A phased approach to drug development.

It is possible in the UK to carry out a phased approach to drug development that is designed to assist in the selection of a candidate drug for development from a series of compounds; minimize the amount of animal work (toxicological and metabolic) necessary to permit early evaluation in man; introduce a more rational basis for decision making by setting criteria that must be satisfied before entry can be made into the next phase of development; obtain safety, pharmacokinetic, and possibly dynamic data in man within about 16 months of receipt of the compound.

The metabolism of [14C]N-ethoxycarbonyl-3-morpholinosydnonimine (molsidomine) in man.

[14C]-N-Ethoxycarbonyl-3-morpholinosydnonimine (molsidomine, Corvaton) was administered orally at a dose of 2 mg per subject to eight healthy male volunteers. Maximal plasma concentrations of total radioactivity of 32.4 +/- 6.4 ng equiv./ml (mean +/- S.D.) were detected compared with maximum plasma concentrations of 14.1 +/- 5.9 ng/ml (mean +/- S.D.) of molsidomine. In both cases these were attained at 0.5 h after dosing. From the peak, concentrations of parent drug fell rapidly with a half-life of 1.25 +/- 0.38 h (mean +/- S.D.). In contrast, total radioactivity declined more slowly with a terminal half-life of 138 +/- 42.7 h (mean +/- S.D.). The bulk of the radiolabel was rapidly excreted as metabolites in the urine, with over 85% of the dose recovered in the first 24 h. The main urinary radiolabelled metabolites appeared, from chromatographic evidence, to be similar to those previously identified in animals, namely N-morpholinosydnonimine, N-cyanomethylamino-N-(2'-hydroxyethyl)glycine and (N-cyanomethylenamino-2-aminoethoxy)-acetic acid.

The metabolism of [14C]N-ethoxycarbonyl-3-morpholinosydnonimine (molsidomine) in laboratory animals.

[14C]N-Ethoxycarbonyl-3-morpholinosydnonimine (molsidomine, Corvaton) was found to be extensively metabolized following oral dosing to rat and dog and intravenous dosing to rabbit. The majority of the radiolabel was rapidly excreted in the urine with the main radiolabelled components being characterized as acidic metabolites resulting from oxidative metabolism of the morpholine ring. A new metabolite, (N-cyanomethylenamino-2-aminoethoxy)-acetic acid, was identified and shown to be a major component of the 14C-labelled urinary metabolites in all three species. However, the previously identified metabolite, N-cyanomethylenaminomorpholine-2-one (compound D) was not detected and may therefore have been formed artefactually in the earlier studies. The long terminal half-life for plasma radioactivity observed in previous studies was shown to be the result of the production of small amounts of 14C-thiocyanate from the nitrile-containing metabolites of molsidomine.

Disposition of cefotaxime in rat, dog and man.

The excretory pathway for the elimination of 14C-cefotaxime (14C-HR 756) was found to be the same for rat, dog and man with elimination into the urine being the most important route, accounting for greater than 80% of the dosed radioactivity. The amounts of unchanged cefotaxime eliminated in the urine ranged from 20-32% in rat and dog to 56% in man. The major metabolite in each species was the microbiologically active desacetyl cefotaxime, which was present in both plasma and urine. Two further metabolites, recently identified as the stereoisomeric forms of the opened beta-lactam ring form of desacetyl cefotaxime lactone were also found in the urine of dog and man.

Whole body autoradiographic and quantitative tissue distribution studies with 14C-cefotaxime in the rat.

The absorption, distribution and elimination of radioactivity following intravenous (i.v.) or intramuscular (i.m.) administration of 14C-cefotaxime (14C-HR 756) to the rat has been examined by qualitative and quantitative techniques. After i.v. and i.m. doses to male albino animals radioactivity was extensively distributed throughout the body and rapidly eliminated with a predominant half-life of approximately 30 to 40 min. Maximum plasma levels for the i.m. dose were reached within 20 min and approximately 85% of the dose was recovered from the urine (74%) and faeces (11%) within 8 h after dosing. In all quantitative studies 100 +/- 5% of the dose was recovered within 24 h. Whole body autoradiography studies showed good distribution of radioactivity from the blood into the tissues including lung, liver, kidney, heart, bone marrow and the gastrointestinal tract. Lowest levels were seen in the eye and brain. There was limited placental transfer of radioactivity in 14-day pregnant animals although by day 18 of the gestation period radioactivity was detected in the foetus but distribution into individual organs and tissues could not be seen. There was no evidence to show that retention of radioactivity in pigmented tissues had occurred nor was there any suggestion of accumulation of radioactivity in any organ or tissue as a consequence of multiple dosing with 14C-cefotaxime.

Cefotaxime: sites and pathway of metabolic conversion.

This paper examines the metabolism of 14C-cefotaxime (14C-HR 756) in the rat and attempts to identify the metabolic events in this and other species. In rat desacetyl cefotaxime is usually the major excretion product and sole metabolite of cefotaxime. However, when renal elimination of this metabolite is prevented by bilateral nephrectomy, two further metabolites, the stereoisomeric opened beta-lactam ring forms of desacetyl cefotaxime lactone, are also produced. These metabolites are found in the urine of man and dogs dosed with cefotaxime. It is suggested that generation of these metabolites is dependent on formation of the lactone form of the desacetyl metabolite. Evidence is also presented showing that these metabolic conversions occur in the liver.

Metabolism of loprazolam in rat, dog and man in vivo.

The metabolism of 14C-loprazolam has been studied in rat, dog and man in vivo. In rat, the major metabolic pathways were hydroxylation on the benzodiazepine ring, and reduction and acetylation of the nitro group. Both metabolites were identified by co-chromatography with standards, and were present in urine and bile conjugated with glucuronic acid. In both dog and human urine and bile significant amounts of the piperazine-N-oxide were found. This N-oxide was identified by co-chromatography with authentic compound and by mass spectroscopy. Both loprazolam and the dog biliary metabolites were hydrolysed spontaneously to polar material. Neither treatment with beta-glucuronidase nor incubation with gut microflora had any further effect. Only polar metabolites were found in dog and human faeces. The principal non-polar material found in rat plasma was the diazepine-hydroxy compound, and little loprazolam was present. Significant levels of loprazolam and lower levels of an unidentified metabolite were found in ether extracts of dog and human plasma. Both the piperazine-N-oxide and loprazolam were found in similar quantities in chloroform extracts of human plasma, and at two hours after dosage, the N-oxide and loprazolam accounted for greater than 90% of the radioactivity present in the plasma.

Disposition of 14C-loprazolam in animals and man.

The disposition of 14C-loprazolam has been studied in rat, dog, cynomolgus monkey and man using oral and parenteral dosing. In all species 14C was excreted principally in the faeces irrespective of the route of administration. In surgically prepared animals, 46% dose (rat) or 60% (dog) was excreted in bile and, together with urinary excretion, indicates that approx. two-thirds of an oral dose was absorbed. In rat there was relatively little enterohepatic circulation (approximately 26%) compared to dog (approximately 73%). Whole-body autoradiography and tissue-distribution studies in rat showed that 14C was distributed principally in liver and intestine, and was eliminated within 24 h. 14C was found in brain of rat and dog; in dog concn. levels were higher in white matter than in grey matter. In studies using pigmented animals, 14C was associated with the uveal tract of the eye and with other melanin-containing tissues. This was reversible and was eliminated from the eye of rat with a half-life of 3.4 d. Blood and plasma concn. of 14C and of unchanged loprazolam declined relatively rapidly in rat. In dog, cynomolgus monkey and man, total 14C concn. in blood fell more slowly than unchanged loprazolam. Less than 85% of loprazolam was protein bound in rat, dog or human plasma in vitro.

The metabolism of 7-ethoxycoumarin and 7-hydroxycoumarin by rat and hairless mouse skin strips.

The metabolism of 7-ethoxycoumarin and 7-hydroxycoumarin was studied in rat and hairless mouse skin strips. These preparations supported de-ethylation, sulphation and glucuronidation reactions. The de-ethylation reaction was inducible in both species by pretreatment with either 5,6-benzoflavone or 3-methylcholanthrene. The hairless mouse strips exhibited a greater basal de-ethylase activity than rat strips, although the latter was the more responsive to inducers. Accompanying the increase in de-ethylation activity was a change in the pattern of metabolites, with a large increase in the percentage of the unconjugated metabolite. When 7-hydroxycoumarin was employed as the primary substrate the glucuronide was the major metabolite formed by strips from both species. The glucuronidation and sulphation activities were unchanged by 3-methylcholanthrene pretreatment.

Cytochrome P-450 dependent deethylase activity in rat and hairless mouse skin microsomes.

Microsomal fractions were prepared from rat and hairless mouse skin. The method of preparation was validated by studying the distribution of succinate dehydrogenase, acid phosphatase and UDP-glucuronosyltransferase. Induction of oxidative deethylation activities by 5,6-benzoflavone and 3-methylcholanthrene was investigated. Preparations from hairless mouse skin exhibited higher basal activities but the enzymes were less responsive than those of rat skin to inducers. Species differences were observed in the extent of induction between topical and i.p. administration of 5,6-benzoflavone, the former route being more effective in the hairless mouse and the latter route most effective in the rat. Generally oxidative deethylation activity increased linearly with protein concn up to 2-3 mg protein/ml. The only exception was rat skin microsomes prepared from animals pretreated with 5,6-benzoflavone, where linearity was observed only to 0.75 mg protein/ml above which oxidative deethylation activity decreased with increasing protein concn. The inhibition of 7-ethoxycoumarin deethylase by various compounds was investigated; the activity in hairless mouse skin exhibited a greater sensitivity to water-soluble solvents than that in rat skin microsomes. Both hairless mouse and rat skin 7-ethoxycoumarin deethylase were sensitive to inhibition by 5,6-benzoflavone, 7,8-benzoflavone and metyrapone.

UDP-glucuronosyltransferase activity in rat-and hairless mouse skin-microsomes.

1. Cutaneous UDP-glucuronosyltransferase activity (E.C.2.4.1.17) was demonstrated in rat- and hairless mouse-skin microsomes using 1-naphthol as substrate. 2. Addition of the detergent Brij 35 increased the activity by approximately twofold in both species. 3. Inhibitor studies demonstrated that under the assay conditions used any UDP-glucuronic acid pyrophosphatase or beta-glucuronidase present did not interfere with the conjugation reaction. 4. Substrate inhibition was observed in hairless mouse-skin preparations and biphasic response to increasing naphthol concentration was seen in rat-skin microsomes. 5. The apparent Km values were considerably lower than those reported for liver. The sp. activity (per mg microsomal protein) in unactivated rat-skin microsomes was about 50% of that reported in unactivated rat-liver microsomes. 6. Pretreatment with 3-methylcholanthrene resulted in a small increase in cutaneous UDP-glucuronosyltransferase activities in both species.

The disposition and metabolism of 5-(4,5-dihydro-2-phenylbenz[e]indol-3-yl)salicylic acid (fendosal) in the rat, mouse, rabbit, do, rhesus monkey, and man.

The disposition and metabolism of 5-(4,5-dihydro-2-phenylbenz[e]indol-3-yl)salicylic acid (fendosal) a new salicylate-type analgesic, has been studied in the rat, mouse, rabbit, dog, rhesus monkey, and man. Animals were given single oral or parenteral doses at levels of 5, 10, or 50 mg/kg; human volunteers received 200 mg orally. In all species, virtually all radioactivity was excreted in the feces. Biliary excretion accounted for approximately 50% of an oral dose in the rat and dog. Enterohepatic circulation was demonstrated in the rat. The compound was fairly rapidly absorbed in all species except the rhesus monkey. The principal excretion products found in all species were unchanged fendosal and a monohydroxylated metabolite, the latter being present both in the free state and as a glucuronide. A minor metabolite, present only in man and rhesus monkey, was tentatively identified as a dihydroxylated metabolite. These compounds were, however, detected only in unpurified samples. During the isolation and purification procedure, oxidation occurred, resulting in the production of the corresponding dehydrogenated derivatives, which were the actual materials whose structures were elucidated.

Absorption and disposition of [14C]-molsidomine in laboratory animals.

14C-Labelled N-ethoxycarbonyl-3-morpholinosyndnonomine(14C-molsidomine, Corvaton) was administered orally to mouse, rat, rabbit, dog and rhesus monkey, and i.v. to rat and dog, at a dose level of 6 mg/kg-1. The rates and routes of excretion of radioactivity were determined. The oral dose was well-absorbed in all species and most (greater than 75%) of the radioactive dose was excreted in urine. In rat and dog less than 1% of the dose was present as expired 14CO2. In dog and rhesus monkey, small amounts of radioactivity were eliminated relatively slowly. In rat, dog and rhesus monkey, the three species examined in detail, radioactivity was generally distributed throughout the body 4-10 days after dosing. Levels were highest in liver, pelt, blood and the gastrointestinal tract. Whole-body autoradiographic studies in rat and rhesus monkey showed that at 4 days post-dose, radioactivity was highest in stomach wall and was distributed into other tissues in lesser amounts. In view of its mode of action, it was interesting that radioactivity was also associated with the heart muscle and aorta walls.

Species differences in the disposition and metabolism of 6,11-dihydro-11-oxodibenz[be]oxepin-2-acetic acid (isoxepac) in rat, rabbit, dog, rhesus monkey, and man.

The disposition and metabolism of 6,11-dihydro-11-oxodibenz[be]oxepin-2-acetic acid (isoxepac), a new nonsteroidal anti-inflammatory agent, has been studied in rat, rabbit, dog, rhesus monkey, and man. Animals were given single oral or parenteral doses of 5 or 50 mg/kg; man received approximately 3 mg/kg orally. Fecal excretion of radioactivity occurred in the rat (26--37%) and dog (33--49%), whereas in the other species elimination was mainly urinary (less than 83%). Biliary excretion accounted for 18--52% of the dose in the rat and dog. Enterohepatic circulation was demonstrated in both species. Plasma of all species was found to contain mainly unchanged isoxepac. The compound was rapidly eliminated from plasma of dog, rhesus monkey and man, but was more slowly eliminated in rat and rabbit. In the rabbit and dog the principal metabolites were the glycine and taurine conjugates of isoxepac, respectively, whereas in the rhesus monkey and man, isoxepac was excreted unchanged or as the glucuronide.

Further absorption studies with the anti-diarrhoeal agent ethacridine lactate in the dog.

2-Ethoxy-6,9-diaminoacridine lactate (ethacridine lactate, Rivanol, Metifex) has been administered orally to the dog once daily for 14 days, tritium labelled matterial having been given on days 1 and 14. The extent and rates of urinary excretion of radioactivity and the peak plasma levels and total radioactivity half-lives following the radiolabelled doses on days 1 and 14 were essentially the same. There was no significant change following multiple dosing in the level of urinary acridine-like material as determined fluorimetrically, which compared to approximately 0.01% of the dose found in the 0--24 h urine. It was concluded that, following oral administration of 3H-ethacridine lactate (5 mg/kg), less than 0.1% of the dose is absorbed as acridine-like material. Multiple dosing for 14 days does not alter this very low degree of oral absorption. In a separate study tritiated ethacridine lactate (30 microgram/kg) was administered i.v. to the dog. Approximately 84% of the radioactivity was eliminated in the 0--72 h post dose period, the majority of it being excreted via the faeces. There was a rapid loss of radioactivity from the plasma, followed by a long terminal phase in which acridine-like material was estimated to have a half-life of about 15 h.

Absorption studies with the anti-diarrhoeal agent ethacridine lactate in laboratory animals and man.

Tritiated 2-ethoxy-6,9-diaminoacridine (ethacridine lactate, Rivanol) has been orally administered to rat, dog, rabbit and man, and the distribution of radioactivity between urine and faeces determined. Complete recoveries of radioactivity were obtained from rat, rabbit and man. From direct measurement of levels of radioactivity, the maximum percentage of the dose excreted via the urine varied from 1.7% (man) to 6.1% (dog). Tritiated water accounted for nearly 50% of this amount. The low levels of radioactivity in plasma of dog and man and in the bile of rats following administration of 3H-ethacridine lactate suggested that there was only a low degree of oral absorption and substantiated previous reports that the drug is essentially unabsorbed in man. From fluorimetric measurements and the determination of levels of radioactivity in ether extracts of urine, it could be concluded that for all species less than 0.1% of the dose appeared in the urine as acridine-like material.