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

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. 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. 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. 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). 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. Simcyp® simulations suggested a low (CYP2C9 and CYP2C8) or moderate (CYP3A4/5) potential for drug-drug interactions. The potential for autoinhibition was low. Overall, GNE-617 exhibited acceptable preclinical properties and projected human PK and dose estimates.

Prediction of Drug-Drug Interactions Arising from CYP3A induction Using a Physiologically Based Dynamic Model.

Using physiologically based pharmacokinetic modeling, we predicted the magnitude of drug-drug interactions (DDIs) for studies with rifampicin and seven CYP3A4 probe substrates administered i.v. (10 studies) or orally (19 studies). The results showed a tendency to underpredict the DDI magnitude when the victim drug was administered orally. Possible sources of inaccuracy were investigated systematically to determine the most appropriate model refinement. When the maximal fold induction (Indmax) for rifampicin was increased (from 8 to 16) in both the liver and the gut, or when the Indmax was increased in the gut but not in liver, there was a decrease in bias and increased precision compared with the base model (Indmax = 8) [geometric mean fold error (GMFE) 2.12 vs. 1.48 and 1.77, respectively]. Induction parameters (mRNA and activity), determined for rifampicin, carbamazepine, phenytoin, and phenobarbital in hepatocytes from four donors, were then used to evaluate use of the refined rifampicin model for calibration. Calibration of mRNA and activity data for other inducers using the refined rifampicin model led to more accurate DDI predictions compared with the initial model (activity GMFE 1.49 vs. 1.68; mRNA GMFE 1.35 vs. 1.46), suggesting that robust in vivo reference values can be used to overcome interdonor and laboratory-to-laboratory variability. Use of uncalibrated data also performed well (GMFE 1.39 and 1.44 for activity and mRNA). As a result of experimental variability (i.e., in donors and protocols), it is prudent to fully characterize in vitro induction with prototypical inducers to give an understanding of how that particular system extrapolates to the in vivo situation when using an uncalibrated approach.

In vitro and in vivo characterization of CYP inhibition by 1-aminobenzotriazole in rats.

1-Aminobenzotriazole (ABT) is a non-isoform specific, time-dependent inhibitor of cytochrome P450 (CYP) enzymes used extensively in preclinical studies to determine the relative contribution of oxidative metabolism. Although ABT has been widely used, the extent and duration of its inhibitory effect is not well understood. The purpose of this study is to characterize ABT inhibition of CYP in rats at both the hepatic and intestinal levels. In vivo studies using midazolam (p.o. and i.v.), as a probe for CYP activity, demonstrated that CYP inhibition was not complete even at the highest dose (300 mg/kg). Additional in vivo studies demonstrated that even at 26 h following ABT administration, there was significant CYP inhibition remaining. In vitro studies, conducted in both rat liver microsomes and rat hepatocytes, confirm that ABT is a time-dependent inhibitor of rat CYP orthologs. However, in rat liver microsomes, there was more than 15% CYP activity remaining following a 60 min preincubation at 2 mm ABT and 5-10% of CYP activity was remaining in rat hepatocytes suspended in rat plasma following a 60 min preincubation at 2 mm ABT. 1-Aminobenzotriazole is a useful tool in elucidating the oxidative component of metabolism in preclinical species; however, conclusions made from the preclinical use of ABT should not operate under the assumption that CYP enzymatic activity is completely inhibited. Copyright © 2016 John Wiley & Sons, Ltd.

1-Aminobenzotriazole coincubated with (S)-warfarin results in potent inactivation of CYP2C9.

1-Aminobenzotriazole (ABT) is a nonselective, mechanism-based inactivator of cytochrome P450 (P450) and a useful tool compound to discern P450- from non-P450-mediated metabolism. ABT effectively inactivates major human P450 isoforms, with the notable exception of CYP2C9. Here we propose that ABT preferentially binds to the warfarin-binding pocket in the CYP2C9 active-site cavity; thus, ABT bioactivation and subsequent inactivation is not favored. Therefore, coincubation with (S)-warfarin would result in displacement of ABT from the warfarin-binding pocket and subsequent binding to the active site, converting ABT into a potent inactivator of CYP2C9. To test this hypothesis, in vitro studies were conducted using various coincubation combinations of ABT and (S)-warfarin or diclofenac to modulate the effectiveness of CYP2C9 inactivation by ABT. Coincubation of ABT with (S)-warfarin (diclofenac probe substrate) resulted in potent inactivation, whereas weak inactivation was observed following coincubation of ABT with diclofenac [(S)-warfarin probe substrate]. The kinetic parameters of time-dependent inhibition of ABT for CYP2C9 in the absence and presence of (S)-warfarin (20 µM) were 0.0826 and 0.273 min(-1) for kinact and 3.49 and 0.157 mM for KI, respectively. In addition, a 73.4-fold shift was observed in the in vitro potency (kinact/KI ratio), with an increase from 23.7 ml/min/mmol (ABT alone) to 1740 ml/min/mmol [ABT with (S)-warfarin (20 µM)]. These findings were supported by in silico structural modeling, which showed ABT preferentially binding to the warfarin-binding pocket and the displacement of ABT to the active site in the presence of (S)-warfarin.

Discovery of potent and efficacious urea-containing nicotinamide phosphoribosyltransferase (NAMPT) inhibitors with reduced CYP2C9 inhibition properties.

Potent, reversible inhibition of the cytochrome P450 CYP2C9 isoform was observed in a series of urea-containing nicotinamide phosphoribosyltransferase (NAMPT) inhibitors. This unwanted property was successfully removed from the described inhibitors through a combination of structure-based design and medicinal chemistry activities. An optimized compound which did not inhibit CYP2C9 exhibited potent anti-NAMPT activity (17; BC NAMPT IC50=3 nM; A2780 antiproliferative IC50=70 nM), good mouse PK properties, and was efficacious in an A2780 mouse xenograft model. The crystal structure of this compound in complex with the NAMPT protein is also described.

Drug-drug interaction potential of marketed oncology drugs: in vitro assessment of time-dependent cytochrome P450 inhibition, reactive metabolite formation and drug-drug interaction prediction.

PURPOSE: To evaluate 26 marketed oncology drugs for time-dependent inhibition (TDI) of cytochrome P450 (CYP) enzymes. Evaluate TDI-positive drugs for potential to generate reactive intermediates. Assess clinical drug-drug interaction (DDI) risk using static mechanistic models. METHODS: Human liver microsomes and CYP-specific probes were used to assess TDI in a dilution shift assay followed by generation of K(I) and k(inact). Reactive metabolite trapping studies were performed with stable label probes. Static mechanistic model was used to predict DDI risk using a 1.25-fold AUC increase as a cut-off for positive DDI. RESULTS: Negative TDI across CYPs was observed for 13/26 drugs; the rest were time-dependent inhibitors of, predominantly, CYP3A. The k(inact)/K(I) ratios for 11 kinase inhibitors ranged from 0.7 to 42.2 ml/min/µmol. Stable label trapping agent-drug conjugates were observed for ten kinase inhibitors. DDI predictions gave no false negatives, one true negative, four false positives and three true positives. The magnitude of DDI was overestimated irrespective of the inhibitor concentration selected. CONCLUSIONS: 13/26 oncology drugs investigated showed TDI potential towards CYP3A, formation of reactive metabolites was also observed. An industry standard static mechanistic model gave no false negative predictions but did not capture the modest clinical DDI potential of kinase inhibitors.

Application of IVIVE and PBPK modeling in prospective prediction of clinical pharmacokinetics: strategy and approach during the drug discovery phase with four case studies.

Prospective simulations of human pharmacokinetic (PK) parameters and plasma concentration-time curves using in vitro in vivo extrapolation (IVIVE) and physiologically based pharmacokinetic (PBPK) models are becoming a vital part of the drug discovery and development process. This paper presents a strategy to deliver prospective simulations in support of clinical candidate nomination. A three stage approach of input parameter evaluation, model selection and multiple scenario simulation is utilized to predict the key components influencing human PK; absorption, distribution and clearance. The Simcyp® simulator is used to illustrate the approach and four compounds are presented as case studies. In general, the prospective predictions captured the observed clinical data well. Predicted C(max) was within 2-fold of observed data for all compounds and AUC was within 2-fold for all compounds following a single dose and three out of four compounds following multiple doses. Similarly, t(max) was within 2-fold of observed data for all compounds. However, C(last) was less accurately captured with two of the four compounds predicting C(last) within 2-fold of observed data following a single dose. The trend in results was towards overestimation of AUC and C(last) , this was particularly apparent for compound 2 for which clearance was likely underestimated via IVIVE. The prospective approach to simulating human PK using IVIVE and PBPK modeling outlined here attempts to utilize all available in silico, in vitro and in vivo preclinical data in order to determine the most appropriate assumptions to use in prospective predictions of absorption, distribution and clearance to aid clinical candidate nomination.

BB-301: a silence and replace AAV-based vector for the treatment of oculopharyngeal muscular dystrophy.

Oculopharyngeal muscular dystrophy (OPMD) is a rare autosomal dominant disease that results from an alanine expansion in the N-terminal domain of Poly-A Binding Protein Nuclear-1 (PABPN1). We have recently demonstrated that a two-vector gene therapy strategy significantly ameliorated the pathology in a mouse model of OPMD. This approach entailed intramuscular injection of two recombinant adeno-associated viruses (AAVs), one expressing three short hairpin RNAs (shRNAs) to silence both mutant and wild-type PABPN1 and one expressing a codon-optimized version of PABPN1 that is insensitive to RNA interference. Here we report the continued development of this therapeutic strategy by delivering "silence and replace" sequences in a single AAV vector named BB-301. This construct is composed of a modified AAV serotype 9 (AAV9) capsid that expresses a unique single bifunctional construct under the control of the muscle-specific Spc5-12 promoter for the co-expression of both the codon-optimized PABPN1 protein and two small inhibitory RNAs (siRNAs) against PABPN1 modeled into microRNA (miRNA) backbones. A single intramuscular injection of BB-301 results in robust inhibition of mutant PABPN1 and concomitant replacement of the codon-optimized PABPN1 protein. The treatment restores muscle strength and muscle weight to wild-type levels as well as improving other physiological hallmarks of the disease in a mouse model of OPMD.

Physiologically based pharmacokinetic modeling as a tool to predict drug interactions for antibody-drug conjugates.

BACKGROUND AND OBJECTIVES: Monomethyl auristatin E (MMAE, a cytotoxic agent), upon releasing from valine-citrulline-MMAE (vc-MMAE) antibody-drug conjugates (ADCs), is expected to behave like small molecules. Therefore, evaluating the drug-drug interaction (DDI) potential associated with MMAE is important in the clinical development of ADCs. The objective of this work was to build a physiologically based pharmacokinetic (PBPK) model to assess MMAE-drug interactions for vc-MMAE ADCs. METHODS: A PBPK model linking antibody-conjugated MMAE (acMMAE) to its catabolite unconjugated MMAE associated with vc-MMAE ADCs was developed using a mixed 'bottom-up' and 'top-down' approach. The model was developed using in silico and in vitro data and in vivo pharmacokinetic data from anti-CD22-vc-MMAE ADC. Subsequently, the model was validated using clinical pharmacokinetic data from another vc-MMAE ADC, brentuximab vedotin. Finally, the verified model was used to simulate the results of clinical DDI studies between brentuximab vedotin and midazolam, ketoconazole, and rifampicin. RESULTS: The pharmacokinetic profile of acMMAE and unconjugated MMAE following administration of anti-CD22-vc-MMAE was well described by simulations using the developed PBPK model. The model's performance in predicting unconjugated MMAE pharmacokinetics was verified by successful simulation of the pharmacokinetic profile following brentuximab vedotin administration. The model simulated DDIs, expressed as area under the concentration-time curve (AUC) and maximum concentration (C max) ratios, were well within the two-fold of the observed data from clinical DDI studies. CONCLUSIONS: This work is the first demonstration of the use of PBPK modelling to predict MMAE-based DDI potential. The described model can be extended to assess the DDI potential of other vc-MMAE ADCs.

Discovery and Biological Profiling of Potent and Selective mTOR Inhibitor GDC-0349.

Aberrant activation of the PI3K-Akt-mTOR signaling pathway has been observed in human tumors and tumor cell lines, indicating that these protein kinases may be attractive therapeutic targets for treating cancer. Optimization of advanced lead 1 culminated in the discovery of clinical development candidate 8h, GDC-0349, a potent and selective ATP-competitive inhibitor of mTOR. GDC-0349 demonstrates pathway modulation and dose-dependent efficacy in mouse xenograft cancer models.

Evaluation of time-dependent cytochrome p450 inhibition in a high-throughput, automated assay: introducing a novel area under the curve shift approach.

Early in the drug discovery process, the identification of cytochrome P450 (CYP) time-dependent inhibition (TDI) is an important step for compound optimization. Here we describe a high-throughput, automated method for the evaluation of TDI utilizing human liver microsomes and conventional CYP-specific mass spectrometer-based probes in a 384-well format. One of the key differences from other published TDI assays is the use of a shift in area the under curve of the percent activity remaining versus inhibitor concentration plot (AUC shift) rather than the traditional fold-shift in IC50, to determine the magnitude of TDI. An AUC shift of < 15% suggests negative TDI and > 15% suggests potential TDI. This AUC shift was used to achieve quantitative data reporting, even in the case of weak inhibitors for which IC50 values cannot be quantified. An Agilent Technologies BioCel 1200 System was programmed such that the TDI liability of up to 77 test compounds, incubated at four test concentrations, with and without NADPH in the pre-incubation, can be analyzed in a single run. The detailed automated methodology, assay validation, data reporting and the novel TDI AUC shift approach to describe magnitude of TDI are presented.

Discovery of novel PI3-kinase δ specific inhibitors for the treatment of rheumatoid arthritis: taming CYP3A4 time-dependent inhibition.

PI3Kδ is a lipid kinase and a member of a larger family of enzymes, PI3K class IA(α, ß, δ) and IB (γ), which catalyze the phosphorylation of PIP2 to PIP3. PI3Kδ is mainly expressed in leukocytes, where it plays a critical, nonredundant role in B cell receptor mediated signaling and provides an attractive opportunity to treat diseases where B cell activity is essential, e.g., rheumatoid arthritis. We report the discovery of novel, potent, and selective PI3Kδ inhibitors and describe a structural hypothesis for isoform (α, ß, γ) selectivity gained from interactions in the affinity pocket. The critical component of our initial pharmacophore for isoform selectivity was strongly associated with CYP3A4 time-dependent inhibition (TDI). We describe a variety of strategies and methods for monitoring and attenuating TDI. Ultimately, a structure-based design approach was employed to identify a suitable structural replacement for further optimization.

Potent non-benzoquinone ansamycin heat shock protein 90 inhibitors from genetic engineering of Streptomyces hygroscopicus.

Inhibition of the protein chaperone Hsp90 is a promising new approach to cancer therapy. We describe the preparation of potent non-benzoquinone ansamycins. One of these analogues, generated by feeding 3-amino-5-chlorobenzoic acid to a genetically engineered strain of Streptomyces hygroscopicus, shows high accumulation and long residence time in tumor tissue, is well-tolerated upon intravenous dosing, and is highly efficacious in the COLO205 mouse tumor xenograft model.