Pharmacokinetics of the novel, high-affinity and selective dopamine D3 receptor antagonist SB-277011 in rat, dog and monkey: in vitro/in vivo correlation and the role of aldehyde oxidase.

1. In vitro studies with the selective dopamine D3 receptor antagonist SB-277011 were conducted in liver microsomes and homogenates from rat, dog, cynomolgus monkey and human to correlate the rate of metabolism with the in vivo pharmacokinetics of the compound in rat, dog and cynomolgus monkey. 2. In the presence of NADPH, SB-277011 was relatively stable in the presence of liver microsomes from rat, dog, cynomolgus monkey and human with an intrinsic clearance (CLi) of < 2 ml min(-1) g(-1) liver for all species. In total liver homogenates, SB-277011 was metabolized at a similar rate in rat and dog (CLi < 2 ml min(-1) g(-1) liver) to that in liver microsomes but in cynomolgus monkey and human (CLi = 9.9 and 45 ml min(-1) g(-1) liver, respectively) the intrinsic clearance was approximately 6- and 35-fold higher, respectively, than that in liver microsomes. 3. In the absence of NADPH, SR-277011 was rapidly cleared in liver homogenates from cynomolgus monkey and human (CLi = 7.4 and 27 ml min(-1) g(-1) liver, respectively) demonstrating that a significant pathway of metabolism of this compound was via an NADPH-independent non-microsomal oxidative route. This pathway was sensitive to inhibition with isovanillin suggesting that the enzyme responsible was aldehyde oxidase. 4. The in vivo pharmacokinetics showed that the plasma clearance of SB-277011 was low in rat (20 ml min(-1) kg(-1)), moderate in dog (14 ml min(-1) kg(-1)) and high in cynomolgus monkey (58 ml min(-1)kg(-1)), which is consistent with the in vitro findings and demonstrated a greater capacity for the monkey to metabolize this compound. The oral bioavailability of SB-277011 in rat, dog and cynomolgus monkey was 35, 43 and 2%, respectively. Given the high clearance of this compound in cynomolgus monkey, the low oral bioavailability is probably as a result of high first-pass elimination, specifically by aldehyde oxidase, rather than poor absorption. 5. The high in vitro clearance of SB-277011 in human liver homogenates and the involvement of aldehyde oxidase in the metabolism of SB-277011 indicates that the bioavailability of the compound is likely to be low in human.

Characterization of the cytochrome P450 enzymes involved in the in vitro metabolism of rosiglitazone.

AIMS: To identify the human cytochrome P450 enzyme(s) involved in the in vitro metabolism of rosiglitazone, a potential oral antidiabetic agent for the treatment of type 2 diabetes-mellitus. Method The specific P450 enzymes involved in the metabolism of rosiglitazone were determined by a combination of three approaches; multiple regression analysis of the rates of metabolism of rosiglitazone in human liver microsomes against selective P450 substrates, the effect of selective chemical inhibitors on rosiglitazone metabolism and the capability of expressed P450 enzymes to mediate the major metabolic routes of rosiglitazone metabolism. Result The major products of metabolism following incubation of rosiglitazone with human liver microsomes were para-hydroxy and N-desmethyl rosiglitazone. The rate of formation varied over 38-fold in the 47 human livers investigated and correlated with paclitaxel 6alpha-hydroxylation (P<0.001). Formation of these metabolites was inhibited significantly (>50%) by 13-cis retinoic acid, a CYP2C8 inhibitor, but not by furafylline, quinidine or ketoconazole. In addition, both metabolites were produced by microsomes derived from a cell line transfected with human CYP2C8 cDNA. There was some evidence for CYP2C9 playing a minor role in the metabolism of rosiglitazone. Sulphaphenazole caused limited inhibition (<30%) of both pathways in human liver microsomes and microsomes from cells transfected with CYP2C9 cDNA were able to mediate the metabolism of rosiglitazone, in particular the N-demethylation pathway, albeit at a much slower rate than CYP2C8. Rosiglitazone caused moderate inhibition of paclitaxel 6alpha-hydroxylase activity (CYP2C8; IC50=18 microm ), weak inhibition of tolbutamide hydroxylase activity (CYP2C9; IC50=50 microm ), but caused no marked inhibition of the other cytochrome P450 activities investigated (CYP1A2, 2A6, 2C9, 2C19, 2D6, 2E1, 3A and 4A). Conclusion CYP2C8 is primarily responsible for the hydroxylation and N-demethylation of rosiglitazone in human liver; with minor contributions from CYP2C9.

Ketoconazole and sulphaphenazole as the respective selective inhibitors of P4503A and 2C9.

1. The potential of ketoconazole and sulphaphenazole to inhibit specific P450 enzyme activities (1A2, 2A6, 2B6, 2C9/8, 2C19, 2D6, 2E1, 3A and 4A) was investigated using human liver microsomes. 2. Ketoconazole demonstrated an inhibitory effect on cyclosporine oxidase and testosterone 6 beta-hydroxylase activities, with mean IC50's of 0.19 and 0.22 microM respectively. Ketoconazole inhibition of the other P450 activities investigated was significantly less, as illustrated by IC50's of at least a magnitude higher. 3. Sulphaphenazole was shown to have an inhibitory effect on tolbutamide hydroxylase activity, with a mean IC50 of 0.8 microM in incubations containing 100 microM tolbutamide. Sulphaphenazole (at concentrations of up to 100 microM) did not exhibit any significant inhibition of the other enzyme activities investigated. 4. Ketoconazole and sulphaphenazole are the respective selective inhibitors of P4503A and 2C9. Ketoconazole at 1 microM and sulphaphenazole at 10 microM can be used to establish the involvement of P4503A and 2C9 respectively in oxidative reactions in human liver microsomes.

Characterisation of the cytochrome P450 enzymes involved in the in vitro metabolism of granisetron.

1. The metabolism of granisetron was investigated in human liver microsomes to identify the specific forms of cytochrome P450 responsible. 2. 7-hydroxy and 9'-desmethyl granisetron were identified as the major products of metabolism following incubation of granisetron with human liver microsomes. At low, clinically relevant, concentrations of granisetron the 7-hydroxy metabolite predominated. Rates of granisetron 7-hydroxylation varied over 100-fold in the human livers investigated. 3. Enzyme kinetics demonstrated the involvement of at least two enzymes contributing to the 7-hydroxylation of granisetron, one of which was a high affinity component with a Km of 4 microM. A single, low affinity, enzyme was responsible for the 9'-desmethylation of granisetron. 4. Granisetron caused no inhibition of any of the cytochrome P450 activities investigated (CYP1A2, CYP2A6, CYP2B6, CYP2C9/8, CYP2C19, CYP2D6, CYP2E1 and CYP3A), at concentrations up to 250 microM. 5. Studies using chemical inhibitors selective for individual P450 enzymes indicated the involvement of cytochrome P450 3A (CYP3A), both pathways of granisetron metabolism being very sensitive to ketoconazole inhibition. Correlation data were consistent with the role of CYP3A3/4 in granisetron 9'-desmethylation but indicated that a different enzyme was involved in the 7-hydroxylation.

Lauric acid as a model substrate for the simultaneous determination of cytochrome P450 2E1 and 4A in hepatic microsomes.

In vitro techniques have been utilized to investigate the microsomal enzymes involved in the metabolism of lauric acid and to establish conditions in which it can be used as a model substrate for both cytochrome P450 4A and cytochrome P450 2E1 in human liver microsomes. Studies of enzyme kinetics of lauric acid omega-hydroxylation in human liver microsomes indicated the involvement of more than one enzyme in this pathway, a relatively low Km enzyme with a Km of 22 microM +/- 12 (n = 8) and a high Km enzyme with a Km an order of magnitude higher (550 microM +/- 310, n = 7). The apparent Vmax for this component correlated with the rate of cyclosporin metabolism and was highly sensitive to ketoconazole inhibition. These results indicated that this enzyme was a member of the 3A subfamily. The activity associated with the low Km enzyme (P450 4A) did not correlate with P450 1A2, 2A6, 2C9/8, 2C19, 2D6, 2E1, or 3A activities in a bank of human liver microsomes and was not appreciably inhibited by ketoconazole, furafylline, quinidine, sulfaphenazole, or diethyldithiocarbamate (DDC). Lauric acid omega-1 hydroxylation demonstrated simple Michaelis-Menten kinetics in each of the human liver microsomal samples examined, with a Km of 130 microM +/- 42 (n = 8). This activity was highly correlated with chlorzoxazone 6-hydroxylation in human liver microsomes (r = 0.98, n = 14, p < 0.001) and was inhibited by both DDC and chlorzoxazone. Additionally, rats treated with the P450 2E1 inducer isoniazid demonstrated a 3-fold increase in lauric acid omega-1 hydroxylation relative to the control group. Thus, the lauric acid hydroxylation assay, at a substrate concentration of 20 microM, appears to be an effective and specific P450 model substrate capable of determining simultaneously P450 4A and P450 2E1 related activities in hepatic microsomal samples.

The role of activation of neutrophils and microvascular pressure in acute pulmonary edema.

Activated polymorphonuclear neutrophils (PMN) can mediate vascular injury in the lung. This study compared activated aggregate PMN (emboli) to activated PMN that were previously adhered to the microvasculature (non-embolic) in the isolated perfused rat lung. Permeability and microvascular pressure (Pmv), components of PMN-induced edema, were examined by continuous measurement of wet weight, pulmonary arterial and left atrial pressures, and by intermittent determination of double occlusion pressure. PMN that were activated with phorbol myristate acetate and then perfused into the lung formed aggregates that lodged primarily in the precapillary bed, increasing arterial resistance. Although these PMN had minimal direct contact with the capillary endothelium, edema rapidly developed and Pmv was progressively elevated. If PMN were allowed to adhere in the capillary bed, a minimal and nonprogressive increase in Pmv and lung weight occurred. When these adherent PMN were then activated, there was a progressive rise in both Pmv and lung weight. The free radical scavenger catalase prevented this edema formation but not the rise in pressure. In control lungs with matched elevation of Pmv, edema did not develop. In another group of lungs with activation of pre-adherent PMN in which Pmv was maintained at control levels, edema formation was greatly delayed. These data show that: (1) the activated PMN free radical products alone caused permeability injury in the lung because neither contact of the PMN with the capillary endothelium nor embolization was necessary, and (2) increased Pmv does not cause edema but greatly increases the rate of edema formation when the endothelium is injured.