The Effects of B1344, a Novel Fibroblast Growth Factor 21 Analog, on Nonalcoholic Steatohepatitis in Nonhuman Primates

Nonalcoholic steatohepatitis (NASH), a more progressive type of nonalcoholic fatty liver disease (NAFLD), is characterized by >5% hepatic steatosis, inflammation caused by immune cells infiltration, and hepatocyte injury (1). NASH is associated with an increased incidence of cirrhosis, hepatocellular carcinoma, and cardiovascular disease, which largely increases the mortality rate of patients (2,3). There are currently no approved medications for treating fatty liver disease and NASH, and weight loss by lifestyle intervention is the primary way for the management of NASH (1). Although several clinical trials of pharmacotherapies for NASH treatment have emerged (4,5), the development of effective therapeutic agents is an urgent need.

Fibroblast growth factor 21 (FGF21), an important metabolic regulator, acts on the receptor complex consisting of FGF receptor (FGFR) and coreceptor βKlotho in vivo (6–8). In humans, the levels of FGF21 in circulation are increased in individuals with both NAFLD and NASH (9–11), suggesting potential roles of FGF21 in the pathogenesis and progression of fatty liver disease. Several studies have shown beneficial effects of FGF21 on the improvement of NAFLD and NASH. Administration of FGF21 attenuates hepatic steatosis and insulin resistance in mice with high-fat diet–induced obesity (12,13) and reverses methionine- and choline-deficient (MCD) diet–induced NASH in mice (13,14). In contrast, the lack of FGF21 remarkably aggravates hepatic steatosis, inflammation, fibrosis, and hepatocytes injury in MCD diet–induced NASH rodent models (14,15). Furthermore, FGF21 analogs have been reported to improve the metabolic parameters related to steatohepatitis through enhancing the fatty acid oxidation in mitochondria and decreasing the expression of interleukin 17A in T-helper 17 cells (16,17). In obese nonhuman primates, FGF21 and its analogs also lower the levels of triglycerides (TGs) in circulation and show the favorable effects on lipoprotein profiles (18,19). In a recent phase 2 clinical trial, an analog of FGF21 improved the hepatic fat fraction and circulation biomarkers of fibrosis in patients with NASH, although the effects on liver histology remain unknown (20). This evidence suggests that FGF21 and the agents mimicking its activity may be developed into pharmacotherapeutic targets for NASH treatment.

Given that the development of wild-type FGF21 as a drug is challenging due to its short half-life of ∼1 h in rodents and 0.5–2 h in nonhuman primates (18,21), many modification strategies have been developed to improve the properties of the human FGF21 protein (22). B1344 is a site-specific polyethylene glycolylated (PEGylated) human FGF21 analog with the functional domain containing β10 to β12 motifs replaced by that of mouse FGF21 and the N-terminal domain containing an alanine insertion (23). The reengineering of FGF21 leads to significantly improved biopharmaceutical properties, extended half-life and pharmacokinetics, and reduced immunogenicity (23). Administration of B1344 stimulates glucose uptake and lowers blood glucose levels and the lipid profile in a rodent model of type 2 diabetes (23,24). These in vivo and in vitro analysis have demonstrated that the biological properties of B1344 are essentially identical to human FGF21 (23).

In this study, we report beneficial effects of B1344 on lowering hepatic steatosis, fibrosis, and inflammation and on protecting against the progression of NASH in rodents and nonhuman primates. Administration of B1344 caused a profound reduction of hepatic fat content and body weight in obese cynomolgus monkeys with NAFLD. Metabolic benefits of B1344 on blood glucose, glucose tolerance, and the plasma lipid profile were also observed in the monkeys. Moreover, the anti-NASH effects were also observed in an MCD diet–induced NASH rodent model. These results demonstrate the impressive effects of B1344 on NASH and metabolic parameters and may represent novel therapeutics for treating these diseases in humans.

Male cynomolgus monkeys >8 years old were housed individually under a 12:12-h light/dark cycle at controlled temperature and humidity. The diets for primates were provided daily and water was accessed ad libitum. The monkeys were fed three meals daily containing one high-fat diet meal (46.5 g pelleted diet containing 3.81 kcal/g at 9:00 a.m.–10:00 a.m.; 150 g apples containing 0.52 kcal/g at 2:00 p.m.–3:00 p.m.; and 100 g high-fat diet containing 4.12 kcal/g at 4:00 p.m.–5:00 p.m.) during the study. The remaining food was removed and weighed after every meal for energy intake calculation.

More than 200 monkeys were initially screened, and 40 obese monkeys that met the enrollment criteria of hepatic fat content of >10% and body weight of >8 kg were acclimated for 3 weeks before B1344 administration. The monkeys were treated with 0.5 mg/kg B1344 twice weekly (BIW), 1 mg/kg B1344 BIW, 2 mg/kg B1344 BIW, 2 mg/kg B1344 once weekly (QW), or vehicle by subcutaneous injection for 11 weeks, followed by an additional 4-week washout period. Day 1 was the 1st day of dosing. Body weight was measured and blood samples were collected weekly. The liver biopsy specimens of monkeys treated with 2 mg/kg B1344 BIW and vehicle were collected on day 88. The hepatic fat fraction was measured by MRI (Siemens 3.0T MAGNETOM Verio), which was performed on all 40 monkeys as the baseline value when the monkeys were enrolled, and on the monkeys treated with 2 mg/kg B1344 BIW or vehicle on day 48 and day 69. Body mass composition was determined by DEXA (QDR Series X-Ray Bone Densitometer Discovery WI; Hologic, Bedford, MA) at the baseline and on day 45 after the first B1344 injection. During the washout period, energy intake, body weight, and other metabolic parameters were measured. The monkey study was approved by the Institutional Animal Care and Use Committee at Kunming Biomed International, Yunnan, China.

Male C57BL/6 mice (8 weeks old) were purchased from Shanghai Laboratory Animal Co. Ltd, Shanghai, China, and were housed under a 12:12-h light/dark cycle at controlled temperature. Mice were fed an MCD diet (A02082002B; Research Diets) for 2 weeks and then randomized into six groups. Mice were injected subcutaneously daily with 0.125 mg/kg or 2 mg/kg B1344, or vehicle (PBS) for 2 or 4 weeks, as indicated. The mice were euthanized, and the blood and livers were collected immediately. All mouse experimental protocols were approved by Institutional Animal Care and Use Committee at the Shanghai Institute of Nutrition and Health, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, China.

A 16–18-gauge biopsy needle was used to collect two percutaneous liver tissue samples from the monkeys under direct ultrasound guidance to ensure accurate localization and needle placement at the intended liver tissue area for biopsy. The needle was removed, and bleeding was stopped by applying pressure over the biopsy site. Liver biopsy specimens were collected in monkeys treated with 2 mg/kg B1344 BIW and vehicle on day 88 after the first injection and fixed in 10% neutral formalin for paraffin wax embedding. For mice, livers were fixed in 10% neutral formalin overnight and embedded in paraffin wax. The liver sections (5 μm) were cut and mounted on glass slides for hematoxylin and eosin (H&E) staining and Sirius Red staining, as previously described (13). The liver sections were analyzed by H&E staining, Sirius Red staining, and immunohistochemistry analysis of myeloperoxidase (MPO) and F4/80, which was described previously (13,25).

Rat L6 cells were cultured in DMEM containing 4,500 mg/L glucose supplemented with 10% FBS, 100 units/mL penicillin, and 100 μg/mL streptomycin, maintained in a humidified atmosphere of 5% CO₂ at 37°C, and passaged every 2 days by trypsinization. Cells were seeded and transfected with FLAG-tagged FGFR1c and βKlotho of human, mouse, or monkey origin using Lipofectamine 3000 transfection reagent (Life Technologies) according to the manufacturer’s protocol. Cells were treated with human FGF21 or B1344 for 25 min before collection. The whole-cell lysates were used for immunoblot analysis of phosphorylated extracellular signal–regulated kinase (ERK)1/2 and total ERK1/2. The immunoblotting analysis was performed as described previously (25,26).

Data for the monkey study are expressed as mean ± SD. One-way ANOVA or repeated-measures ANOVA was applied to analyze the difference of means, and the Fisher least significant difference method was used for post hoc comparison of means. A two-tailed α-value of 0.05 was considered significant for all statistical tests. All analyses were conducted using XLSTAT 2016 software. Data for the pharmacokinetic parameters are represented as mean ± SD. Data for the other studies are expressed as mean ± SEM. Statistical significance was evaluated using the unpaired two-tailed Student t test and among more than two groups by analysis of one-way ANOVA. Differences were considered significant at a P value of <0.05.

All data generated or analyzed during this study are included in the published article and its Supplementary Material. The data sets analyzed during the present study are available from the corresponding authors on reasonable request.

B1344 is a novel FGF21 analog that has been designed and characterized with improved biopharmaceutical and biological properties and extended half-life (23). As shown in Fig. 1A–D, to investigate the efficacies of B1344 on stimulating signaling from the βKlotho/FGFR1c receptor complex, analysis was performed in rat L6 myoblast cells that express low levels of endogenous FGFRs and do not respond to FGF treatment. Expression plasmids containing human, monkey, or mouse βKlotho and FGFR1c were cotransfected into L6 myoblast cells, followed by treatment with B1344, human FGF21, or vehicle. Phosphorylation of ERK was detected to determine the activation of βKlotho/FGFR-1c signaling. B1344 was sufficient to activate human, monkey, and mouse FGFR signaling, as evidenced by phosphorylation levels of ERK, similar to that of FGF21. Interestingly, B1344 appears to have stronger potency and higher maximal responses compared with that of FGF21. These results indicate that the long-acting PEGylated B1344 has improved ability to activate FGFR signaling and potential biological activity in vitro.

To investigate the role of B1344 on the liver, independent of obesity, the MCD diet was used to induce a lean model of fatty liver disease, which recapitulated pathologic phenotype observed in NASH but did not have body weight gain or insulin resistance. After 2 weeks of the MCD diet, mice were treated with B1344 for a short term of 14 days or a long term of 28 days, which was administered daily in a dose of 0.125 mg/kg or 2 mg/kg subcutaneously (Fig. 2A). Similar rates of body weight loss were observed in mice that received vehicle or B1344 with different time or doses (Supplementary Fig. 1). Next, to examine the progression of steatohepatitis, histological analysis of liver sections stained with H&E and Sirius Red was performed. The degree of steatosis, lobular inflammation, and hepatocellular ballooning were scored according to a criteria presented in a well-recognized grading system (27). For the short-term injection, the NASH Clinical Research Network (CRN) score in vehicle-treated mice was 6, which is considered diagnostic of NASH (27). Notably, B1344 administration was found to improve hepatic steatosis, inflammation, and ballooning, with a significantly lower CRN score in a dose-dependent manner. Moreover, Sirius Red staining revealed that B1344 treatment reversed the pericellular fibrosis (Fig. 2B and C). The alleviation of steatohepatitis exerted by B1344 administration was observed in mice that received the long-term injection, although the dosage effects were not obvious (Fig. 2D and E).

Consistent with the histological analysis, B1344 decreased the hepatic TG levels in mice treated with both short- and long-term B1344 (Fig. 3A). There were no significant differences in hepatic cholesterol content between vehicle- and B1344-treated mice (Fig. 3B). Either dose of B1344 treatment reduced circulating alanine aminotransferase (ALT) and aspartate aminotransferase (AST), which indicated the B1344 protection of hepatocyte injury (Fig. 2C and D). Strikingly, expression of genes related to hepatic inflammation, such as IL-1β, monocyte chemotactic protein 1, and tumor necrosis factor-α, was significantly reduced in the livers of mice treated with B1344. In addition, the macrophage markers CD68 and F4/80 were also decreased by B1344 treatment (Fig. 3E). These results suggested B1344 suppressed the hepatic inflammation and innate immune cells infiltration. Consistently, mRNA levels of genes involved in liver fibrosis were downregulated in B1344-treated mice, suggesting reduced collagen deposition and liver fibrosis (Fig. 3F). Notably, no obvious changes of FGFR1 or βKlotho expression levels were observed in the livers of the MCD diet-fed mice by administration of B1344 (Supplementary Fig. 1B), supporting the hypothesis of mammalian target of rapamycin complex 1 (mTORC1) inhibition in mediating the FGF21–βKlotho signal’s improvements in NASH (13). Taken together, these data indicate that B1344 treatment improves hepatic lipid accumulation, hepatocyte damage, inflammation, and fibrosis and prevents the progression of NASH in mice.

To further investigate the beneficial effects of B1344 on NASH, a study in obese male cynomolgus monkeys with NAFLD was conducted. Forty monkeys were divided into five groups based on several metabolic parameters, such as body weight, fasting blood glucose, and liver fat content. B1344 and vehicle were administered subcutaneously QW or BIW for 78 days, with the doses of 0.5 mg/kg, 1 mg/kg, and 2 mg/kg, and then the monkeys entered a washout period for 28 days (Fig. 4A). The body weight of animals administered with B1344 decreased progressively and reached the maximal reduction on day 78 after the first injection. Even the animals treated with 0.5 mg/kg showed a significant decrease in body weight by 10%. The body weight was maintained after the cession of dosing, with a slight rebound (Fig. 4B). Consistently, the body mass analyzed by the DEXA on day 45 decreased significantly in animals treated with B1344 (Fig. 4C). Compared with baseline, B1344 also caused a reduction of fat mass (Fig. 4D). Notably, a slight reduction of food intake that is presented as total energy intake was observed (Fig. 4E), which suggests that food intake was not the primary cause of the weight loss.

The fasting plasma lipid parameters were also measured once weekly. Plasma TG levels were reduced significantly and plateaued in monkeys treated with B1344 on day 8 after the first injection, although no obvious difference was observed among the monkeys treated with the four doses of B1344. The effects of B1344 on TG reduction were gradually attenuated, and TG levels reversed to baseline on day 92 during the washout period in animals that received various doses of B1344 (Fig. 4E). In addition, administration of B1344 significantly decreased VLDL cholesterol on day 8 in monkeys, and the effects were maintained until the washout period, in which VLDL levels gradually increased and returned to the basal level on day 92 (Fig. 4F). Notably, LDL cholesterol levels were not significantly changed by administration of B1344 (Supplementary Fig. 2A). Administration of 0.5 mg/kg B1344 caused a 31% increase in HDL cholesterol, whereas 1 mg/kg and 2 mg/kg dosing had little effect (Supplementary Fig. 2B).

Consistent with the previous reports (18,19,28), the plasma adiponectin level was increased significantly on day 22 in monkeys treated with B1344 at 0.5 mg/kg BIW, 1 mg/kg BIW, 2 mg/kg BIW, and 2 mg/kg QW. The effects were diminished in the washout period, and the adiponectin levels had decreased to baseline levels at the end of the study (Fig. 4H). The measurement of bone mineral density was performed by DEXA. As shown in Fig. 4I, compared with vehicle, no obvious changes of the bone mineral density were observed in monkeys treated with different doses of B1344, which is consistent with previous observation showing that FGF21 is not a major mediator for bone homeostasis in mice (29). Taken together, these data suggest B1344 treatment decreased the body weight and improved the plasma lipid profile in the cynomolgus monkeys.

To further understand the effects of B1344 on the progression of steatohepatitis in obese monkeys with NAFLD, MRI was performed to quantify the liver fat content. On the baseline images, liver fat content was ∼20% and comparable between the monkeys treated with vehicle (19.20 ± 2.51%) and 2 mg/kg BIW B1344 (19.37 ± 6.97%). Treatment with 2 mg/kg BIW significantly reduced the hepatic fat content to 11.33 ± 5.95%, a profound reduction of ∼40%, but the effects were not observed in monkeys treated with vehicle (Fig. 5A and B).

To investigate whether B1344 resulted in improved hepatic histological changes, the liver biopsy specimens were taken from monkeys treated with 2 mg/kg BIW or vehicle on day 88 after the first injection. The hepatic sections were stained with H&E for histological analysis, and the degree of steatosis, inflammation, and fibrosis was scored according to the standard criteria for evaluation of NAFLD activity and NASH progression (Fig. 5C–E). Consistent with the MRI results, administration of 2 mg/kg BIW decreased the scores of hepatic steatosis significantly compared with monkeys treated with vehicle. Unlike the NASH mouse model, B1344 treatment showed moderate effects on improving inflammation and hepatocyte ballooning in obese monkeys with NAFLD. However, treatment with 2 mg/kg BIW decreased the scores of fibrosis remarkably, which corresponded to the results of Sirius Red staining of liver sections. These data provide direct evidence that B1344 is capable of preventing the progression of NASH and especially improving hepatic lipid accumulation and fibrosis in cynomolgus monkeys with NAFLD.

Because the inflammation is a key event that leads to the development of NASH and many drugs in phase 3 trials or those with the results from phase 2 trials target inflammation in the liver (4,5), further studies were performed to evaluate the level of inflammation and degree of hepatocyte injury. Hepatic neutrophil and macrophage infiltration represent inflammation in NASH. Expression levels of MPO, a neutrophil marker, were determined in hepatic sections with immunohistochemistry analysis (Fig. 6A). Although the infiltration of neutrophils was low in the liver of monkeys, administration of 2 mg/kg BIW B1344 caused a remarkable reduction of infiltration of MPO-positive neutrophils. The macrophage marker F4/80 was used to distinguish the macrophages that migrated into the liver (Fig. 6B). Consistent with the neutrophils, 2 mg/kg BIW dosing prevented infiltration of macrophages, even though the hepatic inflammation was at a low grade in the cynomolgus monkeys with NAFLD. No discernible differences were found in circulating ALT and AST after 78 days of B1344 treatment (Fig. 6C and D), which is probably because of the mild hepatocyte damage at baseline in the obese monkeys.

The plasma B1344 concentration-time profile of the studied doses on day 1 and day 78 after the first injection are presented in Fig. 7A and B. In the day 1 profile, monkeys treated with 0.5 mg/kg BIW, 1 mg/kg BIW, and 2 mg/kg BIW were administered B1344 at 120 h for the second time, and the data collected from those three groups of monkeys at 120 h and 168 h were not used for pharmacokinetic analysis. The mean pharmacokinetic parameters are summarized in Table 1. After the first injection, peak concentrations were observed at 16–27 h post dose. Then, the B1344 concentrations declined in parallel across the 0.5 mg/kg to 2 mg/kg dose range. The maximum plasma concentration (C_max) and area under the curve from time 0 to infinity (AUC_0–∞) increased proportionally with the dose increase. The mean values for B1344 clearance ranged from 3.82 to 9.19 mL/h/kg, and the terminal elimination half-life (t_1/2) ranged from 17.6 to 50.9 h. After the injection on day 78, peak concentrations were reached at 7–27 h, which were consistent with the time to reach the maximum concentration (t_max) after the first administration. However, the C_max was higher than that observed after the day 1 injection, which ranged from 4- to 28-fold across doses. The C_max and AUC_0–∞ were increased as the dose increased, except for monkeys treated with 2 mg/kg BIW, which showed an abnormally low level. The systemic clearance ranged from 0.12 to 1.55 mL/h/kg, which was lower than the first administration. The terminal t_1/2 was approximately twofold longer than the first administration, which ranged from 58.6 to 106 h.

The effects of B1344 on glucose metabolism in cynomolgus monkeys were also tested, and the fasting blood glucose, hemoglobin A_1c (HbA_1c), and insulin were monitored weekly throughout the treatment period (Supplementary Table 1). Consistent with the previous reports (18,28,30), B1344 administration decreased fasting blood glucose after 8 days of treatment, and 1 mg/kg BIW and 2 mg/kg BIW dosing decreased it significantly (Supplementary Fig. 3A). Compared with monkeys treated with vehicle, HbA_1c was remarkably reduced by B1344 after 22 days after the first injection and was maintained at a lower level until the end of the washout period (Supplementary Fig. 3B). Administration of 1 mg/kg BIW, 2 mg/kg BIW, and 2 mg/kg QW B1344 for 78 days also decreased the fasting insulin levels (Supplementary Fig. 3C), which suggested a potential improvement of insulin sensitivity by B1344. Moreover, to investigate effects of B1344 on systemic glycemic homeostasis, intravenous glucose tolerance tests were performed after 45 days of treatment. Compared with vehicle, monkeys treated with various doses of B1344 showed improvement in glucose clearance (Supplementary Fig. 3D and E). Interestingly, as shown in Supplementary Fig. 3F and G, administration of B1344 did not cause an induction of insulin levels at the first phase after the glucose injection, suggesting an insulin-sensitizing effect of B1344 on lowering blood glucose instead of stimulating insulin secretion. These results are consistent with previous observations showing improved insulin-sensitizing effects of FGF21 (12,13,31–33). These results suggest insulin-sensitizing and glycemic-control efficacies of B1344 in cynomolgus monkeys with NAFLD.

The current study reports that 2 mg/kg B1344 treatment twice a week decreased the hepatic fat fraction and alleviated fibrosis and innate immune cells infiltration in livers in cynomolgus monkeys with ∼20% hepatic fat content. Moreover, administration of B1344 at 0.125 mg/kg or 2 mg/kg once daily significantly improved hepatic steatosis and attenuated hepatocyte injury, inflammation, and fibrosis in MCD diet–fed mice with NASH. Overall, B1344, the novel FGF21-based candidate, has potent effects on preventing the progression of NASH in mice and nonhuman primates.

One of the most important findings is the translation of B1344s effects on lowering NASH in nonhuman primate species. The salutary effects of B1344 on liver metabolism are likely independent of body weight loss. First, the liver fat reduction was not correlated with body weight loss in monkeys treated with B1344 at 2 mg/kg BIW. The monkeys that lost the least body weight showed a greater reduction of hepatic fat content compared with the monkeys that lost the most body weight (data not shown). Moreover, B1344 protects against steatohepatitis in mice fed the MCD diet that does not cause body weight gain, which is consistent with the previous observations in MCD diet–fed mice treated with FGF21 (13,14). Future studies are needed to investigate the effects of B1344 on NASH phenotypes in mice fed NASH diets, such as a high fat, fructose, and cholesterol diet (34–36). Together, these results further support an improvement of liver metabolism by B1344 independent of body weight loss.

Although the food intake was slightly decreased by B1344 in the dose of 0.5 mg/kg BIW, there is no apparent dose response because no obvious changes were observed in monkeys treated with other doses of 1 mg/kg BIW, 2 mg/kg BIW, and 2 mg/kg QW. These results suggest that reduction of food intake is not solely responsible for body weight loss, supporting the potential action of B1344 in increasing energy expenditure. These results are also consistent with previous observations showing enhanced energy expenditure in rodents or monkeys by FGF21 or its analog (12,37,38). Future investigations are needed to determine the relative contribution of energy expenditure and food intake to the body weight-lowering effects of B1334 in nonhuman primates, for example, by pair-feeding the control animal to exactly match the food intake of monkeys treated with B1344.

The impact of B1344 treatment in obese monkeys on lowering circulating TG and VLDL cholesterol occurred and reached the maximum effect after 7 days of treatment, which is correlated with an induction of HDL cholesterol levels. The findings that LDL cholesterol levels were not significantly changed by administration of B1344 is consistent with previous findings of FGF21 or its analogs in obese rhesus or cynomolgus monkeys (19,30). Although administration of FGF21 appears to cause a reduction of LDL cholesterol levels in diabetic cynomolgus monkeys (18), the differences may be attributed to the pharmacokinetic properties of molecules or the heterogeneity of nonhuman primates. Overall, administration of B1344 appears to improve the lipid profiles in obese monkeys. Given that dyslipidemia management is important in the clinic for the improvement of NASH patients (2), B1344 may have the potential for treating NASH.

Administration of B1344 is correlated with an induction of circulating adiponectin, which is consistent with previous reports in animal models and clinical trials (19,39–42). Whether adiponectin mediates the metabolic effects of FGF21 is controversial, however, because administration of FGF21 to adiponectin ^−/− mice resulted in the same effects on improving insulin sensitivity and energy expenditure compared with wild-type mice (43). Recent studies showed that FGF21 acts as an inhibitor of the mTORC1 complex and functions as an autocrine/paracrine in the modulation of hepatic insulin sensitivity and glucose homeostasis (13,44). Future studies are needed to determine the downstream targets that directly mediate the anti-NASH effects of FGF21 or B1344 in the liver. It will also be interesting to investigate the potential roles of B1344 in the central nervous system by the measurement of cerebrospinal fluid and its effects on circulating corticosterone levels in future studies.

To our knowledge, this is the first study to assess FGF21 analog administration for treating NASH in nonhuman primate species that underwent liver biopsy. The efficacies of B1344 on lowering NASH are consistent with the recent clinical trial showing pegbelfermin, a PEGylated human FGF21 analog, reduced the hepatic fat fraction in patients with NASH (20). Notably, the absence of weight loss was observed, and examination of liver biopsy specimens was not included in that trial. Consistent with the previous observations in monkey and human studies (18,19,42), B1344 administration was associated with a substantial reduction of body weight and improvements of the lipid profile in monkeys with NAFLD. Given the prolonged half-life and preservation time and improved ability to activate FGFR signaling, it is possible that therapeutic effects of B1344, the PEGylated human FGF21 analog, is superior to native FGF21 in improving biopharmaceutical properties, including liver metabolism. This is supported by the findings that B1344 was more potent in inducing glucose uptake in vitro and alleviating hyperglycemia in db/db mice compared with the wild-type human FGF21 (23). Future studies are needed to demonstrate this hypothesis. Importantly, B1344 has shown reduced immunogenicity, as demonstrated by reduced expression of anti-modified FGF21 IgG levels in rabbits (23). The remarkable efficacy and safety of B1344 in cynomolgus monkeys have been evidenced in the study. Future clinical studies are needed to evaluate the efficacy, safety, and immunogenicity of B1344 in humans. Taken together, these results suggest that B1344 may have superior effects on improvement of liver metabolism and metabolic parameters and likely represents an effective and safe therapeutic strategy for treating NASH and related metabolic diseases.

In summary, as evidenced by the histological analysis of the liver biopsy specimens, our study shows the robust effects of B1344 on the improvement of NASH progression. The hepatic fat fraction, inflammation, and fibrosis are significantly alleviated by B1344 in rodents and nonhuman primates, which suggest that B1344 exerts its beneficial effects on parameters relative to NASH. This study provides evidence to support the conclusion that B1344 may be a potential candidate for NASH treatment and warrants further discoveries and development of B1344 in the clinic.

Acknowledgments. The authors are grateful to Chen Chen, Guoyong Jia, and Ruijing Huang (Tasly Pharmaceutical, Tianjin, China) for formulating B1344 samples. The authors thank Zhonghui Weng (Shanghai Institute of Nutrition and Health, Chinese Academy of Sciences) for animal studies.

Funding. This work was supported in part by grants from the National Key R&D Program of China (2019YFA0802502), National Natural Science Foundation of China (81925008), and Strategic Priority Research Program of the Chinese Academy of Sciences (XDA12030210) and by grants from National Science and Technology Major Projects (2019ZX09201001-001-001) to J.G. and the Shanghai Science and Technology Commission (19140903300) to Y.L.

Duality of Interest. This study was funded in part by Tasly Pharmaceutical Co., Ltd. No other potential conflicts of interest relevant to this article were reported.

Author Contributions. A.C., J.L., X.M., and Y.L. contributed to experiment design. A.C., S.J., F.M., G.W., P.T., T.W., and J.C. contributed to the acquisition and analysis of data. A.C., X.M., and Y.L. wrote the manuscript. Y.X. and Z.L. reviewed the manuscript. J.G., X.M., and Y.L. obtained the funding. J.H. provided reagents and material support. X.M. and Y.L. are the guarantors of this work and, as such, had full access to all the data in the study and take responsibility for the integrity of the data and the accuracy of the data analysis.

Prior Presentation. Parts of this study were presented in abstract form at the at the 80th Scientific Sessions of the American Diabetes Association, 12–16 June 2020.