Application of (1)H- and (19)F-NMR spectroscopy in the investigation of the urinary and biliary excretion of 3,5-, 2,4-ditrifluoromethylbenzoic and pentafluorobenzoic acids in rat

1. The metabolism and excretion of 2,4-, 3,5-ditrifluoromethyl- and pentafluorobenzoic acids were studied in the bile-cannulated rat using (1)H- and (19)F-NMR spectroscopy following intraperitoneal administration at 50 mg kg(-1). 2. Pentafluorobenzoic acid was excreted in the urine entirely unchanged. No detectable compound or metabolites were eliminated in the bile. A total of 63.5 +/- 6.7% of the dose was recovered in the 24-h collection period. 3. In the case of 2,4-ditrifluromethyl benzoic acid, 83.9 +/- 5.2% of the dose was recovered in the 24h after administration, with about 52% being excreted in the urine and 32% in the bile. The majority of the material present in the urine was unchanged parent compound. In bile, some 60% of the compound-related material excreted was present as transacylated ester glucuronide conjugates. 4. For 3,5-ditrifluoromethylbenzoic acid, 49.6 +/- 5.3% of the dose was recovered in the 24-h collection period, with about 22% being excreted in the urine and 28% in the bile. The material excreted in both the urine and bile was a mixture of the parent acid and transacylated ester glucuronides. 5. Urinary excretion in bile-cannulated animals was similar to that found in studies using non-cannulated animals dosed at 100mg kg(-1).     

Pharmacokinetics of a novel surface-active agent,purified poloxamer 188, in rat,rabbit, dog and man

Purified poloxamer 188 (PP188) is a non-ionic, block copolymer surfactant that is currently being evaluated clinically in sickle cell disease vaso-occlusive crisis and acute chest syndrome and preclinically in spinal cord injury and muscular dystrophy. This paper describes the pharmacokinetics of PP188 in rats, pregnant rats, pregnant rabbits, dogs and humans. Plasma protein binding interaction studies demonstrated no clinically significant effects on narcotic analgesics, hydroxyurea, warfarin, diazepam or digitoxin, but an increase in free fraction for propranolol. The plasma concentrations increased proportionate with increasing dose in all species tested. Renal clearance accounted for 90% of total plasma clearance in man. A single metabolite was detected and quantified in the plasma from dogs and humans that was cleared more slowly than parent drug. Allometric scaling of plasma clearance and volume of distribution at steady-state (Vss) across species provided good predictions of the pharmacokinetic parameters in humans. Based on the comparative pharmacokinetics of PP188 in rat, rabbit, dog and man, all three animal species were appropriate models for evaluating various aspects of PP188's toxicological profile.     

Allometric relationships between the pharmacokinetics of propofol in rats, children and adults

AIMS: Allometric equations have proven useful for the extrapolation of animal data to determine pharmacokinetic parameters in man. It has been proposed that these equations are also applicable over the human size range including the paediatric population. The aim of this work was to study the relationship between various pharmacokinetic parameters for propofol and body weight using data from rats, children and adults. Furthermore, the utility of allometric scaling is evaluated by the prediction of propofol concentrations in humans based on data obtained in the rat. METHODS: The relationship between the pharmacokinetic parameters of propofol obtained in rats, children and adults was analyzed by plotting the logarithmically transformed parameters against the corresponding logarithmically transformed body weights. In addition, based on allometric equations, pharmacokinetic parameters obtained in rats were scaled to humans. These parameters were used to simulate propofol concentrations in long-term sedated critically ill patients using NONMEM. Simulated concentrations were then compared with actually observed concentrations in humans. RESULTS: The relationship between pharmacokinetic parameters of propofol from rats, children and adults was in good agreement with those from the literature on allometric modelling. For clearance, intercompartmental clearance, central volume of distribution and peripheral volume of distribution, the power parameters were 0.78, 0.73, 0.98 and 1.1, respectively, and r2 values for the linear correlations were 0.990, 0.983, 0.977 and 0.994, respectively. On the basis of data obtained after a single bolus injection in the rat, adequate predictions of propofol concentrations in critically ill patients can be made using allometric equations, despite the long-term nature of the use of the drug, the large number of infusion changes per day and/or differences in state of health and age. CONCLUSIONS: For propofol, allometric scaling has proved to be valuable for cross species extrapolation. Furthermore, the use of the allometric equation between adults and children seems to be an adequate tool for the development of rational dosing schemes for children of varying body weights, and requires further study.     

Assessment of chimeric mice with humanized livers in new drug development: generation of pharmacokinetics,metabolism and toxicity data for selecting the final candidate compound

1. Chimeric mice with humanized livers are expected to be a novel tool for new drug development. This review discusses four applications where these animals can be used efficiently to collect supportive data for selecting the best compound in the final stage of drug discovery.

2. The first application is selection of the final compound based on estimated pharmacokinetic parameters in humans. Since chimeric mouse livers are highly repopulated with human hepatocytes, hepatic clearance values in vivo could be used preferentially to estimate pharmacokinetic profiles for humans.

3. The second is prediction of human-specific or disproportionate metabolites. Chimeric mice reproduce human-specific metabolites of drugs under development to conform to ICH guidance M3(R2), except for compounds that were extensively eliminated by co-existing mouse hepatocytes.

4. The third is identifying metabolites with distinct pharmacokinetic profiles in humans. Slow metabolite elimination specifically in humans increases its exposure level, but if its elimination is faster in laboratory animals, the animal exposure level might not satisfy ICH guidance M3(R2).

5. Finally, two examples of reproducing acute liver toxicity in chimeric mice are introduced. Integrated pharmacokinetics, metabolism and toxicity information are expected to assist pharmaceutical scientists in selecting the best candidate compound in new drug development.     

Comparison of predictability for human pharmacokinetics parameters among monkeys,rats, and chimeric mice with humanised liver

1.?The aim of the present study was to evaluate the usefulness of chimeric mice with humanised liver (PXB mice) for the prediction of clearance (CL_t) and volume of distribution at steady state (Vd_ss), in comparison with monkeys, which have been reported as a reliable model for human pharmacokinetics (PK) prediction, and with rats, as a conventional PK model.

2.?CL_t and Vd_ss values in PXB mice, monkeys and rats were determined following intravenous administration of 30 compounds known to be mainly eliminated in humans via the hepatic metabolism by various drug-metabolising enzymes. Using single-species allometric scaling, human CL_t and Vd_ss values were predicted from the three animal models.

3.?Predicted CL_t values from PXB mice exhibited the highest predictability: 25 for PXB mice, 21 for monkeys and 14 for rats were predicted within a three-fold range of actual values among 30 compounds. For predicted human Vd_ss values, the number of compounds falling within a three-fold range was 23 for PXB mice, 24 for monkeys, and 16 for rats among 29 compounds. PXB mice indicated a higher predictability for CL_t and Vd_ss values than the other animal models.

4.?These results demonstrate the utility of PXB mice in predicting human PK parameters.     

Preclinical pharmacokinetics of the novel PI3K inhibitor GDC-0941 and prediction of its pharmacokinetics and efficacy in human

1. The phosphatidylinositol 3-kinase (PI3K) pathway is a major determinant of cell cycling and proliferation. Its deregulation is associated with the development of many cancers.

2. GDC-0941, a potent and selective inhibitor of PI3K, was characterised preclinically in in vitro and in vivo studies.

3. Plasma protein binding was extensive, with free fraction less than 7%, and blood-to-plasma ratio ranged from 0.6 to 1.2 among the species tested. GDC-0941 human hepatic clearance was predicted to be moderate by liver microsomal incubations. GDC-0941 had high permeability in Madin-Darby canine kidney cells.

4. The clearance of GDC-0941 was high in mouse (63.7?mL/min/kg), rat (49.3?mL/min/kg) and cynomolgus monkey (58.6?mL/min/kg), and moderate in dog (11.9?mL/min/kg). The volume of distribution ranged from 2.52?L/kg in rat to 2.94?L/kg in monkey. Oral bioavailability ranged from 18.6% in monkey to 77.9% in mouse.

5. Predicted human clearance and volume of distribution using allometry were 6?mL/min/kg and 2.9?L/kg, respectively. The human efficacious doses were predicted based on results from preclinical pharmacokinetic studies and xenograft models.

6. GDC-0941 preclinical characterisation and predictions of its properties in human supported its progression towards clinical development. GDC-0941 is currently in phase II clinical trials.

     

Quantitative studies on the urinary metabolic fate of 2-chloro-4-trifluoromethylaniline in the rat using 19F-NMR spectroscopy and directly coupled HPLC-NMR-MS

1. The metabolism and urinary excretion of 2-chloro-4-trifluoromethylaniline has been studied in the rat using 19F-NMR spectroscopy and directly coupled HPLC-NMR-MS methods. The compound was dosed to three male Sprague-Dawley rats (50 mg kg(-1) i.p.) and urine collected over 0-8, 8-24 and 24-48 h post-dosing. 2. A total urinary recovery of 56.3+/-2.2% of the dose was achieved up to 48 h after dosing. The major metabolite in the urine was identified as 2-amino-3-chloro-5-trifluoromethylphenylsulphate accounting for a total of 33.5+/-2.2% of the dose. 3. Further metabolites detected and characterized included 2-chloro-4-trifluoromethylphenylhydroxylamine glucuronide (13.2+/-0.5% of the dose), 2-amino-3-chloro-5-trifluoromethylphenylglucuronide (3.8+/-0.4% of the dose) and 2-chloro-4-trifluoromethylaniline-N-glucuronide (3.6+/-0.1% of the dose). Several minor metabolites were also found and identified, including 2-chloro-4-trifluoromethylphenylsulphamate, which together accounted for 2.1+/-0.4% of the dose. 4. Directly coupled HPLC-NMR-MS and 19F-NMR spectroscopy is shown to provide an efficient approach for the unequivocal and rapid determination of the quantitative urinary metabolic fate and excretion balance of a fluorinated xenobiotic without the necessity for specific radiolabelling.     

Quantification of urinary excretion of ethosuximide enantiomers and their major metabolites in the rat.

A chiral gas chromatographic assay has been developed for quantitative analysis of ethosuximide and its major metabolites in rat urine. The extraction procedure was found to be precise and reproducible. Recovery was in the range of 94-98%, intraday CV(%) was 0.92% for (S)-ethosuximide (50 microg/ml) and 0.51% for (R)-ethosuximide (50 microg/ml). Interday CV(%) was 1.12% for (S)-ethosuximide and 0.72% for (R)-ethosuximide. The limit of detection was determined to be around 0.01 microg/ml for each enantiomer. Following administration of rac-ethosuximide by i.v., i.p. and oral routes, unchanged ethosuximide was detected in urine up to 72 h after drug administration. The appearance of all detected metabolites occurred within 24 h of drug administration. Significantly more (S)-ethosuximide was excreted unchanged than (R)-ethosuximide with all three routes studied. A substantial amount of the drug was eliminated as the 2-(1-hydroxyethyl)-2-methylsuccinimide (2 pairs of diastereoisomers). Much less drug was eliminated as the 2-ethyl-3-hydroxy-2-methylsuccinimide with only one diastereoisomer observed. Examination of the one pair of diastereoisomers of 2-(1-hydroxyethyl)-2-methylsuccinimide that was resolved showed preferential excretion of one isomer. Comparison of both pairs of diastereoisomers showed that one pair was formed in preference to the other with a ratio of approximately 0.8:1. It is concluded that stereoselective metabolism of ethosuximide occurs.     

Preclinical metabolism and pharmacokinetics of NVS-CRF38, a potent and orally bioavailable corticotropin-releasing factor receptor 1 antagonist

Abstract

1.?The pharmacokinetic properties and metabolism of NVS-CRF38 [7-(3,5-dimethyl-1H-1,2,4-triazol-1-yl)-3-(4-methoxy-2-methylphenyl)-2,6-dimethylpyrazolo[5,1-b]oxazole], a novel corticotropin-releasing factor receptor 1 (CRF₁) antagonist, were determined in vitro and in animals.

2.?NVS-CRF38 undergoes near complete absorption in rats and dogs. In both species the compound has low hepatic extraction and is extensively distributed to tissues.

3.?In rat and human hepatic microsomes and cryopreserved hepatocytes from rat, dog, monkey and human, NVS-CRF38 was metabolised to form O-desmethyl NVS-CRF38 (M7) and several oxygen adducts (M1, M3, M4, M5 and M6). In hepatocytes further metabolites were observed, specifically the carboxylic acid (M2) and conjugates (sulphate and glucuronide) of M7.

4.?Formation of primary metabolites in hepatocytes was blocked by the cytochrome P450 enzyme (P450) suicide inhibitor 1-aminobenzotriazole, implicating P450 enzymes in the primary metabolism of this compound.

5.?NVS-CRF38 is weakly bound to plasma proteins from rat (fu_b?=?0.19), dog (fu_b?=?0.25), monkey (fu_b?=?0.20) and humans (fu_b?=?0.23). Blood-to-plasma partition for NVS-CRF38 approaches unity in rat and human blood.

6.?The hepatic clearance of NVS-CRF38 in humans is predicted to be low (extraction ratio?～?0.2) based on scaling from drug depletion profiles in hepatic microsomes.     

Metabolization of 14C-N-acetylpenicillamine and 14C-cysteine in relation to 1-chloro-2,4-dinitrobenzene and sulphobromophtalein conjugation and biliary excretion in the rat

Upon intraperitoneal administration of N-(1-14C)-acetylpenicillamine (NAPA) to rats, at a dose of 1-2 mmol/kg body weight, a 14C-labelled metabolite of NAPA together with unchanged NAPA were excreted in bile. The NAPA metabolite was characterized by acid hydrolysis and thin-layer chromatography. Rats previously depleted of liver glutathione by intraperitoneal injection of cyclohexene oxide, 2.5 mmol/kg body weight, were given intraperitoneal injections of 14C-cysteine, 0.5 mmol/kg, or 14C-NAPA, 1-2 mmol/kg, together with 1-chloro-2,4-dinitrobenzene (DNB), 1 mmol/kg, or bromosulphophtalein (BSP), 0.5 mmol/kg body weight. Simultaneous administration of 14C-cysteine and DNB or BSP lead to rapid incorporation of 14C-activity in glutathione and glutathione conjugates of DNB and BSP being excreted in bile. Upon simultaneous administration of 14C-NAPA and DNB or BSP, 14C-labelled metabolite of NAPA not conjugated to DNB or BSP, and unchanged NAPA, together with non-labelled glutathione conjugates of DNB or BSP, were excreted in bile. This is interpreted as indicating that neither NAPA nor NAPA metabolite are capable of forming conjugates of DNB or BSP in rat liver.     

Short term effects and urinary excretion of the new diuretic,indapamide, in normal subjects

Summary The pharmacology, toxicology and kinetics of a new diuretic indapamide, have been studied in six normal volunteers following a single oral dose of 40 mg. Pronounced diuresis was found, commencing three hours after ingestion, with a peak urinary flow at four to six hours, and continuing for a total of thirty-six hours. A fall in systolic standing blood pressure occurred twenty four hours after ingestion, coincident with the period of maximum dehydration. Free water clearance rose, accompanied by increased urinary losses of Na⁺, K⁺ and Cl^– and alkalinisation of the urine comparable to the actions of benzothiadiazines. Total urinary losses of Ca²⁺, Mg²⁺ and PO ₄ ^3– rose in spite of a fall in urinary concentrations of these ions. The Ca²⁺ effect compares with the acute ionic effects of other diuretics. No renal, hepatic or haematological toxic effect was demonstrated. The blood sugar level was not disturbed. Serum uric acid rose to abnormal levels although the change did not reach statistical significance. — A thin layer chromatographic method, with a sensitivity limit of 0.1 µg/ml., has been developed for the assay of indapamide in urine. The urinary excretion rates of the volunteers measured over forty-eight hours indicate that the drug is rapidly absorbed with a peak excretion, 2.9±1.3 µg/min occurring three hours after ingestion. The drug is eliminated bi-phasically with an initial short rapid elimination followed by a slower exponential decline with a mean elimination half-life of 10.3 ± 3.9 h. The mean urinary excretion of unchanged indapamide over forty-eight hours was 4.4±1.4% of the administered dose. — It is concluded that indapamide is an effective long-acting diuretic with comparable action to the benzothiadiazine diuretics, but without an effect on blood sugar level in single doses in normal subjects. In contrast with other diuretics, indapamide appears to be extensively metabolised in man, and its longer duration of action to be related to a longer elimination half-life.     

Metabolism of 3-chloro-4-fluoroaniline in rat using [14C]-radiolabelling, 19F-NMR spectroscopy,HPLC-MS/MS,HPLC-ICPMS and HPLC-NMR

The metabolic fate of 3-chloro-4-fluoroaniline was investigated in rat following intraperitoneal (i.p.) administration at 5 and 50?mg?kg^?1 using a combination of HPLC-MS, HPLC-MS/MS, ¹⁹F-NMR spectroscopy, HPLC-NMR spectroscopy and high-pressure liquid chromatography-inductively coupled plasma mass spectrometry (HPLC-ICPMS) with ³⁵Cl and ³⁴S detection. The metabolism of 3-chloro-4-fluoroaniline at both doses was rapid and extensive, to a large number of metabolites, with little unchanged compound excreted via the urine. Dosing at 5?mg?kg^?1 with [¹⁴C]-labelled compound enabled the comparison of standard radioassay analysis methods with ¹⁹F-NMR spectroscopy. ¹⁹F-NMR resonances were only readily detectable in the 0–12?h post-dose samples. Dosing at 50?mg?kg^?1 allowed the facile and specific detection and quantification of metabolites by ¹⁹F-NMR spectroscopy. Metabolite profiling was also possible at this dose level using HPLC-ICPMS with ³⁵Cl-specific detection. The principal metabolites of 3-chloro-4-fluoroaniline were identified as 2-amino-4-chloro-5-fluorophenyl sulfate and 2-acetamido-4-chloro-5-fluorophenyl glucuronide. N-acetylation and hydroxylation followed by O-sulfation were the major metabolic transformations observed.     

Effect of toluene and xylenes on liver glutathione and their urinary excretion as mercapturic acids in the rat

Administration of toluene and xylenes to rats caused a decrease in liver glutathione concentration. The effect was most pronounced after the administration of o-xylene. 26% of the initial glutathione level was found three hours after treatment with o-xylene (4.0 mmoles/kg).No in vitro conjugation of o-xylene with glutathione was observed, neither spontaneously nor in the presence of 105,000 g supernatant from rat liver homogenate, containing glutathione S-transferases. Thus, a metabolite of o-xylene, which is not formed during incubation with 105,000 g supernatant, reacts with glutathione.A thioether was isolated from urine of rats given o-xylene; the compound was identified as o-methylbenzyl mercapturic acid by GC-MS and NMR. Chromatographic evidence was found for the presence of benzyl mercapturic acid in the urine of toluene-treated rats. The amounts of mercapturic acids excreted in the urine after administration of toluene, p-xylene, m-xylene, and o-xylene were 0.4–0.7, 0.6, 1.3, and 10–21% of the dose, respectively.These results demonstrate the involvement of a thusfar unknown pathway in the biotransformation of toluene and xylenes.     

The metabolism and excretion of [C] 2- and 4-chlorobenzoic acids in the rat

The metabolic fate of [¹⁴C]-labelled 2 and 4-chlorobenzoic acids (2- and 4-CBA) has been determined in the rat following intraperitoneal (i.p.) administration at 100 mg/kg to male rats. The major route of elimination for both 2- and 4-CBA was urine with >80% of the dose recovered in the initial 0–24 h after administration. Glycine conjugation was found to be the dominant metabolic fate for both [¹⁴C] 2- and 4-CBA however, the position of chloro substitution had a clear effect on the extent of metabolism via this route with ortho substitution reducing the extent of metabolism via this pathway.     

Plasma pharmacokinetics and urinary and biliary excretion of a new potent tripeptide HIV-1 protease inhibitor,KNI-272, in rats after intravenous administration

The pharmacokinetic (PK) characteristics of KNI-272, a potent and selective HIV-1 protease inhibitor, were evaluated in rats after intravenous (IV) administration. The effect of dose on KNI-272 plasma kinetics, and the urinary and biliary elimination kinetics of KNI-272, were examined. After IV administration of 10.0 mg kg^?1 KNI-272, the mean terminal elimination half-life, t_1/2λz, was 3.49 ± 0.19 (SE) h, the total plasma clearance, CL_tot, was 15.1 ± 1.2 mL min^?1 and the distribution volume at steady state, V_d,ss, was 3790±280 mL kg^?1. On the other hand, after 1.0mg kg^?1 IV administration, t_d,ss, was 3.04±0.11 h, CL_tot was 15.9±0.2mL min^?1, and V_d,ss was 6950±600 mL kg^?1. The PK parameters of KNI-272 after IV administration showed that the disposition of KNI-272 in the rat plasma is linear within the dose range from 1.0 to 10.0mg kg^?1. Using an equilibrium dialysis method, the plasma binding of KNI-272 was measured in vitro. The free fractions were 17.7 ± 0.6%, 12.1±1.5%, and 13.8 ± 1.4% at the total concentration ranges of 9.898 ± 0.097 μg mL^?1, 0.888 ± 0.008 μg mL^?1, and 0.470±0.55 μg mL^?1, respectively. The percentages of the dose excreted into the urine and bile as the unchanged form were 1.20 ± 1.06% and 1.61 ± 0.32% at 1.0mg kg^?1 dose, and 0.164 ± 0.083% and 1.42 ± 0.26% at 10.0 mg kg^?1 dose, respectively. The renal clearance (CL_R) and the biliary clearance (CL_B) were calculated to be 0.191 and 0.256mL min^?1 for 1.0mg kg^?1, and 0.0248 and 0.215 mL min^?1 for 10.0 mg kg^?1, respectively. When comparing these values with the CL_tot values, the urinary and biliary excretion of KNI-272 are minor disposition routes.     

Efficacy, tissue distribution and biliary excretion of methyl (3R*,5S*)-(E)-3,5-dihydroxy-9,9-diphenyl-6,8-nonadienoate (CP-83101), a hepatoselective inhibitor of HMG-CoA reductase activity in the rat

Methyl (3R*,5S*)-(E)-3,5-dihydroxy-9,9-diphenyl-6,8-nonadienoate, CP-83101, was identified as a potent competitive inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase activity, inhibiting enzyme activity in vitro with an IC50 of 8.5 +/- 0.7 microM and a Ki with respect to HMG-CoA of 2.6 microM. CP-83101 also inhibited rat hepatic sterol biosynthesis by 39 +/- 7% at a dose of 100 mg/kg. [3H]CP-83101, administered orally to rats, exhibited peak plasma levels at approximately 1 hr that declined thereafter with an apparent half-time of 2-3 hr. Peak tissue levels also occurred 1 hr following oral administration of [3H]CP-83101. The decline in radioactivity in the liver, however, was considerably slower than that noted in blood, whereas the half-life in non-hepatic tissues was approximately 1 hr. Liver/blood ratios of 14, and liver/lens ratios of greater than 3000, following oral administration of [3H]CP-83101, were similar to those previously reported for other HMG-CoA reductase inhibitors, suggesting a high degree of tissue selectivity. In addition, liver/adrenal and liver/ovary ratios were approximately 1000 at all time points examined between 30 min and 24 hr following oral [3H]CP-83101 administration, indicating a high specificity for hepatic versus other steroidogenic tissues. Evaluation of intravenous versus oral administration of the water-soluble, free acid, sodium salt of [3H]CP-83101 in bile duct canulated rats indicated that approximately 20% of orally administered CP-83101 is absorbed from the gastrointestinal tract, and that absorbed CP-83101 is cleared rapidly from the plasma via the liver and from the liver via the bile. In addition, several lines of evidence suggest that CP-83101 may undergo enterohepatic recirculation. Agents of this synthetic series may thus possess advantages over other HMG-CoA reductase inhibitors with respect to tissue kinetics and specificity.     

Circadian variations in the pharmacokinetics, tissue distribution and urinary excretion of nifedipine after a single oral administration to rats

Circadian variations in the pharmacokinetics, tissue distribution and urinary excretion of nifedipine were examined in fasted rats after administering a single oral dose at three different dosing times (08:00 am, 16:00 pm, 00:00 am). The plasma concentrations, the areas under the plasma concentration-time curve from zero to 6 h (AUC(0-6 h)) and the peak plasma concentration (C(max)) were significantly higher in the rats dosed at 08:00 am (immediately inactive), and was lower at 16:00 pm (most inactive) and 00:00 am (most active). The time to reach the C(max) (T(max)) was the shortest in the rats dosed at 08:00 am. It was very interesting to observe the double peak phenomena in the plasma concentration profiles, showing a larger peak followed by a smaller peak. There was a dosing time dependency on the tissue distribution 30 min after administration, showing a similar tendency to the pharmacokinetic behavior. However, there was no distinct dosing time dependency observed at 2 h after administration due to the extensive disposition. The cumulative urine excretion of nifedipine in the rats dosed at 08:00 am was significantly higher (about two-fold) than in those dosed at 16:00 pm and 00:00 am. The pharmacokinetics of nifedipine in the rats was consistent with that observed in human subjects in terms of the day-night clock time but the biological time was the opposite, as marked by the rest-activity cycles. These results may help to explain the circadian time-dependency of nifedipine pharmacokinetics.     

Absorption,distribution, metabolism and excretion of the novel SARM GTx-024 [(S)-N-(4-cyano-3-(trifluoromethyl)phenyl)-3-(4-cyanophenoxy)-2-hydroxy-2-methylpropanamide] in rats

Abstract

1. GTx-024, a novel selective androgen receptor modulator, is currently being investigated as an oral treatment for muscle wasting disorders associated with cancer and other chronic conditions.

2. Absorption of GTx-024 was rapid and complete, with high oral bioavailability. A wide tissue distribution of [¹⁴C]GTx-024 derived radioactivity was observed. [¹⁴C]GTx-024-derived radioactivity had a moderate plasma clearance (117.7 and 74.5?mL/h/kg) and mean elimination half-life of 0.6?h and 16.4?h in male and female rats, respectively.

3. Fecal excretion was the predominant route of elimination, with ～70% of total radioactivity recovered in feces and 21–25% in urine within 48?h. Feces of intact rats contained primarily unchanged [¹⁴C]GTx-024 (49.3–64.6%). Metabolites were identified in urine and feces resulting from oxidation of the cyanophenol ring (M8, 17.6%), hydrolysis and/or further conjugation of the amide moiety (M3, 8–12%) and the cyanophenol ring (M4, 1.3–1.5%), and glucuronidation of [¹⁴C]GTx-024 at the tertiary alcohol (M6, 3.5–3.7%). There was no quantifiable metabolite in plasma.

4. In summary, in the rat GTx-024 is completely absorbed, widely distributed, biotransformed through several metabolic pathways, and eliminated in feces primarily as an unchanged drug.     

Preclinical assessment of the absorption,distribution, metabolism and excretion of GDC-0449 (2-chloro-N-(4-chloro-3-(pyridin-2-yl)phenyl)-4-(methylsulfonyl)benzamide), an orally bioavailable systemic Hedgehog signalling pathway inhibitor

1. GDC-0449 (2-chloro-N-(4-chloro-3-(pyridin-2-yl)phenyl)-4-(methylsulfonyl)benzamide) is a potent, selective Hedgehog (Hh) signalling pathway inhibitor being developed for the treatment of various cancers.

2. The in vivo clearance of GDC-0449 was estimated to be 23.0, 4.65, 0.338, and 19.3?ml min^?1 kg^?1 in mouse, rat, dog and monkeys, respectively. The volume of distribution ranged from 0.490 in rats to 1.68 l kg^?1 in mice. Oral bioavailability ranged from 13% in monkeys to 53% in dogs. Predicted human clearance using allometry was 0.096–0.649?ml min^?1 kg^?1 and the predicted volume of distribution was 0.766 l kg^?1.

3. Protein binding was extensive with an unbound fraction less than or equal to 6%, and the blood-to-plasma partition ratio ranged from 0.6 to 0.8 in all species tested. GDC-0449 was metabolically stable in mouse, rat, dog and human hepatocytes and had a more rapid turnover in monkey hepatocytes.

4. Proposed metabolites from exploratory metabolite identification in vitro (rat, dog and human liver microsomes) and in vivo (dog and rat urine) include three primary oxidative metabolites (M1–M3) and three sequential glucuronides (M4–M6). Oxidative metabolites identified in microsomes M1 and M3 were formed primarily by P4503A4/5 (M1) and P4502C9 (M3).

5. GDC-0449 was not a potent inhibitor of P4501A2, P4502B6, P4502D6, and P4503A4/5 with IC₅₀ estimates greater than 20?μM. K_i’s estimated for P4502C8, P4502C9 and P4502C19 and were 6.0, 5.4 and 24?μM, respectively. An evaluation with Simcyp® suggests that GDC-0449 has a low potential of inhibiting P4502C8 and P4502C9. Furthermore, GDC-0449 (15?μM) was not a potent P-glycoprotein/ABCB1 inhibitor in MDR1-MDCK cells.

6. Overall, GDC-0449 has an attractive preclinical profile and is currently in Phase II clinical trials.