Pegbelfermin in Patients With Nonalcoholic Steatohepatitis and Stage 3 Fibrosis (FALCON 1): A Randomized Phase 2b Study.

BACKGROUND & AIMS: Pegbelfermin is a polyethlene glycol-conjugated analog of human fibroblast growth factor 21, a nonmitogenic hormone that regulates energy metabolism. This phase 2b study evaluated 48-week pegbelfermin treatment in patients with nonalcoholic steatohepatitis (NASH) and stage 3 (bridging) fibrosis. METHODS: The FALCON 1 study (NCT03486899) was a multicenter, randomized (1:1:1:1), double-blind, placebo-controlled study. Patients with biopsy-confirmed NASH and stage 3 fibrosis (N = 197) received weekly subcutaneous pegbelfermin (10, 20, or 40 mg) or placebo injections for 48 weeks. The week 24 primary endpoint was a ≥1-point decrease in fibrosis score without NASH worsening or NASH improvement without fibrosis worsening; pegbelfermin dose response was assessed using a Cochran-Armitage trend test across proportions (1-sided α = 0.05). Secondary/exploratory endpoints included histological and noninvasive measures of steatosis, fibrosis, and liver injury/inflammation. RESULTS: At week 24, the primary endpoint was met by 14% (placebo) vs 24%-31% (pegbelfermin arms); statistical significance was not reached due to lack of pegbelfermin dose response (P = .134). At weeks 24 and 48, more patients who received pegbelfermin had ≥30% relative reductions in hepatic fat fraction (magnetic resonance imaging-proton density fat fraction) vs placebo, although no differences reached statistical significance. In the pegbelfermin arms, improvements in liver fibrosis (magnetic resonance elastography and N-terminal type III collagen propeptide) and liver injury/inflammation (alanine aminotransferase, aspartate aminotransferase) were observed vs placebo. Adverse events occurred at similar frequencies across arms. No treatment-related serious adverse events were observed. CONCLUSIONS: The FALCON 1 study did not meet its primary endpoint; a ≥1-point decrease in fibrosis score without NASH worsening or NASH improvement without fibrosis worsening assessed via biopsy. Pegbelfermin was generally well tolerated during 48 weeks of treatment.

Pegbelfermin in Patients With Nonalcoholic Steatohepatitis and Compensated Cirrhosis (FALCON 2): A Randomized Phase 2b Study.

BACKGROUND & AIMS: Pegbelfermin is a polyethylene glycol-conjugated analog of human fibroblast growth factor 21, a nonmitogenic hormone that regulates energy metabolism. This phase 2b study evaluated 48-week pegbelfermin treatment in patients with nonalcoholic steatohepatitis (NASH) with compensated cirrhosis. METHODS: FALCON 2 (NCT03486912) was a randomized (1:1:1:1), double-blind, placebo-controlled study. Eligible adults had biopsy-confirmed NASH and stage 4 fibrosis. Pegbelfermin (10, 20, or 40 mg) or placebo was injected subcutaneously once weekly. The primary endpoint was 1 or more stages of improvement in the NASH Clinical Research Network fibrosis score without NASH worsening at week 48; pegbelfermin dose response was assessed using a Cochran-Armitage trend test across proportions (1-sided α = .05). Additional endpoints included histologic and noninvasive measures of steatosis, fibrosis, and liver injury/inflammation. RESULTS: Overall, 155 patients were randomized, and 154 patients received treatment. At week 48, 24% to 28% of the pegbelfermin arms had primary endpoint responses vs 31% of the placebo arm (P = .361). Nonalcoholic fatty liver disease activity score improvements were more frequent with pegbelfermin vs placebo and were driven primarily by reduced lobular inflammation. Numerically higher proportions of the pegbelfermin arms had liver stiffness (magnetic resonance elastography) and steatosis (magnetic resonance imaging-proton density fat fraction) improvements vs placebo; these differences were not statistically significant. Mean N-terminal type III collagen propeptide, alanine aminotransferase, and aspartate aminotransferase values were numerically lower in the 20- and/or 40-mg pegbelfermin arms compared with placebo. Serious adverse events were more frequent with pegbelfermin vs placebo, although none were treatment related. One patient (40-mg pegbelfermin) discontinued treatment because of a treatment-emergent adverse event (worsening ascites). CONCLUSIONS: FALCON 2 did not meet its primary endpoint of 1 or more stages of improvement in the NASH Clinical Research Network fibrosis without NASH worsening assessed via biopsy. Pegbelfermin generally was well tolerated in this advanced NASH population.

Pegbelfermin selectively reduces secondary bile acid concentrations in patients with non-alcoholic steatohepatitis.

BACKGROUND & AIMS: Increased serum bile acids (BAs) have been observed in patients with non-alcoholic steatohepatitis (NASH). Pegbelfermin (PGBF), a polyethylene glycol-modified (PEGylated) analogue of human fibroblast growth factor 21 (FGF21), significantly decreased hepatic steatosis and improved fibrosis biomarkers and metabolic parameters in patients with NASH in a phase IIa trial. This exploratory analysis evaluated the effect of PGBF on serum BAs and explored potential underlying mechanisms. METHODS: Serum BAs and 7α-hydroxy-4-cholesten-3-one (C4) were measured by HPLC-mass spectrometry (MS) using serum collected in studies of patients with NASH (NCT02413372) and in overweight/obese adults (NCT03198182) who received PGBF. Stool samples were collected in NCT03198182 to evaluate faecal BAs by liquid chromatography (LC)-MS and the faecal microbiome by metagenetic and metatranscriptomic analyses. RESULTS: Significant reductions from baseline in serum concentrations of the secondary BA, deoxycholic acid (DCA), and conjugates, were observed with PGBF, but not placebo, in patients with NASH; primary BA concentrations did not significantly change in any arm. Similar effects of PGBF on BAs were observed in overweight/obese adults, allowing for an evaluation of the effects of PGBF on the faecal microbiome and BAs. Faecal transcriptomic analysis showed that the relative abundance of the gene encoding choloylglycine hydrolase, a critical enzyme for secondary BA synthesis, was reduced after PGBF, but not placebo, administration. Furthermore, a trend of reduction in faecal secondary BAs was observed. CONCLUSIONS: PGBF selectively reduced serum concentrations of DCA and conjugates in patients with NASH and in healthy overweight/obese adults. Reduced choloylglycine hydrolase gene expression and decreased faecal secondary BA levels suggest a potential role for PGBF in modulating secondary BA synthesis by gut microbiome. The clinical significance of DCA reduction post-PGBF treatment warrants further investigation. LAY SUMMARY: Pegbelfermin (PGBF) is a hormone that is currently being studied in clinical trials for the treatment of non-alcoholic fatty liver disease. In this study, we show that PGBF treatment can reduce bile acids that have previously been shown to have toxic effects on the liver. Additional studies to understand how PGBF regulates bile acids may provide additional information about its potential use as a treatment for fatty liver.

BMS-986263 in patients with advanced hepatic fibrosis: 36-week results from a randomized, placebo-controlled phase 2 trial.

BACKGROUND AND AIMS: Hepatic fibrosis secondary to HCV infection can lead to cirrhosis and hepatic decompensation. Sustained virologic response (SVR) is possible with direct-acting antiviral drug regimens; however, patients with advanced fibrosis have an increased risk for HCC. Heat shock protein 47 (HSP47), a key collagen chaperone, has been implicated in fibrosis development. We evaluated the efficacy and safety of BMS-986263, a lipid nanoparticle delivering small interfering RNA designed to degrade HSP47 mRNA, for the treatment of advanced fibrosis. APPROACH AND RESULTS: NCT03420768 was a Phase 2, randomized (1:1:2), placebo-controlled trial conducted at a hepatology clinic in the United States. Patients with HCV-SVR (for ≥ 1 year) and advanced fibrosis received once-weekly i.v. infusions of placebo or BMS-986263 (45 or 90 mg) for 12 weeks. The primary endpoint was ≥ 1 METAVIR stage improvement at Week 12; key secondary endpoints included Ishak score improvement, pharmacokinetics, fibrosis biomarkers, and safety. All 61 patients completed treatment, and 2/15 (13%, placebo), 3/18 (17%, 45 mg), and 6/28 (21%, 90 mg) had METAVIR improvements of ≥ 1 stage at Week 12. Five patients in the 90-mg arm had Ishak improvements by ≥ 2 stages. BMS-986263 plasma concentrations increased in a generally dose-proportional fashion between BMS-986263 doses, with no notable accumulation with weekly dosing. All adverse events (AEs) were mild or moderate in intensity; most treatment-related AEs were infusion-related reactions in the BMS-986263 arms. At baseline, collagen levels were low, indicating low levels of fibrogenesis in these patients. CONCLUSIONS: In patients with HCV-SVR, BMS-986263 administration was generally well tolerated through Week 36 and resulted in METAVIR and Ishak score improvements. Further evaluation of BMS-986263 in patients with active fibrogenesis is warranted.

PRO-C3, a Serological Marker of Fibrosis, During Childhood and Correlations With Fibrosis in Pediatric NAFLD.

Nonalcoholic fatty liver disease (NAFLD) is a common chronic liver disease in children and may lead to cirrhosis requiring liver transplant. Thus, prompt diagnosis of advanced fibrosis is essential. Our objectives were to examine PRO-C3 (a neo-epitope pro-peptide of type III collagen formation) levels across childhood/adolescence and associations with advanced fibrosis in pediatric NAFLD. This cross-sectional study included 88 children and adolescents with biopsy-proven NAFLD (mean age: 13.9 ± 2.9 years, 71% male) and 65 healthy participants (11.8 ± 4.5 years, 38% male). PRO-C3, and the bone remodeling biomarkers C-terminal telopeptide of type I collagen (CTX-I; bone resorption) and osteocalcin (N-MID; bone formation), were measured in serum by enzyme-linked immunosorbent assay. Fibrosis was assessed by liver biopsy in participants with NAFLD, who were categorized as having advanced (Ishak score ≥ 3) or none/mild fibrosis (Ishak score ≤ 2). Overall, PRO-C3 was similar in participants with NAFLD (median [interquartile range]: 20.6 [15.8, 25.9] ng/mL) versus healthy participants (19.0 [13.8, 26.0] ng/mL), but was significantly lower in older adolescents ≥ 15 years old (16.4 [13.0, 21.2] ng/mL) compared with children ≤ 10 years old (22.9 [18.1, 28.4] ng/mL; P < 0.001) or 11-14 years old (22.4 [18.3, 31.2] ng/mL; P < 0.001). PRO-C3 was also directly correlated with levels of CTX-I and N-MID (r = 0.64 and r = 0.62, respectively; both P < 0.001). Among participants with NAFLD, PRO-C3 was higher in those with advanced fibrosis (median [IQR]: 28.5 [21.6, 37.6]) compared with none/mild fibrosis (20.3 [18.2, 22.8]; P = 0.020) in models adjusted for age, sex, and body mass index z-score. However, associations were attenuated after additionally adjusting for bone-remodeling CTX-I (P = 0.09) or N-MID (P = 0.08). Conclusion: Collectively, these findings show that PRO-C3 levels are higher in children with advanced fibrosis in NAFLD, but are also influenced by age and pubertal growth spurt, assessed by bone remodeling biomarkers, and therefore may not be a reliable biomarker for liver fibrosis in pediatric NAFLD until late adolescence.

Determining a healthy reference range and factors potentially influencing PRO-C3 - A biomarker of liver fibrosis.

BACKGROUND & AIMS: Progressive fibrosis has been identified as the major predictor of mortality in patients with non-alcoholic fatty liver disease (NAFLD). Several biomarkers are currently being evaluated for their ability to substitute the liver biopsy as the reference standard. Recent clinical studies in NAFLD/NASH patients support the utility of PRO-C3, a marker of type III collagen formation, as a marker for the degree of fibrosis, disease activity, and effect of treatment. Here we establish the healthy reference range, optimal sample handling conditions for both short- and long-term serum storage, and robustness for the PRO-C3 assay. METHODS: PRO-C3 was measured in 269 healthy volunteers and in 222 NAFLD patients. Robustness of the PRO-C3 assay was measured according to Clinical and Laboratory Standards Institute standards and included validation of interference, precision, and reagent stability, whilst sample stability was defined for storage at different temperatures and for 3 freeze-thaw cycles. Fibrosis scoring was based on histological assessments and used as a reference for the diagnostic ability of PRO-C3 to discriminate between patients with different levels of fibrosis. RESULTS: Robustness of the PRO-C3 analysis validated by interference, precision, and reagent stability was found to be within the predefined acceptance criteria. The healthy reference range was determined to be 6.1-14.7 ng/ml. Levels of PRO-C3 were not affected by sex, age, BMI, or ethnicity. Levels of PRO-C3 were able to identify patients with clinically significant fibrosis and advanced fibrosis (AUC = 0.83 (95% CI [0.77-0.88], p <0.0001), and AUC = 0.79 (95% CI [0.73-0.85], p <0.0001), respectively). CONCLUSIONS: The assay proved to be robust and sample stability was found to comply with hospital sample handling requirements. PRO-C3 measured in samples from patients with NAFLD/NASH was diagnostic for significant and advanced liver fibrosis. LAY SUMMARY: We showed that PRO-C3 levels were stable under conditions conforming with hospital sample-handling requirements. We determined a healthy reference range and showed that PRO-C3 levels were not associated with sex, age, BMI, or ethnicity. Finally, we provide further evidence of an association of PRO-C3 with increasing liver fibrosis.

SOMAscan Proteomics Identifies Serum Biomarkers Associated With Liver Fibrosis in Patients With NASH.

Nonalcoholic steatohepatitis (NASH) is a major cause of liver-related morbidity and mortality worldwide. Liver fibrosis stage, a key component of NASH, has been linked to the risk of mortality and liver-related clinical outcomes. Currently there are no validated noninvasive diagnostics that can differentiate between fibrosis stages in patients with NASH; many existing tests do not reflect underlying disease pathophysiology. Noninvasive biomarkers are needed to identify patients at high-risk of NASH with advanced fibrosis. This was a retrospective study of patients with histologically proven NASH with fibrosis stages 0-4. The SOMAscan proteomics platform was used to quantify 1,305 serum proteins in a discovery cohort (n = 113). In patients with advanced (stages 3-4) versus early fibrosis (stages 0-2), 97 proteins with diverse biological functions were differentially expressed. Next, fibrosis-stage classification models were explored using a machine learning-based approach to prioritize the biomarkers for further evaluation. A four-protein model differentiated patients with stage 0-1 versus stage 2-4 fibrosis (area under the receiver operating characteristic curve [AUROC] = 0.74), while a 12-protein classifier differentiated advanced versus early fibrosis (AUROC = 0.83). Subsequently, the model's performance was validated in two independent cohorts (n = 71 and n = 32) with similar results (AUROC = 0.74-0.78). Our advanced fibrosis model performed similarly to or better than Fibrosis-4 index, aspartate aminotransferase-to-platelet ratio index, and nonalcoholic fatty liver disease (NAFLD) fibrosis score-based models for all three cohorts. Conclusion: A SOMAscan proteomics-based exploratory classifier for advanced fibrosis, consisting of biomarkers that reflect the complexity of NASH pathophysiology, demonstrated similar performance in independent validation cohorts and performed similarly or better than Fibrosis-4 index, aspartate aminotransferase-to-platelet ratio index, and NAFLD fibrosis score. Further studies are warranted to evaluate the clinical utility of these biomarker panels in patients with NAFLD.

The FALCON program: Two phase 2b randomized, double-blind, placebo-controlled studies to assess the efficacy and safety of pegbelfermin in the treatment of patients with nonalcoholic steatohepatitis and bridging fibrosis or compensated cirrhosis.

BACKGROUND: Nonalcoholic steatohepatitis (NASH) is the progressive form of nonalcoholic fatty liver disease (NAFLD); no approved therapies for NASH currently exist. Pegbelfermin (PGBF), a human fibroblast growth factor 21 analog, has metabolic effects that may provide benefit for patients with NASH. DESIGN: The FALCON 1 and 2 studies are phase 2b, multicenter, double-blind, placebo-controlled, randomized trials to assess safety and efficacy of PGBF treatment in patients who have histologically-confirmed NASH with stage 3 liver fibrosis (FALCON 1; NCT03486899) or compensated cirrhosis (FALCON 2; NCT03486912). In both studies, randomized patients receive once weekly subcutaneous injections of PGBF (10, 20, or 40 mg) or placebo during a 48-week treatment period and are then followed for an additional 4 weeks. ENDPOINTS: The primary efficacy endpoint for FALCON 1 is the proportion of patients who achieve ≥1 stage improvement in fibrosis (by NASH CRN fibrosis score) without NASH worsening or NASH improvement (≥2 point decrease in NAFLD Activity Score) without fibrosis worsening at Week 24. For FALCON 2, the primary efficacy endpoint is ≥1 stage improvement in fibrosis without NASH worsening at Week 48. Key safety endpoints for both studies include incidence and frequency of adverse events, bone mineral density and immunogenicity. SUMMARY: Previous clinical trial data show that PGBF can reduce hepatic fat and improve metabolic factors and biomarkers of hepatic injury and fibrosis. The FALCON studies aim to evaluate PGBF treatment specifically in patients with NASH and advanced fibrosis, who are at greatest risk of poor clinical outcomes over time.

Population Pharmacokinetic Analysis of BMS-986166, a Novel Selective Sphingosine-1-Phosphate-1 Receptor Modulator, and Exposure-Response Assessment of Lymphocyte Counts and Heart Rate in Healthy Participants.

Sphingosine-1-phosphate (S1P) binding to the S1P-1 receptor (S1P1R) controls the egress of lymphocytes from lymphoid organs and targets modulation of immune responses in autoimmune diseases. Pharmacologic modulation of S1P receptors has been linked to heart rate reduction. BMS-986166, a prodrug of the active phosphorylated metabolite BMS-986166-P, presents an improved cardiac safety profile in preclinical studies compared to other S1P1R modulators. The pharmacokinetics, safety, and pharmacodynamics of BMS-986166 versus placebo after single (0.75-5.0 mg) and repeated (0.25-1.5 mg/day) oral administration were assessed in healthy participants after a 1-day lead-in placebo period. A population model was developed to jointly describe BMS-986166 and BMS-986166-P pharmacokinetics and predict individual exposures. Inhibitory sigmoid models described the relationships between average daily BMS-986166-P concentrations and nadir of time-matched (day -1) placebo-corrected heart rate on day 1 (nDDHR, where DD represents ∆∆) and nadir of absolute lymphocyte count (nALC). Predicted decreases in nDDHR and nALC were 9 bpm and 20% following placebo, with maximum decreases of 10 bpm in nDDHR due to drug effect, and approximately 80% in nALC due to drug and placebo. A 0.5-mg/day dose regimen achieves the target 65% reduction in nALC associated with a 2-bpm decrease in nDDHR over placebo.

Profiling and targeting connective tissue remodeling in autoimmunity - A novel paradigm for diagnosing and treating chronic diseases.

Connective tissue (ConT) remodeling is an essential process in tissue regeneration, where a balanced replacement of old tissue by new tissue occurs. This balance is disturbed in chronic diseases, often autoimmune diseases, usually resulting in the buld up of fibrosis and a gradual loss of organ function. During progression of liver, lung, skin, heart, joint, skeletal and kidney diseasesboth ConT formation and degradation are elevated, which is tightly linked to immune cell activation and a loss of specific cell types and extracellular matrix (ECM) structures that are required for normal organ function. Here, we address the balance of key general and organ specific components of the ECM during homeostasis and in disease, with a focus on collagens, which are emerging as both structural and signaling molecules harbouring neoepitopes and autoantigens that are released during ConT remodeling. Specific collagen molecular signatures of ConT remodeling are linked to disease activity and stage, and to prognosis across different organs. These signatures accompany and further drive disease progression, and often become detectable before clinical disease manifestation (illness). Recent advances allow to quantify and define the nature of ConT remodeling via blood-based assays that measure the levels of well-defined collagen fragments, reflecting different facets of ConT formation and degradation, and associated immunological processes. These novel serum assays are becoming important tools of precision medicine, to detect various chronic and autoimmune diseases before their clinical manifestation, and to non-invasively monitor the efficacy of a broad range of pharmacological interventions.

Urinary CD80 Discriminates Among Glomerular Disease Types and Reflects Disease Activity.

INTRODUCTION: Heterogeneity of nephrotic diseases and a lack of validated biomarkers limits interventions and reduces the ability to examine outcomes. Urinary CD80 is a potential biomarker for minimal change disease (MCD) steroid-sensitive nephrotic syndrome (NS). We investigated and validated a CD80 enzyme-linked immunosorbent assay (ELISA) in urine in a large cohort with a variety of nephrotic diseases. METHODS: A commercial CD80 ELISA was enhanced and analytically validated for urine. Patients were from Mayo Clinic (307) and Nephrotic Syndrome Study Network Consortium (NEPTUNE; 104) as follows: minimal change disease (MCD, 56), focal segmental glomerulosclerosis (FSGS, 92), lupus nephritis (LN, 25), IgA nephropathy (IgAN, 20), membranous nephropathy (MN, 49), autosomal dominant polycystic kidney disease (ADPKD, 10), diabetic nephropathy (DN; 106), pyuria (19), and controls (34). Analysis was by Kruskal-Wallis test, generalized estimating equation (GEE) models, and receiver operating characteristic (AUC) curve. RESULTS: Urinary CD80/creatinine values were highest in MCD compared to other glomerular diseases and were increased in DN with proteinuria >2 compared to controls (control = 36 ng/g; MCD = 139 ng/g, P < 0.01; LN = 90 ng/g, P < 0.12; FSGS = 66 ng/g, P = 0.18; DN = 63, P = 0.03; MN = 69 ng/g, P = 0.33; ng/g, P = 0.07; IgA = 19 ng/g, P = 0.09; ADPKD = 42, P = 0.36; and pyuria 31, P = 0.20; GEE, median, P vs. control). In proteinuric patients, CD80 concentration appears to be independent of proteinuria levels, suggesting that it is unrelated to nonspecific passage across the glomeruli. CD80/creatinine values were higher in paired relapse versus remission cases of MCD and FSGS (P < 0.0001, GEE). CONCLUSION: Using a validated ELISA, urinary CD80 levels discriminate MCD from other forms of NS (FSGS, DN, IgA, MN) and primary from secondary FSGS.

The safety and pharmacokinetics of a novel, selective S1P1R modulator in healthy participants.

Background: Safety, pharmacokinetics and pharmacodynamics of BMS-986166, a novel sphingosine-1-phosphate receptor 1 modulator, were assessed.Research design and methods: Two double-blind, placebo-controlled, randomized Phase l studies were conducted in healthy participants. In the single ascending dose study (N = 70), BMS-986166 was administered as a single dose, upwardly titrated daily doses or a single dose in participants who were fed, fasted or administered famotidine. In the multiple ascending dose study (N = 32), BMS-986166 was administered daily for 28 days. Safety, pharmacokinetics and pharmacodynamics (absolute lymphocyte count [ALC]) were assessed.Results: BMS-986166 was generally well tolerated; treatment-related adverse events were mild. Dose-related, clinically insignificant reductions in time-matched heart rate were recorded versus placebo. Pharmacokinetics were linear and stationary with approximately dose-related increases in blood exposure of BMS-986166. Decreases in ALC percent change from baseline with multiple doses of BMS-986166 versus placebo were dose-related. Between Day 0 and 35, median nadir lymphocyte reductions were 53.7%, 75.9% and 81.9% with 0.25-, 0.75- and 1.5-mg BMS-986166 doses. ALC recovery began 14, 14-21 and 7 days after last dose of 0.25, 0.75 and 1.5 mg.Conclusions: BMS-986166 was generally well tolerated in this population and warrants further investigation.Trial registration: ClinicalTrials.gov: NCT02790125, NCT03038711.

Collagen biology and non-invasive biomarkers of liver fibrosis.

There is an unmet need for high-quality liquid biomarkers that can safely and reproducibly predict the stage of fibrosis and the outcomes of chronic liver disease (CLD). The requirement for such markers has intensified because of the high global prevalence of diseases such as non-alcoholic fatty liver disease (NAFLD). In particular, there is a need for diagnostic and prognostic tools, as well as predictive biomarkers that reflect the efficacy of interventions, as described by the BEST criteria (Biomarkers, EndpointS, and other Tools Resource). This review covers the various liver collagens, their functional role in tissue homeostasis and delineates the common nomenclature for biomarkers based on BEST criteria. It addresses the common confounders affecting serological biomarkers, and describes defined collagen epitope biomarkers that originate from the dynamic processes of extracellular matrix (ECM) remodelling during liver injury.

Pharmacokinetic, Pharmacodynamic, and Safety Profile of a Novel Anti-CD28 Domain Antibody Antagonist in Healthy Subjects.

We report pharmacokinetics, pharmacodynamics, and safety of a novel anti-CD28 domain antibody antagonist (lulizumab pegol) in healthy subjects following single- or multiple-dose administration. A minimal anticipated biological effect level approach was used to select a 0.01 mg starting dose for a single-ascending-dose (SAD), double-blind, first-in-human study. Part 1 included 9 intravenous (IV; 0.01-100 mg) and 3 subcutaneous (SC; 9-50 mg) doses or placebo. In part 2, a keyhole limpet hemocyanin (KLH) immunization was performed in 16 subjects/panel, who received 1 of 3 IV doses (9-100 mg) or placebo. In a double-blind, multiple-ascending-dose (MAD) study, subjects received SC lulizumab 6.25 mg every 2 weeks, 12.5 mg weekly, 37.5 mg weekly, or placebo. Among 180 treated subjects, 169 completed the studies. Peak concentrations and areas under the curve from time 0 to infinity increased dose proportionally. Estimated SC bioavailability was 68.2%. Receptor occupancy of approximately ≥80% was maintained for ≥2 weeks at ≥9-mg doses (SAD) and throughout the dosing interval (MAD). IV doses ≥9 mg inhibited antibody production against KLH for 2 weeks. No significant cytokine or immune cell changes were observed. No immunogenicity responses persisted, and there was no correlation to adverse events. Headache occurred in 21 SAD and 4 MAD subjects receiving lulizumab; in the MAD study 5 lulizumab subjects experienced infections. Lulizumab IV or SC was safe at all doses studied, without evidence of cytokine release.

Discovery and Clinical Evaluation of MK-8150, A Novel Nitric Oxide Donor With a Unique Mechanism of Nitric Oxide Release.

BACKGROUND: Nitric oxide donors are widely used to treat cardiovascular disease, but their major limitation is the development of tolerance, a multifactorial process to which the in vivo release of nitric oxide is thought to contribute. Here we describe the preclinical and clinical results of a translational drug development effort to create a next-generation nitric oxide donor with improved pharmacokinetic properties and a unique mechanism of nitric oxide release through CYP3A4 metabolism that was designed to circumvent the development of tolerance. METHODS AND RESULTS: Single- and multiple-dose studies in telemetered dogs showed that MK-8150 induced robust blood-pressure lowering that was sustained over 14 days. The molecule was safe and well tolerated in humans, and single doses reduced systolic blood pressure by 5 to 20 mm Hg in hypertensive patients. Multiple-dose studies in hypertensive patients showed that the blood-pressure-lowering effect diminished after 10 days, and 28-day studies showed that the hemodynamic effects were completely lost by day 28, even when the dose of MK-8150 was increased during the dosing period. CONCLUSIONS: The novel nitric oxide donor MK-8150 induced significant blood-pressure lowering in dogs and humans for up to 14 days. However, despite a unique mechanism of nitric oxide release mediated by CYP3A4 metabolism, tolerance developed over 28 days, suggesting that tolerance to nitric oxide donors is multifactorial and cannot be overcome solely through altered in vivo release of nitric oxide. CLINICAL TRIAL REGISTRATION: URL: http://www.clinicaltrials.gov. Unique identifiers: NCT01590810 and NCT01656408.

Optical imaging biomarkers of drug-induced vascular injury.

Drug-induced vascular injury (DIVI), defined as arterial medial degeneration/necrosis usually associated with perivascular inflammation, is frequently observed in the mesenteric arteries of rats but the relevance to humans remains a hurdle for drug development. Here, we describe the evaluation of commercially available optical imaging biomarkers using a rat DIVI model. Male Sprague Dawley rats were administered 10 mg/kg/day of a proprietary soluble guanylate cyclase activator (sGCa). Optical agents, AngioSense for the detection of vessel permeability, MMPSense for the detection of activated matrix metalloproteinases (MMPs), and IntegriSense for the detection of αvß3 integrin, were injected via tail vein 24 hours before fluorescence (FL) ex vivo imaging. Imaging found a statistically significant difference in FL from all optical agents between treated and vehicle groups (p < .05). Mesenteric arteries were further analyzed by histopathology, flow cytometry, and immunohistochemistry. Histopathology confirmed perivascular inflammation and/or arterial medial degeneration in the sGCa-treated animals. Flow cytometry of digested arteries revealed myeloid cells as a main source of MMPSense signal. Immunohistochemical analysis further identified elevated MMP-9 expression within arterial walls and surrounding tissue of treated animals. Together, these data demonstrate that MMPSense and AngioSense are sensitive optical imaging biomarkers for the quantification of DIVI in rat mesenteric arteries.

Evaluation of selective inhibitors of 11ß-HSD1 for the treatment of hypertension.

In an effort to understand the origin of blood-pressure lowering effects observed in recent clinical trials with 11ß-HSD1 inhibitors, we examined a set of 11ß-HSD1 inhibitors in a series of relevant in vitro and in vivo assays. Select 11ß-HSD1 inhibitors reduced blood pressure in our preclinical models but most or all of the blood pressure lowering may be mediated by a 11ß-HSD1 independent pathway.

The target of ezetimibe is Niemann-Pick C1-Like 1 (NPC1L1).

Ezetimibe is a potent inhibitor of cholesterol absorption that has been approved for the treatment of hypercholesterolemia, but its molecular target has been elusive. Using a genetic approach, we recently identified Niemann-Pick C1-Like 1 (NPC1L1) as a critical mediator of cholesterol absorption and an essential component of the ezetimibe-sensitive pathway. To determine whether NPC1L1 is the direct molecular target of ezetimibe, we have developed a binding assay and shown that labeled ezetimibe glucuronide binds specifically to a single site in brush border membranes and to human embryonic kidney 293 cells expressing NPC1L1. Moreover, the binding affinities of ezetimibe and several key analogs to recombinant NPC1L1 are virtually identical to those observed for native enterocyte membranes. KD values of ezetimibe glucuronide for mouse, rat, rhesus monkey, and human NPC1L1 are 12,000, 540, 40, and 220 nM, respectively. Last, ezetimibe no longer binds to membranes from NPC1L1 knockout mice. These results unequivocally establish NPC1L1 as the direct target of ezetimibe and should facilitate efforts to identify the molecular mechanism of cholesterol transport.

Small molecule antagonists of complement receptor type 3 block adhesion and adhesion-dependent oxidative burst in human polymorphonuclear leukocytes.

The leukocyte integrin complement receptor type 3 (CR3, Mac-1, CD11b/CD18) is the predominant beta(2) integrin receptor of polymorphonuclear leukocytes (PMNs). This cell surface receptor plays a central role in innate immunity against pathogens as well as being a major cellular effector of inflammation and tissue injury. Two small molecules, compounds 1 and 2, have been identified, that interact with CR3 and prevent CR3 from binding to its natural ligand, C3bi. Compounds 1 and 2 have IC(50) values of 0.14 and 0.33 microM, respectively, for the inhibition of binding of monomeric C3bi-alkaline phosphatase to immobilized CR3. Both compounds also inhibit binding of CR3 to biotinylated sheep red blood cells opsonized with C3bi, with IC(50) values in the micromolar range. Inhibition of ligand binding by the compounds is not easily reversed and requires light, suggesting the formation of a covalent adduct through photoactivation. Compounds 1 and 2 also inhibit adhesion of human PMNs to fibrinogen in response to tumor necrosis factor (TNF) or PMA, with IC(50) values of 2.5 to >10 microM. They block the adhesion-dependent production of H(2)O(2) stimulated by TNF or phorbol 12-myristate 13-acetate (PMA) with IC(50) values of 0.2 to 0.8 microM and 1 to 3 microM, respectively. Limited structure-activity relationship studies based on compound 2 indicate the importance of the two benzothiazole rings, an ethyl side chain, and the length of the carbon chain linking the rings. Further modification of these groups may help in making compounds appropriate for in vivo studies.