. The total amount of ABZSO measured was 0.341 +/- 0.04 nmol/cm with 0.176 +/- 0.03 nmol/cm in mesenteric blood. The metabolite secretion to intestinal lumen was 0.165 +/- 0.05 nmol/cm. Intestinal sulfoxidation was induced by repeated administration of ABZ and ABZ coadministered with surfactants, especially polysorbate 80. The enantioselectivity of the in vitro intestinal sulfoxidation of ABZ showed that the relative contribution of P-450 and flavin-containing monooxygenase was quite similar, but after the induction by ABZ coadministered with polysorbate 80, the cytochrome P-450 system contribution was significantly increased. The appearance of ABZSO in mesenteric blood clearance was also increased under these conditions. (+info)
Role of tyrosine 265 of alanine racemase from Bacillus stearothermophilus.
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Tyrosine 265 (Y265) of Bacillus stearothermophilus is believed to serve as a catalytic base specific to the L-enantiomer of a substrate amino acid by removing (or returning) an alpha-hydrogen from (or to) the isomer on the basis of the X-ray structure of the enzyme [Stamper, C.G., Morollo, A.A., and Ringe, D. (1998) Biochemistry 37, 10438-10443]. We found that the Y265-->Ala mutant (Y265A) enzyme is virtually inactive as a catalyst for alanine racemization. We examined the role of Y265 further with beta-chloroalanine as a substrate with the expectation that the Y265A mutant only catalyzes the alpha,beta-elimination of the D-enantiomer of beta-chloroalanine. However, L-beta-chloroalanine also served as a substrate; this enantiomer was rather better as a substrate than its antipode. Moreover, the mutant enzyme was as equally active as the wild-type enzyme in the elimination reaction. These findings indicate that Y265 is essential for alanine racemization but not for beta-chloroalanine elimination. (+info)