Almokalant glucuronidation in human liver and kidney microsomes: evidence for the involvement of UGT1A9 and 2B7.

1. Almokalant, a class III antiarrythmic drug, is metabolized to form isomeric glucuronides identified in human urine. Synthesis of the total glucuronide was studied in human liver and kidney microsomes. Recombinant UDP-glucuronosyltransferases (UGTs) were screened for activity and kinetic analysis was performed to identify the isoform(s) responsible for the formation of almokalant glucuronide in man. 2. From a panel of recombinant isoforms used, both UGT1A9 and 2B7 catalysed the glucuronidation of almokalant. The Km values in both instances were similar with 1.06 mM for the 1A9 and 0.97 mM for the 2B7. Vmax for 1A9 was fourfold higher than that measured for UGT2B7, 92 compared with 21 pmol min(-1) mg(-1), respectively, but UGT1A9 was expressed at approximately twofold higher level than the UGT2B7 in the recombinant cell lines. Therefore, the contribution of UGT2B7 to almokalant glucuronidation could be as significant as that of UGT1A9 in man. 3. Liver and kidney microsomes displayed similar Km values to the cloned expressed UGTs, with the liver and kidney microsomes at 1.68 and 1.06 mM almost identical to the 1A9. 4. The results suggest a significant role for UGT1A9 and 2B7 in the catalysis of almokalant glucuronidation.

Tolerance and effects of almokalant, a new selective Ik blocking agent, on ventricular repolarization and on sino-atrial and atrioventricular nodal function in the heart: a study in healthy, male volunteers utilizing transesophageal atrial stimulation.

Almokalant, (4-(3-ethyl(3-propylsulfinyl)propyl)amino)-2-hydroxy-propoxy)- benzonitrile), is a newly developed Ik channel blocker that exhibits pure class III effects. Using a noninvasive approach with transesophageal atrial stimulation (TAS), we wished to identify the dose of almokalant, given as an intravenous bolus infusion, that prolonged ventricular repolarization in the healthy human heart to an extent of potential clinical interest. Furthermore, we defined the electrophysiological effects of this dose on the heart, as well as the pharmacokinetics, safety, and tolerance throughout a wide dosing range. In the titration part, increasing doses were given to identify the dose that produced a reproducible QTend prolongation of approximately 20%. This dose (12.8 mumol) was then given in a placebo-controlled, double-blind, cross-over fashion. In the double-blind part, almokalant significantly prolonged the QTend intervals during sinus rhythm and during TAS at 100 beats/min and increased the effective refractory period of the atria (AERP). There was no alteration in either the cardiac conduction (PQ and QRS), or blood pressure (BP) sinus node function, or the ERP of the atrioventricular (AV) node. Therefore, almokalant exhibited pure class III effects with no signs of beta-blockade or unwanted hemodynamic effects. The plasma concentration-time curve showed a biexponential decrease with a terminal half-life (t1/2) of approximately 3 h. There was a large interindividual variation in the plasma concentration at the end of infusion, Cmax. This variability diminished considerably 60 min after infusion, and the pharmacokinetic characteristics studied appeared to be proportional to the dose. The drug was well tolerated, and the only side effect noted was a brief metallic taste after a dose of 25.6 mumol. Corresponding to high plasma peak values, T-wave morphology changes of short duration were observed, sometimes with the development of pronounced, biphasic T waves.

Induction of rhythm abnormalities in the fetal rat heart. A tentative mechanism for the embryotoxic effect of the class III antiarrhythmic agent almokalant.

OBJECTIVES: The aim was to test the hypothesis that the recently reported embryotoxic effect of class III antiarrhythmic agents may be a result of electrophysiological disturbances induced by these agents. METHODS: Comparative studies of drug effects in the adult and fetal rat were performed using three experimental models: (1) effects of almokalant upon pregnancy and fetal mortality in rats given daily doses of 0, 10, 50, 100, or 400 mumol.kg-1 orally in the diet on days 6-15 of pregnancy; (2) effects of d-sotalol (1-1000 microM), almokalant (0.1-100 microM) and dofetilide (0.01-10 microM) on the adult and fetal cardiac action potential in vitro; (3) voltage clamp recordings in single fetal and adult ventricular myocytes superfused with almokalant (0.5 microM). RESULTS: In the groups of rats treated with 100 and 400 mumol.kg-1, respectively, the body weight gain was decreased from day 12 of gestation, and there were no viable fetuses at termination of pregnancy. In atrial as well as ventricular tissue, the class III agents induced a concentration dependent prolongation of the fetal action potential duration, accompanied by a reduction in heart rate and eventually the appearance of rhythm abnormalities and/or early afterdepolarisations. The adult action potential duration remained unaffected. An almokalant sensitive current (probably the delayed rectifier, IK) could be evoked both in the fetal and in the adult ventricular cells. CONCLUSIONS: Class III antiarrhythmic agents were shown to induce fetal mortality and rhythm abnormalities in the rat heart. Although they do not prove a causal relationship between these effects, our observations may have implications for the clinical use of class III antiarrhythmic agents in women of childbearing potential.

Stereoselective metabolism of felodipine in liver microsomes from rat, dog, and human.

Felodipine, a 1,4-dihydropyridine derivative, is a potent, vasoselective calcium antagonist that is used clinically as a racemic mixture of two stereoisomers. In the rat, dog, and human, the bioavailability of an oral dose is about 15% because of high first-pass metabolism. Oxidation of the dihydropyridine ring to the corresponding achiral, pharmacologically inactive pyridine metabolite is the predominant metabolic step. In the liver, this metabolism is catalyzed by cytochrome P-450. In the present study, the metabolism of (R)- and (S)- felodipine was compared in vitro in liver microsomes from rats, dogs, and humans. Slightly higher rates of metabolism were found for the (S)-enantiomer in rat and dog liver microsomes. However, no significant differences were observed in the Michaelis-Menten parameters, Vmax or KM. In human liver microsomes, the (R)-enantiomer was metabolized more readily than (S)-felodipine. The mean value of KM was lower for (R)-felodipine, while the Vmax values of the enantiomers were similar. The intrinsic clearance, defined as the ratio of Vmax and KM, was about two times higher for (R)-felodipine. Assuming complete absorption and that the bioavailability is determined by the first-pass metabolism in the liver, these in vitro results suggest that the bioavailability of (S)-felodipine in vivo is about two times higher than that of (R)-felodipine.

Oxidation of dihydropyridine calcium channel blockers and analogues by human liver cytochrome P-450 IIIA4.

A series of 21 different 4-substituted 2,6-dimethyl-3-(alkoxycarbonyl)-1,4-dihydropyridines was considered with regard to oxidation to pyridine derivatives by human liver microsomal cytochrome P-450 (P-450). Antibodies raised against P-450 IIIA4 inhibited the microsomal oxidation of nifedipine and felodipine to the same extent, as did cimetidine and the mechanism-based inactivator gestodene. Gestodene was approximately 10(3) times more effective an inhibitor than cimetidine, on a molar basis. When rates of oxidation of the 1,4-dihydropyridines were compared to each other in different human liver microsomal preparations, all were highly correlated with each other with the exceptions of a derivative devoid of a substituent at the 4-position and an N1-CH3 derivative. A P-450 IIIA4 cDNA clone was expressed in yeast and the partially purified protein was used in reconstituted systems containing NADPH-cytochrome P-450 reductase and cytochrome b5. This system catalyzed the oxidation of all of the 1,4-dihydropyridines except the two for which poor correlation was seen in the liver microsomes. Principal component analysis supported the view that most of these reactions were catalyzed by the same enzyme in the yeast P-450 IIIA4 preparation and in the different human liver microsomal preparations, or by a closely related enzyme showing nearly identical properties of catalytic specificity and regulation. The results indicate that the enzyme P-450 IIIA4 is probably the major human catalyst involved in the formal dehydrogenation of most but not all 1,4-dihydropyridine drugs.

Contribution of the intestine to the first-pass metabolism of felodipine in the rat.

The systemic availability of intraduodenally (i.d.) administered felodipine in the rat is about 10%. The purpose of this study was to determine to what extent intestinal metabolism contributes to the first-pass elimination of felodipine in the rat. Four different types of experiments were performed. 1) [3H]Felodipine was given i.v. and i.p.; 2) the uptake of i.p. administered [3H]felodipine by the lymph was studied for 3 hr after dosing; 3) portal blood was collected quantitatively for 40 min after i.d. administration of [3H]felodipine; and 4) the in vitro metabolism of felodipine was studied in intestinal cell suspensions. The mean bioavailability of the i.p. dose was approximately 48%. The uptake via the lymph was negligible as an insignificant amount of the radioactive i.p. dose was recovered in lymph from a main lymph vessel in the peritoneal cavity. An average of 21 +/- 12% of given radioactive dose was recovered in portal blood during the first 40 min after i.d. dosing. The recovered radioactivity was to 40 to 70% due to felodipine and 9 to 16% was due to dehydro-felodipine. These results indicate that substantial first-pass elimination occurs in the intestine of the rat. Further support for gastrointestinal metabolism of felodipine in the rat was obtained from incubations with intestinal cells.

Biotransformation of felodipine in liver microsomes from rat, dog, and man.

The biotransformation of the calcium antagonist felodipine was investigated in liver microsomes from rat, dog, and man. The metabolites were quantified and identified by gradient elution reverse phase liquid chromatography, liquid scintillation analysis, and GC/MS. Ten metabolites were identified, including the pyridine analogue of felodipine, two carboxylic mono acids, two ester lactones, as well as the corresponding open hydroxy acid forms, and a lactonic compound with a carboxylic acid group. The presence of two decarboxylated products was also verified. Metabolites with an intact dihydropyridine nucleus were not detected. The total pool of metabolites formed in vitro was more lipophilic than that excreted in urine from the same species. The metabolic pathways were similar in the three species studied, although quantitative differences were observed. Comparison between incubations with liver microsomes from male and female rats indicated that the females metabolized felodipine more slowly than the males. From a more detailed quantitative analysis of eight metabolites, in relation to incubation time, it was apparent that the hydroxylation of the 2- and 6-methyl groups occurred at a faster rate (0.027 min-1) than did the ester hydrolysis (0.016 min-1). These hydroxy metabolites rearranged spontaneously to lactones. The results from this study indicate that the open hydroxy acid metabolites were formed enzymatically from the corresponding lactones. A metabolic scheme for the overall metabolism of felodipine is given and discussed with reference to the in vivo situation.

In vivo pharmacokinetics of felodipine predicted from in vitro studies in rat, dog and man.

The elimination of felodipine in liver microsomes from dog and man were characterized by Km and Vmax. The results were compared with previous data reported for rat. In all species studied, felodipine was primarily metabolized to its corresponding pyridine analogue. The elimination rate order was rat greater than dog greater than man. The same species difference was observed in vivo for the oral plasma clearance which was; rat 26 1/hr/kg, dog 7.5 1/hr/kg and man 4.3 1/hr/kg. The intrinsic hepatic clearance of felodipine was predicted in vitro from Vmax over Km. The in vitro values were not significantly different from those observed in vivo. Felodipine is a high-clearance drug and the in vivo extraction ratios were about the same in all species: rat 0.80, dog 0.83 and man 0.84. The extraction ratios predicted from the in vitro studies, rat 0.91, dog 0.70 and man 0.80, agreed well with those observed in vivo.

Quantitative relationships between structure and microsomal oxidation rate of 1,4-dihydropyridines.

The oxidation rate of a series of 4-phenyl substituted 1,4-dihydropyridine esters was investigated in dog liver microsomes and was evaluated in relation to physiochemical properties as well as to antihypertensive effect. The relative rate of microsomal oxidation was mainly dependent on the substituents of the aromatic ring and almost unaffected by small changes in the ester substituents. A 20-fold variation in the microsomal oxidation rate was observed within a group of dichloro-substituted analogues. Generally, the highest oxidation rate was found with 2',6'-disubstituted derivatives, while compounds with substituents in position 4' exhibited longer half-lives. The oxidation rate increased with increased steric bulk, increased lipophilicity and increased electron withdrawal of the substituents in position 2'. The energies of the highest occupied molecular orbital (HOMO) were calculated and correlated with the oxidation rate of some of the dihydropyridine analogues. The antihypertensive effect appeared to be restricted to compounds with oxidation rates within a narrow range, indicating the unlikelihood of increasing the duration of the pharmacologic effect by stabilisation of the dihydropyridine system.

Oxidation of 1,4-dihydropyridines by prostaglandin synthase and the peroxidic function of cytochrome P-450. Demonstration of a free radical intermediate.

Oxidation of 1,4-dihydropyridines by the hydroperoxidic function of cytochrome P-450 and prostaglandin synthase was investigated using felodipine as a model substance. Nifedipine and the 2,6-dichlorophenyl analogue of felodipine were used in some experiments with similar results. Felodipine was metabolized to a pyridine metabolite in vitro when incubated with liver microsomes and cumene hydroperoxide, as well as with ram seminal vesicle microsomes and arachidonic acid. The oxidation of 1,4-dihydropyridines is proposed to proceed via formation of a free radical intermediate, judging from EPR analysis with the spin trap POBN. When reduced glutathione was added the EPR signal was decreased as well as the formation of the pyridine metabolite while an oxidation of glutathione was observed. This effect was due to a reduction of the radical intermediate back to felodipine by glutathione. Felodipine interacts with the hydroperoxidase activity of prostaglandin synthase, since the pyridine metabolite was formed also when 15-hydroperoxyeicosatetraenoic acid was used as a substitute for arachidonic acid. Indomethacin could only inhibit the metabolism of felodipine when arachidonic acid was used as substrate. The cooxidation of felodipine by prostaglandin synthase is associated with an increased metabolism of arachidonic acid. This was further supported by a stimulated oxygen consumption and an increased formation of prostaglandin E2.

Cytochrome P-450-dependent oxidation of felodipine--a 1,4-dihydropyridine--to the corresponding pyridine.

Felodipine, a 1,4-dihydropyridine diester, is metabolized to its corresponding pyridine analogue by rat-liver microsomes. Kinetic studies showed similar Km, Vmax and t1/2 for the formation of the pyridine metabolite and the disappearance of felodipine, indicating that oxidation of felodipine to the corresponding pyridine analogue is the major pathway of metabolism. Response to inhibitors such as CO, SKF 525-A and metyrapone indicates participation of cytochrome P-450 in the aromatization of felodipine. Phenobarbital pretreatment markedly increased the metabolism of felodipine and its pyridine analogue. Felodipine pretreatment had no effect on the cytochrome P-450 concn. in rat-liver microsomes, nor on the rate of its own metabolism, but a slight increase was observed in the rate of metabolism of four standard substrates.

Stereoselective disposition of RS-tocainide in man.

The disposition of RS-tocainide in three healthy volunteers has been studied after oral administration of a pseudoracemic mixture containing S(+) [3H] tocainide as a radioactive tracer together with a therapeutic dose of the racemate. Analytical methods based on HPLC have been developed to measure S(+) and R(-) tocainide in urine samples. Selected ion detection has been used for quantification of a tocainide conjugate. The radioactive dose was efficiently absorbed and mainly cleared via the kidneys. The elimination half-life of RS-tocainide was found to be 14.3 hours. The elimination half-lives of the two stereoisomers of tocainide differed significantly, i.e. R(-) tocainide 10 hours, and S(+) tocainide 16.7 hours. The observed t1/2 for the tocainide conjugate of 10.3 hours was close to that of R(-) tocainide, indicating that the metabolite was preferably formed from the R(-) stereoisomer of tocainide. Of the given dose, between 45 and 70% can be accounted for.