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

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

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

Although reversible CYP3A inhibition testing is well established to predict the drug to drug interaction potential of clinical candidates it is only recently that time dependent inhibition (TDI) became the focus of drug designers. This particular inhibition mechanism was indeed the source of a few documented late stage failures and is strongly suspected to participate in the frequently disqualifying liver toxicities in pre-clinical species. Because of the specific challenges of measuring enzyme inactivation rates and its characteristic parameters kinact and KI, a great deal of variability can be found in the literature. In this paper we measured these parameters for 63 known CYP3A inactivators using a single robust method. Taking advantage of its specific design and miniaturisation, a screening assay based on inactivation rate (kobs) at 10 µM test article concentration was validated versus the current gold standard assay, the IC50 shift. More than 375 World Drug Index compounds have been profiled and used to exemplify the specific advantages of this alternative method. Using an empirically defined positive/negative kobs bin of 0.02 min-1, 4% of WDI drugs only were found positive. This proportion increased to more than 20% when in-house lead optimization molecules were considered, emphasizing the importance of filtering this property out to make good 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 modelling

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

the Novartis internal working group came up with an internal guidance. We would like to publish this guidance together with external collaborators. Therefore , we seek OAK clearance on the guidance to share it with external collaborators from AZ, UCB and University of Basel. No current running Novartis Projects are mentioned in the guidance

CYP4F Enzymes are Responsible for the Elimination of Fingolimod (FTY720), a Novel Treatment of Relapsing Multiple Sclerosis

Fingolimod is a novel drug in clinical development 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 (Km) of 183 µM and a maximum velocity (Vmax) 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 (CYP) enzymes or of flavin-containing monooxygenase (FMO). Among 21 recombinant human CYP 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 (Ki) of 0.74 µM (and by recombinant CYP4F2 with IC50 of 1.6 µM), while there was only a slight inhibition found with azamulin and none with troleandomycin. An antibody against CYP4F2 was able to inhibit almost completely the metabolism of fingolimod 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

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

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

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

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

An integrated approach for the identification and target validation of anti-fungal 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