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.

     

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.

     

Pharmacokinetics,disposition and lipid-modulating activity of 5-{2-[4-(3,4-difluorophenoxy)-phenyl]-ethylsulfamoyl}-2-methyl-benzoic acid,a potent and subtype-selective peroxisome proliferator-activated receptor α agonist in preclinical species and human

1. 5-{2-[4-(3,4-Difluorophenoxy)-phenyl]-ethylsulfamoyl}-2-methyl-benzoic acid (1) is a novel, potent, and selective agonist of the peroxisome proliferator-activated receptor alpha (PPAR-α).

2. In preclinical species, compound 1 demonstrated generally favourable pharmacokinetic properties. Systemic plasma clearance (CL_p) after intravenous administration was low in Sprague–Dawley rats (3.2?±?1.4?ml min^?1 kg^?1) and cynomolgus monkeys (6.1?±?1.6?ml min^?1 kg^?1) resulting in plasma half-lives of 7.1?±?0.7?h and 9.4?±?0.8?h, respectively. Moderate bioavailability in rats (64%) and monkeys (55%) was observed after oral dosing. In rats, oral pharmacokinetics were dose-dependent over the dose range examined (10 and 50?mg kg^?1).

3. In vitro metabolism studies on 1 in cryopreserved rat, monkey, and human hepatocytes revealed that 1 was metabolized via oxidation and phase II glucuronidation pathways. In rats, a percentage of the dose (approximately 19%) was eliminated via biliary excretion in the unchanged form.

4. Studies using recombinant human CYP isozymes established that the rate-limiting step in the oxidative metabolism of 1 to the major primary alcohol metabolite M1 was catalysed by CYP3A4.

5. Compound 1 was greater than 99% bound to plasma proteins in rat, monkey, mouse, and human.

6. No competitive inhibition of the five major cytochrome P450 enzymes, namely CYP1A2, P4502C9, P4502C19, P4502D6 and P4503A4 (IC₅₀’s?>?30 μM) was discerned with 1.

7. Because of insignificant turnover of 1 in human liver microsomes and hepatocytes, human clearance was predicted using rat single-species allometric scaling from in vivo data. The steady-state volume was also scaled from rat volume after normalization for protein-binding differences. As such, these estimates were used to predict an efficacious human dose required for 30% lowering of triglycerides.

8. In order to aid human dose projections, pharmacokinetic/pharmacodynamic relationships for triglyceride lowering by 1 were first established in mice, which allowed an insight into the efficacious concentrations required for maximal triglyceride lowering. Assuming that the pharmacology translated in a quantitative fashion from mouse to human, dose projections were made for humans using mouse pharmacodynamic parameters and the predicted human pharmacokinetic estimates.

9. First-in-human clinical studies on 1 following oral administration suggested that the human pharmacokinetics/dose predictions were in the range that yielded a favourable pharmacodynamic response.

     

Preclinical stereoselective disposition and toxicokinetics of two novel MET inhibitors

1. The R- and S-enantiomer of N-(4-(3-(1-ethyl-3,3-difluoropiperidin-4-ylamino)-1H-pyrazolo[3,4-b]pyridin-4-yloxy)-3-fluorophenyl)-2-(4-fluorophenyl)-3-oxo-2,3-dihydropyridazine-4-carboxamide are novel MET kinase inhibitors that have been investigated as potential anticancer agents. The effect of the chirality of these compounds on preclinical in vivo pharmacokinetics and toxicity was studied.

2. The plasma clearance for the S-enantiomer was low in mice and monkeys (23.7 and 7.8?mL min^?1 kg^?1, respectively) and high in rats (79.2?mL min^?1 kg^?1). The R/S enantiomer clearance ratio was 1.5 except in rats (0.49). After oral single-dose administration at 5?mg kg^?1 the R/S enantiomer ratio of AUC_inf was 0.95, 1.9 and 0.41 in mice, rats and monkeys, respectively.

3. In an oral single-dose dose-ranging study at 200 and 500?mg kg^?1 and multi-dose toxicity study in mice plasma AUC exposure was approximately 2- to 3-fold higher for the R-enantiomer compared to the S-enantiomer. Greater toxicity of the S-enantiomer was observed which appeared to be due to high plasma C_min values and tissue concentrations approximately 24?h after the final dose.

4. Both enantiomers showed low to moderate permeability in MDCKI cells with no significant efflux, no preferential distribution into red blood cells and similar plasma protein binding in vitro.

5. Overall, the differences between the enantiomers with respect to low dose pharmacokinetics and in vitro properties were relatively modest. However, toxicity results warrant further development of the R-enantiomer over the S-enantiomer.

     

Metabolism,pharmacokinetics and excretion of the GABAA receptor partial agonist [14C]CP-409,092 in rats

1. The metabolism and excretion of a GABA_A partial agonist developed for the treatment of anxiety, CP-409,092; 4-oxo-4,5,6,7-tetrahydro-1H-indole-3-carboxylic acid (4-methylaminomethyl-phenyl)-amide, were studied in rats following intravenous and oral administration of a single doses of [¹⁴C]CP-409,092.

2. The pharmacokinetics of CP-409,092 following single intravenous and oral doses of 4 and 15?mg kg^?1, respectively, were characterized by high clearance of 169?±?18?ml min^?1 kg^?1, a volume of distribution of 8.99?±?1.46 l kg^?1, and an oral bioavailability of 2.9% ± 3%.

3. Following oral administration of 100?mg kg^?1 [¹⁴C]CP-409,092, the total recovery was 89.1% ± 3.2% for male rats and 89.3% ± 0.58% for female rats. Approximately 87% of the radioactivity recovered in urine and faeces were excreted in the first 48?h. A substantial portion of the radioactivity was measured in the faeces as unchanged drug, suggesting poor absorption and/or biliary excretion. There were no significant gender-related quantitative/qualitative differences in the excretion of metabolites in urine or faeces.

4. The major metabolic pathways of CP-409,092 were hydroxylation(s) at the oxo-tetrahydro-indole moiety and oxidative deamination to form an aldehyde intermediate and subsequent oxidation to form the benzoic acid. The minor metabolic pathways included N-demethylation and subsequent N-acetylation and oxidation.

5. The present work demonstrates that oxidative deamination at the benzylic amine of CP-409,092 and subsequent oxidation to form the acid metabolite seem to play an important role in the metabolism of the drug, and they contribute to its oral clearance and low exposure.

     

Bioavailability and pharmacokinetics of S-propargyl-L-cysteine,a novel cardioprotective agent,after single and multiple doses in Beagle dogs

1. As a novel hydrogen sulfide-modulated agent, S-propargyl-L-cysteine (SPRC) is proven to be a potent cardioprotective candidate. Bioavailability and pharmacokinetics of SPRC (20?mg/kg) in beagle dogs after oral and intravenous administrations were investigated in this study. Plasma concentrations of SPRC were measured by a LC-MS/MS method.

2. Intravenous administration of SPRC (single dose) to beagle dogs gave a mean plasma half-life of 14.7?h, mean clearance of 0.4?ml min^?1 kg^?1 and mean apparent volume of distribution of 0.56?L/kg. Single oral administration was completely, fast absorbed (T_max= 0.33?±?0.20?h) with a mean absolute availability of 112% and mean plasma half-life of 16.5?h.

3. Multiple oral administration (once daily for 10 consecutive days) of SPRC to dogs resulted in steady state plasma drug concentration being reached after seven doses and didn’t cause obvious accumulation. No significant difference was found between the single and multiple pharmacokinetic parameters.

     

Pharmacokinetics and anti-inflammatory pharmacodynamics of prednisolone in cynomolgus monkey

1. The purpose was to investigate whether the pharmacokinetics and pharmacodynamics of prednisolone in the non-human primate was an appropriate surrogate for man.

2. After single intravenous doses of 0.03, 0.3, and 3?mg kg^?1, prednisolone demonstrated a dose-dependent clearance and volume of distribution. When corrected for concentration-dependent protein binding, the free clearance was linear at the tested dose levels. The protein binding-corrected volume of distribution was similar across doses. The serum half-life was estimated as being between 2 and 4?h. Prednisolone exhibits near complete inhibition of the cytokines TNF-α, IL-1β, IL-6 and IL-8 with very similar IC₅₀ estimates from 0.09 to 0.16 μg ml^?1 (from 0.24 to 0.44 μM).

3. The monkey demonstrated a similar pharmacokinetics–pharmacodynamics profile of prednisolone when compared with man (from the literature).

     

Mechanism-based inhibition of human cytochrome P4503A4 by domperidone

1. Domperidone was evaluated in direct and time-dependent cytochrome P450 (CYP) 3A inhibition assays in human liver microsomes with midazolam and testosterone as probe substrates.

2. Domperidone was found to be a modest mechanism-based inhibitor of human and rat CYP3A. For human CYP3A, the inactivation constant (K_I) is 12 μM, and the maximum inactivation rate (k_inact) is 0.037?min^?1.

3. A rat interaction study was conducted between midazolam and either a single dose or five daily doses of domperidone. Although a single oral dose of 10?mg kg^?1 domperidone did not affect the pharmacokinetics of 10?mg kg^?1 oral midazolam, five daily oral doses of domperidone almost doubled the area under the plasma concentration versus time curve (AUC) of midazolam, and increased the maximum plasma concentration (C_max) of midazolam by 72%.

4. Based on the simulation and rat in vitro–in vivo extrapolation, it is predicted that co-administration of domperidone in humans could modestly increase (approximately 50%) the exposure of drugs that are primarily cleared by CYP3A.

     

Role of individual human cytochrome P450 enzymes in the in vitro metabolism of hydromorphone

1. The aim was to identify the individual human cytochrome P450 (CYP) enzymes responsible for the in vitro N-demethylation of hydromorphone and to determine the potential effect of the inhibition of this metabolic pathway on the formation of other hydromorphone metabolites.

2. Hydromorphone was metabolized to norhydromorphone (apparent K_m = 206?? 822?μM, V_max = 104 ? 834?pmol?min^?1?mg^?1 protein) and dihydroisomorphine (apparent K_m = 62 ? 557?μM, V_max = 17 ? 122?pmol?min^?1?mg^?1 protein) by human liver microsomes.

3. In pooled human liver microsomes, troleandomycin, ketoconazole and sulfaphenazole reduced norhydromorphone formation by an average of 45, 50 and 25%, respectively, whereas furafylline, quinidine and omeprazole had no effect. In an individual liver microsome sample with a high CYP3A protein content, troleandomycin and ketoconazole inhibited norhydromorphone formation by 80%.

4. The reduction in norhydromorphone formation by troleandomycin and ketoconazole was accompanied by a stimulation in dihydroisomorphine production.

5. Recombinant CYP3A4, CYP3A5, CYP2C9 and CYP2D6, but not CYP1A2, catalysed norhydromorphone formation, whereas none of these enzymes was active in dihydroisomorphine formation.

6. In summary, CYP3A and, to a lesser extent, CYP2C9 catalysed hydromorphone N-demethylation in human liver microsomes. The inhibition of norhydromorphone formation by troleandomycin and ketoconazole resulted in a stimulation of microsomal dihydroisomorphine formation.

     

In vitro stability and metabolism of O2′, O3′, O5′-tri-acetyl-N6-(3-hydroxylaniline) adenosine in rat,dog and human plasma: Chemical hydrolysis and role of plasma esterases

1. O2′, O3′, O5′-tri-acetyl-N⁶-(3-hydroxylaniline)adenosine (WS070117), a new structure-type lipid regulator, is being developed in pre-clinical study. In order to monitor drug kinetics it is essential to understand pre-analytical factors that may affect drug assay.

2. In vitro stability and metabolism were investigated using high-performance liquid chromatography (HPLC) method in this study. The hydrolysis products were identified by HPLC–mass spectrometry (MS)/MS method. The esterases involved in WS070117 hydrolysis was assigned via inhibition rate assay.

3. It was found that WS070117 was chemically unstable in alkaline solutions compared to acidic and near neutral solutions. Enzymatic hydrolysis was even more rapid. Hydrolytic rate constants differ between species, being 4.24, 5.96?×?10^?3 and 6.85?×?10^?2 min^?1 in rat, dog and human plasma at 37°C, respectively. The hydrolysis was catalyzed by plasma esterase because NaF (sodium fluoride: a general esterase inhibitor) inhibited WS070117 hydrolysis and metabolite production. Hydrolysis was fast in rat plasma and was catalysed by carboxylesterase and butyrylcholinesterase. In dog plasma, carboxylesterase, butyrylcholinesterase and paraoxonase were mainly responsible. Butyrylcholinesterase was the major esterase involved in WS070117 hydrolysis in human plasma. The WS070117 hydrolysis in plasma proceeded by gradual loss of acetyl groups.

4. The knowledge of in vitro drug stability and metabolic pathways identified in this study will be essential for future pre-clinical and clinical pharmacokinetics studies.

     

Comparison of intrinsic metabolic clearance in fresh and cryopreserved human hepatocytes

1. We compared the intrinsic clearance (CL_int) of a number of substrates in suspensions of fresh and cryopreserved human hepatocytes from seven donors.

2. CL_int values for a cocktail incubation of phenacetin, diclofenac, diazepam, bufuralol, midazolam, and hydroxycoumarin were 4.9?±?3.4, 18?±?7.2, 5.1?±?4.9, 6.3?±?3.3, 9.8?±?5.8 and 22?±?14?μl min^?1/10⁶ cells, respectively, and they correlated well with corresponding CL_int values using cryopreserved hepatocytes from 25 different donors.

3. CL_int values of each cocktail substrate and 20 AstraZeneca new chemical entities were compared in fresh and cryopreserved hepatocytes from the same three donors. There was a statistically significant correlation between CL_int in fresh and cryopreserved hepatocytes for each of the three livers (p?int values was 1.03.

4. In conclusion, the results add further support to the use of cryopreserved human hepatocytes as a screening model for the intrinsic clearance of new chemical entities.

     

Pharmacokinetics and first-pass effects of ϵ-acetamidocaproic acid after administration of zinc acexamate in rats

1. Zinc acexamate (ZAC) is ionized to zinc and ?-acetamidocaproic acid (AACA). Thus, the pharmacokinetics and tissue distribution of zinc and AACA after intravenous (50?mg kg^?1) and oral (100?mg kg^?1) administration of ZAC were evaluated in rats. Also the pharmacokinetics of AACA after intravenous (10, 20, 30, and 50?mg kg^?1) and oral (20, 50, and 100?mg kg^?1) administration of ZAC and the first-pass extractions of AACA at a ZAC dose of 20?mg kg^?1 were evaluated in rats.

2. After oral administration of ZAC (20?mg kg^?1), approximately 0.408% of the oral dose was not absorbed, the F value was approximately 47.1%, and the hepatic and gastrointestinal (GI) first-pass extractions of AACA were approximately 8.50% and 46.4% of the oral dose, respectively. The incomplete F value of AACA was mainly due to the considerable GI first-pass extraction in rats.

3. Affinity of rat tissues to zinc and AACA was low—the tissue-to-plasma (T/P) ratios were less than unity. The equilibrium plasma-to-blood cells partition ratios of AACA were independent of initial blood ZAC concentrations of 1, 5, and 10?µg ml^?1—the mean values were 0.481, 0.490, and 0.499, respectively. The bound fractions of zinc and AACA to rat plasma were 96.6% and 39.0%, respectively.

     

Pharmacokinetics of lipoyl vildagliptin,a novel dipeptidyl peptidase IV inhibitor after oral administration in rats

1. The pharmacokinetics of lipoyl vildagliptin, a novel dipeptidyl peptidase IV (DPP IV) inhibitor, was studied in rats after oral administration for developing it as an antidiabetic agent.

2. A liquid chromatography-tandem mass spectroscopy (LC-MS/MS) method was developed to determine lipoyl vildagliptin in rat plasma. After an overnight fasting, rats were orally given lipoyl vildagliptin. Following a single oral dose of 25, 50, and 100?mg·kg^?1, T_max values were from 1.25 to 1.84?h, CL/F values were around 100?l h^?1 kg^?1. In the dose range, C_max values (63.9–296?μg·l^?1) and AUC_0–∞values (260–1214?μg·h·l^?1) were proportional to the doses.

3. In conclusion, this LC-MS/MS method for the determination of lipoyl vildagliptin in rat plasma was selective and sensitive. In rats, lipoyl vildagliptin displayed linear pharmacokinetics after a single oral dose in the range of 25–100?mg·kg^?1. Lipoyl vildagliptin might have very high CL/F values and V_d/F values, which indicated that the bioavailability of this drug might be low or lipoyl vildagliptin might distribute extensively or accumulate in tissues in view of its high liposolubility.

     

Prediction of animal clearance using naïve Bayesian classification and extended connectivity fingerprints

1. In silico models were developed for predicting high animal clearance using naïve Bayesian classification and extended connectivity fingerprints. Validation and test sets were created from a structurally diverse database of mouse, rat, dog, and monkey clearance (CL) representing approximately 20 000 unique compounds. Model performance was compared with experimental predictors used widely in drug discovery, namely in vitro intrinsic clearance (CL_i) and CL from a lower preclinical species.

2. The Bayesian model for dog CL was a better predictor than experimental rat or mouse CL. The Bayesian model for rat CL performed at least as well as mouse CL. Bayesian models outperformed mouse, rat, and monkey CL_i for predicting mouse, rat, and monkey CL, respectively.

3. These models can be used to optimize chemical libraries, direct new chemical synthesis and increase efficiency of screening cascades for lead optimization while reducing overall drug discovery cost, time and animal usage.

     

Pharmacokinetics of PSC 833 (valspodar) in its Cremophor EL formulation in rat

1. Valspodar is a P-glycoprotein inhibitor widely used in preclinical and clinical studies for overcoming multidrug resistance. Despite this, the pharmacokinetics of valspodar in rat, a commonly used animal model, have not been reported. Here, we report on the pharmacokinetics of valspodar in Sprague–Dawley rats following intravenous and oral administration of its Cremophor EL formulation, which has been used for humans in clinical trials.

2. After intravenous doses, valspodar displayed properties of slow clearance and a large volume of distribution. Its plasma unbound fraction was around 15% in the Cremophor EL formulation used in the study. After 10?mg kg^?1 orally it was rapidly absorbed with an average maximal plasma concentration of 1.48?mg l^?1 within approximately 2?h. The mean bioavailability of valspodar was 42.8%.

3. In rat, valspodar showed properties of low hepatic extraction and wide distribution, similar to that of its structural analogue cyclosporine A.

     

Absorption,distribution, metabolism and excretion of peginesatide,a novel erythropoiesis-stimulating agent,in rats

1. The pharmacokinetics (PK) (absorption, distribution, metabolism, excretion) of peginesatide, a synthetic, PEGylated, investigational, peptide-based erythropoiesis-stimulating agent (ESA), was evaluated in rats. The PK profile was evaluated at 0.1–5 mg·kg^?1 IV using unlabeled or [¹⁴C]-labeled peginesatide. Mass balance, tissue distribution and metabolism were evaluated following IV administration of 5 mg·kg^?1 [¹⁴C]-peginesatide, with tissue distribution also evaluated by quantitative whole-body autoradiography (QWBA) following an IV dose of 17 mg·kg^?1 [¹⁴C]-peginesatide.

2. Plasma clearance was slow and elimination was biphasic with unchanged peginesatide representing >90% of the total radioactivity of the total radioactive exposure. Slow uptake of the radiolabeled compound from the vascular compartment into the tissues was observed.

3. Biodistribution to bone marrow and extramedullary hematopoietic sites, and to highly vascularized lymphatic and excretory tissues occurred.

4. A predominant degradation event to occur in vivo was the loss of one PEG chain from the branched PEG moiety to generate mono-PEG.

5. Renal excretion was the primary mechanism (41%) of elimination, with parent molecule (67%) the major moiety excreted.

6. In conclusion, elimination of [¹⁴C]-peginesatide-derived radioactivity was extended, retention preferentially occurred at sites of erythropoiesis (bone marrow), and urinary excretion was the primary elimination route.

     

Metabolism-related liabilities of a potent histone deacetylase (HDAC) inhibitor and relevance of the route of administration on its metabolic fate

1. Compound A [1-methyl-N-{(1S)-1-[5-(2-naphthyl)-1H-imidazol-2-yl]-7-oxooctyl}piperidine-4-carboxamide is a potent class I histone deacetylase (HDAC) inhibitor that demonstrated good antiproliferative activity against human tumour cell lines of different origin.

2. This compound showed high in vivo clearance in rats (160?ml min^?1 kg^?1) due to metabolism. The main metabolite detected in urine after intravenous dosing was characterized as a dihydrohydroxy S-mercapturic acid conjugate. Following oral dosing, however, the mercapturic acid derivative was no longer the main metabolite but the major metabolites were mono- and di-glucuronide conjugates of oxidized species having a mass shift of +34 m/z with respect to the parent.

3. Comparison of plasma concentration after intra-arterial infusion and intravenous infusion and incubation with microsomes from different tissues (liver, kidney, small intestine and lung) in the presence of β-nicotinamide adenine dinucleotide phosphate (NADPH) indicated that the compound was highly cleared by the lung.

4. Oxidation of the naphthalene moiety was demonstrated to be the cause of the high in vivo clearance of compound A and the potential for bioactivation of this group was flagged.

     

Effect of ursodeoxycholic acid on the pharmacokinetics of midazolam and CYP3A in the liver and intestine of rats

1. The elimination half-life of midazolam administered intravenously (5 mg kg^?1) or orally (15 mg kg^?1) was significantly decreased by 70% and 73%, respectively, 24 h after a single oral administration of ursodeoxycholic acid (UDCA, 300 mg kg^?1) in rats. In the liver there was a significant enhancement of the hydroxylation of midazolam in the microsomes and expression of cytochrome P450 (CYP) 3A1 messenger RNA (mRNA) and CYP3A2 mRNA.

2. The C_max and area under the curve (AUC)_0–∞ of midazolam were significantly (1.8–2.3 fold) increased by the single oral treatment with UDCA (100 and 300 mg kg^?1). Thus, the oral bioavailability, estimated from the AUC_0–∞, of midazolam administered intravenously and orally was significantly (1.8- and 2.3-fold, respectively) increased by the treatment with UDCA.

3. Repeated administration of UDCA (300 mg kg^?1 day^?1) for 7 days did not alter the pharmacokinetics of midazolam administered intravenously or orally, and the expression of mRNA for CYP3As in the rat liver.

4. The study has shown that a single administration of UDCA in rats induces significant hepatic CYP3A activity and increases significantly the oral bioavailability of midazolam. Such effects on the pharmacokinetics of midazolam were little observed on the repeated administration of UDCA.

     

Preclinical assessment of the ADME,efficacy and drug-drug interaction potential of a novel NAMPT inhibitor

1. GNE-617 (N-(4-((3,5-difluorophenyl)sulfonyl)benzyl)imidazo[1,2-a]pyridine-6-carboxamide) is a potent, selective nicotinamide phosphoribosyltransferase (NAMPT) inhibitor being explored as a potential treatment for human cancers.

2. Plasma clearance was low in monkeys and dogs (9.14?mL min^?1?kg^?1 and 4.62?mL min^?1?kg^?1, respectively) and moderate in mice and rats (36.4?mL min^?1?kg^?1 and 19.3?mL min^?1?kg^?1, respectively). Oral bioavailability in mice, rats, monkeys and dogs was 29.7, 33.9, 29.4 and 65.2%, respectively.

3. Allometric scaling predicted a low clearance of 3.3?mL min^?1?kg^?1 and a volume of distribution of 1.3?L kg^?1 in human.

4. Efficacy (57% tumor growth inhibition) in Colo-205 CRC tumor xenograft mice was observed at an oral dose of 15?mg/kg BID (AUC?=?10.4?µM h).

5. Plasma protein binding was moderately high. GNE-617 was stable to moderately stable in vitro. Main human metabolites identified in human hepatocytes were formed primarily by CYP3A4/5. Transporter studies suggested that GNE-617 is likely a substrate for MDR1 but not for BCRP.

6. Simcyp^® simulations suggested a low (CYP2C9 and CYP2C8) or moderate (CYP3A4/5) potential for drug-drug interactions. The potential for autoinhibition was low.

7. Overall, GNE-617 exhibited acceptable preclinical properties and projected human PK and dose estimates.

     

In vivo metabolism and kinetics of ethylene glycol monobutyl ether and its metabolites, 2-butoxyacetaldehyde and 2-butoxyacetic acid,as measured in blood,liver and forestomach of mice

1. Ethylene glycol monobutyl ether (EGBE) causes forestomach hyperplasia and neoplasia in mice when administered chronically by inhalation.

2. The study was initiated to test the physiologically based pharmacokinetic (PBPK) model prediction that 2-butoxyacetaldehyde (BAL), a transient, labile intermediate in the oxidation of EGBE to butoxyacetic acid (BAA), is unlikely to achieve concentrations sufficient to cause DNA damage in target tissues.

3. Male and female B6C3F1 mice were administered a high oral dose of EGBE (600?mg?kg^?1), and tissues were collected at 5, 15, 45 and 90?min following the dose. The tissues were processed for determination of EGBE, BAL and BAA by gas chromatography-mass spectrometry.

4. BAL was detected at low concentrations in all tissues sampled and at all time points following EGBE administration (about 0.3–33?μM). BAL concentrations were highest in the initial samples (5?min) in all tissues and declined from that point.

5. BAL concentrations in liver and forestomach tissues corresponded to the peak concentrations predicted by an already published PBPK model, and are higher than BAL concentrations that could be achieved by inhalation exposure to EGBE.

6. Mouse inhalation exposure to EGBE is therefore unlikely to generate BAL concentrations in tissues sufficient to initiate a carcinogenic response.