Randomized, Controlled Trial of the FGF21 Analogue Pegozafermin in NASH.

BACKGROUND: Pegozafermin is a long-acting glycopegylated (pegylated with the use of site-specific glycosyltransferases) fibroblast growth factor 21 (FGF21) analogue in development for the treatment of nonalcoholic steatohepatitis (NASH) and severe hypertriglyceridemia. The efficacy and safety of pegozafermin in patients with biopsy-proven noncirrhotic NASH are not well established. METHODS: In this phase 2b, multicenter, double-blind, 24-week, randomized, placebo-controlled trial, we randomly assigned patients with biopsy-confirmed NASH and stage F2 or F3 (moderate or severe) fibrosis to receive subcutaneous pegozafermin at a dose of 15 mg or 30 mg weekly or 44 mg once every 2 weeks or placebo weekly or every 2 weeks. The two primary end points were an improvement in fibrosis (defined as reduction by ≥1 stage, on a scale from 0 to 4, with higher stages indicating greater severity), with no worsening of NASH, at 24 weeks and NASH resolution without worsening of fibrosis at 24 weeks. Safety was also assessed. RESULTS: Among the 222 patients who underwent randomization, 219 received pegozafermin or placebo. The percentage of patients who met the criteria for fibrosis improvement was 7% in the pooled placebo group, 22% in the 15-mg pegozafermin group (difference vs. placebo, 14 percentage points; 95% confidence interval [CI], -9 to 38), 26% in the 30-mg pegozafermin group (difference, 19 percentage points; 95% CI, 5 to 32; P = 0.009), and 27% in the 44-mg pegozafermin group (difference, 20 percentage points; 95% CI, 5 to 35; P = 0.008). The percentage of patients who met the criteria for NASH resolution was 2% in the placebo group, 37% in the 15-mg pegozafermin group (difference vs. placebo, 35 percentage points; 95% CI, 10 to 59), 23% in the 30-mg pegozafermin group (difference, 21 percentage points; 95% CI, 9 to 33), and 26% in the 44-mg pegozafermin group (difference, 24 percentage points; 95% CI, 10 to 37). The most common adverse events associated with pegozafermin therapy were nausea and diarrhea. CONCLUSIONS: In this phase 2b trial, treatment with pegozafermin led to improvements in fibrosis. These results support the advancement of pegozafermin into phase 3 development. (Funded by 89bio; ENLIVEN ClinicalTrials.gov number, NCT04929483.).

Aldafermin in patients with non-alcoholic steatohepatitis (ALPINE 2/3): a randomised, double-blind, placebo-controlled, phase 2b trial.

BACKGROUND: Non-alcoholic steatohepatitis (NASH) is characterised by hepatic steatosis, inflammation, and injury, and is associated with an increased risk of liver transplantation and death. NASH affects more than 16 million people in the USA, and there is no approved therapy. The aim of this study was to evaluate the safety and efficacy of aldafermin, an engineered analogue of the gut hormone fibroblast growth factor 19 (FGF19). METHODS: In this randomised, double-blind, placebo-controlled, phase 2b study (ALPINE 2/3) in patients with biopsy-confirmed NASH and stage 2 or 3 fibrosis, we randomly assigned patients stratified by fibrosis stage in a 1:1:1:1 ratio to receive placebo, aldafermin 0·3 mg, 1·0 mg, or 3·0 mg once daily for 24 weeks at 30 study sites in the USA. Patients, investigators, the funder, and all other staff, were masked to treatment assignment throughout the study. The primary endpoint was an improvement in liver fibrosis of at least one stage with no worsening of NASH at week 24. Analyses were done by intention-to-treat. This trial is registered with ClinicalTrials.gov, number NCT03912532, and has been completed. FINDINGS: Between May 16, 2019, and Sept 4, 2020, 786 patients were screened, of whom 171 were randomly assigned to a treatment group and included in the intention-to-treat population: 43 in the 0·3 mg aldafermin group, 42 in the 1·0 mg group, 43 in the 3·0 mg group, and 43 in the placebo group. In total, 145 (85%) of patients completed treatment. At week 24, among patients with biopsies at both baseline and week 24, was seven (19%) of 36 patients in the placebo group, 11 (31%) of 36 in the 0·3 mg aldafermin group (difference 90% CI 12% [-9 to 33]; p=0·11), five (15%) of 34 patients in the 1·0 mg group (difference -5% [-24 to 13]; p=0·80), and 11 (30%) of 37 patients in the 3·0 mg group (difference 10% [-9 to 30]; p=0·12) had an improvement in liver fibrosis of at least one stage with no worsening of NASH, without meeting the prespecified significance for dose response (p=0·55). Adverse events were mostly mild or moderate in severity. Diarrhoea occurred in six (14%) of 43 patients in the placebo group, three (7%) of 43 patients in the 0·3 mg aldafermin group, five (12%) of 41 patients in the 1·0 mg group, and ten (23%) of 43 patients in the 3·0 mg group. Incidences of serious adverse events and discontinuations owing to adverse events were similar between groups. INTERPRETATION: Aldafermin was generally well tolerated but did not produce a significant dose response on fibrosis improvement of at least one stage with no worsening of NASH, despite positive effects on a number of secondary endpoints. The findings of this trial may have implications for the design of future NASH trials. FUNDING: NGM Biopharmaceuticals.

Pegbelfermin, a PEGylated FGF21 analogue, has pharmacology without bone toxicity after 1-year dosing in skeletally-mature monkeys.

Pegbelfermin (PGBF) is a PEGylated fibroblast growth factor 21 (FGF21) analogue in development for treatment of nonalcoholic steatohepatitis (NASH). Mouse models highlight potential utility of FGF21 in NASH, but also suggest negative effects on bone, though these findings are confounded by profound FGF21-related decreases in body mass/growth. This study aimed to profile PGBF-related bone effects in adult nonhuman primates after long-term, clinically-relevant exposures. Adult male cynomolgus monkeys received weekly subcutaneous PGBF (0.3, 0.75 mg/kg) or control injections for 1 year (n = 5/group). Assessments included body weight, clinical chemistry, adiponectin levels, bone turnover biomarkers, skeletal radiography, pharmacokinetics, immunogenicity, and histopathology. Bone densitometry and body composition were evaluated in vivo and/or ex vivo with dual-energy x-ray absorptiometry, peripheral quantitative computed tomography, and biomechanical strength testing. After 1 year of PGBF administration, there was clear evidence of sustained PGBF pharmacology in monkeys (peak increase in serum adiponectin of 1.7× and 2.35× pretest at 0.3 and 0.75 mg/kg PGBF, respectively) and decreased body weight compared with control at exposures comparable to those tested in humans. At 0.75 mg/kg PGBF, pharmacologically-mediated reductions in lean mass, lean area, and fat area were observed relative to controls. There were no PGBF-related effects on bone biomarkers, radiography, densitometry, or strength. Together, these data demonstrate that PGBF did not adversely alter bone metabolism, density, or strength following 1 year of dosing at clinically relevant (0.7-2.2× human AUC[0-168 h] at 20 mg once weekly), pharmacologically-active exposures in adult monkeys, suggesting a low potential for negative effects on bone quality in adult humans.

Synthesis of tritium labeled 30-KDa PEG and conjugation to form [3 H]Pegbelfermin.

Non-alcoholic steatohepatitis (NASH) is the most chronic liver condition in the western population and is fueled by the obesity and type 2 diabetes epidemic. Pegbelfermin (1), a PEGylated human fibroblast growth factor 21 (FGF21) analogue, has previously been shown to improve markers of metabolism and liver fibrosis in obese patients with type 2 diabetes. Radiolabeled Pegbelfermin was needed to access the accumulation of intact drug and metabolized PEG. In an effort to accomplish both goals with one labeled synthesis, the isotopic label was positioned in the PEG. A total of 21 mCi of tritium labeled Pegbelfermin was synthesized having a specific activity of 21.6 Ci/mmol for use in animal studies.

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.

Evolving Role for Pharmacotherapy in NAFLD/NASH.

Nonalcoholic fatty liver disease (NAFLD) is a highly prevalent, dynamic disease that occurs across the age spectrum and can lead to cirrhosis and hepatocellular carcinoma. There are currently no US Food and Drug Administration (FDA) approved treatments for NAFLD; however, this is a field of active research. This review summarizes emerging pharmacotherapies for the treatment of adult and pediatric NAFLD. Investigated pharmacotherapies predominantly target bile acid signaling, insulin resistance, and lipid handling within the liver. Three drugs have gone on to phase III trials for which results are available. Of those, obeticholic acid is the single agent that demonstrates promise according to the interim analyses of the REGENERATE trial. Obeticholic acid showed reduction of fibrosis in adults with nonalcoholic steatohepatitis (NASH) taking 25 mg daily for 18 months (n = 931, reduction in fibrosis in 25% vs. 12% placebo, P < 0.01). Ongoing phase III trials include REGENERATE and MAESTRO-NASH, which investigates thyroid hormone receptor-ß agonist MGL-3196. Outcomes of promising phase II trials in adults with NASH are also available and those have investigated agents, including the fibroblast growth factor (FGF)19 analogue NGM282, the GLP1 agonist liraglutide, the FGF21 analogue Pegbelfermin, the sodium glucose co-transporter 2 inhibitor Empagliflozin, the ketohexokinase inhibitor PF-06835919, the acetyl-coenzyme A carboxylase inhibitor GS-0976, and the chemokine receptor antagonist Cenicriviroc. Completed and ongoing clinical trials emphasize the need for a more nuanced understanding of the phenotypes of subgroups within NAFLD that may respond to an individualized approach to pharmacotherapy.

FGF19 and FGF21: In NASH we trust.

OBJECTIVE: FGF19 and FGF21 have shown therapeutic promise since their discovery, attested by the fact there are at least 5 assets that activate the FGFR/KLB pathway and one FGF19 analog in clinical development. METHODS: We performed a detailed analyses of published preclinical and clinical data to offer insights into the mechanism of action, as well as PK/PD and efficacy data of the clinical assets. RESULTS: Scouring the literature, we offer mechanistic insights from preclinical data using rodents and non-human primates and pharmacodynamic data from clinical studies. CONCLUSION: The basic and applied science around endocrine FGFs has evolved exponentially over the years with FGF19 and FGF21 analogs are now entering Phase 3 clinical research.

Targeting of Secretory Proteins as a Therapeutic Strategy for Treatment of Nonalcoholic Steatohepatitis (NASH).

: Nonalcoholic steatohepatitis (NASH) is defined as a progressive form of nonalcoholic fatty liver disease (NAFLD) and is a common chronic liver disease that causes significant worldwide morbidity and mortality, and has no approved pharmacotherapy. Nevertheless, growing understanding of the molecular mechanisms underlying the development and progression of NASH has suggested multiple potential therapeutic targets and strategies to treat this disease. Here, we review this progress, with emphasis on the functional role of secretory proteins in the development and progression of NASH, in addition to the change of expression of various secretory proteins in mouse NASH models and human NASH subjects. We also highlight secretory protein-based therapeutic approaches that influence obesity-associated insulin resistance, liver steatosis, inflammation, and fibrosis, as well as the gut-liver and adipose-liver axes in the treatment of NASH.

Pegbelfermin (BMS-986036): an investigational PEGylated fibroblast growth factor 21 analogue for the treatment of nonalcoholic steatohepatitis.

Introduction: Nonalcoholic fatty liver disease (NAFLD) is the most common liver disease worldwide and is strongly associated with obesity and insulin resistance. NAFLD refers to a spectrum of disorders ranging from asymptomatic hepatic steatosis (nonalcoholic fatty liver, NAFL) to nonalcoholic steatohepatitis (NASH), which increases the risk of developing more severe forms of liver disease such as progressive fibrosis, cirrhosis, and liver cancer. Currently, there are no food and drug administration (FDA) approved drugs to treat NASH. Pegbelfermin (BMS-986036) is a PEGylated fibroblast growth factor 21 (FGF21) analogue that is under investigation for the treatment of NASH.Areas covered: We reviewed the (pre)clinical pegbelfermin studies and compared these with other studies that assessed FGF21 and FGF21 analogues in the treatment of NASH.Expert opinion: With no FDA approved treatments available for NASH, there is an urgent need for novel therapies. Pegbelfermin is a systemic treatment with pleiotropic effects on various tissues. Short-term adverse effects are limited, but more research is required to study potential long-term safety issues. In a phase 2a trial, pegbelfermin has shown promising improvements in several NASH related outcomes. However, clinical trials demonstrating long-term benefits on hard outcomes such as liver histology, cirrhosis development, or survival are required for further validation.

Multiple therapeutic targets in rare cholestatic liver diseases: Time to redefine treatment strategies.

Primary biliary cholangitis and primary sclerosing cholangitis are rare diseases affecting the bile ducts and the liver. The limited knowledge of their pathogenesis leads to limited therapeutic options. Nevertheless, the landscape of novel therapies for these cholangiopathies is now rapidly changing, providing new treatment opportunities for patients and clinicians involved in their care. The aim of this review is to summarize the evidence of novel molecules under investigation for primary biliary cholangitis and primary sclerosing cholangitis and to discuss how they can potentially change current treatment paradigms.

Pegbelfermin (BMS-986036), PEGylated FGF21, in Patients with Obesity and Type 2 Diabetes: Results from a Randomized Phase 2 Study.

OBJECTIVE: Obesity and type 2 diabetes mellitus (T2DM) are risk factors for nonalcoholic fatty liver disease, including nonalcoholic steatohepatitis. This study assessed pegbelfermin (BMS-986036), recombinant PEGylated human fibroblast growth factor 21 (FGF21), in patients with obesity and T2DM predisposed to fatty liver. METHODS: In this randomized, double-blind, placebo-controlled study, patients with T2DM and BMI of 30 to 50 kg/m2 received subcutaneous pegbelfermin (1, 5, or 20 mg daily or 20 mg weekly; n = 96) or placebo (n = 24) for 12 weeks. Primary end points were safety, tolerability, and change in HbA1c. Additional end points included insulin sensitivity, lipids, adiponectin, and disease progression biomarkers. RESULTS: There were no significant effects of pegbelfermin versus placebo on HbA1c. Pegbelfermin 20 mg/d significantly improved high-density lipoprotein cholesterol (P = 0.015) and triglycerides (P = 0.037). All pegbelfermin regimens significantly increased adiponectin levels; 20-mg daily and weekly regimens decreased serum PRO-C3. Most adverse events were mild; the most frequent adverse events were injection-site bruising and diarrhea. CONCLUSIONS: Twelve-week pegbelfermin treatment did not impact HbA1c concentrations, but QW and higher daily doses were associated with improved metabolic parameters and fibrosis biomarkers in patients with obesity and T2DM predisposed to fatty liver. These results support evaluation of pegbelfermin in patients with obesity-related metabolic diseases (e.g., nonalcoholic steatohepatitis).

Pegbelfermin (BMS-986036), a PEGylated fibroblast growth factor 21 analogue, in patients with non-alcoholic steatohepatitis: a randomised, double-blind, placebo-controlled, phase 2a trial.

BACKGROUND: Pegbelfermin (BMS-986036), a PEGylated human fibroblast growth factor 21 (FGF21) analogue, has previously been shown to improve markers of metabolism and liver fibrosis in obese patients with type 2 diabetes. In this phase 2a study, we aimed to evaluate the safety and efficacy of pegbelfermin in patients with non-alcoholic steatohepatitis. METHODS: In this multicentre, randomised, double-blind, placebo-controlled, parallel-group, phase 2a study, we recruited adults (aged 21-75 years) with a body-mass index of at least 25 kg/m2, biopsy-confirmed non-alcoholic steatohepatitis (fibrosis stage 1-3), and a hepatic fat fraction of at least 10% when assessed by magnetic resonance imaging-proton density fat fraction. These patients were enrolled at 17 medical centres in the USA. Eligible patients were stratified by type 2 diabetes status and they were randomly assigned (1:1:1) by a computer-based system to receive subcutaneous injections of placebo once a day, 10 mg pegbelfermin once a day, or 20 mg pegbelfermin once a week, all for 16 weeks. Participants, the study team administering treatment, and investigators analysing outcomes (who were independent of the study team and had no further involvement) were masked to treatment groups. The primary outcomes were safety and the absolute change in hepatic fat fraction after 16 weeks of treatment. All patients who were randomly assigned to groups and received the study drug or placebo were included in the primary analyses. This trial was registered with ClinicalTrials.gov, number NCT02413372. FINDINGS: Between May 12, 2015, and Aug 4, 2016, 184 overweight or obese patients with non-alcoholic steatohepatitis were screened for study inclusion. Of these, 95 (52%) patients were excluded because they no longer met study criteria and 80 (43%) patients entered the placebo lead-in phase. After further exclusions, 75 (94%) patients were randomly assigned to groups, received at least one dose of treatment (25 patients to receive 10 mg pegbelfermin once a day; 24 patients to receive 20 mg pegbelfermin once a week, and 26 patients to receive placebo), and were included in the primary analysis. A prespecified interim analysis at week 8 showed a greater than expected change in the primary outcome and supported early closing of patient enrolment, since this analysis indicated that the full planned sample size was not needed. We observed a significant decrease in absolute hepatic fat fraction in the group receiving 10 mg pegbelfermin daily (-6·8% vs -1·3%; p=0·0004) and in the group receiving 20 mg pegbelfermin weekly (-5·2% vs -1·3%; p=0·008) compared with the placebo group. Most adverse events were mild; the most common events were diarrhoea in eight (16%) of 49 patients treated with pegbelfermin and two (8%) of 26 patients treated with placebo and nausea in seven (14%) patients treated with pegbelfermin and two (8%) patients treated with placebo. There were no deaths, discontinuations due to adverse events, or treatment-related serious adverse events. INTERPRETATION: Treatment with subcutaneously administered pegbelfermin for 16 weeks was generally well tolerated and significantly reduced hepatic fat fraction in patients with non-alcoholic steatohepatitis. Further study of pegbelfermin is warranted in patients with non-alcoholic steatohepatitis. Additional studies that use liver biopsies would allow for the assessment of pegbelfermin's effects on liver histology. Moreover, further studies should allow assessments of the safety and effectiveness of pegbelfermin in a larger number of patients. FUNDING: Bristol-Myers Squibb.

Current Treatment Options for Primary Biliary Cholangitis.

Primary biliary cholangitis is a progressive, autoimmune disease of the interlobular bile ducts, leading to secondary damage of hepatocytes that may progress to cirrhosis and liver failure. Until recently, the only approved treatment was ursodeoxycholic acid. However, 40% of patients do not have an adequate response. Obeticholic acid was approved for treatment as add-on therapy in this group of patients. Off-label use of fibrates has also been reported to be effective. Several new therapies are in development and may further add to the treatment options available to patients with primary biliary cholangitis.

Work in Progress: Drugs in Development.

Primary biliary cholangitis is an archetypal autoimmune disease that causes cholestasis, fibrosis, and liver failure. Ursodeoxycholic acid and obeticholic acid are approved for its treatment. Not all patients respond, some are intolerant, many have ongoing symptoms, and new therapies are required. Herein we describe drugs in development and potential future biological targets. We consider compounds acting on the farnesoid X receptor/fibroblast growth factor 19 pathway, fibrates and other agonists of the peroxisome proliferator-activated receptor family, transmembrane-G-protein-receptor-5 agonists, and several immunological agents. We also consider the roles of bile acid reuptake inhibitors, nalfurafine, and fibrates in pruritus management.

NGM282 for treatment of non-alcoholic steatohepatitis: a multicentre, randomised, double-blind, placebo-controlled, phase 2 trial.

BACKGROUND: Non-alcoholic steatohepatitis is a chronic liver disease characterised by the presence of hepatic steatosis, inflammation, and hepatocellular injury, for which no Food and Drug Administration (FDA)-approved treatment exists. FGF19 is a hormone that regulates bile acid synthesis and glucose homoeostasis. We aimed to assess the safety and efficacy of NGM282, an engineered FGF19 analogue, for the treatment of non-alcoholic steatohepatitis. METHODS: In this randomised, double-blind, placebo-controlled, phase 2 study, we recruited patients aged 18-75 years with biopsy-confirmed non-alcoholic steatohepatitis as defined by the non-alcoholic steatohepatitis clinical research network histological scoring system, from hospitals and gastroenterology and liver clinics in Australia and the USA. Key eligibility criteria included a non-alcoholic fatty liver disease activity score of 4 or higher, stage 1-3 fibrosis, and at least 8% liver fat content. Patients were randomly assigned (1:1:1) via a web-based system and stratified by diabetic status to receive either 3 mg or 6 mg subcutaneous NGM282 or placebo. The primary endpoint was the absolute change from baseline to week 12 in liver fat content. Responders were patients who achieved a 5% or larger reduction in absolute liver fat content as measured by MRI-proton density fat fraction. Efficacy analysis was by intention to treat. This trial is registered with ClinicalTrials.gov, number NCT02443116. FINDINGS: Between July 14, 2015, and Aug 30, 2016, 166 patients were screened across 18 sites in Australia and the USA. 82 patients were randomly assigned to receive 3 mg NGM282 (n=27), 6 mg NGM282 (n=28), or placebo (n=27). At 12 weeks, 20 (74%) patients in the 3 mg dose group and 22 (79%) in the 6 mg dose group achieved at least a 5% reduction in absolute liver fat content from baseline (relative risk 10·0 [95% CI 2·6-38·7] vs 11·4 [3·0-43·8], respectively; p<0·0001 for both comparisons) versus two (7%) in the placebo group. Overall, 76 (93%) of 82 patients experienced at least one adverse event, most of which were grade 1 (55 [67%]), and only five (6%) were grade 3 or worse. The most commonly (≥10%) reported adverse events were injection site reactions (28 [34%]), diarrhoea (27 [33%]), abdominal pain (15 [18%]), and nausea (14 [17%]). These adverse events were reported more frequently in the NGM282 groups compared with the placebo group. No life-threatening events or patient deaths occurred during the study. INTERPRETATION: NGM282 produced rapid and significant reductions in liver fat content with an acceptable safety profile in patients with non-alcoholic steatohepatitis. Further study of NGM282 is warranted in this patient population. FUNDING: NGM Biopharmaceuticals.

Antiangiogenic activity of semisynthetic biotechnological heparins: low-molecular-weight-sulfated Escherichia coli K5 polysaccharide derivatives as fibroblast growth factor antagonists.

OBJECTIVE: Low-molecular-weight heparin (LMWH) exerts antitumor activity in clinical trials. The K5 polysaccharide from Escherichia coli has the same structure as the heparin precursor. Chemical and enzymatic modifications of K5 polysaccharide lead to the production of biotechnological heparin-like compounds. We investigated the fibroblast growth factor-2 (FGF2) antagonist and antiangiogenic activity of a series of LMW N,O-sulfated K5 derivatives. METHODS AND RESULTS: Surface plasmon resonance analysis showed that LMW-K5 derivatives bind FGF2, thus inhibiting its interaction with heparin immobilized to a BIAcore sensor chip. Interaction of FGF2 with tyrosine-kinase receptors (FGFRs), heparan sulfate proteoglycans (HSPGs), and alpha(v)beta3 integrin is required for biological response in endothelial cells. Similar to LMWH, LMW-K5 derivatives abrogate the formation of HSPG/FGF2/FGFR ternary complexes by preventing FGF2-mediated attachment of FGFR1-overexpressing cells to HSPG-bearing cells and inhibit FGF2-mediated endothelial cell proliferation. However, LMW-K5 derivatives, but not LMWH, also inhibit FGF2/alpha(v)beta3 integrin interaction and consequent FGF2-mediated endothelial cell sprouting in vitro and angiogenesis in vivo in the chick embryo chorioallantoic membrane. CONCLUSIONS: LMW N,O-sulfated K5 derivatives affect both HSPG/FGF2/FGFR and FGF2/alpha(v)beta3 interactions and are endowed with FGF2 antagonist and antiangiogenic activity. These compounds may provide the basis for the design of novel LMW heparin-like angiostatic compounds.

Engineering of an FGF-proteoglycan fusion protein with heparin-independent, mitogenic activity.

In the absence of heparan sulfate (HS) on the surface of target cells, or free heparin (HP) in the vicinity of their receptors, fibroblast growth factor (FGF) family members cannot exert their biological activity and are easily damaged by proteolysis. This limits the utility of FGFs in a variety of applications including treatment of surgical, burn, and periodontal tissue wounds, gastric ulcers, segmental bony defects, ligament and spinal cord injury. Here we describe an FGF analog engineered to overcome this limitation by fusing FGF-1 with HS proteoglycan (PG) core protein. The fusion protein (PG-FGF-1), which was expressed in Chinese hamster ovary cells and collected from the conditioned medium, possessed both HS and chondroitin sulfate sugar chains. After fractionation, intact PG-FGF-1 proteins with little affinity to immobilized HP and high-level HS modification, but not their heparitinase or heparinase digests, exerted mitogenic activity independent of exogenous HP toward HS-free Ba/F3 transfectants expressing FGF receptor. Although PG-FGF-1 was resistant to tryptic digestion, its physiological degradation with a combination of heparitinase and trypsin augmented its mitogenic activity toward human endothelial cells. The same treatment abolished the activity of simple FGF-1 protein. By constructing a biologically active proteoglycan-FGF-1 fusion protein, we have demonstrated an approach that may prove effective for engineering not only FGF family members, but other HP-binding molecules as well.

Prevention of gastric ulcer relapse induced by indomethacin in rats by a mutein of basic fibroblast growth factor.

We found indomethacin aggravates healed gastric ulcers (ulcer relapse) in rats. In the present study, we examined the effects of human basic fibroblast growth factor (bFGF) mutein CS23 (TGP-580) and histamine H2-receptor antagonists (H2-RAs) on ulcer relapse in this model. In male SD rats, gastric ulcers were induced in the antrum by injection of acetic acid. Indomethacin (1 mg/kg/day) given s.c. for 2 weeks starting 4 weeks after the operation aggravated the healed ulcer; the areas with and without indomethacin were 4.8 +/- 1.4 and 0.4 +/- 0.3 mm2, respectively. Drugs were given orally once daily for 4 weeks starting 2 days after the operation or for the 2-week indomethacin administration period. Treatment with ranitidine (100 mg/kg), cimetidine (100 mg/kg) and TGP-580 (0.1 mg/kg) for 4 weeks accelerated the healing. The aggravation by indomethacin was significantly inhibited by pretreatment with TGP-580 and mildly inhibited by cimetidine but not ranitidine. When the drugs were co-administered with indomethacin for 2 weeks, the aggravation was significantly prevented by ranitidine and mildly inhibited by cimetidine and TGP-580. Both TGP-580 and H2-RAs can prevent the ulcer relapse induced by indomethacin but via different modes of action: TGP-580 inhibits relapse mainly by acting on the process of healing, while H2-RAs act mainly on the process of aggravation.