Safety and Efficacy of Efruxifermin in Combination With a GLP-1 Receptor Agonist in Patients With NASH/MASH and Type 2 Diabetes in a Randomized Phase 2 Study.

BACKGROUND & AIMS: In phase 2 studies, efruxifermin, an Fc-FGF21 analog, significantly reduced steatohepatitis and fibrosis in patients with non-alcoholic steatohepatitis, now called metabolic dysfunction-associated steatohepatitis (MASH), for which there is no approved treatment. Type 2 diabetes (T2D) and obesity are prevalent among patients with MASH and increasingly treated with glucagon-like peptide-1 receptor agonists (GLP-1RAs). This study evaluated the safety and efficacy of efruxifermin in patients with MASH, fibrosis, and T2D taking a GLP-1RA. METHODS: Cohort D was a double-blind, placebo-controlled, phase 2b study in adults with T2D and MASH with fibrosis (F1-F3) on stable GLP-1RA therapy randomized (2:1) to receive efruxifermin 50 mg or placebo, once weekly for 12 weeks. The primary endpoint was safety and tolerability of efruxifermin added to a stable dose of GLP-1RA. Secondary endpoints included changes in hepatic fat fraction (HFF), markers of liver injury and fibrosis, and metabolic parameters. RESULTS: Adults (N = 31) with T2D and MASH fibrosis (F1-F3) on a stable GLP-1RA (semaglutide, 48.4%; dulaglutide, 45.2%; liraglutide, 6.5%) received efruxifermin 50 mg (n = 21) or placebo (n = 10) for 12 weeks. The addition of efruxifermin to a GLP-1RA appeared safe and well-tolerated. The most frequent efruxifermin-related adverse events were mild to moderate gastrointestinal events. One patient receiving efruxifermin discontinued due to nausea, and another withdrew consent. There were no treatment-related serious adverse events. After 12 weeks, efruxifermin reduced HFF by 65% (P < .0001 vs placebo) compared with a 10% reduction for placebo (GLP-1RA alone). Efruxifermin also improved noninvasive markers of liver injury, fibrosis, glucose, and lipid metabolism while maintaining GLP-1RA-mediated weight loss. CONCLUSIONS: The tolerability profile of efruxifermin added to GLP-1RA appeared comparable to that of either drug alone, while also significantly reducing HFF and noninvasive markers of fibrosis in patients with MASH and T2D. Liver health in patients already on a GLP-1RA may be further improved by addition of efruxifermin. CLINICALTRIALS: gov, Number: NCT05039450.

An integrated analysis of fecal microbiome and metabolomic features distinguish non-cirrhotic NASH from healthy control populations.

BACKGROUND AND AIMS: There is great interest in identifying microbiome features as reliable noninvasive diagnostic and/or prognostic biomarkers for non-cirrhotic NASH fibrosis. Several cross-sectional studies have reported gut microbiome features associated with advanced NASH fibrosis and cirrhosis, where the most prominent features are associated with cirrhosis. However, no large, prospectively collected data exist establishing microbiome features that discern non-cirrhotic NASH fibrosis, integrate the fecal metabolome as disease biomarkers, and are unconfounded by BMI and age. APPROACH AND RESULTS: Results from shotgun metagenomic sequencing performed on fecal samples prospectively collected from 279 US patients with biopsy-proven NASH (F1-F3 fibrosis) enrolled in the REGENERATE I303 study were compared to those from 3 healthy control cohorts and integrated with the absolute quantification of fecal bile acids. Microbiota beta-diversity was different, and BMI- and age-adjusted logistic regression identified 12 NASH-associated species. Random forest prediction models resulted in an AUC of 0.75-0.81 in a receiver operator characteristic analysis. In addition, specific fecal bile acids were significantly lower in NASH and correlated with plasma C4 levels. Microbial gene abundance analysis revealed 127 genes increased in controls, many involving protein synthesis, whereas 362 genes were increased in NASH many involving bacterial environmental responses (false discovery rate < 0.01). Finally, we provide evidence that fecal bile acid levels may be a better discriminator of non-cirrhotic NASH versus health than either plasma bile acids or gut microbiome features. CONCLUSIONS: These results may have value as a set of baseline characteristics of non-cirrhotic NASH against which therapeutic interventions to prevent cirrhosis can be compared and microbiome-based diagnostic biomarkers identified.

Non-invasive evaluation of response to obeticholic acid in patients with NASH: Results from the REGENERATE study.

BACKGROUND & AIMS: Non-alcoholic steatohepatitis (NASH) is a chronic, progressive fibrotic liver disease that can lead to cirrhosis. While liver biopsy is considered the reference standard for the histologic diagnosis of NASH and staging of fibrosis, its use in clinical practice is limited. Non-invasive tests (NITs) are increasingly being used to identify and stage liver fibrosis in patients with NASH, and several can assess liver-related outcomes. We report changes in various NITs in patients treated with obeticholic acid (OCA) or placebo in the phase III REGENERATE study. METHODS: Patients with NASH and fibrosis stage F2 or F3 (n = 931) were randomized (1:1:1) to receive placebo, OCA 10 mg, or OCA 25 mg once daily. Various NITs based on clinical chemistry and/or imaging were evaluated at baseline and throughout the study. RESULTS: Rapid, sustained reductions from baseline in alanine aminotransferase (ALT), aspartate aminotransferase (AST), and gamma-glutamyltransferase levels, as well as in Fibrosis-4 (FIB-4), FibroTest, FibroMeter, and FibroScan-AST scores were observed in OCA-treated vs. placebo-treated patients. Reduction in liver stiffness by vibration-controlled transient elastography was observed in the OCA 25 mg group vs. the placebo group at Month 18. NIT changes were associated with shifts in histologic fibrosis stage. The greatest improvements were observed in patients with ≥1-stage fibrosis improvement; however, improvements in ALT, AST, FIB-4, and FibroTest were also observed in OCA-treated patients whose histologic fibrosis remained stable. CONCLUSIONS: Based on the REGENERATE Month 18 interim analysis, rapid and sustained improvements in various NITs were observed with OCA treatment. Dynamic changes in selected NITs separated histologic responders from non-responders. These results suggest that NITs may be useful in assessing histologic response to OCA therapy. CLINICALTRIALS. GOV NUMBER: NCT02548351 LAY SUMMARY: Non-alcoholic steatohepatitis (NASH) is a chronic, progressive liver disease that can lead to cirrhosis. To diagnose and assess liver fibrosis (scarring) in patients with NASH, non-invasive tests (NITs) are increasingly being used rather than liver biopsy, which is invasive, expensive, and can be risky. In the REGENERATE study, which is evaluating the effects of obeticholic acid vs. placebo in patients with NASH, various NITs were also evaluated. This analysis shows that improvements in levels of certain blood components, as well as favorable results of ultrasound imaging and proprietary tests of liver function, were associated with improvements in liver fibrosis after treatment with obeticholic acid, suggesting that NITs may be useful alternatives to liver biopsy in assessing NASH patients' response to therapy.

Obeticholic acid for the treatment of non-alcoholic steatohepatitis: interim analysis from a multicentre, randomised, placebo-controlled phase 3 trial.

BACKGROUND: Non-alcoholic steatohepatitis (NASH) is a common type of chronic liver disease that can lead to cirrhosis. Obeticholic acid, a farnesoid X receptor agonist, has been shown to improve the histological features of NASH. Here we report results from a planned interim analysis of an ongoing, phase 3 study of obeticholic acid for NASH. METHODS: In this multicentre, randomised, double-blind, placebo-controlled study, adult patients with definite NASH, non-alcoholic fatty liver disease (NAFLD) activity score of at least 4, and fibrosis stages F2-F3, or F1 with at least one accompanying comorbidity, were randomly assigned using an interactive web response system in a 1:1:1 ratio to receive oral placebo, obeticholic acid 10 mg, or obeticholic acid 25 mg daily. Patients were excluded if cirrhosis, other chronic liver disease, elevated alcohol consumption, or confounding conditions were present. The primary endpoints for the month-18 interim analysis were fibrosis improvement (≥1 stage) with no worsening of NASH, or NASH resolution with no worsening of fibrosis, with the study considered successful if either primary endpoint was met. Primary analyses were done by intention to treat, in patients with fibrosis stage F2-F3 who received at least one dose of treatment and reached, or would have reached, the month 18 visit by the prespecified interim analysis cutoff date. The study also evaluated other histological and biochemical markers of NASH and fibrosis, and safety. This study is ongoing, and registered with ClinicalTrials.gov, NCT02548351, and EudraCT, 20150-025601-6. FINDINGS: Between Dec 9, 2015, and Oct 26, 2018, 1968 patients with stage F1-F3 fibrosis were enrolled and received at least one dose of study treatment; 931 patients with stage F2-F3 fibrosis were included in the primary analysis (311 in the placebo group, 312 in the obeticholic acid 10 mg group, and 308 in the obeticholic acid 25 mg group). The fibrosis improvement endpoint was achieved by 37 (12%) patients in the placebo group, 55 (18%) in the obeticholic acid 10 mg group (p=0·045), and 71 (23%) in the obeticholic acid 25 mg group (p=0·0002). The NASH resolution endpoint was not met (25 [8%] patients in the placebo group, 35 [11%] in the obeticholic acid 10 mg group [p=0·18], and 36 [12%] in the obeticholic acid 25 mg group [p=0·13]). In the safety population (1968 patients with fibrosis stages F1-F3), the most common adverse event was pruritus (123 [19%] in the placebo group, 183 [28%] in the obeticholic acid 10 mg group, and 336 [51%] in the obeticholic acid 25 mg group); incidence was generally mild to moderate in severity. The overall safety profile was similar to that in previous studies, and incidence of serious adverse events was similar across treatment groups (75 [11%] patients in the placebo group, 72 [11%] in the obeticholic acid 10 mg group, and 93 [14%] in the obeticholic acid 25 mg group). INTERPRETATION: Obeticholic acid 25 mg significantly improved fibrosis and key components of NASH disease activity among patients with NASH. The results from this planned interim analysis show clinically significant histological improvement that is reasonably likely to predict clinical benefit. This study is ongoing to assess clinical outcomes. FUNDING: Intercept Pharmaceuticals.

Factors Associated With Histologic Response in Adult Patients With Nonalcoholic Steatohepatitis.

BACKGROUND & AIMS: Nonalcoholic steatohepatitis (NASH) is a leading cause of liver transplantation, and many trials are underway to evaluate potential therapies. The farnesoid X receptor ligand obeticholic acid in the NASH treatment trial evaluated the effects of obeticholic acid vs placebo on histologic response (defined as decrease in nonalcoholic fatty liver disease activity score [NAS] by ≥2, with no worsening of fibrosis); 45% of patients had a histologic response to obeticholic acid (25 mg), and 21% had a response to placebo (P < .01). We performed a secondary analysis of data from this trial to identify clinical parameters associated with a histologic response. METHODS: We used a logistic regression model with a stepwise selection procedure to identify baseline and early on-treatment factors associated with a histologic response at 72 weeks. Baseline demographics, liver histology, medical history, concomitant medications, cardiometabolic parameters, and serum biochemistry, as well as the changes over the course of the trial (at weeks 12 and 24), were evaluated as potential predictors of a histologic response. The model was cross-validated by a jackknife method, and performance was evaluated with the area under the receiver operating characteristic curve. RESULTS: The logistic regression model found that obeticholic acid treatment, baseline NAS > 5, baseline triglyceride level ≤ 154 mg/dL, baseline international normalized ratio ≤ 1, baseline aspartate aminotransferase level ≤ 49 U/L, and a decrease in alanine aminotransferase level at week 24 by 17 U/L or more, to be significantly associated with histologic response (area under the receiver operating characteristic curve, 0.83; 95% confidence interval, 0.77-0.89; P < .0001). CONCLUSIONS: In a secondary analysis of data from a clinical trial of obeticholic acid in patients with NASH, we identified routine clinical and laboratory parameters during the first 24 weeks of treatment (such as baseline NAS, triglyceride levels, and a decrease in alanine aminotransferase level) to significantly associate with histologic markers of response.

Relationship between three commonly used non-invasive fibrosis biomarkers and improvement in fibrosis stage in patients with non-alcoholic steatohepatitis.

BACKGROUND & AIMS: Non-invasive biomarkers are needed for monitoring changes in liver histology in patients with non-alcoholic steatohepatitis (NASH). Obeticholic acid (OCA) was shown to improve fibrosis in patients with NASH in the FLINT trial; a post hoc analysis of these data was performed to determine the relationship between 3 non-invasive fibrosis markers and liver fibrosis improvement. METHODS: In the Phase 2b FLINT trial, patients were randomised (1:1) to receive 25 mg OCA or placebo once daily for 72 weeks. Aspartate aminotransferase:platelet ratio index (APRI), fibrosis-4 (FIB-4) index and non-alcoholic fatty liver disease fibrosis score (NFS) were evaluated in serum at baseline and weeks 24, 48, 72 and 96. Liver biopsies were obtained at baseline and 72 weeks. RESULTS: In patients with fibrosis improvement at week 24, scores were reduced by a median of 34% for APRI, 10% for FIB-4 and 4% for NFS. Reductions in APRI (P = 0.015) and FIB-4 (P = 0.036), but not NFS (P = 0.201) at week 24, significantly correlated with ≥1-stage improvement in histologic fibrosis at week 72. Reductions in APRI at week 72 were significantly correlated with fibrosis improvement at week 72 (P = 0.012). Patients receiving OCA had significant reductions in all markers compared with patients receiving placebo at week 72 [APRI and FIB-4 (P < 0.0001); NFS (P < 0.05)]. CONCLUSIONS: Readily available non-invasive markers may predict improvement in liver fibrosis in patients with NASH. Upon external confirmation and further refinement in larger populations, these markers may serve as surrogate endpoints in NASH clinical trials.

CONTROL: A randomized phase 2 study of obeticholic acid and atorvastatin on lipoproteins in nonalcoholic steatohepatitis patients.

BACKGROUND & AIMS: Nonalcoholic steatohepatitis (NASH) is a chronic and severe form of nonalcoholic fatty liver disease that can progress to cirrhosis and hepatocellular carcinoma and is a risk factor for cardiovascular disease. Although NASH has no approved treatments, obeticholic acid (OCA), a synthetic bile acid and farnesoid X receptor (FXR) agonist, was shown to improve histological features of NASH and fibrosis. Considering that FXR activation influences plasma lipoprotein concentrations, the Combination OCA aNd sTatins for monitoRing Of Lipids (CONTROL) study evaluated how statins can regulate lipoprotein metabolism with OCA treatment in patients with NASH. METHODS: This randomized, double-blind, placebo-controlled, phase 2 study began with a 5-week screening/statin washout; 84 patients with NASH were randomly assigned (1:1:1:1) to receive placebo or 5 mg, 10 mg or 25 mg OCA once daily during the 16-week double-blind phase. Concurrent once daily atorvastatin (10 mg/days) was initiated at Week 4 with subsequent titration. Enrolled patients had biopsy-confirmed diagnosis of NASH with no evidence of hepatic decompensation. Plasma was collected to analyse lipoprotein parameters. RESULTS: At Week 4, all OCA groups had an increase from baseline in mean low-density lipoprotein cholesterol (LDLc) and mean LDL particle concentration (LDLpc), mostly owing to large, less atherogenic LDLc particles. Atorvastatin 10 mg decreased LDLc and LDLpc levels below baseline in all OCA groups by Week 8; higher doses did not provide additional clinical benefits. CONCLUSIONS: The CONTROL study showed that OCA-induced increases in LDLc in patients with NASH were mitigated with atorvastatin. The combination of OCA and atorvastatin was generally safe and well tolerated (NCT02633956).

A randomized, double-blind, placebo-controlled phase IIa trial of efruxifermin for patients with compensated NASH cirrhosis.

Background & Aims: Efruxifermin has shown clinical efficacy in patients with non-alcoholic steatohepatitis (NASH) and F1-F3 fibrosis. The primary objective of the BALANCED Cohort C was to assess the safety and tolerability of efruxifermin in patients with compensated NASH cirrhosis. Methods: Patients with NASH and stage 4 fibrosis (n = 30) were randomized 2:1 to receive efruxifermin 50 mg (n = 20) or placebo (n = 10) once-weekly for 16 weeks. The primary endpoint was safety and tolerability of efruxifermin. Secondary and exploratory endpoints included evaluation of non-invasive markers of liver injury and fibrosis, glucose and lipid metabolism, and changes in histology in a subset of patients who consented to end-of-study liver biopsy. Results: Efruxifermin was safe and well-tolerated; most adverse events (AEs) were grade 1 (n = 7, 23.3%) or grade 2 (n = 19, 63.3%). The most frequent AEs were gastrointestinal, including transient, mild to moderate diarrhea, and/or nausea. Significant improvements were noted in key markers of liver injury (alanine aminotransferase) and glucose and lipid metabolism. Sixteen-week treatment with efruxifermin was associated with significant reductions in non-invasive markers of fibrosis including Pro-C3 (least squares mean change from baseline [LSMCFB] -9 µg/L efruxifermin vs. -3.4 µg/L placebo; p = 0.0130) and ELF score (-0.4 efruxifermin vs. +0.4 placebo; p = 0.0036), with a trend towards reduced liver stiffness (LSMCFB -5.7 kPa efruxifermin vs. -1.1 kPa placebo; n.s.). Of 12 efruxifermin-treated patients with liver biopsy after 16 weeks, 4 (33%) achieved fibrosis improvement of at least one stage without worsening of NASH, while an additional 3 (25%) achieved resolution of NASH, compared to 0 of 5 placebo-treated patients. Conclusions: Efruxifermin appeared safe and well-tolerated with encouraging improvements in markers of liver injury, fibrosis, and glucose and lipid metabolism following 16 weeks of treatment, warranting confirmation in larger and longer term studies. Lay summary: Cirrhosis resulting from non-alcoholic steatohepatitis (NASH), the progressive form of non-alcoholic fatty liver disease, represents a major unmet medical need. Currently there are no approved drugs for the treatment of NASH. This proof-of-concept randomized, double-blind clinical trial demonstrated the potential therapeutic benefit of efruxifermin treatment compared to placebo in patients with cirrhosis due to NASH. Clinical Trial Number: NCT03976401.

Safety and efficacy of once-weekly efruxifermin versus placebo in non-alcoholic steatohepatitis (HARMONY): a multicentre, randomised, double-blind, placebo-controlled, phase 2b trial.

BACKGROUND: Fibroblast growth factor 21 (FGF21) regulates metabolism and protects cells against stress. Efruxifermin is a bivalent Fc-FGF21 analogue that replicates FGF21 agonism of fibroblast growth factor receptor 1c, 2c, or 3c. The aim of this phase 2b study was to assess its efficacy and safety in patients with non-alcoholic steatohepatitis (NASH) and moderate (F2) or severe (F3) fibrosis. METHODS: HARMONY is a multicentre, randomised, double-blind, placebo-controlled, 96-week, phase 2b trial that was initiated at 41 clinics in the USA. Adults with biopsy-confirmed NASH, defined by a non-alcoholic fatty liver disease activity score (NAS) of 4 or higher and scores of 1 or higher in each of steatosis, ballooning, and lobular inflammation, with histological stage F2 or F3 fibrosis, were randomly assigned (1:1:1), via an interactive response system, to receive placebo or efruxifermin (28 mg or 50 mg), subcutaneously once weekly. Patients, investigators, pathologists, site staff, and the sponsor were masked to group assignments during the study. The primary endpoint was the proportion of patients with improvement in fibrosis of at least 1 stage and no worsening of NASH, based on analyses of baseline and week 24 biopsies (liver biopsy analysis set [LBAS]). A sensitivity analysis evaluated the endpoint in the full analysis set (FAS), for which patients with missing biopsies were considered non-responders. This trial is registered with ClinicalTrials.gov, NCT04767529, and is ongoing. FINDINGS: Between March 22, 2021, and Feb 7, 2022, 747 patients were assessed for eligibility and 128 patients (mean age 54·7 years [SD 10·4]; 79 [62%] female and 49 male [38%]; 118 [92%] white; and 56 [41%] Hispanic or Latino) were enrolled and randomly assigned to receive placebo (n=43), efruxifermin 28 mg (n=42; two randomised patients were not dosed because of an administrative error), or efruxifermin 50 mg (n=43). In the LBAS (n=113), eight (20%) of 41 patients in the placebo group had an improvement in fibrosis of at least 1 stage and no worsening of NASH by week 24 versus 15 (39%) of 38 patients in the efruxifermin 28 mg group (risk ratio [RR] 2·3 [95% CI 1·1-4·8]; p=0·025) and 14 (41%) of 34 patients in the efruxifermin 50 mg group (2·2 [1·0-5·0]; p=0·036). Based on the FAS (n=128), eight (19%) of 43 patients in the placebo group met this endpoint versus 15 (36%) of 42 in the efruxifermin 28 mg group (RR 2·2 [95% CI 1·0-4·8]; p=0·033) and 14 (33%) of 43 in the efruxifermin 50 mg group (1·9 [0·8-4·3]; p=0·123). The most frequent efruxifermin-related adverse events were diarrhoea (16 [40%] of 40 patients in the efruxifermin 28 mg group and 17 [40%] of 43 patients in efruxifermin 50 mg group vs eight [19%] of 43 patients in the placebo group; all events except one were grade 1-2) and nausea (11 [28%] patients in the efruxifermin 28 mg group and 18 [42%] patients in the efruxifermin 50 mg group vs ten [23%] patients in the placebo group; all grade 1-2). Five patients (two in the 28 mg group and three in the 50 mg group) discontinued due to adverse events. Serious adverse events occurred in four patients in the 50 mg group; one was defined as drug related (ulcerative esophagitis in a participant with a history of gastro-oesophageal reflux disease). No deaths occurred. INTERPRETATION: Efruxifermin improved liver fibrosis and resolved NASH over 24 weeks in patients with F2 or F3 fibrosis, with acceptable tolerability, supporting further assessment in phase 3 trials. FUNDING: Akero Therapeutics.

Inhibition of the insulin-like growth factor receptor-1 tyrosine kinase activity as a therapeutic strategy for multiple myeloma, other hematologic malignancies, and solid tumors.

Insulin-like growth factors and their receptor (IGF-1R) have been implicated in cancer pathophysiology. We demonstrate that IGF-1R is universally expressed in various hematologic (multiple myeloma, lymphoma, leukemia) and solid tumor (breast, prostate, lung, colon, thyroid, renal, adrenal cancer, retinoblastoma, and sarcoma) cells. Specific IGF-1R inhibition with neutralizing antibody, antagonistic peptide, or the selective kinase inhibitor NVP-ADW742 has in vitro activity against diverse tumor cell types (particularly multiple myeloma), even those resistant to conventional therapies, and triggers pleiotropic antiproliferative/proapoptotic molecular sequelae, delineated by global transcriptional and proteomic profiling. NVP-ADW742 monotherapy or its combination with cytotoxic chemotherapy had significant antitumor activity in an orthotopic xenograft MM model, providing in vivo proof of principle for therapeutic use of selective IGF-1R inhibitors in cancer.

Janus kinase inhibitor INCB20 has antiproliferative and apoptotic effects on human myeloma cells in vitro and in vivo.

Protein tyrosine kinases of the Janus kinase (JAK) family are associated with many cytokine receptors, which, on ligand binding, regulate important cellular functions such as proliferation, survival, and differentiation. In multiple myeloma, JAKs may be persistently activated due to a constant stimulation by interleukin (IL)-6, which is produced in the bone marrow environment. INCB20 is a synthetic molecule that potently inhibits all members of the JAK family with a 100- to 1,000-fold selectivity for JAKs over >70 other kinases. Treatment of multiple myeloma cell lines and patient tumor cells with INCB20 resulted in a significant and dose-dependent inhibition of spontaneous as well as IL-6-induced cell growth. Importantly, multiple myeloma cell growth was inhibited in the presence of bone marrow stromal cells. The IL-6 dependent cell line INA-6 was particularly sensitive to the drug (IC50<1 micromol/L). Growth suppression of INA-6 correlated with an increase in the percentage of apoptotic cells and inhibition of signal transducer and activator of transcription 3 phosphorylation. INCB20 also abrogated the protective effect of IL-6 against dexamethasone by blocking phosphorylation of SHP-2 and AKT. In contrast, AKT phosphorylation induced by insulin-like growth factor-I remained unchanged, showing selectivity of the compound. In a s.c. severe combined immunodeficient mouse model with INA-6, INCB20 significantly delayed INA-6 tumor growth. Our studies show that disruption of JAKs and downstream signaling pathways may both inhibit multiple myeloma cell growth and survival and overcome cytokine-mediated drug resistance, thereby providing the preclinical rationale for the use of JAK inhibitors as a novel therapeutic approach in multiple myeloma.

Alosetron versus traditional pharmacotherapy in clinical practice: effects on resource use, health-related quality of life, safety and symptom improvement in women with severe diarrhea-predominant irritable bowel syndrome.

OBJECTIVE: Severe diarrhea-predominant irritable bowel syndrome (IBS-D) is associated with decreased health-related quality of life (HRQOL) and increased health care costs. Treatment recommendations for IBS-D often start with traditional pharmacotherapy (TP), with escalation to alosetron, rifaximin or eluxadoline if there is no success. There has been no previous head-to-head clinical trial comparing IBS-D treatment outcome for alosetron versus TP. This study, GSK protocol S3B30020, evaluated resource use, work productivity, health-related quality of life and global symptom response in women with IBS-D who were treated with alosetron or TP. METHODS: A total of 1956 patients who met criteria for severe IBS-D were randomized to treatment with alosetron 1 mg twice daily (BID) or only TP for up to 24 weeks. Work productivity and resource use were evaluated by standard questionnaires, HRQOL by the IBSQOL instrument and IBS symptoms by the Global Improvement Scale (GIS). RESULTS: Compared to only TP, alosetron-treated patients reported: (1) fewer clinic/office visits for any health problem (p = .0181) or for IBS-D (p = .0004); (2) reduced use of over-the-counter medications for IBS-D (p < .0001); (3) fewer days of lost work productivity (p < .0001); (4) decreased restriction of social and outdoor activities (p < .0001); and (5) greater global improvement in IBS-D symptoms (p < .0001). Alosetron treatment improved HRQOL scores for all domains (p < .0001). Incidence of adverse events during alosetron use was not remarkable and was similar to that previously reported. CONCLUSIONS: Alosetron 1 mg BID significantly reduced health care utilization and lost productivity, and significantly improved global IBS symptoms, HRQOL, and participation in outdoor and social activities compared with treatment response to TP.

REGENERATE: Design of a pivotal, randomised, phase 3 study evaluating the safety and efficacy of obeticholic acid in patients with fibrosis due to nonalcoholic steatohepatitis.

BACKGROUND: Nonalcoholic steatohepatitis (NASH) is a chronic, progressive, and severe form of nonalcoholic fatty liver disease. In FLINT, obeticholic acid (OCA) treatment improved multiple histological NASH features. The design and endpoints of REGENERATE, an ongoing phase 3 study, further evaluate OCA treatment in patients with fibrosis due to NASH. AIMS: The Month 18 interim analysis assesses the effect of OCA on liver histology, defined as improvement of fibrosis by ≥1 stage with no worsening of NASH or resolution of NASH with no worsening of fibrosis. The end-of-study analyses evaluate the effect of OCA on mortality, liver-related clinical outcomes, and long-term safety. METHODS: REGENERATE is a pivotal, long-term study of ~2400 patients with NASH, including ~2100 patients with stage 2 or 3 liver fibrosis. Additionally, ~300 patients with stage 1 fibrosis and ≥1 accompanying comorbidity are included to gather information on the safety of OCA and liver disease progression. Patients are randomised 1:1:1 to receive placebo or OCA (10 or 25 mg). A liver biopsy evaluation occurs at screening, Months 18 and 48, and end of study. The duration of the study is dependent upon accrual of a predetermined number of clinical outcome events. CONCLUSIONS: REGENERATE is designed in conjunction with regulatory authorities to support regulatory approvals in NASH. This robust phase 3 study assesses the effect of OCA on liver histology as a surrogate for transplant-free survival and liver-related outcomes, including progression to cirrhosis and mortality, and will ultimately assess clinical benefit through specific evaluation of these outcomes. CLINICAL TRIAL REGISTRATION: ClinicalTrials.gov with the identifier NCT02548351.

Effects of PS-341 on the activity and composition of proteasomes in multiple myeloma cells.

Multiple myeloma is a B-cell malignancy for which no curative therapies exist to date, despite enormous research efforts. The remarkable activity of the proteasome inhibitor bortezomib (PS-341, Velcade) observed in clinical trials of patients with relapsed refractory myeloma has led to investigations of the role of the ubiquitin-proteasome pathway in the pathogenesis of myeloma. Here we report a biochemical analysis of proteasome activity and composition in myeloma cells exposed to PS-341 in the presence or absence of cytokines present in the bone marrow milieu. We observed that the myeloma cell lines MM1.S, RPMI8226, and U266 contain active immunoproteasomes, the amount of which is enhanced by IFN-gamma and tumor necrosis factor-alpha. Using a radiolabeled active site-directed probe specific for proteasome catalytic subunits, we show that PS-341 targets the beta5 and beta1 subunits in a concentration-dependent manner. Furthermore, PS-341 also targeted the corresponding catalytic subunits of the immunoproteasome, beta5i and beta1i, respectively. These data suggest that PS-341 targets both normal and immunoproteasome species to a similar extent in myeloma cells.

Blockade of Hsp27 overcomes Bortezomib/proteasome inhibitor PS-341 resistance in lymphoma cells.

Bortezomib (PS-341), a selective inhibitor of proteasome, induces apoptosis in various tumor cells, but its mechanism of action is unclear. Treatment with PS-341 induces apoptosis in SUDHL6 (DHL6), but not SUDHL4 (DHL4), lymphoma cells. Microarray analysis shows high RNA levels of heat shock protein-27 (Hsp27) in DHL4 versus DHL6 cells, which correlates with Hsp27 protein expression. Blocking Hsp27 using an antisense strategy restores the apoptotic response to PS-341 in DHL4 cells; conversely, ectopic expression of wild-type Hsp27 renders PS-341-sensitive DHL6 cells resistant to PS-341. These findings provide the first evidence that Hsp27 confers PS-341 resistance.

Caveolin-1 is required for vascular endothelial growth factor-triggered multiple myeloma cell migration and is targeted by bortezomib.

We recently demonstrated that caveolae, vesicular flask-shaped invaginations of the plasma membrane, represent novel therapeutic targets in multiple myeloma. In the present study, we demonstrate that vascular endothelial growth factor (VEGF) triggers Src-dependent phosphorylation of caveolin-1, which is required for p130(Cas) phosphorylation and multiple myeloma cell migration. Conversely, depletion of caveolin-1 by antisense methodology abrogates p130(Cas) phosphorylation and VEGF-triggered multiple myeloma cell migration. The proteasome inhibitor bortezomib both inhibited VEGF-triggered caveolin-1 phosphorylation and markedly decreased caveolin-1 expression. Consequently, bortezomib inhibited VEGF-induced multiple myeloma cell migration. Bortezomib also decreased VEGF secretion in the bone marrow microenvironment and inhibited VEGF-triggered tyrosine phosphorylation of caveolin-1, migration, and survival in human umbilical vascular endothelial cells. Taken together, these studies demonstrate the requirement of caveolae for VEGF-triggered multiple myeloma cell migration and identify caveolin-1 in multiple myeloma cells and human umbilical vascular endothelial cells as a molecular target of bortezomib.

Proteasomal degradation of topoisomerase I is preceded by c-Jun NH2-terminal kinase activation, Fas up-regulation, and poly(ADP-ribose) polymerase cleavage in SN38-mediated cytotoxicity against multiple myeloma.

Topoisomerase I inhibitors are effective anticancer therapies and have shown activity in hematologic malignancies. Here we show for the first time that SN38, the potent active metabolite of irinotecan, induces c-Jun NH(2)-terminal kinase activation, Fas up-regulation, and caspase 8-mediated apoptosis in multiple myeloma (MM) cells. Proteasomal degradation of nuclear topoisomerase I has been proposed as a resistance mechanism in solid malignancies. SN38-induced proteasomal degradation of topoisomerase I was observed during SN38-mediated cytotoxicity against MM.1S myeloma cell line but occurred after c-Jun NH(2)-terminal kinase activation, Fas up-regulation, and poly(ADP-ribose) polymerase cleavage and failed to protect cells from apoptosis. Differential toxicity was observed against MM cells versus bone marrow stromal cells, and SN38 inhibited adhesion-induced up-regulation of MM cell proliferation when MM cells adhere to bone marrow stromal cells. In addition, SN38 directly inhibited constitutive and inducible interleukin 6 and vascular endothelial growth factor secretion by bone marrow stromal cells. Synergy was observed when SN38 was used in combination with doxorubicin, bortezomib, as well as poly(ADP-ribose) polymerase inhibitor NU1025 and Fas-activator CH11. These findings have clinical significance, because identification of downstream apoptotic signaling after topoisomerase I inhibition will both elucidate mechanisms of resistance and optimize future combination chemotherapy against MM.

Insulin-like growth factor-1 induces adhesion and migration in human multiple myeloma cells via activation of beta1-integrin and phosphatidylinositol 3'-kinase/AKT signaling.

Insulin-like growth factor-1 (IGF-I) is a growth and survival factor in human multiple myeloma (MM) cells. Here we examine the effect of IGF-I on MM cell adhesion and migration, and define the role of beta1 integrin in these processes. IGF-I increases adhesion of MM.1S and OPM6 MM cells to fibronectin (FN) in a time- and dose-dependent manner, as a consequence of IGF-IR activation. Conversely, blocking anti-beta1 integrin monoclonal antibody, RGD peptide, and cytochalasin D inhibit IGF-I-induced cell adhesion to FN. IGF-I rapidly and transiently induces association of IGF-IR and beta1 integrin, with phosphorylation of IGF-IR, IRS-1, and p85(PI3-K). IGF-I also triggers phosphorylation of AKT and ERK significantly. Both IGF-IR and beta1 integrin colocalize to lipid rafts on the plasma membrane after IGF-I stimulation. In addition, IGF-I triggers polymerization of F-actin, induces phosphorylation of p125(FAK) and paxillin, and enhances beta1 integrin interaction with these focal adhesion proteins. Importantly, using pharmacological inhibitors of phosphatidylinositol 3'-kinase (PI3-K) (LY294002 and wortmannin) and extracellular signal-regulated kinase (PD98059), we demonstrate that IGF-I-induced MM cell adhesion to FN is achieved only when PI3-K/AKT is activated. IGF-I induces a 1.7-2.2 (MM.1S) and 2-2.5-fold (OPM6) increase in migration, whereas blocking anti-IGF-I and anti-beta1 integrin monoclonal antibodies, PI3-K inhibitors, as well as cytochalasin D abrogate IGF-I-induced MM cell transmigration. Finally, IGF-I induces adhesion of CD138+ patient MM cells. Therefore, these studies suggest a role for IGF-I in trafficking and localization of MM cells in the bone marrow microenvironment. Moreover, they define the functional association of IGF-IR and beta1 integrin in mediating MM cell homing, providing the preclinical rationale for novel treatment strategies targeting IGF-I/IGF-IR in MM.

Blockade of ubiquitin-conjugating enzyme CDC34 enhances anti-myeloma activity of Bortezomib/Proteasome inhibitor PS-341.

The ubiquitin-conjugating enzyme CDC34 (UBC3) is linked to cell cycle progression in diverse cell types; however, its role in multiple myeloma (MM) pathogenesis is unclear. Here, we show that CDC34 is highly expressed in patient MM cells and MM cell lines versus normal cells. Blocking CDC34 using a dominant-negative strategy enhances the anti-MM activity of Bortezomib/Proteasome inhibitor PS-341, dexamethasone (Dex) and 2-Methoxyestradiol (2ME2). The expression of wild-type CDC34 reduces Dex-induced cytotoxicity in MM cells. Moreover, inhibition of CDC34 enzymatic activity abrogates interleukin-6-induced protection against Dex-induced apoptosis. Together, these findings provide evidence that (1) CDC34 expression is associated with growth and survival of MM cells and (2) blocking CDC34 activity not only enhances anti-MM activity of Bortezomib and 2ME2 but also overcomes IL-6-triggered Dex-resistance.