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It is almost impossible to design a bioavailable, long half-life and safe drug without a good understanding of both the principles of, and specific risks associated with, drug metabolism. Designing potent compounds with limited metabolic liabilities has proven to be one of the key challenges of drug discovery. The reasons for this, and some potential solutions, are explored in this chapter. The issues described also include the propensity for rapid metabolism, the risks associated with reactive metabolites and the origins of metabolically derived drug–drug interactions. The factors influencing the metabolism of drugs, the challenges of adopting prodrug strategies and exploiting active metabolites are also discussed, as are the complexities of metabolism–transport interplay. The chapter aims to provide a good understanding of drug metabolism and its relevance and value in support of drug research and development.

Drug metabolism is the chemical alteration of a drug via a biological system to aid its elimination by increasing its hydrophilicity. Drug metabolism occurs in two phases: Phase 1 includes oxidative reactions such as those undertaken by the cytochrome P450s, but also includes reduction and hydrolysis. Phase II reactions involve conjugations with endogenous molecules such as glucuronic acid, sulphate, amino acids and glutathione. Most drug metabolism occurs in the liver but can take place in other tissues including the gastrointestinal tract, kidney and lung. Drug metabolism is studied in vitro using enzyme systems prepared from liver and in vivo throughout the drug discovery and development process. LC-MS/MS, and where appropriate radiolabeled drugs are used in these studies. For example, it is important to avoid extensive first pass metabolism for early drug selection but also to ensure all human metabolites are qualified in toxicology studies according to the MIST guidelines for product registration. Some metabolites may be active, with the same or different pharmacology, and/or toxic, and in some cases reactive metabolites cause idiosyncratic toxicity. Thus metabolite identification and the relationship to efficacy and toxicity need to be understood as quickly as possible. Since drug metabolism is mediated by enzymes, there is potential for natural variability and for co-administered drugs to interact, either by enzyme inhibition or induction. Methods to investigate this and transporter interactions are, therefore, discussed with respect to drug discovery decisions and Regulatory Guidelines. The chapter provides an overview of all these aspects of drug metabolism and its relevance and value in support of drug R and D.

The pharmacokinetics and metabolism of intravenously infused 14C-fotemustine (about 100 mg/m2) were examined in 2 cancer patients. Plasma levels of radioactivity increased to a maximum of 4.1 and 5.5 micrograms equivalents per g when the infusion stopped then declined triexponentially with mean half-lives of about 1/2, 10 and 80 h for the initial, mid and terminal phases, respectively. Plasma levels of intact drug were lower, with maximum levels of 1.1 and 2.8 micrograms/ml, and declined monophasically with a half-life of about 24 min. Plasma clearance was high (1426 and 764 ml/min) with the volume of distribution based on areas of 47.7 and 26.4 l. Most of the radioactivity was eliminated in urine (50.1 and 61.3%) over 7 days with smaller amounts in the feces (6.8 and 0.3%) and only minimal quantities (under 0.1%) as expired carbon dioxide. Metabolites of fotemustine were identified as chloroethanol and N-nitroso-1-imidazolone-ethyl-diethylphosphonate in plasma and as 1-hydantoin-ethyl-diethyl-phosphonate and acetic acid in urine.

1. The comparative metabolism of fenfluramine was investigated in mouse, rat, dog and man following a single oral dose of 14C-(+/-)-fenfluramine hydrochloride (1 mg/kg), and also in rat after eight consecutive 12-h subcutaneous doses (24 mg/kg). 2. Main route of excretion of radioactivity in all species and at all doses was into urine (80%), with only minor amounts of radioactivity found in faeces. 3. From all species examined a total of 11 metabolites were observed in urine and plasma by t.l.c. and h.p.l.c. analysis and no metabolite was present in the plasma which was not present in urine. 4. All species dealkylate fenfluramine to the active metabolite norfenfluramine, to a relative greater or lesser extent, with plasma metabolic ratios (norfenfluramine/fenfluramine) showing inter-animal variation (ratdogmouse = man). 5. These differences are due to the efficient deamination of both compounds to polar inactive metabolites in man, with less dealkylation and lower plasma levels of norfenfluramine compared with the other species studied. 6. In conclusion, major species differences in the metabolism of (+/-)-fenfluramine, both qualitative and quantitative were observed, and no one species had a similar metabolic profile to that found in man.

1. A novel anticancer vinca alkaloid derivative (I) has been given as an i.v. bolus to cancer patients, using four different dosage regimens with dose levels ranging from 0.04 to 0.84 mg/m2 (equivalent to between 0.12 and 1.35 mg per dose), and the pharmacokinetics determined up to 72 h after dosing. In addition, secondary effects of leukopenia and neutropenia, were related to drug exposure using a sigmoid Emax model. 2. Plasma levels of I declined in a triphasic manner with a terminal half-life of approximately 50 h; most drug elimination (55%) being associated with the terminal phase. 3. Clearance of I was relatively low (245 +/- 160 ml/min) and remained constant with increasing doses. Initial distribution volume was low (approximately 71) but once distribution was complete, it was comparatively high (327 +/- 2121). 4. Both leukopenia and neutropenia were fitted successfully to a sigmoid Emax model showing that these effects were related to the total exposure to the drug. The Hill constant was less than 1, indicating a relatively shallow exposure/response curve and a predictable, graded increase in response with increasing I exposure, rather than a sudden quantal response. 5. Pharmacokinetically, I shows some similarities to other vinca alkaloids in its plasma level decline profile, although there are some notable differences which can be exploited clinically. In addition, the ability to model both leukopenia and neutropenia to the exposure to I, provides a valuable tool in the design of the most appropriate dosage regimen for the drug, as well as for dose adjustment taking into account inter-individual variations.

1. Methods of interspecies extrapolation using physiological models and allometric scaling have been reviewed with their possible application to drug development, both for candidate drug selection and the interpretation of toxicokinetic data. 2. Physiological models offer a mechanistic approach to extrapolation from one species to another, examining individual components which interrelate to produce the characteristics of the whole system. Tissues of interest are arranged in anatomical order based on blood circulation, and the disposition of a drug can be simulated with knowledge of tissue size (volume), tissue perfusion (blood flow), drug permeability, binding of the drug between the tissue and blood (partition), as well as elimination. Using this approach the behaviour of the drug under different conditions, such as dose route, disease state or animal species, can be predicted. 3. The alternative approach of allometric scaling is an empirical examination of relationships between size, time and its consequences. A regression of the logarithm of the pharmacokinetic parameter and the logarithm of the body weight of the animal species produces a linear relationship which enables the value of pharmacokinetic parameters in any animal species to be calculated from the product of an allometric coefficient and the body weight to a power function. 4. Whilst this technique gives acceptable predictions for the pharmacokinetics of those drugs eliminated renally, or which are blood flow-dependent, there is poor prediction for humans for low clearance drugs primarily eliminated by the mixed-function oxidase system. This appears to be a result of differences in maturation, and can be corrected for by including a brain weight or maximum life-span potential term into the allometric equation. 5. Of the two approaches described for extrapolation of pharmacokinetics between animal species, physiological models tend to be resource-demanding and costly, with more frequent failures, but can be invaluable for examining target organ exposure and for the targeting of drugs as in cancer chemotherapy. For routine drug development, however, allometric scaling is potentially more useful since it uses data which are routinely obtained and the calculations are relatively simple. 6. The problems of intraspecies scaling from high-dose data to low-dose predictions are discussed with respect to current models of dose levels. A new approach is proposed using a modified Hill equation based on drug exposure, which should allow for a more meaningful determination of the toxicity of a compound with different drug exposures.(ABSTRACT TRUNCATED AT 400 WORDS)

The distribution and excretion of radioactivity from [14C]-fotemustine was examined in mice with melanomas at different stages of development to determine whether the disease state substantially alters the disposition of the drug and its metabolites. Normal BDF1 mice and mice that had been subcutaneously grafted with B16 melanoma either 1, 3, 7, 14 or 21 days previously were used. The animals were killed at either 5 min, or at 3, 24 or 96 h after receiving an intravenous dose of [14C]-fotemustine (20 mg/kg) and were examined either by whole-body autoradiography or by liquid scintillation counting of excreta and tissues of interest. The majority of the [14C]-fotemustine dose was excreted in the urine, with similar amounts being measured in both non-tumourous animals (61.6% +/- 13.1%) and tumourous mice grafted 14 days previously (67.2% +/- 5.7%). Small amounts of radioactivity, again similar in both non-tumourous and tumourous mice, were recovered in the faeces (5.4% +/- 5.6% and 3.6% +/- 1.8%, respectively) and as carbon dioxide (7% +/- 3.5% and 6.4% +/- 1%, respectively), with minimal amounts being expired as chloroethanol (less than 1%). When mice were examined 5 min after dosing, there was extensive tissue distribution accounting for 75% +/- 10% of the dose. The highest concentrations determined by both whole-body autoradiography and liquid scintillation counting were measured in the excretory organs, with 33 and 28 micrograms Eq/g being found in the liver and kidney, respectively. High levels were also seen in the lung and plasma (19.8 and 19.5 micrograms Eq/g, respectively). Analysis of variance indicated that groups of tissues, such as the excretory organs, blood and plasma or the pigmented tissues, showed distinct but inconsistent patterns. Only tumours at 14 and 21 days of development were suitable for examination, and these showed levels of 12.1 micrograms Eq/g; however, the tumour-to-plasma ratio increased from between approx. 0.5 and 0.6 at 5 min to approx. 2 at 96 h after dosing, suggesting retention within the melanoma, whereas the ratio for the femur remained at approx. 1. Whole-body autoradiography showed that the distribution in the tumour was not uniform, but rather was concentrated in the peripheral area (presumably viable cells) as opposed to the central necrotic region. Thus, the high and sustained concentration of radioactivity found in the active cells of the melanoma may provide an explanation for the high efficacy of the drug.

1. An analytical method for a novel nitrosourea, fotemustine, has been developed using solid-phase extraction and h.p.l.c. with u.v. detection. As part of the development, different methods for stabilising fotemustine after sample collection have been investigated. The method has been successfully applied to pharmacokinetic studies in monkeys and man. 2. Providing plasma was separated immediately from blood and frozen within 3 min of collection, negligible degradation of fotemustine occurred. The samples could then be stored at -20 degrees C in the dark for up to six days particularly if thawing prior to analysis was accelerated using a 50 degrees C water-bath so that it was complete within 3 min. Equivalent results were also obtained with samples stabilised with 0.1 M citric acid immediately after the preparation of plasma. 3. The analytical method showed good precision with a within-day variation ranging between +/- 10.7% at the lowest concentration investigated (0.1 micrograms ml-1) to 2.0% at 50.0 micrograms ml-1. The accuracy of measurement was from 108.9% to 97.6% at 0.1 and 50.0 micrograms ml-1 respectively and the response was linear up to 50 micrograms ml-1. The minimum level of quantitation was 20 ng ml-1. 4. After a single intravenous bolus dose of [14C]fotemustine (100 mg m-2) to Cynomolgus monkeys, intact drug levels rapidly declined (t1/2 12.6 +/- 0.5 min) although the half-life of radioactivity (approx 100 h) was much longer. The plasma clearance of fotemustine was 225 +/- 63 ml min-1 with a volume of distribution based on area of 4.1 +/- 1.2 litres. 5. As with monkey, plasma levels of intact fotemustine in a patient given [14C]-drug as a 1 h constant rate intravenous infusion (approx. 100 mg m-2), declined rapidly but with a half-life of 23.2 min. Again, the half-life for total radioactivity was considerably longer (30.8 h). The plasma clearance was 1426 ml min-1 and the volume of distribution based on area was 47.71.

An animal model has been developed to investigate the potentials of various beta-lactam antibiotics for inducing (or producing) disulfiram-like effects. The method, which measures the rate of 14CO2 production in rats after [14C]ethanol administration, is simple to operate and sensitive. On the basis of available clinical information the model appears to be highly predictive for the likely incidence of disulfiram-like side effects in humans. Rats were pretreated intravenously with beta-lactam antibiotics (420 or 500 mg/kg-1) 18 h before ethanol administration or with N-methyl tetrazole thiol (NMTT; 1-methyl-5-mercaptotetrazole) at 96 mg kg-1, 6 h before ethanol administration. The rate of 14CO2 production was decreased to 70 to 80% of control levels by NMTT and the NMTT-containing beta-lactam antibiotics moxalactam, cefamandole, and cefoperazone. Cefotaxime, cephalothin, and cefuroxime which do not contain the NMTT side chain had no significant effect on 14CO2 production. Oral administration of moxalactam (500 mg kg-1) and NMTT (96 mg kg-1) 18 and 6 h, respectively, before ethanol administration significantly decreased 14CO2 production. Intravenous administration of moxalactam (500 mg kg-1) to rats with cannulated bile ducts 18 h before ethanol administration had no statistically significant effect on 14CO2 production, although the rate of 14CO2 production was decreased to 89% of the control level. The effect of dose level and dose interval was also investigated by using moxalactam. The results obtained support the hypothesis that disulfiram-like side effects associated with beta-lactam antibiotics are mediated by NMTT which is released and reabsorbed from the gut after biliary elimination of the parent beta-lactam antibiotic. The time course of inhibition of ethanol metabolism by moxalactam appears to differ from that of disulfiram.

The pharmacokinetics of oral loprazolam and pharmacodynamic responses on the morning following nightly drug administration were examined after single and after seven consecutive 1 mg doses in six non-fasting healthy subjects. The serum concentration-time profiles for unchanged loprazolam measured by a specific high pressure liquid chromatography/gas chromatography (h.p.l.c./g.c.) method and for benzodiazepine material measured by radioimmunoassay (RIA) were qualitatively similar although RIA levels were consistently higher. Approximate elimination half-life of unchanged loprazolam after single doses was 8.0 h. For RIA measured material, approximate half-life was 11.7 h following acute administration and 12.8 h after seven consecutive doses. Compared to results after single doses, maximum serum concentration and AUC were greater following 1 week's treatment. Using RIA results, the increases were 27.2% (95% CL 6.9 to 47.4%) and 35.1% (95% CL 15.8 to 54.3%) respectively and using h.p.l.c./g.c. data, 11% (95% CL - 22.6% to 44.5%) and 41% (95% CL - 50.9 to 133.0%). After repeated doses of loprazolam, there were no significant changes with respect to results after single doses in psychomotor function assessed objectively by critical flicker fusion threshold or choice reaction time, or in sleep quality or behaviour on awakening assessed subjectively by 10 cm analogue scales. Mean time to maximum serum concentration was 4.95 h with considerable inter-individual variability (range 1-12 h) and there was a lag time of 1-1.5 h before drug was detectable in blood.(ABSTRACT TRUNCATED AT 250 WORDS)

Subjects were each given either a 25, 50 or 100 mg intravenous loading dose of oxpentifylline followed by an intravenous infusion at a constant rate of 1.5 mg/min for 3 h. Plasma levels of oxpentifylline were measured to obtain information on its pharmacokinetics and to establish which of the loading doses gave the most rapid attainment of the steady state plasma levels of intact drug. Oxpentifylline kinetics were best described by a two compartment model giving a characteristic dip in the plasma level versus time curves before steady state was reached when either the 50 or 100 mg loading doses, followed by the constant intravenous infusion, were given. The terminal half-life of oxpentifylline was 1.02 +/- 0.86 h, reflecting a very high clearance of the drug (approx. 3 000 to 6 000 ml/min). The high clearance could be attributed to extrahepatic metabolism occurring in blood which was observed in vitro using whole blood but not plasma. The clearance of a reduced metabolite of oxpentifylline was less than that of the intact drug, although the half-life was similar (0.83 +/- 0.18 h). Of the three loading doses tested, only the highest showed any side effects, these being transient and occurring within a 5 to 10 min period after dosing and appeared to correlate with the high initial plasma levels of the drug. The 25 mg loading dose gave initial plasma levels generally below the final steady state levels, whilst the 50 mg loading was the closest to giving immediate steady state plasma levels of oxpentifylline.

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.

1. A specific and sensitive gas chromatographic assay is described for the measurement of d-fenfluramine and its de-ethylated metabolite, d-norfenfluramine, in biological fluids, together with some data on its application to the oral pharmacokinetics of the drug. 2. The analytical method developed has advantages over the previously described methods since it uses nitrogen specific detection and, when applied routinely, enables smaller sample volumes to be used (typically 1 ml of plasma) with a shorter chromatography time and an improved sensitivity (minimum quantifiable level of 2.5 ng ml-1). 3. Peak plasma concentrations of 22 and 24 ng ml-1 of intact drug were reached at 4 h after an oral dose of 14C-d-fenfluramine hydrochloride (30 mg) given to two volunteers as part of a metabolism and disposition study. Subsequently, concentrations of intact drug declined monoexponentially with a half-life of approximately 13 h. Peak concentrations of 10 and 8 ng ml-1 of the metabolite, d-norfenfluramine, were reached after 4 and 6 h and were maintained as a plateau for a further 4-6 h. Assessment of the half-life of the metabolite could not be made because of lack of data on the terminal portion of the curves. 4. The urinary excretion of d-fenfluramine (6.0 and 10.6% of the dose) and d-norfenfluramine (5.8 and 8.8% of the dose) was low, indicating extensive metabolism of the parent drug.

Six healthy fasted volunteers each received oral doses of a placebo, 1 mg and 2 mg loprazolam with one week between treatments using a double-blind balanced crossover design. Serum samples were obtained at selected times after dosing for measurement of loprazolam using a combined high-performance liquid and gas chromatographic assay. Drug effect was also measured at the corresponding times using self-assessment scales and psychomotor tests. The serum levels of loprazolam followed a somewhat irregular shape with secondary and tertiary peaks possibly associated with food intake. The maximum serum levels of loprazolam following 1 mg and 2 mg doses of the drug (6.0 +/- 2.6 and 11.3 +/- 2.9 ng/ml, respectively) occurred at approximately one hour after dosing. Both the maximum serum levels and the area under the curve of loprazolam measured to six hours increased in direct proportion to dose. Statistically significant drug effects were seen after 2 mg loprazolam, although the subjects also appeared sedated after 1 mg doses. There appeared to be a good correlation between the logarithm of the serum concentration of loprazolam and effect which suggested that the hypnotic activity of the drug was not mediated via a long-lived metabolite. A threshold serum concentration associated with evident sedation was observed at approximately 3 ng/ml.

The pharmacokinetics of cefotaxime were investigated in human volunteers given constant intravenous infusions, intravenous bolus, and intramuscular doses of the drug. After intravenous dosing, the plasma levels of cefotaxime declined in a biphasic manner with a terminal half-life varying between 0.92 and 1.65 hr. Moreover, the pharmacokinetics were linear up to at least a 2.0 g dose for volume of distribution based on area (23.3-31.3 l), plasma clearance (249-2.88 ml/min), and renal clearance (151-177 ml/min). Renal tubular secretion of intact cefotaxime and each of its metabolites was demonstrated by its interaction with probenecid, although the ratio of drug to metabolites ultimately excreted in urine after probenecid was similar to that seen normally (54 +/- 6, 19 +/- 4, 6.5 +/- 0.7 and 5.5 +/- 0.7% for cefotaxime, DACM, M2, and M3, respectively, when calculated as a percentage of the dose). The observed half-lives of DACM, M2, and M3 were 2.3 +/- 0.4, 2.2 +/- 0.1 and 2.2 hr, respectively. However, when the true half-life of DACM was calculated (0.83 +/- 0.23 hr) it was not only significantly shorter than that observed but also shorter than that for intact cefotaxime. The plasma clearance of DACM (744 +/- 226 ml/min) was much higher than that of cefotaxime while the volume of distribution was of a similar order (56 +/- 24 l). When administered intramuscularly, there was good absorption of cefotaxime from the site of injection (92-94%) giving maximum plasma levels of the drug of between 30 and 35 mg/l at approximately 40 min after dosing. Thereafter, the plasma levels of cefotaxime declined in a monophasic manner with a half-life (1.0-1.2 hr) similar to that of the terminal half-life seen after intravenous administration. Lidocaine had no significant effect on either its absorption or elimination kinetics.

Almitrine bismesylate, a new compound developed for the treatment of chronic obstructive lung disease, is thought to improve blood gases by a direct action on the carotid body (Laubie and Schmitt, 1980) and by improving ventilation - perfusion mismatching (Rigaud et al., 1981)). Following carotid artery injection of almitrine in experimental animals, a delay of more than 2 minutes is observed before there is an increase in the amplitude of carotid sinus nerve discharge (Laubie and Diot, 1972), but this then persists for several hours. This is in contrast to other compounds which stimulate the chemoreceptor within seconds of administration, but with a short lasting activity. Indeed, the mechanism by which almitrine acts on the carotid body, following administration of the drug under hypoxic and normoxic conditions, is unknown, despite investigations with a wide range of agonists and antagonists (Hanson, 1985) and the direct measurement of catecholamines in the chemoreceptor (Pallot, 1983). It is also of some interest that the action of almitrine is specific to the carotid body with little other demonstrable pharmacological activity (Evrard, 1984). To more fully understand this specific pharmacological activity of almitrine, we have investigated the uptake of almitrine into the carotid body over time in the rat in comparison with other tissues, to determine whether this process is rapid and specific for the unchanged drug and/or its metabolites, and to establish whether there is a particular affinity for chemoreceptor tissue.

1. [14C]Metronidazole was rapidly absorbed from the gastro-intestinal tract of rats giving maximum blood levels of radioactivity, equivalent to 6-4 and 6-7 mug metronidazole per ml blood, 1 h after oral dosing. 2. There was rapid equilibration between blood and most tissues, although radioactivity was concentrated in the liver, kidney, gastro-intestinal tract and vaginal secretions. 3. The half-life of clearance of radioactivity from the majority of tissues was between 3 and 4 h, although it was longer in the skin (8 h) and gastro-intestinal tract (14 h). 4. Fourteen radioactive excretion products were detected in rat urine and all the major products were identified. These all contained a nitro group and represented 97 degrees of the total radioactivity excreted in urine. 5. Unchanged metronidazole was secreted throughout the entire length of the gastro-intestinal tract and into the vagina of rats. 6. A hypothesis has been proposed to explain the high clinical efficacy of metronidazole in treating trichomonal and amoebic infections.

(1984). The Melanin Binding of Drugs and Its Implications. Drug Metabolism Reviews: Vol. 15, No. 5-6, pp. 1183-1212.

Die Behandlung mit Pentoxifyllin (Trental(R)) hat sich als wirksame Medikation bei Gefaskrankheiten mit Beteiligung der Mikrozirkulation erwiesen. Um insbesondere bei schweren Fallen einen raschen therapeutischen Effekt zu erzielen, werden haufig Pentoxifyllin-Infusionen gegeben. Steady state Plasmaspiegel sollen moglichst rasch erreicht werden, wozu eine entsprechende Initialdosis erforderlich ist. Aufgrund der am Menschen gewonnenen kinetischen paten wurde errechnet, das mit einer Initialdosis von 44 ± 20 mg Pentoxifyllin und anschliesender i.v. Infusionsgeschwindigkeit von 1.3 mg/min unmittelbare steady state Plasmaspiegel von 300 ng/ml zu erwarten sind.