Species-differences in disposition and reductive metabolism of methoxymorpholinodoxorubicin (PNU 152243), a new potential anticancer agent.

Plasma pharmacokinetics, excretion balance and urinary metabolites of methoxymorpholino doxorubicin (MMDX) were investigated in male and female rats and in female dogs after i.v. administration of the(14)C-labelled drug. The mean total recovery of radioactivity in 96 h (urine plus faeces) was approximately 74 and 60% dose in male and female rats, respectively, while in female dogs approximately 72% dose was recovered in 336 h. Most of the radioactivity was present in faeces, with the urinary elimination accounting for only 3-4% dose in rats and dogs. These data suggest that biliary excretion is an important route of elimination of MMDX and/or its metabolites in both species. No differences were observed in the urinary metabolic profile of male and female rats. Two main peaks were present in radiochromatograms of urine from rats and dogs, i.e. MMDX and its 13-dihydro metabolite (MMDX-ol), accounting for approximately 25 and 20% of total radioactivity in 0-24-h urine in rats and 30 and 36% in dogs. The MMDX-ol/MMDX ratio in dog urine was higher than that observed in rat urine. No aglycones were detected in the urine samples from either species. In the rat, the plasma concentration-time profile suggested that the disposition of MMDX, MMDX-ol and total radioactivity is not sex-dependent. MMDX was the major species present in the systemic circulation; its AUC (0-96 h) accounted for 70% of total plasma radioactivity with the sum of AUC (MMDX) plus AUC (MMDX-ol) accounting for 77% of total radioactivity. In the dog, the sum of AUC (MMDX) plus AUC (MMDX-ol) amounted to 8% of radioactivity AUC(0-t(z) indicating that an important proportion of other(s) unknown metabolite(s) is present in dog plasma. Plasma levels of MMDX-ol in the rat were approximately 10-fold lower than those of the parent compound, whereas they were three times higher than those of MMDX in the dog. These data show that the reduction of the 13-keto group of MMDX is species-dependent, and occurs preferentially in the dog compared to the rat.

The effect of bidisomide (SC-40230), a new class Ia/Ib antiarrhythmic agent, on defibrillation energy requirements in dogs with healed myocardial infarctions.

UNLABELLED: Bidisomide is a Class Ia/Ib antiarrhythmic agent with activity against ventricular and supraventricular arrhythmias. The potential for bidisomide to increase defibrillation threshold (DFT) was tested in anesthetized dogs with healed left ventricular infarcts (> or = 10 days). Defibrillation patches were attached to each ventricle and shocks were delivered via an external cardioverter/defibrillator. Three groups were studied: placebo (saline), canine therapeutic bidisomide (TB, 2-5 micrograms/mL plasma concentration) and supratherapeutic bidisomide (STB, 6-14 micrograms/mL). Each animal received only one treatment. An abbreviated DFT curve was determined before and after treatment. Heart rate, blood pressure, PR, QRS, infarct size, and hematocrit were also measured before and after treatment. DFT was significantly increased (average +3 to +5 joules [J], P < 0.05) by TB and STB. TB (5/5) did not increase DFT beyond 40 J. In 6/7 experiments, STB did not increase DFT beyond 40 J. Placebo (n = 6) had no significant effect on DFT. Infarct size (mean = 11% of the left ventricle) was not significantly different between groups. Heart rate and QRS were not significantly altered but blood pressure was significantly decreased (16%-31% systolic, 29%-45% diastolic) and hematocrit was significantly increased (19% to 25%) in all groups. PR was significantly increased by STB only. CONCLUSION: therapeutic and supratherapeutic doses of bidisomide slightly but significantly increased DFT (3-5 J) in a canine infarcted heart model.

Simple in vitro method to characterize antiarrhythmic agents.

A simple, nonmicroelectrode method was developed for the in vitro identification and characterization of potential antiarrhythmic agents. To evaluate the method, standard antiarrhythmic agents from three different classifications (I, III, IV) were tested in isolated right ventricular guinea pig papillary muscles for their effect on developed tension (DT), excitability (EX), and effective refractory period (ERP). ERP was measured with the use of paired field stimuli. Depression or reversal of the force frequency relationship was an index of an agent's effect on DT. A shift in the stimulus strength-duration relationship was an index of an agent's effect on EX. A computer program was developed for data handling and analysis. Disopyramide phosphate (D, 3.0 x 10(-5) M), sotalol (S, 3.0 x 10(-5) M), clofilium phosphate (C, 1.0 x 10(-5) M), and N-acetyl procainamide hydrochloride (N, 3.0 x 10(-5) M) significantly prolonged ERP (+20, +35, +24, +16 ms, respectively), while verapamil (V, 3.0 x 10(-7) M) and the distilled water vehicle (W) did not. D and V significantly decreased DT (-78% and -57% at 1 Hz, respectively) while W, S, C, and N did not. Only D decreased EX. These data correspond well with findings in other models reported in the literature, supporting the use of this simple in vitro method for identification and characterization of potential antiarrhythmic agents.

The quantitative disposition of 3-O-methyl-(+)-[U-14C]catechin in man following oral administration.

Three young male volunteers excreted 51.5, 33.6 and 55.9% of a single 2 g oral dose of radiolabelled 3-O-methyl-(+)-catechin in urine within 120 h of administration. Excretion of the unchanged compound, however, accounted for only 0.7, 0.1 and 0.2% of the dose. The major urinary metabolites were glucuronides of 3,3'-O-dimethyl-(+)-catechin (15.8% dose) and a glucuronide and a sulphate of 3-O-methyl-(+)-catechin (11.4% and 10.6% dose, respectively). No evidence for demethylation was obtained. 3-O-Methyl-(+)-catechin was detected and measured in plasma by h.p.l.c. 0.5-12 h after administration. Peak levels (11-18 micrograms/ml) were attained within two hours of administration and the half-life of removal from plasma was approx. 140 min. Metabolism of 3-O-methyl-(+)-catechin in man was found to vary significantly from other species, as methylated metabolites (3'-O-methyl ethers) in man represented only about 53% of the urinary metabolites, whereas in the mouse, rat and marmoset, 3'-O-methylation was almost quantitative (Hackett and Griffiths 1981). The glucuronic acid conjugates of 3-O-methyl-(+)-catechin detected in man also differed from those previously reported in the rat. Additionally, sulphate conjugation of 3-O-methyl-(+)-catechin was observed in man although not in other species. 3-O-Methyl-(+)-catechin, unlike (+)-catechin, did not undergo ring fission.

The prevention by (+)-Cyanidanol-3 of hepatitis-induced changes in the disposition of imipramine in the rat.

The administration of (+)-Cyanidanol-3 [+)-catechin) to the rat using a subchronic dosing regime based on that currently used in the therapy of acute viral hepatitis in man, largely prevented the changes in the disposition of a single dose of [14C]imipramine hydrochloride induced by the hepatotoxin, D-(+)-galactosamine hydrochloride in rats. Complete return to normal pharmacokinetics was not attained due to interaction between (+)-Cyanidanol-3 and imipramine. Biliary excretion of imipramine metabolites was 79.3% of the dose in control rats. This was reduced to 69.3 and 39.8% by the separate administration of (+)-catechin and galactosamine respectively. Concurrent administration of (+)-Cyanidanol-3 and galactosamine resulted in 64.8% of the imipramine dose appearing in bile. These results were reflected in changes in faecal and renal excretion of imipramine metabolites in surgically unmodified rats in which galactosamine injection caused an elevation of urinary excretion from 31.0 to 69.8% of the imipramine dose. Concurrent Cyanidanol administration reduced the effect of galactosamine so that only 46.9% was excreted in urine. These changes were due to decreased biliary excretion and increased renal excretion of the glucuronide conjugates of 2-hydroxyimipramine, 2-hydroxydesmethylimipramine and 10-hydroxyimipramine. None of the treatments used impaired the overall ability of the rat to metabolize imipramine, although the plasma clearance of imipramine was reduced by 42% as a result of galactosamine administration and by 21% during treatment with (+)-catechin alone or combined catechin and galactosamine treatment.

The metabolism and excretion of [14C]imipramine in an experimental hepatitis.

The effect of experimental hepatitis, induced by i.p. D-galactosamine, on the metabolism and excretion of [14C]imipramine in the rat is reported. The major consequence was an increase of the conjugated metabolites of imipramine excreted in urine, resulting in a four-fold increase in 2-hydroxyimipramine glucuronide and two-fold increases in 2-hydroxydesmethylimipramine glucuronide and 10-hydroxyimipramine glucuronide. The total excretion of 14C-labelled metabolites in urine of galactosamine-treated rats was 69% dose compared with 37% in untreated animals. Faecal excretion of [14C]imipramine metabolites was lowered from 68% dose in untreated animals to 27% in animals with induced hepatitis. Induction of a galactosamine hepatitis decreased markedly the biliary excretion of imipramine metabolites in bile duct-cannulated rats; 80% dose was excreted in bile in normal rats, and 35.5% in rats with hepatitis. Plasma clearance of imipramine, after i.v. dosage, was decreased by 60% and clearance of metabolites (excluding imipramine) by 40%, in galactosamine hepatitis; the pharmacokinetics changed from a two- to a single-compartment system reflecting decreased extraction and/or metabolism, by the liver. The clinical implications of these findings are discussed in relation to the hazard attending the use of imipramine in patients suffering from liver disease.

The effects of an experimental hepatitis on the metabolic disposition of 3-o-(+)-[14C]methylcatechin in the rat.

The induction of an experimental hepatitis did not affect the overall ability of the rat to metabolize the flavanol 3-O-(+)-[14C]methylcatechin by methylation or glucuronidation. The induction of hepatitis did cause a significant increase in metabolite excretion in urine (from 52% of the dose in control rats to 88% in hepatitis). Fecal excretion was correspondingly depressed (44 to 4% of the dose). In bile duct-cannulated rats, the induction of hepatitis prior to 3-O-(+)-[14C]methylcatechin administration resulted in low 14C excretion (38%) in bile (cf. 58% in bile of controls). The data obtained indicate that following induction of hepatitis biliary metabolites reabsorbed from the intestine are not reexcreted in bile in an enterohepatic cycle as in the normal rat but are excreted via the kidney. Induction of hepatitis did not affect the fast clearance of unchanged 3-O-methyl-(+)-catechin from plasma but plasma clearance of the metabolites was reduced from 112 to 89 ml/hr.

The metabolism and excretion of (+)-[14C]cyanidanol-3 in man following oral administration.

Following administration of a single dose of [U14C]cyanidanol-3 to human volunteers, a mean of 55% of the dose of 14C was excreted in urine; 90% of urine 14C was excreted within 24 h of drug administration. The major urinary metabolites were the glucuronides of (+)-catechin and 3'-O-methyl-(+)-catechin, and the sulphate of the latter. These three conjugates collectively accounted for three quarters of urine 14C. Urinary excretion of unchanged (+)-cyanidanol-3 was 0.1-1.4% dose. (+)-Cyanidanol-3 and metabolites containing the intact flavanol ring system accounted for 90% of urine 14C. Ring scission was, under these conditions, a minor metabolic pathway resulting in the excretion of small amounts of 3-hydroxybenzoic acid, 3-hydroxyhippuric acid and 3-hydroxyphenylpropionic acid. Unchanged (+)-cyanidanol-3 was detected in plasma between 30 min and 12 h after administration. Metabolites (as total 14C) persisted in plasma for at least 120 h after administration.

The disposition of 3-O-methyl-(+)-catechin in the rat and the marmoset following oral administration.

The tissue distribution of an alkyl substituted flavanol, 3-0-methyl-(+)-catechin, has been investigated. Following oral administration, 3-0-methyl[14C]-(+)-catechin was well absorbed, peak levels of serum radioactivity for three dose levels being recorded within one hour of administration. Despite extensive absorption, it has been demonstrated that over the period 0-24 h after administration much of the radioactivity in the carcasses of rats was associated with the contents of the alimentary canal. This appears to be largely due to the enterohepatic circulation of the major metabolite 3,3'-0-dimethylcatechin glucuronide, since the present study indicates that some 60% of biliary excreted metabolites are reabsorbed in the first enterohepatic circulation. Metabolism of 3-0-methyl-(+)-catechin was rapid since the unchanged compound was detected in serum only at high dose levels and trace amounts only of 3-0-methyl-(+)-catechin were detected in intestinal contents 3 h after dosing. Radiometric examination of tissue samples following 3-0-[14C]methyl-(+)-catechin administration indicated that maximal tissue levels were observed at 30 min. After 6 h only trace amounts were detectable.

Metabolism and excretion of the liver-protective agent (+)-catechin in experimental hepatitis.

1. Following oral administration of [U-14C](+)-catechin to rats with galactosamine-hepatitis, the biliary and faecal elimination of (+)-catechin metabolites was decreased compared with that in normal animals, Renal excretion of (+)-catechin metabolites was enhanced in galactosamine-hepatitis. 2. Although the biliary metabolites were present in similar proportions in galactosamine-hepatitis animals and controls, the major urinary metabolite, 3'-O-methyl (+)-catechin sulphate, was markedly decreased whereas 3'-O-methyl(+)-catechin glucuronide was increased by over 100%. 3. The total overall excretion of 3'-O-methyl (+)-catechin conjugates in rats with galactosamine-hepatitis was similar to that in normal animals indicating that catechol-O-methyltransferase activity is not significantly depressed in galactosamine-hepatitis. 4. Clearance of radioactivity from the blood following i.v. administration of [U-14C]-(+)-catechin was prolonged in galactosamine-hepatitis. 5. Liver perfusion experiments demonstrated depressed glucuronylation of (+)-catechin metabolites in galactosamine-hepatitis, whereas in liver homogenates synthesis of glucuronide conjugates of (+)-catechin metabolites was enhanced. 6. Lung slices were able to metabolize (+)-catechin and the lung is proposed as an extrahepatic site of (+)-catechin metabolism of increased importance in galactosamine-hepatitis. 7. The effects of galactosamine-hepatitis upon the structure of the hepatocyte plasma membrane are discussed in relation to decreased biliary excretion and glucuronylation.

The metabolism and excretion of 3-palmitoyl-(+)-catechin in the rat.

1. Oral administration of 3-palmitoyl-(+)-[U-14C]catechin to rats resulted in the excretion of 63% of the dose in urine, 24% in faeces and 7% as 14CO2 in the 17 days following dosing. Persistent levels of 14C were noted in tissues, and 28 days after dosing 3.7% of the dose was present in the animal body. 2. Biliary excretion accounted for 28.2% of the dose in 48h after dosing. The major biliary metabolite was 3'-O-methyl-(+)-catechin glucuronide. These experiments also demonstrated the dependence of 3-palmitoyl-(+)-catechin on the presence of bile in the intestine for absorption. 3. Studies in vitro showed that intestinal micro-organisms were unable to metabolize 3-palmitoyl-(+)-catechin. The compound did, however, undergo deesterification in plasma and also in liver-perfusion studies. In the latter experiments, conjugates of the liberated (+)-catechin were also formed. 4. Urinary metabolites were predominantly (80%) conjugates of (+)-catechin and 3'-O-methyl-(+)-catechin. Ring scission products and 14CO2 were also excreted but not from rats with ligated bile ducts. These metabolites are therefore considered to arise from biliary metabolites not containing the ester linkage.

The metabolism and excretion of 3-O-methyl-(+)-catechin in the rat, mouse, and marmoset.

Following oral or intravenous administration to the rat, 3-O-methyl-(+)-catechin was metabolized by methylation of a B-ring phenolic hydroxyl group to 3,3'(or 4')-dimethyl-(+)-catechin, which was further conjugated with glucuronic acid and excreted both in urine and bile. Small amounts of unchanged 3-O-methyl-(+)-catechin was excreted in rat urine following parenteral but not oral administration. Excretion of radioactivity following parenteral administration of 3-O-[14C]methyl-(+)-catechin to the rat and marmoset was similar (approximately 50% in urine and 35% in feces). The mouse excreted 79% of radioactivity in urine and 22% in feces. Radioactivity in mouse and marmoset urine was associated with free dimethyl-(+)-catechin rather than the glucuronide conjugate prevalent in rat urine. Following oral administration to the marmoset, urinary excretion of 14C was lower than in the rat and again the major metabolite was the free dimethyl-(+)-catechin. In bile duct-cannulated rats approximately half of administered radioactivity was excreted in bile. In each experiment more than 85% of the 14C in bile was in the form of dimethylcatechin glucuronide. It is concluded that biological methylation is the major route of 3-O-methyl(+)-catechin metabolism in all species investigated, and that the compound does not undergo ring fission as has been reported in respect of the parent compound, (+)-catechin.

The bilary excretion of flavanones in the rat.

1. The major biliary metabolites of flavanones in the rat have been identified by chromatographic and spectral methods. 2. Evidence is presented that flavanones and flavanone glycosides, following oral or parenteral administration, undergo glucuronylation and are selectively excreted via the bile. 3. Flavanone glycosides but not unconjugated aglycones may be excreted to a significant extent in bile. 4. The percentage of parenterally administered flavanones excreted in bile varies inversely with the amount administered.

The metabolism and excretion of 7-mono-0-(beta-hydroxyethyl) rutoside in the dog.

Following i.v. administration of mono-HR to the beagle, plasma levels of both mono-HR and its glucuronide conjugates fell rapidly, neither being detectable 8 h after injection. Following oral administration of 14C-mono-HR, mono-HR-glucuronide was detected in plasma, confirming the absorption of mono-HR, and low levels of 14C were detectable up to 72 h after dosage. Following either oral or i.v. administration of mono-HR, the major route of excretion was fecal elimination of the compound as its aglycone form. Urinary excretion was slight being less than 15% following i.v. dosage and 4% following oral administration. Metabolism of mono-HR was confined to glucuronidation and hydrolytic cleavage of the glycoside side chain. Ring fission products of mono-HR were not detected.

Determination of individual hydroxyethyl rutosides in various animal body fluids by thin-layer chromatography and scanning densitometry.

A method is described for the accurate determination in plasma of the beta-hydroxyethylrutosides by measurement of the fluorescence of their borocitrate complexes by scanning densitometry following separation by thin-layer chromatography. A modification is also described for estimation of individual hydroxyethylrutosides and their glucuronide conjugates in samples of bile and urine.

Hepatic clearance and disposition of hydroxyethylrutosides.

Although salts of rutin on i-v admin. undergo insolubilization giving rise to concretions and suppurative inflammation in the liver this was not observed in respect of the vaso-active hydroxyethylrutosides (Pfeifer et al., 1970). I-v admin. of 3',4',7-tri0-(beta-hydroxy[14C]ethyl)-rutoside (tri-HR) and 7-mono0-(beta-hydroxy[14C]ethyl)rutoside (mono-HR) to mice showed rapid biliary excretion (approx. 71% within 24 h). Approx. 2/3 of the dose was subsequently excreted in faeces and ca. 25% in urine over 72 h. Autoradiography and scintilation counting showed short term concentration in the liver over the initial 4 h period but at 72 h less than 0.22% of tri-HR and 0.59% of mono-HR was detectable in hepatic tissue. Carcasses of mice killed at 72 h contained less than 7% of the initial dose which was mainly present in intestinal contents.

The disposition and metabolism of 3',4',7-tri-O-(beta-hydroxyethyl)rutoside and 7-mono-O-(beta-hydroxyethyl)rutoside in the mouse.

1. Following intravenous administration of 3',4',7-tri-O-(beta-hydroxy[14C2]ethyl)rutoside or 7-mono-O-(beta-hydroxy[14C2]ethyl)rutoside to male mice, 68% of the dose of each is excreted in faeces as the corresponding hydroxyethyl-quercetin within 72 h of dosage. Mean urinary excretions of mono- and tri-hydroxyethylrutosides in 72 h were 27 and 21% respectively. Unchanged rutosides and their glucuronides were detected in urine. 2. In biliary-cannulated animals, the mean biliary excretion of both tri- and mono-hydroxyethylrutosides was 71%, in 24 h of dosage. In both cases most 14C was excreted in 3 h, as unchanged rutosides and glucuronide conjugates. 3. Fall of blood 14C concn, was rapid for both compounds. Neither compound was detected in brain but there was short-term accumulation in liver and kidney, and 2--3 h after dosage, most 14C for both compounds was associated with the gastro-intestinal contents. 4. Animals killed 72 h after dosage of either compound contained less than 7% of dose, mostly in the colon and caecal contents. 5. Foetuses removed 3 h after dosage of either compound to the dams did not contain 14C; foetuses removed 5 min after dosage contained low levels of 14C, substantially below the maternal blood level and equiv. to less than 0-1% of dose in each case. No 14C was detected in amniotic fluid.