Perspective on high-throughput bioanalysis to support in vitro assays in early drug discovery.

As the desire for a shortened design/make/test/learn cycle increases in early drug discovery, the pressure to rapidly deliver drug metabolism pharmacokinetic data continues to rise. From a bioanalytical standpoint, in vitro assays are challenging because they are amenable to automation and thus capable of generating a high number of samples for analysis. To keep up with analysis demands, automated method development workflows, rapid sample analysis approaches and efficient data analysis software must be utilized. This work provides an outline of how we implemented those three aspects to provide bioanalytical support for in vitro drug metabolism pharmacokinetic assays, which include developing hundreds of mass spectrometry methods and analyzing thousands of samples per week, while delivering a median bioanalytical turnaround time of 1-2 business days.

The gut microbiota regulates white adipose tissue inflammation and obesity via a family of microRNAs.

The gut microbiota is a key environmental determinant of mammalian metabolism. Regulation of white adipose tissue (WAT) by the gut microbiota is a process critical to maintaining metabolic fitness, and gut dysbiosis can contribute to the development of obesity and insulin resistance (IR). However, how the gut microbiota regulates WAT function remains largely unknown. Here, we show that tryptophan-derived metabolites produced by the gut microbiota controlled the expression of the miR-181 family in white adipocytes in mice to regulate energy expenditure and insulin sensitivity. Moreover, dysregulation of the gut microbiota-miR-181 axis was required for the development of obesity, IR, and WAT inflammation in mice. Our results indicate that regulation of miR-181 in WAT by gut microbiota-derived metabolites is a central mechanism by which host metabolism is tuned in response to dietary and environmental changes. As we also found that MIR-181 expression in WAT and the plasma abundance of tryptophan-derived metabolites were dysregulated in a cohort of obese human children, the MIR-181 family may represent a potential therapeutic target to modulate WAT function in the context of obesity.

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.

Activation of the Amino Acid Response Pathway Blunts the Effects of Cardiac Stress.

BACKGROUND: The amino acid response (AAR) is an evolutionarily conserved protective mechanism activated by amino acid deficiency through a key kinase, general control nonderepressible 2. In addition to mobilizing amino acids, the AAR broadly affects gene and protein expression in a variety of pathways and elicits antifibrotic, autophagic, and anti-inflammatory activities. However, little is known regarding its role in cardiac stress. Our aim was to investigate the effects of halofuginone, a prolyl-tRNA synthetase inhibitor, on the AAR pathway in cardiac fibroblasts, cardiomyocytes, and in mouse models of cardiac stress and failure. METHODS AND RESULTS: Consistent with its ability to inhibit prolyl-tRNA synthetase, halofuginone elicited a general control nonderepressible 2-dependent activation of the AAR pathway in cardiac fibroblasts as evidenced by activation of known AAR target genes, broad regulation of the transcriptome and proteome, and reversal by l-proline supplementation. Halofuginone was examined in 3 mouse models of cardiac stress: angiotensin II/phenylephrine, transverse aortic constriction, and acute ischemia reperfusion injury. It activated the AAR pathway in the heart, improved survival, pulmonary congestion, left ventricle remodeling/fibrosis, and left ventricular function, and rescued ischemic myocardium. In human cardiac fibroblasts, halofuginone profoundly reduced collagen deposition in a general control nonderepressible 2-dependent manner and suppressed the extracellular matrix proteome. In human induced pluripotent stem cell-derived cardiomyocytes, halofuginone blocked gene expression associated with endothelin-1-mediated activation of pathologic hypertrophy and restored autophagy in a general control nonderepressible 2/eIF2α-dependent manner. CONCLUSIONS: Halofuginone activated the AAR pathway in the heart and attenuated the structural and functional effects of cardiac stress.

Soluble epoxide hydrolase inhibition exerts beneficial anti-remodeling actions post-myocardial infarction.

BACKGROUND: A contributory role for soluble epoxide hydrolase (sEH) in cardiac remodeling post-myocardial infarction (MI) has been suggested; however effects of sEH inhibition following MI have not been evaluated. In this study, we examined in vivo post-MI anti-remodeling effects of a novel sEH inhibitor (GSK2188931B) in the rat, and evaluated its direct in vitro effects on hypertrophy, fibrosis and inflammation. METHODS AND RESULTS: Post-MI administered GSK2188931B (80 mg/kg/d in chow) for 5 weeks improved left ventricular (LV) ejection fraction compared to vehicle-treated (Veh) rats (P<0.01; Sham 65 ± 2%, MI+Veh 30 ± 2%, MI+GSK 43 ± 2%) without affecting systolic blood pressure. Percentage area of LV tissue sections stained positive for picrosirius red (PS) and collagen I (CI) were elevated in LV non-infarct zone (P<0.05; NIZ; PS: Sham 1.46 ± 0.13%, MI+Veh 2.14 ± 0.22%, MI+GSK 1.28 ± 0.14%; CI: Sham 2.57 ± 0.17%, MI+Veh 5.06 ± 0.58%, MI+GSK 2.97 ± 0.34%) and peri-infarct zone (P<0.001; PIZ; PS: Sham 1.46 ± 0.13%, MI+Veh 9.06 ± 0.48%, MI+GSK 6.31 ± 0.63%; CI: Sham 2.57±0.17%, MI+Veh 10.51 ± 0.64%, MI+GSK 7.77 ± 0.57%); GSK2188931B attenuated this increase (P<0.05). GSK2188931B reduced macrophage infiltration into the PIZ (P<0.05). GSK2188931B reduced AngII- and TNFα-stimulated myocyte hypertrophy, AngII- and TGFß-stimulated cardiac fibroblast collagen synthesis, including markers of gene expression ANP, ß-MHC, CTGF and CI (P<0.05). GSK2188931B reduced TNFα gene expression in lipopolysaccharide (LPS)-stimulated monocytes (P<0.05). CONCLUSION: sEH inhibition exerts beneficial effects on cardiac function and ventricular remodeling post-MI, and direct effects on fibrosis and hypertrophy in cardiac cells. These findings suggest that sEH is an important contributor to the pathological remodeling following MI, and may be a useful target for therapeutic blockade in this setting.

Soluble epoxide hydrolase inhibition does not prevent cardiac remodeling and dysfunction after aortic constriction in rats and mice.

Epoxyeicosatrienoic acids, substrates for soluble epoxide hydrolase (sEH), exhibit vasodilatory and antihypertrophic activities. Inhibitors of sEH might therefore hold promise as heart failure therapeutics. We examined the ability of sEH inhibitors GSK2188931 and GSK2256294 to modulate cardiac hypertrophy, fibrosis, and function after transverse aortic constriction (TAC) in rats and mice. GSK2188931 administration was initiated in rats 1 day before TAC, whereas GSK2256294 treatment was initiated in mice 2 weeks after TAC. Four weeks later, cardiovascular function was assessed, plasma was collected for drug and sEH biomarker concentrations, and left ventricle was isolated for messenger RNA and histological analyses. In rats, although GSK2188931 prevented TAC-mediated increases in certain genes associated with hypertrophy and fibrosis (α-skeletal actin and connective tissue growth factor), the compound failed to attenuate TAC-induced increases in left ventricle mass, posterior wall thickness, end-diastolic volume and pressure, and perivascular fibrosis. Similarly, in mice, GSK2256294 did not reverse cardiac remodeling or systolic dysfunction induced by TAC. Both compounds increased the sEH substrate/product (leukotoxin/leukotoxin diol) ratio, indicating sEH inhibition. In summary, sEH inhibition does not prevent cardiac remodeling or dysfunction after TAC. Thus, targeting sEH seems to be insufficient for reducing pressure overload hypertrophy.

Design and synthesis of polyethylene glycol-modified biphenylsulfonyl-thiophene-carboxamidine inhibitors of the complement component C1s.

Complement C1s protease inhibitors have potential utility in the treatment of diseases associated with activation of the classical complement pathway such as humorally mediated graft rejection, ischemia-reperfusion injury (IRI), vascular leak syndrome, and acute respiratory distress syndrome (ARDS). The utility of biphenylsulfonyl-thiophene-carboxamidine small-molecule C1s inhibitors are limited by their poor in vivo pharmacokinetic properties. Pegylation of a potent analog has provided compounds with good potency and good in vivo pharmacokinetic properties.

Voltage-gated Na+ channel blockers reduce functional bladder capacity in the conscious spontaneously hypertensive rat.

OBJECTIVE: To evaluate the consequence of pharmacologic inhibition of voltage-gated Na(+) channels (Nav) in the conscious rat, based on Nav having been implicated as modulators of rodent urodynamics using knockout as well as antisense oligodeoxynucleotide approaches. METHODS: The urodynamic response to standard Nav blockers, lamotrigine, amitriptyline, mexiletine, and carbamazepine were evaluated using conscious, continuous-filling cystometry in spontaneously hypertensive rats (SHRs). As a selectivity evaluation, the activity of the Nav blockers at muscarinic receptors was assessed via effect on carbachol-evoked bladder contractions. RESULTS: Lamotrigine, amitriptyline, mexiletine, and carbamazepine decreased peak micturition pressure, micturition interval, and void volume. These effects were markedly similar to observations with muscarinic antagonists. Therefore, we evaluated the selectivity of these agents against bladder muscarinic receptors. Lamotrigine, mexiletine, and carbamazepine had no effect on muscarinic bladder contractions, whereas amitriptyline displayed a robust antagonism of carbachol-induced contractility. CONCLUSION: Three Nav blockers--lamotrigine, mexiletine, and carbamazepine--demonstrated a reduction in micturition pressure and functional bladder capacity, similar to previous observations with muscarinic antagonists. These 3 Nav blockers are free of muscarinic antagonism, consistent with their cystometric effects being mediated via their Nav blocking activities. The negative findings reported here with Nav blockers suggest that Nav channel blockade is unlikely to reflect an improved treatment strategy for bladder disorders over currently prescribed muscarinic antagonists.

Orally efficacious thrombin inhibitors with cyanofluorophenylacetamide as the P2 motif.

2-Cyano-6-fluorophenylacetamide was explored as a novel P2 scaffold in the design of thrombin inhibitors. Optimization around this structural motif culminated in 14, which is a potent thrombin inhibitor (K(i)=1.2nM) that exhibits robust efficacy in canine anticoagulation and thrombosis models upon oral administration.

Pharmacokinetic properties of TDP4815 after single intravenous and oral administrations to rat, rabbit, monkey, dog and in vitro drug metabolism.

The pharmacokinetics of TDP4815 was evaluated in rats, rabbits, dogs and monkeys. After intravenous administration, TDP4815 achieved C(O) of 3255 ng/ml in rats at 5 mg/kg, 9066 ng/ml in rabbits and 7858 ng/ml in monkeys at 6 mg/kg, and 4457 ng/ml in dogs at 3 mg/kg. The clearance (C(L)) was 3105, 1692, 835 and 640 ml/h/kg in rats, rabbits, monkeys and dogs, respectively. The volume of distribution (V(Z)) was more than 3861 ml/kg in all species, except 1915 ml/kg in monkeys. The oral bioavailability was rabbit >rat> monkey compared at 100 mg/kg, but it was much higher in dogs (>64%) after oral administrations. The calculated intrinsic clearance data suggested that the clearance of dog and human was restricted by binding to the plasma protein, and the clearance of rat and monkey was dependent on both the free fraction of plasma protein binding and the liver blood flow rate. The unbound hepatic intrinsic clearance of monkey was close to its C(L) suggesting that the hepatic clearance was an important excretion in monkeys. The poor oral bioavailability in the monkey may be related to the extensive glucuronidation. The V(Z).kg and C(L).kg in test species showed good correlation with the animal body weights (R(2)=0.87 and 0.96).

A functional radioreceptor assay of alpha-V-beta-3 (alphavbeta3) inhibitors in plasma: application as an ex vivo pharmacodynamic model.

Development of alphavbeta3-integrin inhibitors has been hampered by a lack of pharmacodynamic endpoints to identify doses that inhibit alphavbeta3 in vivo. To address this need, we developed an alphavbeta3 radioreceptor assay (RRA) that could be performed in 100% plasma. The RRA was based on 125I-echistatin binding to plate-immobilized alphavbeta3. Small molecule alphavbeta3 inhibitors efficiently competed echistatin binding to alphavbeta3 when the assay was carried out in buffer. However, when carried out in 100% plasma, the RRA revealed a 45 to >3000-fold loss in compound potencies. The losses in potency reflected, in part, the high plasma protein binding by the compounds examined. The RRA was adapted as an ex vivo pharmacodynamic model. Echistatin binding was measured in the presence of plasma harvested at timed intervals from rats dosed with select compounds. Using this pharmacodynamic model, compound and dose selection was optimized for further testing in models of corneal angiogenesis. Moderate anti-angiogenic activity was achieved when rats were dosed sufficient to achieve sustained (>50%) plasma inhibition through the trough interval. Thus, the RRA provided a simple technique to rank order compound potency in plasma, and could find general use as an ex vivo pharmacodynamic assay to select compounds and doses for preclinical and clinical proof-of-principle studies.

Design, synthesis, and biological evaluation of novel potent and selective alphavbeta3/alphavbeta5 integrin dual inhibitors with improved bioavailability. Selection of the molecular core.

A novel series of potent and selective alpha(v)beta(3)/alpha(v)beta(5) dual( )()inhibitors was designed, synthesized, and evaluated against several integrins. These compounds were synthesized through a Mitsunobu reaction between the guanidinium mimetics and the corresponding central templates. Guanidinium mimetics with enhaced rigidity (i.e., (2-pyridylamino)propoxy versus the 2-(6-methylamino-2-pyridyl)ethoxy) led to improved activity toward alpha(v)beta(3). Exemplary oral bioavailability in mice was achieved using the indole central scaffold. Although, oral bioavailability was maintained when the indole molecular core was replace with the bioisosteric benzofuran or benzothiophene ring systems, it was found to not significantly impact the integrin activity or selectivity. However, the indole series displayed the best in vivo pharmacokinetic properties. Thus, the indole series was selected for further structure-activity relationships to obtain more potent alpha(v)beta(3)/alpha(v)beta(5) dual antagonist with improved oral bioavailability.

Oxyguanidines. Part 2: Discovery of a novel orally active thrombin inhibitor through structure-based drug design and parallel synthesis.

Through structure-based drug design and parallel synthesis, we have discovered a novel series of nonpeptidic phenyl-based thrombin inhibitors using oxyguanidines as guanidine bioisosteres. These compounds have been found to be highly potent, highly selective, and orally bioavailable.

Oxyguanidines: application to non-peptidic phenyl-based thrombin inhibitors.

Although thrombin has been extensively researched with many examples of potent and selective inhibitors, the key characteristics of oral bioavailability and long half-life have been elusive. We report here a novel series non-peptidic phenyl-based, highly potent, highly selective and orally bioavailable thrombin inhibitors using oxyguanidines as guanidine-mimetics.