GDF15 analogs and methods for reducing body weight and / or reducing food intake

Abstract

The present invention relates to GDF15 analogs and methods for reducing body weight and / or reducing food intake. In particular, the present invention relates to fusion proteins comprising a half-life prolonging protein, a linker and a GDF15 protein, which fusion proteins are useful as GDF15 agonists. These GDF15 agonists are useful for treating obesity, reducing body weight, reducing food intake, or reducing appetite.

Description

[0001] This application is a divisional application of the original patent application filed on November 19, 2019, with application number 201980089716.4 (international application number PCT / IB2019 / 059945) and entitled "GDF15 analogues and methods for reducing weight and / or reducing food intake". Technical Field

[0002] This invention relates to GDF15 fusion proteins. Specifically, this invention relates to fusion proteins comprising a half-life-extending protein, a linker, and a GDF15 protein, nucleic acids encoding the fusion protein, expression vectors, recombinant cells thereof, and pharmaceutical compositions comprising the fusion protein. Methods for using said fusion protein to reduce body weight and / or decrease food intake are provided. Background Technology

[0003] GDF15, a member of the TGFβ family, is a secreted protein that circulates as a 25 kDa homodimer in plasma. In most individuals, plasma GDF15 levels range between 150 pg / ml and 1150 pg / ml (Tsai et al., J Cachexia Sarcopenia Muscle. 2012, 3:239-243). High plasma GDF15 levels are associated with weight loss due to anorexia and cachexia due to cancer, renal failure, and heart failure. In clinical trials, GDF15 levels were an independent predictor of insulin resistance in obese non-diabetic subjects (Kempf et al., Eur. J. Endo. 2012, 167:671–678). Studies on twins have shown that differences in GDF15 levels within twins are associated with differences in BMI within those twins, suggesting that GDF15 acts as a long-term regulator of energy homeostasis (Tsai et al., PLoS One, 2015, 10(7):e0133362).

[0004] Numerous reports have demonstrated improved glucose tolerance and insulin sensitivity in mouse models following treatment with GDF15 protein. Transgenic mice in two separate strains overexpressing GDF15 showed reduced body weight and fat mass, and improved glucose tolerance (Johnen et al., Nat. Med. 2007, 13: 1333-1340; Macia et al., PLoS One. 2012, 7: e34868; Chrysovergis et al., Int. J. Obesity. 2014, 38: 1555-1564). Increased systemic energy expenditure and oxidative metabolism have been reported in GDF15 transgenic mice (Chrysovergis et al., 2014, Id.). These are accompanied by increased expression of thermogenic genes in brown adipose tissue and increased expression of lipolysis genes in white adipose tissue. Mice lacking the GDF15 gene showed increased body weight and fat mass (Tsai et al., PLoS One. 2013, 8(2): e55174). When the Fc fusion of GDF15 was administered weekly to an obese cynomolgus model over a six-week period, the Fc fusion of GDF15 was shown to reduce body weight and improve glucose tolerance and insulin sensitivity in the obese cynomolgus model (WO2013 / 113008).

[0005] It is believed that the effects of GDF15 on body weight may be mediated by reducing food intake and potentially increasing energy expenditure. GDF15 may improve glycemic control through both weight-dependent and potentially non-dependent mechanisms.

[0006] In summary, these observations suggest that increased GDF15 levels may be beneficial as a therapy for metabolic diseases. There is a need in the art for GDF15-based compositions that can be used to treat or prevent metabolic diseases, disorders, or conditions.

[0007] Current treatments for obesity include dietary and behavioral interventions, pharmacological therapy, and bariatric surgery. Lifestyle interventions (including diet and increased physical activity) form the basis of any weight loss effort and can be effective in achieving weight loss in the short term (3 to 6 months). However, in most cases, weight loss achieved through lifestyle interventions is not sustainable in the long term, and only 5%–10% of individuals are able to maintain significant weight loss over time (Fisher BL and Schauer P., Am J Surg. 2002; 184:9S–16S; Rueda-Claussen CF et al., Annu. Rev. Nutr. 2015; 35:475–516). When lifestyle changes are ineffective in achieving significant weight loss, pharmacological therapy is recommended as a second-line treatment. Medications approved for chronic weight management in the US and EU include orlistat (a gastrointestinal lipase inhibitor), naltrexone / bupropion (a combination of an opioid antagonist and dopamine and norepinephrine reuptake inhibitors), and liraglutide (a glucagon-like peptide-1 receptor agonist); lorcaserin (a selective 5-HT2C receptor agonist) and phentermine / topiramate (a combination of a sympathomimetic agonist and an antiepileptic agent) are also available in the US. Additionally, phentermine, along with several other anorexia nervosa medications (including amphetamine, benzphenamine, and benzodiazepine), are registered in the US for short-term use (up to 12 weeks). In combination with behavioral interventions, these pharmacological agents have variable efficacy, resulting in additional weight loss ranging from 2% to 10% of initial body weight. Furthermore, the use of pharmacological agents may be limited by side effects, including gastrointestinal effects (i.e., nausea, vomiting, flatulence, diarrhea), neuropsychiatric effects (i.e., cognitive impairment, disordered sleep), and increased heart rate (depending on the specific agent). Due to these inherent limitations of available pharmacological approaches (limited efficacy, safety profile, and a non-responder rate ranging from 30% to 65%), there is an unmet medical need for more effective, well-tolerated, and safer pharmacological therapies for obesity that can also improve obesity-related comorbidities such as cardiovascular disease, type 2 diabetes, and hypertension. While bariatric surgery (gastric banding, sleeve gastrectomy, and Roux-en-Y gastric bypass) can result in greater and more lasting weight loss than medical therapies (weight loss ranging from approximately 15% to 30% after 10 years) and can produce significant health improvements and reduce mortality in patients with severe obesity, perioperative complications (e.g., venous thromboembolism) and postoperative complications (e.g., nausea, dumping syndrome, malabsorption of fat-soluble vitamins) are possible. Furthermore, given the limitations of both cost and surgical capacity in most healthcare systems, bariatric surgery may only be suitable for a small percentage of eligible patients (Rueda-Claussen CF et al., Annu. Rev. Nutr. 2015; 35:475-516).Therefore, there is a need for more effective and well-tolerated long-term weight management therapies that may also positively impact obesity-related comorbidities such as hypertension, dyslipidemia, and type 2 diabetes. Summary of the Invention

[0008] This invention addresses this need by providing the GDF15 agonist FP2, which represents a novel mechanism of action for reducing food intake and achieving weight loss. In non-clinical pharmacology and safety studies, FP2 has demonstrated beneficial pharmacological effects and promising safety profiles, qualifying it as a candidate for transition to clinical development.

[0009] In one aspect, the present invention provides a method for reducing the weight of a subject, the method comprising administering a composition comprising a fusion protein containing SEQ ID NO:92 and at least one pharmaceutically acceptable carrier or diluent, wherein the fusion protein is administered in a dose ranging from about 0.8 mg to about 90 mg, and wherein the subject's weight is about 80 kg or higher. In one aspect of the invention, the subject is overweight. In one aspect of the invention, the subject has a weight of about 25 kg / m². 2 Or a higher BMI. In one aspect of the invention, the subject has a BMI of 25 kg / m². 2 Up to 29.9 kg / m 2 The BMI range. In one aspect of the invention, the fusion protein is administered at a dose selected from the following: about 0.8 mg, about 2.5 mg, about 7.5 mg, about 15 mg, about 30 mg, about 60 mg, and about 90 mg. In one aspect of the invention, the fusion protein is administered at a dose range from about 0.01 mg / kg to about 1.08 mg / kg. In one aspect of the invention, the fusion protein is administered at a dose selected from the following: about 0.01 mg / kg, about 0.03 mg / kg, about 0.09 mg / kg, about 0.18 mg / kg, about 0.36 mg / kg, about 0.72 mg / kg, and about 1.08 mg / kg.

[0010] In one aspect of the invention, the fusion protein is administered via subcutaneous injection.

[0011] In one aspect of the invention, the fusion protein is administered to the subject once a week.

[0012] In one aspect, the present invention provides a method for reducing food intake in a subject, the method comprising administering a composition comprising a fusion protein containing SEQ ID NO:92 and at least one pharmaceutically acceptable carrier or diluent, wherein the fusion protein is administered at a dose ranging from about 0.8 mg to about 90 mg, and wherein the subject's weight is 80 kg or higher. In one aspect of the invention, the subject is overweight. In one aspect of the invention, the subject has a weight of 25 kg / m². 2 Or a higher BMI. In one aspect of the invention, the subject has a BMI of 25 kg / m². 2 Up to 29.9 kg / m 2 The BMI range. In one aspect of the invention, the fusion protein is administered at a dose selected from the following: about 0.8 mg, about 2.5 mg, about 7.5 mg, about 15 mg, about 30 mg, about 60 mg, and about 90 mg. In one aspect of the invention, the fusion protein is administered at a dose range from about 0.01 mg / kg to about 1.08 mg / kg. In one aspect of the invention, the fusion protein is administered at a dose selected from the following: about 0.01 mg / kg, about 0.03 mg / kg, about 0.09 mg / kg, about 0.18 mg / kg, about 0.36 mg / kg, about 0.72 mg / kg, and about 1.08 mg / kg.

[0013] In one aspect of the invention, the fusion protein is administered via subcutaneous injection.

[0014] In one aspect of the invention, the fusion protein is administered to the subject once a week.

[0015] The present invention also provides the following solutions.

[0016] 1. A method for reducing the weight of a subject, the method comprising administering a composition comprising a fusion protein containing SEQ ID NO:92 and at least one pharmaceutically acceptable carrier or diluent, wherein the fusion protein is administered at a dose ranging from 0.8 mg to 90 mg, and wherein the subject's weight is 80 kg or higher.

[0017] 2. The method according to scheme 1, wherein the subject is overweight.

[0018] 3. The method according to scheme 2, wherein the subject has a BMI of 25 kg / m2 or greater.

[0019] 4. The method according to scheme 3, wherein the subject has a BMI in the range of 25 kg / m2 to 29.9 kg / m2.

[0020] 5. The method according to Scheme 1, wherein the fusion protein is administered in a dose selected from the following: 0.8 mg, 2.5 mg, 7.5 mg, 15 mg, 30 mg, 60 mg and 90 mg.

[0021] 6. The method according to Scheme 5, wherein the fusion protein is administered at a dose of 0.8 mg.

[0022] 7. The method according to Scheme 5, wherein the fusion protein is administered at a dose of 2.5 mg.

[0023] 8. The method according to Scheme 5, wherein the fusion protein is administered at a dose of 7.5 mg.

[0024] 9. The method according to Scheme 5, wherein the fusion protein is administered at a dose of 15 mg.

[0025] 10. The method according to Scheme 5, wherein the fusion protein is administered at a dose of 30 mg.

[0026] 11. The method according to Scheme 5, wherein the fusion protein is administered at a dose of 60 mg.

[0027] 12. The method according to Scheme 5, wherein the fusion protein is administered at a dose of 90 mg.

[0028] 13. The method according to Scheme 1, wherein the fusion protein is administered via subcutaneous injection.

[0029] 14. The method according to Scheme 1, wherein the composition is administered to the subject once a week.

[0030] 15. A method for reducing food intake of a subject, the method comprising administering a composition comprising a fusion protein containing SEQ ID NO:92 and at least one pharmaceutically acceptable carrier or diluent, wherein the fusion protein is administered at a dose ranging from 0.8 mg to 90 mg, and wherein the subject weighs 80 kg or more.

[0031] 16. The method according to scheme 15, wherein the subject is overweight.

[0032] 17. The method according to scheme 16, wherein the subject has a weight of 25 kg / m² or greater.

[0033] BMI.

[0034] 18. The method according to scheme 17, wherein the subject has a BMI in the range of 25 kg / m2 to 29.9 kg / m2.

[0035] 19. The method according to Scheme 15, wherein the fusion protein is administered in a dose selected from the following: 0.8 mg, 2.5 mg, 7.5 mg, 15 mg, 30 mg, 60 mg and 90 mg.

[0036] 20. The method according to scheme 19, wherein the fusion protein is administered at a dose of 0.8 mg.

[0037] 21. The method according to scheme 19, wherein the fusion protein is administered at a dose of 2.5 mg.

[0038] 22. The method according to scheme 19, wherein the fusion protein is administered at a dose of 7.5 mg.

[0039] 23. The method according to scheme 19, wherein the fusion protein is administered at a dose of 15 mg.

[0040] 24. The method according to scheme 19, wherein the fusion protein is administered at a dose of 30 mg.

[0041] 25. The method according to scheme 19, wherein the fusion protein is administered at a dose of 60 mg.

[0042] 26. The method according to scheme 19, wherein the fusion protein is administered at a dose of 90 mg.

[0043] 27. The method according to Scheme 15, wherein the fusion protein is administered via subcutaneous injection.

[0044] 28. The method according to scheme 15, wherein the composition is administered to the subject once a week.

[0045] 29. A method for reducing the weight of a subject, the method comprising administering a composition comprising a fusion protein containing SEQ ID NO:92 and at least one pharmaceutically acceptable carrier or diluent, wherein the composition is administered at a dose in the range of 0.01 mg / kg to 1.08 mg / kg.

[0046] 30. The method according to claim 29, wherein the composition is administered in a dose selected from the following: 0.01 mg / kg, 0.03 mg / kg, 0.09 mg / kg, 0.18 mg / kg, 0.36 mg / kg,

[0047] 0.72 mg / kg and 1.08 mg / kg.

[0048] 31. The method according to Scheme 29, wherein the composition is administered at a dose of 0.01 mg / kg.

[0049] 32. The method according to Scheme 29, wherein the composition is administered at a dose of 0.03 mg / kg.

[0050] 33. The method according to Scheme 29, wherein the composition is administered at a dose of 0.09 mg / kg.

[0051] 34. The method according to Scheme 29, wherein the composition is administered at a dose of 0.18 mg / kg.

[0052] 35. The method according to Scheme 29, wherein the composition is administered at a dose of 0.36 mg / kg.

[0053] 36. The method according to Scheme 29, wherein the composition is administered at a dose of 0.72 mg / kg.

[0054] 37. The method according to Scheme 29, wherein the composition is administered at a dose of 1.08 mg / kg.

[0055] 38. The method according to Scheme 29, wherein the fusion protein is administered via subcutaneous injection.

[0056] 39. The method according to scheme 29, wherein the composition is administered to the subject once a week.

[0057] 40. A method for reducing food intake in a subject, the method comprising administering a composition comprising a fusion protein containing SEQ ID NO:92 and at least one pharmaceutically acceptable carrier or diluent, wherein the composition is administered at a dose ranging from 0.01 mg / kg to 1.08 mg / kg.

[0058] 41. The method according to claim 40, wherein the composition is administered in a dose selected from the following: 0.01 mg / kg, 0.03 mg / kg, 0.09 mg / kg, 0.18 mg / kg, 0.36 mg / kg,

[0059] 0.72 mg / kg and 1.08 mg / kg.

[0060] 42. The method according to Scheme 41, wherein the composition is administered at a dose of 0.01 mg / kg.

[0061] 43. The method according to Scheme 41, wherein the composition is administered at a dose of 0.03 mg / kg.

[0062] 44. The method according to Scheme 41, wherein the composition is administered at a dose of 0.09 mg / kg.

[0063] 45. The method according to Scheme 41, wherein the composition is administered at a dose of 0.18 mg / kg.

[0064] 46. ​​The method according to Scheme 41, wherein the composition is administered at a dose of 0.36 mg / kg.

[0065] 47. The method according to Scheme 41, wherein the composition is administered at a dose of 0.72 mg / kg.

[0066] 48. The method according to Scheme 41, wherein the composition is administered at a dose of 1.08 mg / kg.

[0067] 49. The method according to scheme 41, wherein the fusion protein is administered via subcutaneous injection.

[0068] 50. The method according to scheme 41, wherein the composition is administered to the subject once a week.

[0069] Other aspects, features, and advantages of the invention will become apparent from the following disclosure, including a detailed description of the invention and its preferred embodiments, as well as the appended claims. Attached Figure Description

[0070] The above-described invention and the following detailed description of the invention will be better understood when read in conjunction with the accompanying drawings. It should be understood that the invention is not limited to the precise embodiments shown in the drawings.

[0071] In the attached diagram:

[0072] Figure 1A and Figure 1B The crystal structure of GDF15 is shown, in which the disulfide pairing of the first cysteine ​​residue and the second cysteine ​​residue (C1-C2) forms a loop at the N-terminus of the protein.

[0073] Figure 2 The effect of subcutaneous administration of fusion proteins according to embodiments of the invention (e.g., fusion proteins FP1 (SEQ ID NO: 60) and 6xHis-FP1 (SEQ ID NO: 26 with a 6xHis tag attached to the N-terminus)) on food intake in C57BL / 6 mice is illustrated, depicting cumulative food intake at 24 hours post-administration. Values ​​shown in bars are percentage reductions compared to the solvent (PBS) group ± SEM; except in the FP1 16 nmol / kg group where N = 9, N = 8 animals per group for all groups. * - p < 0.05, compared to solvent; p-values ​​were calculated using one-way ANOVA and Tukey's multiple comparison test.

[0074] Figure 3The effects of subcutaneous administration of FP1 and 6xHis-FP1 (SEQ ID NO:26 with a 6xHis tag at the N-terminus) on food intake in Sprague-Dawley rats are shown, depicting cumulative food intake at 48 hours post-administration. Values ​​shown in bars are percentage reductions compared to the solvent (PBS) group ± SEM; N = 8 animals per group. * - p < 0.05, compared to solvent; p-values ​​were calculated using one-way ANOVA and Tukey's multiple comparison test.

[0075] Figure 4 The changes in body weight in diet-induced obese (DIO) mice during FP1 treatment are shown. Arrows indicate the time (days) after subcutaneous administration following the initial dose (day 0); N = 8 animals per group. * - p < 0.05, for the FP1 1 nmol / kg group compared to the solvent; # - p < 0.05, for the FP1 10 nmol / kg group compared to the solvent; p-values ​​were calculated using two-way RM ANOVA and Tukey's multiple comparison test.

[0076] Figure 5A and Figure 5B Blood glucose levels in DIO mice during an oral glucose tolerance test (OGTT) 14 days after administration of FP1 every 3 days (q3d) are shown, expressed as area under the curve. N = 8 animals per group. * - p < 0.05, relative to the solvent in the FP1 1 nmol / kg group; p-values ​​were calculated using one-way ANOVA and Tukey's multiple comparison test.

[0077] Figure 6 The ingested blood glucose levels of DIO mice during FP1 treatment are shown. N = 8 animals per group. * - p < 0.05, compared to solvent; p-values ​​were calculated using two-dimensional RM ANOVA and Tukey's multiple comparison test.

[0078] Figure 7 This paper presents an assessment of a 4-hour fasting homomorphic regulation model of insulin resistance (HOMA-IR) in DIO mice after 14 days of FP1 treatment. N = 8 animals per group. *-p<0.05, compared to solvent; p-values ​​were calculated using one-way ANOVA and Tukey's multiple comparison test.

[0079] Figure 8The changes in body weight of ob / ob mice during treatment with FP1 every 3 days (qd3) are shown. Arrows indicate the time (days) after subcutaneous administration following the initial dose (day 0); N = 9 animals per group. * - p < 0.05, for the FP1 10 nmol / kg group compared to the solvent; # - p < 0.05, for the FP1 1 nmol / kg group compared to the solvent; p-values ​​were calculated using two-way RM ANOVA and Tukey's multiple comparison test.

[0080] Figure 9 Blood glucose levels in ob / ob mice during FP1 treatment are shown. Arrows indicate the time (days) after subcutaneous administration following the initial dose (day 0); N = 9 animals per group. * - p < 0.05, for the FP1 10 nmol / kg group compared to the solvent; # - p < 0.05, for the FP1 1 nmol / kg group compared to the solvent; p-values ​​were calculated using two-way RM ANOVA and Tukey's multiple comparison test.

[0081] Figure 10 The mean (± standard deviation, SD) of serum drug concentration-time distribution of FP1 after intravenous (IV) and subcutaneous (SC) administration of 2 mg / kg in C57Bl / 6 mice is shown.

[0082] Figure 11 The mean (±SD) serum drug concentration-time distribution of FP1 following IV and SC administration of 2 mg / kg in Sprague-Dawley rats is shown.

[0083] Figure 12 The mean (±SD) serum drug concentration-time distribution of FP1 following IV and SC administration of 1 mg / kg in cynomolgus monkeys is shown, as determined by immunoassay.

[0084] Figure 13 Serum concentrations (ng / mL) of FP1 as a complete dimer over time following a single IV administration in cynomolgus monkeys are shown, as determined by immunoaffinity (IA) capture LCMS analysis.

[0085] Figure 14 Serum concentrations (ng / mL) of FP1 as a intact dimer over time following a single SC administration in cynomolgus monkeys are shown, as determined by immunoaffinity capture LCMS analysis.

[0086] Figure 15 The concentrations of FP1 in plasma obtained from two human subjects (Sub) after in vitro incubation for 0, 4, 24, and 48 hours, as determined by immunoassay, are shown and expressed as a percentage of the initial concentration.

[0087] Figure 16 The average concentration of FP1 as a complete dimer in plasma obtained from two human subjects (Sub) after in vitro incubation for 0, 4, 24 and 48 hours is shown as a percentage of time 0, as determined by intact mass immunoaffinity capture LCMS analysis.

[0088] Figure 17 Acute food intake in lean C57BL6N male mice before and after administration of various N-terminal deletion variants of GDF15 is shown. (SEQ ID NO: 92, 111, and 112, compared to wild-type fusions without deletion (SEQ ID 26 with a 6xHis tag at the N-terminus)). N = 8 animals per group; *-p < 0.05, compared to solvent; p-values ​​were calculated using two-way RMANOVA and Tukey's multiple comparison test.

[0089] Figure 18 The effect of a single dose of FP2 on food intake in C57BL / 6 mice is shown; specifically, cumulative food intake at 24 hours post-administration is shown. Values ​​shown in bars are percentage reductions relative to the PBS group (mean ± SEM); N = 8 animals per group for all groups except in 6xHis-FP1 where N = 6. **-p < 0.01, ***-p < 0.001, ****-p < 0.0001; p-values ​​were calculated using two-way ANOVA and Dunnetts' multiple comparison test.

[0090] Figure 19 Cumulative food intake measured in Sprague-Dawley rats 24 hours after administration of a single dose of FP2 is shown. Values ​​shown in bars are percentage reductions (mean ± SEM) compared to the PBS group; N = 8 animals per group. **-p < 0.01, p-values ​​were calculated using two-way ANOVA and Tukey's multiple comparison test.

[0091] Figure 20 The percentage change in body weight of DIO mice during treatment with FP2 at q3d is shown. Arrows indicate the time of subcutaneous FP2 injection; N = 6 animals per group; *-p, 0.05, p-values ​​were calculated using two-way ANOVA and Tukey's multiple comparison test compared to the solvent;

[0092] Figure 21A and Figure 21BThe area under the curve (AUC) for blood glucose concentration levels in DIO mice during the OGTT test 14 days after FP2 administration on a q3d basis is shown. *-p<0.05, using one-way ANOVA and Tukey's multiple comparison test, with n=8 animals per group.

[0093] Figure 22A Plasma insulin levels in DIO mice during the OGTT are shown 8 days after administration of FP2 on a q3d basis. *-p<0.05, solvents compared to FP2 (0.3 nmol / kg); FP2 (10 nmol / kg); and rosiglitazone. #-p<0.05, compared to rosiglitazone (10 mg / kg), using two-dimensional RM ANOVA and Tukey's multiple comparison test.

[0094] Figure 22B The AUCs for plasma insulin levels in DIO mice during the OGTT are shown after 8 days of FP2 administration on a q3d basis. * - p < 0.05, compared to the solvent; # - p < 0.05, compared to rosiglitazone.

[0095] Figure 23 The ingested blood glucose levels in DIO mice were shown 8 days after administration of FP2 on a q3d basis. *-p<0.05, compared to the solvent, using two-dimensional RM ANOVA and Tukey's multiple comparison test, n=8 animals per group.

[0096] Figure 24 Fasting HOMA-IR in DIO mice after 14 days of treatment with FP2 on a q3d basis followed by a 5-hour fast on day 14 is shown. *-p<0.05, compared to solvent, using one-way ANOVA and Tukey's multiple comparison test for n = 8 animals per group.

[0097] Figure 25 Serum FP2 concentrations in C57Bl / 6 mice following intravenous (IV) and subcutaneous (SC) administration of 2 mg / kg are shown. Values ​​represent mean ± SD (n = 5 samples at each time point).

[0098] Figure 26 Serum FP2 concentrations are shown in Sprague Dawley rats following intravenous (IV) and subcutaneous (SC) administration of 2 mg / kg. N = 5 samples at each time point.

[0099] Figure 27Plasma concentrations of FP2 in cynomolgus monkeys are shown as analyzed by immunoassay. Values ​​represent the mean ± SD for n=3, except for IV on day 22 (528 hours) when n=2. IV – intravenous, SC – subcutaneous.

[0100] Figure 28 Plasma concentrations of FP2 as a complete dimer in cynomolgus monkeys, as analyzed by LCMS, are shown. Values ​​represent mean ± SEM for n=3, except for n=2 for subcutaneous (SC) administration at 168 hours, n=1 for SC administration at 120 hours and 432 hours, and n=1 for IV-venous administration at 168 hours and 432 hours.

[0101] Figure 29 The in vitro stability (normalized percentage recovery) of FP2 in human plasma over 48 hours, as measured by immunoassay, is shown.

[0102] Figure 30 The in vivo stability (normalized percentage recovery) of FP2 in human plasma over 48 hours is shown by full LC / MS.

[0103] Figure 31 Daily food intake (g) before and after a single administration of FP1 in cynomolgus monkeys is shown. *-p<0.05, relative to the solvent at 10 nmol / kg FP1;

[0104] Figure 32 The percentage change in body weight before and after a single dose of FP1 in cynomolgus monkeys is shown. *-p<0.05, for 10 nmol / kg FP1 compared to the solvent; #-p<0.05, for 3 mg / kg compared to the solvent, using two-way RM ANOVA and Tukey's multiple comparison test for n = 8 animals per group.

[0105] Figure 33 Daily food intake (g) in cynomolgus monkeys before and after a single administration of FP2 is shown. *-p<0.05, compared to solvent, using two-dimensional RM ANOVA and Tukey's multiple comparison test, for n = 8 animals per group.

[0106] Figure 34 The percentage change in body weight before and after a single dose of FP2 in cynomolgi is shown. *-p<0.05, for 10 nmol / kg FP2 compared to the solvent, #-p<0.05, for 3 nmol / kg FP2 compared to the solvent, &-p<0.05, for 1 nmol / kg FP2 compared to the solvent, were calculated using two-dimensional RM ANOVA and Tukey's multiple comparison test for n = 8 animals per group.

[0107] Figure 35 Food intake in spontaneously obese cynomolgus monkeys during a 12-week long-term period of weekly subcutaneous administration of FP2 is shown, normalized to the percentage reduction relative to baseline, and calculated as the mean daily food intake during the week prior to administration. Data are presented as mean ± SEM, where N animals are below the curve; for N=2, error bars are ± range.

[0108] Figure 36 The body weight (change from baseline) of spontaneously obese cynomolgus monkeys is shown as a percentage of change during a long period of 12 weeks of weekly subcutaneous administration of FP2. Data are presented as mean ± SEM, where N animals are below the curve; for N=2, error bars are ± range.

[0109] Figure 37 Serum concentrations (nM) of FP2, measured by immunoassay, are shown in spontaneously obese cynomolgus monkeys during a long period of 12 weeks of weekly subcutaneous administration of FP2. Data are presented as mean ± SEM, where N animals with detectable exposure are below the curve; for N=2, error bars are ± range.

[0110] Figure 38 A schematic overview of the study is shown. DG – Dosing group. Detailed Implementation

[0111] The background and description throughout this specification reference or describe various publications, articles, and patents; the full text of each of these references is incorporated herein by reference. Discussions of documents, actions, materials, devices, articles, etc., included in this specification are intended to provide context for the invention. Such discussions are not intended to acknowledge that any or all of these matters constitute prior art with respect to any disclosed or claimed invention.

[0112] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Otherwise, certain terms used herein have the meanings set forth in this specification. All patents, published patent applications, and publications referenced herein are incorporated herein by reference as if fully set forth herein. It should be noted that, unless the context clearly indicates otherwise, the singular forms “a” and “the” as used herein and in the appended claims include plural references.

[0113] The present invention relates to a fusion protein comprising: (a) a half-life extended protein, (b) a linker, and (c) a GDF15 protein, wherein the fusion protein is arranged in the order (a)-(b)-(c) from the N-terminus to the C-terminus.

[0114] It has been found that the fusion protein according to embodiments of the present invention (comprising a half-life-extending protein, a linker, and a GDF15 protein) results in an increased half-life of the GDF15 protein, and the fusion protein of the present invention exhibits suitability as a therapeutic agent for the treatment and prevention of metabolic diseases, disorders, or conditions. Such effects include, but are not limited to, weight loss, increased glucose tolerance, and improved insulin sensitivity in animals administered the fusion protein.

[0115] As used herein, the term "fusion protein" refers to a protein having two or more parts covalently linked together, each of which is derived from a different protein.

[0116] The fusion protein according to embodiments of the present invention may include any GDF15 protein. As used herein, the term "GDF15 protein" refers to any naturally occurring wild-type growth differentiation factor 15 protein or a functional variant thereof. The GDF15 protein may be derived from any mammal, such as human or other suitable mammals, such as mice, rabbits, rats, pigs, dogs, or primates. In a specific embodiment, the GDF15 protein is the human GDF15 protein or a functional variant thereof. In a preferred embodiment, the GDF15 protein is the mature GDF15 protein or a functional variant thereof.

[0117] As used herein, the term "mature GDF15 protein" refers to the portion of the GDF15 precursor protein released from the full-length protein after intracellular cleavage at the RXXR furin-like cleavage site. Mature GDF15 protein is secreted as a homodimer linked by disulfide bonds. In one embodiment of the invention, the mature GDF15 protein (quick-call GDF15(197-308) (SEQ ID NO:6)) comprises amino acids 197-308 of the full-length human GDF15 protein.

[0118] As used herein, a "functional variant" refers to a variant of the parent protein that has a high or significant sequence identity with the parent protein and retains at least one of the biological activities of the parent protein. According to this disclosure, functional variants of the parent protein can be prepared by means known in the art. Functional variants may include one or more modifications to the amino acid sequence of the parent protein. Modifications may alter the physicochemical properties of the polypeptide, for example, by improving the polypeptide's thermal stability, altering substrate specificity, changing the optimal pH, etc. Such modifications may also alter the biological activity of the parent protein, provided that these modifications do not destroy or eliminate all the biological activities of the parent protein. Modifications may also be the deletion or insertion of one or more amino acids.

[0119] According to another embodiment of the invention, functional variants of the parent protein include the deletion of one or more amino acids and / or the insertion of one or more amino acids into the parent protein. For example, functional variants of the mature GDF15 protein may include the deletion of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more amino acids and / or the insertion of these numbers of amino acids into the mature GDF15 protein, preferably the deletion of 1 to 30 amino acids at the N-terminus of the mature GDF15 protein.

[0120] According to an embodiment of the present invention, the fusion protein of the present invention comprises a GDF15 protein having at least 90% identical amino acid sequence to that of mature GDF15 (such as GDF15(197-308)(SEQ ID NO:6)); or having at least 90% identical amino acid sequence to a truncated amino acid sequence at the N-terminus of mature GDF15 (such as GDF15(200-308)(SEQ ID NO:7), GDF15(201-308)(SEQ ID NO:8), GDF15(202-308)(SEQ ID NO:9), GDF15(203-308)(SEQ ID NO:10) or GDF15(211-308)(SEQ ID NO:11)). The GDF15 protein may have at least one of the substitutions, insertions, and deletions of SEQ ID NO:6, 7, 8, 9, 10, or 11, provided that it retains at least one of the biological activities of the GDF15 protein, such as its effects on food intake, blood glucose levels, insulin resistance, and body weight.

[0121] In a specific embodiment, the fusion protein of the present invention comprises a GDF15 protein having the amino acid sequence of SEQ ID NO:11, which includes, but is not limited to, the amino acid sequences of SEQ ID NO:6, 7, 8, 9, 10 or 11.

[0122] Any suitable half-life-extending protein can be used in the fusion protein according to embodiments of the present invention. As used herein, the term "half-life-extending protein" can be any protein or fragment thereof known for extending the half-life of a protein to which it is fused. Examples of such half-life-extending proteins include, but are not limited to, human serum albumin (HSA), the constant fragment domain (Fc) of immunoglobulin (Ig), or transferrin (Tf). In embodiments of the present invention, the half-life-extending protein comprises HSA or a functional variant thereof. In a specific embodiment of the present invention, the half-life-extending protein comprises an amino acid sequence having at least 90% identity with SEQ ID NO:1. In a preferred embodiment of the present invention, the half-life-extending protein comprises HSA or a functional variant thereof, wherein the cysteine ​​residue at position 34 of the HSA has been substituted with serine or alanine.

[0123] In a specific embodiment, the fusion protein of the present invention comprises a half-life extended protein having an amino acid sequence selected from SEQ ID NO:1-3.

[0124] Any suitable linker can be used in the fusion protein according to embodiments of the invention. As used herein, the term "linker" refers to a connecting portion containing a peptide linker. Preferably, the linker helps ensure proper folding, minimizes steric hindrance, and does not significantly interfere with the structure of each functional component within the fusion protein. In some embodiments of the invention, the peptide linker comprises 2 to 120 amino acids. For example, peptide linkers include 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, or 120 amino acids.

[0125] In embodiments of the invention, the linker enhances the flexibility of the fusion protein component. In a specific embodiment of the invention, the linker may be a flexible linker comprising the sequence (GGGGS)n (including but not limited to GS-(GGGGS)n or AS-(GGGGS)n-GT), wherein n is 2 to 20, such as 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15.

[0126] In other embodiments of the invention, the connector is structured. In specific embodiments of the invention, the connector may be a structured connector comprising the sequence (AP)n or (EAAAK)n (including but not limited to AS-(AP)n-GT or AS-(EAAAK)n-GT), where n is 2 to 20, such as 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15. In other embodiments of the invention, the connector comprises the sequence (GGGGA). n (PGGGS) n (AGGGS) n Or GGS-(EGKSSGSGSESKST) n -GGS, where n is 2 to 20.

[0127] In embodiments of the invention, the fusion protein comprises an amino acid sequence having at least 90% sequence identity with SEQ ID NO:5, 25-30, 36-37, 40, 48, 55-56, 59-60, or 64-75. In a specific embodiment of the invention, the fusion protein comprises an amino acid sequence selected from SEQ ID NO:5, 25-30, 36-37, 40, 48, 55-56, 59-60, and 64-75. In a more specific embodiment of the invention, the fusion protein comprises an amino acid sequence selected from SEQ ID NO:5, 25-30, 40, 55-56, 55-56, 59-60, and 70. In a more specific embodiment of the invention, the fusion protein comprises an amino acid sequence of SEQ ID NO:92, SEQ ID NO:60, or SEQ ID NO:26. The fusion protein may also include small extensions at the amino or carboxyl termini of the protein, such as tags for easy purification, such as polyhistidine tags, antigenic epitopes, or binding domains.

[0128] The fusion proteins disclosed herein can be characterized or evaluated for their GDF15 bioactivity, including but not limited to effects on food intake, oral glucose tolerance tests, measurement of blood glucose levels, insulin resistance analysis, weight changes, pharmacokinetic analysis, toxicokinetic analysis, immunoassay and mass spectrometry analysis of the level and stability of the full-length fusion protein, and in vitro stability analysis in human plasma.

[0129] The present invention also provides isolated nucleic acid molecules encoding the fusion protein of the present invention. In embodiments of the invention, the isolated nucleic acid molecule encodes a fusion protein comprising an amino acid sequence having at least 90% sequence identity with SEQ ID NO:5, 25-30, 36-37, 40, 48, 55-56, 59-60, 64-75, or 92. In a specific embodiment, the isolated nucleic acid molecule encodes a fusion protein comprising an amino acid sequence selected from SEQ ID NO:5, 25-31, 36-37, 40, 48, 55-56, 59-60, 64-75, and 92. In a more specific embodiment, the isolated nucleic acid molecule encodes a fusion protein comprising an amino acid sequence selected from SEQ ID NO:5, 25-30, 40, 55-56, 59-60, 70, and 92. In a more specific embodiment, the isolated nucleic acid molecule comprises the nucleotide sequences of SEQ ID NO:76-91, 95, and 110.

[0130] According to other embodiments of the present invention, the nucleic acid molecule encoding the fusion protein may be in an expression vector. Expression vectors include, but are not limited to, vectors for recombinant protein expression and vectors such as viral vectors for delivering nucleic acids to a subject for expression in the subject's tissues. Examples of viral vectors suitable for the present invention include, but are not limited to, adenovirus vectors, adeno-associated virus vectors, lentiviral vectors, etc. Vectors may also be non-viral vectors. Examples of non-viral vectors include, but are not limited to, plasmids, bacterial artificial chromosomes, yeast artificial chromosomes, bacteriophages, etc. Vectors may include any elements that establish the conventional function of the expression vector, such as promoters, ribosome-binding elements, terminators, enhancers, selection markers, or origins of replication.

[0131] According to other embodiments of the invention, the nucleic acid molecule encoding the fusion protein may be a codon optimized in accordance with the present disclosure using methods known in the art to improve recombinant expression in a desired host cell (such as human embryonic kidney (HEK) or Chinese hamster ovary (CHO) cell).

[0132] The present invention also provides host cells comprising nucleic acid molecules encoding the fusion protein of the present invention. Host cells include, but are not limited to, host cells for recombinant protein expression and host cells for delivering nucleic acids to a subject for expression in the subject's tissues. Examples of host cells suitable for the present invention include, but are not limited to, HEK or CHO cells.

[0133] In another general aspect, the present invention relates to a method for obtaining the fusion protein of the present invention. In one general aspect, the method comprises: (1) culturing a host cell containing a nucleic acid molecule encoding the fusion protein under conditions for producing the fusion protein, and (2) recovering the fusion protein produced by the host cell. The fusion protein may be further purified using methods known in the art.

[0134] In some embodiments, the fusion protein is expressed in a host cell, and the fusion protein derived therefrom is purified using a combination of one or more standard purification techniques, including but not limited to affinity chromatography, size exclusion chromatography, ultrafiltration, and dialysis. Preferably, the fusion protein is purified to be free of any proteases.

[0135] The present invention also provides a pharmaceutical composition comprising the fusion protein of the present invention and a pharmaceutically acceptable carrier.

[0136] The present invention also provides a composition comprising a nucleic acid molecule encoding the fusion protein of the present invention and a pharmaceutically acceptable carrier. The composition containing the nucleic acid molecule encoding the fusion protein of the present invention may include a delivery solvent for introducing the nucleic acid molecule into cells to express the fusion protein. Examples of nucleic acid delivery vectors include liposomes, biocompatible polymers (including natural and synthetic polymers), lipoproteins, peptides, polysaccharides, lipopolysaccharides, artificial viral envelopes, metal particles, and bacterial, viral (such as baculoviruses, adenoviruses, and retroviruses), bacteriophages, granules, plasmids, fungal vectors, and other recombinant vectors commonly used in the art and described as being expressed in a variety of eukaryotic hosts.

[0137] The present invention also relates to kits comprising the pharmaceutical compositions of the present invention. The kit may comprise a first container having the dried fusion protein of the present invention and a second container having an aqueous solution mixed with the dried fusion protein prior to administration to a subject, or a single container comprising the liquid pharmaceutical composition of the present invention. The kit may comprise a single-dose administration unit or multiple-dose administration units of the pharmaceutical composition of the present invention. The kit may also comprise one or more pre-filled syringes (e.g., liquid syringes and lyophilized syringes). The kit may also include its instructions for use. The instructions may describe the use and properties of the substances provided in the kit and may be tailored to the specific metabolic disorder to be treated.

[0138] This invention also relates to the use of the pharmaceutical compositions described herein to treat or prevent metabolic diseases, disorders, or conditions, such as type 2 diabetes, elevated glucose levels, elevated insulin levels, obesity, dyslipidemia, diabetic nephropathy, ischemic myocardial injury, congestive heart failure, or rheumatoid arthritis. According to embodiments of the invention, a method of treating or preventing a metabolic disease, disorder, or condition in a subject requiring treatment comprises administering to the subject a therapeutically effective amount or a preventatively effective amount of the pharmaceutical composition of the invention. Any pharmaceutical composition described herein may be used in the methods of the invention, including pharmaceutical compositions comprising a fusion protein of the invention or pharmaceutical compositions comprising a nucleic acid encoding a fusion protein.

[0139] This article provides a method for reducing the weight of a subject, the method comprising administering a composition comprising a fusion protein containing SEQ ID NO:92 and at least one pharmaceutically acceptable carrier or diluent, wherein the fusion protein is administered at a dose ranging from about 0.8 mg to about 90 mg, and wherein the subject weighs 80 kg or more.

[0140] In some embodiments, the subject is overweight. In some embodiments of the invention, the subject has a weight of 25 kg / m². 2 Or a larger BMI, and in some implementations, the subject has a BMI of 25 kg / m². 2 Up to 29.9 kg / m 2 BMI within the range.

[0141] According to certain embodiments, the fusion protein is administered in doses selected from the following: about 0.8 mg, about 2.5 mg, about 7.5 mg, about 15 mg, about 30 mg, about 60 mg, and about 90 mg. In some embodiments, the fusion protein is administered at a dose of about 0.8 mg. In other embodiments, the fusion protein is administered at a dose of about 2.5 mg. In other embodiments, the fusion protein is administered at a dose of about 7.5 mg. In other embodiments, the fusion protein is administered at a dose of about 15 mg. In other embodiments, the fusion protein is administered at a dose of about 30 mg. In other embodiments, the fusion protein is administered at a dose of about 60 mg. In other embodiments, the fusion protein is administered at a dose of about 90 mg.

[0142] According to certain embodiments, the fusion protein is administered at a dose range of about 0.01 mg / kg to about 1.08 mg / kg. In some such embodiments, the fusion protein is administered at a dose selected from the following: about 0.01 mg / kg, about 0.03 mg / kg, about 0.09 mg / kg, about 0.18 mg / kg, about 0.36 mg / kg, about 0.72 mg / kg, and about 1.08 mg / kg. In some embodiments, the fusion protein is administered at a dose of about 0.01 mg / kg. In other embodiments, the fusion protein is administered at a dose of about 0.03 mg / kg. In other embodiments, the fusion protein is administered at a dose of about 0.09 mg / kg. In other embodiments, the fusion protein is administered at a dose of about 0.18 mg / kg. In other embodiments, the fusion protein is administered at a dose of about 0.36 mg / kg. In other embodiments, the fusion protein is administered at a dose of about 0.72 mg / kg. In other embodiments, the fusion protein is administered at a dose of approximately 1.08 mg / kg.

[0143] According to certain embodiments of the invention, the fusion protein is administered via subcutaneous injection.

[0144] According to certain embodiments of the present invention, the fusion protein is administered to the subject once a week.

[0145] This article provides a method for reducing food intake in subjects, the method comprising administering a composition comprising a fusion protein containing SEQ ID NO:92 and at least one pharmaceutically acceptable carrier or diluent, wherein the composition is administered at a dose ranging from about 0.8 mg to about 90 mg, and wherein the subject weighs 80 kg or more.

[0146] In some embodiments, the subject is overweight. In some embodiments of the invention, the subject has a weight of 25 kg / m². 2 Or a larger BMI, and in some implementations, the subject has a BMI of 25 kg / m². 2 Up to 29.9 kg / m 2 BMI within the range.

[0147] According to certain embodiments, the fusion protein is administered in doses selected from the following: about 0.8 mg, about 2.5 mg, about 7.5 mg, about 15 mg, about 30 mg, about 60 mg, and about 90 mg. In some embodiments, the fusion protein is administered at a dose of about 0.8 mg. In other embodiments, the fusion protein is administered at a dose of about 2.5 mg. In other embodiments, the fusion protein is administered at a dose of about 7.5 mg. In other embodiments, the fusion protein is administered at a dose of about 15 mg. In other embodiments, the fusion protein is administered at a dose of about 30 mg. In other embodiments, the fusion protein is administered at a dose of about 60 mg. In other embodiments, the fusion protein is administered at a dose of about 90 mg.

[0148] According to certain embodiments, the fusion protein is administered at a dose range of about 0.01 mg / kg to about 1.08 mg / kg. In some such embodiments, the fusion protein is administered at a dose selected from the following: about 0.01 mg / kg, about 0.03 mg / kg, about 0.09 mg / kg, about 0.18 mg / kg, about 0.36 mg / kg, about 0.72 mg / kg, and about 1.08 mg / kg. In some embodiments, the fusion protein is administered at a dose of about 0.01 mg / kg. In other embodiments, the fusion protein is administered at a dose of about 0.03 mg / kg. In other embodiments, the fusion protein is administered at a dose of about 0.09 mg / kg. In other embodiments, the fusion protein is administered at a dose of about 0.18 mg / kg. In other embodiments, the fusion protein is administered at a dose of about 0.36 mg / kg. In other embodiments, the fusion protein is administered at a dose of about 0.72 mg / kg. In other embodiments, the fusion protein is administered at a dose of approximately 1.08 mg / kg.

[0149] According to certain embodiments of the invention, the fusion protein is administered via subcutaneous injection.

[0150] According to certain embodiments of the present invention, the fusion protein is administered to the subject once a week.

[0151] When used to refer to a range of values, cutoff values, or specific values, the term "about" is used to indicate that the listed value may differ from the specified value by up to 10%. Therefore, the term "about" is used to cover variations of ±10% or less, ±5% or less, ±1% or less, ±0.5% or less, ±5% or less, or ±0.1% or less from the specified value.

[0152] As used herein, “subject” means any animal, specifically a mammal, and most specifically a human, that will be treated or has been treated by methods according to embodiments of the invention. As used herein, the term “mammal” covers any mammal. Examples of mammals include, but are not limited to, cows, horses, sheep, pigs, cats, dogs, mice, rats, rabbits, guinea pigs, non-human primates (NHPs) (such as monkeys or apes), humans, and more specifically, humans.

[0153] As used in this article, "overweight" refers to being overweight. Various parameters are used to determine whether a subject is overweight compared to a healthy reference individual, including the subject's age, height, sex, and health status. For example, a subject can be considered overweight or obese by assessing their body mass index (BMI), which is calculated by dividing the subject's weight (in kilograms) by the square of their height (in meters). A BMI of 18.5 kg / m² is considered ideal. 2 Up to 24.9 kg / m 2 Adults within this range are considered to have a normal weight; a BMI of 25 kg / m² is considered normal. 2 and 29.9 kg / m 2 Adults between these age ranges can be considered overweight (pre-obese); BMI is 30 kg / m². 2 Adults of advanced age or older can be considered obese. Increased appetite often leads to being overweight.

[0154] "Metabolic disease, disorder, or condition" refers to any disorder associated with abnormal metabolism. Examples of metabolic diseases, disorders, or conditions that can be treated by the methods of the present invention include, but are not limited to, type 2 diabetes, elevated glucose levels, elevated insulin levels, obesity, overweight, dyslipidemia, diabetic nephropathy, ischemic myocardial injury, congestive heart failure, or rheumatoid arthritis.

[0155] As used herein, the terms “treatment,” “being treated,” and “being treated” refer to administering the composition to a subject to achieve a desired therapeutic or clinical effect in the subject. In one embodiment, the terms “treatment,” “being treated,” and “being treated” refer to administering the pharmaceutical composition of the invention to slow, alleviate, or decelerate the progression or development of metabolic disorders such as type 2 diabetes, elevated glucose levels, elevated insulin levels, obesity, dyslipidemia, diabetic nephropathy, ischemic myocardial injury, congestive heart failure, or rheumatoid arthritis.

[0156] According to embodiments of the present invention, the pharmaceutical composition of the present invention can be administered to a subject by any method known to those skilled in the art according to this disclosure, such as intramuscular, subcutaneous, oral, intravenous, skin, mucous membrane (e.g., intestine), intranasal, or intraperitoneal administration routes. In a specific embodiment, the pharmaceutical composition of the present invention is administered to a subject by intravenous or subcutaneous injection.

[0157] As used herein, “once a week” administration is performed within a single day. Preferably, “once a week” administration is performed in a single step, such as a single injection.

[0158] In some embodiments, the present invention provides a method for reducing the weight of a subject using a clinically proven safe and effective dose of a GDF15 fusion protein having a sequence comprising SEQ ID NO:92, wherein the clinically proven safe and effective dose is a single subcutaneous (SC) injection administered to a subject weighing 80 kg or more at a dose ranging from 0.8 mg to 90 mg.

[0159] In some embodiments, the present invention provides a clinically safe and clinically effective dose of GDF15 fusion protein having a sequence including SEQ ID NO:92 for a method of reducing food intake in a subject, wherein the clinically safe and clinically effective dose is a single subcutaneous (SC) injection administered to a subject weighing 80 kg or more at a dose ranging from 0.8 mg to 90 mg.

[0160] According to the invention as defined herein, the term "clinically proven safe," when referring to treatment with a dose of GDF15 fusion protein having the sequence comprising SEQ ID NO:92, means a favorable risk-benefit ratio having a relatively low or reduced frequency and / or low or reduced severity of adverse events, said adverse events including adverse vital signs (heart rate, systolic and diastolic blood pressure, body temperature), adverse standard clinical laboratory tests (hematology, clinical chemistry, urinalysis, lipids, coagulation), allergic reactions / hypersensitivity reactions, adverse local injection site reactions, or adverse EKG.

[0161] According to the invention as defined herein, the terms “clinically proven effective” or “clinically proven efficacy” when they refer to terms such as a dose, dosing regimen, or treatment of a GDF15 fusion protein having the sequence comprising SEQ ID NO:92, mean reduced food intake, decreased appetite level, reduced food palatability as assessed by using a questionnaire, or reduced weight.

[0162] As used herein, a reduction in body weight is a reduction of at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 11%, at least 12%, at least 13%, at least 14%, at least 15%, at least 16%, at least 17%, at least 18%, at least 19%, at least 20%, or any amount thereof.

[0163] As used herein, a reduction in food intake is defined as a reduction of at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 11%, at least 12%, at least 13%, at least 14%, at least 15%, at least 16%, at least 17%, at least 18%, at least 19%, at least 20%, at least 21%, at least 22%, at least 23%, at least 24%, at least 25%, at least 26%, at least 27%, at least 28%, at least 29%, at least 30%, or any amount thereof. Food intake can be measured by measuring the calories consumed based on the grams consumed and the nutritional content of each food item.

[0164] As used herein, unless otherwise specified, the term "clinically proven" (used alone or to modify the terms "safe" and / or "effective") may mean that a clinical trial has demonstrated its effectiveness, wherein the clinical trial has met the standards of the U.S. Food and Drug Administration, the EMEA, or the relevant national regulatory agency. For example, a clinical study may be a well-sized, randomized, double-blind study designed to clinically confirm the efficacy of a drug. In some implementations, "clinically proven" indicates that it has been demonstrated through clinical trials that have met the standards of the U.S. Food and Drug Administration, the EMEA, or the relevant national regulatory agency for Phase I clinical trials.

[0165] Implementation Plan

[0166] 1. A method for reducing the weight of a subject, the method comprising administering a composition comprising a fusion protein containing SEQ ID NO:92 and at least one pharmaceutically acceptable carrier or diluent, wherein the fusion protein is administered at a dose ranging from 0.8 mg to 90 mg, and wherein the subject's weight is 80 kg or higher.

[0167] 2. The method according to implementation plan 1, wherein the subject is overweight.

[0168] 3. The method according to implementation scheme 2, wherein the subject has a BMI of 25 kg / m2 or greater.

[0169] 4. The method according to embodiment 3, wherein the subject has a BMI in the range of 25 kg / m2 to 29.9 kg / m2.

[0170] 5. The method according to embodiment 1, wherein the fusion protein is administered in a dose selected from the following: 0.8 mg, 2.5 mg, 7.5 mg, 15 mg, 30 mg, 60 mg and 90 mg.

[0171] 6. The method according to embodiment 5, wherein the fusion protein is administered at a dose of 0.8 mg.

[0172] 7. The method according to embodiment 5, wherein the fusion protein is administered at a dose of 2.5 mg.

[0173] 8. The method according to embodiment 5, wherein the fusion protein is administered at a dose of 7.5 mg.

[0174] 9. The method according to embodiment 5, wherein the fusion protein is administered at a dose of 15 mg.

[0175] 10. The method according to embodiment 5, wherein the fusion protein is administered at a dose of 30 mg.

[0176] 11. The method according to embodiment 5, wherein the fusion protein is administered at a dose of 60 mg.

[0177] 12. The method according to embodiment 5, wherein the fusion protein is administered at a dose of 90 mg.

[0178] 13. The method according to embodiment 1, wherein the fusion protein is administered via subcutaneous injection.

[0179] 14. A method for reducing the weight of a subject, the method comprising administering a composition comprising a fusion protein containing SEQ ID NO:92 and at least one pharmaceutically acceptable carrier or diluent, wherein the fusion protein is administered at a dose ranging from 0.01 mg / kg to 1.08 mg / kg.

[0180] 15. The method according to embodiment 14, wherein the fusion protein is administered at a dose selected from the following: 0.01 mg / kg, 0.03 mg / kg, 0.09 mg / kg, 0.18 mg / kg, 0.36 mg / kg, 0.72 mg / kg and 1.08 mg / kg.

[0181] 16. The method according to embodiment 15, wherein the fusion protein is administered at a dose of 0.01 mg / kg.

[0182] 17. The method according to embodiment 15, wherein the fusion protein is administered at a dose of 0.03 mg / kg.

[0183] 18. The method according to embodiment 15, wherein the fusion protein is administered at a dose of 0.09 mg / kg.

[0184] 19. The method according to embodiment 15, wherein the fusion protein is administered at a dose of 0.18 mg / kg.

[0185] 20. The method according to embodiment 15, wherein the fusion protein is administered at a dose of 0.36 mg / kg.

[0186] 21. The method according to embodiment 15, wherein the fusion protein is administered at a dose of 0.72 mg / kg.

[0187] 22. The method according to embodiment 15, wherein the fusion protein is administered at a dose of 1.08 mg / kg.

[0188] 23. The method according to embodiment 14, wherein the fusion protein is administered via subcutaneous injection.

[0189] 24. The method according to embodiment 14, wherein the composition is administered to the subject once a week.

[0190] 25. The method according to embodiment 1, wherein the composition is administered to the subject once a week.

[0191] 1A. A method for reducing food intake in a subject, the method comprising administering a composition comprising a fusion protein containing SEQ ID NO:92 and at least one pharmaceutically acceptable carrier or diluent, wherein the fusion protein is administered at a dose ranging from 0.8 mg to 90 mg, and wherein the subject weighs 80 kg or more.

[0192] 2A. The method according to implementation scheme 1A, wherein the subject is overweight.

[0193] 3A. The method according to embodiment 2A, wherein the subject has a BMI of 25 kg / m2 or greater.

[0194] 4A. The method according to embodiment 3A, wherein the subject has a BMI in the range of 25 kg / m2 to 29.9 kg / m2.

[0195] 5A. The method according to embodiment 1A, wherein the fusion protein is administered in a dose selected from the following: 0.8 mg, 2.5 mg, 7.5 mg, 15 mg, 30 mg, 60 mg and 90 mg.

[0196] 6A. The method according to embodiment 5A, wherein the fusion protein is administered at a dose of 0.8 mg.

[0197] 7A. The method according to embodiment 5A, wherein the fusion protein is administered at a dose of 2.5 mg.

[0198] 8A. The method according to embodiment 5A, wherein the fusion protein is administered at a dose of 7.5 mg.

[0199] 9A. The method according to embodiment 5A, wherein the fusion protein is administered at a dose of 15 mg.

[0200] 10A. The method according to embodiment 5A, wherein the fusion protein is administered at a dose of 30 mg.

[0201] 11A. The method according to embodiment 5A, wherein the fusion protein is administered at a dose of 60 mg.

[0202] 12A. The method according to embodiment 5A, wherein the fusion protein is administered at a dose of 90 mg.

[0203] 13A. The method according to embodiment 1A, wherein the fusion protein is administered via subcutaneous injection.

[0204] 14A. A method for reducing food intake in a subject, the method comprising administering a composition comprising a fusion protein containing SEQ ID NO:92 and at least one pharmaceutically acceptable carrier or diluent, wherein the fusion protein is administered at a dose ranging from 0.01 mg / kg to 1.08 mg / kg.

[0205] 15A. The method according to embodiment 14A, wherein the fusion protein is administered at a dose selected from the following: 0.01 mg / kg, 0.03 mg / kg, 0.09 mg / kg, 0.18 mg / kg, 0.36 mg / kg, 0.72 mg / kg and 1.08 mg / kg.

[0206] 16A. The method according to embodiment 15A, wherein the fusion protein is administered at a dose of 0.01 mg / kg.

[0207] 17A. The method according to embodiment 15A, wherein the fusion protein is administered at a dose of 0.03 mg / kg.

[0208] 18A. The method according to embodiment 15A, wherein the fusion protein is administered at a dose of 0.09 mg / kg.

[0209] 19A. The method according to embodiment 15A, wherein the fusion protein is administered at a dose of 0.18 mg / kg.

[0210] 20A. The method according to embodiment 15A, wherein the fusion protein is administered at a dose of 0.36 mg / kg.

[0211] 21A. The method according to embodiment 15A, wherein the fusion protein is administered at a dose of 0.72 mg / kg.

[0212] 22A. The method according to embodiment 15A, wherein the fusion protein is administered at a dose of 1.08 mg / kg.

[0213] 23A. The method according to embodiment 15A, wherein the fusion protein is administered via subcutaneous injection.

[0214] 24A. The method according to embodiment 14A, wherein the composition is administered to the subject once a week.

[0215] 25A. The method according to embodiment 1A, wherein the composition is administered to the subject once a week.

[0216] 1B. A method for reducing the weight of a subject, the method comprising administering to the subject once weekly a composition comprising a fusion protein containing SEQ ID NO:92 and at least one pharmaceutically acceptable carrier or diluent, wherein the fusion protein is administered at a dose ranging from 0.8 mg to 90 mg, and wherein the subject's weight is 80 kg or higher.

[0217] 2B. The method according to implementation scheme 1B, wherein the subject is overweight.

[0218] 3B. The method according to embodiment 2B, wherein the subject has a BMI of 25 kg / m2 or greater.

[0219] 4B. The method according to embodiment 3B, wherein the subject has a BMI in the range of 25 kg / m2 to 29.9 kg / m2.

[0220] 5B. The method according to embodiment 1B, wherein the fusion protein is administered in a dose selected from the following: 0.8 mg, 2.5 mg, 7.5 mg, 15 mg, 30 mg, 60 mg and 90 mg.

[0221] 6B. The method according to embodiment 5B, wherein the fusion protein is administered at a dose of 0.8 mg.

[0222] 7B. The method according to embodiment 5B, wherein the fusion protein is administered at a dose of 2.5 mg.

[0223] 8B. The method according to embodiment 5B, wherein the fusion protein is administered at a dose of 7.5 mg.

[0224] 9B. The method according to embodiment 5B, wherein the fusion protein is administered at a dose of 15 mg.

[0225] 10B. The method according to embodiment 5B, wherein the fusion protein is administered at a dose of 30 mg.

[0226] 11B. The method according to embodiment 5B, wherein the fusion protein is administered at a dose of 60 mg.

[0227] 12B. The method according to embodiment 5B, wherein the fusion protein is administered at a dose of 90 mg.

[0228] 13B. The method according to embodiment 1B, wherein the fusion protein is administered via subcutaneous injection.

[0229] 14B. A method for reducing the weight of a subject, the method comprising administering to the subject once weekly a composition comprising a fusion protein containing SEQ ID NO:92 and at least one pharmaceutically acceptable carrier or diluent, wherein the fusion protein is administered at a dose ranging from 0.01 mg / kg to 1.08 mg / kg.

[0230] 15B. The method according to embodiment 14B, wherein the fusion protein is administered at a dose selected from the following: 0.01 mg / kg, 0.03 mg / kg, 0.09 mg / kg, 0.18 mg / kg, 0.36 mg / kg, 0.72 mg / kg and 1.08 mg / kg.

[0231] 16B. The method according to embodiment 15B, wherein the fusion protein is administered at a dose of 0.01 mg / kg.

[0232] 17B. The method according to embodiment 15B, wherein the fusion protein is administered at a dose of 0.03 mg / kg.

[0233] 18B. The method according to embodiment 15B, wherein the fusion protein is administered at a dose of 0.09 mg / kg.

[0234] 19B. The method according to embodiment 15B, wherein the fusion protein is administered at a dose of 0.18 mg / kg.

[0235] 20B. The method according to embodiment 15B, wherein the fusion protein is administered at a dose of 0.36 mg / kg.

[0236] 21B. The method according to embodiment 15B, wherein the fusion protein is administered at a dose of 0.72 mg / kg.

[0237] 22B. The method according to embodiment 15B, wherein the fusion protein is administered at a dose of 1.08 mg / kg.

[0238] 23B. The method according to embodiment 22B, wherein the fusion protein is administered via subcutaneous injection.

[0239] 1C. A method for reducing food intake in a subject, the method comprising administering to the subject once weekly a composition comprising a fusion protein containing SEQ ID NO:92 and at least one pharmaceutically acceptable carrier or diluent, wherein the fusion protein is administered at a dose ranging from 0.8 mg to 90 mg, and wherein the subject weighs 80 kg or more.

[0240] 2C. The method according to embodiment 1C, wherein the subject is overweight.

[0241] 3C. The method according to embodiment 2C, wherein the subject has a BMI of 25 kg / m2 or greater.

[0242] 4C. The method according to embodiment 3C, wherein the subject has a BMI in the range of 25 kg / m2 to 29.9 kg / m2.

[0243] 5C. The method according to embodiment 1C, wherein the fusion protein is administered in a dose selected from the following: 0.8 mg, 2.5 mg, 7.5 mg, 15 mg, 30 mg, 60 mg and 90 mg.

[0244] 6C. The method according to embodiment 5C, wherein the fusion protein is administered at a dose of 0.8 mg.

[0245] 7C. The method according to embodiment 5C, wherein the fusion protein is administered at a dose of 2.5 mg.

[0246] 8C. The method according to embodiment 5C, wherein the fusion protein is administered at a dose of 7.5 mg.

[0247] 9C. The method according to embodiment 5C, wherein the fusion protein is administered at a dose of 15 mg.

[0248] 10C. The method according to embodiment 5C, wherein the fusion protein is administered at a dose of 30 mg.

[0249] 11C. The method according to embodiment 5C, wherein the fusion protein is administered at a dose of 60 mg.

[0250] 12C. The method according to embodiment 5C, wherein the fusion protein is administered at a dose of 90 mg.

[0251] 13C. The method according to embodiment 1C, wherein the fusion protein is administered via subcutaneous injection.

[0252] 14C. A method for reducing food intake in a subject, the method comprising administering to the subject once weekly a composition comprising a fusion protein containing SEQ ID NO:92 and at least one pharmaceutically acceptable carrier or diluent, wherein the fusion protein is administered at a dose ranging from 0.01 mg / kg to 1.08 mg / kg.

[0253] 15C. The method according to embodiment 14C, wherein the fusion protein is administered at a dose selected from the following: 0.01 mg / kg, 0.03 mg / kg, 0.09 mg / kg, 0.18 mg / kg, 0.36 mg / kg, 0.72 mg / kg and 1.08 mg / kg.

[0254] 16C. The method according to embodiment 15C, wherein the fusion protein is administered at a dose of 0.01 mg / kg.

[0255] 17C. The method according to embodiment 15C, wherein the fusion protein is administered at a dose of 0.03 mg / kg.

[0256] 18C. The method according to embodiment 15C, wherein the fusion protein is administered at a dose of 0.09 mg / kg.

[0257] 19C. The method according to embodiment 15C, wherein the fusion protein is administered at a dose of 0.18 mg / kg.

[0258] 20C. The method according to embodiment 15C, wherein the fusion protein is administered at a dose of 0.36 mg / kg.

[0259] 21C. The method according to embodiment 15C, wherein the fusion protein is administered at a dose of 0.72 mg / kg.

[0260] 22C. The method according to embodiment 15C, wherein the fusion protein is administered at a dose of 1.08 mg / kg.

[0261] 23C. The method according to embodiment 22C, wherein the fusion protein is administered via subcutaneous injection.

[0262] Example

[0263] The following embodiments of the present invention are intended to further illustrate the nature of the invention. It is believed that those skilled in the art can use the foregoing description and the following exemplary embodiments to prepare and utilize the present invention and practice the claimed methods. It should be understood that the following embodiments do not limit the invention, and the scope of the invention is defined by the appended claims.

[0264] Example 1: Design of fusion molecules containing GDF15—GDF15 truncation effect

[0265] Like other members of the TGFβ family, GDF15 is synthesized as a precursor protein that forms a dimer in the endoplasmic reticulum and undergoes furin cleavage to produce secreted mature GDF15 (amino acids 197-308). The secreted mature GDF15 homodimer is approximately 25 kDa, and each monomer has the potential to form up to four intramolecular disulfide bonds, with individual intermolecular disulfide bonds linking the homodimer components.

[0266] The crystal structure of GDF15 was determined and described in this invention. Figure 1A and Figure 1B The crystal structure indicates that the C-terminus of mature GDF15 is embedded at the dimer junction, while the N-terminus is exposed. This exposed end allows fusion proteins, such as half-life-extending proteins, to attach to the N-terminus of GDF15.

[0267] This crystal structure also depicts a novel disulfide bond pairing mode for the cysteine ​​residues in GDF15. While TGFβ1 exhibits C1-C3 and C2-C7 pairing (i.e., pairing between its first and third cysteine ​​residues and between its second and seventh cysteine ​​residues), GDF15 exhibits C1-C2 and C3-C7 pairing (see [link to crystal structure]). Figure 1A and Figure 1B This unique disulfide pairing results in a loop formed by the C1-C2 pairing, located at the N-terminus of the protein and away from the cysteine ​​knot containing other disulfide bonds. This structure predicts that the N-terminus of GDF15 may not be critical for dimer formation or overall protein folding, and that GDF15 and its N-terminal fusion molecules can tolerate the deletion of C1 and C2, residues within the C1-C2 loop, or even the deletion of residues from the C-terminus to the N-terminus of C2.

[0268] Example 2: Design of fusion molecules containing GDF15—connector effect

[0269] Different linkers between HSA and GDF15 molecules were evaluated. Flexible linkers containing the sequence (GGGGS)n and structured linkers containing the sequences (AP)n or (EAAAK)n, where n ranged from 2 to 20, were evaluated.

[0270] The biophysical properties of fusion proteins containing different linkers, their effects on dietary intake in lean mice, their mouse pharmacokinetic (PK) values, and their in vitro stability in human blood were compared. Results for the tested linker variants are shown in Table 1. The molecule containing SEQ ID NO:31 (which contains the (EAAAK)8 linker) showed aggregation by HPLC. The remaining seven linker variants in Table 1 were shown not to aggregate.

[0271] Table 1: Summary of Joint Variation Analysis

[0272]

[0273] *-Attach a 6xHis tag at the N-terminus for purification purposes.

[0274] The linker stability of these variants was also assessed through in vivo studies in mice and in vitro stability studies in human whole blood and plasma samples. Results from these studies were analyzed using two assays. Linker integrity was assessed by measuring the presence of two molecules on either side of the linker using an immunoassay with anti-GDF15 capture and anti-HSA detection antibody pairs. A broader picture of overall molecular integrity was analyzed using liquid chromatography-mass spectrometry (LC-MS) analysis of different alternative peptide sequences from HSA and GDF15. This immunoassay showed stable PK profiles for all linker variants and no loss of spiked plasma sample concentrations for any linker variant observed within 48 hours. LC-MS results were consistent with the immunoassay, showing that the alternative peptides from different parts of the HSA and GDF15 molecules were intact. PK profiles of the linker variants analyzed by LC-MS using the alternative peptides showed similar trends among the different linker variants, all of which were detectable at day 7. All variants in Table 1, except SEQ ID 31, exhibited desirable biophysical properties and PK values.

[0275] The in vivo activity of the adaptor variants was evaluated through a food intake study in lean mice. Table 2 shows the effect of the adaptor variants on the efficacy of the fusion protein in reducing food intake. The adaptor had a significant effect on efficacy. For the flexible (GGGGS)n adaptor, increasing the adaptor length from 2 to 4 to 8 significantly increased the efficacy of the fusion protein. For the more rigid (AP)n adaptor, the trend was less pronounced, indicating that the degree of freedom of the GDF15 molecule within the fusion protein plays a crucial role in its efficacy.

[0276] Table 2: Effects of the linker on the in vivo efficacy of the HSA-GDF15 fusion protein in lean mice

[0277] SEQ ID NO* connector % Food intake decreased (average) 25 <![CDATA[AS(GGGGS)2GT]]> 28.8 5 <![CDATA[GS(GGGGS)4]]> 40.5 26 <![CDATA[AS(GGGGS)8GT]]> 60.7 27 <![CDATA[AS(AP)5GT]]> 48.2 28 <![CDATA[AS(AP) 10 GT]]> 66.2 29 <![CDATA[AS(AP) 20 GT]]> 55.1 30 <![CDATA[AS(EAAAK)4GT]]> 51.9

[0278] *-Attach a 6xHis tag at the N-terminus for purification purposes.

[0279] Example 3: Design of fusion molecules containing GDF15—HSA mutation effect

[0280] A recombinant protein, human serum albumin, with an extended half-life, was designed with fusion of the protein to the N-terminus of GDF15 via a linker. This design was intended to allow the GDF15 dimerization junction to remain undisturbed and to allow the formation of native interchain disulfide bonds, thereby producing a GDF15 homodimer with an HSA fusion extending from each GDF15 arm. Using this approach, only a single gene is required to generate the HSA-GDF15 homodimer.

[0281] Natural human serum albumin contains 35 cysteine ​​(Cys,C) residues that form 17 disulfide bonds, with Cys-34 being the only free cysteine ​​residue in the molecule. This free Cys-34 has been shown to function as a free radical scavenger by capturing various reactive oxygen species (ROS) and reactive nitrogen species (RNS). Therefore, this free Cys residue is mutated to minimize the risk of heterogeneity due to oxidation.

[0282] The free cysteine ​​residue at position 34 of HSA was mutated to either serine or alanine, and GDF15 fusion molecules with the HSA(C34S) or HSA(C34A) mutations were analyzed. Both molecules were purified using a three-step purification method: (i) ion exchange chromatography, (ii) hydrophobic interaction chromatography, and (iii) size exclusion chromatography. HPLC analysis upon initial formation showed that both molecules were pure and free of aggregation (Table 3).

[0283] However, by HPLC, the fusion protein containing the HSA(C34A) mutation (SEQ ID NO:48) showed aggregation two weeks after its generation, while the fusion protein containing the HSA(C34S) mutation (SEQ ID NO:40) still did not aggregate after four weeks.

[0284] Table 3: Effect of HSA C34 mutation on fusion protein aggregation

[0285] SEQ ID NO HSA mutation % aggregation during purification % aggregation 2 weeks after purification 40 C34S 0 0 48 C34A 0 33.29

[0286] Example 4: Protease cleavage tendency on GDF15

[0287] The inventors observed that the arginine residue at amino acid position 198 of GDF15 (R198) is susceptible to protease degradation within the HSA-GDF15 fusion molecule. This degradation leads to a heterogeneous population and is undesirable for therapeutic compositions. Cleavage can be prevented by a mixture of protease inhibitors. Purification methods to remove the protease were investigated. Table 4 lists two types of HSA affinity columns used for the purification of the HSA-GDF15 fusion protein as measured by HPLC. During purification, the HSA-GDF15 fusion protein purified by both methods was 100% pure and intact. At low concentrations (2–5 mg / mL), the protein purified by both methods remained intact throughout the 4-week testing period. However, at high concentrations (40–50 mg / mL), antibody-based HSA resin (CaptureSelect) produced a protease-free protein that remained intact throughout the 4-week testing period. HSA ligand-based resin (Albupure) produced initially intact protein but exhibited degradation over time when stored at high concentrations. The addition of a protease inhibitor mixture (PI) and EDTA completely prevented the degradation of the high-concentration HSA-GDF15 fusion protein group purified using Albupure resin. Therefore, the purification method plays a crucial role in the production of stable therapeutic compositions. The absence of corresponding degradation in vivo or in vitro indicates that degradation of the fusion protein in vivo is not a problem once the therapeutic composition is protease-free. Therefore, purification methods capable of effectively removing potential proteases during the production process (e.g., purification methods using CaptureSelect resin) are key to the successful preparation of homogeneous, intact, and stable GDF15 therapeutic agents.

[0288] Table 4: Protease cleavage of the HSA-GDF15 fusion protein can be eliminated through sample purification methods.

[0289]

[0290] Example 5: N-terminal deletion variant of GDF15

[0291] Figure 1A and Figure 1B The crystal structure of GDF15 described in the study predicts that the N-terminus of GDF15 involving deletion variants is not critical for dimer formation and overall protein folding. It also predicts that such N-terminal deletions should not affect any potential receptor interactions. The in vivo activity of various HSA-GDF15 fusion proteins containing deletions of the N-terminus of GDF15 was tested.

[0292] A GDF15 N-terminal deletion variant was designed, removing the protease cleavage site at GDF15(R198). Following the R198 residue, potential deamidation sites exist at residues N199-G200, which are also detrimental to substrate deamidation in therapeutic compositions. The GDF15 N-terminal deletion simultaneously removes both the proteolytic cleavage site and the deamidation site. The resulting GDF15 deletion variants incorporated into fusion proteins along with HSA include GDF15(201-308; SEQ ID NO:8), GDF15(202-308; SEQ ID NO:9), and GDF15(211-308; SEQ ID NO:11). In vivo studies in mice showed that the GDF15 N-terminal deletion variants still retain activity in reducing food intake. Figure 17 Experimental results confirmed that this type of GDF15 N-terminal deletion variant is correctly expressed in vivo, forms a suitable dimer, and is active.

[0293] Example 6: Inactive mutants of GDF15

[0294] Table 5 lists 12 GDF15 mutants that eliminate GDF15 activity in vivo and recognize its functional epitope. The mutants include five single mutants, two double mutants, and five triple mutants. The biophysical properties and activities of the HSA-GDF15 fusion protein containing these mutations were characterized (Table 5). Of the 12 mutants, one was not expressed, and four formed aggregates over time, indicating that the mutations disrupted protein folding and biophysical properties. Of the remaining seven mutants, four contained single mutations of GDF15, and these mutants were tested in mice with reduced food intake compared to wild-type. Three single mutants (I89R, I89W, and W32A) lost in vivo activity, while the remaining mutant (Q60W) had the same activity as wild-type. These results suggest that the I89R, I89W, or W32A mutations disrupt the interaction between the receptor / co-receptor and GDF15, indicating that the functional epitope of GDF15 is located around residues I89 and W32. The mutation number is based on the presence of mature GDF15 in the fusion protein. For example, "1" refers to the first amino acid of mature GDF15 (SEQ ID NO:6), and "89" refers to the 89th amino acid of mature GDF15 protein.

[0295] Table 5: Overview of the biophysical properties and activities of fusion proteins containing the GDF15 mutant

[0296] SEQ ID NO Mutations in GDF15 Biophysical characteristics active 5 wild type Good expression, stable Wild-type active 64 I89R Good expression, stable Completely loses its activity 65 I89W Good expression, stable Completely loses its activity 66 L34A, S35A, R37A Good expression, stable 67 V87A, I89A, L98A Good expression, unstable 68 L34A, S35A, I89A Good expression, stable 69 V87A, I89A Good expression, stable 70 Q60W Good expression, stable Activity is the same as wild type 71 W32A Good expression, stable Completely loses its activity 72 W29A Good expression, unstable 73 Q60A, S64A, R67A Good expression, unstable 74 W29A, Q60A, I61A No expression 75 W29A, W32A Good expression, unstable

[0297] *-Attach a 6xHis tag at the N-terminus for purification purposes.

[0298] Example 7: Expression and Purification Methods

[0299] Express

[0300] For expressions of 20 ml and more, use Expi293. TM HEK Expi293 grown in expression medium TM Cells were used for expression. Cells were grown at 37°C with shaking at 125 RPM in 8% CO2. Expi293 was used. TM The expression kit was used to transfect cells at a rate of 2.5 × 10⁶ cells / ml. For each liter of transfected cells, 1 mg of total DNA was diluted in 25 mL of Opti-MEM, and 2.6 mL of Expi293 was added. TM The reagent was diluted in 25 mL of Opti-MEM and incubated at room temperature for 5 minutes. The diluted DNA and diluted Expi293 reagent were mixed and incubated at room temperature for 20 minutes. The DNA complex was then added to the cells. The cells were placed in a shaking incubator overnight. The day after transfection, 5 mL of Enhancer 1 from the kit was diluted to 50 mL of Enhancer 2 from the kit, and the total volume of both enhancers was added to the cells. The transfected cells were returned to the incubator for 4 days until harvest. The cells were concentrated by centrifugation at 6,000 g for 30 minutes and then filtered through a 0.2 μm filter before the purification step.

[0301] Expression was also performed in CHO cells. The plasmid was purified and characterized. Prior to transfection, a 200 μg aliquot of plasmid DNA containing the HSA-GDF15 coding region was linearized by digestion with an Acl I restriction enzyme. Digestion with this restriction endonuclease ensured the removal of the ampicillin resistance gene. Two 15 μg aliquots of linearized DNA were transfected into two 1 × 10⁷ CHO cell lines (designated transfection pool A and transfection pool B) using a BTX ECM 830 cell fusion transfection instrument (Harvard Apparatus, Holliston, MA). Cells were electroporated three times in 4 mm cuvettes at 250 volts with 15 ms pulse lengths and 5 s pulse intervals. Transfected cells were transferred to MACH-1+ L-glutamine in shake flasks and incubated for 1 day. Transfection pools A and B were centrifuged, resuspended in MACH-1+ MSX, and transferred to shake flasks for 6 days. Cells were generated by fusion of transfected HSA-protein from transfection pool A and transfection pool B and seeded in methylcellulose on day 8 after electroporation.

[0302] purification

[0303] A two-step purification method using CaptureSelect resin and size exclusion chromatography was employed. The purified Expi293 from transiently transfected cells was purified... TM Cell supernatant was loaded onto a pre-equilibrated HSA CaptureSelect column (CaptureSelect human albumin affinity matrix from ThermoFisher Scientific) with an approximate capacity of 10 mg protein per mL of resin (PBS, pH 7.2). After loading, unbound protein was removed by washing the column with 10 column volumes (CV) of PBS at pH 7.2. HSA-GDF15 bound to the column was eluted with 10 CV of 2M MgCl2 in 20 mM Tris (pH 7.0). The peak fractions were combined, filtered (0.2 μL), and dialyzed at 4°C with PBS at pH 7.2. After dialyzing, the protein fraction was filtered again (0.2 μL) and concentrated to an appropriate volume before loading onto a 26 / 60 Superdex 200 column (GE Healthcare). The high-purity protein fraction eluted from the size exclusion chromatography (SEC) column (as determined by SDS-PAGE) was combined. Protein concentration was determined by absorbance at 280 nm using a BioTek Synergy HTTM spectrophotometer. The quality of the purified protein was assessed by SDS-PAGE and analytical size-exclusion HPLC (SE-HPLC, Dionex HPLC system). LAL assay was used. The Associates of Cape Cod (ACC) measures endotoxin levels.

[0304] A two-step purification method using AlbuPure resin and SEC was also employed. The HSA-GDF15 fusion protein was purified at room temperature using AlbuPure resin (ProMetic BioSciences Ltd) that selectively binds HSA with immobilized synthetic triazine ligands. The expression supernatant was applied to the AlbuPure resin. The resin was then washed, first with 4CV PBS pH 7.2, then with 4CV of 50 mM Tris pH 8.0, 150 mM NaCl buffer. HSA-GDF15 bound to the column was eluted with 4CV PBS pH 7.2 containing 100 mM sodium caprylate. The protein-containing fraction was concentrated to 10 mL using a 30,000 kDa molecular weight cutoff spin concentrator (Amicon) and then applied to a 26 / 60 Superdex S200 pg column (GE) equilibrated in PBS pH 7.2 buffer. The SEC fraction containing the HSA-GDF15 homodimer was identified by SDS-PAGE and pooled for analysis. Protein purity was assessed using SDSPAGE and SE-HPLC.

[0305] Examples 8 through 14 and 19 relate to the characterization of an exemplary fusion protein of the present invention having the amino acid sequence SEQ ID NO: 60. This fusion protein is a fully recombinant protein present as a homodimer of HSA fused with mature human GDF15 via a 42-amino acid linker GS-(GGGGS)8 consisting of glycine and serine residues. The predicted molecular weight of this fusion protein is 162,696 Daltons, and a single native free cysteine ​​residue at position 34 of the HSA has been mutated to a serine residue. For simplicity, this particular HSA-GDF15 fusion protein is referred to simply as “FP1” in the following examples. In some of the following examples, a 6xHis-tagged FP1 variant (6xHis-FP1, SEQ ID NO: 26) containing the AS-(GGGGS)x8-GT linker is used for comparison.

[0306] Example 8: Effect of FP1 on food intake in C57B1 / 6 mice

[0307] The purpose of this experiment was to demonstrate the dose-response effect of FP1 on inhibiting food intake in C57Bl / 6 mice.

[0308] Male C57Bl / 6 mice were acclimatized to BioDAQ cages for at least 72 hours. The mice were then divided into six groups of eight mice each based on their food intake over the first 24 hours. Animals were weighed between 4:00 PM and 5:00 PM and administered either a solvent or a composition containing FP1 via subcutaneous injection. Food weight changes for each cage were continuously recorded using the BioDAQ system for 48 hours post-injection. 6xHis-FP1 was used for comparisons in this study.

[0309] result( Figure 2 Table 6 shows the average cumulative food intake over a given time interval. The results indicated that subcutaneous administration of FP1 to C57BL / 6 mice significantly suppressed food intake compared to solvent-treated animals at all tested doses and time points. 6xHis-FP1 at a dose of 8 nmol / kg reduced food intake.

[0310] Table 6: Effects of subcutaneous FP1 administration on food intake in C57BL / 6 mice; figures are shown at 12, 24 and 24 days post-administration. Cumulative food intake over 48 hours

[0311]

[0312] Data are expressed as mean ± SEM.

[0313] *p≤0.05, relative to PBS; **p≤0.01, relative to PBS; ***p≤0.001, relative to PBS; ****p≤0.0001, relative to PBS

[0314] One-way ANOVA-Tukey multiple comparison test; n = 8 / group

[0315] Example 9: Effect of FP1 on food intake in Sprague-Dawley rats

[0316] The aim of this experiment was to demonstrate the dose-response effect of FP1 on the inhibition of food intake in Sprague Dawley rats.

[0317] Male Sprague Dawley rats were acclimatized to BioDAQ cages for at least 72 hours. The rats were then divided into six groups of eight based on their food intake over the first 24 hours. Animals were weighed between 4:00 PM and 5:00 PM and administered either a solvent or a composition containing the fusion protein via subcutaneous injection. Food weight changes were continuously recorded for each cage using the BioDAQ system for 48 hours post-injection. Comparisons were performed using 6xHis-FP1 in this study.

[0318] The results are shown in Figure 3And as shown in Table 7. Compared with solvent-treated animals, subcutaneous administration of FP1 at doses of 2.5 nmol / kg and 10 nmol / kg inhibited food intake. The inhibition reached statistical significance only at the highest dose (10 nmol / kg) tested at 24 and 48 hours post-administration. FP1 at a dose of 8 nmol / kg reduced food intake, and the effect was significant at 24 and 48 hours.

[0319] Table 7: Effects of subcutaneous FP1 administration on food intake in Sprague-Dawley rats; the effects after administration are shown. Cumulative food intake at 12, 24 and 48 hours

[0320]

[0321] Data are expressed as mean ± SEM.

[0322] *p≤0.05, relative to PBS; **p≤0.01, relative to PBS

[0323] One-way ANOVA-Tukey multiple comparison test; n = 8 / group

[0324] Example 10: Effects of FP1 on glucose homeostasis and body weight in diet-induced obese (DIO) mice

[0325] The aim of this experiment was to evaluate the effects of FP1 on food intake, body weight, and glucose homeostasis during a two-week treatment period in DIO C57Bl / 6 mice.

[0326] Male DIO mice were weighed and FP1 was administered subcutaneously at 2 mL / kg every 3 days (q3d) on days 0, 3, 6, 9, and 12. In a similar regimen, the solvent and rosiglitazone treatment groups received PBS at the prescribed doses. Control rosiglitazone was provided in the diet at a free-access rate of 0.015%. Mouse and food weights were recorded daily. A blood glucose meter (One Glucose Meter) was used. Glucose was measured at Lifescan (Milpitas, CA). Fat body mass and lean body mass of conscious mice were quantified by time-domain NMR (TD-NMR) using a Bruker Mini-Spec LF110. For the oral glucose tolerance test (OGTT), mice were fasted for 4 hours. Blood glucose was measured at 0, 30, 60, 90, and 120 minutes after oral administration of 2 g / kg glucose (10 mL / kg) via tail cutting. Insulin was measured at 0, 30, and 90 minutes after glucose administration.

[0327] At the end of the study, mice were euthanized by CO2 inhalation, and terminal blood samples were collected. Serum was placed in 96-well plates on moist ice and then stored at -80°C. The liver was removed, and fat content relative to the total mass of the liver portion was assessed using TD-NMR and a Bruker MiniSpec mq60 according to the manufacturer's instructions.

[0328] An assessment of fasting homeostasis model for insulin resistance (HOMA-IR) based on the product of fasted glucose (mg / dL) and insulin (mU / L) divided by a factor of 405.

[0329] Treatment of DIO mice with FP1 q3d at 1 nmol / kg and 10 nmol / kg reduced body weight (Table 8) and food intake (Table 9). As described below, these reductions were statistically significant only at certain time points.

[0330] In DIO mice, Fp1 reduced body weight at doses of 1 nmol / kg (from day 2 to day 14) and 10 nmol / kg (from day 1 to day 14) (Table 8 and 10 nmol / kg). Figure 4 Significant reductions in food intake were observed on days 1 and 2 at the 1 nmol / kg dose and on days 1, 8, and 9 at the 10 nmol / kg dose (Table 9).

[0331] Table 8: Body weight change (%) in DIO mice during FP1 treatment

[0332]

[0333] Table 9: Daily food intake (gm) of DIO mice during FP1 treatment

[0334]

[0335] In the OGTT conducted on day 14 of the study, FP1 significantly reduced glucose levels at all time points after time 0, when all three doses were tested, compared to the solvent-treated animals (Table 10). This was further quantified as the total area under the curve (AUC) and ΔAUC, which were significantly lower compared to all three solvent doses tested (Table 10 and ). Figure 5A (and Table 5B).

[0336] Table 10: Blood glucose (mg / dL) levels in DIO mice during OGTT 114 days after FP1 administration at q3d.

[0337]

[0338] Blood glucose levels were measured at the start of the study (day 0), day 7, and day 13 (Table 11 and 13). Figure 6 On day 13 of the study, FP1 at doses of 1 nmol / kg and 10 nmol / kg significantly reduced blood glucose levels.

[0339] Table 11: Blood glucose levels during FP1 treatment in DIO mice with q3d (female) intake

[0340]

[0341] Data are expressed as mean ± SEM, n = 8 animals per group.

[0342] * = p < 0.05, compared with the solvent treatment group.

[0343] For FP1, plasma insulin levels during OGTT were significantly higher than in the corresponding solvent group at 30 minutes with a dose of 0.1 nmol / kg, and lower at the same time points at doses of 1 nmol / kg and 10 nmol / kg (Table 12). Insulin drift during OGTT, as measured by total AUC, was higher in the solvent group with FP1 at a dose of 0.1 nmol / kg (Table 12), and lower at doses of 1 nmol / kg and 10 nmol / kg. In both cases, statistical significance was achieved only at the lowest dose. Mice treated with 1 and 10 nmol / kg FP1 had lower insulin levels at the 90-minute time point; however, this effect did not achieve statistical significance. HOMA-IR, used as a measure of insulin sensitivity, was measured on day 14 of the study. At this time point, FP1 at 10 nmol / kg reduced HOMA-IR or improved insulin sensitivity (Table 13 and 14). Figure 7 ).

[0344] Table 12: Plasma insulin (pg / L) in DIO mice during OGTT 114 days after FP1 administration at q3d. mL) level

[0345]

[0346] Data are expressed as mean ± SEM, n = 8 animals per group.

[0347] * = p < 0.05, compared with the solvent treatment group.

[0348] Table 13: Fasting HOMA-IR in DIO mice after 14 days of FP1q3d treatment

[0349]

[0350] Data are expressed as mean ± SEM.

[0351] n = 8 animals per group.

[0352] * = p < 0.05, compared with the solvent treatment group.

[0353] The weight loss achieved on day 13 did not result in any measurable change in absolute fat mass or fat mass percentage at any dose (Table 14). At a dose of 10 nmol / kg, absolute lean body mass was significantly reduced. This reduction was not observed when expressed as lean body mass percentage. Liver weight was measured during final autopsy on day 15 of the study (Table 15). At a dose of 10 nmol / kg, FP1 reduced absolute liver weight and liver weight as a percentage of body weight. A reduction was observed at a dose of 1 nmol / kg, but this was not statistically significant for either parameter. Liver fat was measured on biopsy by NMR (Table 16). At doses of 1 nmol / kg and 10 nmol / kg, the FP1 fusion protein reduced liver fat content, expressed as a percentage of liver biopsy weight. The reduction was significant at higher doses.

[0354] Table 14: Body composition of DIO mice after 13 days of FP1q3d treatment

[0355]

[0356] Data are expressed as mean ± SEM, n = 8 animals per group.

[0357] * = p < 0.05, compared with the solvent treatment group.

[0358] Table 15: Liver weight of DIO mice after 15 days of FP1q3d treatment

[0359]

[0360] Data are expressed as mean ± SEM, n = 8 animals per group.

[0361] * = p < 0.05, compared with the solvent treatment group.

[0362] Table 16: Liver fat content of DIO mice measured 15 days after FP1q3d treatment.

[0363]

[0364] Data are expressed as mean ± SEM, n = 8 animals per group.

[0365] * = p < 0.05, compared with the solvent treatment group.

[0366] Example 11: Effects of FP1 on blood glucose levels and body weight in ob / ob mice

[0367] The purpose of this experiment was to evaluate the effects of FP1 on body weight and blood glucose levels in obese, hyperglycemic, and leptin-deficient ob / ob mice after 8 days of treatment.

[0368] Male ob / ob mice were weighed and FP1 was administered subcutaneously at 2 mL / kg every 3 days (q3d) on days 0, 3, and 6. Mouse and food weights were recorded daily. Glucose was measured daily using a glucometer. At the end of the study, mice were euthanized and terminal blood samples were collected.

[0369] Compared to solvent-treated mice, FP1 at a dose of 1 nmol / kg significantly reduced the body weight (expressed as a percentage of starting body weight) of ob / ob mice from day 2 to day 8. Compared to solvent-treated mice, FP1 at a dose of 10 nmol / kg significantly reduced the body weight (expressed as a percentage of starting body weight) of ob / ob mice from day 1 to day 8 (Table 17 and...). Figure 8 ).

[0370] Table 17: Changes in body weight (%) during FP1q3d treatment in ob / ob mice

[0371]

[0372] Data are expressed as mean ± SEM, n = 8 animals per group.

[0373] * = p < 0.05, compared with the solvent treatment group.

[0374] Compared to solvent-treated mice, FP1 at a dose of 10 nmol / kg reduced blood glucose levels in ob / ob mice on days 1 and 2, and days 4 through 8 of the study. A reduction in blood glucose was observed at 1 nmol / kg; however, this effect did not reach statistical significance (Table 18 and...). Figure 9 ).

[0375] Table 18: Blood glucose levels after feeding during FP1q3d treatment in ob / ob mice

[0376]

[0377] Example 12: Pharmacokinetics of Multiple Species

[0378] Mouse pharmacokinetics

[0379] Female C57Bl / 6 mice were administered FP1 at doses of 2 mg / kg IV and SC in PBS (pH 7). Blood samples and treatment serum were collected, and drug concentrations were measured up to 7 days after both administration routes. FP1 concentrations were determined using an immunoassay. Serum drug concentration-time curves are summarized in Tables 19 and 20, and... Figure 10 As shown in the image.

[0380] Table 19: Changes in serum FP1 concentration (nM) over time after a single SC administration in female C57Bl / 6 mice.

[0381]

[0382] Table 20: Changes in serum FP1 concentration (nM) over time after a single IV administration in female C57Bl / 6 mice.

[0383]

[0384] Pharmacokinetic analysis showed that the terminal half-life of FP1 in C57Bl / 6 mice after SC and IV administration was 1.67 days and 1.57 days, respectively (Table 21). The mean bioavailability of FP1 after SC administration was ~71%.

[0385] Table 21: Mean (±SD) pharmacokinetic parameters of FP1 after administration of 2 mg / kg IV and SC in female C57Bl / 6 mice number

[0386]

[0387] Note: *Tmax (median)

[0388] Rat Pharmacokinetics

[0389] Female Sprague Dawley rats were administered FP1 at doses of 2 mg / kg IV and SC in PBS (pH 7). Blood samples and treatment serum were collected, and drug concentrations were measured up to 7 days after both administration routes. FP1 concentrations were determined using an immunoassay. Serum drug concentration-time curves are summarized in Tables 22 and 23, and... Figure 11 As shown in the image.

[0390] Table 22: Serum concentration (nM) of FP1 over time after a single SC administration in female Sprague-Dawley rats change.

[0391]

[0392] Table 23: Serum concentration (nM) of FP1 over time after a single IV administration in female Sprague-Dawley rats change

[0393]

[0394] *Duplicate analysis confirmed the results

[0395] Pharmacokinetic analysis showed that the terminal half-lives of FP1 in Sprague Dawley rats after SC and IV administration were 1.34 and 1.51 days, respectively (Table 24). The mean bioavailability of FP1 after SC administration was ~23%.

[0396] Table 24: Mean (±SD) pharmacokinetics of FP1 after administration of 2 mg / kg IV and SC in Sprague-Dawley rats Learning parameters

[0397]

[0398] Note: *Tmax (median)

[0399] Monkey pharmacokinetics

[0400] FP1 was administered to juvenile cynomolgus macaques (Macacafascicularis) at doses of 1 mg / kg IV and SC in PBS (pH 7). Blood samples and treatment serum were collected, and drug concentrations were measured using an immunoassay bioanalytical method 21 days after both administration routes. Serum drug concentration-time curves are summarized in Tables 25 and 26, and... Figure 12 As shown in the image.

[0401] Table 25: Serum concentration (nM) of FP1 over time after a single SC administration in cynomolgus monkeys, determined by immunoassay. Changes

[0402]

[0403] Table 26: Serum concentration (nM) of FP1 over time after a single IV administration in cynomolgus monkeys, determined by immunoassay. Changes

[0404]

[0405] Pharmacokinetic analysis showed that the terminal half-life of FP1 in cynomolgus monkeys after SC and IV administration were 8.5 and 9.2 days, respectively, and the mean bioavailability after SC administration was ~88% (Table 27).

[0406] Table 27: Mean (±SD) pharmacokinetic parameters of FP1 after administration of 1 mg / kg IV and SC in cynomolgus monkeys .

[0407]

[0408] Note: *Tmax (median)

[0409] Immunoaffinity capture LCMS analysis was used to quantify the concentration of intact dimers present in cynomolgus monkey serum after IV and SC administration (Tables 28 and 29). Figure 13 and Figure 14 The concentrations determined by this method are similar to those determined by immunoassay (IA), indicating that FP1, as a complete dimer cycle, has no detectable metabolic potential in cynomolgus monkeys.

[0410] Figure 28 : For example, as determined by immunoaffinity capture LCMS analysis in cynomolgus monkeys after a single IV administration Serum concentration (ng / mL) of FP1 as a complete dimer over time.

[0411] Days Complete MS data of dimers (pooled samples) IA data (average) 0.00 0 0 0.04 33347 34537 0.25 29686 30328 1.00 28787 24089 2.00 17249 19449 3.00 16827 17272 5.00 16159 16282 7.00 11124 13674 10.00 8746 10973 14.00 5328 6879 18.00 3857 4629 22.00 2252 3984

[0412] Figure 29 : For example, as determined by immunoaffinity capture LCMS analysis in cynomolgus monkeys after a single SC administration Serum concentration (ng / mL) of FP1 as a complete dimer over time. .

[0413] Days Complete MS data of dimers (pooled samples) IA data (average) 0.00 0 0 0.25 9625 8959 1.00 15799 14219 2.00 17671 17373 3.00 19130 17476 5.00 12284 15633 7.00 10808 12971 10.00 8910 10143 14.00 5814 7034 18.00 4074 5054 22.00 2967 3694

[0414] The concentrations of the analytes in cynomolgus monkey serum after administration of IV and SC were also measured by immunoaffinity capture-trypsin digestion LC-MS / MS analysis (Tables 30 and 31). Selected trypsin peptides, namely ALV (ALVLIAFAQYLQQSPFEDHVK), ASL (ASLEDLGWADWVLSPR), and TDT (TDTGVSLQTYDDLLAK), were located within FP1 near the N-terminus of the HSA region, the N-terminus of GDF15, and the C-terminus of GDF15, respectively. Monitoring peptides were used as substitutes for FP1. The concentrations of all substitute peptides were similar to each other, and the concentrations were measured by immunoassay, demonstrating that the GDF15 sequence in FP1 remained intact and was linked to the intact HSA sequence in vivo.

[0415] Table 30: Identification of single-dose immunoaffinity capture-trypsin digestion LC-MS / MS analysis in cynomolgus monkeys Serum concentrations (ng / mL) of alternative peptides representing various regions of FP1 after IV administration.

[0416]

[0417] Table 31: Identification of single-dose immunoaffinity capture-trypsin digestion LC-MS / MS analysis in cynomolgus monkeys Serum concentrations (ng / mL) of alternative peptides representing various regions of FP1 after SC administration.

[0418]

[0419] Human plasma stability assay

[0420] The aim of this study was to analyze the in vitro stability of FP1 in human plasma. Fresh, non-frozen human plasma was generated from heparinized blood from two subjects (one male and one female) by centrifugation. FP1 was incubated in the matrix at 37°C with gentle mixing for 0, 4, 24, and 48 hours. The concentration of FP1 was determined using an immunoassay. The mean percentage difference from the initial concentration (0 hours) ranged from -4.1 to -12.9 and did not increase over time, indicating that FP1 is stable in human plasma in vitro for up to 48 hours (Table 32 and 33). Figure 15 ).

[0421] Table 32: In vitro temperature in plasma obtained from two human subjects (Sub), as determined by immunoassay The concentration of FP1 (μg / mL) after 0, 4, 24 and 48 hours of incubation.

[0422]

[0423] Immunoaffinity capture LCMS was used to quantify the concentration of intact dimers present in human plasma after incubation. The concentrations determined by this method were stable over time (0, 4, 24, and 48 hours), demonstrating that FP1 remains an intact dimer in human plasma in vitro up to 48 hours (Table 33 and 34). Figure 16 ).

[0424] Table 33: As determined by immunoaffinity capture LCMS analysis in plasma obtained from two human subjects The mean FP1 concentration (μg / mL) as a complete dimer after in vitro incubation for 0, 4, 24, and 48 hours and its percentage relative to the initial concentration. difference

[0425] Dimer concentration (ug / mL) % difference 0 hours 15.8 100.0 4 hours 15.8 100.1 24 hours 15.9 100.9 48 hours 15.2 96.0

[0426] Examples 15-19 relate to the characterization of an exemplary fusion protein of the present invention, described in Example 5, having the amino acid sequence SEQ ID NO:92 (encoded by nucleotide sequences SEQ ID NO:95 (codon optimization 1) and SEQ ID NO:110 (codon optimization 2)). This fusion protein is a fully recombinant protein existing as a homodimer of the fusion of HSA (C34S) with a deletion variant of mature human GDF15 (201-308; SEQ ID NO:8), the fusion being carried out via a 42-amino acid linker consisting of glycine and serine residues GS-(GGGGS)8. The single native free cysteine ​​residue at position 34 of HSA has been mutated to serine. For simplicity, this particular HSA-GDF15 fusion protein is referred to as “FP2” in the following examples.

[0427] SEQ ID NO:92:

[0428] DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQSPFEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLD ELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRAKTYETTLEKCCAAADPH ECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGLGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSDHCPLGPGRCCRLHTVRASLEDLGWADWVLSPREVQVTMCIGACPSQFRAANMHAQIKTSLHRLKPDTVPAPCCVPASYNPMVLIQKTDTGVSLQTYDDLLAKDCHCI

[0429] Example 13: In vitro agonist efficacy of FP2

[0430] The in vitro agonist potency of FP2 was assessed using a cell-based pAKT assay in SK-N-AS cells stably overexpressing the human GDF15 receptor (GFRAL). GFRAL activity was determined by measuring phosphorylated AKT (Ser473) levels in stably transfected SK-N-AS human neuroblastoma cells (ATCC) overexpressing human GFRAL. AKT phosphorylation was measured after treatment of GFRAL-expressing cells with various concentrations of the assay using a phosphorylated AKT (Ser473) assay kit (Cisbio, Beford, MA) according to the manufacturer's instructions. EC50 was calculated using Prism statistical software (GraphPad Software, San Diego). 50 Value. FP2 has a half-maximum effective concentration (EC50) of 2.908 ± 0.239 nM (N = 3). 50 Activation of pAKT. Natural GDF15 was used as a control and EC50 was calculated at 0.153 ± 0.008 nM (N = 3). 50 Demonstrates agonist activity.

[0431] Example 14: Effect of FP2 on food intake in C57Bl / 6 mice

[0432] The ability of FP2 to reduce food intake in male C57Bl / 6 mice after a single dose was evaluated. Male C57Bl / 6N mice (10–12 weeks old) obtained from Taconic Biosciences (Hudson, NY) were used in this study. Mice were housed individually in a temperature-controlled room with a 12-hour light / dark cycle (6 a.m. / 6 p.m.) and free access to water and food. Male C57Bl / 6 mice were acclimatized to BioDAQ cages for at least 72 hours; then, they were divided into 6 groups of 8 mice based on their food intake over the most recent 24 hours. Animals were weighed between 4:00 p.m. and 5:00 p.m. and administered the solvent or compound via subcutaneous injection. Food weight changes in each cage were continuously recorded using the BioDAQ system for 48 hours following compound administration. 6xHis-FP1 was used as a comparison in this study.

[0433] At all tested dose levels, FP2 had a significant effect on reducing food intake at 12, 24, and 48 hours post-administration (Table 34). At all time points and at all dose levels in mice (Table 35), the percentage change in food intake relative to PBS was reduced.

[0434] Table 34: Effect of a single dose of FP2 on food intake in C57Bl / 6 mice over 48 hours

[0435]

[0436] Data are expressed as mean ± SEM.

[0437] Specifically: *p≤0.05, relative to PBS

[0438] **p≤0.01, relative to PBS

[0439] ***p≤0.001, relative to PBS

[0440] ****p≤0.0001, relative to PBS

[0441] Statistical analysis used: ANOVA and Dunnett's multiple comparison test.

[0442] n = 8 per group, except for 6xHis-FP1 8nmol / kg (n = 6).

[0443] Table 35: Percentage reduction in food intake (relative to solvent) after a single dose of FP2 within 48 hours in C57Bl / 6 mice The influence of ratio

[0444]

[0445] The anorexia of FP2 is expressed as a relative decrease in food intake compared to the corresponding PBS control.

[0446] Data are expressed as mean ± SEM.

[0447] Specifically: *p≤0.05, relative to PBS

[0448] **p≤0.01, relative to PBS

[0449] ***p≤0.001, relative to PBS

[0450] ****p≤0.0001, relative to PBS

[0451] Statistical analysis used: ANOVA and Dunnett's multiple comparison test.

[0452] n = 8 per group, except for 6xHis-FP1 8nmol / kg (n = 6).

[0453] Example 15: Effect of FP2 on food intake in Sprague-Dawley rats

[0454] The ability of FP2 to reduce food intake and weight gain in male Sprague-Dawley rats was evaluated after a single dose. Animals, weighing 200–225 g, were obtained from Charles River Labs (Wilmington, MA) and administered within one week postpartum. They were housed one per cage on α-dry bedding and plastic tubing for enrichment in a temperature-controlled indoor environment with a 12-hour light / dark cycle. They were allowed free access to water and fed a laboratory rodent diet; irradiation certified. Rodent diet 20,5K75* (provided by Purina Mills, St. Louis, MO via ASAP Quakertown, PA). Animal weight was collected and recorded for each rat prior to administration.

[0455] Animals were acclimatized in BioDAQ cages for at least 72 hours; then, rats were divided into 6 groups of 8 rats each based on their food intake over the most recent 24 hours. Animals were weighed between 4:00 PM and 5:00 PM and administered the solvent or compound via subcutaneous injection. Food weight changes in each cage were continuously recorded using the BioDAQ system for 48 hours following compound administration. 6XHis-FP1 was used as a comparison in this study.

[0456] A dose-dependent decrease in food intake was tested after a single dose of FP2. No significant difference in food intake was observed at a dose of 0.3 nmol / kg. A significant effect of reduced food intake at 1 nmol / kg was observed at 12 hours, but not at 24 or 48 hours. Significant decreases in food intake were observed at all time points at dose levels of 3 nmol / kg and 10 nmol / kg (Table 36). Figure 19 At all time points and at all dose levels (Table 37), the percentage change in food intake relative to PBS was reduced.

[0457] Table 36: Effect of a single dose of FP2 on food intake in Sprague-Dawley rats over 48 hours .

[0458]

[0459] Data are expressed as mean ± SEM.

[0460] Specifically: *p≤0.05, relative to PBS

[0461] **p≤0.01, relative to PBS

[0462] ***p≤0.001, relative to PBS

[0463] Statistical analysis used: ANOVA and Dunnett's multiple comparison test.

[0464] n = 8 / group

[0465] Table 37: Food intake (relative to solvent) in Sprague-Dawley rats after a single dose of FP2 over 48 hours. Reduce the percentage effect .

[0466]

[0467] The anorexia of FP2 is expressed as a relative decrease in food intake compared to the corresponding PBS control.

[0468] Data are expressed as mean ± SEM.

[0469] Specifically: *p≤0.05, relative to PBS

[0470] **p≤0.01, relative to PBS

[0471] ***p≤0.001, relative to PBS

[0472] Statistical analysis used: ANOVA and Dunnett's multiple comparison test.

[0473] n = 8 / group

[0474] Example 16: Effects of FP2 on food intake, body weight, and glucose in diet-induced obese (DIO) C57Bl / 6 mice Steady-state effects

[0475] This study evaluated the ability of FP2 to reduce food intake and body weight and improve glucose homeostasis in male DIO C57Bl / 6 mice over an 8-day period with repeated administration. Male DIOC57Bl / 6 mice (21 weeks old, fed a high-fat diet for 15 weeks) obtained from Taconic Biosciences (Hudson, NY) were used for this study. Mice were housed individually in a temperature-controlled room with a 12-hour light / dark cycle (6 a.m. / 6 p.m.) and allowed free access to water, fed with Research Diet D12492 (Research Diets, New Brunswick, NJ). Mice were acclimatized to the mouse house for >1 week prior to the experiment. The endpoints of this study were measurements of food intake, body weight, body composition, and oral glucose tolerance test (OGTT, blood glucose). Animals were weighed and grouped by body weight (BW) the day before administration. FP2 was administered to mice via subcutaneous injection. Animals given FP2 received the compound on days 0, 3, 6, 9, and 12. During these days, the solvent group and the rosiglitazone group also received sterile PBS via subcutaneous injection. Rosiglitazone was administered in the diet at an ad libitum rate of 0.015% w / w. Body weight and food intake were recorded daily for fifteen days. Blood glucose was measured on days 0, 7, and 13. An oral glucose tolerance test (OGTT) was performed on day 14. Insulin levels were measured at selected time points during the OGTT. Mice were euthanized with CO2, and peripheral blood samples were collected via cardiac puncture for exposure on day 15. Separate pharmacokinetic (PK) groups were performed, with three mice per dose group, for a total of 15 mice.

[0476] Exposure-response (ER) analysis of FP2 in DIO mice

[0477] On the final study day when pharmacokinetic (PK) samples were obtained, most animals in the pharmacodynamic (PD) (efficacy) group had undetectable drug concentrations, likely due to immunogenicity. Therefore, the mean PK curve from the PK group, rather than individual PK curves from the PD group, was used to assess the exposure-response for percentage weight changes from baseline at corresponding dose levels in the PD group (from days 3, 6, and 9, respectively). This method assumes that the PK group behaves similarly to the PD group in terms of drug exposure.

[0478] Use E max The model (GraphPad Prism 6, log(agonist) versus response) correlates exposure with response data (log-transformed drug concentration). The Hill slope is set to 1. Note that although E max Different estimates (E) max The EC values ​​were -4.26%, -8.18%, and -9.85% respectively, but on days 3, 6, and 9, the model-fitted EC values ​​were...10 To EC 50 The values ​​were within 2. Some animals also showed loss of drug exposure on day 9 due to potential ADA formation; therefore, ER parameter estimates based on day 9 data should be interpreted with caution.

[0479] The effects of two weeks of FP2 exposure on food intake, body weight, glucose homeostasis, and hepatic fat content were assessed in diet-induced obese male C57Bl / 6 mice. Slot exposure was maintained between 1.7 nM and 3.3 nM FP2 in the 0.3 nmol / kg treatment group, between 7.1 nM and 14 nM in the 1.0 nmol / kg treatment group, between 20.8 nM and 41.6 nM in the 3.0 nmol / kg treatment group, and between 28.5 nM and 112.9 nM FP2 in the 10 nmol / kg treatment group until day 9 in the study PK group (n = 2 or 3, Table 49). After day 9, despite continued q3d administration, a decrease in circulating levels was observed in most animals (Table 49). Consistent with this accelerated clearance, most animals in the study PD group had undetectable circulating FP2 levels on day 15 (Table 50).

[0480] Compared with solvent therapy, FP2 q3d treatment in DIO mice reduced food intake (Table 38) and body weight (Tables 39, 40, and 50). Figure 20 ) and blood glucose levels after eating (Table 43 and Figure 23 For 0.3 nmol / kg on days 2, 5, and 8; for 1.0 nmol / kg from day 1 to day 7; for 3.0 nmol / kg on days 1, 2, 4 to 6, and 8; and for 10.0 nmol / kg on days 1, 3 to 6, 8, and 9, a significant decrease in food intake was observed. For 0.3 nmol / kg from day 5 to day 13; for 1.0 nmol / kg and 10.0 nmol / kg from day 3 to day 13; and for 3.0 nmol / kg from day 4 to day 13, significant changes in body weight percentage were observed. For 0.3 nmol / kg starting from day 8; for 1.0 nmol / kg starting from day 6; for 3.0 nmol / kg starting from day 7; and for 10.0 nmol / kg starting from day 5, significant changes in body weight in grams were observed. For animals at a dose level of 3.0 nmol / kg, the reduction in blood glucose levels after feeding was significant on day 7, and for animals at dose levels of 3.0 and 10.0 nmol / kg, it was significant on day 13.

[0481] Compared with solvent treatment during oral glucose screening, DIO mice treated with FP2 q3d had improved glucose tolerance on day 14 (Table 41). Figure 21A and Figure 21B For the 0.3 nmol / kg group, glucose levels decreased significantly at 30 minutes; for the 1.0 nmol / kg group, glucose levels decreased significantly at 60 and 120 minutes; for the 3.0 nmol / kg group, glucose levels decreased significantly at 120 minutes; and for the 10.0 nmol / kg group, glucose levels decreased significantly at 30, 90, and 120 minutes. The total area under the curve was significant for all dose groups. During glucose screening, insulin levels were significantly lower at 30 minutes in the 0.3 and 10.0 nmol / kg groups (Table 42). Figure 22A and Figure 22B Furthermore, compared with solvent-treated animals, fasting HOMA-IR was significantly reduced in DIO mice after 14 days of treatment with FP2q3d at 10.0 nmol / kg, indicating improved insulin sensitivity (Table 44 and 10.0 nmol / kg). Figure 24 ).

[0482] Body composition was measured by MRI on days 1 and 13 prior to the start of the study (Tables 47 and 48). DIO mice treated with FP2 at 1.0 nmol / kg and 10.0 nmol / kg showed a significant reduction in adipose body mass on day 13; however, lean body mass remained unchanged for any treatment group. On day 13, the 10.0 nmol / kg treatment group showed a significantly increased percentage of lean body mass and a significantly decreased percentage of adipose body mass compared to the solvent treatment group. From day -1 to day 13, significant changes in lean body mass were observed in the 0.3 nmol / kg, 1.0 nmol / kg, and 10.0 nmol / kg treatment groups, and significant changes in lean body mass percentage were observed in the 1.0 nmol / kg, 3.0 nmol / kg, and 10.0 nmol / kg treatment groups. Compared to the solvent treatment, significant changes in both adipose body mass and lean body mass percentage were observed in all treatment groups from day -1 to day 13.

[0483] There was no significant difference in endogenous mouse GDF15 serum levels between solvent-treated animals and mice treated with FP2 q3d for 15 days (Table 46).

[0484] Conclusion: The results indicate that higher drug exposure on days 3, 6, and 9 was generally associated with greater percentage weight change from baseline at the population level in the study dose groups.

[0485] Exposure to FP2 over two weeks resulted in reduced food intake, weight loss, decreased blood glucose, improved glucose tolerance, and improved insulin sensitivity in DIO mice. Significant reductions in food intake were achieved over multiple days at 1.0 nmol / kg, 3.0 nmol / kg, and 10.0 nmol / kg q3d. Significant weight loss began three to five days after the start of the study. Following administration of FP2 at 3.0 and 10.0 nmol / kg q3d, blood glucose levels were significantly reduced on day 13 after food intake. A significant reduction in fasting HOMA-IR, representing insulin sensitivity, was achieved 14 days after administration of 10.0 nmol / kg FP2 q3d. On day 13, a significant increase in lean body mass percentage and a significant decrease in fat body mass percentage were observed in DIO mice treated with 10.0 nmol / kg FP2 q3d.

[0486] Table 38: Effect of FP2 on daily food intake (g) during 13 days of treatment. .

[0487]

[0488] The numerical value represents the mean ± SEM of each data point for each of the 8 animals in each group, except when n = 7 is represented by ^.

[0489] *-p<0.05, relative to solvent

[0490] Statistical analyses used: two-way ANOVA, RM, Tukey's multiple comparison test

[0491] Table 39: Effect of FP2 on percentage change in body weight during 13 days of treatment.

[0492]

[0493] The numerical values ​​represent the mean ± SEM of each data point for each of the 8 animals in each group.

[0494] *p<0.05 relative to the solvent.

[0495] Statistical analyses used: two-way ANOVA, RM, Tukey's multiple comparison test

[0496] Table 40: Effect of FP2 on body weight change (g) during 13 days of treatment.

[0497]

[0498] The numerical values ​​represent the mean ± SEM of each data point for each of the 8 animals in each group.

[0499] *-p<0.05, relative to solvent

[0500] Statistical Analysis: Two-way ANOVA, RM, and Tukey's multiple comparison test

[0501] Table 41: Effect of FP2 on blood glucose (mg / dL) levels during OGTT 14 days after treatment

[0502]

[0503] The numerical values ​​represent the mean ± SEM of each data point for each of the 8 animals in each group.

[0504] *-p<0.05, relative to solvent statistical analysis: two-way ANOVA RM and Tukey's multiple comparison test were applied to glucose values;

[0505] One-way ANOVA and Tukey's multiple comparison test were applied to AUC.

[0506] Table 42: Effect of FP2 on insulin (ng / ml) levels during OGTT 14 days after treatment

[0507]

[0508] The numerical values ​​represent the mean ± SEM of each data point for each of the 8 animals in each group.

[0509] *-p<0.05, relative to solvent statistical analysis: two-way ANOVA RM and Tukey's multiple comparison test were applied to insulin values;

[0510] One-way ANOVA and Tukey's multiple comparison test were applied to AUC.

[0511] Table 43: Effect of FP2 on blood glucose (mg / dL) levels after food intake

[0512]

[0513] The numerical values ​​represent the mean ± SEM of each data point for each of the 8 animals in each group.

[0514] *-p<0.05, relative to solvent

[0515] Statistical Analysis: Two-way ANOVA, RM, and Tukey's multiple comparison test

[0516] Table 44: Fasting HOMA-IR in DIO mice after 14 days of FP2q3d treatment

[0517]

[0518] The numerical values ​​represent the mean ± SEM of each data point for each of the 8 animals in each group.

[0519] *-p<0.05, relative to solvent

[0520] Statistical Analysis: One-way ANOVA and Tukey's Multiple Comparison Test

[0521] Table 45: Liver weight of DIO mice after 15 days of FP2q3d treatment

[0522]

[0523] The values represent the mean ± SEM of each data point for 8 animals per group

[0524] * - p < 0.05, relative to vehicle

[0525] Statistical analysis: one - way ANOVA, Tukey's multiple comparison test

[0526] Table 46: Serum GDF15 (pg / mL) levels in DIO mice after 15 days of treatment with FP2q3d.

[0527]

[0528] The values represent the mean ± SEM of each data point for 8 animals per group

[0529] * - p < 0.05, relative to vehicle

[0530] Statistical analysis: one - way ANOVA, Tukey's multiple comparison test

[0531] Table 47: Effects of FP2q3d on body composition (g) as measured by MRI in DIO mice

[0532]

[0533] The values represent the mean ± SEM of each data point for 8 animals per group

[0534] *p < 0.05, relative to vehicle

[0535] Statistical analysis: one - way ANOVA, Tukey's multiple comparison test

[0536] Table 48: Effect of FP2q3d on body composition (%) as measured by MRI in DIO mice

[0537]

[0538] The values represent the mean ± SEM of each data point for 8 animals per group

[0539] *p < 0.05, relative to vehicle

[0540] Statistical analysis: one - way ANOVA, Tukey's multiple comparison test

[0541] Table 49: Serum FP2 exposure (nM) in the PK group during q3d treatment in DIO mice.

[0542]

[0543] Data are expressed as concentration per animal

[0544] <LOQ = below limit of quantification; LOQ is 0.494 nM

[0545] **Values at Days 3, 6, 9, and 12 are immediately before the next dose

[0546] Table 50: Terminal serum exposure (nM) of FP2 after 15 days of treatment with q3d in DIO mice.

[0547]

[0548] Data are expressed as concentration per animal.

[0549] <LOQ = below limit of quantification; LOQ is 0.494 nM

[0550] Example 17: Pharmacokinetics and Immune Response of FP2 in Multiple Species

[0551] Mouse pharmacokinetics

[0552] When administered subcutaneously to female C57Bl / 6 mice, the pharmacokinetic properties of FP2 were evaluated. FP2 was administered subcutaneously (n = 5 samples per time point) and intravenously (n = 5 samples per time point) at a dose level of 2.0 mg / kg in PBS (pH 7.3 - 7.5) to female C57Bl / 6 mice (Sage Laboratories, St Louis, MO). Samples at the last time point were collected by terminal bleeding. Blood samples were collected, serum was processed, and drug concentrations were measured over up to 168 hours. The level of FP2 was measured using an immunoassay. The drug concentration curves in plasma are summarized in Tables 51 and 52 and are shown in Figure 25 shown below.

[0553] Pharmacokinetic analysis of FP2 in C57Bl / 6 mice demonstrated that the terminal half - lives after IV and SC administration were approximately 1.51 days and 1.76 days, respectively, and the mean bioavailability after SC administration was approximately 61%.

[0554] Table 51: Serum concentrations (ng / ml) of FP2 after a single subcutaneous (SC) administration in C57Bl / 6 mice.

[0555]

[0556] Table 52: Serum concentrations (ng / ml) of FP2 after a single intravenous (IV) administration in C57Bl / 6 mice.

[0557]

[0558] Table 53: Pharmacokinetic parameters of FP2 in C57Bl / 6 mice after administration of 2 mg / kg IV and 2 mg / kg SC. .

[0559]

[0560] Rat Pharmacokinetics

[0561] FP2 was administered subcutaneously (n=5 samples per time point) and intravenously (n=5 samples per time point) to female Sprague-Dawley mice (Sage Laboratories, St. Louis, MO) at a dose level of 2.0 mg / kg (pH 7.3–7.5) in PBS. Samples at the last time point were collected via terminal blood flow. Blood samples were collected, serum was processed, and drug concentrations were measured over a maximum of 168 hours. FP2 levels were measured using an immunoassay. Plasma drug concentration curves are summarized in Tables 54 and 55, and are presented in... Figure 26 The pharmacokinetic parameters calculated from these data are shown in Table 56.

[0562] Pharmacokinetic analysis of FP2 in Sprague Dawley rats showed that the terminal half-life after IV administration and SC administration was approximately 1.46 days and approximately 1.37 days, respectively, and the mean bioavailability after SC administration was approximately 28%.

[0563] Table 54: Serum concentrations (ng / ml) of FP2 following a single subcutaneous (SC) administration in Sprague-Dawley rats.

[0564]

[0565] Table 55: Serum concentrations (ng / ml) of FP2 following a single intravenous (IV) administration in Sprague-Dawley rats.

[0566]

[0567] Table 56: Pharmacokinetic parameters of FP2 in Sprague-Dawley rats after administration of 2 mg / kg IV and 2 mg / kg SC number .

[0568]

[0569] Monkey pharmacokinetics

[0570] FP2 was dissolved in PBS (pH 7.0–7.6) and administered subcutaneously at 1 mg / kg and intravenously at 1 mg / kg to each of three male cynomolgus monkeys. Blood samples were collected, serum was processed, and drug concentrations were measured over a maximum of 21 days.

[0571] The pharmacokinetics (PK) of FP2 were characterized in cynomolgus monkeys following single administration of IV (1.0 mg / kg) and SC (1.0 mg / kg). Plasma drug concentration-time curves after SC administration for immunoassay and LCMS analysis are summarized in Tables 57 and 58, respectively, and after IV administration for immunoassay and LCMS analysis are summarized in Tables 59 and 60, respectively. The immunoassay data were... Figure 27 Plotting in the middle, and LCMS data in... Figure 28 The Chinese side indicated that...

[0572] Using results from immunoassay analysis, the mean terminal half-life (t1 / 2) of FP2 based on NCA was approximately 7.05 days after IV administration and approximately 8.51 days after SC administration. Mean PK parameters after IV and SC administration are summarized in Table 61. Using results from immunoassay bioanalysis, the mean terminal half-life (t1 / 2) of FP2 estimated using a non-compartmental model was approximately 7.05 days after IV administration and approximately 8.51 days after SC administration. Mean bioavailability (F%) of FP2 is based on AUC. 0-last The estimate is approximately 98.5%, and is based on the AUC in cynomolgus monkeys after SC administration. 0-inf The estimate is approximately 109.2%.

[0573] Table 57: Plasma concentrations of FP2 (ng / ml) measured by immunoassay after a single SC administration in cynomolgus monkeys. .

[0574]

[0575] N / A = Not applicable

[0576] Table 58: Plasma concentrations of FP2 (ng / ml) measured by LCMS after a single SC administration in cynomolgus monkeys.

[0577]

[0578] -= Initial run failed; insufficient samples for repeat analysis.

[0579] # = Tubes with incorrect markings; samples excluded from analysis. N / A = Not applicable.

[0580] Table 59: Plasma concentrations (ng / ml) of FP2 measured by immunoassay after a single IV dose in cynomolgus monkeys.

[0581]

[0582] N / A = Not applicable

[0583] Table 60: Plasma concentrations of FP2 (ng / ml) measured by LCMS after a single IV dose in cynomolgus monkeys.

[0584]

[0585] -=Initial run failed, insufficient samples for repeat analysis. N / A=Not applicable.

[0586] # = Tubes with incorrect markings; samples excluded from analysis.

[0587] Table 61: Mean (±SD) pharmacokinetic parameters of FP2 after administration of 1 mg / kg IV and SC in cynomolgus monkeys .

[0588]

[0589] The PK parameter is the average value of NCA based on immunoassay PK data.

[0590] *Tmax (median)

[0591] Human plasma stability assay

[0592] The in vitro stability of FP2 in fresh heparinized plasma was assessed at 37°C for up to 48 hours. Fresh, non-frozen human plasma was generated from heparinized blood from two subjects (one male and one female) by centrifugation. FP2 was incubated in the matrix at 37°C with gentle mixing for 0, 4, 24, and 48 hours. The concentration of FP2 was determined using an immunoassay. The concentration of intact dimers present in the matrix was quantified under assay conditions using independent immunoaffinity capture followed by LCMS.

[0593] In this immunoassay, the percentage recovery from the initial concentration ranged from 104.8% to 94.1%, and did not decrease over time, indicating that FP2 is stable in human plasma for up to 48 hours in vitro. Figure 29 (See Table 62). LCMS showed that the concentration remained stable over time, demonstrating that FP2 maintained an intact dimer in human plasma for up to 48 hours in vitro. Figure 30 (and Table 63).

[0594] Table 62: In vitro stability (normalized) of FP2 (ng / ml) in human plasma measured by immunoassay over 48 hours (Recycling percentage) .

[0595]

[0596] Table 63: In vitro stability of FP2 (ng / ml) in human plasma over 48 hours by complete LC / MS measurement (normalized return) (percentage) .

[0597]

[0598] Example 18: The efficacy of FP1 and FP2 in cynomolgus monkeys

[0599] The effects of a single dose of FP1 and FP2 on food intake and body weight in juvenile cynomolgus monkeys were evaluated.

[0600] FP1 was administered subcutaneously to juvenile cynomolgus monkeys at three dose levels: 1 nmol / kg, 3 nmol / kg, and 10 nmol / kg. A solvent treatment group was also included. Animals were treated blindly. The study lasted for 6 weeks: baseline food intake measurements and data collection for 2 weeks, and data collection for 4 weeks following the single compound administration. Plasma drug exposure was measured on days 1, 7, 14, 21, and 28 post-administration.

[0601] Compared with solvent therapy, treatment with a single dose of FP1 in cynomolgus monkeys reduced food intake and body weight. Figures 31 to 32At a dose level of 10 nmol / kg, a significant reduction in daily food intake was observed on days 4, 5, 6, and 8 through 12. Figure 31 At the 10 nmol / kg dose level, the weekly average daily food intake was significantly reduced over the 2-week period following administration. At the 3 nmol / kg dose level, the weekly average food intake was significantly reduced compared to the pre-administration weekly average at week 2, and at the 10 nmol / kg dose level, it was significantly reduced compared to the pre-administration weekly average at weeks 1 and 2. At the 3 nmol / kg dose level, a significant reduction in the percentage change in body weight from day 0 was observed on day 28, and at the 10 nmol / kg dose level, significant reductions in the percentage change in body weight were observed on days 14, 21, and 28. Figure 32 ).

[0602] FP2 was administered subcutaneously to juvenile cynomolgus monkeys at three dose levels: 1 nmol / kg, 3 nmol / kg, and 10 nmol / kg. A solvent treatment group was also included. Animals were treated blindly. The study lasted 11 weeks: 5 weeks for baseline food intake measurements and data collection, 1 week for treatment, and 5 weeks for clearance phase data collection. Plasma drug exposure was measured on days 1, 7, 14, 21, 28, 35, and 42 post-administration.

[0603] Compared with solvent therapy, treatment with a single dose of FP2 in cynomolgus monkeys reduced food intake and body weight. Figures 33 to 34 At the 3 nmol / kg dose level, a significant reduction in daily food intake was observed on days 3, 5–8, 10, and 12, and at the 10 nmol / kg dose level, a significant reduction in daily food intake was observed on days 3–38 and 40. Figure 33 At the 3 nmol / kg dose level, the weekly average daily food intake was significantly reduced in the first week after administration, and this was also significantly reduced in weeks 1 through 6 at the 10 nmol / kg dose level. In the second week after administration, the 3 nmol / kg dose level showed a significant decrease in the percentage of weekly average daily food intake compared to the week prior to administration, and the 10 nmol / kg dose level showed a significant decrease in the percentage of weekly average daily food intake compared to the week prior to administration in weeks 1 through 6. A significant decrease in the percentage of body weight change from day 0 was observed at the 1 nmol / kg dose level from day 21 to day 42, at the 3 nmol / kg dose level from day 14 to day 42, and at the 10 nmol / kg dose level from day 7 to day 42. Figure 33 ).

[0604] Example 19: Efficacy of multiple doses of FP2 in cynomolgus monkeys

[0605] The efficacy of FP2 was evaluated by weekly subcutaneous injection into an initially spontaneously overweight cynomolgus monkey population (aged 8–20 years and weighing 8.0 kg–11.9 kg) at three dose levels: 0.3 nmol / kg, 1 nmol / kg, and 10 nmol / kg. Food consumption was measured daily, body weight weekly, and animals were clinically assessed daily. Compared to solvent treatment, treatment with FP2 at 12-week doses reduced food intake in overweight cynomolgus monkeys. Figure 35 ) and weight ( Figure 36 The circulating FP2 concentration was determined by immunoassay. Figure 37 Loss of FP2 exposure was observed in some animals at a later time point, presumably due to the development of anti-drug antibodies (ADA): figures show data collected up to the point prior to exposure loss (defined as a ≥40% reduction in previously measured trough serum drug concentration in the same animal). No treatment-related adverse effects were observed throughout the study.

[0606] Example 20: Joint thermal stability

[0607] Thermal stability studies were conducted on various linkers connecting HSA and GDF15. To assess the likelihood of fragmentation and aggregation, HSA-GDF15 fusion proteins with various linkers were diluted to 10 mg / ml. After the addition of EDTA and methionine, the samples were incubated at 40 °C for 14 days. The samples were then diluted to a concentration of 1 mg / ml and evaluated using size exclusion high-performance liquid chromatography (SE-HPLC). The percentages of intact protein, aggregates, and fragments were quantified for these proteins. Table 64 shows that the HSA-GDF15 protein with linkers composed of AP repeats is the most stable for fragments under thermal stress.

[0608] To assess whether these linkers affect the interaction between GDF15 and its receptor, immunoassays or anti-GDF15 or anti-HSA assays were performed on the GFRAL-Fc fusion protein coated on plates using monoclonal antibodies against GDF15 (Janssen) and HSA (Kerafast, Inc., Boston, MA). These assays showed that all these linker variants in Table 66 exhibited similar binding to the receptor.

[0609] Table 64. SE-HPLC results after 14 days of thermal stress .

[0610] SEQ ID NO connector Aggregates (%) whole(%) Fragment (%) 113 <![CDATA[GS(GGGGS)8]]> 3.33 84.44 12.22 115 <![CDATA[GA(GGGGA)8]]> 3.51 87.98 8.5 117 <![CDATA[(AP) 10 ]]> 1.64 98.36 0 119 <![CDATA[(AP) 12 ]]> 2.36 97.64 0 121 <![CDATA[GGS-(EGKSSGSGSESKST)3-GGS]]> 1.67 85.24 13.09 123 <![CDATA[GS(PGGGS)8]]> 2.96 88.12 8.91 125 <![CDATA[GS(AGGGS)8]]> 3.44 86.22 10.34 127 <![CDATA[GGS-(EGKSSGSGSESKST)2-GGS]]> 1.71 91.17 7.12

[0611] Example 21: Clinical Trial Protocol

[0612] A double-blind, placebo-controlled, randomized, single-dose escalation study was conducted to investigate the safety, tolerability, pharmacokinetics (including absolute bioavailability), and immunogenicity of subcutaneously administered FP2 in overweight or otherwise healthy subjects.

[0613] Solution 64739090EDI1001; Phase 1

[0614] EudraCT ID: 2018-000324-34

[0615] abbreviation

[0616] ADA anti-drug antibodies

[0617] ALT (alanine aminotransferase)

[0618] Anti-HCV hepatitis C antibody

[0619] AST aspartate aminotransferase

[0620] Area under the AUC curve

[0621] BA bioavailability

[0622] BLQ below the minimum quantifiable concentration

[0623] BMI (Body Mass Index)

[0624] BP (blood pressure)

[0625] BPM (beats per minute)

[0626] BUN (blood urea nitrogen)

[0627] BW weight

[0628] CNS (Central Nervous System)

[0629] CRF Case Report Form (the electronic form used in this study)

[0630] CRU Clinical Research Unit

[0631] CV (Cardiovascular)

[0632] DCF Data Clarification Table

[0633] DG dosage group

[0634] DIO (Diet-Induced Obesity)

[0635] DRC Data Review Committee

[0636] Diagnostic and Statistical Manual of Mental Disorders (DSM-V), 5th Edition

[0637] EC 50 Half-maximum effective concentration

[0638] ECG (electrocardiogram)

[0639] eCRF Electronic Case Report Form

[0640] EDC Electronic Data Capture

[0641] EMA (European Medicines Agency)

[0642] EU European Union

[0643] FcRn neonatal Fc receptor

[0644] FDA (U.S. Food and Drug Administration)

[0645] FIH First-in-human trial

[0646] FSH (Follicle-Stimulating Hormone)

[0647] Good Clinical Practice (GCP)

[0648] GDF15 (Growth Differentiation Factor 15)

[0649] GFRAL GDNF family receptor α-like, GDF15 receptor

[0650] GGT γ-glutamyltransferase

[0651] GLP (Good Laboratory Practice)

[0652] HA Health Bureau

[0653] HbA1c (hemoglobin A1c)

[0654] HBsAg (Hepatitis B surface antigen)

[0655] hCG (human chorionic gonadotropin)

[0656] HCV (Hepatitis C Virus)

[0657] HDL (High-density lipoprotein)

[0658] HED human equivalent dose

[0659] HIV (Human Immunodeficiency Virus)

[0660] HR heart rate

[0661] HSA human serum albumin

[0662] IAC Interim Analysis Committee

[0663] IB Researcher's Handbook

[0664] ICF Informed Consent Form

[0665] ICH International Harmonization Meeting

[0666] IEC Independent Ethics Committee

[0667] IMP Research Medical Products

[0668] IRB Institutional Review Committee

[0669] IV. Intravenous

[0670] IVRS Interactive Sound Response System

[0671] IWRS Interactive Network Response System

[0672] LC-MS / MS (Liquid Chromatography / Mass Spectrometry / Mass Spectrometry)

[0673] LDL (low-density lipoprotein)

[0674] LLOQ lower limit of quantitation

[0675] MABEL Minimum Expected Biological Effect Level

[0676] MedDRA Regulatory Activities Medical Dictionary

[0677] Maximum recommended starting dose for MRSD

[0678] MRU Medical Resource Utilization

[0679] n is the number (size of the subsample).

[0680] N is the total sample size.

[0681] NAb neutralizing antibody

[0682] NAFLD (Non-alcoholic fatty liver disease)

[0683] NASH (Non-Alcoholic Steatohepatitis)

[0684] NBE New biological entity

[0685] No adverse effects were observed at NOAEL.

[0686] PAD drug active dose

[0687] PAP (Papanicolaou) smear

[0688] PD efficacy

[0689] PI (Principal Investigator)

[0690] PK Pharmacokinetics

[0691] PQC Product Quality Complaints

[0692] PRO patient-reported results (applicable to paper or electronic results of this study)

[0693] PSA (prostate-specific antigen)

[0694] QEWP-5 Questionnaire on Eating and Weight Patterns - 5

[0695] RBC (red blood cells)

[0696] RET GFRAL signal transduction co-receptor

[0697] SAD single escalation dose

[0698] SAE Serious Adverse Events

[0699] SBP (systolic blood pressure)

[0700] SC subcutaneous

[0701] SD Sprague-Dawley

[0702] SUSAR suspected to be an unexpectedly serious adverse reaction

[0703] Type 2 diabetes mellitus (T2DM)

[0704] Adverse events caused by TEAE treatment

[0705] TK Toxicity Kinetics

[0706] TSH (Thyroid Stimulating Hormone)

[0707] ULN Normal Upper Limit

[0708] US

[0709] VAS (Visual Analog Scale)

[0710] WBC (white blood cells)

[0711]

[0712]

[0713]

[0714]

[0715]

[0716]

[0717]

[0718]

[0719]

[0720]

[0721] 1. Introduction

[0722] Growth differentiation factor 15 (GDF15) is a circulating protein factor that exists as a 25 kDa dimer in human plasma. Both published and internal data support its role in regulating energy balance, which is a major factor affecting energy (i.e., food intake).

[0723] Subcutaneous (SC) administration of FP2 resulted in reduced food intake and subsequent body weight (BW) loss in rodents and non-human primates. Furthermore, SC treatment with FP2 led to improved glucose homeostasis and improved insulin resistance in diet-induced obese (DIO) mice, likely due to weight loss. FP2 works by binding to the recently identified GDF15 receptor, a GDNF family receptor α-like (GFRAL) receptor (primarily expressed in the final regions of the central nervous system (CNS)). 5 (17,24,15) plays its role. It is assumed that FP2 will reduce food intake in obese subjects and subsequently lead to weight loss, which will also lead to improvement in obesity-related comorbidities. 1.1. Background Technology

[0725] 1.1.1. Non-clinical research

[0726] Pharmacological characteristics

[0727] The in vitro agonist potency of FP2 was assessed using a cell-based pAKT assay in SK-N-AS cells stably overexpressing the human GFRAL receptor. FP2 achieved a half-maximal effective concentration (EC50) of 2.908 ± 0.239 nM (N = 3). 50 Activation of pAKT (see Example 14). Natural GDF15 was used as a control and EC50 was calculated at 0.153 ± 0.008 nM (N = 3). 50 Demonstrates agonist activity.

[0728] The ability of FP2 to reduce food intake was evaluated in multiple species. A single SC administration of FP2 severely suppressed food intake in male C57Bl / 6 mice (see Example 14) and Sprague-Dawley (SD) rats (see Example 15). Compared with solvent-treated animals, a single SC administration of FP2 to initially spontaneously overweight cynomolgus monkeys resulted in reduced food intake and subsequent significant weight loss up to 4 weeks after administration (Example 18).

[0729] In DIO mice, repeated administration of FP2 every 3 days over a 2-week period reduced food intake and body weight, and improved glucose tolerance and insulin sensitivity as measured by a homeostasis model of insulin resistance (see Example 16). Compared to solvent therapy, weekly administration of FP2 to an initially spontaneously overweight cynomolgus monkey population over a 12-week period resulted in a significant reduction in food intake and body weight (see Example 19). Loss of exposure was observed at later time points in some animals, presumably due to the development of anti-drug antibodies (ADA). No treatment-related adverse effects were noted throughout the study.

[0730] Safety Pharmacology

[0731] In accordance with the International Council for Harmonisation of Technical Requirements for the Environment (ICH) S6(R1) guidelines, safety pharmacology endpoints (cardiovascular [CV]-, respiratory [CNS]-, and central nervous system [CNS]-) were assessed in cynomolgus monkeys (Study 8372593) and SD rats (Study 8371098) as part of a Good Laboratory Practice (GLP) 4-week repeated-dose toxicity study. In addition, an independent CV safety pharmacology study was conducted in cynomolgus monkeys equipped with telemetry instruments (Study T-2017-044).

[0732] In summary, IV and SC administration of FP2 continued until the highest dose had no effect on CV endpoints, core body temperature, respiratory rate, neurological or behavioral endpoints.

[0733] toxicology

[0734] Non-clinical safety studies (see Table 65) were conducted in accordance with GLP, 21 CFR, Part 58, and / or as part of the OECD-GLP principles in countries where they are part of the Organization for Economic Cooperation and Development (OECD) Mutual Acceptance of Data process, and included appropriate documentation. The FP2 test material (lot number CVC_PCM01) used for non-clinical safety studies was considered representative of clinical test material.

[0735] Table 65: Overview of toxicological studies using FP2

[0736]

[0737] Keywords: biw = twice a week; IV = intravenous; qw = once a week; SC = subcutaneous; SD = Sprague-Dawley.

[0738] Repeated administration of FP2 was generally well tolerated in cynomolgus monkeys and SD rats. No deaths or obvious clinical signs were observed. Some findings (e.g., reduced food intake and body weight) were considered to be a result of the intended mode of action and were not considered adverse effects.

[0739] Related species selection

[0740] Since FP2 is a fully recombinant fusion protein of human GDF15 and HSA domains linked by a short peptide composed of natural amino acids, the toxicology procedures were designed primarily in accordance with ICH Guideline S6(R1), Preclinical Safety Evaluation of Biotechnology-Derived Pharmaceuticals.

[0741] In terms of relevant animal species identification, the GDF15 fraction of FP2 is the bioactive component; however, the HSA fraction primarily increases FP2 exposure by extending its half-life through its interaction with the neonatal Fc receptor (FcRn). The GDF15 receptor (GFRAL) and the GFRAL-signaling co-receptor (RET) have recently been identified. 17,24,5,15 .

[0742] Computer-simulated amino acid sequence homology analysis of the bioactive component (GDF15), its receptors (GFRAL and RET), its half-life extended component (HSA), and albumin receptor (FcRn) in different species revealed the highest degree of similarity (95%-100%) between humans and monkeys (i.e., cynomolgus monkeys) and a fairly high degree of similarity (78%-100%) between humans and rats (Table 66).

[0743] Table 66: Sequence homology among species

[0744]

[0745] Keywords: Cyno = cynomolgus monkey; FcRn = neonatal Fc receptor; GFRAL = GDNF family receptor α-like; RET = GFRAL signaling co-receptor; *Non-rodent species were selected for toxicity testing and **Rodent species were selected for toxicity testing.

[0746] In vitro binding assays indicated that FP2 bound to recombinant GFRAL fusion proteins from humans, rats, and cynomolgus monkeys, with affinity between humans and cynomolgus monkeys in the 2-fold range and between humans and rats in the 5-fold range. Furthermore, tissue expression analysis of the GFRAL receptor in different species revealed comparable expression patterns in rats, monkeys, and humans (primarily in the last region of the hindbrain). 24 .

[0747] In vivo pharmacodynamic (PD) studies confirmed the putative PD effect of FP2 in both cynomolgus monkeys and rats (e.g., reduction in food intake and body weight).

[0748] However, single-dose PK studies also confirmed that cynomolgus monkeys (T... 1 / 2 (approximately 7-9 days) and SD rats (T 1 / 2 Some significant differences in PK were observed between the two groups (approximately 1-2 days), due to the lower affinity of human HSA for rat FcRn receptors but similar affinity of human HSA for cynomolgus monkey FcRn.

[0749] Therefore, the cynomolgus monkey was considered the most relevant / predictable animal species and was chosen as the non-rodent species for a first-in-human (FIH) trial-initiated non-clinical safety study. Choosing rats as the rodent toxicology species for PK has some limitations.

[0750] Pharmacokinetic characteristics

[0751] The pharmacokinetics and toxicokinetics (TK) of FP2 were characterized in rodents and lean cynomolgus monkeys after a single dose and after up to 4 weeks of prolonged administration. The median time to reach maximum concentration (TK) was also determined for mice, rats, and cynomolgus monkeys. maxThe estimated elimination half-lives were 1 day, 1 day, and 1.67 days, respectively. The clearance rate of FP2 (approximately 25 mL / day / kg in rodents and 5 mL / day / kg in monkeys) and elimination half-lives (approximately 1.5 days and 7.1 days in rodents and monkeys, respectively) differed significantly between monkeys and rodents, presumably due to differences in the affinity of HSA for rodent or monkey FcRn (i.e., HSA has a lower binding affinity for rodent FcRn than for human FcRn, but its affinity for monkey FcRn is similar to that for human FcRn). Therefore, monkeys are considered a more predictive species for FP2 PK in humans than in rodents. The predicted elimination half-life for a 90 kg human is approximately 12 to 17 days.

[0752] Following administration by SC and IV, FP2 was shown to be stable as an intact dimer in human plasma for up to 48 hours in vitro and stable in vivo in cynomolgus monkeys. Metabolism of intact FP2 is expected to occur via the standard proteolytic pathway.

[0753] 1.1.2. Clinical Research

[0754] This will be the first administration of FP2 in humans; therefore, no clinical experience is available.

[0755] Human pharmacokinetics and immunogenicity

[0756] To date, no human-to-human comparison studies have been conducted using FP2.

[0757] Efficacy / Safety Studies

[0758] To date, no clinical studies have been conducted using FP2.

[0759] 2. Objectives and Hypotheses

[0760] 2.1 Objectives

[0761] 2.1.1. Part 1: Incremental Single Dose

[0762] In overweight (BMI ≥ 25 kg / m² to ≤ 29.9 kg / m²) or otherwise healthy subjects, after a single escalation of the SC dose to FP2:

[0763] Main objectives

[0764] To evaluate the safety and tolerability of subcutaneous (SC) administration of FP2.

[0765] Secondary objectives

[0766] • Evaluate the PK of FP2.

[0767] • Assess the immunogenicity of FP2 based on potential ADA formation and the possible formation of antibodies against endogenous GDF15.

[0768] • Assess pharmacodynamic (PD) endpoints, such as body weight and food intake.

[0769] Exploratory goals

[0770] • Assess whether the administration of FP2 is associated with changes in PD endpoints (such as appetite level) and food palatability using the Visual Analogue Scale (VAS) questionnaire.

[0771] • Assess whether the endogenous level of GDF15 is associated with the PD endpoint.

[0772] • Assess whether the PK of FP2 is associated with the PD endpoint.

[0773] 2.1.2. Part 2: Absolute Bioavailability

[0774] In overweight (BMI ≥ 25 kg / m² to ≤ 29.9 kg / m²) or otherwise healthy subjects, after a single intravenous (IV) dose of FP2:

[0775] Main objectives

[0776] The absolute SC bioavailability of FP2 was estimated by administering a single short-term IV infusion over 30 minutes (at a constant rate) to age-, sex-, and weight-matched subjects (matched with those who participated in one of the aforementioned escalating SC dose groups in Part 1).

[0777] Secondary objectives

[0778] Assess the safety and tolerability of IV-administered FP2.

[0779] 2.2. Hypothesis

[0780] Given that the primary objectives are safety and tolerability, no formal statistical hypothesis testing has been conducted for this study. All other analyses will be exploratory.

[0781] 3. Research Design and Basic Principles

[0782] Research Design Overview

[0783] This is the first-in-human (FIH) study of FP2. The study has two parts and will be conducted in overweight, otherwise healthy subjects at a single research center. Part 1 is a randomized, double-blind, placebo-controlled study evaluating the safety, tolerability, and pharmacokinetics (PK) of FP2 administered as a single, escalating SC dose. Part 2 is an open-label, single-arm study evaluating systemic exposure and PK of FP2 administered as a single-dose, short-term IV infusion over 30 minutes (constant rate).

[0784] The plan includes a maximum of approximately 62 overweight individuals (BMI ≥ 25 kg / m²). 2 Up to ≤29.9kg / m 2 Otherwise healthy male and female (non-fertile age) subjects participated in this study (Part 1 and Part 2). In Part 1 of this study, up to approximately 56 subjects were randomly assigned, and in Part 2, approximately 6 subjects were assigned.

[0785] Eligibility for subjects will be screened between day -28 and day -3. Eligible subjects will be admitted to the Clinical Research Unit (CRU) on day -2 for a baseline safety assessment. On days -1 and -3, subjects will have their food intake measured over 24 hours and will complete a VAS questionnaire to assess appetite grades and food palatability. Subjects will receive the study drug on day 1 and will remain continuously in the CRU for safety, tolerability, PK / ADA, and PD assessments until the morning of day 5, at which point they may be released. Subjects will be required to return to the CRU for outpatient visits at week 1 (day 7), week 2 (day 14), week 3 (day 21), week 4 (day 28), week 6 (day 42), week 8 (day 56), week 10 (day 70), week 12 (day 84), and at the end of the study visit (7 to 10 days later). The total study duration for each subject will be approximately 17 weeks.

[0786] Figure 38 The diagrams designed for this study are provided in the document.

[0787] 3.1.1. Part 1: Single Incremental Dose

[0788] Overweight or otherwise healthy subjects will be studied sequentially across up to seven dose groups (DGs) (eight subjects per DG). Within each DG, six subjects will be randomized to FP2 and two subjects will be randomized to a matched placebo; therefore, the ratio of active drug to placebo will be 3:1 for each dose level (see Table 67). Four male and four female subjects (three randomized to receive the active substance and one to receive the placebo from each sex group) will participate in the first DG in Part 1, where the undiluted study drug (i.e., 50 mg / mL of the undiluted formulation) will be administered to allow subjects to be matched to the corresponding IV dose group in Part 2 of the study.

[0789] The planned dose escalation scheme for FP2 is described in Table 67 below. The doses administered during the study period are based on the average dose method calculated for individuals weighing 80 kg, as specified in column “FP2 (mg SC)”:

[0790] Table 67: Planned FP2 Dose Levels in Part 1 .

[0791]

[0792] The treatment will be double-blind and randomized at each dose level.

[0793] For each dose level, subjects will be divided into four subgroups (n = up to 2 / subgroup) and administered on different days. Two sentinel subjects will be administered simultaneously on the same day (one placebo, one FP2) and will complete a 72-hour safety monitoring period before subsequent subjects in the DG can be administered. After reviewing the safety data, up to two additional subjects may be administered daily (approximately 2 hours apart) until all subjects have completed dosing. There will be at least 10 days between the last subject in the previous group and the first subject in the next DG.

[0794] Following each completed dose level, the sponsor and principal investigator (PI) will review preliminary safety and PK data to determine the next planned dose level. Each dose escalation decision will be based on blinded preliminary safety, tolerability, and PK data collected in all subjects within a given DG at least 72 hours after dosing. The minimum number of evaluable subjects required for dose escalation review (i.e., subjects who have completed the study procedure at least 72 hours after dosing) will be N = 7 for each DG.

[0795] The expected mean serum exposure (C) without FP2 administration max or AUC 0-48hr This dose will exceed the minimum unobserved adverse effect level (NOAEL) exposure in a 1-month GLP toxicology study in the most relevant species (i.e., cynomolgus monkeys).

[0796] 3.1.2. Part 2: Absolute Bioavailability

[0797] Part 2 assesses healthy overweight (BMI ≥ 25 kg / m²). 2 Up to ≤29.9kg / m 2 This is an open-label, single-arm study of systemic exposure and PK in male and female subjects administered as a single-dose IV infusion of FP2 over 30 minutes (at a constant rate). PK data from Part 2 will be used to determine the absolute bioavailability of the SC FP2 formulation.

[0798] Part 2 will recruit 6 overweight individuals (BMI ≥ 25 kg / m²). 2 Up to ≤29.9kg / m 2 ), or otherwise healthy male (n=3) and female (n=3) subjects. Subjects will be matched with subjects in the SC dose group of Part 1 (possibly DG5; 30 mg, the first dose group, in which 50 mg / mL of undiluted FP2 preparation will be used) in terms of sex, age (±5 years), and weight (±5 kg). Part 2 may begin before the completion of Part 1 of the study after the sponsor and PI have reviewed the preliminary blinded safety and tolerability data of the aforementioned DG from Part 1 of the study.

[0799] Each eligible subject in Part 2 will receive a single IV dose of FP2 administered over 30 minutes via an indwelling catheter in a suitable forearm vein as a short-term infusion at a constant rate. The IV dose for Part 2 will be selected based on the preliminary safety and PK data from Part 1. The selected IV dose will not exceed one-third of the dose already assessed as well-tolerated in Part 1 to account for expected differences in maximum exposure levels at IV administration and potential incomplete bioavailability of the SC preparation. For details, see Section 3.5, Dosage Selection, Part 2 of Example 21. For safety monitoring, one sentinel subject will be administered first, and a 72-hour safety monitoring period will be completed before subsequent subjects can be administered. The remaining 5 subjects will be subdivided into subgroups (administered at least 24 hours apart) so that no more than 2 subjects will be administered daily (approximately 2 hours apart).

[0800] 3.2. Basic Principles of Research Design

[0801] 3.2.1. General Research Design Considerations

[0802] The proposed study was a single-increment, double-blind, randomized, placebo-controlled FIH trial conducted in overweight, otherwise healthy adult subjects.

[0803] The (SAD) trial was used to evaluate the safety, tolerability, PK, immunogenicity, and PD (i.e., food intake, body weight, appetite grade, and food palatability) of FP2 (a fully recombinant homodimer of GDF15 fused with HSA).

[0804] This study has been designed to comply with relevant regulatory guidelines for first-in-human trials and other early clinical development studies (EMA Guidance EMEA / CHMP / SWP / 28367 / 07Rev.1, 2017; FDA Guidance for Industry, 2005).

[0805] Regarding the anticipated systemic safety, based on available nonclinical data and pharmacological characteristics, and in accordance with the EMA's "Guideline on strategies to identify and mitigate risks for first-in-human clinical trials with investigational medicinal products,"... 4 According to the criteria outlined in the document, FP2 is not considered a “high-risk” new biological entity (NBE).

[0806] Key trial design elements, such as the determination of the safe starting dose (based on the minimum expected biological effect level [MABEL], pharmacologically active dose [PAD], and NOAEL data), the definition of dose escalation strategies, and the definition of stopping criteria, comply with current scientific, medical, and ethical standards and requirements (see Sections 3.3, 3.4, 3.5, and 3.6 of Example 21) and are consistent with the design of other current FIH trials investigating comparable products with similar objectives.

[0807] The targeted patient population is well-defined and will be carefully selected based on a comprehensive set of applicable inclusion and exclusion criteria (see Subject Population in Section 4 of the protocol). All subjects will be monitored through regular safety follow-ups for 13 weeks following administration.

[0808] The study is designed and will be conducted in a dedicated CRU under medical monitoring conditions that ensure a high probability of early detection of adverse events and appropriate treatment interventions (if necessary).

[0809] 3.2.2. Blinding, Control Group, Study Phase / Period, Treatment Group

[0810] Part 1

[0811] The double-blind, placebo-controlled, randomized study design allows for the optimal practical assessment of the safety and tolerability characteristics of FP2 by minimizing potential biases during data collection and clinical endpoint assessment. Placebo control will be used in Part 1 to assess the frequency and magnitude of possible changes in clinical endpoints in the absence of active treatment. Randomization will be used to minimize bias in assigning subjects to the treatment group and increase the likelihood of a balanced distribution of known and unknown subject attributes (e.g., demographics and baseline characteristics) between the treatment groups.

[0812] Part 2

[0813] Part 2 is an open-label, single-arm study design that will provide formulation-independent IV PK data on the disposal of FP2 that are not otherwise available and will be used to estimate the absolute bioavailability (BA) of the SC FP2 dosage form.

[0814] 3.2.3. Research Group

[0815] The basic principle for recruiting overweight, otherwise healthy subjects in the study is as follows:

[0816] FP2 will be administered via the SC route, and the absorption characteristics (i.e., rate and extent of absorption) of the drug from the SC tissue may differ between different subjects (e.g., males vs. females) and groups (e.g., lean vs. overweight vs. obese subjects). Therefore, this study aims to determine the initial human PK in the relevant groups so that repeated dose PK and dose selection in groups approaching the target population can be reliably predicted before exposure to overweight or obese subjects in longer-term trials.

[0817] Because the screening criteria will exclude subjects with clinically significant conditions known to be more prevalent in overweight individuals (e.g., type 2 diabetes mellitus, hypertension), the subject risk for overweight, otherwise healthy subjects is considered comparable to that for lean, healthy subjects.

[0818] Recruiting overweight or otherwise healthy subjects allows for a preliminary assessment of the safety and PD effects of FP2 in a subject population (such as food intake, weight, appetite grade, and food palatability) that is close to or equivalent to the study population expected to participate in Phase 2.

[0819] 3.2.4. Pharmacokinetics and Pharmacodynamics

[0820] The timing and duration of PK sampling in this study were based on non-clinical PK data, including allometric growth model predictions. Using this information, a frequent blood sample collection schedule will allow for a complete characterization of PK curves and provide the data needed to define key PK parameters required to support further clinical development.

[0821] Food intake was assessed throughout the 24-hour period of the study (before administration and at the expected T). max Monitoring and weight will allow for the assessment of potential reductions in food intake (i.e., reduced calorie intake) and possible weight loss during single-dose administration of FP2. Frequent completion of the VAS questionnaire will allow for characterization of changes in appetite behavior that may be associated with reduced food intake and weight loss during single-dose treatment with FP2.

[0822] 3.2.5. Safety and Tolerability

[0823] Most findings documented in a 1-month GLP toxicology study in rats and monkeys with FP2 were considered minor for the observed significant reduction in food intake and weight loss, which is the hypothetical target pharmacology for this class of drugs. Overall, FP2 was considered well-tolerated in the toxicology studies, and no findings were noted that would require dedicated monitoring.

[0824] Therefore, safety monitoring in this study will include a range of standard safety assessments, such as vital signs (heart rate, systolic and diastolic blood pressure, body temperature), standard clinical laboratory tests (hematology, clinical chemistry, urinalysis, lipids, coagulation), physical examination, monitoring of signs and symptoms / adverse events [TEAEs] occurring during treatment, including allergic reactions / hypersensitivity reactions and local injection site reactions, and documentation of a series of standard 12-lead ECGs. Continuous lead II ECG monitoring will also be conducted in Part 2.

[0825] 3.2.6. Immunogenicity

[0826] Since the immunogenic potential of NBE is part of its overall safety profile, the potential immunogenicity of FP2 will be monitored through continuous quantification of ADA and screening for antibodies that can target the formation of endogenous GDF15.

[0827] 3.2.9. IV Administration (Part 2)

[0828] Part 2 of the study is an open-label, single-arm study to evaluate the systemic exposure and PK of FP2 administered as a single-dose IV infusion over 30 minutes (constant rate) to overweight, otherwise healthy subjects matched for age, sex, and weight to an appropriate SC reference group (the first dose group of the undiluted study drug in Part 1). Part 2 will provide formulation-independent IV PK data on the disposition of FP2 that are not otherwise available and will be used to estimate the absolute BA of the SC FP2 dosage form. The six subjects who received the IV infusion of FP2 in Part 2 are routinely used to assess the absolute BA and serve as a benchmark for evaluating the drug quality properties of the SC dosage form.

[0829] 3.3. Dosage Selection and Elevation Principles (Part 1)

[0830] An indirect response PK / PD model between FP2 PK and food intake (combined with a physiological representation of the relationship between food intake and body weight (BW)) was developed to simultaneously characterize the PK and PD (changes in food intake and BW from baseline) of FP2 at three study dose levels in a repeated-dose study in overweight cynomolgus monkeys (see Example 19). In this study, food intake decreased most significantly from week 2 to week 3 and showed a dose-dependent decay with continued treatment; however, body weight decreased continuously until week 4 (in the 1 nmol / kg group) or week 7 (in the 10 nmol / kg group), after which it plateaued. To characterize these observations, a novel physiology-based PK / PD model was developed to describe treatment-induced changes in both food intake (FI) and therefore body weight (BW) by including items describing compensatory changes in food intake and energy expenditure in response to weight loss. Changes in BW were described as the longitudinal effect of changes in food intake and energy expenditure over time. This PK / PD model was able to describe food intake and BW trajectory in a 12-week study and provide an exposure-response relationship for FP2 in overweight cynomolgus monkeys. The BW reduction-dependent compensation food intake item in the model allows parameters of the drug's effect on food intake to remain constant over time for a given exposure. The semi-mechanistic model developed in cynomolgus monkeys enabled further translational modeling based on the known relationship between human energy intake and BW changes, where results showed that for a given % reduction in energy intake, humans had a greater % reduction in BW than cynomolgus monkeys. The modeling results also quantitatively support the mechanism of action of FP2, and that BW reduction is primarily driven by drug-induced reduction in food intake in overweight cynomolgus monkeys. This modeling approach was used to determine PAD and effective clinical dose / exposure in humans.

[0831] Assuming that the PK / PD relationship of FP2, along with physiologically relevant parameters and SC bioavailability, can be transferred between overweight cynomolgus monkeys and humans, the preliminary predicted human weekly SC dose, conferring a 20% reduction in food intake at week 12, is approximately 0.08 mg / kg (approximately 0.5 nmol / kg). This is based on published literature. 13,12,11 According to model simulations, a dose providing a 20% reduction in food intake at week 12 after weekly SC administration corresponds to a weight loss of more than 10% after one year of treatment.

[0832] 3.3.1. Adjustment of starting dose

[0833] Based on toxicological (NOAEL) and pharmacological (PAD) data, and in accordance with relevant regulatory guidelines for first-in-human trials.3,6 The starting dose for this study was selected.

[0834] The NOAEL dose used in the 1-month toxicity study in rats and cynomolgus monkeys was 100 mg / kg for rats and 50 mg / kg for cynomolgus monkeys. The human equivalent dose (HED) was calculated by normalizing the dose to body surface area. The maximum recommended starting dose (MRSD) was calculated using a default safety factor of 10 to provide a safety margin for protecting human subjects receiving the initial clinical dose. 6 As reflected in the exposure ratio calculations described below, using a safety factor of 10, the MRSD for FP2 is calculated to be 1.6 mg / kg BW. For an individual weighing 80 kg, the MRSD dose is calculated to be 128 mg.

[0835] Based on its pharmacology, FP2 is expected to function similarly to endogenous GDF15 to reduce food intake, leading to weight loss. However, the mechanism of action and non-clinical safety profile of FP2 suggest that it does not meet the criteria for being considered a high-risk product.

[0836] Since repeated-dose cynomolgus monkey studies showed drug activity at much lower doses (i.e., reduced food intake), a PAD-based approach should be used to guide MRSD. To this end, human PK parameters for FP2 were predicted using a fixed exponential allometric growth scale for PK parameter estimation via modeling, based on body weight following a single SC administration in overweight cynomolgus monkeys.

[0837] Based on this, a PK / PD model for FP2 was developed using PK and PD data at three study dose levels in cynomolgus monkeys. This model predicted that a single SC dose of 0.05 mg / kg (approximately 0.3 nmol / kg) would result in a reduction of approximately 10% of maximum food intake, which was considered a meaningful threshold indicative of pharmacological activity. Since the predicted reduction in food intake associated with 0.05 mg / kg was not considered critical for subject safety, and no other known safety-critical PD effects were observed, a safety factor of 5 (instead of the default safety factor of 10) was applied to the model-estimated 0.05 mg / kg dose. This safety factor was chosen also considering the 2-fold in vitro binding affinity of FP2 to the recombinant GFRAL fusion protein in humans and cynomolgus monkeys, as well as comparable tissue expression patterns of the GFRAL receptor in monkeys and humans, resulting in an MRSD of 0.01 mg / kg. Since a flat-dose approach based on 80 kg body weight would be used in this study, the PAD-based MRSD was calculated to be 0.8 mg (0.01 mg / kg × 80 kg). This indicates that the MRSD based on PAD is approximately 160 times lower than the MRSD based on NOAEL.

[0838] The PAD-based MRSD is expected to produce a maximum serum drug concentration of approximately 0.6 nM at a dose of 0.01 mg / kg, which is 13 times higher than the upper limit of the normal range for endogenous GDF15 (i.e., approximately 0.046 nM or 1.15 ng / mL). 2 It was also 5 times lower than the median level of endogenous GDF15 found in pregnant women without complications (i.e., about 3.2 nM or 80,000 pg / mL). 20 This concentration value, when adjusted for differences in: 1) human GFRAL receptor binding affinity (FP2 binding affinity is 11-fold lower than endogenous GDF15); and 2) power differences from in vitro functional assays (EC5 of FP2 relative to endogenous GDF15 in pAKT functional assays using rhGFRAL-expressing SK-N-AS cells). 50 The value decreased by approximately 19-fold, resulting in the predicted maximum concentration of FP2 in humans after a single SC dose of 0.01 mg / kg being within 1.2 times the normal upper limit range of endogenous GDF15 in lean individuals.

[0839] In addition, C is expected to come from the starting dose of 0.01 mg / kg. max EC50 levels lower than those measured using pAKT function assays in cells expressing rhGFRAL. 10 .

[0840] 3.3.2. Maximum dose

[0841] NOAEL doses in 4-week GLP rat or cynomolgus monkey toxicology studies resulted in mean C max The values ​​were 415 μg / mL (days 1–4, females and males) and 1,117 μg / mL (days 22–29, females and males), respectively, and the mean AUC values ​​were 883 μg / mL (days 1–4, males and females) and 6,341 μg / mL (days 22–29, males and females), respectively. No mean drug exposure (by C) was observed between male and female cynomolgus monkeys depicted by sex. max Significant differences were observed in drug exposure (as assessed by Cmax and AUC) and other TK parameters in female rats. 第1-4天 The assessed drug exposure trend was slightly higher than that in male rats. The expected average Cw of the maximum dose of 1.08 mg / kg BW planned in Part 1 was... max The AUC exposure was compared with the mean C at NOAEL doses in cynomolgus monkeys. maxThe AUC exposure was approximately 97 to 21 times lower. As Part 1 of this study progresses, PK data from each sequential DG will be used to simulate systemic exposure to FP2 and further refine these exposure estimates. Subsequent doses may be adjusted based on a review of the emerging safety, tolerability, and PK data, but will not exceed the planned maximum dose.

[0842] The CV safety study in instrumented cynomolgus monkeys did not confirm any meaningful findings at the highest tested SC dose of up to 50 mg / kg, and therefore is not considered a limit to the planned maximum exposure outlined above.

[0843] 3.4. Dosage escalation

[0844] The planned dose range in Part 1 will allow characterizing the dose that is expected to provide a safety margin (≥10-fold) of expected repeated dose exposure from the therapeutically effective dose to be explored in future studies in obese subjects, and will account for potential increased exposure in special populations (e.g., subjects with kidney and liver impairment) and environments (e.g., drug-drug interaction studies, thorough QT / QTc studies, etc.).

[0845] The proposed dose escalation strategy follows this concept: approximately a 3-fold dose escalation after the first two dose escalation steps reaches the third dose level in the study, at which point the previous dose levels have been shown to be safe and the exposure level (C) has been determined. max and AUC 0-72小时 This shows a roughly proportional dose-to-p-value (or less-than-proportional) increase in exposure. Following the same guidelines, the third, fourth, and fifth dose escalation steps at dose levels 4, 5, and 6 will consist of approximately a 2-fold dose escalation, while all subsequent dose escalation steps (if any) will be planned with approximately a 50% dose escalation (Table 3).

[0846] Following each completed dose level, initial safety and PK data will be reviewed by the sponsor and PI to determine the next planned dose level escalation. Each dose escalation decision will be based on blinded preliminary safety, tolerability, and PK data collected in all subjects in a given dose group at least 72 hours post-dose, and preliminary PK data obtained at least 72 hours post-dose. The minimum number of evaluable subjects required for review (i.e., subjects who have completed the study procedure at least 72 hours post-dose) will be N=7 for each dose group. Any additional clinically relevant information from 72 hours post-dose up to the review meeting will be communicated by the investigator at the review meeting. As the study progresses, cumulative safety data from previous dose groups will be reviewed periodically and as part of each dose escalation decision meeting. If the planned dose is supported by preliminary PK, safety, and / or tolerability data from previous doses, it may be modified and may be reduced or repeated, but not increased, unless a substantial modification to the study protocol is published and submitted to a competent Health Authority (HA) and Independent Ethics Committee (IEC).

[0847] There will be at least a 10-day interval between the last subject in the previous dose group and the first subject in the next dose group.

[0848] The NOAEL dose in the 4-week GLP rat or cynomolgus monkey toxicology study will be used to guide the upper limit of targeted exposure in the study (see Section 3.3.2 of Example 21 for details).

[0849] 3.5. Dosage Selection Part 2

[0850] The dose intensity for Part 2 will be selected based on the preliminary safety and PK data from Part 1, and will not exceed one-third of the dose assessed as well-tolerated in Part 1 to provide approximately 3-fold safety margin, provided that the FP2 SC BA in overweight human subjects is substantially lower than that established in lean cynomolgus monkeys (99% absolute BA after a single IV dose of 1.0 mg / kg). This is in accordance with the C... max The value was only slightly higher than the C value observed when the same dose was administered at SC. max The dose is approximately 2 times higher, therefore the expected 3-fold reduction in well-tolerated SC dose also corresponds to the expected maximum safe exposure (C) for constant-rate IV infusion of FP2 over 30 minutes. maxThis provides sufficient margin. For example, if the 30 mg SC dose administered in dose group 5 is considered safe and well-tolerated, the IV dose in part 2 can be selected based on that DG and will be either 30 mg or 1 / 3 of 10 mg. This would be based on the assumption that the absolute BA after SC administration is low to approximately 33% in overweight subjects, and therefore the 10 mg IV dose intensity will ensure that the AUC after IV administration will not exceed the AUC obtained after 30 mg SC administration. Because BA is determined by AUC (not C... max The calculations and the 33% BA assumption are conservative; therefore, an IV dose intensity of 1 / 3 of the SC dose may produce a lower C than 30 mg SC. max (i.e., the infusion is finished.)

[0851] 3.6. Stop Criteria

[0852] 3.6.1. Individual Termination Criteria - Part 1

[0853] Since Part 1 of the study only involves a single SC administration of FP2, individual discontinuation criteria for the study drug (such as dosing cessation) are not applicable. However, if a randomized subject withdraws from the study, he / she will not receive the study drug (see Section 10.2 of Example 21).

[0854] 3.6.2. Individual Termination Criteria - Part 2

[0855] In cases of medically significant adverse events (AEs), the IV infusion will be discontinued, indicating that the subject is at potential risk as determined by the investigator. Such significant medical AEs include, but are not limited to, the following findings:

[0856] In two consecutive measurements (15 minutes apart) following the first occurrence, the subject had an absolute QT of ≥500 ms corrected according to the Fridericia formula (QTcF) or an increase in QTcF of >60 ms relative to baseline.

[0857] Subjects with tachycardia were defined as having a resting supine heart rate >100 bpm, based on continuous heart rate monitoring lasting at least 15 minutes after the first occurrence.

[0858] Subjects with bradycardia were defined as having a resting supine heart rate <45 bpm, based on continuous heart rate monitoring lasting at least 15 minutes after the first occurrence.

[0859] Subjects who developed hypertension were defined as having a resting supine systolic blood pressure (SBP) of 180 mmHg or higher, and for at least 15 minutes after the first occurrence.

[0860] If a subject experiences a severe or serious adverse event, and the researchers deem it to be in the best interest of the subject to discontinue the IV infusion for safety reasons.

[0861] Once discontinued for any of those reasons, the administration of the study drug is considered definitively terminated (i.e., it will not be restarted). The reason for the discontinuation of the study drug will be documented.

[0862] 3.6.3. Research Termination Criteria

[0863] If at any time the study is to proceed (within the completed DG or progressing to a higher dose level), it will be suspended:

[0864] - Two subjects withdrew from the study (regardless of dose group or period) due to experiencing any medically significant or serious adverse event (SAE). The adverse events were caused by...

[0865] - The investigator assessed it as possibly, very likely, or extremely likely to be related to the investigational drug, or

[0866] -1 subject experienced an SAE that the investigator assessed as possibly, very likely, or extremely likely to be associated with the study drug.

[0867] An internal DRC, independent of the clinical research team, can be assembled (see Section 11.8). The purpose of the DRC will be to review all unblinded safety data. Following this in-depth safety review, one of the following recommendations will be made:

[0868] The study will continue as planned (i.e., there are no significant security concerns).

[0869] The study will continue by repeating the current dose in a larger number of subjects.

[0870] Continue the study at a dose between the current dose and the next planned dose, or at a dose between the current dose and the previous lower dose.

[0871] The study was terminated.

[0872] 4. Subject population

[0873] Screening for eligible subjects will be performed within 28 days prior to administration of the study drug.

[0874] For Part 1 and Part 2, at least two additional reserve subjects will be admitted on day -2 of hospitalization and will undergo all assessments prior to dosing to ensure that the full dose group is randomized.

[0875] If a replacement is required, additional subjects may be recruited; each replacement subject will complete the entire study of the subject they are replacing according to the randomization schedule (see Section 5, Study Drug Allocation and Blinding). No subject may participate in more than one dose group or part of this study. Reserve subjects may be rescreened to participate in another dose group of the study.

[0876] In Part 1, four males and four females (each sex group randomly assigned to three active substances and one placebo) will participate in the first dose group, where the undiluted study drug (i.e., 50 mg / mL of undiluted formulation) will be administered to allow subjects to be matched with the corresponding IV dose group in Part 2 of the study.

[0877] The inclusion and exclusion criteria for recruiting participants in this study are described in the following two sections. If there are any questions regarding the inclusion or exclusion criteria below, the researchers will consult with the appropriate sponsor representative and resolve any issues before recruiting participants for this study. Waivers are not permitted.

[0878] For a discussion of the statistical considerations for subject selection, see Section 11.2, Sample Size Determination, in Example 21.

[0879] 4.1. Inclusion Criteria

[0880] Each potential participant will meet all of the following criteria to participate in the study:

[0881] Ages 18 to 45

[0882] (Including the end value) male or female.

[0883] Body Mass Index (BMI) between 25.0 kg / m² 2 and 29.9 kg / m 2 (Including the extreme values) and weight ≥80kg.

[0884] Based on physical examination, medical history, vital signs, clinical laboratory tests, and a healthy 12-lead ECG performed at screening and at baseline (day -2 and / or day -1). If any results are abnormal, the subject may be included only if the investigator determines that the abnormality or deviation from the normal range is not clinically significant. This decision will be documented in the subject's source document and drafted by the investigator.

[0885] He or she will sign an informed consent form (ICF) indicating that he or she understands the purpose of the study and the required procedures and is willing to participate in the study.

[0886] Women of childbearing age will be defined as:

[0887] Postmenopause

[0888] Postmenopausal status is defined as the absence of menstruation for at least 12 months without alternative medical cause, and a follicle-stimulating hormone (FSH) level (>40 IU / L or mIU / mL) at screening within the postmenopausal range. However, if a subject has been amenorrhea for less than 12 months, two FSH measurements (one from the subject's medical record) are required to confirm postmenopausal status. All women should have a negative serum beta-human chorionic gonadotropin (hCG) pregnancy test at screening; and a negative urine pregnancy test on day -2 of admission.

[0889] Permanent infertility

[0890] Permanent infertility is caused by procedures such as hysterectomy, bilateral salpingectomy, bilateral tubal occlusion / ligation, and bilateral oophorectomy, or by other means of preventing pregnancy, as documented in medical records. All women should have a negative serum hCG pregnancy test at screening and a negative urine pregnancy test on day -2 of gestation.

[0891] The resting heart rate (after the subject has been lying supine for 5 minutes) should be between 50 and 90 beats per minute (bpm). If the heart rate is outside this range, a maximum of two repeat assessments are permitted.

[0892] Blood pressure (after the subject has been lying supine for 5 minutes) should be between 90 mmHg and 140 mmHg systolic (inclusive) and not higher than 90 mmHg diastolic. If blood pressure is outside this range, up to two repeat assessments are permitted.

[0893] Men will consent to use condoms (including those who have undergone vasectomy), even if their partners become pregnant (to ensure the fetus is not exposed to the study drug through vaginal absorption), and will not donate sperm during the study and for three months after its conclusion. In addition to condoms, male study participants should encourage their female partners to use effective methods of contraception (such as prescription oral contraceptives, injections, intrauterine devices, double barrier methods, and contraceptive patches).

[0894] We are willing to abide by the prohibitions and restrictions stipulated in the research plan.

[0895] Subjects will enjoy and typically consume the food items provided for the 24-hour food intake assessment (at least one of the main course and side dish from the lunch and dinner menus) and will have a habitual eating pattern of 3 main meals per day (breakfast, lunch, and dinner).

[0896] If a participant agrees to provide an optional DNA sample for the study, he or she will sign a separate informed consent form. Refusal to provide an optional DNA sample for the study does not preclude the participant from participating in the study.

[0897] 4.2. Exclusion Criteria

[0898] Any potential participant who meets any of the following criteria will be excluded from participation in this study:

[0899] A history of significant illness or medical disorder or a currently active significant illness or medical disorder, including (but not limited to) cardiovascular diseases (including arrhythmias, myocardial infarction, stroke, peripheral vascular disease), endocrine or metabolic disorders (e.g., diabetes, hyperthyroidism / hypothyroidism, severe hypertriglyceridemia [>400 mg / dL]), hematological disorders (e.g., von Willebrand's disease or other bleeding disorders), respiratory diseases, liver or gastrointestinal diseases, neurological or psychiatric diseases, ophthalmic disorders (including retinal disorders or cataracts), adenoid disorders, skin disorders, kidney disorders, or any other disease that the investigator deems appropriate to exclude the subject or that may interfere with the interpretation of the study results.

[0900] Previous surgical treatment for obesity or recent weight changes (≥5%) due to diet (including screening for commercial weight loss programs or pharmacological treatments within the past 6 months).

[0901] Life history of any eating disorder or high risk of eating disorder (using Questionnaire-5 on eating and weight patterns [QEWP-5]). 25 See Appendix 1.

[0902] A life history of malignant tumors or a family history of susceptibility to malignant tumors is defined as at least two close relatives on the same side of the family (defined as parents, siblings, children, grandparents, aunts, uncles, nephews, nieces) having the same type of cancer, or a close relative having more than one type of cancer.

[0903] Close relatives who had cancer at a young age (<50 years old), or close relatives who had cancer in either of a pair of organs (e.g., both kidneys), or more than one child in a sibling family who had cancer, or male relatives who had breast cancer, or cancer that had occurred in many generations (e.g., grandfather, father, son).

[0904] A history of abnormal or positive results from routine cancer screening tests (e.g., prostate-specific antigen [PSA] in men, Pap smear or mammogram in women).

[0905] Genetic syndromes that predispose individuals to cancer (e.g., BRCA1 and BRCA2, Lynch syndrome, familial polyposis syndrome, Li-Fraumeni syndrome, and multiple endocrine neoplasia syndrome).

[0906] At screening or on day -2 of admission, aspartate aminotransferase (AST) and alanine aminotransferase (ALT) exceeded the upper limit of normal (ULN) of the clinical laboratory reference range.

[0907] Total bilirubin exceeding 1.5 times the ULN (i.e., 1.5 x ULN) was used to explain the slightly elevated bilirubin levels in subjects with Gilbert syndrome (a harmless congenital non-hemolytic low-grade hyperbilirubinemia due to UGT1A1 polymorphism).

[0908] At screening or on day -2, hemoglobin, hematocrit, or red blood cell count were below the lower limit of the normal range of the clinical laboratory reference range.

[0909] At screening or on day 2, abnormal fasting blood glucose (i.e., >125 mg / dL or >6.9 mmol / L; matrix plasma from venous blood samples) and / or hemoglobin A1c (HbA1c) were detected. 1c (i.e., >6.4% [high performance liquid chromatography] or >42 mmol / mol Hb). Blood glucose measurements may be repeated during screening if dietary inadequacy is suspected of not meeting the required overnight fasting period.

[0910] Serum creatinine levels above the clinical laboratory reference range at screening or at day -2 admission are considered ULN.

[0911] At the time of screening, the thyroid-stimulating hormone (TSH) level was outside the normal limit of the clinical laboratory reference range.

[0912] Do not take any prohibited therapies, pre-study and concomitant therapies for up to 30 days before the first dose of the study drug program.

[0913] A history of drug or alcohol abuse according to the Diagnostic and Statistical Manual of Mental Disorders (5th Edition) (DSM-V) criteria within 2 years prior to screening, or a positive test result for alcohol or drug abuse (including but not limited to barbiturates, opioids, cocaine, cannabinoids, amphetamines, and benzodiazepines) at the time of screening or on day-2 of admission.

[0914] Known allergies, hypersensitivity, or intolerances to any excipients of FP2 (see Section 2.3 of the IB for formulation information).

[0915] Donate blood or blood products (approximately 450 mL) or lose a significant amount of blood within two months prior to the first administration of the investigational drug.

[0916] Before the first dose of the investigational drug program, receive the investigational drug (including investigational vaccines) or use an invasive investigational medical device within one month or within a period less than 10 times the drug's half-life (whichever is longer).

[0917] Pregnant or breastfeeding, or planning to become pregnant during this study or within 12 months after the last dose of the study drug.

[0918] I plan to become a father during the study or within 12 months after the last dose of the study drug.

[0919] A history of positive hepatitis B surface antigen (HBsAg) or hepatitis C antibody (anti-HCV) or other clinically active liver disease, or a positive HBsAg or anti-HCV test during screening.

[0920] A history of positive human immunodeficiency virus (HIV) antibodies, or a positive HIV test result during screening.

[0921] Subjects who have undergone major surgery (e.g., requiring general anesthesia) within 6 months prior to screening, or who have not yet fully recovered from surgery, or who are scheduled to undergo surgery during the intended participation period of the study or within 12 months of the last dose of the study drug, may participate. Note: Subjects scheduled for surgery under local anesthesia may participate if the investigator consents.

[0922] Subjects who smoke (or have an equivalent) and / or have used nicotine-based products within 3 months prior to administration of the study drug, or who tested positive for cotinine at screening or on day-2 of admission.

[0923] Drinks that average more than 1,200 mL (i.e., 5 cups, combined total volume) per day of tea / coffee / cocoa / cola / caffeinated beverages (e.g., energy drinks).

[0924] Subjects who were strict vegetarians or vegetarians had food allergies or food intolerances.

[0925] Psychological and / or emotional problems that would invalidate informed consent or limit a subject’s ability to meet research requirements.

[0926] Subjects were unable or unwilling to undergo multiple venipunctures due to poor tolerance or difficulty in accessing the vein.

[0927] The researchers believe that participation in this study is not in the best interests of the participants (e.g., may harm their health) or may prevent, limit, or obscure any condition that may prevent, limit, or confuse the assessments specified in the protocol.

[0928] Employees of the sponsor, researcher, or research site who are directly involved in the proposed research or other research under the guidance of the researcher or research site, as well as family members of the employee, researcher, or sponsor.

[0929] Subjects live in institutions under orders from courts or regulatory agencies.

[0930] Randomization was performed in the previous dose group of this study.

[0931] 4.3. Prohibitions and Restrictions

[0932] Potential participants must be willing and able to comply with the following prohibitions and restrictions during the research process to be eligible to participate:

[0933] Agreement to comply with all requirements (e.g., contraception requirements) that will be met during the study, as indicated in the inclusion and exclusion criteria.

[0934] Strenuous exercise can affect study-specified assessment and safety laboratory results; therefore, throughout the study, from three (3) days before screening until the end of the study visit, strenuous exercise (e.g., long-distance running of 5 km / day, weightlifting, or any physical activity that the subject is not accustomed to) should be avoided.

[0935] Participants will be instructed to avoid donating blood for at least 3 months after the study is completed (i.e., the end-of-study visit).

[0936] No alcohol or alcoholic beverages are permitted for at least 24 hours prior to screening and before admission to the CRU on day -2, until the end of the decision-of-presence period on day 5, and at least 24 hours prior to all other outpatient clinical visits. During the remaining days of the study, alcohol consumption should be limited to a maximum of 24 grams per day for men (i.e., 0.5 L beer / day or 0.25 L wine / day or 3 glasses [2 cL / glass] of alcohol / day).

[0937] The maximum daily intake for women is 12 grams (equivalent to 0.25L of beer / day, 0.125L of wine / day, or 1.5 glasses [2cL / glass] of alcohol / day).

[0938] From 48 hours before Day 1 until the end of the accommodation period on Day 5, participants may not consume any food or beverage containing grape juice, Serbian oranges (including any orange marmalade), or quinine (e.g., tonic water).

[0939] From 48 hours before day 1 (day of study drug administration) until day 5, participants will avoid any products containing methylxanthine (e.g., chocolate bars or beverages, coffee, tea, cola, or energy drinks). During the remaining days between screening and follow-up, participants will be instructed not to consume more than 1,200 mL of tea / coffee / cocoa / cola per day (5 cups, combined total volume).

[0940] At least 72 hours before admission to the CRU, participants will be instructed to abstain from poppy seed consumption, as this can interfere with drug screening.

[0941] From three months prior to administration of the study drug until the end of the study, smoking (or equivalent) and / or using nicotine-based products are not permitted.

[0942] During the clinic phase, participants will not consume any food or beverages other than those provided by the research site staff.

[0943] Participants agreed to follow the contraceptive requirements as indicated in the inclusion criteria. There is no information regarding the effects of FP2 on sperm or its effects in the body, nor on fetal development. Importantly, participants and their partners should not become pregnant during the study or for up to 3 months after the study. Participants should inform the investigators whether their partners became pregnant during the study or within 3 months after the study's completion (i.e., the end-of-study visit).

[0944] 5. Study drug allocation and blinding.

[0945] Part 1

[0946] On day 1, prior to administration of the study drug, all eligible participants will be randomized. A computer-generated randomization schedule will be provided by the sponsor and kept at the CRU pharmacy.

[0947] Within each DG, participants will be randomly assigned to active treatment (FP2) or placebo based on a computer-generated randomization plan prepared prior to the study by or under the supervision of the sponsor. Randomization will be balanced using blocks of randomized changes. A total of 6 participants will receive FP2 and 2 participants will receive placebo within each DG. For DGs in Part 1 that will be matched with Part 2, 4 women and 4 men will be recruited, and each sex group will be randomly assigned in a 3:1 ratio (3 FP2 participants and 1 placebo participant).

[0948] For each DG, subjects will be randomly assigned to four subgroups (n = up to 2 per subgroup) and administered on different days. On day 1 of each DG, the first subgroup of 2 subjects (the sentinel group) will be randomized in a 1:1 ratio to either FP2 or placebo and will be administered at approximately the same time on the same day to allow for assessment of safety and tolerability over a period of up to 72 hours. Any adverse events reported / observed in subjects administered in the first subgroup that could affect the administration of the remaining subjects in the DG will be communicated to the sponsor before randomization and administration of additional subjects. The remaining 6 subjects (1 placebo, 5 FP2) will be randomized in a 5:1 ratio to either FP2 or placebo (5 FP2 and 1 placebo). Following the administration of the sentinel subjects, the remaining 6 subjects in each DG will be administered in groups of 2 (administered approximately 2 hours apart) over approximately 3 days (at least 24 hours apart).

[0949] At CRU, the unblinding pharmacist will prepare individual subject doses of the investigational drug according to the randomization schedule and will apply blinding labels before dispensing and masking the syringe to avoid accidental unblinding due to solution color. Administration of the investigational drug will be performed by researchers who are not involved in any safety assessments of the study. Each subject will be provided with a sealed randomization code containing coded details of the investigational drug. These sealed codes will be kept together in a limited access area accessible 24 hours a day. All randomization codes (open or sealed) will be collected after the subjects have completed their study.

[0950] Data on investigational drug allocations that may potentially lead to unblinding (i.e., serum concentrations of the investigational drug, anti-drug antibodies in FP2 data, investigational drug preparation / interpretable data, treatment allocation) will be handled with particular care to ensure uninformed integrity and minimize the possibility of bias. This may include establishing special provisions, such as separating data under consideration where deemed appropriate by the investigator, clinical team, or other personnel, until the database is locked and unblinded. Any site or research team access to PK data will be anonymized (i.e., if individual subject data is available, only group-level data and / or virtual subject numbers will be assigned).

[0951] Under normal circumstances, unblinding should not occur until all participants have completed the study and the database is finalized. Otherwise, specific emergency treatment / action plans can only be determined by understanding the participant's treatment status, thus breaking the unawareness of individual study participants. In such cases, researchers can identify the investigational drug by opening the sealed code in an emergency. If possible, researchers are advised to contact the sponsor or its designated personnel before unblinding to discuss the specific situation. Maintain 24 / 7 telephone contact with the sponsor or its designated personnel. If the unawareness is broken, the sponsor must be notified as soon as possible. The date, time, and reason for unblinding will be recorded in the source document.

[0952] Subjects whose drug allocation has been unblinded will return for scheduled evaluations.

[0953] Generally, randomization codes are only fully disclosed once the study is completed and the clinical database is locked. However, for unblinding DRC reviews, randomization codes, and, if necessary, the conversion of randomization codes to the treatment and placebo groups, will be disclosed to authorized parties.

[0954] Part 2

[0955] Since Part 2 is an open-label treatment with a single dose, and all subjects will receive the same IV dose of FP2, randomization or other special provisions for treatment allocation are not required.

[0956] Subjects will be selected based on sex, age (±5 years), and weight (±5 kg) to match individual subjects from the reference SC dose group in Part 1 of the study.

[0957] Part 1 and Part 2

[0958] For both Part 1 and Part 2, and for each DG, at least two additional reserve subjects will be admitted on Day 2 and will undergo all assessments prior to dosing to ensure that the full dose group is randomized.

[0959] Randomization numbers will be assigned sequentially to eligible subjects, starting with 1001 in Part 1 and 3001 in Part 2. Additional subjects may be recruited as replacements to ensure that in Part 1, at least 7 subjects per dose group complete the minimum 72-hour post-dose study procedure, and in Part 2, 6 subjects complete the study procedure within a timeframe equivalent to at least two half-lives of FP2 (determined based on PK data from the previous dose groups in Part 1). Replacement subjects will be assumed to be replacing subjects receiving the same treatment and will be assigned a new randomization number equal to the randomization number of the replaced subject, but with the first digit replaced by '2' in Part 1 and '4' in Part 2. For example, subject 1004 in Part 1 will be replaced by subject 2004, and subject 3006 in Part 2 will be replaced by subject 4006. In Part 2, all subjects will receive FP2 on an open-label basis.

[0960] 6. Dosage and administration

[0961] 6.1 Research Drugs

[0962] FP2 is provided as a sterile injectable solution to be stored at -40°C and protected from light. The solution has a brownish-yellow appearance and a concentration of 50 mg / mL of FP2 in 10 mM sodium phosphate, 8% sucrose, and 0.04% polysorbate 20 at pH 6.5. FP2 is provided frozen in R2 glass vials with a 1.2 mL fill volume (Table 68).

[0963] The preparation buffer used in the FP2 preparation will be provided for this study, and will also be used as a placebo preparation and as a diluent in the preparation of the initial FP2 SC doses (DG 1 to DG 4). It is a sterile, clear solution consisting of 10 mM sodium phosphate, 8.0% sucrose, and 0.04% polysorbate 20. The preparation buffer will be used to prepare the placebo injection. The preparation buffer will be provided frozen in R2 glass vials with a 1.2 mL fill volume.

[0964] Table 68: Description of the investigational drug

[0965]

[0966] Due to the visual difference between the FP2 drug product (a brownish-yellow solution) and the placebo (a clear, colorless solution), the unblinding pharmacist will follow the instructions in the Investigational Medical Product (IMP) Handling Manual to maintain the study’s ignorance and prevent researchers and subjects from seeing the product in Part 1.

[0967] Detailed instructions on dosage preparation, administration procedures, and storage conditions for the investigational drug will be provided separately to the research site in a separate guidance document.

[0968] Part 1 Dosing :

[0969] The investigational drug will be administered by designated, trained, and qualified site personnel who are independent of the research team and not involved in any other aspect of the clinical trial. Following an overnight fast of at least 10 hours, the investigational drug (FP2 or placebo) will be administered on Day 1 as a single SC dose (maximum volume 2 mL) in the lower right quadrant of the abdomen using either a 1.0 mL insulin syringe (DG 1, DG 2, and DG 5) or a 2.0 mL syringe (DG 3, DG 4, DG 6, and DG7). For each dose group, subjects will be divided into four subgroups (n = up to 2 per subgroup) and administered on different days. Two sentinel subjects will be administered first on the same day at approximately the same time (1 placebo, 1 FP2) and will complete a 72-hour safety monitoring period before subsequent subjects in the DG can be administered. After reviewing the blinded safety data, up to two additional subjects may be administered daily in an alternating manner (approximately 2 hours apart, with one subject being administered at approximately 7 a.m. and another at approximately 9 a.m.) until all subjects in the dose group have completed their dosing.

[0970] Time zero (0) is the time for studying drug injection.

[0971] All SC injections will be performed on the anterior abdominal wall, avoiding the 2-inch (approximately 5 cm) area around the umbilicus. Prior to any injection, the person in charge at the site will check / palpate the planned injection sites. Injections should not be performed in abdominal wall areas assessed as abnormal.

[0972] Physicians with experience and training in emergency medicine and emergency equipment (including ready-to-use medications for treating allergic reactions) will be readily available in the application room during the administration of investigational drugs.

[0973] Part 2 Dosing :

[0974] Using an administration device with a filter, via a separate line, an automated infusion device (Braun) is administered via an indwelling catheter placed in a suitable forearm vein. The Compact S (or equivalent device) administers a single dose of FP2 at a constant infusion rate over 30 minutes. Dosing will be performed at approximately the same time each day, but in an staggered manner (one subject at approximately 7:00 AM and one subject at approximately 9:00 AM each day). Two sentinel subjects will be administered first, followed by two subjects at least 24 hours after the two sentinel subjects, and then the final two subjects at least 24 hours later. One sentinel subject will be administered first and will complete a 72-hour safety monitoring period before subsequent subjects can be administered. The remaining five subjects will be subdivided into groups (administered at least 24 hours apart) so that no more than two subjects will be administered each day (approximately 2 hours apart).

[0975] Time zero (0) is the start time of the IV infusion of the drug under study.

[0976] Since no placebo comparison is expected for IV administration of FP2, blinding measures for the investigational drug are not required.

[0977] Physicians with experience and training in emergency medicine and emergency equipment (including ready-to-use medications for treating allergic reactions) will be readily available in the application room during the administration of investigational drugs.

[0978] There may be technical issues requiring a temporary suspension of study drug administration (e.g., malfunction of the indwelling catheter, or the need to place a new catheter). In such cases, the infusion should be resumed as soon as possible, and the time and reason for the suspension should be recorded.

[0979] 7. Treatment adherence

[0980] The investigational drug will be administered by qualified research site personnel as a SC injection (Part 1) or as an IV infusion (Part 2), and details of each administration will be recorded in an electronic data collection system where applicable [Part 1 SC: date of injection, time of injection, volume of injection, injection site; Part 2 IV: start and stop times of IV infusion and volume of infusion].

[0981] 8. Pre- and concomitant therapies

[0982] Pre-study therapy administered up to 30 days prior to the first dose of the study drug will be recorded. Throughout the study, no therapy (prescription or nonprescription medications, including vaccines, vitamins, mineral supplements, nutritional supplements, herbal supplements [including St. John's Wort, garlic extract, and herbal teas]) is permitted within 30 days prior to the planned first dose of the study drug, except for acetaminophen. If a subject requires prescription or nonprescription medication during the study, the subject may enroll or continue in the study with the consent and approval of the sponsor (or designated person) and the principal investigator.

[0983] If the administration of any concomitant therapies becomes necessary, it will be reported in the appropriate section of the electronic case report form (eCRF). The recorded information will include a description of the drug, duration of treatment, dosing regimen, route of administration, and instructions.

[0984] Acetaminophen is permitted until 3 days prior to administration of the study drug. Throughout the study, up to three 500mg doses of acetaminophen per day and no more than 3 grams per week are permitted for the treatment of headaches or other pains.

[0985] From the first dose of the study drug until the final visit of the study, accompanying therapies will be recorded throughout the study. After the final visit, accompanying therapies should also be recorded only in conjunction with new or worsening adverse events and serious adverse events that meet the criteria.

[0986] 9. Research Evaluation

[0987] 9.1. Research Procedure

[0988] 9.1.1. Overview

[0989] The time and event schedule summarizes the frequency and timing of PK, immunogenicity, PD, exploratory biomarkers, pharmacogenomics and safety measurements applicable to this study.

[0990] The meal and VAS questionnaire time and event schedule specifies the time arrangement for the meal and VAS questionnaire.

[0991] If multiple assessments are scheduled at the same time point, and / or if one or more assessments are scheduled at the same time as a meal, the following order of procedures is recommended: vital signs, ECG, PK, blood draw, VAS questionnaire for appetite grades, meal, and VAS questionnaire for food palatability (after the first bite of food). Blood collection for PK assessment should be as close as possible to the designated time. When ECG is to be performed at the same time point as PK, the PK specimen should be obtained immediately after the ECG is completed. Other measurements may be performed earlier than the designated time point if necessary. The order of multiple assessments at the same time point should be the same throughout the study. The actual date and time of the assessment will be recorded in the source document and eCRF.

[0992] When feasible (except during IV infusion), vital signs (i.e., blood pressure [BP], heart rate [HR]) should be recorded from the opposite arm from which the blood sample was taken.

[0993] Pregnancy testing will be performed on all women at screening and throughout the study. Serum pregnancy testing will be performed at screening, and urine pregnancy testing will be available at all other points in the time and event schedule.

[0994] Table 69: Blood volumes to be collected from each subject (Parts 1 and 2 of the study)

[0995]

[0996] Approximate total e 315

[0997] a The calculation is the number of samples multiplied by the blood volume of each sample.

[0998] b Serum chemistry includes serological tests (HBsAg, anti-HCV antibody, HIV 1 and 2 antibodies) and serum β-hCG pregnancy tests.

[0999] c Only in female subjects.

[1000] d Blood samples will only be collected from subjects who have already agreed to provide optional DNA samples for the study.

[1001] For safety reasons or due to technical issues with the sample, duplicate or unplanned samples may be taken. Note: Indwelling intravenous catheters may be used for blood sample collection. [If a mandarin (occluder) is used, blood loss due to disposal is not expected.]

[1002] These volumes can be adjusted in the final laboratory manual, provided that the maximum blood volume drawn from each subject in this study does not exceed 500 mL (variations in blood collection tube size or availability are permissible). Additional blood samples may be collected based on emerging data for further safety, immunogenicity, or PK assessments if necessary, but the total blood volume collected from individual subjects during this study will not exceed the amount stated in the study protocol without prior approval from the Independent Ethics Committee (IEC) and the health authority. Duplicate or unplanned samples may be obtained for safety reasons or due to technical issues requiring prior IEC and health authority approval.

[1003] For each subject, the maximum blood volume drawn in this study will not exceed 500 mL. The total blood volume to be collected from each subject will be approximately 315 mL.

[1004] 9.1.2. Screening Period (Part 1 and Part 2)

[1005] Potential participants will be screened within 28 days prior to Day 1 administration of the study drug to determine their eligibility for study participation. If a participant meets the recruitment criteria, he / she will be admitted to the CRU on Day 2.

[1006] Before any research procedure is initiated, the PI (or designated researcher) will review and explain the written ICF to each participant. Research procedures (including fasting for research laboratory tests) can not be performed until the participant has signed the ICF. For screening visits, all participant-reported assessments should be conducted before any testing, procedure, or counseling to discontinue any participants who do not meet these entry criteria.

[1007] The researcher (or designated researcher) will also review and interpret the written ICF of the optional gene study sample prior to the pharmacogenomics blood sampling.

[1008] All adverse events (whether serious or minor) will be reported from the date of receipt of the signed and dated informed consent form until the final study procedure at the end of the final study visit, and will also be reported through direct questioning at specific time points. (See the time and event schedule.)

[1009] Retesting for outliers that could lead to exclusion will only be permitted once. Retesting may be performed during unplanned visits. If any screening tests are repeated, the test results will meet eligibility requirements and will be available for review by researchers before admission to the CRU (e.g., day -2).

[1010] 9.1.3. Inpatient Treatment Period (Part 1 and Part 2)

[1011] Day -2 and Day -1 (baseline)

[1012] Eligible participants will be admitted to the CRU on day -2 and will undergo a baseline safety assessment (day -2) and baseline ECG collection (matched to the ECG time on day 1) on day -1, as specified in the time and event schedule. On day -1, participants will also undergo a 24-hour food intake measurement and will complete a VAS questionnaire to assess appetite grades and food palatability.

[1013] Day 1 / Randomization and Dosing

[1014] After confirming that all recruitment criteria were met, eligible subjects were randomized on day 1, just before administration of the study drug.

[1015] The study drug will be administered under the supervision of the PI or his / her designated personnel. Refer to the time and event schedule for details of the study procedures and timelines.

[1016] For each dose level in Parts 1 and 2, subjects will be subdivided and administered on different days, such that no more than two subjects will be administered each day. Two sentinel subjects in Part 1 (one placebo, one FP2) and one sentinel subject in Part 2 (FP2) will be administered first, and a 72-hour safety monitoring period will be completed before subsequent subjects in the dose groups are eligible for administration. After reviewing the safety data, up to two additional subjects will be administered daily in an staggered manner (approximately 2 hours apart) until all subjects have completed dosing.

[1017] Day 3 to Day 5

[1018] On day 3, the 24-hour food intake measurement and VAS questionnaire administration will be repeated.

[1019] Subjects will remain in the CRU continuously for safety, tolerability, PK, and PD assessments until the morning of day 5, when they will be released upon completion of the study assessment.

[1020] 9.1.4. Outpatient Period (Part 1 and Part 2)

[1021] Subjects will return to the CRU for fasting (at least 10 hours) for safety, tolerability, pharmacokinetic, PD, and immunogenicity assessments, as detailed in the time and event schedule. Completion of the study end visit constitutes the end of subject participation in the study. All reasonable attempts should be made to conduct outpatient visits at the predetermined time points (i.e., the specific number of days for each visit), but a window of ±1 day is allowed up to week 4 (day 28) for the visit, and a window of ±3 days is allowed for the remaining outpatient visits up to the study end visit. All subsequent visits should be scheduled relative to the date of the first study drug dose (day 1), not relative to the date of any previously rescheduled visit.

[1022] Early exit

[1023] If a participant withdraws from the study for any reason before the end of the outpatient period, a study termination assessment should be obtained.

[1024] 9.2. Pharmacokinetics and Immunogenicity

[1025] Venous blood samples will be collected over time, as specified in the time and event schedule. PK sampling time may be adjusted based on initial PK data from previous dose groups (e.g., if FP2 serum concentration is below the lower limit of quantitation [(LLOQ]], later sampling times may be omitted). The actual date and time of each PK and immunogenic blood sample (ADA) collection will be recorded on the eCRF in the electronic data collection system. Subjects who prematurely terminate their study participation should have the final evaluation sample collected at the termination time.

[1026] 9.2.1. Evaluation

[1027] Samples collected for analyzing FP2 serum concentrations and antibodies against FP2 may be used to further evaluate the safety or efficacy in addressing issues that arise during or after the study period, to further characterize immunogenicity, or to evaluate relevant biomarkers. Subject confidentiality will be maintained.

[1028] 9.2.2. Analysis Procedure

[1029] Pharmacokinetics

[1030] Serum samples were analyzed by the sponsor or under the sponsor's supervision using a validated, specific, and sensitive immunoassay to determine the concentration of FP2.

[1031] Immunogenicity

[1032] The sponsor, or under its supervision, will use validated assays to detect and characterize serum anti-FP2 antibodies and potential antibodies against endogenous GDF15. All samples collected for ADA detection will also be evaluated against FP2 serum concentrations to enable the interpretation of antibody data.

[1033] 9.2.3. Pharmacokinetic Parameters

[1034] Pharmacokinetic parameters of FP2 will be calculated using non-regional analysis of serum concentration-time curves. Pharmacokinetic parameters following a single FP2 administration will include, but are not limited to, the following:

[1035] C max : The highest observed serum concentration.

[1036] T max Time to reach the observed maximum serum concentration.

[1037] AUC inf The area under the curve of serum concentration versus time from time zero to the infinitely large extrapolated terminal phase.

[1038] AUC last Area under the serum concentration curve against time from time zero to the time corresponding to the final quantifiable concentration.

[1039] T 1 / 2 Terminal half-life.

[1040] CL: Total systemic clearance (IV only).

[1041] CL / F: Apparent total systemic clearance after extravascular administration (SC only);

[1042] V z : Distribution volume based on terminal phase (IV only).

[1043] V z / F: Apparent volume of distribution in the terminal phase after extravascular application (SC only).

[1044] F(%): Absolute SC bioavailability, expressed as a percentage, calculated as the fraction of the systemically available dose. Absolute bioavailability is calculated using the following formula.

[1045]

[1046] 9.2.4. Immunogenicity assessment

[1047] Anti-FP2 antibodies will be evaluated in blood samples collected from all participants according to the time and event schedule. Additionally, blood samples should be collected from participants who withdraw from the study at the end-of-study visit. These samples will be tested by the sponsor or designated personnel.

[1048] Blood samples will be screened for antibodies binding to FP2, and the titer of confirmed positive samples will be reported. Further analyses may be performed to further characterize the immunogenicity of FP2.

[1049] 9.3. Pharmacodynamic Evaluation

[1050] Weight will be measured in duplicate in the morning, before breakfast, and after excretion, as detailed in the time and event schedule. Subjects will be weighed using a calibrated scale while wearing an smock or light indoor clothing and without shoes.

[1051] Twenty-four-hour food intake will be assessed on Day -1 and Day 3 by providing four meals (breakfast, lunch, snack, and dinner) served at the same times on these two days. Meals will be standardized between days (the meals on Day -1 and Day 3 will be the same). Meals will also be standardized among participants so that the composition and portion sizes will be identical for all participants and will be based on local preferences and guidelines assessed by the study dietitian. Each item in each meal will be weighed before and after consumption, and the grams consumed for each food item will be calculated and recorded. Calories consumed will be estimated based on the grams consumed for each food item and its nutrient content. Changes in 24-hour calorie intake from Day -1 to Day 3 will be evaluated. For days when food intake is not recorded (Day -2, Day 1, Day 2, Day 4, and Day 5), each meal (including breakfast and snack) should differ from the meals provided on Day -1 and Day 3.

[1052] The VAS questionnaire assessing appetite levels (hunger, craving, nausea, and satiety) will be administered hourly during wakefulness on Day -1 and Day 3, every 3 hours during wakefulness on Days 1, 2, 4, and 5, and immediately before and at the end of each meal (Day -1 to Day 5). The VAS questionnaire assessing food palatability (which may indicate possible food aversion) will include questions about the pleasantness of food odors, the taste of the meal, and its texture, and will be completed by the subjects immediately after they have finished the first bite of the main course at each meal (Day -1 to Day 5). For a detailed description of the timing of the VAS questionnaire administration, refer to the Food Intake and VAS Questionnaire Time and Event Schedule.

[1053] 9.6. Safety Evaluation

[1054] 9.6.2. Clinical laboratory testing

[1055] Fasting blood samples will be collected for chemistry, hematology, coagulation, and lipid analysis, as well as urine samples for urinalysis. Subjects will fast overnight for at least 10 hours prior to all laboratory sample collection. Reference times and event schedules will be provided for the timing of clinical laboratory assessments. Investigators will review laboratory reports, document this review, and record any clinically relevant changes occurring during the study in the adverse events section of the eCRF. Laboratory reports will be submitted along with the source documents.

[1056] The following tests will be conducted by a local laboratory:

[1057] hematological examination

[1058] -Hemoglobin-platelet count

[1059] -Hematocrit-Reticulocyte percentage

[1060] - Red blood cell (RBC) count

[1061] - Differential white blood cell (WBC) count

[1062] Note: WBC evaluation may include any abnormal cells, which will subsequently be reported by the laboratory. RBC evaluation may include abnormalities in RBC count, RBC parameters, or RBC morphology, which will subsequently be reported by the laboratory. Additionally, any other abnormal cells in the blood smear will also be reported.

[1063] Coagulation test

[1064] - Activated partial thromboplastin time (aPTT) - Prothrombin time (PT)

[1065] Chemical test

[1066]

[1067]

[1068] *Only when total bilirubin is elevated

[1069] lipid test

[1070] -Total cholesterol-High-density lipoprotein (HDL)-cholesterol

[1071] - Triglycerides - Low-density lipoprotein (LDL) - Cholesterol

[1072] Urine analysis

[1073]

[1074] If the test strip results are abnormal, a microscope will be used to measure the precipitate.

[1075] In microscopic examination, the laboratory may also report observations in addition to the presence of WBCs, RBCs, and exfoliated material.

[1076] Other laboratory tests

[1077] The following laboratory tests will be conducted at the times indicated in the time and event schedule:

[1078] All women will undergo serum and urine pregnancy tests (β-hCG).

[1079] FSH (female only), TSH and HbA1c

[1080] Serological tests (HIV 1 and 2 antibodies, HBsAg and anti-HCV antibodies)

[1081] Urine drug screening (amphetamine, barbiturates, benzodiazepines, cannabinoids, cocaine, opioids, methadone) and cotinine.

[1082] breathalyzer test

[1083] 9.6.3. Electrocardiogram

[1084] The collection criteria will be a 12-lead ECG as specified in the time and event schedule. Each ECG will be printed and stored in the subject's medical file at the study site. Furthermore, the ECG data will be electronically transmitted to a dedicated ECG laboratory (Nabios GmbH, Munich) for centralized analysis, in accordance with ICH E14 recommendations. Details regarding ECG analysis methods, reading schedules and delivery, and the format of dose escalation meeting results will be detailed in a separate ECG manual.

[1085] After the subject has been quietly resting in a supine position for at least 5 minutes and has avoided speaking or moving their arms or legs, a 12-lead ECG will be captured in triplicate at time points less than 2 minutes apart. During ECG collection, the subject should be in a quiet, undisturbed environment (e.g., television, mobile phone). If multiple assessments are scheduled at the same time points, and / or if one or more assessments are scheduled at the same time as a meal, the following sequence is recommended: vital signs, ECG, PK, blood draw, VAS questionnaire for appetite grading, meal, and VAS questionnaire for food palatability (after the first bite of food). Blood collection for PK assessment should be kept as close as possible to the designated time. When ECG is to be performed at the same time point as PK, the PK specimen should be obtained immediately after ECG completion. Other measurements may be performed earlier than the designated time points if necessary. The order of multiple assessments at the same time points should be consistent throughout the study.

[1086] ECGs will be performed in triplicate at each predetermined time point for a more accurate assessment of QTc interval variation. ECGs obtained on day -1 should be timed to match those obtained on day 1. At each time point requiring triplicate ECGs, three separate ECG lines should be obtained as closely and consecutively as possible, but no more than 2 minutes apart. The complete set of triplicate ECGs should be completed in less than 4 minutes.

[1087] The average of the triplicate measurements at each time point on day -1 will be used as the baseline value for time matching of the corresponding parameter for each subject on day 1.

[1088] In addition to the off-site ECG reading process, each ECG should be reviewed for potential clinical safety relevance findings (e.g., QTc interval prolongation) after collection by a qualified research site physician. Any pathological findings and corresponding medical interventions (if any) will be documented in the eCRF.

[1089] 9.6.4. Continuous Lead IIECG Monitoring (Part 2 only)

[1090] Continuous lead II ECG monitoring will be performed only on Day 1, in Part 2, from 30 minutes before the start of the IV infusion until 2 hours after completion of the infusion. Continuous lead II ECG monitoring may be extended at the investigator's (or designated) discretion. This data will be used for real-time visual monitoring, and any abnormalities detected by the ECG monitoring device or by the investigator will be printed out and retained as source data. An unplanned 12-lead ECG measurement should also be performed as soon as possible after an abnormality is identified. Any clinically significant abnormality will be recorded as an adverse event.

[1091] 9.6.5. Vital signs

[1092] Blood pressure and heart rate (HR) measurements will be performed in the supine position using a fully automated oscilloscope. Manual techniques will only be used when automated equipment is unavailable. Vital signs should be measured using the opposite arm from which blood samples were collected (except during IV infusions).

[1093] Blood pressure and heart rate (HR) measurements should only be taken after resting undisturbed in a quiet environment (e.g., away from television, mobile phones) for at least 5 minutes. Individual BP and HR measurements will be taken and recorded at all time points.

[1094] The same method used for body temperature measurement (i.e., tympanic cavity) will be used for all measurements of all subjects throughout the duration of the study.

[1095] The timing of these checks is specified in the time and event schedule.

[1096] 9.6.6. Physical Examination

[1097] A complete physical examination includes routine medical tests, including: general appearance, neurology, eyes, ears, nose and throat, thyroid, cardiovascular, respiratory system, abdomen / gastrointestinal tract, liver, musculoskeletal system, and dermatology.

[1098] A simple physical examination includes evaluating the skin, respiratory system, CV system, abdomen (liver, spleen), and CNS.

[1099] The timing of these examinations is specified in the time and event schedule. New, clinically significant findings that are not noticed during screening (in the investigator's opinion) will be captured as adverse events (AEs).

[1100] 9.6.7. Allergic reactions / General hypersensitivity reactions

[1101] All subjects will be observed and carefully monitored during the study for the development of any allergic reactions and for any infusion reactions during the administration of the IV study drug in Part 2.

[1102] During the administration of the investigational drug, physicians will have it readily available at the site at any time.

[1103] All subjects will be carefully monitored for symptoms of infusion reactions during and after IV administration, as indicated in the time and event schedule. Investigators should use clinical judgment to assess the severity of any infusion reaction.

[1104] If an infusion reaction is observed, treatment may be administered, depending on the nature of the allergic reaction and the severity of symptoms, such as oral acetaminophen and / or oral / IV antihistamines and / or inhaled β-agonists and / or IV corticosteroids and / or IV epinephrine. The following precautions should be taken during Part 2 IV administration of the study drug:

[1105] Before infusion can begin, appropriate personnel, medications (such as adrenaline, inhaled beta-agonists, antihistamines, and corticosteroids) and other requirements such as infusion solutions for treating allergic reactions (including anaphylactic reactions) will be available.

[1106] If a subject has a moderate or severe infusion response, the infusion should be terminated immediately and the subject should be treated appropriately in accordance with institutional guidelines.

[1107] Subjects whose study drug administration was discontinued due to infusion reactions will be required to return for all scheduled visits until the end of the study for necessary evaluations.

[1108] The study of reactions following drug administration may occur 1 to 21 days after infusion or injection, and the signs and symptoms presented may be variable and not always obvious (including, but not limited to, myalgia and / or arthralgia with fever and / or rash [which do not indicate signs and symptoms of other recognized clinical syndromes], and may be accompanied by other symptoms, including pruritus, facial, hand, or lip edema, dysphagia, urticaria, sore throat, and / or headache). Any anaphylactic or hypersensitivity reaction should be documented as an AE, and the type of reaction should be indicated. In cases where a subject experiences a delayed infusion or injection reaction, the subject will be asked to provide additional unplanned samples (urine, serum, and plasma for inflammatory markers). Each attempt should be made to collect these samples as close as possible to the onset of the adverse event suggesting a delayed hypersensitivity reaction. These samples will be used to try and understand the etiology of the symptoms. In cases where a skin reaction (i.e., rash) has been observed, the subject will be asked to allow a skin biopsy, again for better understanding of the cause of the rash.

[1109] 9.6.8. Local injection site reaction (Part 1)

[1110] At the time points indicated in the time and event schedule, the local injection site response will be evaluated at the injection site following administration of the study drug to the SC.

[1111] Any adverse reactions (e.g., pain, erythema, and / or induration) should be documented and characterized based on the following four parameters as described in the "Toxicity Grading Scale for Healthy Adult and Adolescent Volunteers Enrolled in Preventive Vaccine Clinical Trials"7: 1) Pain intensity, 2) Tenderness, 3) Erythema / redness, and 4) Induration / swelling. See Table 70 below for details.

[1112] Table 70: Toxicity Rating Scale for Infusion Reactions and Local Injection Site Reactions

[1113]

[1114] ER = Emergency Room

[1115] a. In addition to rating the local response at the point of maximum single diameter, measurements should be recorded as a continuous variable. b. Functional scales and actual measurements should be used to evaluate and rate induration / swelling.

[1116] All local injection site reactions that produce visual results (e.g., redness, induration, swelling) captured as AE should be photographed along with a measuring tape for later evaluation.

[1117] 9.7. Sample Collection and Processing

[1118] The actual date and time of sample collection will be recorded in the eCRF. If blood samples are collected via an indwelling cannula, a suitable amount (1 mL) of serum-blood fluid slightly larger than the dead space volume of the lock will be removed from the cannula and discarded before each blood sample collection. After blood sample collection, the cannula will be flushed with 0.9% sodium chloride and filled with a volume equal to the dead space volume of the lock. If mandarin (occluder) is used, blood loss due to disposal is not expected. Heparin flushing is not permitted.

[1119] For the timing and frequency of all sample collections, refer to the time and event schedule.

[1120] Instructions for the collection, handling, storage, and transport of samples can be found in the laboratory manual that will be provided. The collection, handling, storage, and transport of samples will be carried out under specified (and where applicable) controlled temperature conditions, as indicated in the laboratory manual.

[1121] 10. Subjects complete / discontinue study treatment / withdraw from the study

[1122] 10.1. Completed

[1123] If a participant has completed the assessment by the end of the study, he or she is considered to have completed the study.

[1124] 10.2. Termination of research treatment / Withdrawal from the research

[1125] Participants will automatically withdraw from the study for any of the following reasons:

[1126] Loss of follow-up

[1127] Agree to withdraw

[1128] die

[1129] If a participant withdraws from the study for any reason before the end of the outpatient period, a study termination assessment should be obtained. If the participant wishes to return for any visit until the study completion visit, any additional information will be collected and any procedures will be performed as permitted.

[1130] If a participant becomes unavailable for follow-up, the research site staff will make every reasonable effort to contact the participant and determine the reason for discontinuation / withdrawal. Follow-up actions will be documented.

[1131] When a subject withdraws before completing the study, the reason for withdrawal should be recorded in the eCRF and the source document. The study drug allocated to the withdrawn subject may not be allocated to another subject. The withdrawn subject will be replaced.

[1132] This is permissible if the PI or sponsor believes (e.g., for safety or tolerability reasons) that discontinuing further study participation is in the best interest of the participant, but the participant should still complete the study evaluation until the end of the study visit.

[1133] 11. Statistical Methods

[1134] Statistical analysis will be conducted by the sponsor or within the sponsor's authority.

[1135] Due to the exploratory and descriptive nature of the study, there are no plans for a formal hypothesis test.

[1136] 11.1. Subject Information

[1137] All subjects who received at least a partial dose of the study drug will be included in the descriptive statistics and analysis of safety.

[1138] 11.2. Sample Size Determination

[1139] No PK data were available for FP2; therefore, no formal sample size calculation was performed for this initial FIH Phase 1 study. The planned number of subjects for each dose group is consistent with the standard sample size in a single-escalation Phase 1 study of a new drug (where the primary objective is safety and tolerability, and one of the secondary objectives is to assess PK curves). In each dose group, 6 subjects will be randomized to FP2 and 2 subjects will be randomized to placebo; therefore, the ratio of active study drug to placebo in each dose group will be 3:1. The anticipated planned number of subjects provides sufficient information on the safety, tolerability, and pharmacokinetics of FP2 to allow for evaluation of the potential for further clinical development. Two subjects receiving placebo in each dose group should be sufficient to allow assessment of safety and tolerability at each dose level, and placebo subjects will be combined at study completion, if appropriate, for data analysis purposes. If combining placebo subjects is not feasible due to issues such as heterogeneous variance, alternative statistical methods (logarithmic transformation, etc.) may be explored to make all data available for analysis.

[1140] The sample size of six subjects treated with FP2 at each dose level was sufficient to estimate the probability of a safety signal (e.g., hypersensitivity reaction). Assuming the true risk of a safety signal is approximately 10%, six subjects receiving the active drug are allowed to detect at least one event with a probability of approximately 47%; although the assumed risk is approximately 50%, the probability of detecting at least one event is approximately 98%.

[1141] For pharmacokinetic parameters, the six subjects (three men and three women) who received IV administration of FP2 in Part 2 are expected to provide sufficient precision to estimate absolute bioavailability. The sample size of the six subjects is expected to provide sufficient precision to estimate the FP2 AUC after IV administration and to serve as a benchmark for assessing the drug quality properties of the SC dosage form.

[1142] 11.3. Pharmacokinetic Analysis

[1143] All subjects who received at least one dose of the study drug and had at least one PK and immunogenic sample collected after administration will be included in the analysis and reporting of PK data. If a subject's data does not allow for accurate assessment of PK (e.g., incomplete administration of the study drug; missing information on administration and sampling times; insufficient concentration data for PK parameter calculation), the subject will be excluded from the PK analysis.

[1144] Descriptive statistics (mean, median, standard deviation, and coefficient of variation) will be used to summarize FP2 serum concentrations at each sampling time point, as well as FP2 PK parameters for each DG. In the summary statistics, all serum concentrations below the minimum quantifiable concentration (BLQ) will be entered as zero, and all subjects and samples excluded from the PK analysis will be clearly recorded. PK parameters will also be summarized by DG.

[1145] Mean and / or median serum FP2 concentration distributions will be plotted on a full distribution after administration of the study drug. For mean plotting, the BLQ value will be set to zero. For individual serum concentration-time curves, the BLQ value will be set to zero up to the first measured concentration and will be removed from the analysis after the last measured concentration.

[1146] Statistical analysis of PK data will be performed on all subjects who received at least one FP2 dose. Geometric mean C max The AUC will be plotted relative to dose for a visual assessment of dose proportionality. The C-value can be transformed using the natural logarithm. max Linear regression analysis (efficacy model) using AUC data was used to examine the dose-proportionality of FP2 after SC administration. Additional analyses of the data can be performed as needed.

[1147] Absolute SC bioavailability will be calculated as a fraction of the systemically available administered dose, expressed as a percentage. To examine individual bioavailability, the individual dose-normalized AUC from SC administration will be calculated. inf Dose-normalized AUC from IV administration group inf The ratio of the geometric mean to the AUC, expressed as a percentage. A partial AUC may be used if deemed appropriate. (0-4周) Replace AUC inf The absolute SC bioavailability of FP2 will be derived by calculating the average of the above individual absolute SC bioavailability. Ninety-five percent confidence intervals and plotting of individual absolute SC bioavailability can also be explored.

[1148] 11.4. Immunogenicity Analysis

[1149] The incidence of anti-FP2 antibodies was aggregated for all subjects who received at least one dose of FP2 and had an appropriate sample for detecting FP2 (i.e., subjects who obtained at least one sample after FP2 administration).

[1150] A list of subjects who are positive for FP2 antibodies will be provided. For subjects who are positive for FP2 antibodies, the maximum antibody titer against FP2 will also be reported.

[1151] For subjects who are positive for antibodies against FP2 and have samples that can be used to assess neutralizing antibodies (NAb) against FP2, the incidence of NAbs for FP2 will be aggregated.

[1152] 11.5. Pharmacodynamic analysis (body weight, food intake) and exploratory studies

[1153] PD assessment (VAS) and biomarkers

[1154] Pharmacodynamic analyses will be performed on all subjects who received at least one dose of the investigational drug (FP2 or placebo) and underwent at least one PD assessment after treatment. For each dose, descriptive statistics will be calculated at each time point for each PD endpoint (e.g., weight, food intake) and exploratory PD outcome (VAS), as well as biomarker parameters. Parameters will be presented graphically: 1) for each subject, and 2) for each endpoint of the visual assessment of dose-related effects, displayed as mean + / - standard deviation (or other appropriate summary measure) of dose relative to the planned sampling time. Additional descriptive statistical analyses may include changes and percentage changes in selected pharmacodynamic and exploratory biomarkers relative to baseline (i.e., before dosing).

[1155] Mixed-effects models suitable for single-dose escalation, sequential panel designs can be used to analyze pharmacodynamics and exploratory biomarkers. Mixed-effects models may include fixed factors for treatment, access, treatment-by-access interactions, baseline, and baseline-by-access interactions, as well as a random factor for subjects. Baseline covariates may also be included in the model (e.g., age, sex, weight, etc.). If appropriate, the mean squared error from a linear model and a reference t-distribution can be used to obtain 90% confidence intervals for the differences in the least-squares mean change relative to baseline (treatment – ​​combined placebo) and the mean change. The least-squares mean and 90% confidence interval for the changes in PD / biomarker endpoints relative to baseline (before dosing) can also be calculated from the treatment group.

[1156] Further exploratory analyses may be conducted if necessary. Before performing analysis in the linear mixed-effects model described above, the area under the curve (trapezoidal method) or time-weighted average can be used to summarize the PD parameters (such as VAS) over time within the access.

[1157] 11.6. Pharmacokinetic / Pharmacodynamic Analysis

[1158] The relationship between pharmacokinetic (PK) and drug-prone (PD) data can be explored. Where appropriate, serum drug concentrations and corresponding PD measurements can be plotted to evaluate their relationship. If deemed suitable, an appropriate model can be applied to describe the exposure-effect relationship.

[1159] The PK / PD relationship can be studied graphically, and further analysis can be performed using appropriate statistical methods if deemed suitable. PK exposure (C max and / or AUC inf PD variables (e.g., weight, food intake, VAS) can be examined graphically. If the graphical representation of the relationship is deemed reasonable, appropriate models can be used to perform statistical analysis on these data.

[1160] PK exposure (C max and / or AUC inf The relationships between these variables and safety variables (e.g., ECG response) can be explored graphically. If the graphical representation of the relationships is deemed reasonable, appropriate models can be used to perform statistical analysis on these data.

[1161] 11.7. Security Analysis

[1162] Safety data reports for all subjects who received at least one dose of FP2 or placebo will include the incidence and type of adverse events, as well as absolute values ​​and variations in the following: blood pressure, heart rate, clinical laboratory data, and 12-lead ECG data from pre-dose to the final post-dose time point.

[1163] Adverse events

[1164] The verbatim terms used by researchers to identify adverse events in the eCRF will be coded using the Medical Dictionary of Regulatory Activities (MedDRA). Adverse events occurring with the study drug are those that occurred during the intervention phase or as a result of a pre-existing condition that had worsened from baseline. All reported adverse events will be included in the analysis. For each adverse event, the percentage of subjects who experienced at least one occurrence of a given event will be aggregated across the intervention groups. Furthermore, comparisons between intervention groups will be provided, where appropriate.

[1165] Summary, list, dataset, or subject narratives may be provided, as appropriate, for those subjects who died, discontinued the study drug due to adverse events, or experienced severe or serious adverse events.

[1166] Clinical laboratory testing

[1167] Laboratory data will be summarized by type of laboratory test. Reference ranges and significantly abnormal results (as specified in the statistical analysis plan) will be used in the summary of laboratory data. Descriptive statistics for each laboratory analyte will be calculated at baseline, and the observed values ​​and changes from baseline will be calculated at each planned time point. Pre- and post-intervention cross-tabulation (with categories of below-normal, within-normal, and above-normal) will present changes from baseline results. A list of subjects with any laboratory results outside the reference range will be provided. A list of subjects with any significantly abnormal laboratory results will also be provided.

[1168] Electrocardiogram (ECG)

[1169] The ECG variables to be analyzed using the following correction methods are HR, PR interval, QRS interval, QT interval, and corrected QT (QTc) interval: QT is corrected according to the Bazett formula (QTcB) and QT is corrected according to the Fridericia formula (QTcF). 1,19,14,16

[1170] At each planned time point, descriptive statistics on QTc interval and changes from baseline will be summarized. The percentage of subjects with QTc intervals >450ms, >480ms, or >500ms, and the percentage of subjects with QTc interval increases from baseline of >30ms or >60ms will be summarized.

[1171] Report all clinically relevant abnormalities in the ECG waveform that change from baseline readings (e.g., changes in T-wave morphology or the presence of U-waves).

[1172] If applicable, linear or nonlinear mixed-effects modeling methods can be used to quantify the relationship between serum concentrations of JNJ-64379090 and the primary endpoint ΔΔQTc (placebo-corrected change in QTc relative to time-matched baseline).

[1173] vital signs

[1174] At each predetermined time point, descriptive statistics of heart rate (HR) and blood pressure (systolic and diastolic) (supine) values, as well as changes from baseline, will be summarized. The percentage of subjects whose values ​​exceed clinically important limits will be summarized.

[1175] physical examination

[1176] At each predetermined time point, descriptive statistics of changes from the baseline will be summarized.

[1177] 11.8. Mid-term Analysis / Data Review Committee

[1178] There are no plans to conduct interim analyses. However, for the purpose of making dose escalation decisions, the research team may conduct a blinded data review after each dose group is completed.

[1179] 12. Adverse Event Reporting

[1180] Timely, accurate, and complete reporting and analysis of safety information from clinical studies are crucial for protecting subjects, investigators, and sponsors, and are mandated by global regulatory bodies. Sponsors have established standard operating procedures (SOPs) that comply with global regulatory requirements to ensure the proper reporting of safety information; all clinical studies conducted by the sponsor or its affiliates will be conducted in accordance with those procedures.

[1181] Methods for detecting adverse events and serious adverse events

[1182] When adverse events (AEs) or severe adverse events (SAEs) are detected, care will be taken not to introduce bias. Open-ended and non-dominant verbal questions to the subjects are the preferred method for inquiring about the occurrence of adverse events.

[1183] adverse events solicited

[1184] AEs (Adverse Events) are predefined local and systemic events that subjects are explicitly asked about. In this study, local injection site reactions (such as pain or itching) will be solicited as specified in the time and event schedule.

[1185] Unsolicited adverse events

[1186] Unsolicited adverse events (AEs) are all spontaneously reported adverse events (i.e., adverse events that were not explicitly asked of the subject).

[1187] 12.1. Definition

[1188] 12.1.1. Definition and Classification of Adverse Events

[1189] Adverse events

[1190] An adverse event is any adverse medical event that occurs in a clinical study subject who has received a drug (investigational or non-investigational) product. An adverse event is not necessarily causally related to the intervention. Therefore, an adverse event can be any unfavorable and unexpected sign (including abnormal findings), symptom, or illness that is temporarily associated with the use of a drug (investigational or non-investigational) product, whether or not it is related to the drug (investigational or non-investigational) product. (As defined by the International Council for the Concordance of Pharmaceutical Regulatory Affairs [ICH]).

[1191] This includes any new events or events that worsen in severity or frequency compared to baseline, or abnormal results from diagnostic procedures, including abnormal laboratory tests.

[1192] The sponsor collects adverse events starting from the signing of the ICF.

[1193] Serious adverse events

[1194] According to the ICH and EU guidelines on pharmaceutical safety for human use, a serious adverse event is any adverse medical event at any dosage.

[1195] Caused death

[1196] It is life-threatening (the subject was at risk of death at the time of the event. This does not refer to an event that, hypothetically, could have resulted in death if it were more severe).

[1197] Hospitalization or extension of existing hospital stay required

[1198] Leading to persistent or significant loss of ability / disability

[1199] It is a congenital abnormality / birth defect

[1200] It is suspected transmission via any infectious agent from pharmaceutical products.

[1201] It is of great medical importance*

[1202] *Medical and scientific judgment should be used to determine whether the rapid reporting system also applies to other situations, such as significant medical events that may not immediately endanger life or lead to death or hospitalization but may endanger the subject or require intervention to prevent one of the other outcomes listed in the above definition. These should generally be considered serious.

[1203] If a serious and unexpected adverse event occurs and there is evidence of a causal relationship between the study intervention and the event (e.g., death due to an allergic reaction), the event is reported as a serious and unexpected suspected adverse event, even if it is a component of the study endpoint (e.g., all-cause mortality).

[1204] Unlisted (unexpected) adverse events / Reference safety information

[1205] If the nature or severity is inconsistent with the applicable product reference safety information, the adverse event is considered unlisted. For FP2, the predictability of an adverse event will be determined by whether it is listed in the Investigator's Manual.

[1206] Adverse events associated with the use of intervention

[1207] If an adverse event is considered to be associated with the use of intervention if, according to the definitions listed in Section 12.1.2, the attribute is possible, very likely, or extremely likely.

[1208] No clinical studies have been conducted using FP2, therefore no data are available regarding the effects of FP2 in humans.

[1209] 12.1.2. Attribute Definition

[1210] Unrelated

[1211] Adverse events unrelated to the use of the intervention.

[1212] suspicious

[1213] Adverse events that are more likely to have alternative explanations, such as concomitant medication, concomitant disease, or temporal relationship, suggest that a causal relationship is unlikely.

[1214] possible

[1215] Adverse events may be due to the use of the intervention. Alternative explanations (e.g., concomitant medications, comorbidities) are uncertain. The temporal relationship is plausible; therefore, causation cannot be ruled out.

[1216] Very likely

[1217] Adverse events may be due to the use of the intervention. The temporal relationship is suggestive (e.g., confirmed by de-stimulation). Alternative explanations are unlikely, such as concomitant medications or comorbidities.

[1218] Very likely

[1219] The adverse event was listed as a possible adverse reaction and could not be reasonably explained by alternative explanations (e.g., concomitant medication, concomitant disease). The temporal relationship was highly suggestive (e.g., it was confirmed by de-excitation and re-excitation).

[1220] 12.1.3. Severity Criteria

[1221] The severity level will be assessed using the following general category descriptions:

[1222] Mild: Symptoms are easily tolerable, causing minimal discomfort and not interfering with daily activities.

[1223] Moderate: There is discomfort sufficient to interfere with normal activities.

[1224] Severe: Extreme pain leading to severe functional impairment or disability. Prevents normal daily activities.

[1225] Researchers should use clinical judgment to assess the severity of events that subjects did not directly experience (e.g., laboratory abnormalities).

[1226] 12.1. Special Reporting Circumstances

[1227] Safety incidents of concern related to the sponsor's research intervention that may require expedited reporting and / or safety evaluation include, but are not limited to:

[1228] Excessive intervention by the sponsor

[1229] Suspicious abuse / misuse of sponsor's research interventions

[1230] Accidental or unintentional exposure to sponsor research intervention

[1231] Drug errors involving the sponsor's products (whether the subject / patient was exposed to the sponsor's research intervention, such as name confusion).

[1232] Special reporting situations should be recorded in the eCRF. Any special reporting situations that meet the criteria for a serious adverse event should be recorded on the serious adverse event page of the eCRF.

[1233] 12.3. Procedure

[1234] 12.3.1. All Adverse Events

[1235] All adverse events and special reporting situations, whether serious or minor, will be reported from the date of receipt of a signed and dated ICF until the completion of the subject's last study-related procedure (which may include contact for safety follow-up). Serious adverse events will be reported using a serious adverse event form, including those voluntarily reported to the investigator at the end-of-study visit. The sponsor will evaluate any safety information voluntarily reported by investigators beyond the timeframe specified in the protocol.

[1236] All events that meet the definition of a serious adverse event will be reported as serious adverse events, regardless of whether they are part of the assessment specified in the protocol.

[1237] All adverse events will be documented in the source document and eCRF using medical terminology, regardless of their severity, severity, or presumed relationship to the study intervention. Where possible, diagnoses should be given when signs and symptoms are caused by common causes (e.g., cough, runny nose, sneezing, sore throat, and head congestion should be reported as “upper respiratory tract infection”). Investigators will document their views on the relationship between adverse events and the study therapy in the eCRF. All actions required for adverse event management will be documented in the source document and reported as per sponsor instructions.

[1238] 14. Research drug information

[1239] 14.1. Studying the physical description of drugs

[1240] The FP2 provided for this study was a sterile, brownish-yellow solution with a concentration of 50 mg / mL at pH 6.5 in 10 mM sodium phosphate, 8% sucrose, and 0.04% polysorbate 20.

[1241] FP2 is provided frozen in glass vials with a 1.2 mL fill volume.

[1242] The preparation buffer provided for this study is a sterile, clear solution consisting of 10 mM sodium phosphate, 8.0% sucrose, and 0.04% polysorbate 20. This preparation buffer will be used to prepare the placebo injection and as a diluent in the preparation of the initial FP2 SC dose.

[1243] The preparation buffer is provided frozen in glass vials with a 1.2 mL fill volume.

[1244] Its manufacturing and supply will be the responsibility of the sponsor. A list of excipients is provided above.

[1245] 14.2. Packaging

[1246] The investigational drug (FP2 and preparation buffer for diluting FP2, as well as a placebo) will be provided in bulk. The investigational drug will be stored in a locked pharmacy and will only be transferred to appropriately qualified members of the research team for administration after the preparation of the IV infusion (Part 2) or the blinded preparation of the SC injection (Part 1).

[1247] 14.3. Marking

[1248] The study suggests that drug labels will include information on compliance with applicable regulatory requirements.

[1249] 14.4. Preparation, Processing and Storage

[1250] All research drugs will be stored at a controlled temperature range of -30°C to -40°C and protected from light.

[1251] For the initial four planned SC dose groups, the drug product will be diluted to the required concentration using preparation buffer before use (0.5 mg / mL for DG 1; 5.0 mg / mL for DG 2 and DG 3; and 10.0 mg / mL for DG 4). The drug product for IV administration will be diluted to a concentration of 10 mg / mL and administered under in-line filtration.

[1252] References

[1253] 1. Bazett HC (1920). An analysis of the time-relationship of electrocardiograms. Heart. 1920;7:353-380.

[1254] 2. Breit SN (2011), Johnen H, Cook AD, Tsai VWW, Mohammad MG, Kuffner T et al. The TGF-β superfamily cytokine, MIC-1 / GDF15: A pleotrophic cytokine with roles in inflammation, cancer and metabolism. Growth Factors. 2011;29(5):187-195.

[1255] 3. EMA Guidance. EMEA CHMP SWP 28367 07 Rev.1 (2017). Guideline on strategies to identify and mitigate risks for first-in-human and early clinical trials with investigational medicinal products. European Medicines Agency; Committee for Medicinal Products for Human Use (CHMP); July 20, 2017.

[1256] 4. EMA Guidance. EMEA / CHMP / SWP / 28367 / 07 (2007). Guideline on strategies to identify and mitigate risks for first-in-human clinical trials with investigational medicinal products. European Medicines Agency; Committee for Medicinal Products for Human Use (CHMP); July 19, 2007.

[1257] 5. Emmerson PJ(2017), Wang F, Du Y, Liu Q, Pickard RT, Gonciarz MD et al. The metabolic effects of GDF15 are mediated by the orphan receptor GFRAL. Nat. Med. 2017;(10):1215 - 1219.

[1258] 6. FDA Guidance for Industry(2005). Estimating the maximum safe starting dose in initial clinical trials for therapeutics in adult healthy volunteers. US Dep of Health and Human Services. Center for Drug Evaluation and Research(CDER, July 2005).

[1259] 7. FDA Guidance for Industry(2007). Toxicity Grading Scale for Healthy Adult and Adolescent Volunteers Enrolled in Preventive Vaccine Clinical Trials. US Dep of Health and Human Services. Center for Biologics Evaluation and Research(CBER, September 2007).

[1260] 8. Fejzo MS(2018), Sazonova OV, Sathirapongsasuti JF, Hallgrímsdóttir IB, 23andMe Research Team, Vicic V et al. Placenta and appetite genes GDF15 and IGFBP7 are associated with hyperemesis gravidarum. Nature Communications. 2018;9:1178 - 1186.

[1261] 9. Fisher BL (2002) and Schauer P. Medical and surgical options in the treatment of severe obesity. Am J Surg. 2002;184:9S-16S.

[1262] 10. Fryar CD (2012), Carroll MD, Ogden CL. Prevalence of overweight, obesity, and extreme obesity among adults: United States, trends 1960–1962 through 2009–2010, 2012. National Center for Health Statistics. Available from http: / / www.cdc.gov / nchs / data / hestat / obesity_adult_09_10 / obesity_adult_09_10.htm Accessed on May 23, 2018.

[1263] 11. Gennemark P (2017), Tragardh M, Linden D, Ploj K, Johansson A, Turnbull A et al. Translational modeling to guide study design and dose choice in obesity exemplified by AZD1979, a melanin-concentrating hormone receptor 1 antagonist. CPT Pharmacometrics Syst Pharmacol. 2017;6:458-468.

[1264] 12. Gobel B (2014), Sanghvi A, and Hall KD. Quantifying energy intake changes during obesity pharmacotherapy. Obesity. 2014;22(10):2105-2108.

[1265] 13. Hall KD (2011), Sacks G, Chandramohan D, Chow C, Wang YC, Gortmaker SL, and Swinburn BA. Quantification of the effect of energy imbalance on bodyweight. Lancet. 2011;378:826–837.

[1266] 14. Hodges M (1983), Salerno D, Erlien D. Bazett’s QT correction reviewed: evidence that a linear QT correction for heart rate is better. J Am Coll Cardiol. 1983;1:694.

[1267] 15. Hsu JY (2017), Crawley S, Chen M, Ayupova DA, Lindhout DA, et al. Non-homeostatic body weight regulation through a brainstem-restricted receptor for GDF15. Nature. 2017;550(7675):255-259.

[1268] 16. International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use. ICH Harmonized Tripartite Guideline E14: Clinical evaluation of QT / QTc interval prolongation and proarrhythmic potential for non-antiarrhythmic drugs. ICH 12 May 2005.

[1269] 17. Mullican SE (2017), Lin-Schmidt X, Chin C-N, Chavez JA, Furman JL et al. GFRAL is the receptor for GDF15 and the ligand promotes weight loss in mice and nonhuman primates. Nat. Med. 2017;(10):1150 - 1157.

[1270] 18. Rueda-Clausen CF (2015), Ogunleye AA, and Sharma AM. Health benefits of long-term weight-loss maintenance. Annu. Rev. Nutr. 2015;35:475 - 516.

[1271] 19. Sagie A (1992), Larson MG, Goldberg RJ, Bengston JR, Levy D. An improved method for adjusting the QT interval for heart rate (the Framingham Heart Study). Am J Cardiol. 1992;70:797 - 801.

[1272] 20. Sugulle M (2009), Dechend R, Herse F, Weedon-Fekjaer SM, Johnsen GM et al. Circulating and placental Growth-Differentiation Factor 15 in preeclampsia and in pregnancy complicated by diabetes mellitus. Hypertension. 2009;54:106 - 112.

[1273] 21. The GBD Obesity Collaboration (2013). Global, regional and national prevalence of overweight and obesity in children and adults 1980 - 2013: a systematic analysis for the Global Burden of Disease Study 2013. Lancet. 2014; 384: 766–781.

[1274] 22. Wolfe BM (2016), Kvach E, and Eckel RH. Treatment of obesity: weight loss and bariatric surgery. Circ Res. 2016; 118: 1844 - 1855.

[1275] 23. World Health Organization (2012), Blood Donor Selection: Guidelines on Assessing Donor Suitability for Blood Donation. Geneva. Available from: https: / / www.ncbi.nlm.nih.gov / books / NBK138212 / Accessed on June 21, 2018.

[1276] 24. Yang L (2017), Chang C, Sun Z, Madsen D, Zhu H, Padkjaer S, et al. GFRAL is the receptor for GDF15 and is required for the anti - obesity effects of the ligand. Nat. Med. 2017; (10): 1158 - 1166.

[1277] 25.Yanovski SZ(2015),Marcus MD,Wadden TA,Walsh BT.The Questionnaireon Eating and Weight Patterns5(QEWP-5):An Updated Screening Instrument for Binge Eating Disorder.Int J Eat Disord.2015;48(3):259–261.

[1278] Example 22: Clinical Trial Results

[1279] Clinical trials were conducted according to the protocol described in Example 21, with the following differences: (1) Part 2 (absolute bioavailability) was not studied; (2) the inclusion criteria limiting the age of participants were changed to raise the upper age limit to 55 years; and (3) the exclusion criteria based on participants with a positive urine cotinine test were modified to allow the inclusion of light and intermittent smokers who were willing to quit smoking during the internal period. Dosing of groups 1-6 was completed, while dosing of group 7 was underway. Forty-eight subjects in six groups (groups 1-6) divided into groups of eight subjects each had been exposed to a single dose of FP2 or a matched placebo. In each group, FP2 was administered to 6 subjects and a matched placebo was administered to 2 subjects. The following is a summary of the doses of FP2 (or matched placebo) administered to each group 1-6 (Table 71):

[1280] Table 71. Dosing in Groups 1-6 .

[1281] group dose Group 1 0.8mg Group 2 2.5mg Group 3 7.5mg Group 4 15mg Group 5 30mg Group 6 60mg

[1282] Blinded safety and tolerability information is available for all administered subjects (groups 1-6). Pooled pharmacokinetic (PK) and pharmacodynamic (PD) results at the group level are available for groups 1-5.

[1283] Safety and tolerability: The treatment is generally considered safe and well-tolerated.

[1284] Pharmacokinetics

[1285] Pharmacokinetic data for groups 1-5 are available. To increase the dose of FP2, exposure was increased in approximately dose-proportional manner. T max It appeared on the 6th day, and T 1 / 2 The duration is approximately 12 days, therefore weekly dosing is recommended.

[1286] Table 72. Pharmacokinetics of FP2 at a single escalation dose .

[1287]

[1288] C 72 Indicate C max 72%

[1289] Pharmacodynamics

[1290] Food intake was assessed at baseline (day -1) and again on day 3 following administration of the study drug. T values ​​were observed in the first four groups of this SAD study. max It takes about 6 days, and the plasma concentration of FP2 on day 3 is about 70%-80% of the concentration reached on day 6.

[1291] To assess food intake, subjects were provided with four standard meals (breakfast, lunch, snack, and dinner); each subject received the same meals (same food items and the same quantities) for two days (i.e., day-1 and day 3). The calories consumed from the four meals were added together to obtain the total calories consumed at baseline (day-1) and on day 3. The change in food intake from baseline to day 3 was used as an exploratory measure of the pharmacokinetic mode of action of FP2.

[1292] Changes in food intake were variable in both placebo and active drug subjects, with changes of approximately ±20% from baseline on day 3 in groups 1 through 4. Nevertheless, four of the six subjects treated with 30 mg (group 5) FP2 experienced reductions in food intake exceeding 50% (range 55%–96%). In contrast, no subjects in the overall placebo group (10 subjects) achieved a reduction in food intake >30%. The two subjects in this same dose group (group 5, 30 mg) who showed the largest reductions in food intake (-85% and -96% reductions) were also the subjects who achieved the highest exposure to FP2. The median percentage change in food intake from baseline to day 3 post-dose is given in Table 73 for both placebo and FP2 subjects.

[1293] Table 73. Food intake of subjects treated with placebo or escalating doses of FP2 from baseline (day -1) to day 3 Changes in intake .

[1294]

[1295] Although the present invention has been described in detail with reference to its specific embodiments, it will be apparent to those skilled in the art that various changes and modifications can be made without departing from the spirit and scope of the invention.

Claims

1. A method for reducing the weight of a subject, the method comprising administering a composition comprising a fusion protein containing SEQ ID NO:92 and at least one pharmaceutically acceptable carrier or diluent, wherein the fusion protein is administered at a dose ranging from 0.8 mg to 90 mg, and wherein the subject's weight is 80 kg or higher.

2. The method of claim 1, wherein the subject is overweight.

3. The method of claim 2, wherein the subject has a BMI of 25 kg / m2 or greater.

4. The method of claim 3, wherein the subject has a BMI in the range of 25 kg / m2 to 29.9 kg / m2.

5. The method of claim 1, wherein the fusion protein is administered in a dose selected from the following: 0.8 mg, 2.5 mg, 7.5 mg, 15 mg, 30 mg, 60 mg and 90 mg.

6. The method of claim 5, wherein the fusion protein is administered at a dose of 0.8 mg.

7. The method of claim 5, wherein the fusion protein is administered at a dose of 2.5 mg.

8. The method of claim 5, wherein the fusion protein is administered at a dose of 7.5 mg.

9. The method of claim 5, wherein the fusion protein is administered at a dose of 15 mg.

10. The method of claim 5, wherein the fusion protein is administered at a dose of 30 mg.

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