BET bromodomain inhibitors regulate keratinocyte plasticity.

Although most acute skin wounds heal rapidly, non-healing skin ulcers represent an increasing and substantial unmet medical need that urgently requires effective therapeutics. Keratinocytes resurface wounds to re-establish the epidermal barrier by transitioning to an activated, migratory state, but this ability is lost in dysfunctional chronic wounds. Small-molecule regulators of keratinocyte plasticity with the potential to reverse keratinocyte malfunction in situ could offer a novel therapeutic approach in skin wound healing. Utilizing high-throughput phenotypic screening of primary keratinocytes, we identify such small molecules, including bromodomain and extra-terminal domain (BET) protein family inhibitors (BETi). BETi induce a sustained activated, migratory state in keratinocytes in vitro, increase activation markers in human epidermis ex vivo and enhance skin wound healing in vivo. Our findings suggest potential clinical utility of BETi in promoting keratinocyte re-epithelialization of skin wounds. Importantly, this novel property of BETi is exclusively observed after transient low-dose exposure, revealing new potential for this compound class.

New Perspectives on Drug-Induced Liver Injury Risk Assessment of Acyl Glucuronides.

Drug-induced liver injury (DILI) remains one of the key challenges in drug development due to the mechanisms of action being multifactorial in nature. This is particularly the case for idiosyncratic DILI which occurs in a very low frequency in humans (e.g., 1:10,000). Despite perceptions that acyl glucuronide metabolites are defacto risks for DILI, scientific evidence suggests that acyl glucuronide formation alone does not pose an increased risk compared to other drug metabolites. This applies in particular to those acyl glucuronides which are not reactive and do not form covalent adducts with proteins. The goal of this paper is to provide guidance on preclinical and clinical strategies to evaluate the potential for acyl glucuronide formation to contribute to DILI. A key element of our proposed safety assessment is to investigate whether a particular acyl glucuronide is reactive or not and whether systemic exposure in humans can be demonstrated in animal toxicology studies following administration of the parent drug. While standard animal toxicology studies can identify overtly hepatotoxic compounds, these studies are not predictive for drugs that produce idiosyncratic forms of DILI. In addition, we do not recommend conducting toxicology studies of administered individual acyl glucuronides due to differences in pharmacokinetic and dispositional properties from the endogenously produced metabolites. Once a drug candidate has entered clinical trials, the focus should be on clinical safety data and emerging risk-benefit analysis.

Discovery of LOU064 (Remibrutinib), a Potent and Highly Selective Covalent Inhibitor of Bruton's Tyrosine Kinase.

Bruton's tyrosine kinase (BTK), a cytoplasmic tyrosine kinase, plays a central role in immunity and is considered an attractive target for treating autoimmune diseases. The use of currently marketed covalent BTK inhibitors is limited to oncology indications based on their suboptimal kinase selectivity. We describe the discovery and preclinical profile of LOU064 (remibrutinib, 25), a potent, highly selective covalent BTK inhibitor. LOU064 exhibits an exquisite kinase selectivity due to binding to an inactive conformation of BTK and has the potential for a best-in-class covalent BTK inhibitor for the treatment of autoimmune diseases. It demonstrates potent in vivo target occupancy with an EC90 of 1.6 mg/kg and dose-dependent efficacy in rat collagen-induced arthritis. LOU064 is currently being tested in phase 2 clinical studies for chronic spontaneous urticaria and Sjoegren's syndrome.

Design of Potent and Selective Covalent Inhibitors of Bruton's Tyrosine Kinase Targeting an Inactive Conformation.

Bruton's tyrosine kinase (BTK) is a member of the TEC kinase family and is selectively expressed in a subset of immune cells. It is a key regulator of antigen receptor signaling in B cells and of Fc receptor signaling in mast cells and macrophages. A BTK inhibitor will likely have a positive impact on autoimmune diseases which are caused by autoreactive B cells and immune-complex driven inflammation. We report the design, optimization, and characterization of potent and selective covalent BTK inhibitors. Starting from the selective reversible inhibitor 3 binding to an inactive conformation of BTK, we designed covalent irreversible compounds by attaching an electrophilic warhead to reach Cys481. The first prototype 4 covalently modified BTK and showed an excellent kinase selectivity including several Cys-containing kinases, validating the design concept. In addition, this compound blocked FcγR-mediated hypersensitivity in vivo. Optimization of whole blood potency and metabolic stability resulted in compounds such as 8, which maintained the excellent kinase selectivity and showed improved BTK occupancy in vivo.

Fixing clearance as early as lead optimization using high throughput in vitro incubations in combination with exact mass detection and automatic structure elucidation of metabolites.

New enabling MS technologies have made it possible to elucidate metabolic pathways present in ex vivo (blood, bile and/or urine) or in vitro (liver microsomes, hepatocytes and/or S9) samples. When investigating samples from high throughput assays the challenge that the user is facing now is to extract the appropriate information and compile it so that it is understandable to all. Medicinal chemist may then design the next generation of (better) drug candidates combining the needs for potency and metabolic stability and their synthetic creativity. This review focuses on the comparison of these enabling MS technologies and the IT tools developed for their interpretation.

An integrated approach for identification and target validation of antifungal compounds active against Erg11p.

Systemic life-threatening fungal infections represent a significant unmet medical need. Cell-based, phenotypic screening can be an effective means of discovering potential novel antifungal compounds, but it does not address target identification, normally required for compound optimization by medicinal chemistry. Here, we demonstrate a combination of screening, genetic, and biochemical approaches to identify and characterize novel antifungal compounds. We isolated a set of novel non-azole antifungal compounds for which no target or mechanism of action is known, using a screen for inhibition of Saccharomyces cerevisiae proliferation. Haploinsufficiency profiling of these compounds in S. cerevisiae suggests that they target Erg11p, a cytochrome P450 family member, which is the target of azoles. Consistent with this, metabolic profiling in S. cerevisiae revealed a buildup of the metabolic intermediates prior to Erg11p activity, following compound treatment. Further, human cytochrome P450 is also inhibited in in vitro assays by these compounds. We modeled the Erg11p protein based on the human CYP51 crystal structure, and in silico docking of these compounds suggests that they interact with the heme center in a manner similar to that of azoles. Consistent with these docking observations, Candida strains carrying azole-resistant alleles of ERG11 are also resistant to the compounds in this study. Thus, we have identified non-azole Erg11p inhibitors, using a systematic approach for ligand and target characterization.

Discovery of 3-(2,6-dichloro-3,5-dimethoxy-phenyl)-1-{6-[4-(4-ethyl-piperazin-1-yl)-phenylamino]-pyrimidin-4-yl}-1-methyl-urea (NVP-BGJ398), a potent and selective inhibitor of the fibroblast growth factor receptor family of receptor tyrosine kinase.

A novel series of N-aryl-N'-pyrimidin-4-yl ureas has been optimized to afford potent and selective inhibitors of the fibroblast growth factor receptor tyrosine kinases 1, 2, and 3 by rationally designing the substitution pattern of the aryl ring. On the basis of its in vitro profile, compound 1h (NVP-BGJ398) was selected for in vivo evaluation and showed significant antitumor activity in RT112 bladder cancer xenografts models overexpressing wild-type FGFR3. These results support the potential therapeutic use of 1h as a new anticancer agent.

CYP3A time-dependent inhibition risk assessment validated with 400 reference drugs.

Although reversible CYP3A inhibition testing is well established for predicting the drug-drug interaction potential of clinical candidates, time-dependent inhibition (TDI) has become the focus of drug designers only recently. Failure of several late-stage clinical candidates has been attributed to TDI, and this mechanism is also suspected to play a role in liver toxicities often observed in preclinical species. Measurement of enzyme inactivation rates (k(inact) and K(I)) is technically challenging, and a great deal of variability can be found in the literature. In this article, we have evaluated the TDI potential for 400 registered drugs using a high-throughput assay format based on determination of the inactivation rate (k(obs)) at a single concentration of test compound (10 µM). The advantages of this new assay format are highlighted by comparison with data generated using the IC50 shift assay, a current standard approach for preliminary assessment of TDI. With use of an empirically defined positive/negative k(obs) bin of 0.02 min⁻¹, only 4% of registered drugs were found to be positive. This proportion increased to more than 20% when in-house lead optimization molecules were considered, emphasizing the importance of identifying this property in selection of promising drug candidates. Finally, it is suggested that the data and technology described here may be a good basis for building structure-activity relationships and in silico modeling.

CYP4F enzymes are responsible for the elimination of fingolimod (FTY720), a novel treatment of relapsing multiple sclerosis.

Fingolimod (FTY720, Gilenya, 2-amino-2-[2-(4-octylphenyl)ethyl]-1,3-propanediol) is a novel drug recently approved in the United States for the oral treatment of relapsing multiple sclerosis. The compound is eliminated predominantly by ω-hydroxylation, followed by further oxidation. The ω-hydroxylation was the major metabolic pathway in human liver microsomes (HLM). The enzyme kinetics in HLM were characterized by a Michaelis-Menten affinity constant (K(m)) of 183 µM and a maximum velocity (V(max)) of 1847 pmol/(min · mg). Rates of fingolimod metabolism by a panel of HLM from individual donors showed no correlation with marker activities of any of the major drug-metabolizing cytochrome P450 (P450) enzymes or of flavin-containing monooxygenase (FMO). Among 21 recombinant human P450 enzymes and FMO3, only CYP4F2 (and to some extent CYP4F3B) produced metabolite profiles similar to those in HLM. Ketoconazole, known to inhibit not only CYP3A but also CYP4F2, was an inhibitor of fingolimod metabolism in HLM with an inhibition constant (K(i)) of 0.74 µM (and by recombinant CYP4F2 with an IC(50) of 1.6 µM), whereas there was only a slight inhibition found with azamulin and none with troleandomycin. An antibody against CYP4F2 was able to inhibit the metabolism of fingolimod almost completely in HLM, whereas antibodies specific to CYP2D6, CYP2E1, and CYP3A4 did not show significant inhibition. Combining the results of these four enzyme phenotyping approaches, we demonstrated that CYP4F2 and possibly other enzymes of the CYP4F subfamily (e.g., CYP4F3B) are the major enzymes responsible for the ω-hydroxylation of fingolimod, the main elimination pathway of the drug in vivo.

Metabolic soft spot identification and compound optimization in early discovery phases using MetaSite and LC-MS/MS validation.

Metabolic stability is a key property to enable drugs to reach therapeutic concentrations. Microsomal clearance assays are used to dial out labile compounds in early discovery phases. However, because they do not provide any information on soft spots, the rational design of more stable compounds remains challenging. A robust soft spot identification procedure combining in silico prediction ranking using MetaSite and mass-spectrometric confirmation is described. MetaSite's first rank order predictions were experimentally confirmed for only about 55% of the compounds. For another 29% of the compounds, the second (20%) or the third (9%) rank order predictions were detected. This automatic and high-throughput reprioritization of a likely soft-spot increases the likelihood of working on the right soft spot from about 50% to more than 80%. With this information, the structure-metabolism relationships are likely to be understood faster and earlier in drug discovery.

Evaluation of fluorescence- and mass spectrometry-based CYP inhibition assays for use in drug discovery.

The potential for metabolism-related drug-drug interactions by new chemical entities is assessed by monitoring the impact of these compounds on cytochrome P450 (CYP) activity using well-characterized CYP substrates. The conventional gold standard approach for in vitro evaluation of CYP inhibitory potential uses pooled human liver microsomes (HLM) in conjunction with prototypical drug substrates, often quantified by LC-MS/MS. However, fluorescent CYP inhibition assays, which use recombinantly expressed CYPs and fluorogenic probe substrates, have been employed in early drug discovery to provide low-cost, high-throughput assessment of new chemical entities. Despite its greatly enhanced throughput, this approach has been met with mixed success in predicting the data obtained with the conventional gold standard approach (HLM+LC-MS). The authors find that the predictivity of fluorogenic assays for the major CYP isoforms 3A4 and 2D6 may depend on the quality of the test compounds. Although the structurally more optimized marketed drugs yielded acceptable correlations between the fluorogenic and HLM+LC-MS/MS assays for CYPs 3A4, 2D6, and 2C9 (r2 = 0.5-0.7; p < 0.005), preoptimization, early discovery compounds yielded poorer correlations (r2 < or = 0.2) for 2 of these major isoforms, CYPs 3A4 and 2D6. Potential reasons for the observed differences are discussed.

High-throughput in vitro profiling assays: lessons learnt from experiences at Novartis.

This article reviews the use of a selection of in vitro assays implemented at Novartis and intends to address exposure and safety in early drug discovery. The authors' own experience, based on a large number of 'real' drug discovery compounds, is described to reflect on what has worked, where improvement is needed and how to best use the data for decision making. Possible strategies are discussed, and guidelines are provided on how to organise assays, extract value and contribute knowledge from the data.

Pimecrolimus: absorption, distribution, metabolism, and excretion in healthy volunteers after a single oral dose and supplementary investigations in vitro.

The absorption and disposition of pimecrolimus, a calcineurin inhibitor developed for the treatment of inflammatory skin diseases, was investigated in four healthy volunteers after a single oral dose of 15 mg of [(3)H]pimecrolimus. Supplementary information was obtained from in vitro experiments. Pimecrolimus was rapidly absorbed. After t(max) (1-3 h), its blood concentrations fell quickly to 3% of C(max) at 24 h, followed by a slow terminal elimination phase (average t(1/2) 62 h). Radioactivity in blood decreased more slowly (8% of C(max) at 24 h). The tissue and blood cell distribution of pimecrolimus was high. The metabolism of pimecrolimus in vivo, which could be well reproduced in vitro (human liver microsomes), was highly complex and involved multiple oxidative O-demethylations and hydroxylations. In blood, pimecrolimus was the major radiolabeled component up to 24 h (49% of radioactivity area under the concentration-time curve(0-24) h), accompanied by a large number of minor metabolites. The average fecal excretion of radioactivity between 0 and 240 h amounted to 78% of dose and represented predominantly a complex mixture of metabolites. In urine, 0 to 240 h, only about 2.5% of the dose and no parent drug was excreted. Hence, pimecrolimus was eliminated almost exclusively by oxidative metabolism. The biotransformation of pimecrolimus was largely catalyzed by CYP3A4/5. Metabolite pools generated in vitro showed low activity in a calcineurin-dependent T-cell activation assay. Hence, metabolites do not seem to contribute significantly to the pharmacological activity of pimecrolimus.

A locally active antiinflammatory macrolide (MLD987) for inhalation therapy of asthma.

One of the characteristic features of asthma is a persistent pulmonary inflammation, with increased numbers of eosinophils and activated T-lymphocytes in the airways. T-helper cells of the Th2 phenotype play a pivotal role in the pathogenesis of asthma, and they are believed to orchestrate the asthmatic response by releasing a wide repertoire of cytokines. Herein, we describe the design, synthesis, and evaluation in models of allergic asthma of a locally active T-cell modulator, MLD987 (1). Compound 1 is a potent immunosuppressant that inhibits the activation, proliferation, and release of cytokines from T-cells with IC(50) values in the low nanomolar range. In a Brown-Norway rat model of allergic asthma, 1, when given into the airways by intratracheal administration (ED(50) = 1 mg/kg) or by inhalation (ED(50) = 0.4 mg/kg), potently reduced the influx of leukocytes into bronchoalveolar lavage fluid samples obtained from antigen-challenged animals. In contrast, 1 had an appreciably weaker activity in this model when given orally or intravenously. Pharmacokinetic evaluation in rat and rhesus monkey showed that 1 had both a low oral (2-4%) and a low pulmonary (7%, monkey) bioavailability. These findings are consistent with a local site of action of the compound and rule out that its antiinflammatory activity in the lung was caused by systemically absorbed material, which had been swallowed during inhalation or which had entered the circulation via the airways. Local administration and the metabolically soft structure of 1, which favors rapid systemic metabolism to less immunosuppressive metabolite 2, are the main reasons for the low exposure and weak systemic activity of the compound. Administration of a locally active compound such as 1, by inhalation, should reduce systemic side effects. Our results indicate that 1 has the potential to serve as an alternative to inhaled glucocorticosteroids for the long-term therapy of asthma of all grades of severity.

SAR of 2,6-diamino-3,5-difluoropyridinyl substituted heterocycles as novel p38MAP kinase inhibitors.

2,6-Diamino-3,5-difluoropyridinyl substituted pyridinylimidazoles, -pyrroles, -oxazoles, -thiazoles and -triazoles have been identified as novel p38alpha inhibitors. Pyridinylimidazole 11 potently inhibited LPS-induced TNFalpha in mice, showed good efficacy in the established rat adjuvant (ED(50): 10 mg/kg po b.i.d.) and collagen induced arthritis (ED(50): 5 mg/kg po b.i.d.) with disease modifying properties based on histological analysis of the joints.