Quantitative evaluation of hepatic and intestinal induction of CYP3A in clinical practice.

This is the first report quantitatively evaluating the clinical induction of CYP3A in the liver and the intestine.To evaluate hepatic induction, we collected literature data on endogenous biomarkers of hepatic CYP3A induction which we then used to calculate the fold-induction (inducer-mediated change in biomarker level). Literature data on decreases in the area under the curve (AUC) of alfentanil, a CYP3A substrate, caused by CYP3A inducers were also collected. We used the hepatic intrinsic clearance of alfentanil to calculate the hepatic induction ratio (inducer-mediated change in intrinsic clearance). For intestinal induction, the intestinal bioavailability (Fg) of alfentanil was used to calculate the intestinal induction ratio. We determined in vivo maximum induction (Emax) and the average unbound plasma concentration (Cav,u) required for half the maximum induction (EC50) for inducers using an Emax model analysis.In our results, fold-induction was comparable to the induction ratio at several inducer concentrations, and almost the maximum induction was achieved by a therapeutic dose. Induction ratios in the intestine were similar to the liver.Our findings suggest that, by knowing only hepatic induction ratios for common inducers, we can quantitatively predict the decreases in the AUC of substrates by CYP3A induction.

Simple Evaluation Method for CYP3A4 Induction from Human Hepatocytes: The Relative Factor Approach with an Induction Detection Limit Concentration Based on the Emax Model.

We investigated the robustness and utility of the relative factor (RF) approach based on the maximum induction effect (Emax) model, using the mRNA induction data of 10 typical CYP3A4 inducers in cryopreserved human hepatocytes. The RF value is designated as the ratio of the induction detection limit concentration (IDLC) for a standard inducer, such as rifampicin (RIF) or phenobarbital (PB), to that for the compound (e.g., RFRIF is IDLCRIF/IDLCcpd; RFPB is IDLCPB/IDLCcpd). An important feature of the RF approach is that the profiles of the induction response curves on the logarithmic scale remain unchanged irrespective of inducers but are shifted parallel depending on the EC50 values. A key step in the RF approach is to convert the induction response curve by finding the IDLC of a standard inducer. The relative induction score was estimated not only from Emax and EC50 values but also from those calculated by the RF approach. These values showed good correlation, with a correlation coefficient of more than 0.974, which revealed the RF approach to be a robust analysis irrespective of its simplicity. Furthermore, the relationship between RFRIF or RFPB multiplied by the steady-state unbound plasma concentration and the in vivo induction ratio plotted using 10 typical inducers gives adequate thresholds for CYP3A4 drug-drug interaction risk assessment. In light of these findings, the simple RF approach using the IDLC value could be a useful method to adequately assess the risk of CYP3A4 induction in humans during drug discovery and development without evaluation of Emax and EC50.

Preclinical evaluation of the potential for cytochrome P450 inhibition and induction of the selective ALK inhibitor, alectinib.

1. A novel selective anaplastic lymphoma kinase (ALK) inhibitor, alectinib, has shown remarkable efficacy and safety in patients with ALK-positive non-small-cell lung cancer (NSCLC). The purpose of this study was to evaluate in vitro the potential to inhibit and induce cytochrome P450 (CYP) isoforms for alectinib and its major metabolite M4. 2. Alectinib and M4 did not show the meaningful direct inhibition of six major CYP isoforms (CYP1A2, 2B6, 2C9, 2C19, 2D6 and 3A4) in human liver microsomes (HLM). Alectinib, but not M4, competitively inhibited CYP2C8, by which few marketed drugs are exclusively metabolized, with an inhibition constant of 1.98 µM. 3. Out of the seven CYP isoforms in HLM, alectinib and M4 showed time-dependent inhibition (TDI) of only CYP3A4, which suggests low TDI potential due to low inactivation efficiency. 4. Alectinib exhibited quite smaller induction of mRNA expression of CYP1A2, 2B6 and 3A4 genes in human hepatocytes compared to the respective positive controls, suggesting a low potential of enzyme induction. 5. In summary, the risk of alectinib causing drug-drug interactions with coadministered drugs is expected to be low due to the weak potential of CYP inhibition and induction estimated in the preclinical studies.

Discovery of [5-Amino-1-(2-methyl-3H-benzimidazol-5-yl)pyrazol-4-yl]-(1H-indol-2-yl)methanone (CH5183284/Debio 1347), An Orally Available and Selective Fibroblast Growth Factor Receptor (FGFR) Inhibitor.

The fibroblast growth factor receptor (FGFR) family of receptor tyrosine kinases regulates multiple biological processes, such as cell proliferation, migration, apoptosis, and differentiation. Various genetic alterations that drive activation of the receptors and the pathway are associated with tumor growth and survival; therefore, the FGFR family represents an attractive therapeutic target for treating cancer. Here, we report the discovery and the pharmacological profiles of 8 (CH5183284/Debio 1347), an orally available and selective inhibitor of FGFR1, FGFR2, and FGFR3. The chemical modifications, which were guided by 3D-modeling analyses of the inhibitor and FGFRs, led to identifying an inhibitor that is selective to FGFR1, FGFR2, and FGFR3. In in vitro studies and xenograft models in mice, 8 shows antitumor activity against cancer cell lines that harbor genetically altered FGFRs. These results support the potential therapeutic use of 8 as a new anticancer agent.

The fibroblast growth factor receptor genetic status as a potential predictor of the sensitivity to CH5183284/Debio 1347, a novel selective FGFR inhibitor.

The FGF receptors (FGFR) are tyrosine kinases that are constitutively activated in a subset of tumors by genetic alterations such as gene amplifications, point mutations, or chromosomal translocations/rearrangements. Recently, small-molecule inhibitors that can inhibit the FGFR family as well as the VEGF receptor (VEGFR) or platelet-derived growth factor receptor (PDGFR) family displayed clinical benefits in cohorts of patients with FGFR genetic alterations. However, to achieve more potent and prolonged activity in such populations, a selective FGFR inhibitor is still needed. Here, we report the identification of CH5183284/Debio 1347, a selective and orally available FGFR1, FGFR2, and FGFR3 inhibitor that has a unique chemical scaffold. By interacting with unique residues in the ATP-binding site of FGFR1, FGFR2, or FGFR3, CH5183284/Debio 1347 selectively inhibits FGFR1, FGFR2, and FGFR3 but does not inhibit kinase insert domain receptor (KDR) or other kinases. Consistent with its high selectivity for FGFR enzymes, CH5183284/Debio 1347 displayed preferential antitumor activity against cancer cells with various FGFR genetic alterations in a panel of 327 cancer cell lines and in xenograft models. Because of its unique binding mode, CH5183284/Debio 1347 can inhibit FGFR2 harboring one type of the gatekeeper mutation that causes resistance to other FGFR inhibitors and block FGFR2 V564F-driven tumor growth. CH5183284/Debio 1347 is under clinical investigation for the treatment of patients harboring FGFR genetic alterations.

Estimating the clinical risk of hypertension from VEGF signal inhibitors by a non-clinical approach using telemetered rats.

Anti-angiogenic drugs that target Vascular Endothelial Growth Factor (VEGF) signaling pathways caused hypertension as an adverse effect in clinical studies. Since the hypertension may limit the benefit provided for patients, the demand for non-clinical research that predicts the clinical risk of the hypertension has risen greatly. To clarify whether non-clinical research using rats can appropriately estimate the clinical risk of hypertension caused by VEGF signal inhibitors, we investigated the hemodynamic effects and pharmacokinetics (PK) of the VEGF signal inhibitors cediranib (0.1, 3, and 10 mg/kg), sunitinib (5, 10, and 40 mg/kg), and sorafenib (0.1, 1, and 5 mg/kg) in telemetered rats and examined the correlation between the non-clinical and the clinical hypertensive effect. The VEGF signal inhibitors significantly elevated blood pressure (BP) in rats within a few days of the initiation of dosing, and levels recovered after dosing ended. The trend of the hypertension was similar to that in clinical studies. We found that the AUC at which BP significantly increased by approximately 10 mmHg in rats was comparable to the clinical AUC at which moderate to severe hypertension occurred. These results represent correlations between the non-clinical and the clinical hypertensive effect of VEGF signal inhibitors, suggesting that non-clinical research using telemetered rats would be an effective approach to predict the clinical risk of hypertension caused by VEGF signal inhibitors.

Enantioselective synthesis of derivatives and structure-activity relationship study in the development of NA255 as a novel host-targeting anti-HCV agent.

Hepatitis C virus (HCV) infection represents a serious health-care problem. Previously we reported the identification of NA255 from our natural products library using a HCV sub-genomic replicon cell culture system. Herein, we report how the absolute stereochemistry of NA255 was determined and an enantioselective synthetic method for NA255 derivatives was developed. The structure-activity relationship of the NA255 derivatives and rat pharmacokinetic profiles of the representative compounds are disclosed.

Comparison of in vitro metabolic conversion of capecitabine to 5-FU in rats, mice, monkeys and humans--toxicological implications.

Capecitabine is an oral anticancer prodrug which is converted to 5-fluorouracil (5-FU) via 3 enzymatic steps, these being 5'-deoxy-5-fluorocytidine (5'-DFCR), 5'-deoxy-5-fluorouridine (5'-DFUR), and finally 5-FU by carboxylesterase (CES), cytidine deaminase (CDA), and thymidine phosphorylase (TP), respectively. Because rats, mice and monkeys are used for preclinical safety studies, we investigated the in vitro conversion from capecitabine to 5-FU by hepatic and intestinal mucosal microsomes and cytosols, to compare their metabolic activity to that of humans. Capecitabine was hydrolyzed to 5'-DFCR in hepatic and intestinal mucosal microsomes in these animal species. In humans and monkeys, CL(int) (V(max)/K(m)) for the hydrolysis of capecitabine in intestine (expressed as µl/min/g tissue) was much lower than that in hepatic microsomes but, in rats and mice, CL(int) was higher in intestine than in liver. In humans and monkeys, similar K(m) values and inhibition patterns by tetrahydrouridine (THU) a CDA inhibitor, were observed in CDA activity of hepatic and intestinal cytosols. However, rats showed very low CDA activity and mice showed non-Michaelis-Menten kinetics and a different inhibition pattern by THU. K(m) values for TP activity were almost similar in rats, mice, monkeys and humans. In conclusion, it was confirmed that monkeys are a suitable animal model for the safety assessment of capecitabine in terms of metabolic enzymes and it was suggested that higher toxic incidences in mouse small intestine were related to high hydrolytic activity of capecitabine in the small intestine.

Effect of dose regimen on the toxicity of 2'-deoxy-2'-methylidenecytidine (DMDC) in monkeys.

2'-deoxy-2'-methylidenecytidine (DMDC) is a potential anticancer deoxycytidine analog of cytosine arabinoside. Using monkeys, we conducted a 4-week toxicity study with toxicokinetics of DMDC at 1, 3, and 10 mg/kg/day and a dose-regimen study of three different schedules of once-daily administration (5 mg/kg/day) for 1 week every 2 weeks, 2 weeks every 4 weeks, and 3 weeks every 4 weeks. Deaths, myelosuppression, intestinal toxicity, and swelling of palm and sole skin were observed by oral DMDC treatment at 10 mg/kg/day in 4-week repeated toxicity study; however, no skin disorders have been reported in humans. No notable changes were observed at 1 and 3 mg/kg/day. The curves of dose vs. AUC and the AUC at MTD in monkey are similar to those in humans. In the dose-regimen study, all the toxicities were reversible but more severe toxicity was observed with the longer administration periods. One-week interruption showed sufficient recovery of decreased WBC in dosing regimens of 1-week-on/1-week-off and 2-weeks-on/2-weeks-off. A 2-week recovery period was almost sufficient for the recovery of decreased RBC, HCT, and skin disorders in the 2-weeks-on/2-weeks-off regimen. Therefore, once-daily for 2 weeks every 4 weeks was concluded to be the optimal dose regimen. In summary, myelosuppression, intestinal toxicity, and skin disorders were observed in DMDC treatment in monkeys, the relationship between AUC and toxicity in monkeys was close to that in humans, and in preclinical studies, it is advantageous to investigate optimal dose regimens using the appropriate species.

Relationship between AUC of 5'-DFUR and toxicity of capecitabine, fluoropyrimidine carbamate analogs, and 5'-DFUR in monkeys, mice, and rats.

Capecitabine is an oral fluoropyrimidine carbamate which is converted to 5-fluorouracil (5-FU) via 3 enzymatic step to 5'-deoxy-5-fluorocytidine (5'-DFCR), 5'-deoxy-5-fluorouridine (5'-DFUR), and finally 5-FU. We performed 4-week toxicity studies of capecitabine (N(4)-pentyloxycarbonyl-5'-deoxy-5-fluorouridine), galocitabine (trimethoxybenzyl-5'-deoxy-5-fluorocytidine), 4 different fluoropyrimidine carbamate analogs (R=butyl, isopentyl, propyl, or phenethyl), and 5'-DFUR in cynomolgus monkeys with toxicokinetic measurements of intact molecules, 5'-DFCR, and 5'-DFUR. Four-week toxicity data for capecitabine in rats and mice were also obtained for comparison. Capecitabine, galocitabine, butyl, and isopentyl analogs showed similar toxicities in hematopoietic and intestinal organs at 1.0 mmol/kg and the AUCs of 5'-DFUR were approximately 40 to 60 microg*hr/ml. These compounds showed slight toxicity at 0.5 mmol/kg and no toxicity at 0.1 mmol/kg, and AUCs of 5'-DFUR were approximately 30 and 5 microg*hr/ml, respectively. Propyl and phenethyl analogs showed slight toxicity at 1.0 mmol/kg and no toxicity at 0.5 mmol/kg, and AUCs of 5'-DFUR were approximately 30 and 10 microg*hr/ml, respectively. On the other hand, severe and slight-to-moderate toxicity was observed at 0.5 and 0.25 mmol/kg in 5'-DFUR-treated monkeys and AUCs of 5'DFUR were 35.6 and 5.2 microg*hr/ml, respectively. In mice and rats, the toxicity of capecitabine was less than in monkeys relative to dose, but 5'-DFUR AUCs were almost the same. In conclusion, 5'-DFUR AUC correlated with toxicity following oral administration of capecitabine and its analogs in monkeys, mice, and rats, although this relationship is not seen in humans. Capecitabine was less toxic in monkeys than oral 5'-DFUR according to dose (mmol/kg) and 5'-DFUR AUC.

Synthesis and biological activities of benzofuran antifungal agents targeting fungal N-myristoyltransferase.

The C-4 side chain modification of lead compound 1 has resulted in the identification of a potent and selective Candida albicans N-myristoyltransferase (CaNmt) inhibitor RO-09-4609, which exhibits antifungal activity against C. albicans in vitro. Further modification of its C-2 substituent has led to the discovery of RO-09-4879, which exhibits antifungal activity in vivo. The drug design is based on X-ray crystal analysis of a CaNmt complex with benzofuran derivative 4a. The optimization incorporates various biological investigations including a quasi in vivo assay and pharmacokinetic study. The computer aided drug design, synthesis, structure-activity relationships, and biological properties of RO-09-4879 are described in detail.

Design and synthesis of novel benzofurans as a new class of antifungal agents targeting fungal N-myristoyltransferase. Part 3.

A new series of acid-stable antifungal agents having strong inhibitory activity against Candida albicans N-myristoyltransferase (CaNmt) has been developed starting from acid-unstable benzofuranylmethyl aryl ether 2. The inhibitor design is based on X-ray crystallographic analysis of a CaNmt complex with aryl ether 3. Among the new inhibitors, pyridine derivative 8b and benzimidazole derivative 8k showed clear antifungal activity in a murine systemic candidiasis model.

Advantages of in vitro cytotoxicity testing by using primary rat hepatocytes in comparison with established cell lines.

We investigated and compared the cytotoxicity of 16 reference compounds in four in vitro systems: primary cultured rat hepatocytes, hepatoma HepG2 cell line, non-hepatic HeLa and Balb/c 3T3 cell lines. After 24 hr of exposure to the test compounds, the water-soluble tetrazolium salts WST-1 assay was used as an endpoint to evaluate cytotoxicity. Acetaminophen, diclofenac sodium cyclophosphamide and disulfiram displayed from 2 to more than 10 times higher IC50 values in three cell lines than in rat primary cultured hepatocytes. The cytotoxic effects of aspirin, amiodarone, clorfibiric acid, chlorpromazine, erythomycin, lithocholic acid, cisplatin and quinidine in rat hepatocytes were similar or 2 times stronger than those observed in cell lines. Ketoconazole resulted in the lowest IC50 value in the HeLa cell line. The data suggested that the compounds which are known to be metabolism-mediated liver toxicants have a differential hepatotoxicity in vitro and that primary cultured rat hepatocytes could represent a valuable tool for both screening and study of the effects of bio-transformation on the cytotoxicity of new chemical entities and xenobiotics in vitro.

Cassette dosing approach and quantitative structure-pharmacokinetic relationship study of antifungal N-myristoyltransferase inhibitors.

Pharmacokinetic (PK) parameters of N-myristoyltransferase (Nmt) inhibitors were measured, and a multivariate quantitative structure-pharmacokinetic relationship (QSPKR) model for predicting rat elimination half-life (t(1/2)) values was constructed. One hundred seven benzofuran derivatives have been selected as the data set for QSPKR analysis. The correlation between the t(1/2) values and 30 physicochemical descriptors was examined by a stepwise multiple linear regression method. The statistical analysis gives a significant QSPKR model (r = 0.843) with the following three variables: partial negative surface area (PNSA), atomic-based octanol/water partition coefficient (AlogP), and the number of rotational bonds (Rotlbonds). The QSPKR model obtained is predictive and simple, and would give a direction for designing new Nmt inhibitors having good PK profiles.

Design and synthesis of novel benzofurans as a new class of antifungal agents targeting fungal N-myristoyltransferase. Part 2.

Modification of the C-2 position of a benzofuran derivative 6 (RO-09-4609), an N-myristoyltransferase (Nmt) inhibitor, has led us to discover antifungal agents that are active in a murine systemic candidiasis model. The drug design is based on the analysis of a crystal structure of a Candida Nmt complex with 2. The optimization has been guided by various biological evaluations including a quasi in vivo assay and pharmacokinetic analysis.