Role of Hepatic Stellate and Liver Sinusoidal Endothelial Cells in a Human Primary Cell Three-Dimensional Model of Nonalcoholic Steatohepatitis.

Nonalcoholic steatohepatitis (NASH) is an inflammatory and fibrotic liver disease that has reached epidemic proportions and has no approved pharmacologic therapies. Research and drug development efforts are hampered by inadequate preclinical models. This research describes a three-dimensional bioprinted liver tissue model of NASH built using primary human hepatocytes and nonparenchymal liver cells (hepatic stellate cells, liver sinusoidal endothelial cells, and Kupffer cells) from either healthy or NASH donors. Three-dimensional tissues bioprinted with cells sourced from diseased patients showed a NASH phenotype, including fibrosis. More importantly, this NASH phenotype occurred without the addition of disease-inducing agents. Bioprinted tissues composed entirely of healthy cells exhibited significantly less evidence of disease. The role of individual cell types in driving the NASH phenotype was examined by producing chimeric bioprinted tissues composed of healthy cells together with the addition of one or more diseased nonparenchymal cell types. These experiments reveal a role for both hepatic stellate and liver sinusoidal endothelial cells in the disease process. This model represents a fully human system with potential to detect clinically active targets and eventually therapies.

Systematic genetic analysis of early-onset gout: ABCG2 is the only associated locus.

OBJECTIVE: The aim of this study was to examine whether serum urate-associated genetic variants are associated with early-onset gout. METHODS: Participants with gout in the Genetics of Gout in Aotearoa study with available genotyping were included (n = 1648). Early-onset gout was defined as the first presentation of gout <40 years of age. Single nucleotide polymorphisms (SNPs) for the 10 loci most strongly associated with serum urate were genotyped. Allelic association of the SNPs with early-onset gout was tested using logistic regression in an unadjusted model and in a model adjusted for sex, body mass index, tophus presence, flare frequency, serum creatinine and highest serum urate. The analysis was also done in two replication cohorts: Eurogout (n = 704) and Ardea (n = 755), and data were meta-analysed. RESULTS: In the Genetics of Gout in Aotearoa study, there were 638 (42.4%) participants with early-onset gout. The ABCG2 rs2231142 gout risk T-allele was present more frequently in participants with early-onset gout compared with the later-onset group. For the other SNPs tested, no differences in risk allele number were observed. In the allelic association analysis, the ABCG2 rs2231142 T-allele was associated with early-onset gout in unadjusted and adjusted models. Analysis of the replication cohorts confirmed the association of early-onset gout with the ABCG2 rs2231142 T-allele, but not with other serum urate-associated SNPs. In the meta-analysis, the odds ratio (95% CI) for early-onset gout for the ABCG2 rs2231142 T-allele was 1.60 (1.41, 1.83). CONCLUSION: In contrast to other serum urate-raising variants, the ABCG2 rs2231142 T-allele is strongly associated with early-onset gout.

Characterization of Stereoselective Metabolism, Inhibitory Effect on Uric Acid Uptake Transporters, and Pharmacokinetics of Lesinurad Atropisomers.

Lesinurad [Zurampic; 2-(5-bromo-4-(4-cyclopropylnaphthalen-1-yl)-4H-1,2,4-triazol-3-ylthio)], a selective inhibitor of uric acid reabsorption transporters approved for the treatment of gout, is a racemate of two atropisomers. The objective of this investigation was to evaluate the stereoselectivity of metabolism, the inhibitory potency on kidney uric acid reabsorption transporters (URAT1 and OAT4), and the clinical pharmacokinetics of the lesinurad atropisomers. Incubations with human liver microsomes (HLM), recombinant CYP2C9, and recombinant CYP3A4 were carried out to characterize the stereoselective formation of three metabolites: M3 (hydroxylation), M4 (a dihydrodiol metabolite), and M6 (S-dealkylation). The formation of M3 in HLM with atropisomer 1 was approximately twice as much as that with atropisomer 2, whereas formation of M4 with atropisomer 1 was 8- to 12-fold greater than that with atropisomer 2. There were no significant differences in the plasma protein binding among lesinurad and the atropisomers. Following oral administration of 400 mg lesinurad once daily for 14 days to healthy human volunteers, the systemic exposure (C max at steady state and area under the concentration-time curve from time zero to the time of dosing interval) of atropisomer 1 was approximately 30% lower than that of atropisomer 2, whereas renal clearance was similar. In vitro cell-based assays using HEK293 stable cells expressing URAT1 and OAT4 demonstrated that atropisomer 2 was approximately 4-fold more potent against URAT1 than atropisomer 1 and equally active against OAT4. In conclusion, lesinurad atropisomers showed stereoselectivity in clinical pharmacokinetics, metabolism, and inhibitory potency against URAT1.

Association between ABCG2 rs2231142 and poor response to allopurinol: replication and meta-analysis.

Objective: ABCG2 rs2231142 (Q141K) has been reported to be associated with poor response to allopurinol, while there are conflicting data on the association between the genetically independent ABCG2 rs10011796 variant and allopurinol response. The aim of this study was to replicate the association of ABCG2 rs2231142 and rs10011796 with allopurinol response and perform a meta-analysis. Methods: Participants in the Long-term Allopurinol Safety Study Evaluating Outcomes in Gout Patients (LASSO) (n = 299) were studied. In patients with evidence of adherence to allopurinol therapy (plasma oxypurinol >20 µmol/l), good response was defined as serum urate <6 mg/dl on allopurinol ⩽300 mg/day and poor response as serum urate ⩾ 6 mg/dl despite allopurinol >300 mg/day. Association of rs2231142 and rs10011796 with poor response was tested in logistic regression models that included age, sex, BMI, ethnicity and estimated glomerular filtration rate. Results from the LASSO study and a subset of participants in the Genetics of Gout in Aotearoa New Zealand study (n = 296, including 264 from a previously published report) were combined by meta-analysis. Results: There was evidence for association of rs2231142 with allopurinol response [odds ratio (OR) = 2.35, P = 7.3 × 10-4] but not for rs10011796 (OR = 1.21, P = 0.33) in the LASSO cohort using an adjusted logistic regression model. Meta-analysis provided evidence of a significant association of rs2231142 with allopurinol response (OR = 2.43, P = 6.2 × 10-7), but not rs10011796 (OR = 1.06, P = 0.69). Conclusion: This study has confirmed the significant association of ABCG2 rs2231142 with poor response to allopurinol.

Coevolution of URAT1 and Uricase during Primate Evolution: Implications for Serum Urate Homeostasis and Gout.

Uric acid is the highly insoluble end-product of purine metabolism in humans. Serum levels exceeding the solubility threshold can trigger formation of urate crystals resulting in gouty arthritis. Uric acid is primarily excreted through the kidneys with 90% reabsorbed back into the bloodstream through the uric acid transporter URAT1. This reabsorption process is essential for the high serum uric acid levels found in humans. We discovered that URAT1 proteins from humans and baboons have higher affinity for uric acid compared with transporters from rats and mice. This difference in transport kinetics of URAT1 orthologs, along with inability of modern apes to oxidize uric acid due to loss of the uricase enzyme, prompted us to ask whether these events occurred concomitantly during primate evolution. Ancestral URAT1 sequences were computationally inferred and ancient transporters were resurrected and assayed, revealing that affinity for uric acid was increased during the evolution of primates. This molecular fine-tuning occurred between the origins of simians and their diversification into New- and Old-World monkey and ape lineages. Remarkably, it was driven in large-part by only a few amino acid replacements within the transporter. This alteration in primate URAT1 coincided with changes in uricase that greatly diminished the enzymatic activity and took place 27-77 Ma. These results suggest that the modifications to URAT1 transporters were potentially adaptive and that maintaining more constant, high levels of serum uric acid may have provided an advantage to our primate ancestors.

Lesinurad in combination with allopurinol: results of a phase 2, randomised, double-blind study in patients with gout with an inadequate response to allopurinol.

OBJECTIVES: To assess the efficacy and tolerability of lesinurad, an oral selective uric acid reabsorption inhibitor, in combination with allopurinol versus allopurinol alone in patients with gout and an inadequate response to allopurinol. METHODS: Patients (N=227) with an inadequate response to allopurinol, defined as serum urate (sUA) ≥6 mg/dL on ≥2 occasions ≥2 weeks apart despite ≥6 weeks of allopurinol, were randomised 2:1 to 4 weeks of double-blind treatment with lesinurad (200, 400 or 600 mg/day) or matching placebo in combination with their prestudy allopurinol dose (200-600 mg/day). Colchicine prophylaxis for gout flares was required. The primary end point was percent reduction from baseline sUA levels at 4 weeks. A pharmacokinetic substudy was also conducted. Safety was assessed throughout. RESULTS: Patients (n=208) received ≥1 dose of blinded medication. Lesinurad 200, 400 and 600 mg in combination with allopurinol produced significant mean percent reductions from baseline sUA of 16%, 22% and 30%, respectively, versus a mean 3% increase with placebo (p<0.0001, all doses vs placebo). Similar results were observed in patients with mild or moderate renal insufficiency (estimated creatinine clearance 30 to <90 mL/min). The incidence of ≥1 treatment-emergent adverse event was 46%, 48% and 54% with lesinurad 200, 400 and 600 mg, respectively, and 46% with placebo (most frequent, gout flares, arthralgia, headache and nasopharyngitis), with no deaths or serious adverse events. CONCLUSIONS: Lesinurad achieves clinically relevant and statistically significant reductions in sUA in combination with allopurinol in patients who warrant additional therapy on allopurinol alone. TRIAL REGISTRATION NUMBER: NCT01001338.

Urate Handling in the Human Body.

Elevated serum urate concentration is the primary cause of gout. Understanding the processes that affect serum urate concentration is important for understanding the etiology of gout and thereby understanding treatment. Urate handing in the human body is a complex system including three major processes: production, renal elimination, and intestinal elimination. A change in any one of these can affect both the steady-state serum urate concentration as well as other urate processes. The remarkable complexity underlying urate regulation and its maintenance at high levels in humans suggests that this molecule could potentially play an interesting role other than as a mere waste product to be eliminated as rapidly as possible.

Pharmacodynamic, pharmacokinetic and tolerability evaluation of concomitant administration of lesinurad and febuxostat in gout patients with hyperuricaemia.

OBJECTIVE: The aim of this study was to evaluate the pharmacodynamics (PDs), pharmacokinetics (PKs) and safety of lesinurad (selective uric acid reabsorption inhibitor) in combination with febuxostat (xanthine oxidase inhibitor) in patients with gout. METHODS: This study was a phase IB, multicentre, open-label, multiple-dose study of gout patients with serum uric acid (sUA) >8 mg/dl following washout of urate-lowering therapy with colchicine flare prophylaxis. Febuxostat 40 or 80 mg/day was administered on days 1-21, lesinurad 400 mg/day was added on days 8-14 and then lesinurad was increased to 600 mg/day on days 15-21. sUA, urine uric acid and PK profiles were evaluated at the end of each week. Safety was assessed by adverse events, laboratory tests and physical examinations. RESULTS: Initial treatment with febuxostat 40 or 80 mg/day monotherapy resulted in 67% and 56% of subjects, respectively, achieving a sUA level <6 mg/dl. Febuxostat 40 or 80 mg/day plus lesinurad 400 or 600 mg/day resulted in 100% of subjects achieving sUA <6 mg/dl and up to 100% achieving sUA <5 mg/dl. No clinically relevant changes in the PKs of either drug were noted. The combination was well tolerated. CONCLUSION: The clinically important targets of sUA <6 mg/dl and <5 mg/dl are achievable in 100% of patients when combining lesinurad and febuxostat.

Multimodal NASH prognosis using 3D imaging flow cytometry and artificial intelligence to characterize liver cells.

To improve the understanding of the complex biological process underlying the development of non-alcoholic steatohepatitis (NASH), 3D imaging flow cytometry (3D-IFC) with transmission and side-scattered images were used to characterize hepatic stellate cell (HSC) and liver endothelial cell (LEC) morphology at single-cell resolution. In this study, HSC and LEC were obtained from biopsy-proven NASH subjects with early-stage NASH (F2-F3) and healthy controls. Here, we applied single-cell imaging and 3D digital reconstructions of healthy and diseased cells to analyze a spatially resolved set of morphometric cellular and texture parameters that showed regression with disease progression. By developing a customized autoencoder convolutional neural network (CNN) based on label-free cell transmission and side scattering images obtained from a 3D imaging flow cytometer, we demonstrated key regulated cell types involved in the development of NASH and cell classification performance superior to conventional machine learning methods.

Discovery of orally available tetrahydroquinoline-based glucocorticoid receptor agonists.

A series of tetrahydroquinoline derivatives were synthesized and profiled for their ability to act as glucocorticoid receptor selective modulators. Structure-activity relationships of the tetrahydroquinoline B-ring lead to the discovery of orally available GR-selective agonists with high in vivo activity.

Tetrahydroquinoline glucocorticoid receptor agonists: discovery of a 3-hydroxyl for improving receptor selectivity.

We have previously disclosed a series of glucocorticoid receptor (GR) ligands derived from 6-indole-1,2,3,4-tetrahydroquinolines through structure-activity relationship (SAR) of the pendent C6-indole ring. In parallel with this effort, we now report SAR of the tetrahydroquinoline A-ring that identified the importance of a C3 hydroxyl in improving GR selectivity within a series of non-steroidal GR agonists.

Antitumour activity of a potent MEK inhibitor RDEA119/BAY 869766 combined with rapamycin in human orthotopic primary pancreatic cancer xenografts.

BACKGROUND: Combining MEK inhibitors with other signalling pathway inhibitors or conventional cytotoxic drugs represents a promising new strategy against cancer. RDEA119/BAY 869766 is a highly potent and selective MEK1/2 inhibitor undergoing phase I human clinical trials. The effects of RDEA119/BAY 869766 as a single agent and in combination with rapamycin were studied in 3 early passage primary pancreatic cancer xenografts, OCIP19, 21, and 23, grown orthotopically. METHODS: Anti-cancer effects were determined in separate groups following chronic drug exposure. Effects on cell cycle and downstream signalling were examined by flow cytometry and western blot, respectively. Plasma RDEA119 concentrations were measured to monitor the drug accumulation in vivo. RESULTS: RDEA119/BAY 869766 alone or in combination with rapamycin showed significant growth inhibition in all the 3 models, with a significant decrease in the percentage of cells in S-phase, accompanied by a large decrease in bromodeoxyuridine labelling and cell cycle arrest predominantly in G1. The S6 ribosomal protein was inhibited to a greater extent with combination treatment in all the three models. Blood plasma pharmacokinetic analyses indicated that RDEA119 levels achieved in vivo are similar to those that produce target inhibition and cell cycle arrest in vitro. CONCLUSIONS: Agents targeting the ERK and mTOR pathway have anticancer activity in primary xenografts, and these results support testing this combination in pancreatic cancer patients.

Antiinflammatory glucocorticoid receptor ligand with reduced side effects exhibits an altered protein-protein interaction profile.

Glucocorticoids are commonly used antiinflammatory agents whose use is limited by side effects. We have developed a series of glucocorticoid receptor (GR) ligands that retain the strong antiinflammatory activity of conventional glucocorticoids with reduced side effects. We present a compound, LGD5552, that binds the receptor efficiently and strongly represses inflammatory gene expression. LGD5552 bound to GR activates gene expression somewhat differently than glucocorticoids. It activates some genes with an efficacy similar to that of the glucocorticoids. However, other glucocorticoid-activated genes are not regulated by LGD5552. These differences may be because of the more efficient binding of corepressor in the presence of LGD5552, compared with glucocorticoid agonists. This class of nonsteroidal, GR-dependent antiinflammatory drugs may offer a safer alternative to steroidal glucocorticoids in the treatment of inflammatory disease.

The search for safer glucocorticoid receptor ligands.

Steroidal glucocorticoids are commonly used due to their powerful antiinflammatory activity. However, despite their excellent efficacy, severe side effects frequently limit the use of these drugs. The search for novel glucocorticoids with reduced side effects has been intensified by the discovery of new molecular details regarding the function of the glucocorticoid receptor. These new insights may pave the way for novel, safer therapies that retain the efficacy of currently prescribed steroids.

Cell-specific activation of the human skeletal alpha-actin by androgens.

Although it is evident that androgens increase muscle mass and strength, little is known about the critical molecular targets of androgens in skeletal muscle. In rodents, the skeletal alpha-actin gene is a tissue-specific gene expressed only in the levator ani and other skeletal muscles but not in the prostate or preputial gland, the well-known androgen target tissue. We identified tissue-specific androgen-regulated genes in the skeletal muscle in rats after oral administration of androgens and focused on androgen-dependent up-regulation of the skeletal alpha-actin gene. To investigate the mechanism of action, an in vitro system with various cell lines and a series of deletion mutants of the alpha-actin promoter were used. The human skeletal alpha-actin promoter was activated by androgens in the muscle cell line C2C12 but not in the liver, prostate, or breast cancer cell lines in which exogenous human androgen receptor is expressed. The sequence of the promoter is sufficient for cell-specific androgen response, providing a model for the tissue specificity demonstrated in vivo. Using a series of deletion mutants, the androgen response can be maintained using just the proximal promoter region. The importance of androgen regulation of this small portion of the human skeletal alpha-actin promoter was demonstrated by the correlation between muscle and the alpha-actin promoter activity for an array of selective androgen receptor modulators (SARMs), including an orally active SARM LGD2226. Taken together, the results suggest that the regulation of skeletal alpha-actin by androgens/SARMs may represent an important model system for understanding androgen anabolic action in the muscle.

LGD-5552, an antiinflammatory glucocorticoid receptor ligand with reduced side effects, in vivo.

Treatment of inflammation is often accomplished through the use of glucocorticoids. However, their use is limited by side effects. We have examined the activity of a novel glucocorticoid receptor ligand that binds the receptor efficiently and strongly represses inflammatory gene expression. This compound has potent antiinflammatory activity in vivo and represses the transcription of the inflammatory cytokine monocyte chemoattractant protein-1 and induces the antiinflammatory cytokine IL-10. The compound demonstrates differential gene regulation, compared with commonly prescribed glucocorticoids, effectively inducing some genes and repressing others in a manner different from the glucocorticoid prednisolone. The separation between the antiinflammatory effects of LGD-5552 and the side effects commonly associated with glucocorticoid treatment suggest that this molecule differs significantly from prednisolone and other steroids and may provide a safer therapeutic window for inflammatory conditions now commonly treated with steroidal glucocorticoids.

Discovery of nonsteroidal glucocorticoid receptor ligands based on 6-indole-1,2,3,4-tetrahydroquinolines.

A series of nonsteroidal glucocorticoid receptor (GR) ligands based on a 6-indole-1,2,3,4-tetrahydroquinoline scaffold are reported. Structure-activity relationship (SAR) of the pendent indole group identified compound 20 exhibiting good GR binding affinity (K(i)=1.5nM) and 100- to 1000-fold selectivity over MR, PR, and AR while showing activity in an E-selectin repression assay.

Pharmacokinetics, pharmacodynamics, and tolerability of verinurad, a selective uric acid reabsorption inhibitor, in healthy Japanese and non-Asian male subjects.

PURPOSE: Verinurad (RDEA3170) is a selective uric acid reabsorption inhibitor in clinical development for treatment of gout and asymptomatic hyperuricemia. This study evaluated verinurad pharmacokinetics, pharmacodynamics, and tolerability in healthy Japanese and non-Asian adult male subjects. METHODS: This was a Phase I, randomized, single-blind, placebo-controlled study. Panels of 8 Japanese subjects were randomized to receive oral verinurad (2.5-15 mg) or placebo administered as a single dose in a fasted and fed state and as once-daily doses for 7 days in a fed state. Eight non-Asian subjects received verinurad 10 mg as a single dose (fasted and fed) and multiple doses in the fed state. Serial plasma/serum and urine samples were assayed for verinurad and uric acid. Safety was assessed by adverse events and laboratory data. RESULTS: Of 48 randomized subjects, 46 (Japanese, 39; non-Asian, 7) completed the study. Following single or multiple doses in Japanese subjects, maximum plasma concentration (Cmax) and area under the plasma concentration-time curve (AUC) increased in a near dose-proportional manner. Time to Cmax (Tmax) was ~1.25-2.0 hours with fasting. A moderate-fat meal delayed Tmax (range 3.0-5.0 hours) and had a variable effect on AUC (0%-97% increase) and Cmax (0%-26% increase) across the dose groups. Following multiple verinurad 10 mg doses, Cmax and AUC were 38% and 23% higher, respectively, in Japanese vs non-Asian subjects, largely due to body weight differences. Mean reduction of serum urate following multiple verinurad 10 mg doses was 46% and 44% after 24 hours in Japanese and non-Asian subjects, respectively. Verinurad was well tolerated at all doses. CONCLUSION: Verinurad monotherapy lowered serum urate and was well tolerated in both healthy Japanese and non-Asian males, while small differences in plasma pharmacokinetics were observed. These data support further evaluation of once-daily verinurad as a treatment for gout and asymptomatic hyperuricemia.