Metabolism of thymoxamine: identification of metabolites in rat.

Thymoxamine hydrochloride administered by mouth to rats at 25 or 100 mg kg-1 was excreted in the urine as the deacetyl and N-demethyl-deacetyl metabolites. These were completely sulpho- and glucuronoconjugated at 25 mg kg-1 but only partially so at the higher dose. Thymoxamine deacetylation in vitro is catalysed by plasma and hepatic cytosol esterases and the deacetyl metabolite undergoes N-demethylation catalysed by the cytochrome P 450 hepatic microsome mixed function monooxygenase system. Because of the rapidity of the deacetylation it is concluded that thymoxamine is a prodrug leading in vivo to the active deacetyl thymoxamine.

In vitro and in vivo alpha-blocking activity of thymoxamine and its two metabolites.

The alpha-adrenoceptor potency of thymoxamine and its two metabolites deacetylthymoxamine and demethyldeacetylthymoxamine were determined on the contraction of rat vas deferens induced by noradrenaline, the blood pressure increase induced by noradrenaline given i.v. to dogs and the contraction of the nictitating membrane induced by electrical stimulation in cats. In vivo the three drugs were administered at 6.35 x 10(-6) mol kg-1 intravenously. Deacetylthymoxamine presented nearly the same alpha-blocking activity as the parent drug. This was ascribed in vivo to the rapid deacetylation of thymoxamine. Demethyldeacetylthymoxamine was less active. In vitro its pA2 was 6.20 +/- 0.09 compared with 6.75 +/- 0.20 for thymoxamine and 6.57 +/- 0.13 for deacetylthymoxamine. In vivo, it was inactive in dog and less active than the other two drugs soon after its administration in the cat. The oral LD 50 values in the mouse for the three drugs were respectively 0.81, 0.71 and 1.14 mmol kg-1 for thymoxamine, deacetylthymoxamine and demethyldeacetylthymoxamine.

High performance liquid chromatography coupled with radioactivity detection: a powerful tool for determining drug metabolite profiles in biological fluids.

High performance liquid chromatography coupled with continuous radioactivity detection represents an advancement in drug metabolism research. Using radioactive substances labelled in biologically stable positions, all metabolites can be specifically detected by radioactivity measurement. Thus no clean-up of biological fluids is required prior to HPLC. This can prevent artefact formation from unstable metabolites, reduces recovery problems and facilitates quantitation. Separation of highly polar and unpolar metabolites is possible in a single chromatographic run using gradient elution and reversed phase materials. This technique is also well-suited for preparative isolation and purification of metabolites for subsequent structure elucidation. Various metabolite profiles of drugs labelled with carbon-14 or tritium are shown. Metabolites of the following drugs are presented: norfenefrine, etozolin, thymoxamine, naloxone, and levobunolol. We review the general methodology and report our experience with this technique. In principle, this technique may be useful for all biological systems in which tracer techniques are applied.

Metabolism of thymoxamine. III. Structure elucidation of the metabolites and interspecies comparison.

The structures of six metabolites were elucidated using rat urine after intragastric administration of 14C-thymoxamine by means of enzyme incubations, mass spectrometry and synthesis of metabolites: desacetylthymoxamine, N-demethyl-desacetylthymoxamine, the corresponding sulfates and glucuronides. The nature of the conjugates was confirmed by biosynthesis, i.e., co-administration of unlabelled thymoxamine and 35S-sulfate or 14C-glucose. The system high performance liquid chromatography-radioactivity detection was used for interspecies comparison. All biotransformation pathways are seen in rat and man. In dog and cat demethylation is a very minor reaction. Glucuronidation is not observed in the cat.

Metabolism of thymoxamine. II. Studies with 14C-thymoxamine in man.

Thymoxamine is rapidly and completely absorbed in man. Rapid biotransformation is observed after intravenous and oral administration of 40 mg 14C-thymoxamine HCl. No unchanged compound is found in the body. More than 90% of plasma and urine radioactivity could be ascribed to six metabolites: the desacetyl compound (metabolite I), the monodemethylated metabolite I (metabolite II), the sulfate conjugates of I and II (metabolites III and IV) and the glucuronides of I and II (metabolites V and VI). The unconjugated metabolites are observed in plasma only after intravenous administration. Similar patterns for polar metabolites are found in plasma and urine for both routes of administration. The sulfate fraction amounts to about 50-60% and the glucuronide fraction to about 30-40% of the radioactivity, the conjugates of metabolite I being more abundant than those of metabolite II. The elimination of the metabolites is rapid, the half-life of radioactivity elimination being 1.5 h during the first 12 hours and 12 h thereafter. 80% of the radioactivity dose is recovered in the urine within 4 hours. Recovery after four days amounts to 99.8% (i.v.) and 97.7% (oral). The results are discussed with regard to the application of the drug in man, taking into account that not only the unconjugated metabolites but also the sulfate conjugates are pharmacologically active.

Metabolism of thymoxamine. I. Studies with 14C-thymoxamine in rats.

Thymoxamine is rapidly and completely absorbed in rats. It is a prodrug which does not enter the systemic circulation in its unchanged form. After either oral or intravenous administration it undergoes rapid and intense metabolism involving four biotransformation reactions: Enzymatic hydrolysis to the corresponding phenol (metabolite I), Monodemethylation to metabolite II, Sulfate conjugation of I and II (metabolites III and IV) and Conjugation of I and II with glucuronic acid (metabolites V and VI). With these 6 metabolites identified approximately 95% of the radioactivity can be accounted for in plasma, urine and bile. Whereas the systemic availability of I and II is low, III and IV show high bioavailability. Metabolites I to IV are pharmacologically active, while III and IV are less potent than I and II. The radioactivity distribution in tissues is different after oral and intravenous administration consistent with the higher portion of unconjugated metabolites in the body after administration by parenteral route. Although 60% of the labelled compounds is eliminated via bile, the radioactive compounds are almost completely excreted in the urine after both routes of administration. This demonstrates complete reabsorption of the biliary metabolites. Secondary peaks of radioactivity in plasma and organs at 4 hours are explained by the participation of the metabolites in the enterohepatic circulation.

Metabolism of 14C-thymoxamine in rat and man.

1. After oral administration of 14C-thymoxamine to rat and man the total 14C excreted in urine and faeces was determined. 2. Six metabolites were isolated from the excret of man and rat by chemical extraction and identified by g.l.c.-mass spectral analyses. 3. Two other metabolites, highly polar and resistant to enzymic hydrolysis, were isolated by extraction on XAD2 resin and h.p.l.c. analysis. These two metabolites were identified by n.m.r. and by mass spectrometry in the fast atomic bombardment mode. 4. These two major metabolites of thymoxamine in man and rat have been identified and characterized as the sulphate conjugates of desacetyl-thymoxamine and N-monodesmethyl-desacetyl-thymoxamine.

Microbial models of mammalian metabolism. Fungal metabolism of phenolic and nonphenolic p-cymene-related drugs and prodrugs. I. Metabolites of thymoxamine.

This study was undertaken to validate the use of microbial biotransformation systems for drug metabolism studies. Thymoxamine 1 was rapidly hydrolyzed to desacetylthymoxamine (DAT) 2 by numerous fungi. Other known animal metabolites, such as N-desmethyl-desacetylthymoxamine 3 and desacetylthymoxamine-O-sulfate 6, were produced from DAT by Mucor rouxii and Mortierella isabellina. DAT-N-oxide 5, a putative animal microsomal metabolite, was also produced by M. isabellina, in addition, a few strains (such as Actinomucor elegans, Mucor hiemalis, and Mucor janssenii) produced a glycosylated metabolite that was identified by high-resolution 1H- and 13C-NMR, MS, and enzymatic hydrolysis as the corresponding [4-(2-dimethylaminoethoxy)-5-isopropyl-2-methyl-phenyl]-1-beta-D- glucopyranoside 7. A similar glucosylation reaction was observed when thymohydroquinone 10 was incubated with A. elegans. Several strains were able to produce transiently thymohydroquinone from DAT-N-oxide 5, possibly through a beta-elimination mechanism.

Plasma induced biotransformation of thymoxamine and its kinetics.

Thymoxamine, 6-acetoxythymol-2-dimethylaminoethyl ether, is a competitive alpha-adrenoceptor blocking agent used as a vasodilator in the treatment of peripheral vascular disorders. The plasma half-life of the drug in vivo is very short. In vitro experiments showed that thymoxamine had a half-life in human plasma of about only 1 min. By using the compound as a substrate in increasing concentrations from 5-320 microgram/ml in the presence of a fixed, low concentration of plasma (0.1% v/v) at pH 7.4 and 37 degrees a hyperbolic correlation was found between the concentration of thymoxamine and the initial rate of formation of the involved reaction product, which was determined by HPLC. A linearized Michaelis-Menten plot indicated an enzymatic biotransformation. A Km-value of about 0.115 mumol ml-1 and a Vm-value of about 0.0037 mumol ml-1 min.-1 were found. Physostigmine, neostigmine and cinchocaine inhibited the biotransformation competitively. This indicated that plasma cholinesterase was the enzyme responsible. The metabolite was identified as desacetylthymoxamine by HPLC, spectrophotometry and direct inlet mass spectrometry.

Moxisylyte plasma kinetics in humans after intracavernous administration.

Obtaining and sustaining an erection are common problems for the male spinal cord injury patient. Intracavernous injection of vasoactive substances offers a new treatment option but it must be approached with caution in this population. In this work, the use of an alpha-adrenergic blocking agent, moxisylyte, after intracavernous administration for complete paraplegic patients with erectile impotence is described. During this study, the pharmacokinetic profile of moxisylyte has been defined. Unchanged moxisylyte is not found in plasma, this drug is immediately metabolized after administration. Three metabolites were found in plasma: desacetylmoxisylyte (DAM), conjugated DAM, and conjugates of desmethylated DAM (MDAM). Maximum plasma levels of 72.3 ng ml-1, 301.4 ng ml-1, and 88.8 ng ml-1 are obtained 0.22 h, 0.9 h, and 2.08 h after drug administration for these three metabolites, respectively. The elimination half-lives are 0.89 h, 2.16 h, and 5.32 h and the MRT, 1.38 h, 3.23 h, and 8.45 h, respectively. No side-effects were noted, only one patient presented sleepiness. Successful erections (10 to 25 min) were obtained in all patients and no priapism was noted.

Metabolism of 14C-moxisylyte after percutaneous application in hairless rat.

The pharmacokinetics of 14C-thymoxamine (4-(2-dimethylaminoethoxy)-5-isopropyl-2-methyl phenyl acetate, CAS 54-32-0, moxisylyte, Carlytène) were studied in female hairless rats following different administration routes: oral, intravenous or percutaneous. After percutaneous administration, the half-life of elimination of 14C-thymoxamine and its metabolites was longer (t 1/2 = 15 h) than after oral or intravenous administration (t 1/2 = 9 h). The penetration/resorption phenomenon of thymoxamine base mainly located in the horny layer could explain the high value of the pseudo half-life of elimination observed after percutaneous administration. Due to the absorption slower than elimination, this special pharmacokinetics had to be considered as a flip-flop model. The type and proportions of thymoxamine metabolites recovered in plasma varied according to the route of administration. The unconjugated metabolites, desacetyl-thymoxamine (DAT) + desacetyl-desmethyl-thymoxamine (DMAT), were observed only after intravenous or percutaneous administration, they represented 12% and 15%, respectively. They were never observed after oral administration suggesting the existence of a hepatic first-pass metabolism. The other metabolites observed were sulphate conjugates and glucuronides of DAT + DMAT. The values of sulfoconjugates were constant with each administration route (21%), whereas glucuronides increased with oral administration. In conclusion, the pharmacokinetics of percutaneous thymoxamine presented two main features: the drug absorption was high and durable (t 1/2 = 15 h); the cutaneous application allowed to avoid the hepatic first-pass metabolism.

Antinoradrenergic activity of thymoxamine and its metabolites in rats.

The alpha-adrenoceptor blocking activity of thymoxamine and its two metabolites deacetylthymoxamine and N-demethyldeacetylthymoxamine was determined on the norepinephrine-induced contraction of rat vas deferens and thoracic aorta and the blood pressure increase induced by intravenously administered norepinephrine in anesthetized normotensive and pithed rats. In isolated rat vas deferens, the three drugs act as competitive antagonists (thymoxamine pA2 = 6.75;l deacetylthymoxamine pA2 = 6.57; N-demethyldeacetylthymoxamine pA2 = 6.20). At the level of the thoracic aorta, the three drugs act as antagonists with a dual mode of action (thymoxamine pA2 = 6.55--pD2' = 4.70; deacetylthymoxamine pA2 = 6.53--pD2' = 4.70; N-demethyldeacetylthymoxamine pA2 = 5.61--pD2' = 4.63). In anesthetized normotensive or pithed rats, thymoxamine and deacetylthymoxamine produce an antagonism of the hypertensive response to norepinephrine. Thymoxamine and deacetylthymoxamine present nearly the same alpha-blocking activity in vitro and in vivo. N-demethyldeacetylthymoxamine is less active.

Design and evaluation of nitrosylated alpha-adrenergic receptor antagonists as potential agents for the treatment of impotence.

We designed and evaluated a new class of molecules, nitrosylated alpha-adrenergic receptor antagonists, as potential agents for the treatment of impotence. In in vitro studies with human and rabbit corpus cavernosum strips in organ chambers, the alpha-adrenergic receptor antagonists (alpha-ARAs) moxisylyte and yohimbine and their corresponding nitrosylated compounds, SNO-moxisylyte (NMI-221) and SNO-yohimbine (NMI-187), concentration-dependently relaxed endothelin-induced contraction. The nitrosylated compounds were significantly more potent than the parent alpha-ARA. In human tissues, the specific phosphodiesterase type 5 inhibitor zaprinast potentiated the relaxing effects of the nitrosylated compounds. Only nitrosylated compounds induced accumulation of cyclic GMP in rabbit corpus cavernosum strips. Yohimbine and NMI-187 demonstrated a potent alpha2-blocking activity, with no significant differences in pA2 values (8.9 versus 8.2, respectively). Moxisylyte and NMI-221 showed moderate potency in antagonizing phenylephrine contraction, with comparable pA2 values for both molecules (6.5 versus 6.6, respectively). alpha-Adrenergic receptor-binding studies showed similar binding affinities for the alpha-ARA and their corresponding nitrosylated compounds. In vivo, intracavernosal injection of nitrosylated molecules caused greater increases in intracavernosal pressure (NMI-221 versus moxisylyte) that were more long lasting than those of moxisylyte or yohimbine. There were no significant differences between nitrosylated and non-nitrosylated compounds in the magnitude of systemic mean arterial pressure decrease after intracavernosal injection. alpha-ARA and the nitrosylated compounds showed no pain-inducing activity as evaluated with the paw-lick model in mice. In summary, nitrosylated alpha-ARA have the dual functionalities of nitric oxide donors and alpha-ARA. These drugs induced penile erection in animals, suggesting their possible therapeutic value as agents for the local pharmacological treatment of impotence.