Development of an LC-MS/MS assay for quantification of intact INSL3 in rat plasma.

Background: Relatively large disulfide-linked polypeptides can serve as signaling molecules for a diverse array of biological processes and may be studied in animal models to investigate their function in vivo. The aim of this work was to develop an LC-MS/MS assay to measure a model peptide, INSL3, in rat plasma. Results: A dual enrichment strategy incorporating both protein precipitation and solid phase extraction was utilized to isolate INSL3 from rat plasma, followed by targeted LC-MS/MS detection. The method was able to measure full-length INSL3 (6.1 kDa) down to 0.2 ng/ml with acceptable accuracy and precision. Conclusion: The final assay was applied to support an exploratory pharmacokinetic study to evaluate steady-state concentrations of dosed INSL3 in rat plasma.

Design and Optimization of an Acyclic Amine Series of TRPV4 Antagonists by Electronic Modulation of Hydrogen Bond Interactions.

Investigation of TRPV4 as a potential target for the treatment of pulmonary edema associated with heart failure generated a novel series of acyclic amine inhibitors displaying exceptional potency and PK properties. The series arose through a scaffold hopping approach, which relied on use of an internal H-bond to replace a saturated heterocyclic ring. Optimization of the lead through investigation of both aryl regions revealed approaches to increase potency through substituents believed to enhance separate intramolecular and intermolecular H-bond interactions. A proposed internal H-bond between the amine and neighboring benzenesulfonamide was stabilized by electronically modulating the benzenesulfonamide. In the aryl ether moiety, substituents para to the nitrile demonstrated an electronic effect on TRPV4 recognition. Finally, the acyclic amines inactivated CYP3A4 and this liability was addressed by modifications that sterically preclude formation of a putative metabolic intermediate complex to deliver advanced TRPV4 antagonists as leads for discovery of novel medicines.

Discovery of GSK3527497: A Candidate for the Inhibition of Transient Receptor Potential Vanilloid-4 (TRPV4).

GSK3527497, a preclinical candidate for the inhibition of TRPV4, was identified starting from the previously reported pyrrolidine sulfonamide TRPV4 inhibitors 1 and 2. Optimization of projected human dose was accomplished by specifically focusing on in vivo pharmacokinetic parameters CLu, Vdssu, and MRT. We highlight the use of conformational changes as a novel approach to modulate Vdssu and present results that suggest that molecular-shape-dependent binding to tissue components governs Vdssu in addition to bulk physicochemical properties. Optimization of CLu within the series was guided by in vitro metabolite identification, and the poor FaSSIF solubility imparted by the crystalline properties of the pyrrolidine diol scaffold was improved by the introduction of a charged moiety to enable excellent exposure from high crystalline doses. GSK3527497 is a preclinical candidate suitable for oral and iv administration that is projected to inhibit TRPV4 effectively in patients from a low daily clinical dose.

Design and Optimization of Sulfone Pyrrolidine Sulfonamide Antagonists of Transient Receptor Potential Vanilloid-4 with in Vivo Activity in a Pulmonary Edema Model.

Pulmonary edema is a common ailment of heart failure patients and has remained an unmet medical need due to dose-limiting side effects associated with current treatments. Preclinical studies in rodents have suggested that inhibition of transient receptor potential vanilloid-4 (TRPV4) cation channels may offer an alternative-and potentially superior-therapy. Efforts directed toward small-molecule antagonists of the TRPV4 receptor have led to the discovery of a novel sulfone pyrrolidine sulfonamide chemotype exemplified by lead compound 6. Design elements toward the optimization of TRPV4 activity, selectivity, and pharmacokinetic properties are described. Activity of leading exemplars 19 and 27 in an in vivo model suggestive of therapeutic potential is highlighted herein.

4,6-Diaminopyrimidines as Highly Preferred Troponin I-Interacting Kinase (TNNI3K) Inhibitors.

Structure-guided progression of a purine-derived series of TNNI3K inhibitors directed design efforts that produced a novel series of 4,6-diaminopyrimidine inhibitors, an emerging kinase binding motif. Herein, we report a detailed understanding of the intrinsic conformational preferences of the scaffold, which impart high specificity for TNNI3K. Further manipulation of the template based on the conformational analysis and additional structure-activity relationship studies provided enhancements in kinase selectivity and pharmacokinetics that furnished an advanced series of potent inhibitors. The optimized compounds (e.g., GSK854) are suitable leads for identifying new cardiac medicines and have been employed as in vivo tools in investigational studies aimed at defining the role of TNNI3K within heart failure.

Discovery of renin inhibitors containing a simple aspartate binding moiety that imparts reduced P450 inhibition.

Discovery of potent renin inhibitors which contain a simplified alkylamino Asp-binding group and exhibit improved selectivity for renin over Cyp3A4 is described. Structure-function results in this series are rationalized based on analysis of selected compounds bound to renin, and the contribution of each molecular feature leading to the reduced P450 inhibition is quantified.

Novel fusion of GLP-1 with a domain antibody to serum albumin prolongs protection against myocardial ischemia/reperfusion injury in the rat.

BACKGROUND: Glucagon-like peptide-1 (GLP-1) and its mimetics reduce infarct size in the setting of acute myocardial ischemia/reperfusion (I/R) injury. However, the short serum half-life of GLP-1 and its mimetics may limit their therapeutic use in acute myocardial ischemia. Domain antibodies to serum albumin (AlbudAbs) have been developed to extend the serum half-life of short lived therapeutic proteins, peptides and small molecules. In this study, we compared the effect of a long acting GLP-1 agonist, DPP-IV resistant GLP-1 (7-36, A8G) fused to an AlbudAb (GAlbudAb), with the effect of the GLP-1 mimetic, exendin-4 (short half-life GLP-1 agonist) on infarct size following acute myocardial I/R injury. METHODS: Male Sprague-Dawley rats (8-week-old) were treated with vehicle, GAlbudAb or exendin-4. Myocardial ischemia was induced 2 h following the final dose for GAlbudAb and 30 min post the final dose for exendin-4. In a subgroup of animals, the final dose of exendin-4 was administered (1 µg/kg, SC, bid for 2 days) 6 h prior to myocardial ischemia when plasma exendin-4 was at its minimum concentration (C(min)). Myocardial infarct size, area at risk and cardiac function were determined 24 h after myocardial I/R injury. RESULTS: GAlbudAb and exendin-4 significantly reduced myocardial infarct size by 28% and 23% respectively, compared to vehicle (both p < 0.01 vs. vehicle) after I/R injury. Moreover, both GAlbudAb and exendin-4 markedly improved post-ischemic cardiac contractile function. Body weight loss and reduced food intake consistent with the activation of GLP-1 receptors was observed in all treatment groups. However, exendin-4 failed to reduce infarct size when administered 6 h prior to myocardial ischemia, suggesting continuous activation of the GLP-1 receptors is needed for cardioprotection. CONCLUSIONS: Cardioprotection provided by GAlbudAb, a long acting GLP-1 mimetic, following myocardial I/R injury was comparable in magnitude, but more sustained in duration than that produced by short-acting exendin-4. Very low plasma concentrations of exendin-4 failed to protect the heart from myocardial I/R injury, suggesting that sustained GLP-1 receptor activation plays an important role in providing cardioprotection in the setting of acute myocardial I/R injury. Long-acting GLP-1 agonists such as GAlbudAb may warrant additional evaluation as novel therapeutic agents to reduce myocardial I/R injury during acute coronary syndrome.

Discovery of 1-(1,3,5-triazin-2-yl)piperidine-4-carboxamides as inhibitors of soluble epoxide hydrolase.

1-(1,3,5-Triazin-yl)piperidine-4-carboxamide inhibitors of soluble epoxide hydrolase were identified from high through-put screening using encoded library technology. The triazine heterocycle proved to be a critical functional group, essential for high potency and P450 selectivity. Phenyl group substitution was important for reducing clearance, and establishing good oral exposure. Based on this lead optimization work, 1-[4-methyl-6-(methylamino)-1,3,5-triazin-2-yl]-N-{[[4-bromo-2-(trifluoromethoxy)]-phenyl]methyl}-4-piperidinecarboxamide (27) was identified as a useful tool compound for in vivo investigation. Robust effects on a serum biomarker, 9, 10-epoxyoctadec-12(Z)-enoic acid (the epoxide derived from linoleic acid) were observed, which provided evidence of robust in vivo target engagement and the suitability of 27 as a tool compound for study in various disease models.

Comparison of soluble guanylate cyclase stimulators and activators in models of cardiovascular disease associated with oxidative stress.

Soluble guanylate cyclase (sGC), the primary mediator of nitric oxide (NO) bioactivity, exists as reduced (NO-sensitive) and oxidized (NO-insensitive) forms. We tested the hypothesis that the cardiovascular protective effects of NO-insensitive sGC activation would be potentiated under conditions of oxidative stress compared to those of NO-sensitive sGC stimulation. The cardiovascular effects of the NO-insensitive sGC activator GSK2181236A [a low, non-depressor dose, and a high dose which lowered mean arterial pressure (MAP) by 5-10 mmHg] and those of equi-efficacious doses of the NO-sensitive sGC stimulator BAY 60-4552 were assessed in (1) Sprague Dawley rats during coronary artery ischemia/reperfusion (I/R) and (2) spontaneously hypertensive stroke prone rats (SHR-SP) on a high salt/fat diet (HSFD). In I/R, neither compound reduced infarct size 24 h after reperfusion. In SHR-SP, HSFD increased MAP, urine output, microalbuminuria, and mortality, caused left ventricular hypertrophy with preserved ejection fraction, and impaired endothelium-dependent vasorelaxation. The low dose of BAY 60-4552, but not that of GSK2181236A, decreased urine output, and improved survival. Conversely, the low dose of GSK2181236A, but not that of BAY 60-4552, attenuated the development of cardiac hypertrophy. The high doses of both compounds similarly attenuated cardiac hypertrophy and improved survival. In addition to these effects, the high dose of BAY 60-4552 reduced urine output and microalbuminuria and attenuated the increase in MAP to a greater extent than did GSK2181236A. Neither compound improved endothelium-dependent vasorelaxation. In SHR-SP isolated aorta, the vasodilatory responses to the NO-dependent compounds carbachol and sodium nitroprusside were attenuated by HSFD. In contrast, the vasodilatory responses to both GSK2181236A and BAY 60-4552 were unaltered by HSFD, indicating that reduced NO-bioavailability and not changes in the oxidative state of sGC is responsible for the vascular dysfunction. In summary, GSK2181236A and BAY 60-4552 provide partial benefit against hypertension-induced end-organ damage. The differential beneficial effects observed between these compounds could reflect tissue-specific changes in the oxidative state of sGC and might help direct the clinical development of these novel classes of therapeutic agents.

Cassette dosing for pharmacokinetic screening in drug discovery: comparison of clearance, volume of distribution, half-life, mean residence time, and oral bioavailability obtained by cassette and discrete dosing in rats.

The purpose of this investigation was to compare selected pharmacokinetic (PK) parameters obtained by cassette and discrete dosing of compounds in rats. The concordance of PK properties obtained by the two dosing strategies was evaluated for 116 compounds representing various therapeutic programs and diverse chemical structures. The correspondence between cassette- and discrete-dosing-derived PK properties was examined semiquantitatively and qualitatively. For semiquantitative comparison, compounds with cassette-to-discrete PK parameter ratios between 0.5 and 2 (inclusive) were considered to be in agreement. For qualitative comparison, compounds were divided into three categories (low, moderate, and high) based on the value of the PK parameter; compounds that fell into the same category following cassette and discrete dosing were considered to be in agreement. Of the 116 compounds evaluated, 89%, 91%, 80%, and 91% of the compounds were semiquantitatively equivalent for the intravenous PK parameters of clearance (CL), volume of distribution (Vdss), terminal elimination plasma half-life (HL), and mean residence time (MRT), respectively, whereas 79%, 80%, 79%, and 72% were qualitatively similar for CL, Vdss, MRT, and terminal elimination plasma HL, respectively. Following oral administration, bioavailability concordance was 72% when assessed qualitatively and 78% when determined semiquantitatively. Results from these analyses indicate that a cassette dosing strategy is a viable approach to screen compounds for PK properties within a drug discovery setting.

Potent, selective and orally bioavailable dihydropyrimidine inhibitors of Rho kinase (ROCK1) as potential therapeutic agents for cardiovascular diseases.

Recent studies using known Rho-associated kinase isoform 1 (ROCK1) inhibitors along with cellular and molecular biology data have revealed a pivotal role of this enzyme in many aspects of cardiovascular function. Here we report a series of ROCK1 inhibitors which were originally derived from a dihydropyrimidinone core 1. Our efforts focused on the optimization of dihydropyrimidine 2, which resulted in the identification of a series of dihydropyrimidines with improved pharmacokinetics and P450 properties.

Discovery of aminofurazan-azabenzimidazoles as inhibitors of Rho-kinase with high kinase selectivity and antihypertensive activity.

The discovery, proposed binding mode, and optimization of a novel class of Rho-kinase inhibitors are presented. Appropriate substitution on the 6-position of the azabenzimidazole core provided subnanomolar enzyme potency in vitro while dramatically improving selectivity over a panel of other kinases. Pharmacokinetic data was obtained for the most potent and selective examples and one (6n) has been shown to lower blood pressure in a rat model of hypertension.

Development of dihydropyridone indazole amides as selective Rho-kinase inhibitors.

Rho kinase (ROCK1) mediates vascular smooth muscle contraction and is a potential target for the treatment of hypertension and related disorders. Indazole amide 3 was identified as a potent and selective ROCK1 inhibitor but possessed poor oral bioavailability. Optimization of this lead resulted in the discovery of a series of dihydropyridones, exemplified by 13, with improved pharmacokinetic parameters relative to the initial lead. Indazole substitution played a critical role in decreasing clearance and improving oral bioavailability.

Methods for predicting human drug metabolism.

Drug metabolism information is a necessary component of drug discovery and development. The key issues in drug metabolism include identifying: the enzyme(s) involved, the site(s) of metabolism, the resulting metabolite(s), and the rate of metabolism. Methods for predicting human drug metabolism from in vitro and computational methodologies and determining relationships between the structure and metabolic activity of molecules are also critically important for understanding potential drug interactions and toxicity. There are numerous experimental and computational approaches that have been developed in order to predict human metabolism which have their own limitations. It is apparent that few of the computational tools for metabolism prediction alone provide the major integrated functions needed to assist in drug discovery. Similarly the different in vitro methods for human drug metabolism themselves have implicit limitations. The utilization of these methods for pharmaceutical and other applications as well as their integration is discussed as it is likely that hybrid methods will provide the most success.

Novel Rho kinase inhibitors with anti-inflammatory and vasodilatory activities.

Increased Rho kinase (ROCK) activity contributes to smooth muscle contraction and regulates blood pressure homeostasis. We hypothesized that potent and selective ROCK inhibitors with novel structural motifs would help elucidate the functional role of ROCK and further explore the therapeutic potential of ROCK inhibition for hypertension. In this article, we characterized two aminofurazan-based inhibitors, GSK269962A [N-(3-{[2-(4-amino-1,2,5-oxadiazol-3-yl)-1-ethyl-1H-imidazo[4, 5-c]pyridin-6-yl]oxy}phenyl)-4-{[2-(4-morpholinyl)ethyl]-oxy}benzamide] and SB-7720770-B [4-(7-{[(3S)-3-amino-1-pyrrolidinyl]carbonyl}-1-ethyl-1H-imidazo[4,5-c]pyridin-2-yl)-1,2,5-oxadiazol-3-amine], as members of a novel class of compounds that potently inhibit ROCK enzymatic activity. GSK269962A and SB-772077-B have IC50 values of 1.6 and 5.6 nM toward recombinant human ROCK1, respectively. GSK269962A also exhibited more than 30-fold selectivity against a panel of serine/threonine kinases. In lipopolysaccharide-stimulated monocytes, these inhibitors blocked the generation of inflammatory cytokines, such as interleukin-6 and tumor necrosis factor-alpha. Furthermore, both SB-772077-B and GSK269962A induced vasorelaxation in preconstricted rat aorta with an IC50 of 39 and 35 nM, respectively. Oral administration of either GSK269962A or SB-772077-B produced a profound dose-dependent reduction of systemic blood pressure in spontaneously hypertensive rats. At doses of 1, 3, and 30 mg/kg, both compounds induced a reduction in blood pressure of approximately 10, 20, and 50 mm Hg. In addition, administration of SB-772077-B also dramatically lowered blood pressure in DOCA salt-induced hypertensive rats. SB-772077-B and GSK269962A represent a novel class of ROCK inhibitors that have profound effects in the vasculature and may enable us to further evaluate the potential beneficial effects of ROCK inhibition in animal models of cardiovascular as well as other chronic diseases.

Extrapolation of preclinical pharmacokinetics and molecular feature analysis of "discovery-like" molecules to predict human pharmacokinetics.

The prediction of human pharmacokinetics from preclinical species is an integral component of drug discovery. Recent studies with a 103-compound dataset suggested that scaling from monkey pharmacokinetic data tended to be the most accurate method for predicting human clearance. Additionally, interrogation of the two-dimensional molecular properties of these molecules produced a set of associations which predict the likely extrapolative outcome (success or failure) of preclinical data to project human pharmacokinetics. However, a limitation of the previous analyses was the relative paucity of data for typical "discovery-like" molecules (molecular weight >300 and/or clogP >3). The objective of this investigation was to generate preclinical data required for extension of this dataset for additional discovery-like molecules and determine whether the aforementioned findings continue to apply for these molecules. In vivo nonrodent intravenous pharmacokinetic data were generated for 13 molecules, and data for 8 additional molecules were obtained from the literature. Additionally, the various scaling methodologies and molecular features analysis were applied to this new dataset to predict human pharmacokinetics. Whereas the predictive accuracies demonstrated across all of the various methodologies were lower for this higher clearance compound dataset, scaling from monkey liver blood flow continued to be an accurate methodology, and human volume of distribution was similarly well predicted regardless of scaling methodology. Lastly, application of the molecular feature associations, particularly data-dependent associations, afforded an improved predictivity compared with the liver blood flow scaling approaches, and provides insight into the extrapolation of high clearance compounds in the preclinical species to human.

Investigation of the utility of published in vitro intrinsic clearance data for prediction of in vivo clearance.

INTRODUCTION: This study was conducted to compare and contrast published in vitro intrinsic clearance values reported for compounds from different laboratories and the predictivity of these data to project in vivo clearance. METHODS: A total of 103 compounds were selected for investigation and an exhaustive literature search was conducted to identify in vitro intrinsic clearance (CL,i) values for comparative purposes. The simple well-stirred model was used to predict in vivo clearance using these in vitro intrinsic clearance values. RESULTS: Data were available in the literature for <10% of the compounds of interest in rat, dog, monkey, or human S9, as well as <10% for dog or monkey microsomes or hepatocytes. Therefore, this comparative exercise was limited to rat and human microsomes and hepatocytes. Examination of the available CL,i values indicated a substantial (up to 100 s-fold) variation in values reported in the literature; this variability translated into substantial variation in predicted in vivo clearance. DISCUSSION: The literature paucity and variability described here demonstrate the importance of generating experimentally consistent de novo CL,i data for the purpose of method validation or in vitro-in vivo scaling.

Extrapolation of human pharmacokinetic parameters from rat, dog, and monkey data: Molecular properties associated with extrapolative success or failure.

Human pharmacokinetic parameters are often predicted prior to clinical study from in vivo preclinical pharmacokinetic data. Recent data suggest that extrapolation of monkey pharmacokinetic data tends to be the most accurate method for predicting human clearance. In this study, the molecular features of a 103-compound dataset were analyzed to determine whether calculated physiochemical properties may be used to predict the extrapolative success or failure of rat, dog, and monkey data to project human pharmacokinetic parameters. Molecular properties (molecular weight, molar refractivity, log of the octanol-water partition coefficient, polar surface area, hydrogen bond donor/acceptor count, and rotatable bond count) were calculated, and relationships were sought for each preclinical species between extrapolative outcome for human clearance, distributional volume, and mean residence time, and each molecular feature or combination of features. The findings indicated that calculated molecular properties may be used both to predict extrapolative outcome for human pharmacokinetic properties from preclinical animal data, and to prospectively aid in the selection of an appropriate preclinical species in which to generate preclinical data to more reliably project human clearance. These observations demonstrate the utility of a combined computational and in vivo animal testing approach to projecting human pharmacokinetic parameters.

Computational and experimental studies on the distribution of addition and substitution products of the microsomal glutathione transferase 1-catalyzed conjugation of glutathione with fluoroalkenes.

The glutathione transferase-catalyzed reaction of glutathione with haloalkenes results in the formation of addition or substitution products or both. Glutathione conjugates of haloalkenes may be metabolized and excreted at different rates, may follow different metabolic pathways, and may exhibit different toxicities. Microsomal glutathione transferase 1 (MGST1)-catalyzed conjugation of chlorotrifluoroethene, hexafluoropropene, and 2-(fluoromethoxy)-1,1,3,3,3-pentafluoro-1-propene results in differing proportions of addition and substitution products. The aim of the present study was to develop a computational model to predict the outcome of the MGST1-catalyzed reaction of glutathione with haloalkenes. An ab initio computational study of the reaction of ethanethiolate, a surrogate for glutathione, with the chlorotrifluoroethene, hexafluoropropene, and 2-(fluoromethoxy)-1,1,3,3,3-pentafluoro-1-propene was conducted. An empirical study was also conducted to quantify the distribution of addition and substitution products that resulted from the MGST1-catalyzed reaction of glutathione with these fluoroalkenes. The results show that this computational model accurately predicted the distribution of the addition and substitution products that result from the MGST1-catalyzed reaction of glutathione with these fluoroalkenes.

Structure-activity relationship for the biotransformation of haloalkenes by rat liver microsomal glutathione transferase 1.

Many haloalkenes are nephrotoxic in rodents, and experimental evidence supports a glutathione-dependent bioactivation pathway that leads to nephrotoxicity or nephrocarcinogenicity, or both. The reaction of glutathione with haloalkenes is catalyzed by cytosolic glutathione transferases (cGST) and microsomal glutathione transferase 1 (MGST1). The aim of this study was to develop a computational approach to predict the competency of cGST and MGST1 to catalyze the reaction of glutathione with a range of haloalkenes. The hypothesis tested was that the semiempirically computed energy of the lowest unoccupied molecular orbital (E(LUMO)) of a haloalkene may be used to predict the competency of cGST and MGST1 to catalyze its reaction with glutathione. The MGST1- and cGST-catalyzed reaction of glutathione with nine haloalkenes with E(LUMO) values ranging from -1.14 to 0.38 eV was determined experimentally. The data indicated that the E(LUMO) values for haloalkenes were inversely related to the specific activity of the MGST1-catalyzed reaction but not the cGST-catalyzed reaction. These data also demonstrated that MGST1 catalyzed the reaction of glutathione with haloalkenes with E(LUMO) values equal to or more negative than -0.73 eV and that cGST catalyzed the reaction of glutathione with haloalkenes with E(LUMO) values more negative than -0.06 eV.