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Because rifampin (RIF) induces hepatic enzymes and inhibits uptake transporters, dosing a drug that is a dual substrate of enzymes and uptake transporters on the final day of an inducing regimen should exhibit less inductive effect than dosing on the following day in the absence of RIF, since RIF decreases drug uptake into liver. In vitro and in vivo rat studies were conducted using digoxin as a model substrate. Digoxin was administered to an uninduced control group to obtain baseline values. The second group (induced with dexamethasone) received digoxin alone, mimicking administration of a test drug 1 day following completion of an induction regimen, whereas the third group (induced) received digoxin with RIF mimicking the concomitant dosing on the final day of an induction regimen. Results from hepatocyte concentration-time course studies showed that compared with uninduced control (26.9 +/- 1.3 microM . min/mg), digoxin area under the time-concentration curve (AUC) in induced cells when no RIF is present decreased significantly (13.7 +/- 0.9 microM . min/mg; p < 0.01), suggesting induction of Cyp3a. However, digoxin AUC for induced cells in the presence of RIF (27.3 +/- 0.9 microM . min/mg) matched the control. Rat pharmacokinetic studies showed that compared with digoxin clearance in uninduced controls (7.08 +/- 1.57 ml/min/kg), digoxin clearance in induced rats increased 2-fold (15.6 +/- 3.7 ml/min/kg; p < 0.001), but when RIF was coadministered in the induced rats, digoxin clearance (7.14 +/- 1.24 ml/min/kg) overlapped with control. That is, concomitant dosing of RIF and digoxin masked the inductive effect. To observe full inductive effects, test drugs should be administered 1 day after final dosing of RIF to minimize potential organic anion transporting polypeptide inhibition effects.

Atorvastatin (ATV) is primarily metabolized by CYP3A in the liver to form two active hydroxy metabolites. Therefore, the sequential transport system governed by hepatic uptake and efflux transporters is important for the drug disposition and metabolism. Here, we assessed the interaction of ATV with hepatic uptake transporter organic anion transporting polypeptide (Oatp) and efflux transporter multidrug resistance associated protein 2 (MRP2/Mrp2) in vitro and ex situ using the isolated perfused rat liver (IPRL). Rifampicin (RIF) was chosen as an inhibitor for Oatp in both uptake and IPRL studies. Its inhibitory effects on MRP2 and metabolism were also tested using MRP2-overexpressing cells and rat microsomes, respectively. Our results indicate that RIF effectively inhibits the Oatp-mediated uptake of ATV and its metabolites. Inhibition on MRP2-mediated efflux of ATV was also observed at a high RIF concentration. Compared with ATV alone in the IPRL, the area under the curve(s) (AUC) of ATV was significantly increased by RIF, whereas the AUC of both metabolites were also increased in a concentration-dependent manner. However, the extent of metabolism was significantly reduced, as reflected by the reduced amounts of metabolites detected in RIF-treated livers. In conclusion, inhibition of Oatp-mediated uptake seems to be the major determinant for interaction between ATV and RIF. Metabolites of ATV were subject to Oatp-mediated uptake as well, suggesting that they undergo a similar disposition pathway as the parent drug. These data emphasize the relevance of uptake transporter as being one of the major players in hepatic drug elimination, even for substrates that undergo metabolism.

The disposition of digoxin and the influence of the organic anion transporting polypeptide (Oatp)2 inhibitor rifampicin and the P-glycoprotein (P-gp) inhibitor quinidine on its hepatic disposition were examined in the isolated perfused rat liver. Livers from groups of rats were perfused in a recirculatory manner after a bolus dose of digoxin (10 microg), a dual substrate for Oatp2 and P-gp as well as CYP3A. Perfusions of digoxin were also examined in groups of rats in the presence of the inhibitors: rifampicin (100 microM) or quinidine (10 microM). In all experiments, perfusate samples were collected for 60 min. Digoxin and its primary metabolite were determined in perfusate and liver by liquid chromatography/mass spectrometry. The area under the curve (AUC) from 0 to 60 min was determined. The AUC +/- S.D. of digoxin was increased from control (3880 +/- 210 nM x min) by rifampicin (5200 +/- 240 nM x min; p < 0.01) and decreased by quinidine (3220 +/- 340 nM x min; P < 0.05). It is concluded that rifampicin limits the hepatic entrance of digoxin and reduced the hepatic exposure of digoxin to CYP3A by inhibiting the basolateral Oatp2 uptake transport, whereas quinidine increased the hepatic exposure of digoxin to CYP3A by inhibiting the canalicular P-gp transport. These data emphasize the importance of uptake and efflux transporters on hepatic drug metabolism.

CYP3A4-transfected Caco-2 cells were used as an in vitro system to predict the importance of drug metabolism and transport on overall drug absorption. We examined the transport and metabolism of two drugs; midazolam, an anesthetic agent and CYP3A4 substrate, and sirolimus, an immunosuppressant and a dual CYP3A4/P-glycoprotein (P-gp) substrate, in the presence of cyclosporine (CsA, a CYP3A4/P-gp inhibitor) or N-[4-[2-(1,2,3,4-tetrahydro-6,7-dimethoxy-2-isoquinolinyl)-ethyl]-phenyl]-9,10-dihydro-5-methoxy-9-oxo-4-acridine carboxamine (GG918) (an inhibitor of P-gp and not CYP3A4). All major CYP3A4 metabolites were formed in the cells (1-OH > 4-OH midazolam and 39-O-desmethyl > 12-OH > 11-OH sirolimus), consistent with results from human liver microsomes. There was no bidirectional transport of midazolam across CYP3A4-transfected Caco-2 cells, whereas there was a 2.5-fold net efflux of sirolimus (1 microM) that disappeared in the presence of CsA or GG918. No change in the absorption rate or extraction ratio (ER) for midazolam was observed when P-gp was inhibited with GG918. Addition of GG918 had a modest impact on the absorption rate and ER for sirolimus (increased 58% and decreased 25%, respectively), whereas a 6.1-fold increase in the absorption rate and a 75% decrease in the ER were found when sirolimus was combined with CsA. Although both midazolam and sirolimus metabolites were preferentially excreted to the apical compartment, only sirolimus metabolites were transported by P-gp as determined from inhibition studies with GG918. Using CYP3A4-transfected Caco-2 cells we determined that, in contrast to P-gp, CYP3A4 is the major factor limiting sirolimus absorption. The integration of CYP3A4 and P-gp into a combined in vitro system was critical to unveil the relative importance of each biochemical barrier.

Two alternative metabolic pathways, acyl glucuronidation and acyl-CoA formation, are implicated in the generation of reactive acylating metabolites of carboxylic acids. Here, we describe studies that determine the relative importance of these two pathways in the metabolic activation of a model substrate, 2-phenylpropionic acid (2-PPA), in vivo in rats. Male Sprague-Dawley rats were pretreated with and without (-)-borneol (320 mg/kg i.p.), an inhibitor of acyl glucuronidation, or trimethylacetic acid (TMA, 500 mg/kg i.p.), an inhibitor of acyl-CoA formation, before receiving 2-PPA (racemic, 130 mg/kg). After administration of 2-PPA, livers were collected over a 2-h period and analyzed for 2-PPA acyl glucuronidation and 2-PPA-CoA formation by high-performance liquid chromatography. Covalent binding was measured by scintillation counting of washed liver protein precipitates. Results showed that pretreatment with TMA led to a 49% decrease in covalent binding of 2-PPA to liver proteins, when a 64% decrease in the exposure of 2-PPA-CoA was observed. Conversely, 95% inhibition of acyl glucuronidation by (-)-borneol, led to a 23% decrease in covalent binding to protein. These results suggest that metabolic activation by 2-PPA-CoA formation contributes to covalent adduct formation to protein in vivo to a greater extent than metabolic activation by acyl glucuronidation for this model substrate.

P-Glycoprotein (P-gp) has been hypothesized to modulate intestinal drug metabolism by increasing the exposure of drug to intracellular CYP3A through repeated cycles of drug absorption and efflux. The rat single-pass intestinal perfusion model was used to study this interplay in vivo. N-Methyl piperazine-Phe-homoPhe-vinylsulfone phenyl (K77), a peptidomimetic cysteine protease inhibitor (CYP3A/P-gp substrate), and midazolam (CYP3A substrate) were each perfused through a segment of rat ileum alone and with the P-gp inhibitor N-(4-[2-(1,2,3,4-tetrahydro-6,7-dimethoxy-2-isoquinolinyl)-ethyl]-phenyl)-9,10-dihydro-5-methoxy-9-oxo-4-acridine carboxamine (GG918). Samples were obtained continuously from the outlet perfusate and the mesenteric vein at 5-min intervals for 40 to 60 min. The parent drug and two main metabolites of K77 (N-desmethyl and N-oxide) and midazolam (1-OH and 4-OH) were quantitated by liquid chromatography/mass spectrometry. K77 appearance in the mesenteric blood (P(blood) = 5 +/- 3 x 10(-6) cm/s) was increased 3-fold with GG918, whereas midazolam permeability (P(blood) = 1.1 +/- 0.3 x 10(-4) cm/s) was unchanged by GG918. K77 metabolites were preferentially excreted into the lumen, 4-OH midazolam was found equally in lumen and blood, and 1-OH was mainly excreted into blood. The extent of metabolism was estimated by calculating the fraction metabolized = 1 - P(blood)/P(lumen) and the extraction ratio (ER) determined from the direct measurement of known metabolites as ER = sum metabolites(all)/(sum metabolites(all) + drug in blood). When P-gp was inhibited, the fraction metabolized for K77 was decreased (95 to 85%) and the ER tended toward a decrease, whereas no differences in either parameter were observed for midazolam (not a P-gp substrate). These data support a role for P-gp in modulating the extent of intestinal metabolism in vivo by controlling drug access to the enzyme.

Cyclosporine protects the heart against ischemia/reperfusion injury, but its effect on cardiac metabolism is largely unknown. We assessed cyclosporine-induced metabolic changes in the rat heart prior to occlusion using magnetic resonance spectroscopy (MRS) and correlated effects with infarct size in a coronary occlusion/reperfusion model. The two study groups were cyclosporine and cyclosporine + coronary occlusion (n = 20/group). Rats were pretreated with cyclosporine (5, 10, 15, and 25 mg/kg/day) or the vehicle by oral gavage for 3 days (n = 4/dose). On day 4, hearts of rats in the cyclosporine group were excised, and extracted cell metabolites were measured using (1)H and (31)P MRS. The second group was subjected to 30 min of coronary artery occlusion followed by 24 h of reperfusion. Infarct size and area at risk were measured using a double staining method. In the cyclosporine group, cyclosporine reduced cardiac energy metabolism (ATP: r = -0.89, P < 0.001) via depression of oxidative phosphorylation and the Krebs' cycle in a dose-dependent manner. The decrease of ATP levels was positively correlated with changes of NAD(+) (r = 0.89), glutamate (r = 0.95), glutamine (r = 0.84), and glucose concentrations (r = 0.92, all P < 0.002). It was inversely correlated with lactate (r = -0.93, P < 0.001). In the coronary occlusion group, cyclosporine dose dependently reduced the ratio [area of infarct/area of the left ventricle] (r = -0.86, P < 0.01), with 15 mg/kg/day being the most effective cyclosporine dose. The reduction in infarct size correlated with the reduction in oxidative phosphorylation (ATP: r = 0.97; NAD(+): r = 0.82, P < 0.01). The reduction in cardiac energy metabolism before occlusion may be the cause of myocardial preservation during ischemia/reperfusion.

Drug efflux by intestinal P-glycoprotein (P-gp) is known to decrease the oral bioavailability of many CYP3A4 substrates. We hypothesized that the interplay occurring between P-gp and CYP3A4 at the apical membrane would increase the opportunity for drug metabolism. To define the roles of P-glycoprotein (P-gp) and CYP3A4 in controlling the extent of intestinal absorption and metabolism, two substrates were tested. The transport, metabolism, and intracellular levels of N-methyl piperazine-Phe-homoPhe-vinylsulfone phenyl (K77, a cysteine protease inhibitor; P-gp and CYP3A4 substrate) and felodipine (CYP3A4 substrate only) were measured across CYP3A4-transfected Caco-2 cells in the presence of an inhibitor of CYP3A4 and P-gp, cyclosporine (CsA), or an inhibitor of P-gp and not CYP3A4, GG918 (N-[4-[2-(1,2,3,4-tetrahydro-6,7- dimethoxy-2-isoquinolinyl)-ethyl]-phenyl]-9,10-dihydro-5-methoxy-9-oxo-4-acridine carboxamine). The extent of metabolism was measured by calculating the extraction ratio (ER) across the cells, while accounting for intracellular changes occurring with P-gp inhibition. The (A)pical to (B)asolateral and B-->A ERs for K77 were 0.33 and 0.06, respectively. These changed with GG918 to 0.14 and 0.12 and with CsA to 0.06 and 0.04. Felodipine ERs were similar in both directions, 0.26 and 0.24 (A-->B and B-->A), and were unchanged in the presence of GG918 but decreased with CsA (0.14 and 0.11). The K77 absorption rate was increased 5 and 4.2-fold in the presence of CsA and GG918, respectively, whereas no change was observed for felodipine absorption. The decreased A-->B ER and increased absorption of K77 with GG918 suggest that P-gp influences the extent of drug metabolism in the intestine via prolonging the access of drugs to CYP3A4 near the apical membrane and decreasing transport across the cells.

We report the tissue distribution and clinical monitoring of the novel macrolide immunosuppressant SDZ-RAD ¿40-O-(2-hydroxyethyl)-rapamycin and its metabolites in monkey lung transplant recipients as well as its interaction with cyclosporine as the Neoral formulation. After left unilateral lung transplantation, cynomolgus monkeys received by oral administration either 1) 1.5 mg/kg/day SDZ-RAD (n = 4); 2) 100 mg/kg/day cyclosporine (n = 4); 3) 0.3 mg/kg/day SDZ-RAD + 100 mg/kg/day cyclosporine (n = 6); 4) 1.5 mg/kg/day SDZ-RAD + 50 mg/kg/day cyclosporine (n = 5); or 5) SDZ-RAD and cyclosporine doses adjusted according to trough blood concentration measurements (n = 6). At the end of the observation period (usually 29 days after transplantation), and 24 h after the last doses, tissue samples were collected and analyzed with HPLC/mass spectrometry. Gall bladder, pancreas, the transplant lung, cerebellum, kidneys, and spleen had the highest SDZ-RAD concentrations. Coadministration of cyclosporine increased SDZ-RAD concentrations in most tissues as well as tissue-to-blood distribution coefficients. In contrast, SDZ-RAD had only a small effect on cyclosporine blood and tissue concentrations. Rejection in lung grafts in monkeys treated with either of the cyclosporine/SDZ-RAD combinations was significantly less than in the monotherapy groups (P <.002). Histological rejection scores were inversely correlated with SDZ-RAD concentrations in blood (r = -0. 68; P <.001; n = 24), lymph nodes (P = -0.58; P <.003; n = 24), thymus (r = -0.63; P <.001; n = 23) and transplant lung tissue (r = -0.58; P <.003; n = 24). We conclude that, in addition to the synergistic pharmacodynamic interaction, a pharmacokinetic interaction resulting in higher SDZ-RAD tissue concentrations contributed to the significantly better immunosuppressive efficacy when both drugs were combined compared with monotherapy.

We compared the intestinal metabolism of the structurally related 3-hydroxy-3-methylglutaryl-coenzyme A reductase inhibitors lovastatin and pravastatin in vitro. Human small intestinal microsomes metabolized lovastatin to its major metabolites 6'beta-hydroxy (apparent K(m) = 11.2 +/- 3.3 microM) and 6'-exomethylene (apparent K(m) = 22.7 +/- 9.0 microM) lovastatin. The apparent K(m) values were similar for lovastatin metabolism by human liver microsomes. 6'beta-Hydroxylovastatin formation by pig small intestinal microsomes was inhibited with the following inhibition K(i) values: cyclosporine, 3.3 +/- 1.2 microM; ketoconazole, 0.4 +/- 0.1 microM; and troleandomycin, 0.8 +/- 0.9 microM. K(i) values for 6'-exomethylene lovastatin were similar. Incubation of pravastatin with human small intestinal microsomes resulted in the generation of 3'alpha,5'beta, 6'beta-trihydroxypravastatin (apparent K(m) = 4560 +/- 1410 microM) and hydroxypravastatin (apparent K(m) = 5290 +/- 1740 microM). In addition, as in the liver, pravastatin was metabolized in the small intestine by sulfation and subsequent degradation to its main metabolite 3'alpha-iso-pravastatin. It was concluded that lovastatin is metabolized by cytochrome P-450 3A enzymes in the small intestine. Compared with lovastatin, the cytochrome P-450-dependent intestinal intrinsic clearance of pravastatin was >5000-fold lower and cannot be expected to significantly affect its oral bioavailability or to be a significant site of drug interactions.

Neurotoxicity, a crucial side effect of immunosuppressive therapy with cyclosporine, also has been demonstrated in vitro for sirolimus, a novel macrolide immunosuppressant, which is under clinical investigation in combination with cyclosporine. NMR spectroscopy was used to study the separate and combined effects of cyclosporine and sirolimus on cerebral metabolism, both in brain cells and in perfused rat brain slices. The high-energy phosphate metabolism was already affected significantly at cyclosporine concentrations as low as 100 micrograms/liter: phosphocreatine was reduced by 10 +/- 2% [half-maximal inhibition concentration (IC50) = 1850 +/- 600 micrograms/liter], and nucleoside triphosphate was reduced by 11 +/- 5% (IC50 = 1110 +/- 420 micrograms/liter; n = 4, P <.05). At 500 micrograms/liter cyclosporine, N-acetylaspartate and glutamate were decreased by 13 +/- 7% (IC50 = 1100 +/- 330 micrograms/liter) and 22 +/- 9% (IC50 = 360 +/- 220 micrograms/liter; n = 4, P <.05), respectively. As evaluated using an algorithm based on Loewe isobolograms, combination of cyclosporine and sirolimus resulted in a synergetic reduction of high-energy phosphate metabolites. Addition of sirolimus to the perfusion medium increased brain slice concentrations of cyclosporine. It is concluded that cyclosporine significantly reduced high-energy phosphate metabolism in brain tissue at in vivo relevant concentrations. Combination with sirolimus resulted in synergism, which, in part, is explained by a greater distribution of cyclosporine into the brain tissue in the presence of sirolimus.

We investigated the effects of ketoconazole on the oral bioavailability of morpholine-urea-phenylalanine-homophenylalanine-vinylsulfone-phenyl (K02), a vinylsulfone peptidomimetic cysteine protease inhibitor, and a P450 3A (CYP3A) and P-glycoprotein dual substrate, in male Sprague-Dawley rats, so as to evaluate the roles of CYP3A and P-gp in K02 disposition. Male Sprague-Dawley rats (8-10 wk old, n = 3-6) were administered a single dose of K02 (10 mg/kg) i.v. or (30 mg/kg) p.o. with or without a concomitant oral dose of ketoconazole (20 mg/kg). Blood samples were collected from 2 min to 8 h after administration through a implanted jugular vein cannula. K02 plasma concentrations were determined by liquid chromatography/mass spectrometer/mass spectrometer analysis. Ketoconazole markedly raised the area under the curve of orally administered K02 from 9.4 +/- 4.4 to 102 +/- 24 mg . min/liter and decreased K02 oral plasma clearance from 3810 +/- 1620 to 306 +/- 60 ml/min/kg. With concomitant ketoconazole dosing, the changes of AUC of i.v. administered K02 (from 94 +/- 17 to 107 +/- 14 mg . min/liter) and clearance (from 110 +/- 22 to 95 +/- 13 ml/min/kg) were not significant, although K02 oral bioavailability increased from 2.9 +/- 1.4 to 31.0 +/- 7.5% (P < .001). In summary, ketoconazole, a dual inhibitor of CYP3A and P-glycoprotein, can effectively increase K02 oral bioavailability by inhibiting the CYP3A/P-gp absorption barrier in the small intestine.

Small intestinal metabolism and transport of sirolimus, a macrolide immunosuppressant with a low and highly variable oral bioavailability, were investigated using small intestinal microsomes and intestinal mucosa in the Ussing chamber. After incubation of sirolimus with human and pig small intestinal microsomes, five metabolites were detected using high performance liquid chromatography/electrospray-mass spectrometry: hydroxy, dihydroxy, trihydroxy, desmethyl and didesmethyl sirolimus. The same metabolites were generated by human liver microsomes and pig small intestinal mucosa in the Ussing chamber. Anti-CYP3A antibodies, as well as the specific CYP3A inhibitors troleandomycin and erythromycin, inhibited small intestinal metabolism of sirolimus, confirming that, as in the liver, CYP3A enzymes are responsible for sirolimus metabolism in the small intestine. Of 32 drugs tested, only known CYP3A substrates inhibited sirolimus intestinal metabolism with inhibitor constants (Ki) equal to those in human liver microsomes. The formation of hydroxy sirolimus by small intestinal microsomes isolated from 14 different patients ranged from 28 to 220 pmol.min-1.mg-1 microsomal protein. In the Ussing chamber, >99% of the sirolimus metabolites reentered the mucosa chamber against a sirolimus gradient, indicating active countertransport. Intestinal drug metabolism and countertransport into the gut lumen, drug interactions with CYP3A substrates and inhibitors in the small intestine and an 8-fold interindividual variability of the intestinal metabolite formation rate significantly contribute to the low and highly variable bioavailability of sirolimus.

Nitroglycerin (GTN) has been used to treat heart disease for many years. It is generally believed that GTN is a prodrug; however, the mechanism for GTN bioactivation remains unknown. Recent studies, using hepatic microsomes, have suggested the involvement of cytochrome P450 3A (CYP3A) in GTN biotransformation. Here, we used an animal model to test the hypothesis that aortic CYP3A plays a role in the bioactivation of GTN in vivo. Ketoconazole (KCZ), a potent CYP3A inhibitor, was given to rats (50 mg/kg i.p.) 1 hr before a bolus dose of GTN (2 mg/rat i.v.). KCZ decreased GTN-induced cGMP (cyclic guanosine monophosphate) levels by 20 to 30% (P < .05), without affecting basal or S-nitroso, N-acetyl penicillamine-induced levels of cGMP. When rats received dexamethasone (DEX, 30 mg/kg, 4 days i.p.), a strong CYP3A inducer, they exhibited a significant (approximately 50%) higher cGMP response to GTN than the control group. When rats received the combination treatment of both DEX and KCZ, they responded to GTN to the same extent as control rats. Although the effect of KCZ on aortic CYP3A activity cannot be detected (activity in control rats is below the detection limit), KCZ markedly inhibited CYP3A activity in rat livers (2.02 +/- 0.04 vs. 0.31 +/- 0.04 nmol/mg prot/min, P < .05, in control vs. KCZ-treated rats, respectively) and in DEX pretreated rat aorta (0.145 +/- 0.036 vs. 0.042 +/- 0.037 nmol/mg prot/min, P < .05, in rats treated with DEX alone vs. rats treated with both DEX and KCZ, respectively). KCZ did not elicit an effect on aortic glutathione S-transferases, another major metabolic enzyme responsible for GTN biotransformation. DEX enhanced the aortic GST mu activity by 3-fold. However, the activity of GST in aorta did not correlate with the cGMP response to GTN. In conclusion, our results demonstrate that CYP3A activity in aorta is correlated with GTN bioactivation in vivo, but the contribution of this enzyme to overall GTN bioactivation is limited.

The present study investigates the effects of thiol-depleting/ modifying agents on the activity of and tolerance to nitroglycerin (GTN), sodium nitroprusside (SNP) and S-nitroso-N-acetylpenicillamine (SNAP) in an in vivo rat model. Rats were treated with either vehicle (control), GTN (before and after induction of tolerance), diethyl maleate (a thiol-depleting agent) or N-ethylmaleimide (a thiol-modifying agent). The effects of GTN, SNP and SNAP on vascular cyclic GMP levels were investigated before and after each treatment. In addition, plasma and tissue thiol concentrations were measured in the same tissues as used for the determination of cyclic GMP in aorta and inferior vena cava after single and serial i.v. bolus doses of each drug. Depletion of free thiols (glutathione and cysteine) was not found to accompany tolerance development in GTN-treated tolerant rats or to significantly enhance tolerance development or augment its magnitude in diethyl maleate-treated rats. When rats were pretreated with a low single dose of N-ethylmaleimide, where no significant changes in vascular free thiols were observed, significant reduction in GTN- and SNP-induced, but not SNAP-induced, vascular cyclic GMP production was obtained. Considering the differential effects of diethyl maleate (mainly free thiol depletion) and N-ethylmaleimide (mainly proteinous thiol-alkylation) on vascular thiols, these results indicate that depletion of sulfhydryl groups other than those from free glutathione and cysteine seems to be involved in the mechanisms defining GTN and SNP (but not SNAP) action and tolerance. Here we propose that SNAP may act either directly by nitrosation of the heme moiety of the enzyme or via an S-enzyme-S-drug transnitrosation reaction, whereas GTN and SNP actions are mediated by the formation of S-nitrosothiol on the enzyme itself, rather than by activation of the enzyme by free S-nitrosothiols.

The in vivo effects of nitroglycerin (GTN) on the biochemical response [cyclic-3',5'-guanosine monophosphate (cGMP) levels] of vascular (aorta and inferior vena cava, IVC) and other tissues (heart and lung) were studied. GTN caused dose and time-dependent increases in cGMP content of aorta and IVC. Compared to base line, maximal concentrations of 3- to 4-fold the basal levels of cGMP were measured in the aorta, IVC and lung of rats after 2 mg i.v. bolus doses of GTN. Tolerance to GTN, as assessed by the reduction in the cGMP increase in response to an i.v. GTN dose, developed 12 to 18 hr after continuous i.v. infusion (100-200 micrograms/hr) of the drug. Significant differences in the pattern of formation and degradation of cGMP were noted between tolerant and nontolerant rats. Unlike the control IVC tissue, where elevated cGMP was measured throughout the 5-min post GTN administration, in tolerant IVC, cGMP levels were not significantly different from the basal values at time points 3 and 5 min post GTN. These results suggest that IVC is more prone to (or less recoverable from) the state of tolerance than the aorta. Furthermore, when tissue thiols were measured in control and tolerant rats, a complete dissociation was observed between the state of tolerance and the endogenous glutathione and cysteine concentrations. These data constitute strong evidence against an in vivo depletion of tissue thiols as being the primary determinant underlying the development of tolerance during nitrate therapy.

The pharmacokinetics and pharmacodynamics of nitroglycerin (GTN) and its dinitrate metabolites, 1,2-glyceryl dinitrate and 1,3-glyceryl dinitrate (1,2-GDN and 1,3-GDN), were examined in a seven-way crossover study in six conscious dogs. The mean apparent clearance after a low GTN i.v. dose (0.025 mg/kg) was 1440 ml/min/kg (S.D. = 630), which decreased to 686 ml/min/kg (S.D. = 317) after a high GTN i.v. dose (0.25 mg/kg), demonstrating dose-dependent pharmacokinetics. Bioavailability of oral GTN (0.25 mg/kg) was very low (0.015 +/- 0.019). The formation of dinitrate metabolites was rapid and extensive after i.v. and oral dosing of GTN. The pharmacokinetics of 1,2-GDN and 1,3-GDN were very similar. The dinitrates exhibited longer half-lives (approximately 45 min) and lower apparent clearances (approximately 16 ml/min/kg) than those found for GTN. The apparent fractional GTN clearances to 1,2-GDN (CLapp,m1) and 1,3-GDN (CLapp,m2) were also determined. The CLapp,m1 decreased significantly (P less than .05) in going from the low GTN dose (1030 +/- 620 ml/min/kg) to the high dose (425 +/- 145 ml/min/kg). In contrast, CLapp,m2 remained unchanged (low dose: 107 +/- 55 ml/min/kg; high dose: 115 +/- 37 ml/min/kg). The duration of systolic blood pressure decrease after low i.v. GTN doses was very short (approximately 15 min) and significantly longer (approximately 90 min) for high i.v. GTN doses. GTN is about 10 to 12 times more efficient than 1,2-GDN and 1,3-GDN in terms of the maximal systolic blood pressure decrease produced by a therapeutic dose. Oral GTN is pharmacodynamically active and this effect can be attributed to the formation of high levels of dinitrate metabolites.

The disposition of zomepirac (Z) and its acyl glucuronide metabolite were studied in rabbits and guinea pigs to determine if hydrolysis of zomepirac glucuronide (ZG) by tissue esterases occurs in vivo and what effect inhibition of esterases would have on exposure to ZG and subsequent covalent binding to plasma proteins. ZG was hydrolyzed rapidly in vivo by both guinea pigs and rabbits, liberating Z. The effect of inhibition of tissue esterases was determined by administration of phenylmethylsulfonyl fluoride (PMSF) concurrently with i.v. doses of Z or ZG to anesthetized, bile duct ligated guinea pigs. Administration of PMSF decreased the apparent plasma clearance of ZG by 86% and elevated the apparent plasma clearance of Z by 300%. Exposure of the guinea pigs to ZG as measured by the area under the plasma concentration vs. time curve (AUC) was increased substantially by PMSF treatment. Covalent binding of Z to plasma proteins in the guinea pig correlated well with AUC of ZG, but not with AUC of Z. The correlation of Z covalent binding with AUC of ZG in the guinea pig is similar to that found in humans suggesting that the degree to which acyl glucuronides form covalent adducts with proteins in humans may be predictable from studies in animals.

The purposes of this study were: 1) to determine in dogs under steady-state conditions whether prednisolone clearance increases with increasing dose as has been suggested from results obtained under nonsteady-state conditions in man and 2) to determine whether the prednisolone to prednisone conversion, apparently resulting from 11 beta-hydroxydehydrogenase, is saturable. Six dogs were given four different zero-order infusions of prednisolone resulting in steady-state levels from 23 to 5884 ng/ml.Prednisone, total and free prednisolone levels were assessed by high-pressure liquid chromatography and equilibrium dialysis. Total and unbound prednisolone clearances were calculated by dividing the infusion rate by the total or unbound prednisolone concentration at steady state. Both total and unbound prednisolone clearance exhibited a slight tendency to decrease (P < .05) with increasing prednisolone steady-state levels. The conversion of prednisolone to prednisone, defined by the prednisolone/prednisone ratio, exhibited saturable characteristics which, when data for all dogs was fitted to a Michaelis-Menten type equation (r2 = 0.938), gave a Km of 658 ng/ml, i.e., total prednisolone concentration at half-maximal saturation.

Pseudoephedrine is an organic base used in the treatment of upper respiratory tract disorders. Surgical techniques and experimental procedures were developed to study the renal elimination mechanisms for this drug in the rat. The ability to measure renal clearance accurately and to demonstrate renal secretion by a carrier-mediated transport system was verified by comparing results from N'-methylnicotinamide (NMN) excretion studies with literature results. Renal tubular secretion of NMN was shown to be saturable and was inhibited by mepiperphenidol to the same extent as that reported in the literature. Pseudoephedrine was cleared by the kidney at a rate in excess of inulin and close to or possibly greater than renal plasma flow. In addition to filtration and secretion, pseudoephedrine appeared to be subject to pH dependent passive reabsorption. When the secretion of pseudoephedrine was studied in detail, it was found to be nonsaturable for plasma levels of pseudoephedrine ranging from 0.16 to 1.5 microgram/ml. Secretion, however, was inhibited by mepiperphenidol suggesting a carrier-mediated process.

Plasma and urine concentrations of d-tubocurarine (d-TC) were determined and twitch tension was monitored before and after the intravenous administration of d-TC, 0.5 mg/kg in patients anethetized with halothane and nitrous oxide with (N=5) and without (N=5) renal failure. Terminal half lives of d-TC of 231 and 330 minutes predicted from other studies for patients with and without renal function were consistent with plasma concentrations measured. In patients with normal renal function, 38% of unchanged d-TC was eliminated in urine in 25 hours as opposed to 13% in those patients with a newly transplanted kidney. However, we still conclude that a newly transplanted kidney can eliminate d-TC effectively since the kidney was inserted 3.5 hours after administration of d-TC. Absence of renal function significantly prolonged the duration of neuromuscular blockade but did not alter the plasma concentrations of d-TC required for neuromuscular blockade. Thus, prolonged neuromuscular blockade from d-TC in patients with renal failure is due to decreased elimination rate of the drug as a consequence of lack of renal function rather than increased sensitivity.

Pharmacokinetic studies of the disposition of acetaminophen were carried out in 12 chronically catheterized pregnant sheep. Acetaminophen was infused separately into the mother and the fetus on two occasions to steady state. The transplacental clearances from both sides of the placental membrane and the nontransplacental clearances in both the mother and the fetus were then determined using an indirect method, a two-compartment open pharmacokinetic body model. The placental clearance and the nontransplacental clearances were also determined independently by direct methods by measuring the extraction ratio and urinary clearances in both the mother and fetus. Model dependent (indirect) and model independent (direct) methods yielded similar results. Neither acetaminophen glucuronide nor acetaminophen sulfate was transferred across the placenta. However, acetaminophen was transferred in both directions exclusively via a passive diffusion mechanism with an extraction ratio of 0.12 across the placenta. Neither glucuronidation nor sulfation capacity was found in the sheep placenta. However, the conjugation pathways accounted for nearly all the nontransplacental elimination of acetaminophen in the mother. Both transplacental clearance and nontransplacental clearance of acetaminophen in the fetus increased with gestational age. The glucuronidation and sulfation activities were determined in the fetus in utero and found to be 16 and 74% of the respective activities in the pregnant ewe on a body weight basis. The two conjugation clearances did not increase significantly with the gestational age of the fetus during the third trimester.

The disposition of the two major metabolites of acetaminophen, acetaminophen glucuronide (A-G) and acetaminophen sulfate (A-S), was studied in nine chronically catheterized fetal lambs during the third trimester. After a bolus i.v. injection, both conjugates exhibited biphasic log plasma concentration vs. time curves with terminal half-lives of 3.36 to 7.60 hr for A-G and 4.61 to 8.20 hr for A-S. The steady-state volumes of distribution and the total clearances of both conjugates increased with the gestational age of the fetus. The ratios of renal clearance to total clearance were 1.00 +/- 0.06 and 0.95 +/- 0.07, respectively, for A-G and A-S. Neither placental transfer nor systemic hydrolysis of either conjugate was detectable in our experiments. The ratios of renal clearance to glomerular filtration rate, as determined by [14C] inulin renal clearance, were 0.96 +/- 0.06 and 0.67 +/- 0.10 for A-G and A-S, respectively. These ratios corresponded to the free fractions of the two conjugates in the plasma, which were 0.96 +/- 0.03 and 0.64 +/- 0.09, respectively, for A-G and A-S. Our data indicate that transplacental transfer of acetaminophen conjugates is not detectable and both conjugates are predominantly eliminated from the fetal plasma through renal excretion by glomerular filtration in the fetal kidney. As the conjugates are apparently not hydrolyzed in the fetus, they remain in the fetus and the surrounding amniotic fluid until birth.