Small intestinal metabolism of the 3-hydroxy-3-methylglutaryl-coenzyme A reductase inhibitor lovastatin and comparison with pravastatin.

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.

Comparison of cytochrome P-450-dependent metabolism and drug interactions of the 3-hydroxy-3-methylglutaryl-CoA reductase inhibitors lovastatin and pravastatin in the liver.

In an in vitro study, the cytochrome P-450 3A (CYP3A)-dependent metabolism and drug interactions of the 3-hydroxy-3-methylglutaryl-Co A reductase inhibitors lovastatin and pravastatin were compared. Lovastatin was metabolized by human liver microsomes to two major metabolites: 6'beta-hydroxy [Michaelis-Menten constant (Km): 7.8 +/- 2.7 microM] and 6'-exomethylene lovastatin (Km,10.3 +/- 2.6 microM). 6'beta-Hydroxylovastatin formation in the liver was inhibited by the specific CYP3A inhibitors cyclosporine (Ki, 7.6 +/- 2.3 microM), ketoconazole (Ki, 0.25 +/- 0.2 microM), and troleandomycin (Ki, 26.6 +/- 18.5 microM). Incubation of pravastatin with human liver microsomes resulted in the generation of 3'alpha,5'beta, 6'beta-trihydroxy pravastatin (Km, 4,887 +/- 2,185 microM) and hydroxy pravastatin (Km, 20,987 +/- 9,389 microM). The formation rates of 3'alpha,5'beta,6'beta-trihydroxy pravastatin by reconstituted CYP3A enzymes were (1,000 microM pravastatin) 1.9 +/- 0.6 pmol.min-1.pmol CYP3A4 and 0.06 +/- 0.04 pmol.min-1.pmol CYP3A5, and the formation rates of hydroxy pravastatin were 0.12 +/- 0.02 pmol.min-1.pmol CYP3A4 and 0.02 +/- 0.004 pmol.min-1.pmol CYP3A5. The specific CYP3A inhibitors cyclosporine, ketoconazole, and troleandomycin significantly inhibited hydroxy pravastatin formation by human liver microsomes, but only ketoconazole inhibited 3'alpha, 5'beta,6'beta-trihydroxy pravastatin formation, suggesting that other CYP enzymes are involved in its formation. It is concluded that, compared with lovastatin [CLint formation 6'beta-hydroxylovastatin (microl.min-1.mg-1): 199 +/- 248, 6'-exomethylene lovastatin: 138 +/- 104)], CYP3A-dependent metabolism of pravastatin [CLint formation 3'alpha,5'beta, 6'beta-trihydroxy pravastatin (microl.min-1.mg-1): 0.03 +/- 0.03 and hydroxy pravastatin: 0.02 +/- 0.02] is a minor elimination pathway. In contrast to lovastatin, drug interactions with pravastatin CYP3A-catalyzed metabolism cannot be expected to have a clinically significant effect on its pharmacokinetics.

Metabolism and transport of the macrolide immunosuppressant sirolimus 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.

Metabolism of the macrolide immunosuppressant, tacrolimus, by the pig gut mucosa in the Ussing chamber.

1. The macrolide tacrolimus (FK506), used as an immunosuppressant, is a cytochrome P450 (CYP) 3A substrate in the liver. The metabolism of tacrolimus and the transport of its metabolites in the pig gut was studied in the Ussing chamber. Tacrolimus and its metabolites were quantified by h.p.l.c./mass spectrometry. 2. In the Ussing chamber, demethyl, didemethyl, hydroxy and hydroxy-demethyl tacrolimus were generated. Their formation was concentration- and time-dependent. The metabolite pattern was not different from that after incubation of tacrolimus with human small intestinal microsomes. 3. The metabolite formation was highest in the duodenum and declined in the order duodenum > jejunum > ileum > colon > stomach. 4. Since tacrolimus metabolism was inhibited by the specific CYP3A inhibitors, troleandomycin and ketoconazole, we concluded that these enzymes are involved in intestinal metabolism of tacrolimus. 5. Tacrolimus metabolites re-entered the mucosa chamber (> 90%) and passed through the small intestinal preparation into the serosa chamber. 6. It is concluded that tacrolimus is metabolized in the intestine, that the metabolites are able to re-enter the gut lumen and also enter into the portal vein and that small intestinal metabolism and transport is at least in part responsible for the low oral bioavailability of tacrolimus.

Drug interactions and interindividual variability of ciclosporin metabolism in the small intestine.

The undecapeptide ciclosporin is used as immunosuppressant after organ transplantation and for therapy of immune diseases. Low and variable bioavailability of ciclosporin has been attributed to its metabolism in the small intestine. The aim of the present study was to investigate drug interactions and interindividual variability of ciclosporin metabolism in the small intestine. Ciclosporin metabolism was studied in vitro using microsomes isolated from the small intestine of humans and pigs. The metabolites generated were quantified by HPLC and identified by mass spectrometry. Using specific antibodies and inhibitors, we showed that, as in the liver, cytochrome P450 3A (CYP 3A) enzymes are responsible for ciclosporin metabolism in the human small intestine. Of the 28 xenobiotics included in the study, 16 drugs, all well-known CYP 3A inhibitors, inhibited ciclosporin metabolism in the small intestine. In the small intestine of different patients, the rate of metabolism varied by a factor of 10. Ciclosporin was metabolized faster by small intestine microsomes from female (n = 4) than from male (n = 10) patients (p < 0.009).

Metabolism of the immunosuppressant tacrolimus in the small intestine: cytochrome P450, drug interactions, and interindividual variability.

The small intestinal metabolism of tacrolimus, which is used as an immunosuppressant in transplantation medicine, was investigated in this study. Tacrolimus was metabolized in vitro by isolated human, pig, and rat small intestinal microsomes. The metabolites generated were identified by HPLC/MS. Tacrolimus and its metabolites were quantified using HPLC or HPLC/MS. The cytochrome P450 (CYP) enzymes responsible for tacrolimus metabolism in small intestine were identified using specific CYP antibodies and inhibitors. For characterization of the interindividual variability, microsomes were isolated from small intestinal samples of patients who had undergone resection for various reasons. In an in vitro model using pig small intestinal microsomes, 32 drugs were analyzed for their interactions with tacrolimus metabolism. After incubation with human, rat, and pig small intestinal microsomes, the metabolites 13-O-demethyl and 13,15-O-demethyl tacrolimus were identified. The metabolism of tacrolimus by human small intestine was inhibited by anti-CYP3A, troleandomycin, and erythromycin, indicating that, as in the liver, CYP3A enzymes are the major enzymes for tacrolimus metabolism in the human small intestine. Metabolism of tacrolimus by small intestinal microsomes isolated from 14 different patients varied between 24 and 110 pmol/13-O-demethyl tacrolimus/min/mg microsomal protein, with a mean +/- SD of 54.2 +/- 29.2 pmol/min/mg. Of 32 drugs tested, 15 were found to inhibit small intestinal tacrolimus metabolism: bromocryptine, corticosterone, cyclosporine, dexamethasone, ergotamine, erythromycin, ethinyl estradiol, josamycin, ketoconazole, nifedipine, omeprazole, progesterone, rapamycin, troleandomycin, and verapamil. All of these drugs inhibited tacrolimus metabolism by human liver microsomes as well. It is concluded that tacrolimus is metabolized by cytochrome CYP3A enzymes in the small intestine. The rate of the CYP3A enzymatic activities varies about 5 times from patient to patient, and drugs that interfere with the in vitro metabolism of tacrolimus in the liver also inhibit its small intestinal metabolism.

Uptake, 3-, and 6-glucuronidation of morphine in isolated cells from stomach, intestine, colon, and liver of the guinea pig.

Orally administered morphine undergoes a considerable first-pass glucuronidation in animals and humans. However, the respective contribution of the gastrointestinal tract and the liver to the formation of the analgetically highly potent morphine-6-glucuronide (M6G) and the inactive morphine-3-glucuronide (M3G) is still debated. In this study, morphine uptake and biotransformation to M3G and M6G were compared in isolated cells from stomach, intestine, colon, and liver of the guinea pig. Morphine was taken up by all cell types in a time-dependent manner. There was evidence for a carrier-mediated accumulation in liver cells, but not in the other cell types. Morphine was glucuronidated to M3G by gastric, intestinal, colonic, and liver cells, and to M6G by all cell types excepted gastric cells. The M3G/M6G ratio averaged 3.5, 4.7, and 5.4 for colonic, intestinal, and liver cells, respectively. At low (1 microM) morphine concentration, glucuronidation rates for M3G and M6G in intestinal cells (88 and 20 pmol x mg protein-1 x hr-1, respectively) were similar to those in liver cells (133 and 12 pmol x mg protein-1 x hr-1, respectively). At high concentration (100 microM), rates of M3G and M6G formation in liver cells exceeded by 5- to 10-fold those of intestinal or colonic cells. In conclusion, the epithelium of the small and large intestine contributes with the liver to the formation of the active M6G; at the same time, the gastric, intestinal, and colonic epithelia are involved in the inactivation of morphine to M3G.

Mechanism of gastric antisecretory effect of SCH 28080.

The mechanism of the gastric antisecretory action of SCH 28080 has been studied utilizing two different in vitro test systems, isolated and enriched parietal cells from the guinea-pig and guinea-pig gastric membranes purified and enriched with K+/H+-ATPase. In guinea-pig isolated and enriched parietal cells SCH 28080 inhibited the acid response to histamine and high K+ concentrations with IC50 values not significantly different from each other. SCH 28080 inhibited the purified K+/H+-ATPase measured in the presence of 5 mM KCl with an IC50 value of 1.3 microM. Kinetic studies indicated a competitive inhibition of ATPase by SCH 28080 with respect to K+. Studies on Na+/K+-ATPase showed that this enzyme was only slightly depressed by SCH 28080. It is concluded that SCH 28080 acts with high selectivity on the parietal cell K%/H+-ATPase, establishing its antisecretory effect by a competitive interaction with the high affinity K+-site of the gastric ATPase.

The adenylate cyclase-cyclic AMP-protein kinase system in different cell populations of the guinea pig gastric mucosa.

In isolated guinea pig gastric mucous and enriched parietal cells it was tested whether or not cyclic AMP in response to histamine stimulation might reach concentrations sufficiently high to activate an intracellular cyclic AMP-dependent protein kinase and thereby mediate the acid response. Although histamine stimulated parietal cell adenylate cyclase to a greater extent than mucous cell adenylate cyclase, cyclic AMP levels in response to maximal histamine stimulation reached higher levels in mucous than in parietal cells. This had to be attributed to a five times higher phosphodiesterase activity in parietal cell than in mucous cell populations. In the absence of the phosphodiesterase inhibitor isobutylmethylxanthine exposure of the cells to histamine only in mucous cells produced an increase in cyclic AMP-dependent protein kinase activity ratio, but not in parietal cells. Dibutyryl-cyclic AMP induced cyclic AMP accumulation in parietal cell populations was compared to dibutyryl-cyclic AMP induced H+ secretion, as measured by 14C-aminopyrine uptake. A maximal acid response was associated with an intracellular cyclic AMP level of approximately 300 pmol/10(6) cells, which was never reached by maximal histamine stimulation even not in the presence of the phosphodiesterase inhibitor. It is concluded that activation of the parietal cell cyclic AMP-dependent protein kinase is one way for stimulating H+ secretion, but that the acid response elicited by histamine requires another intracellular pathway.

Adenosine inhibition of catecholamine-induced increase in force of contraction in guinea-pig atrial and ventricular heart preparations. Evidence against a cyclic AMP- and cyclic GMP-dependent effect.

The antagonism between adenosine and isoprenaline on force of contraction, cyclic AMP (cAMP) and cyclic GMP (cGMP) content, adenylate cyclase activity and transmembrane action potential in isolated electrically driven atrial and ventricular muscle preparations from guinea-pig hearts was investigated. In atrial preparations adenosine added 5 min after isoprenaline decreased force of contraction. Adenosine abolished completely the positive inotropic effect of isoprenaline. Similarly, adenosine prevented the positive inotropic effect of isoprenaline when both substances were added simultaneously. In ventricular preparations adenosine also decreased the isoprenaline-induced increase in force of contraction. The effect was much smaller than it was in the atria. Adenosine reduced the isoprenaline-induced increase in force of contraction only by about 60%. Adenosine did not at all influence the positive inotropic effect of isoprenaline when both substances were added simultaneously. In both preparations the isoprenaline-induced increase in cAMP content of the intact contracting preparations was not diminished by adenosine. cGMP content remained unchanged too. Adenosine inhibited adenylate cyclase activity in broken cell preparations from both tissues. In atrial preparations the decrease in force of contraction of adenosine in the presence of isoprenaline was accompanied by a shortening of the action potential duration. In ventricular preparations adenosine failed to shorten the action potential. In conclusion, the effects of adenosine to inhibit the stimulatory action of isoprenaline on myocardial force of contraction are not due to changes in the cAMP and/or cGMP content. Instead, adenosine may inhibit a step beyond an increased cAMP level, e.g., may exert an inhibition of protein kinases. However, in the atria, but not in the ventricles, an additional direct effect of adenosine on transmembrane ion currents, most likely an increase in potassium conductance, probably is of even greater importance.

Effect of vanadate on myocardial force of contraction.

Ammonium vanadate (NH4VO3; 50-1000 microM) increases the force of contraction of isolated electrically driven cat papillary muscles in a concentration-dependent manner. The positive inotropic effect (PIE) of NH4VO3 became significant at 50 microM and was maximal at 500 to 1000 microM. It was accompanied by an increase in the rate of force development, in the rate of relaxation and in relaxation time of the isometric contraction. Similar results as with NH4VO3 were also observed in the presence of 1 microM propranolol, 5 microM phentolamine or after reserpine-pretreatment (5 mg/kg i.p.). These results indicate that vanadate produces a direct PIT in ventricular cardiac muscle which is unlikely to be mediated by alpha- or beta-adrenoceptor stimulation. In cat left atrial strips, however, vanadate ions produced a negative inotropic effect through a hitherto unknown mechanism. Vanadate effects similar to those observed in the cat heart were obtained in ventricular and atrial preparations from bovine hearts.

[On the effect of PGE2 and PGF2 alpha on circular and longitudinal myometrium strips of different corpus uteri tissues of the guinea pig in vitro (author's transl)]. / Die Wirkung von PGE2 and PGF2 alpha auf zirkuläre und longitudinale Muskelstreifen verschiedener Uterussegmente des Meerschweinchens in vitro.

The effects of PGE2 and PGF2 alpha on corpus uteri and cervix strips of guinea pigs were studied in vitro. Myometrium strips developed maximal contraction activity at concentrations of 10(-7) M PGE2 and 5 x 10(-7) M PGF2 alpha in the organ bath, respectively. As for corpus uteri myometrium strips, longitudinal (extern) and circular (intern) layers of the organ wall showed comparable Prostaglandin-induced contractions. On the other hand, longitudinal and circular strips from cervix uteri tissue showed adverse Prostaglandin effects: PGE2 relaxed the cervix strips by lowering the muscle tone, the contraction frequency and a complete stop of contractions within most experiments lasting for an average time of 15 min. PGF2 alpha induced strong contractions of the same kind like corpus uteri strips did. The results indicate a role of PGE2 in the opening of the cervix, whereas PGF2 alpha possibly plays a role in the involution of the uterus post partum.

[Observations on prostaglandin effects with myometrium strips of human gravid uterus in vitro. Influence of diclofenac on contractions induced by prostaglandin (author's transl)]. / Beobachtungen über Prostaglandineffekte an Myometriumstreifen vom menschlichen graviden Uterus in vitro. Einfluss von Diclofenac auf Prostaglandin-induzierte Kontraktionen.

The effect of Diclofenac (Voltaren) was studied on PGE2- and PGF2alpha-induced contractions of pregnant human myometrium in vitro. Tests to establish experimental conditions revealed that PGE2 and PGF2alpha affect the myometrium differently from I. and IInd trimenon uteri. PGF2alpha always induced contractions in corpus uteri as well as cervix strips. PGE2 relaxed myometrium strips obtained from superficial muscle layers and from cervix tissue and caused contractions in the myometrium from the central parts of the corpus uteri. Diclofenac (Voltaren) at concentrations of 10(-5) to 10(-4) M inhibited PGE2 and PGF2alpha-induced contractions. The inhibition was demonstrated by planimetric evaluation of the contractions curves. Contraction activity of I. and IInd trimenon myometrium strips was reduced by Diclofenac but not completely inhibited. From uteri at term, three contraction curves showed complete cessation of PGE2-and PGF2alpha-induced contractions by Diclofenac (10(-4) M). The dose-dependent reduction of myometrial contractions induced by exogen prostaglandins indicates that Diclofenac possesses a direct effect on PGE2 and PGF2alpha-induced contractile activity of the uterus.

The positive inotropic effect of phenylephrine in the presence of propranolol. Increase in time to peak force and in relaxation time without increase in c-AMP.

The effects of phenylephrine on the shape of the contraction curve and on the cyclic adenosine 3',5'-monophosphate (c-AMP) content were studied in electrically driven (frequency 0.2 Hz) cat papillary muscles. All experiments were done in the presence of 1 micron propranolol in order to minimize interference from beta-adrenoceptors. 1. Phenylephrine increased the force of contraction in a concentration-dependent manner. Maximal effects (about 200% of control) occurred at 30 micron phenylephrine. 2. The positive inotropic effect (PIE) of phenylephrine was antagonized by phentolamine. Phentolamine, 5 micron, produced a parallel shift of the concentration-response curve for the PIE of phenylephrine by about two log units to the right. 3. The PIE of 30 micron phenylephrine occurred without any detectable increase in the c-AMP levels of the preparations. 4. The PIE of 30 micron phenylephrine developed about three times more slowly than the PIE of an equieffective concentration of isoprenaline. 5. The PIE of phenylephrine was accompanied by significant, concentration-dependent increases in both time to peak force and relaxation time. 6. It is concluded that the PIE of phenylephrine in the presence of propranolol is mediated mainly by a stimulation of alpha-adrenoceptors. It is unlikely to be related to an increase in c-AMP. With respect to time course and influence on the shape of the contraction curve it is qualitatively different from the effects of beta-adrenoceptor stimulation. These data are taken to support the hypothesis that the mechanical effects of alpha- and beta-adrenoceptor stimulating agents on the heart are produced by different mechanisms.