Structure based design of macrocyclic factor XIa inhibitors: Discovery of cyclic P1 linker moieties with improved oral bioavailability.

This manuscript describes the discovery of a series of macrocyclic inhibitors of FXIa with oral bioavailability. Assisted by structure based drug design and ligand bound X-ray crystal structures, the group linking the P1 moiety to the macrocyclic core was modified with the goal of reducing H-bond donors to improve pharmacokinetic performance versus 9. This effort resulted in the discovery of several cyclic P1 linkers, exemplified by 10, that are constrained mimics of the bioactive conformation displayed by the acrylamide linker of 9. These cyclic P1 linkers demonstrated enhanced bioavailability and improved potency.

Discovery of a Parenteral Small Molecule Coagulation Factor XIa Inhibitor Clinical Candidate (BMS-962212).

Factor XIa (FXIa) is a blood coagulation enzyme that is involved in the amplification of thrombin generation. Mounting evidence suggests that direct inhibition of FXIa can block pathologic thrombus formation while preserving normal hemostasis. Preclinical studies using a variety of approaches to reduce FXIa activity, including direct inhibitors of FXIa, have demonstrated good antithrombotic efficacy without increasing bleeding. On the basis of this potential, we targeted our efforts at identifying potent inhibitors of FXIa with a focus on discovering an acute antithrombotic agent for use in a hospital setting. Herein we describe the discovery of a potent FXIa clinical candidate, 55 (FXIa Ki = 0.7 nM), with excellent preclinical efficacy in thrombosis models and aqueous solubility suitable for intravenous administration. BMS-962212 is a reversible, direct, and highly selective small molecule inhibitor of FXIa.

The utility of stable isotope labeled (SIL) analogues in the bioanalysis of endogenous compounds by LC-MS applied to the study of bile acids in a metabolomics assay.

The growing field of biomarker bioanalysis by liquid chromatography mass spectrometry (LC-MS) is challenged with the selection of suitable matrices to construct relevant and valid calibration curves resulting in not only precise but also accurate data. Because surrogate matrices are often employed with the associated concerns about the accuracy of the obtained data, here we present an assay using surrogate analytes in naive biological matrices. This approach is illustrated with the analysis of endogenous bile acids (e-BAs) in serum and plasma using stable isotope-labeled (SIL) analogues as calibration standards to address the matrix concerns. Several deuterated BAs (d-BAs) were used as standards representing respectively grouped e-BAs with structural similarity allowing for the simultaneous bioanalysis of 16 e-BA. The utility of this LC-MS assay employing d-BAs is demonstrated with the analysis of samples resultant of a controlled metabolomics study where a cohort of rats was fed/fasted to investigate the change of e-BAs dependent on food consumption and fasting time.

Novel phenylalanine derived diamides as Factor XIa inhibitors.

The synthesis, structural activity relationships (SAR), and selectivity profile of a potent series of phenylalanine diamide FXIa inhibitors will be discussed. Exploration of P1 prime and P2 prime groups led to the discovery of compounds with high FXIa affinity, good potency in our clotting assay (aPPT), and high selectivity against a panel of relevant serine proteases as exemplified by compound 21. Compound 21 demonstrated good in vivo efficacy (EC50=2.8µM) in the rabbit electrically induced carotid arterial thrombosis model (ECAT).

Orally bioavailable factor Xa inhibitors containing alpha-substituted gem-dimethyl P4 moieties.

In an effort to identify a potential back-up to apixaban (Eliquis®), we explored a series of diversified P4 moieties. Several analogs with substituted gem-dimethyl moieties replacing the terminal lactam of apixaban were identified which demonstrated potent FXa binding affinity (FXa Ki), good human plasma anticoagulant activity (PT EC2x), cell permeability, and oral bioavailability.

Development of a highly sensitive liquid chromatography/tandem mass spectrometry method to quantify total and free levels of a target protein, interferon-gamma-inducible protein-10, at picomolar levels in human serum.

RATIONALE: Liquid chromatography/tandem mass spectrometry (LC/MS/MS) assays are increasingly being used for absolute quantitation of proteins due to high specificity and low cost. However, the major challenge for the LC/MS method is insufficient sensitivity. This paper details the strategies developed to maximize the sensitivity from aspects of chromatography, mass spectrometry, and sample preparation to achieve a highly sensitive LC/MS method. METHODS: The method is based on the LC/MS/MS measurement of a surrogate peptide generated from trypsin digestion of interferon-gamma-inducible protein-10 (IP-10). The sample preparation strategy involved selectively extracting IP-10 and removing high-abundance serum proteins through acidified protein precipitation (PPT). It was revealed in this work that these high-abundance serum proteins, if not separated from the protein of interest, could cause significant ionization saturation and high background noise in selected reaction monitoring (SRM), leading to a 100-fold higher lower limit of quantification (LLOQ). RESULTS: Our method demonstrated that the acidified PPT could be optimized to selectively extract the protein of interest with full recovery of 97% to 103%, while the high-abundance serum proteins could be effectively removed with minimal matrix effect of 90% to 93%. For the first time, a highly sensitive LC/MS method with a LLOQ of 31.62 pM for the quantitation of IP-10 has been achieved, which is a 100-fold improvement over the generic method. CONCLUSIONS: The described method offers excellent sensitivity with advantages of being antibody reagent independent and leads to significant cost and time savings. It has been successfully employed to determine both total and free IP-10 levels in human serum samples. This method development strategy may also be applied to other small proteins.

Application and challenges in using LC-MS assays for absolute quantitative analysis of therapeutic proteins in drug discovery.

As more protein therapeutics enter the drug-discovery pipeline, the traditional ligand-binding assay (LBA) faces additional challenges to meet the rapid and diverse bioanalytical needs in the early drug-discovery stage. The high specificity and sensitivity afforded by LC-MS, along with its rapid method development, is proving invaluable for the analysis of protein therapeutics in support of drug discovery. LC-MS not only serves as a quantitative tool to complement LBA in drug discovery, it also provides structural details at a molecular level, which are used to address issues that cannot be resolved using LBA alone. This review will describe the key benefits and applications, as well as the techniques and challenges for applying LC-MS to support protein quantification in drug discovery.

Bioanalytical strategies for developing highly sensitive liquid chromatography/tandem mass spectrometry based methods for the peptide GLP-1 agonists in support of discovery PK/PD studies.

Highly sensitive liquid chromatography/tandem mass spectrometry (LC/MS/MS)-based methods have been developed and implemented for the quantitative determination of a number of peptides under evaluation in our Glucagon-Like Peptide-1 (GLP-1) discovery program for the treatment of diabetes. These peptides are GLP-1 receptor agonists. Due to the high potency, low dose, and low exposure of these peptides, LC/MS/MS-based methods with Lower Limits of Quantitation (LLOQs) (low picomolar range) were required to support discovery pharmacokinetic/ pharmacodynamic (PK/PD) studies. Compared with small molecules, many of these peptides posed significant bioanalytical challenges in the development of highly sensitive methods because of their parent signal splitting as a result of the formation of multiply charged states, the unfavorable fragmentation patterns for Selected Reaction Monitoring (SRM) transitions due to the generation of a large number of small mass product ions with relative low intensities, and adsorption issues observed during sample preparation. This paper details the strategies developed to maximize the sensitivity and improve LLOQs from aspects of mass spectrometry, chromatography, and sample preparation. A LLOQ of 10 picomolar was achieved for all of the investigated peptides using 100 µL of mouse plasma. This is a 100-fold improvement on LLOQs over generic LC/MS/MS-based methods when the same sample volume and the same mass spectrometer platform were used. The methods have been implemented in the support of discovery PK/PD studies.

Preclinical pharmacokinetics and pharmacodynamics of apixaban, a potent and selective factor Xa inhibitor.

Apixaban is a potent, highly selective, reversible, oral, direct factor Xa (fXa) inhibitor in development for thrombosis prevention and treatment. The preclinical pharmacokinetic (PK) attributes of apixaban feature small volume of distribution (Vd), low systemic clearance (CL), and good oral bioavailability. Apixaban is well absorbed in rat, dog, and chimpanzee, with absolute oral bioavailability of approximately 50% or greater. The steady-state Vd of apixaban is approximately 0.5, 0.2, and 0.17 l/kg in rats, dogs, and chimpanzees, while CL is approximately 0.9, 0.04, and 0.018 l/h/kg, respectively. In vitro metabolic clearance of apixaban is also low. Renal clearance comprises approximately 10-30% of systemic clearance in rat, dog, and chimpanzee. Anti-fXa activity, prothrombin time (PT), and HEPTEST(®) clotting time (HCT) prolongation correlated well with plasma apixaban concentration in rat, dog and chimpanzee. There was no lag time between apixaban plasma concentration and the pharmacodynamic (PD) markers, suggesting a rapid onset of action of apixaban. The PK/PD analyses were performed using an inhibitory E (max) model for anti-fXa assay and a linear model for PT and HCT assays. The IC(50) values for anti-fXa activity were 0.73 ± 0.03 and 1.5 ± 0.15 µM for rat and dog, respectively. The apparent K ( i ) values for PT were approximately 1.7, 6.6, and 4.8 µM for rat, dog and chimpanzee, respectively. The apparent K ( i ) for HCT was approximately 1.3 µM for dog. Apixaban exhibits desirable PK and PD properties for clinical development with good oral bioavailability, small Vd, low CL, and direct, predictable, concentration-dependent PD responses.

Optimization of Exactive Orbitrap™ acquisition parameters for quantitative bioanalysis.

BACKGROUND: Orbitrap™ mass spectrometry has made significant impacts in the qualitative field of mass spectrometry, and it can be a potentially powerful quantitative technique. Because the Orbitrap is a relatively new platform, our understanding of this technology is not as well-versed as other mass spectrometric techniques. RESULTS: An investigation of the optimal acquisition parameters for quantitation was conducted for propranolol, reserpine, leucine enkephalin and neurotensin from mouse plasma samples. The lower limits of quantitation were demonstrated to be 1-3 nM while the quantitation linear dynamic range extends to four orders of magnitude. This level of performance is sufficient for most bioanalytical applications in drug discovery. CONCLUSION: Increasing the ion population in the Orbitrap improves detection and lowers the limit of quantitation, but the upper limit of quantitation can suffer. A better understanding of the operating parameters will help guide us toward better experimental designs and the best conditions for quantitation.

BMS-593214, an active site-directed factor VIIa inhibitor: enzyme kinetics, antithrombotic and antihaemostatic studies.

Factor (F) VIIa in association with tissue factor (TF) is the primary in vivo initiator of blood coagulation and activates FX and FIX to generate thrombin, which plays a key role in the pathogenesis of thrombosis. We evaluated the enzyme kinetics, antithrombotic and antihaemostatic properties of BMS-593214, an active-site, direct FVIIa inhibitor. Studies were conducted in enzymatic assays, and in anesthetised rabbit models of electrically-induced carotid arterial thrombosis (AT), thread-induced vena cava venous thrombosis (VT) and cuticle bleeding time (BT). Antithrombotic efficacy of BMS-593214 given intravenously was evaluated for both the prevention and treatment of AT and VT. BMS-593214 displayed direct, competitive inhibition of human FVIIa in the hydrolysis of a tripeptide substrate with Ki of 5 nM. However, it acted as a noncompetitive inhibitor of the activation of the physiological substrate FX by TF/VIIa with Ki of 9.3 nM. BMS-593214 showed selectivity for FVIIa and exhibited species differences in TF-FVIIa-dependent anticoagulation with similar potency in human and rabbit plasma. BMS-593214 was efficacious in the prevention and treatment models of AT and VT with ED50 values of 1.1 to 3.1 mg/kg. Furthermore, BMS-593214 exhibited a wide therapeutic window with respect to BT. These results suggest that inhibition of FVIIa with small-molecule active-site inhibitors represents a promising antithrombotic approach for the development of new therapies for the prevention and treatment of AT and VT.

Effect of the direct factor Xa inhibitor apixaban in rat models of thrombosis and hemostasis.

Apixaban is an oral, direct, and highly selective factor Xa inhibitor in late-stage clinical development for the prevention and treatment of thromboembolic diseases. Apixaban was evaluated in rat thrombosis and hemostasis models. Thrombosis was produced in the carotid artery by FeCl2 application, in the vena cava by either FeCl2 application or tissue factor injection, and in an arterial-venous shunt. Hemostasis was assessed using cuticle, renal cortex, and mesenteric artery bleeding times. Intravenous apixaban infusions of 0.1, 0.3, 1, and 3 mg/kg per hour increased the ex vivo prothrombin time to 1.24, 1.93, 2.75, and 3.98 times control, respectively. The 0.3, 1, and 3-mg/kg per hour doses inhibited thrombosis in all models. Concentrations for 50% thrombus reduction ranged from 1.84 to 7.57 microM. The 3-mg/kg per hour dose increased cuticle, renal, and mesenteric bleeding times to 1.92, 2.13, and 2.98 times control, respectively. Lower doses had variable (1 mg/kg per hour) or no effect (0.1, 0.3 mg/kg per hour) on hemostasis. Heparin's prolongation of renal and cuticle bleeding time was twice that of apixaban when administered at a dose that approximated apixaban (3 mg/kg per hour) efficacy in arterial thrombosis. In summary, apixaban was effective in a broad range of thrombosis models at doses producing modest increases in multiple bleeding time models.

In silico prediction of biliary excretion of drugs in rats based on physicochemical properties.

Evaluating biliary excretion, a major elimination pathway for many compounds, is important in drug discovery. The bile duct-cannulated (BDC) rat model is commonly used to determine the percentage of dose excreted as intact parent into bile. However, a study using BDC rats is time-consuming and cost-ineffective. The present report describes a computational model that has been established to predict biliary excretion of intact parent in rats as a percentage of dose. The model was based on biliary excretion data of 50 Bristol-Myers Squibb Co. compounds with diverse chemical structures. The compounds were given intravenously at <10 mg/kg to BDC rats, and bile was collected for at least 8 h after dosing. Recoveries of intact parents in bile were determined by liquid chromatography with tandem mass spectrometry. Biliary excretion was found to have a fairly good correlation with polar surface area (r = 0.76) and with free energy of aqueous solvation (DeltaG(solv aq)) (r = -0.67). In addition, biliary excretion was also highly corrected with the presence of a carboxylic acid moiety in the test compounds (r = 0.87). An equation to calculate biliary excretion in rats was then established based on physiochemical properties via a multiple linear regression. This model successfully predicted rat biliary excretion for 50 BMS compounds (r = 0.94) and for 25 previously reported compounds (r = 0.86) whose structures are markedly different from those of the 50 BMS compounds. Additional calculations were conducted to verify the reliability of this computation model.

Metabolism, pharmacokinetics and pharmacodynamics of the factor Xa inhibitor apixaban in rabbits.

Apixaban has similar affinity for human and rabbit factor Xa (FXa). Rabbits are commonly used in development of thrombosis disease models; however, unlike in other species, apixaban demonstrated poor oral bioavailability (F = 3%) and a high clearance rate (2.55 l/h/kg) in rabbits. Oxidative metabolism of [14C] apixaban by liver microsomes was approximately 20 times faster in rabbits than in rats or humans. Following an intravenous (IV) dose of 5 mg/kg, circulating levels of [14C] apixaban decreased from the earliest sampling time (5 min) to undetectable at 4 h. After an oral dose of 30 mg/kg, levels of [14C] apixaban were only detected at 1 and 4 h. Radioactivity profiling showed that apixaban was a significant component in plasma only after IV administration; O-demethyl apixaban (M2), O-demethyl apixaban glucuronide (M14) and O-demethyl apixaban sulfate (M1) were prominent metabolites after both IV and oral administration. Studies of apixaban in rabbits showed a good correlation between apixaban concentrations and inhibition of FXa activity, prolongation of prothrombin time and modified prothrombin time, with no lag time between these ex vivo pharmacodynamic markers and plasma drug levels. The apixaban concentration required for 50% inhibition (IC50) of FXa activity ex vivo (0.22 +/- 0.02 microM) agreed with the IC50 from in vitro experiments in rabbit and human plasma. In summary, apixaban shows similar affinity for human and rabbit FXa. It produces a rapid onset of action, predictable concentration-dependent pharmacodynamic responses, and, unlike rats or humans, a rapid hepatic metabolism in rabbits.

Increasing high-throughput discovery bioanalysis using automated selected reaction monitoring compound optimization, ultra-high-pressure liquid chromatography, and single-step sample preparation workflows.

QuickQuan is an integrated software package for Thermo Scientific triple quadrupole mass spectrometers that allows users to automate routine operations ranging from method development to data processing. QuickQuan automated optimization of compound-selected reaction monitoring (SRM) transitions by evaluating both positive and negative polarities during an infusion. Whichever mode produces the most intense Q1 scan is then carried to product ion spectra. QuickQuan then writes these SRM methods to a shared network database. The total volume of compound needed is 100 microL infused over approximately 1.6 min. The auto-optimization is carried out in 96-well plates and does not require an operator present. The SRM database was shared between two identical TSQ Quantum mass spectrometers. For data acquisition, QuickQuan automatically created a sequence file complete with a data processing method pre-populated with compound IDs and corresponding SRM transitions. To increase throughput we coupled each Finnigan Quantum with ultra-high-pressure liquid chromatography (uHPLC) accomplished using 4x Ultra Flux quaternary pumps that were designed to handle pressures up to 15 000 psi. The total run time for all analyses was 1.2 min using BEH 1.7 microm particle C18 columns. Further time reductions were realized with sample preparation accomplished using Strata Impact protein precipitation plates which provided an in-well protein crash and 0.20 micron filtering in a one-step process. Pharmacokinetic data turnaround time was significantly improved by combining these three techniques of automated method development with the speed efficiency of uHPLC and a single step in well sample preparation.

Highly efficacious factor Xa inhibitors containing alpha-substituted phenylcycloalkyl P4 moieties.

We previously disclosed a series of highly potent FXa inhibitors bearing alpha-substituted (CH(2)NR(1)R(2)) phenylcyclopropyl P4 moieties in the pyrazolodihydropyridone core system. Herein, we describe our continuous SAR efforts in this series. Effects of the C-3 substitution of the pyrazolodihydropyridone core and the alpha-substitution (R group) of the cyclopropyl ring on FXa binding affinity (FXa K(i)), human plasma anticoagulant activity (PT EC(2x)) and permeability are discussed. A set of compounds obtained from optimization of the R group and the C-3 substituent were orally bioavailable in dogs. Furthermore, representative compounds were highly efficacious in the rabbit arterio-venous shunt thrombosis model (EC(50)s=29-81nM).

N-in-1 dosing pharmacokinetics in drug discovery: experience, theoretical and practical considerations.

N-in-1 (or cassette) dosing pharmacokinetics (PK) has been used in drug discovery for rapid assessment of PK properties of new chemical entities. However, because of potential for drug-drug interactions this procedure is still controversial. This study was to retrospectively evaluate the N-in-1 dosing approach in drug discovery with an emphasis on the potential for drug-drug interactions. The systemic clearance, volume of distribution, oral bioavailability, and renal excretion of the 31 lead compounds in rats, dogs or chimpanzees were significantly correlated between the N-in-1 dosing and discrete studies with r values of 0.69, 0.91, 0.53, and 0.83 (p < 0.005 for all), respectively. PK parameters for 11 quality control compounds which were involved in 194 N-in-1 studies for screening approximately 1000 compounds had coefficient of variations of less than 70%. The intrinsic microsomal clearances generated from the N-in-1 and discrete incubations were nearly identical (r = 0.97, p < 0.0001). The intrinsic clearances of quality control compound from the N-in-1 incubations were consistent with its discrete CL(int) estimate (cv: 5.4%). Therefore, N-in-1 dosing is a useful approach in drug discovery to quickly obtain initial PK estimates. Potential drug-drug interactions that result in confounding PK estimates do not occur as frequently as expected.

Discovery of 1-(4-methoxyphenyl)-7-oxo-6-(4-(2-oxopiperidin-1-yl)phenyl)-4,5,6,7-tetrahydro-1H-pyrazolo[3,4-c]pyridine-3-carboxamide (apixaban, BMS-562247), a highly potent, selective, efficacious, and orally bioavailable inhibitor of blood coagulation factor Xa.

Efforts to identify a suitable follow-on compound to razaxaban (compound 4) focused on modification of the carboxamido linker to eliminate potential in vivo hydrolysis to a primary aniline. Cyclization of the carboxamido linker to the novel bicyclic tetrahydropyrazolopyridinone scaffold retained the potent fXa binding activity. Exceptional potency of the series prompted an investigation of the neutral P1 moieties that resulted in the identification of the p-methoxyphenyl P1, which retained factor Xa binding affinity and good oral bioavailability. Further optimization of the C-3 pyrazole position and replacement of the terminal P4 ring with a neutral heterocycle culminated in the discovery of 1-(4-methoxyphenyl)-7-oxo-6-(4-(2-oxopiperidin-1-yl)phenyl)-4,5,6,7-tetrahydro-1H-pyrazolo[3,4-c]pyridine-3-carboxamide (apixaban, compound 40). Compound 40 exhibits a high degree of fXa potency, selectivity, and efficacy and has an improved pharmacokinetic profile relative to 4.

Enantiopure five-membered cyclicdiamine derivatives as potent and selective inhibitors of factor Xa. Improving in vitro metabolic stability via core modifications.

We previously reported a series of enantiopure cis-(1R,2S)-cyclopentyldiamine derivatives as potent and selective inhibitors of Factor Xa (FXa). Herein, we describe our approach to improve the metabolic stability of this series via core modifications. Multiple resulting series of compounds demonstrated similarly high FXa potency and improved metabolic stability in human liver microsomes compared with the cyclopentyldiamide 1. (3R,4S)-Pyrrolidinyldiamide 31 was the best overall compound with human FXa K(i) of 0.50 nM, PT EC(2x) of 2.1 microM in human plasma, bioavailability of 25% and t(1/2)of 2.7h in dogs. Further biochemical characterization of compound 31 is also presented.

Development of a quantitative LC-MS/MS analytical method coupled with turbulent flow chromatography for digoxin for the in vitro P-gp inhibition assay.

Caco-2 cells, the human colon carcinoma cells, are typically used for screening compounds for their permeability characteristics and P-glycoprotein (P-gp) interaction potential during discovery and development. The P-gp inhibition of test compounds is assessed by performing bi-directional permeability studies with digoxin, a well established P-gp substrate probe. Studies performed with digoxin alone as well as digoxin in presence of test compounds as putative inhibitors constitute the P-gp inhibition assay used to assess the potential liability of discovery compounds. Radiolabeled (3)H-digoxin is commonly used in such studies followed by liquid scintillation counting. This manuscript describes the development of a sensitive, accurate, and reproducible LC-MS/MS method for analysis of digoxin and its internal standard digitoxin using an on-line extraction turbulent flow chromatography coupled to tandem mass spectrometric detection that is amendable to high throughput with use of 96-well plates. The standard curve for digoxin was linear between 10 nM and 5000 nM with regression coefficient (R(2)) of 0.99. The applicability and reliability of the analysis method was evaluated by successful demonstration of efflux ratio (permeability B to A over permeability A to B) greater than 10 for digoxin in Caco-2 cells. Additional evaluations were performed on 13 marketed compounds by conducting inhibition studies in Caco-2 cells using classical P-gp inhibitors (ketoconazole, cyclosporin, verapamil, quinidine, saquinavir etc.) and comparing the results to historical data with (3)H-digoxin studies. Similarly, P-gp inhibition studies with LC-MS/MS analytical method for digoxin were also performed for 21 additional test compounds classified as negative, moderate, and potent P-gp inhibitors spanning multiple chemo types and results compared with the historical P-gp inhibition data from the (3)H-digoxin studies. A very good correlation coefficient (R(2)) of 0.89 between the results from the two analytical methods affords an attractive LC-MS/MS analytical option for labs that need to conduct the P-gp inhibition assay without using radiolabeled compounds.