Altering Metabolic Profiles of Drugs by Precision Deuteration 2: Discovery of a Deuterated Analog of Ivacaftor with Differentiated Pharmacokinetics for Clinical Development.

Ivacaftor is currently used for the treatment of cystic fibrosis as both monotherapy (Kalydeco; Vertex Pharmaceuticals, Boston, MA) and combination therapy with lumacaftor (Orkambi; Vertex Pharmaceuticals). Each therapy targets specific patient populations: Kalydeco treats patients carrying one of nine gating mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) protein, whereas Orkambi treats patients homozygous for the F508del CFTR mutation. In this study, we explored the pharmacological and metabolic effects of precision deuteration chemistry on ivacaftor by synthesizing two novel deuterated ivacaftor analogs, CTP-656 (d9-ivacaftor) and d18-ivacaftor. Ivacaftor is administered twice daily and is extensively converted in humans to major metabolites M1 and M6; therefore, the corresponding deuterated metabolites were also prepared. Both CTP-656 and d18-ivacaftor showed in vitro pharmacologic potency similar to that in ivacaftor, and the deuterated M1 and M6 metabolites showed pharmacology equivalent to that in the corresponding metabolites of ivacaftor, which is consistent with the findings of previous studies of deuterated compounds. However, CTP-656 exhibited markedly enhanced stability when tested in vitro. The deuterium isotope effects for CTP-656 metabolism (DV = 3.8, DV/K = 2.2) were notably large for a cytochrome P450-mediated oxidation. The pharmacokinetic (PK) profile of CTP-656 and d18-ivacaftor were assessed in six healthy volunteers in a single-dose crossover study, which provided the basis for advancing CTP-656 in development. The overall PK profile, including the 15.9-hour half-life for CTP-656, suggests that CTP-656 may be dosed once daily, thereby enhancing patient adherence. Together, these data continue to validate deuterium substitution as a viable approach for creating novel therapeutic agents with properties potentially differentiated from existing drugs.

High-throughput screening for glutathione conjugates using stable-isotope labeling and negative electrospray ionization precursor-ion mass spectrometry.

RATIONALE: It has been proposed that the increase in the instances of idiosyncratic adverse drug reactions (IADRs) and black box warnings may be attributed to the occurrence of reactive metabolites. Consequently, a high-throughput screen for reactive metabolites formed from liver microsome extracts with added glutathione (GSH) was developed for use in the early stages of drug discovery. METHODS: To enhance sensitivity and specificity, as well as accelerate data processing, a mixture of a stable-isotope probe consisting of natural GSH (light GSH) and stable-isotope-labeled [(15) N,(13) C(2)] GSH (heavy GSH) at a ratio of 1:1 was used. Any metabolite that reacted with the GSH results in the formation of light and heavy GSH conjugates with a 3 Da difference. Employing a precursor-ion scan using negative ion electrospray ionization (ESI) corresponding to the expected fragments, signals with the appropriate ratio in the precursor ion scan are then further examined. RESULTS: The new method greatly simplifies data collection by assuming molecules containing GSH will fragment to form specific ions. As such, this approach accelerates data processing (and collection) at the risk of missing compounds that do not fragment as expected. The assay was validated with 33 diverse drugs known to form GSH conjugates, 5 drugs known to not form GSH adducts and over 100 samples containing components of the normal in vitro matrix. In all cases data collected matched the expected result. CONCLUSIONS: The observed sensitivity, specificity, and fast data processing make this assay an excellent fit for high-throughput screening of reactive metabolites in the early stages of drug discovery. This method is not intended to eliminate compounds or terminate their development. Instead, it is to bring forward molecules with one less liability and thus a greater probability of ultimate success.

Evaluating LC-MS/MS To Measure Accumulation of Compounds within Bacteria.

A general method for determining bacterial uptake of compounds independent of antibacterial activity would be a valuable tool in antibacterial drug discovery. LC-MS/MS assays have been described, but it has not been shown whether the data can be used directly to inform medicinal chemistry. We describe the evaluation of an LC-MS/MS assay measuring association of compounds with bacteria, using a set of over a hundred compounds (inhibitors of NAD-dependent DNA ligase, LigA) for which in vitro potency and antibacterial activity had been determined. All compounds were active against an efflux-deficient strain of Escherichia coli with reduced LigA activity ( E. coli ligA251 Δ tolC). Testing a single compound concentration and incubation time, we found that, for equipotent compounds, LC-MS/MS values were not predictive of antibacterial activity. This indicates that measured bacteria-associated compound was not necessarily exposed to the target enzyme. Our data suggest that, while exclusion from bacteria is a major reason for poor antibacterial activity of potent compounds, the distribution of compound within the bacterial cell may also be a problem. The relative importance of these factors is likely to vary from one chemical series to another. Our observations provide directions for further study of this difficult issue.

Discovery of VX-509 (Decernotinib): A Potent and Selective Janus Kinase 3 Inhibitor for the Treatment of Autoimmune Diseases.

While several therapeutic options exist, the need for more effective, safe, and convenient treatment for a variety of autoimmune diseases persists. Targeting the Janus tyrosine kinases (JAKs), which play essential roles in cell signaling responses and can contribute to aberrant immune function associated with disease, has emerged as a novel and attractive approach for the development of new autoimmune disease therapies. We screened our compound library against JAK3, a key signaling kinase in immune cells, and identified multiple scaffolds showing good inhibitory activity for this kinase. A particular scaffold of interest, the 1H-pyrrolo[2,3-b]pyridine series (7-azaindoles), was selected for further optimization in part on the basis of binding affinity (Ki) as well as on the basis of cellular potency. Optimization of this chemical series led to the identification of VX-509 (decernotinib), a novel, potent, and selective JAK3 inhibitor, which demonstrates good efficacy in vivo in the rat host versus graft model (HvG). On the basis of these findings, it appears that VX-509 offers potential for the treatment of a variety of autoimmune diseases.

Universal LC-MS method for minimized carryover in a discovery bioanalytical setting.

BACKGROUND: A frequent impediment to accurate quantitation in bioanalytical LC-MS arises from carryover. For many new chemical entities in drug discovery carryover is not limited to the autosampler, but instead arises from several different sources. METHOD: We tested several different columns, injector wash sequences and gradient compositions to understand and eliminate these sources. In many instances carryover was dictated by the elution gradient and column as much as the autosampler hardware and wash protocol. CONCLUSION: Several trends were observed. First, different columns resulted in significantly different amounts of carryover (even for nominally the same column chemistry). Second, a continuous high organic wash of the column was not as effective at removing carryover as cycling between high and low organic mobile phases during the column wash. Combining our observations (column, gradient and autosampler configuration) we devised a short 3-min method that is appropriate for a diverse set of new chemical entities and minimizes carryover while still being sufficiently robust to use in a drug-discovery setting.

Minimizing matrix effects while preserving throughput in LC-MS/MS bioanalysis.

BACKGROUND: Phospholipids are known to cause matrix effects in LC-MS analysis and are not effectively removed by one of the most common method of sample preparation: organic solvent protein precipitation. The objective of this research is to minimize phospholipid interferences chromatographically. RESULTS: In this article we examine several chromatographic approaches and highlight the method we developed that allows for the rapid gradient separation of model drug molecules from phospholipids. CONCLUSION: The new approach (which utilizes a mixture of methanol and acetonitrile as the organic mobile phase on a 2.1 × 20 mm C18 column) minimized phospholipids-related matrix effects in the analysis of plasma samples prepared by protein precipitation and is suitable for high-throughput bioanalysis in drug discovery.

Bioanalytical solutions to acetonitrile shortages.

The acetonitrile shortage during 2008 to 2009 challenged bioanalytical scientists due to the ubiquitous role that acetonitrile plays in sample preparation and analysis. Replacement, reduction and reuse of acetonitrile were the core tenants behind each approach used to tackle the shortage. Sample preparation of biological matrices can be accomplished by protein precipitation using a variety of solvents; methanol is usually the best substitute for acetonitrile. The potential liabilities in using methanol can be handled with appropriate modifications. Often methanol is superior to acetonitrile for both protein precipitation and chromatography if phospholipid interference is a problem. Solvent consumption can be minimized by reducing column dimensions and particle size. Separations can be achieved at greatly reduced run times using sub-2-µm and fused-core particle columns. Emerging technologies, such as desorption ESI, direct analysis in real time and laser diode thermal desorption, eliminate the need for chromatography and achieve significant solvent and time savings. Acetonitrile recyclers can purify HPLC waste for reuse.

In vitro and in vivo isotope effects with hepatitis C protease inhibitors: enhanced plasma exposure of deuterated telaprevir versus telaprevir in rats.

Telaprevir 2 (VX-950), an inhibitor of the hepatitis C virus (HCV(a)) NS3-4A protease, is in phase 3 clinical trials. One of the major metabolites of 2 is its P1-(R)-diastereoisomer, 3 (VRT-394), containing an inversion at the chiral center next to the alpha-ketoamide on exchange of a proton with solvent. Compound 3 is approximately 30-fold less active against HCV protease. In an attempt to suppress the epimerization of 2 without losing activity against the HCV protease, the proton at that chiral site was replaced with deuterium (d). The compound 1 (d-telaprevir) is as efficacious as 2 in in vitro inhibition of protease activity and viral replication (replicon) assays. The kinetics of in vitro stability of 1 and 2 in buffered pH solutions and plasma samples, including human plasma, suggest that 1 is significantly more stable than 2. Oral administration (10 mg/kg) in rats resulted in a approximately 13% increase of AUC for 1.

Method for rapid metabolite profiling of drug candidates in fresh hepatocytes using liquid chromatography coupled with a hybrid quadrupole linear ion trap.

Rapid information on metabolic profiling is required to evaluate the structural liabilities of drug candidates in early drug discovery. In this study, a sensitive and rapid semi-quantitative method was developed to simultaneously monitor the drug candidate and metabolites as well as collect tandem mass (MS/MS) spectra for subsequent metabolite identification. The simultaneous semi-quantitation and identification of metabolites in fresh hepatocytes is achieved using high-performance liquid chromatography (HPLC) coupled with a hybrid quadrupole linear ion trap. The survey experiment consists of monitoring multiple-reaction monitoring (MRM) transitions for the internal standard, the parent, and 48 MRM transitions designed to cover the most common phase I and II biotransformations. An information-dependent acquisition (IDA) method was employed to trigger product ion scans above the MRM signal threshold. Three biotransformations of a lead compound have been identified through enhanced product ion scans and the respective MRM transitions of those metabolites were selected for semi-quantitation. Parent disappearance and formation of the metabolites as a function of incubation time in five different species were monitored by their respective MRM responses. The method provides the necessary sensitivity to detect minor metabolites in a relevant therapeutic concentration range. Enzymatic turnover of the parent and the metabolites in different species are revealed based on the different initial concentrations of the parent. This methodology integrates the parent disappearance, metabolite identification, and the formation of the metabolites along the time course using a single rapid LC/MS/MS analysis. This method can be used as a complementary tool to the conventional method of metabolic profiling. It provides a rapid and sensitive initial profile of the metabolism of potential structural series at the lead selection stage. The method can also be incorporated into the overall metabolite profiling scheme to evaluate the drug candidates in drug discovery.