Calmodulin antagonistic action of new 1,5-benzothiazepines derived from diltiazem.

A series of newly synthesized 1,5-benzothiazepines derived from diltiazem (CAS 42399-41-7) were tested for calmodulin antagonistic activities using Ca(2+)-calmodulin stimulated phosphodiesterase (PDE). Some compounds possessing the benzoyloxy moieties at position 4 of 1,5-benzothiazepine ring of diltiazem showed a dose-dependent inhibitory action with the potencies comparable to that of a calmodulin antagonist, N-(6-aminohexyl)-5-chloro-1-naphthalene-sulfonamide (W-7). In contrast, diltiazem did not exert the inhibitory action at the same concentrations. Further, radioligand binding experiment, using a radiolabeled 1,5-benzothiazepine, showed that these compounds bound to Ca(2+)-calmodulin complex, but not to calmodulin in the presence of EGTA, suggesting that these 1,5-benzothiazepines are new calmodulin antagonists. Some of these compounds inhibited [3H]diltiazem binding to Ca antagonist binding sites in cell membranes of rat cerebral cortex but with a less potent affinities than diltiazem, suggesting that there was no correlation between their anti-calmodulin effect and the binding affinity to Ca antagonist binding sites. In conclusion, new 1,5-benzothiazepines have been demonstrated to have an anti-calmodulin action. These compounds may possess a pharmacological activity based on their anti-calmodulin action in addition to their interaction with Ca channel.

Metabolic fate of the new anti-ulcer drug enprostil in animals. 2nd communication: whole-body autoradiographic distribution of [3H]-enprostil in rats.

The distribution of radioactivity was studied by whole-body autoradiography in rats after oral or intravenous administration of [3H]-enprostil ((+/-)-11a-15a-dihydroxy-9-oxo-16-phenoxy-17,18,19,20-tetranorp r osta-4,5,13(t)- trienoic acid methyl ester, TA-84135) at a dose of 23 micrograms/kg. After oral administration to male rats, radioactivity in almost all the tissues and organs reached a peak within 15 min to 1 h. The highest levels of radioactivity were found in the contents of the stomach and intestine. High levels of radioactivity were also observed in the liver and kidney, and moderate levels were found in the lung, blood, dental pulp and the walls of the stomach. Radioactivity was the lowest in the skeletal muscle, testis, eye and brain. After reaching peak levels, radioactivity in the body decreased gradually, and it was detected only in the excretory organs at 24 h after drug administration. The distribution pattern after the intravenous dose was essentially similar to that after oral administration. The distribution profile of radioactivity in non-pregnant female rats after an oral dose was similar to that in male rats. Placental transfer and excretion in milk of radioactivity was slight. When the affinity of this compound to the melanin-containing tissues such as the uveal tract of the eye and the hair follicle was examined using pigmented rats, no tendency to retention of radioactivity in these tissues was observed.

Comparative study on the disposition of a new orally active dopamine prodrug, N-(N-acetyl-L-methionyl)-O,O-bis(ethoxycarbonyl)dopamine (TA-870) and dopamine hydrochloride in rats and dogs.

The pharmacokinetics of a dopamine derivative, TA-870, and dopamine (DA) after oral administration are compared in rats and dogs. The maximum concentrations of free DA in plasma after oral administration of TA-870 were 150 ng/ml in the rat (30 mg/kg) and 234 ng/ml in the dog (33.5 mg/kg). On the contrary, the maximum plasma concentrations after oral administration of DA at an equimolar dose to TA-870 were 12 ng/ml in the rat (12 mg/kg) and 36 ng/ml in the dog (13.5 mg/kg). The AUC values of free DA in plasma after oral administration of TA-870 (30 or 33.5 mg/kg) were 4-6 times higher than those after DA in both animal species. The peak tissue levels of radioactivity in rats after oral administration of [14C]TA-870 (30 mg/kg) were also 5.5 times higher in the liver and 1-2 times higher in other tissues than those after [14C]DA dose (12 mg/kg). In rats, the main excretion route of radioactivity after oral administration of [14C]TA-870 or DA was via the urine. The total recoveries of radioactivity in the urine and feces were 91-96% of the dose within 24 hr for both compounds. Biliary excretion in rats accounted for 19.8% of the dose of [14C]TA-870 and 12.6% of the dose of [14C]DA within 24 hr. These results demonstrate that TA-870 was well absorbed from the digestive tract, extensively metabolized to dopamine, and proved to be an orally usable dopamine prodrug.

Studies on intestinal lymphatic absorption of drugs. II. Glyceride prodrugs for improving lymphatic absorption of naproxen and nicotinic acid.

A series of alpha- and beta-monoglycerides and triglyceride derivatives of naproxen or nicotinic acid were synthesized and investigated in order to elucidate the molecular form of the derivative with properties that enhanced lymphatic absorption. The lymphatic absorption rate was increased by adjusting the length of an n-alkyl chain introduced between the alpha- or beta-position of glycerol and the drug residue. The alpha- and beta-monoglyceride derivatives (containing an n-alkyl chain) were approximately equal in lymphatic absorption rates, but differed markedly in the concentration of the di- and triglyceride analogues in the lymph lipids. The lymphatic absorption of triglyceride derivatives, drugs combined directly with beta-position of glycerol, was low in comparison with the monoglyceride derivatives. Compared with nicotinic acid, the alpha-monoglyceride derivative (n-alkyl chain length, Cn = 20) of nicotinic acid provided a higher AUC0-8 h value of free nicotinic acid and maintained a lower level of free fatty acids in blood.

Studies on intestinal lymphatic absorption of drugs. I. Lymphatic absorption of alkyl ester derivatives and alpha-monoglyceride derivatives of drugs.

Several alkyl ester derivatives or alpha-monoglyceride derivatives of 3H-labeled compounds, i.e. trimetoquinol, TA-594, acetaminophen, naproxen and nicotinic acid, were synthesized and administered orally to rats cannulated in the thoracic duct. The radioactivity appearing in 24 h-lymph was measured and analyzed by thin-layer chromatography. Most of the alpha-monoglyceride derivatives were absorbed via the intestinal lymphatic system, while the alkyl esters were very poorly absorbed. After oral administration of alpha-monoglyceride derivatives of labeled naproxen and nicotinic acid, the radioactive compounds found in the lymph were mainly monoglyceride, diglyceride and triglyceride analogues, while in plasma the main radioactive compound was the parent drug. It was concluded that alpha-monoglyceride derivatives of drugs were absorbed via the lymphatic system and transported into blood, yielding the parent drug in blood.

Metabolism of diltiazem. I. Structures of new acidic and basic metabolites in rat, dog and man.

The metabolites of diltiazem in the urine of rats, dogs and man and rat bile were investigated. New metabolites including six acidic metabolites (A1-A6) and four basic metabolites (M9, MB, MC, MD) were isolated by high performance liquid chromatography and their structures were characterized by gas chromatography-mass spectrometry. Acidic metabolites A1, A2, A3, A4 and basic metabolite MD identified by comparing their properties with the synthetic compounds; (+)-(2S, 3S)-2-(4-methoxyphenyl)-3-acetoxy-4-oxo-2,3,4,5-tetrahydro-1,5- benzothiazepin-5-acetic acid (A1), 3-deacetyl-A1 (A2), O-demethyl-A1 (A3), O-demethyl-3-deacetyl-A1 (A4) and N-didemethyl-diltiazem (MD). All acidic metabolites have a CH2COOH group which may be formed by oxidative deamination of the CH2CH2N(CH3)2 group of diltiazem.

Metabolism of diltiazem. II. Metabolic profile in rat, dog and man.

The metabolism of diltiazem was studied in male rats and dogs after intravenous administration of 14C-diltiazem.HCl or in men after oral administration of a tablet of diltiazem.HCl. The main metabolites in rat urine, in decreasing order of content, were A4, M6, M4 and M5. The unchanged drug in the urine accounted for only 0.7% of the administered drug based on radioactivity. About 81% of biliary radioactivity in rat was due to water-soluble metabolites and the remainder was mostly acidic metabolites such as A4 and A6. In dog urine, the unchanged drug was most abundant (30.6% of the radioactivity in the 6-h urine), followed by A2, A1 and M2. The main metabolites of diltiazem in rat plasma were A2 and A4, while in dog plasma, the unchanged drug and A2 were found as main components. In human urine, M4, the unchanged drug, A2, MB, MD and M2 were the major components. Acidic metabolites, A1-A4, were found also in human plasma and A2 was the main metabolite found. These results indicated that the main metabolic pathway of diltiazem was an oxidative deamination which is extensive in the rat. Other metabolic pathways recognized in this study were oxidative demethylation, deacetylation, hydroxylation of the aromatic ring and conjugation. Although direct comparison may not be appropriate because of the different routes of administration, the metabolism of diltiazem in man appears to be more similar to that in the dog than that in the rat.

Metabolic fate of diltiazem. Distribution, excretion and protein binding in rat and dog.

Pharmacokinetics of (+)-(2S,3S)-2,3-dihydro-3-acetoxy-2-(4-methoxyphenyl)-5- [2-(dimethylamino)ethyl]-1,5-benzothiazepin-4(5H)-one hydrochloride (diltiazem-HCl, Cardizem, Herbesser) in rats and dogs were investigated using 14C-diltiazem-HCL. The plasma concentration of unchanged drug in rats was 1.78 microgram/ml 1 min after intravenous administration at a dose of 3 mg/kg, and rapidly decreased thereafter with a half-life of 20 min (alpha-phase) and 56 min (beta-phase). In contrast, the plasma concentration of radioactivity in rats increased during 0-2 h in spite of intravenous administration, and thereafter the level of radioactivity in plasma decreased very slow. In dogs, the plasma concentration of unchanged drug was 0.138 micrograms/ml 1 min after intravenous administration at a dose of 0.2 mg/kg, and then decreased with a half-life of 2.5 min (alpha-phase) and 1.68 h (beta-phase). Dog plasma level of radioactivity decreased once after intravenous administration, but increased from 30 min to 1 h. Unchanged drug plasma levels at 1 h after intravenous administration were 6.6 and 35.1% of the plasma radioactivities in rats and dogs, respectively. When 14C-diltiazem-HCl was orally administered, unchanged drug plasma levels in rats and dogs were 1.8 and 12.8% of the plasma radioactivities at 15 min, respectively. Therefore, the first-pass effect was extensive, especially in rats. Rat whole body autoradiograms showed that radioactivity distributed well to tissues and organs in either route of administration. Similar results were obtained by the method of counting the radioactivity in the tissues and organs of rats.(ABSTRACT TRUNCATED AT 250 WORDS)

Mechanism of metabolic cleavage of a furan ring.

We studied the mechanism of metabolic cleavage of a furan ring, using a new hypolipidemic agent, ethyl 2-(4-chlorophenyl)-5-(2-furyl)oxazole-4-acetate (TA-1801), as a model compound. A TA-1801 analogue labeled with deuterium at the 5-position of its furan ring was administered orally to rats. The analysis of urinary metabolites by GC/MS revealed that the deuterium of the furan was retained in the ring-opened metabolite (M3). Metabolic cleavage of furan has been generally considered to proceed by hydroxylation of the 5-position followed by tautomerism and hydrolysis of the resulting 5-hydroxyfuran derivative. However, if the cleavage proceeded by this pathway, the deuterium of the 5-position would be eliminated during hydroxylation. Therefore, we propose that the ring was cleaved directly to form an unsaturated aldehyde, considering the mechanism of oxidation by cytochrome P-450. Although this "intermediate" was not detected in the biological specimens, a synthetic unsaturated aldehyde was transformed to the actual urinary metabolites M2 and M3 (major ring-opened metabolites) in the isolated rat liver.

Metabolism of ethyl 2-(4-chlorophenyl)-5-(2-furyl)-oxazole-4-acetate, a new hypolipidemic agent, in the rat, rabbit, and dog. Glucuronidation of carboxyl group and cleavage of furan ring.

Metabolism of ethyl 2-(4-chlorophenyl)-5-(2-furyl)-oxazole-4-acetate (TA-1801), a new hypolipidemic agent, was studied in the rat, rabbit, and dog. Animals were given a single oral dose of 50 mg/kg TA-1801 labeled with 14C. The first metabolic reaction for TA-1801 was hydrolysis of the ester linkage. The resulting metabolite M1 was found to undergo further biotransformations, i.e. glucuronidation at the carboxyl group and ring cleavage of the furan group. These metabolic pathways were observed in all the species examined, although species differences were seen in the amount of metabolites.

Glucoside formation as a novel metabolic pathway of pantothenic acid in the dog.

Metabolism of pantothenic acid (PaA) in beagle dogs was investigated. The dogs excreted 12.3% of the dose in the urine within 24 hr after a single oral administration of [3H]PaA (3 mg/kg). High performance liquid chromatographic analysis of the urine showed the presence of unchanged vitamin and a major metabolite, which accounted for 60.2 and 39.8% of the urinary radioactivity respectively. Although the metabolite was hydrolyzed by treatment with beta-glucuronidase or acid phosphatase, it was found that this hydrolysis resulted from the actions of beta-glucosidase contained as a contaminant in these enzyme preparations. beta-Glucosidase completely hydrolyzed the metabolite to generate PaA and glucose. The metabolite was isolated and subjected to GC/MS and NMR analyses. It was identical to synthetic PaA beta-glucoside, 4'-O-(beta-D-glucopyranosyl)-D-pantothenic acid. It was shown by the use of dog liver microsomes that PaA underwent beta-glucosidation in the presence of uridine diphosphate glucose (UDPG). It is proposed that beta-glucosidation by UDP-glucosyltransferase is a novel metabolic pathway of PaA in the dog.

Hopantenic acid beta-glucoside as a new urinary metabolite of calcium hopantenate in dogs.

The metabolism of calcium hopantenate (HOPA) was studied in beagle dogs. After oral administration of 14C-labeled HOPA, 25.5% of the administered radioactivity was excreted in the urine within 24 hr, mostly in the form of unchanged drug. The only metabolite, accounting for 4.2% of the radioactivity in the urine, was isolated by HPLC. The metabolite was hydrolyzed by the treatment of beta-glucuronidase (Helix pomatia), acid phosphatase, or beta-glucosidase. These enzyme activities were not inhibited by treatment with D-glucaric acid 1,4-lactone or PO4(3-), but with D-gluconic acid 1,5-lactone, demonstrating that the metabolite is a glucose conjugate. The compound was identified as HOPA-glucoside, 4'-O-(beta-D-glucopyranosyl)-D-hopantenic acid, by GC/MS analyses after derivatization of the metabolite and the synthetic compound. This is the first reported instance of glucose conjugation to a non-acidic hydroxyl group in the metabolism of xenobiotics in mammals.

Metabolic fate of the new cardiotonic denopamine in animals. 1st communication: absorption, distribution and excretion in the rat, rabbit and dog.

Absorption, distribution and excretion of (-)-(R)-1-(p-hydroxyphenyl)-2-[(3,4-dimethoxyphenethyl)amino] ethanol (denopamine, TA-064) a new positive inotropic agent, were studied after oral and intravenous administration of 3H- or 14C-denopamine (5 mg/kg) to different animal species. After oral administration to rats, rabbits and dogs, the time to attain the peak and the maximum concentration of the plasma levels of radioactivity were about 15 min, 4 micrograms eq./ml in rats, 15-45 min, 8 micrograms eq./ml in rabbits and 2-4 h, 2 micrograms eq./ml in dogs, respectively. The plasma denopamine levels in dogs reached the peak (0.34 microgram/ml) at 0.5-3 h after administration, and thereafter gradually decreased with half-lives of 1.6-3.1 h. Following oral administration to rats, the amounts remaining of the parent compound in the digestive tract at 0.5 and 3 h after administration were about 27 and 2% of the dose administered, respectively. This indicated that the compound was rapidly and almost completely absorbed from the intestinal tract. When 3H-denopamine was orally administered to rats, cumulative excretion of radioactivity in the urine and feces within 24 h were about 60 and 32% of the dose, respectively. Almost 100% of the dose were recovered from the urine and feces within 120 h. About 50% of the dose administered were excreted in the bile within 24 h. The occurrence of enterohepatic circulation was indicated in rats. Distribution of radioactivity was investigated in rats by means of whole body autoradiography and the tracer technique.(ABSTRACT TRUNCATED AT 250 WORDS)

Metabolic fate of the new cardiotonic denopamine in animals. 2nd communication: tissue accumulation in the rat after consecutive oral administration of 14C-denopamine.

Accumulation characteristics of (-)-(R)-1-(p-hydroxyphenyl)-2-[(3,4-dimethoxyphenethyl)amino]ethanol (denopamine, TA-064) in organs and tissues were investigated after oral administration of 14C-denopamine to rats once a day (5 mg/kg/d) for 1, 6 and 10 days. The levels of radioactivity in each organ and tissue were measured at various time intervals after the final dose. The levels of radioactivity at 15 min after administration were generally higher in the consecutive-dose groups than in the single-dose group. However, the levels were equilibrated within 6 days of administration. Radioactivity at 24 h after administration decreased to about the detection limit in each group. In addition, half-lives of disappearance of radioactivity from the body were not significantly affected by the consecutive doses. About 100% of the dose was recovered from the urine and feces during consecutive administration periods. Consequently, it was considered that the drug hardly showed retention and accumulation characteristics in the body.

Metabolic fate of the new cardiotonic denopamine in animals. 3rd communication: placental transfer and excretion into milk in the rat.

3H-labelled (-)-(R)-1-(p-hydroxyphenyl)-2-[(3,4-dimethoxyphenethyl)amino]ethanol (denopamine, TA-064) was administered orally to pregnant rats at a dose of 5 or 60 mg/kg on the 14th or 20th day of pregnancy. Irrespective of the doses and the stages of pregnancy, radioactivity in the fetus reached a peak at 30 min after administration, being 1/7 to 1/25 of the maternal plasma levels. Total radioactivity in each fetus at 30 min was estimated to be only about 0.002 to 0.06% of the dose. Disappearance of radioactivity from the fetus, uterus and amniotic fluid was slightly slower than that from the maternal plasma. After 3H-denopamine (5 mg/kg) was orally administered to lactating rats on the 12th day after delivery, radioactivity in milk was generally lower than in the blood of the dams, and time to the peak levels in milk tended to delay as compared with that in the blood. The levels of radioactivity in the blood of the sucklings, nursed by their dams, at 1 h after nursing were about 1/30 of the maternal blood levels, showing a slight transfer of the drug and/or its metabolites via milk. At 24 h after administration, radioactivity levels in the sucklings decreased to near the detection limit. The results of whole body autoradiography were generally consistent with the quantitative data on the placental transfer and excretion into milk.

Metabolic fate of the new cardiotonic denopamine in animals. 4th communication: effects of coadministered drugs on the plasma concentration and metabolism of denopamine in dogs.

Effect of coadministered drugs on plasma level and metabolism of (-)-(R)-1-(p-hydroxyphenyl)-2-[(3,4-dimethoxyphenethyl)amino]ethanol (denopamine, TA-064), a new, orally active, cardiotonic agent, were studied in dogs. Plasma levels of denopamine and urinary excretion of denopamine and its main metabolites were determined after oral administration of denopamine with or without digoxin, furosemide and isosorbide dinitrate. Mean plasma levels of denopamine were slightly lower when denopamine was given with the coadministered drugs than when given alone, but the difference was not statistically significant. No significant differences were also found between the areas under the plasma concentration curve (110 vs. 127 ng/ml X h, with and without the coadministered drugs, respectively), peak plasma concentrations (32.4 vs. 39.6 ng/ml), times to peak concentration (1.2 vs. 1.0 h) and plasma half-lives (2.2 vs. 2.0 h), respectively. In addition, the urinary excretion of denopamine and its main metabolites was not affected by the coadministered drugs.

Penetration into and elimination from the cerebrospinal fluid of diltiazem, a calcium antagonist, in anesthetized rabbits.

Penetration into and elimination from the cerebrospinal fluid (CSF) of diltiazem were studied following intravenous (i.v.) or intra-cisternal (i.c.) administration of 14C-diltiazem to anesthetized rabbits. 14C-Diltiazem rapidly penetrated into the CSF through the blood-CSF barrier after i.v. injection (1 mg/kg). A CSF level of the radioactivity attained the peak (0.13 microgram eq./ml) 5 min after i.v. injection and declined bi-exponentially with rapid initial half-life of 3.8 min (5-15 min) and second half-life of 2.7 h (15 min-8 h). A CSF/plasma ratio was 0.05-0.2 throughout the observation period. After i.c. administration (100 micrograms/body), the CSF level of the radioactivity decreased with a half-life of 7.5 min up to 1 h. The CSF level was maintained above 10(-7) mol/l until 2-3 h. Elimination half-life of 14C-diltiazem after i.c. injection was much more similar to that of 3H2O than 14C-inulin, indicating that the main elimination route of this compound from the CSF is diffusion into the brain, followed by rapid diffusion into the cerebral capillary, rather than filtration from the sub-arachnoid villi. An autoradiographic investigation supported the above route of elimination.

Effect of GTP on the affinity of denopamine, a new cardiotonic agent, for beta-adrenergic receptors of turkey erythrocytes and rat reticulocyte membranes.

Affinities of denopamine, a new cardiotonic agent, and several beta-adrenergic drugs for turkey erythrocyte membranes (TEM) and rat reticulocyte membranes (RRM) which contain homogeneous beta 1- and beta 2-receptors, respectively, were studied by receptor binding. The order of potencies of denopamine and several beta-adrenergic agonists in displacing 3H-dihydroalprenolol binding (Ki, nM) in TEM was isoproterenol (Iso, 27) greater than norepinephrine (Nor, 360) greater than epinephrine (Epi, 860) greater than dobutamine (DB, 1380) greater than denopamine (1540) greater than dopamine (DA, 49500). The order in RRM was Iso (7.3) greater than Epi (58) greater than DB (750) greater than Nor (1090) greater than denopamine (2300) greater than DA (26800). In the presence of GTP, competition curves for full agonists like Iso, Epi and Nor shifted to the low affinity side (Ki values increased by 300-500% in TEM and 200-460% in RRM), and the slopes were steepened in both membrane preparations. The Ki value for denopamine increased only in TEM (70%) and that in RRM was not influenced by GTP. This suggests that denopamine has an agonist property at the beta 1-receptor but not at the beta 2-receptor and that the intrinsic activity at the beta 1-receptor of the drug is lower than full agonists. Affinities of DB and DA for TEM were influenced by GTP as well as those for RRM, although the extent of the rightward shift was less than full agonists.