Safety, pharmacokinetics and pharmacodynamics of obeticholic acid in subjects with fibrosis or cirrhosis from NASH.

BACKGROUND & AIMS: Fibrosis stage is a strong predictor of nonalcoholic steatohepatitis (NASH) outcomes. Two blinded studies evaluated the pharmacokinetics, pharmacodynamics and safety of obeticholic acid (OCA) in subjects with staged NASH fibrosis or cirrhosis. METHODS: Study 747-117 randomized 51 subjects with NASH (fibrosis stages F1-F4) to daily placebo, OCA 10 or OCA 25 mg (1:2:2) for 85 days. Study 747-118 randomized 24 subjects with NASH cirrhosis (F4; Child-Pugh [CP]-A) and normal liver control subjects matched for similar body weight to daily OCA 10 or OCA 25 mg (1:1) for 28 days. Individual and combined study data were analysed. RESULTS: No severe or serious adverse events (AEs) or AEs leading to discontinuation or death occurred. Pruritus was the most frequent AE. Plasma OCA exposure (dose-normalized area under the curve) increased with fibrosis stage but was a relatively poor predictor of hepatic OCA exposure (primary site of action), which remained constant across fibrosis stages F1-F3 and increased 1.8-fold compared with F1 in subjects with cirrhosis due to NASH. Both cohorts showed robust changes in farnesoid X receptor activation markers with OCA treatment and marked decreases in alanine transaminase, aspartate transaminase and gamma-glutamyltransferase. CONCLUSIONS: Despite higher drug exposures in subjects with NASH cirrhosis, short-term daily treatment with OCA 10 or 25 mg was generally safe and well tolerated in subjects with NASH fibrosis or NASH CP-A cirrhosis. Both cohorts experienced improvements in nonhistologic pharmacodynamic markers consistent with previously conducted OCA phase 2 and phase 3 studies in NASH fibrosis.

Farnesoid X Receptor Agonists Obeticholic Acid and Chenodeoxycholic Acid Increase Bile Acid Efflux in Sandwich-Cultured Human Hepatocytes: Functional Evidence and Mechanisms.

The farnesoid X receptor (FXR) is a nuclear receptor that regulates genes involved in bile acid homeostasis. FXR agonists, obeticholic acid (OCA) and chenodeoxycholic acid (CDCA), increase mRNA expression of efflux transporters in sandwich-cultured human hepatocytes (SCHH). This study evaluated the effects of OCA and CDCA treatment on the uptake, basolateral efflux, and biliary excretion of a model bile acid, taurocholate (TCA), in SCHH. In addition, changes in the protein expression of TCA uptake and efflux transporters were investigated. SCHH were treated with 1 µM OCA, 100 µM CDCA, or vehicle control for 72 hours followed by quantification of deuterated TCA uptake and efflux over time in Ca2+-containing and Ca2+-free conditions (n = 3 donors). A mechanistic pharmacokinetic model was fit to the TCA mass-time data to obtain estimates for total uptake clearance (CLUptake), total intrinsic basolateral efflux clearance (CLint,BL), and total intrinsic biliary clearance (CLint,Bile). Modeling results revealed that FXR agonists significantly increased CLint,BL by >6-fold and significantly increased CLint,Bile by 2-fold, with minimal effect on CLUptake Immunoblotting showed that protein levels of the basolateral transporter subunits organic solute transporter α and ß (OSTα and OSTß) in FXR agonist-treated SCHH were significantly induced by >2.5- and 10-fold, respectively. FXR agonist-mediated changes in the expression of other TCA transporters in SCHH were modest. In conclusion, this is the first report demonstrating that OCA and CDCA increased TCA efflux in SCHH, which contributed to reduced intracellular TCA concentrations. Increased basolateral efflux of TCA was consistent with increased OSTα/ß protein expression in OCA- and CDCA-treated SCHH.

Comparative potency of obeticholic acid and natural bile acids on FXR in hepatic and intestinal in vitro cell models.

Obeticholic acid (OCA) is a semisynthetic farnesoid X receptor (FXR) agonist, an analogue of chenodeoxycholic acid (CDCA) which is indicated for the treatment of primary biliary cholangitis (PBC) in combination with ursodeoxycholic acid (UDCA). OCA efficiently inhibits bile acid synthesis and promotes bile acid efflux via activating FXR-mediated mechanisms in a physiologically relevant in vitro cell system, Sandwich-cultured Transporter Certified ™ human primary hepatocytes (SCHH). The study herein evaluated the effects of UDCA alone or in combination with OCA in SCHH. UDCA (≤100 µmol/L) alone did not inhibit CYP7A1 mRNA, and thus, no reduction in the endogenous bile acid pool observed. UDCA ≤100 µmol/L concomitantly administered with 0.1 µmol/L OCA had no effect on bile acid synthesis beyond what was observed with OCA alone. Furthermore, this study evaluated human Caco-2 cells (clone C2BBe1) as in vitro intestinal models. Glycine conjugate of OCA increased mRNA levels of FXR target genes in Caco-2 cells, FGF-19, SHP, OSTα/ß, and IBABP, but not ASBT, in a concentration-dependent manner, while glycine conjugate of UDCA had no effect on the expression of these genes. The results suggested that UDCA ≤100 µmol/L did not activate FXR in human primary hepatocytes or intestinal cell line Caco-2. Thus, co-administration of UDCA with OCA did not affect OCA-dependent pharmacological effects.

Exposure-Response Analysis of Alvocidib (Flavopiridol) Treatment by Bolus or Hybrid Administration in Newly Diagnosed or Relapsed/Refractory Acute Leukemia Patients.

Purpose: To elucidate any differences in the exposure-response of alvocidib (flavopiridol) given by 1-hour bolus or a hybrid schedule (30-minute bolus followed by a 4-hour infusion) using a flavopiridol/cytosine arabinoside/mitoxantrone sequential protocol (FLAM) in patients with acute leukemia. The hybrid schedule was devised to be pharmacologically superior in chronic leukemia based on unbound exposure.Experimental Design: Data from 129 patients in three FLAM studies were used for pharmacokinetic/pharmacodynamic modeling. Newly diagnosed (62%) or relapsed/refractory (38%) patients were treated by bolus (43%) or hybrid schedule (57%). Total and unbound flavopiridol concentrations were fit using nonlinear mixed-effect population pharmacokinetic methodologies. Exposure-response relationships using unbound flavopiridol AUC were explored using recursive partitioning.Results: Flavopiridol pharmacokinetic parameters were estimated using a two-compartment model. No pharmacokinetic covariates were identified. Flavopiridol fraction unbound was 10.9% and not different between schedules. Partitioning found no association between dosing schedule and clinical response. Clinical response was associated with AUC ≥ 780 h*ng/mL for newly diagnosed patients and AUC ≥ 1,690 h*ng/mL for relapsed/refractory patients. Higher exposures were not associated with increases in severe adverse events (≥ grade 3).Conclusions: Pharmacokinetic modeling showed no difference in flavopiridol plasma protein binding for bolus versus hybrid dosing. Further trials in newly diagnosed patients with acute leukemia should utilize the bolus FLAM regimen at the MTD of 50 mg/m2/day. Trials in relapsed/refractory patients should use the hybrid dosing schedule at the MTD (30/60 mg/m2/day) to achieve the higher exposures required for maximal efficacy in this population. Clin Cancer Res; 23(14); 3592-600. ©2017 AACR.

Low-dose pramlintide reduced food intake and meal duration in healthy, normal-weight subjects.

OBJECTIVE: We previously reported that a single preprandial injection (120 microg) of pramlintide, an analog of the beta-cell hormone amylin, reduced ad libitum food intake in obese subjects. To further characterize the meal-related effects of amylin signaling in humans, we studied a lower pramlintide dose (30 microg) in normal-weight subjects. RESEARCH METHODS AND PROCEDURES: In a randomized, double-blind, placebo-controlled, cross-over study, 15 healthy men (age, 24 +/- 7 years; BMI, 22.2 +/- 1.8 kg/m(2)) underwent a standardized buffet meal test on two occasions. After an overnight fast, subjects received a single subcutaneous injection of pramlintide (30 microg) or placebo, followed immediately by a standardized pre-load meal. After 1 hour, subjects were offered an ad libitum buffet meal, and total caloric intake and meal duration were measured. RESULTS: Compared with placebo, pramlintide reduced total caloric intake (1411 +/- 94 vs. 1190 +/- 117 kcal; Delta, -221 +/- 101 kcal; -14 +/- 9%; p = 0.05) and meal duration (36 +/- 2 vs. 31 +/- 3 minutes; Delta, -5.1 +/- 1.4 minutes; p < 0.005). Visual analog scale profiles of hunger trended lower and fullness higher during the first hour after pramlintide administration. In response to the buffet, hunger and fullness changed to a similar degree after pramlintide and placebo, despite subjects on pramlintide consuming 14% fewer kilocalories. Visual analog scale nausea ratings remained near baseline, without differences between treatments. Plasma peptide YY, cholecystokinin, and ghrelin concentrations did not differ with treatment, whereas glucagon-like peptide-1 concentrations after meals were lower in response to pramlintide than to placebo. DISCUSSION: These observations add support to the concept that amylin agonism may have a role in human appetite control.

A high-capacity assay for PPARgamma ligand regulation of endogenous aP2 expression in 3T3-L1 cells.

A novel class of insulin-sensitizing agents, the thiazolidinedines (TZDs), has proven effective in the treatment of type 2 diabetes. These compounds, as well as a subclass of non-TZD insulin-sensitizing agents, have been shown to be peroxisome proliferator-activated receptor (PPAR) gamma agonists. PPARgamma plays a critical role in adipogenesis and PPARgamma agonists have been shown to induce adipocyte differentiation. Here, PPARgamma ligand activity has been assessed in murine 3T3-L1 cells, a commonly used in vitro model of adipogenesis, by measuring their ability to induce adipocyte fatty acid-binding protein (aP2) mRNA expression. In order to perform this task, we have developed a novel, multiwell assay for the direct detection of aP2 mRNA in cell lysates that is based on hybridization of mRNA to target-specific oligonucleotides. These oligonucleotide probes are conjugated to enzymes that efficiently process unique chemical substrates into robust fluorescent products. Ribosomal protein 36B4 mRNA, a gene whose expression is unaffected by adipogenesis, serves as the control in the assay. Two assay formats have been developed, a single analyte assay in which aP2 and 36B4 mRNA expression are assayed in separate lysate aliquots and a dual analyte assay which can measure aP2 and 36B4 mRNA simultaneously. Both forms of the assay have been used to quantify attomole levels of aP2 and 36B4 mRNAs in differentiating 3T3-L1 preadipocytes treated with PPARgamma agonists. The potencies of PPARgamma agonists determined by this novel methodology showed good correlation with those derived from aP2 mRNA slot-blot analysis and PPARgamma transactivation assays. We conclude that the aP2 single and dual analyte assays both provide specific and sensitive measurements of endogenous aP2 mRNA levels that can be used to assess the activity of PPARgamma ligands in 3T3-L1 cells. Since the assay obviates the need for RNA isolation and is performed in an automatable multiwell format, it can serve as a high-throughput, cell-based screen for the identification and characterization of PPARgamma modulators.

Rapid assays for quantitating cytokine gene expression without target amplification.

Many drug discovery efforts are focused on finding candidates that alter gene expression of the cytokines involved in inflammation, allergy, and cell-mediated immunity. Current methods used to evaluate gene expression such as northern blot and RT-PCR are laborious, time-consuming, expensive, and are not conducive to high throughput screening. High Performance Signal Amplification (HPSA( trade mark )) gene expression assays quantitate mRNA targets directly from cell lysate samples using DNA probe hybridization and fluorescent signal amplification. The assay format eliminates the need for RNA purification prior to testing and does not involve target amplification. The 96 or 384-well microplate formats allow the method to be run manually, by a workstation approach, or with full automation. Cellular mRNA levels are quantitated relative to a standard curve comprised of highly purified in vitro RNA calibrators. The analytical sensitivity is in the low attomole (10(-18) mole) range. This technique was used to monitor the transcription patterns of mRNA encoding TNF-alpha, IL-1beta, and Interferon-gamma in human cell lines or primary PBMC treated with inducers such as PMA, ionomycin, and endotoxin. The specificity, precision and reproducibility of the assay are sufficient to provide a reliable screening system. The HPSA gene expression assay system offers a rapid and convenient alternative to more cumbersome, expensive methods.