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

Abstract

To provide GDF15-based compositions that can be used to treat or prevent metabolic diseases, disorders, or conditions.SOLUTION: The present invention provides the fusion proteins containing a half-life extension protein, a linker, and a GDF15 protein which function as GDF15 agonists. These GDF15 agonists may be useful in the treatment of obesity, reduction of body weight, decrease in food intake, or decrease of appetite.SELECTED DRAWING: None

Description

Technical Field

[0001] The present invention relates to a GDF15 fusion protein. Specifically, the present invention relates to a fusion protein comprising a half-life extended protein, a linker, and a GDF15 protein, a nucleic acid encoding the fusion protein and an expression vector, a recombinant cell thereof, and a pharmaceutical composition comprising the fusion protein. A method of reducing body weight and / or reducing food intake using the fusion protein is provided.

Background Art

[0002] GDF15 is a member of the TGFβ family and is a secreted protein that circulates in plasma as a 25 kDa homodimer. The plasma concentration of GDF15 is in the range of 150 - 1150 pg / ml in many individuals (Tsai et al., J Cachexia Sarcopenia Muscle. 2012, 3:239 - 243). High plasma concentrations of GDF15 are associated with anorexia and weight loss due to cachexia in cancer, as well as weight loss in renal insufficiency and heart failure. In clinical trials, GDF15 levels have been identified as an independent predictor of insulin resistance in non - diabetic subjects with obesity (Kempf et al., Eur.J.Endo. 2012, 167:671 - 678). Studies in twins have shown that the difference in GDF15 levels in twin pairs correlates with the difference in BMI of the pair, suggesting that GDF15 functions as a long - term regulator of energy homeostasis (Tsai et al., PLoS One. 2015, 10(7):e0133362).

[0003] ​​​​​​​​​​​​​​​In many reports, treatment with the GDF15 protein has been shown to improve glucose tolerance and insulin sensitivity in mouse models. In two independent lines of transgenic mice overexpressing GDF15, body weight and body fat mass were reduced and glucose tolerance was improved (J ohnen et al., Nat. Med. 2007, 13:1333 - 1340; M acia et al., PLoS One. 2012, 7: e34868; Chrys overgis et al., Int. J. Obesity. 2014, 38:155 5 - 1564). In GDF15 mice, increased whole - body energy consumption and oxidative metabolism have been reported (Chrysovergis et al., 2014, ibid.). These are accompanied by an increase in the expression of thermogenic genes in brown adipose tissue and an increase in lipolytic genes in white adipose tissue. In mice deficient in the GDF15 gene, body weight and body fat mass were increased (Tsai et al., PLoS One. 2013, 8(2): e5 5174). The Fc - fusion protein of GDF15 has been shown to reduce body weight and improve glucose tolerance and insulin sensitivity when administered once weekly for 6 weeks in an obese cynomolgus monkey model (International Publication No. WO2013 / 113008).

[0004] The effect of GDF15 on body weight is thought to be mediated through a reduction in food intake and possibly an increase in energy consumption. GDF15 may improve glycemic control through weight - dependent and possibly non - dependent mechanisms.

[0005] Taking these observations together, increasing the level of GDF15 may be a treatment for It may be useful as a treatment method. For treating or preventing metabolic diseases, disorders, or conditions There is a need in the art for a composition using GDF15 that can be used for

[0006] Current obesity treatments include dietary and behavioral interventions, pharmacotherapy, and bariatric surgery. Lifestyle interventions, including diet and increased physical activity, are fundamental to any weight loss effort and can be effective in achieving weight loss in the short term (3 - 6 months). However, in many cases, the weight loss achieved through lifestyle interventions is not sustained over the long term, and only a small percentage (5 - 10%) of individuals can sustain significant weight loss over the long term. (Fisher BL and Schauer P., Am J Surg.2002 184:9 S - 16 S, Rueda - Claussen C F et al., Annu.Rev.Nutr.2015 35:475 - 516). When lifestyle changes are not effective in achieving significant weight loss, pharmacotherapy is recommended as a second - line treatment. Approved drugs for long - term weight management in the US and EU include orlistat (a gastrointestinal lipase inhibitor), naltrexone / bupropion (a combination of an opioid antagonist and a dopamine / norepinephrine reuptake inhibitor), and liraglutide (a glucagon - like peptide - 1 receptor agonist), and in the US, lorcaserin (a selective 5 - HT C receptor agonist) and phentermine / topiramate (a combination of a sympathomimetic amine and an anticonvulsant) are also available. In addition, phentermine, as well as several other appetite suppressants (diethylpropion, be Furthermore, phentermine, as well as several other appetite suppressants (diethylpropion, be are listed, and in the US, lorcaserin (a selective 5 - HT C receptor agonist) and phentermine / topiramate (a combination of a sympathomimetic amine and an anticonvulsant) are also available. and liraglutide (a glucagon - like peptide - 1 receptor agonist) are listed, and in the US, lorcaserin (a selective 5 - HT C receptor agonist) and phentermine / topiramate (a combination of a sympathomimetic amine and an anticonvulsant) are also available. 2 C receptor agonist) and phentermine / topiramate (a combination of a sympathomimetic amine and an anticonvulsant) are also available. Furthermore, phentermine, as well as several other appetite suppressants (diethylpropion, be Phentermine, and phentermine-topiramate extended-release (Qsymia®, which contains phentermine and topiramate) are registered in the United States for short-term use (up to 12 weeks). In combination with behavioral interventions, these pharmacologic agents have various effects, resulting in additional weight loss in the range of 2% to 10% of initial body weight. Furthermore, the use of the pharmacologic agents is limited by side effects, including gastrointestinal effects (i.e., nausea, vomiting, fullness, diarrhea), neuropsychiatric effects (i.e., cognitive impairment, sleep disorders), and increased heart rate (depending on the specific drug). Due to these inherent limitations of available pharmacologic approaches (limited efficacy, safety profile, and a non-responder rate ranging from 30% to 65%), there remains a clearly unmet medical need for a more effective, better-tolerated, and safer pharmacologic therapy for obesity that may also improve obesity-related comorbidities such as cardiovascular disease, type 2 diabetes, and hypertension. Bariatric surgery (gastric banding, sleeve gastrectomy, and Roux-en-Y gastric bypass) provides weight loss of approximately 15% to 30% at 10 years, which is more sustained than medical therapy and can result in significant health improvements, and can reduce mortality in patients with severe obesity and intraoperative (e.g., venous thromboembolism) and postoperative (e.g., nausea, dumping syndrome, fat-soluble vitamin malabsorption) complications. Furthermore, considering both cost and surgical capacity limitations in many healthcare systems, bariatric surgery can only be offered to a small fraction of eligible patients (Rueda-Claussen CF et al., Annu.Rev.Nutr. 2015; 35:475-516). Therefore, there is a need for a more effective and better-tolerated long-term weight loss therapy that may also have a positive impact on obesity-related comorbidities such as hypertension, dyslipidemia, and type 2 diabetes. and is registered in the United States. In combination with behavioral interventions, these pharmacologic agents have various effects, resulting in additional weight loss in the range of 2% to 10% of initial body weight. Furthermore, the use of the pharmacologic agents is limited by side effects, including gastrointestinal effects (i.e., nausea, vomiting, fullness, diarrhea), neuropsychiatric effects (i.e., cognitive impairment, sleep disorders), and increased heart rate (depending on the specific drug). Due to these inherent limitations of available pharmacologic approaches (limited efficacy, safety profile, and a non-responder rate ranging from 30% to 65%), there remains a clearly unmet medical need for a more effective, better-tolerated, and safer pharmacologic therapy for obesity that may also improve obesity-related comorbidities such as cardiovascular disease, type 2 diabetes, and hypertension. Bariatric surgery (gastric banding, sleeve gastrectomy, and Roux-en-Y gastric bypass) provides weight loss of approximately 15% to 30% at 10 years, which is more sustained than medical therapy and can result in significant health improvements, and can reduce mortality in patients with severe obesity and intraoperative (e.g., venous thromboembolism) and postoperative (e.g., nausea, dumping syndrome, fat-soluble vitamin malabsorption) complications. Furthermore, considering both cost and surgical capacity limitations in many healthcare systems, bariatric surgery can only be offered to a small fraction of eligible patients (Rueda- Claussen CF et al., Annu.Rev.Nutr. 2015; 35 :475-516). Therefore, there is a need for a more effective and better-tolerated long-term weight loss therapy that may also have a positive impact on obesity-related comorbidities such as hypertension, dyslipidemia, and type 2 diabetes. and can reduce mortality in patients with severe obesity and intraoperative (e.g., venous thromboembolism) and postoperative (e.g., nausea, dumping syndrome, fat-soluble vitamin malabsorption) complications. Furthermore, considering both cost and surgical capacity limitations in many healthcare systems, bariatric surgery can only be offered to a small fraction of eligible patients (Rueda- Claussen CF et al., Annu.Rev.Nutr. 2015; 35 :475-516). Therefore, there is a need for a more effective and better-tolerated long-term weight loss therapy that may also have a positive impact on obesity-related comorbidities such as hypertension, dyslipidemia, and type 2 diabetes. Claussen CF et al., Annu.Rev.Nutr. 2015; 35 :475-516). Therefore, there is a need for a more effective and better-tolerated long-term weight loss therapy that may also have a positive impact on obesity-related comorbidities such as hypertension, dyslipidemia, and type 2 diabetes. and type 2 diabetes. Bariatric surgery (gastric banding, sleeve gastrectomy, and Roux-en-Y gastric bypass) provides weight loss of approximately 15% to 30% at 10 years, which is more sustained than medical therapy and can result in significant health improvements, and can reduce mortality in patients with severe obesity and intraoperative (e.g., venous thromboembolism) and postoperative (e.g., nausea, Administrative therapy is required.

Summary of the Invention

Means for Solving the Problems

[0007] The present invention provides FP2, a novel GDF 15 agonist, which satisfies this need. In non- clinical pharmacological and safety trials, FP2 has been shown to exhibit favorable pharmacological effects and promising safety profiles suitable for

[0008] transfer to clinical development. In one aspect, the present invention is a method for reducing the weight of a subject, comprising administering a composition comprising a fusion protein having SEQ ID NO: 92 and at least one pharmaceutically acceptable carrier or diluent, wherein the fusion protein 2 is administered at a dose in the range of about 0.8 mg to about 90 mg, and the weight of the subject is about 80 kg or more. In one aspect of the present 2 invention, the subject is overweight. In one aspect of the present 2 invention, the subject has a BMI of about 25 kg / m or more. In one aspect of the present invention, the subject has a BMI in the range of 25 kg / m to 29.9 kg / m In one aspect of the present invention, the fusion protein is Administered at a dose selected from the group consisting of 18 mg / kg, about 0.36 mg / kg, about 0.72 mg / kg, and about 1.08 mg / kg.

[0009] In one aspect of the present invention, the fusion protein is administered by subcutaneous injection.

[0010] In one aspect of the present invention, the fusion protein is administered to the subject once a week.

[0011] In one aspect, the present invention provides a method for reducing a subject's food intake, the method comprising administering a composition comprising a fusion protein having SEQ ID NO: 92 and at least one pharmaceutically acceptable carrier or diluent, wherein the fusion protein is administered at a dose in the range of about 0.8 mg to about 90 mg, and the subject weighs 80 kg or more. In one aspect of the present invention, the subject is overweight. In one aspect of the present invention, the subject has a BMI of 25 kg / m or more. In one aspect of the present invention, the subject has a BMI in the range of 25 kg / m to 29.9 kg / m . In one aspect of the present invention, the fusion protein is administered at a dose selected from the group consisting of 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 present invention, the fusion protein is administered at a dose in the range of about 0.01 mg 2 / kg to about 1.08 mg / kg. In one aspect of the present invention, the fusion protein is administered at a dose selected from the group consisting of 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 2 / kg. 2

[0012] In one embodiment of the present invention, the fusion protein is administered by subcutaneous injection.

[0013] In one embodiment of the present invention, the fusion protein is administered to the subject once a week.

[0014] Other aspects, features, and advantages of the present invention will become apparent from the following disclosure, including the detailed description of the invention, as well as its preferred embodiments and the appended claims. **Brief Description of the Drawings**

[0015] The above-mentioned "Means for Solving the Problems" and the following "Modes for Carrying Out the Invention" will be better understood when read in conjunction with the accompanying drawings. It should be understood that the present invention is not limited to the embodiments themselves shown in the drawings. The drawings are as follows. It should be understood that the present invention is not limited to the embodiments themselves shown in the drawings.

[0016] The drawings are as follows.

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Mode for Carrying Out the Invention

[0017] In the background art, various publications, papers, and patents are also cited or described throughout this specification. The entire contents of each of these reference documents are incorporated herein by reference. The present specification's discussion of documents, operations, materials, devices, articles, etc. is in the context of the present invention for providing. Such considerations do not recognize that any or all of these things constitute part of the prior art for any invention claimed herein. No.

[0018] 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 belongs. If not, the specific terms used herein have the meaning ascribed to them in this specification. All patents, published patent applications, and publications cited herein are incorporated by reference as if the entire contents thereof were set forth herein. When used in this specification and the appended claims, the singular forms "a", "an", and "the" are to be construed to include the plural referents unless the context clearly dictates otherwise. This invention relates to a fusion protein comprising (a) a half-life extended protein, (b) a linker, and (c) a GDF15 protein, arranged in the order (a)-(b)-(c) from the N-terminus to the C-terminus.

[0019]

[0020] Fusion proteins according to embodiments of the present invention, comprising a half-life extended protein, a linker, and a GDF15 protein, have been found to exhibit metabolic effects that demonstrate their suitability as therapeutics for treating and preventing metabolic diseases, disorders, or conditions. Such effects include, but are not limited to, weight loss, improved glucose tolerance, and improved insulin sensitivity in animals administered the fusion protein.

[0021] ​​ As used herein, the term "fusion protein" refers to a protein having two or more portions that are covalently linked to each other and are derived from different proteins. .

[0022] A fusion protein according to an embodiment of the invention can comprise any GDF15 protein. As used herein, the term "GDF15 protein" refers to a naturally occurring wild-type growth differentiation factor 15 protein or a functional variant thereof. The GDF15 t protein can be derived from any mammal, such as a human or other suitable mammals such as mouse, rabbit, rat, pig, dog, or primate. In certain embodiments, the GDF15 protein is a human GDF15 protein or a functional variant thereof. In a preferred embodiment, the GDF15 protein is a mature GDF15 protein or a functional variant thereof.

[0023] As used herein, the term "mature GDF15 protein" refers to the portion of the GDF15 preproprotein that is released from the full-length protein after intracellular cleavage at the RXXR furin-like cleavage site. The mature GDF15 protein is secreted as a homodimer linked by disulfide bonds. In one embodiment of the invention, the mature GDF15 protein ( abbreviated as GDF15(197 - 308)(SEQ ID NO: 6)) comprises amino acids 197 - 308 of the full-length human GDF15 protein.

[0024] As used herein, the term "functional variant" refers to a variant that has substantial or equivalent sequence identity to the parent protein and retains at least one of the biological activities of the parent protein. protein. Refers to a variant of a protein. Functional variants of the parent protein can be prepared by means well-known in the art in view of this disclosure. The functional variant can include one or more modifications to the amino acid sequence of the parent protein. Such modifications can, for example, improve the thermal stability of the polypeptide, change the substrate specificity, change the optimal pH, and thereby change the physicochemical properties of the polypeptide. Such modifications do not completely lose or abolish all of the biological activity of the parent protein and can also change the biological activity of the parent protein as long as they do not. Such modifications may be deletions or insertions of one or more amino acids. In the art, it can be prepared by well-known means. The functional variant can include one or more modifications to the amino acid sequence of the parent protein. Such modifications can, for example, improve the thermal stability of the polypeptide, change the substrate specificity, change the optimal pH, and thereby change the physicochemical properties of the polypeptide. Such modifications do not completely lose or abolish all of the biological activity of the parent protein and can also change the biological activity of the parent protein as long as they do not. Such modifications may be deletions or insertions of one or more amino acids. In any way, the physicochemical properties of the polypeptide can be changed. Such modifications do not completely lose or abolish all of the biological activity of the parent protein and can also change the biological activity of the parent protein as long as they do not. Such modifications may be deletions or insertions of one or more amino acids. In any way, the physicochemical properties of the polypeptide can be changed. Such modifications do not completely lose or abolish all of the biological activity of the parent protein and can also change the biological activity of the parent protein as long as they do not. Such modifications may be deletions or insertions of one or more amino acids. If such modifications do not completely lose or abolish all of the biological activity of the parent protein, they can also change the biological activity of the parent protein. Such modifications may be deletions or insertions of one or more amino acids. If such modifications do not completely lose or abolish all of the biological activity of the parent protein, they can also change the biological activity of the parent protein. Such modifications may be deletions or insertions of one or more amino acids. It may be.

[0025] According to another embodiment of the present invention, the functional variant of the parent protein includes one or more amino acid deletions and / or insertions relative to the parent protein. For example, functional variants of the mature GDF15 protein can include 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 acid deletions and / or insertions relative to the mature GDF15 protein, preferably including deletions of 1 to 30 amino acids at the N-terminus of the mature GDF15 protein. According to another embodiment of the present invention, the functional variant of the parent protein includes one or more amino acid deletions and / or insertions relative to the parent protein. For example, functional variants of the mature GDF15 protein can include 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 acid deletions and / or insertions relative to the mature GDF15 protein, preferably including deletions of 1 to 30 amino acids at the N-terminus of the mature GDF15 protein. According to another embodiment of the present invention, the functional variant of the parent protein includes one or more amino acid deletions and / or insertions relative to the parent protein. For example, functional variants of the mature GDF15 protein can include 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 acid deletions and / or insertions relative to the mature GDF15 protein, preferably including deletions of 1 to 30 amino acids at the N-terminus of the mature GDF15 protein. According to another embodiment of the present invention, the functional variant of the parent protein includes one or more amino acid deletions and / or insertions relative to the parent protein. For example, functional variants of the mature GDF15 protein can include 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 acid deletions and / or insertions relative to the mature GDF15 protein, preferably including deletions of 1 to 30 amino acids at the N-terminus of the mature GDF15 protein. According to another embodiment of the present invention, the functional variant of the parent protein includes one or more amino acid deletions and / or insertions relative to the parent protein. For example, functional variants of the mature GDF15 protein can include 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 acid deletions and / or insertions relative to the mature GDF15 protein, preferably including deletions of 1 to 30 amino acids at the N-terminus of the mature GDF15 protein. According to another embodiment of the present invention, the functional variant of the parent protein includes one or more amino acid deletions and / or insertions relative to the parent protein. For example, functional variants of the mature GDF15 protein can include 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 acid deletions and / or insertions relative to the mature GDF15 protein, preferably including deletions of 1 to 30 amino acids at the N-terminus of the mature GDF15 protein. It can include.

[0026] According to an embodiment of the present invention, the fusion protein of the present invention has an amino acid sequence that is at least 90% identical to the amino acid sequence of mature GDF15, such as GDF15(197 - 308)(SEQ ID NO: 6), or GDF15(200 - 308)(SEQ ID NO: 7), GDF15(201 - 308)(SEQ ID NO: 8), GDF15(202 - 308)(SEQ ID NO: 9), GDF15(2 According to an embodiment of the present invention, the fusion protein of the present invention has an amino acid sequence that is at least 90% identical to the amino acid sequence of mature GDF15, such as GDF15(197 - 308)(SEQ ID NO: 6), or GDF15(200 - 308)(SEQ ID NO: 7), GDF15(201 - 308)(SEQ ID NO: 8), GDF15(202 - 308)(SEQ ID NO: 9), GDF15(2 According to an embodiment of the present invention, the fusion protein of the present invention has an amino acid sequence that is at least 90% identical to the amino acid sequence of mature GDF15, such as GDF15(197 - 308)(SEQ ID NO: 6), or GDF15(200 - 308)(SEQ ID NO: 7), GDF15(201 - 308)(SEQ ID NO: 8), GDF15(202 - 308)(SEQ ID NO: 9), GDF15(2 08)(SEQ ID NO: 9), GDF15(2 03 - 308 (SEQ ID NO: 10), or GDF15(211 - 308) (SEQ ID NO: 11) and having an amino acid sequence that is at least 90% identical to the amino acid sequence of mature GDF15 with a truncated N-terminus including a GDF15 protein having such an amino acid sequence. The GDF15 protein, as long as it maintains at least one of its biological activities such as its effects on food intake, blood glucose level, insulin resistance, and body weight etc., can have at least one substitution, insertion, and deletion with respect to SEQ ID NOs: 6, 7, 8, 9, 1 0, or 11. In certain embodiments, the fusion protein of the present invention includes a GDF15 protein including, but not limited to, the amino acid sequences of SEQ ID NOs: 6, 7, 8, 9, 10, or

[0027] 11, including the amino acid sequence of SEQ ID NO: 11. In certain embodiments, the fusion protein of the present invention includes a GDF15 protein including the amino acid sequence of SEQ ID NO: 11, which includes, but is not limited to, the amino acid sequences of SEQ ID NOs: 6, 7, 8, 9, 10, or 11.

[0028] Any suitable half-life extending protein can be used in the fusion protein according to the embodiments of the present invention . As used herein, the term "half-life extending protein" refers to any protein or fragment thereof known to extend the half-life of the protein to which the half-life extending protein 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 lobulin (Ig), or transferrin (Tf) . In embodiments of the present invention, the half-life extending protein includes HSA or a functional variant thereof. In certain embodiments of the present invention, the half-life extending protein includes an amino acid sequence that is at least 90% identical to SEQ ID NO: 1. In preferred embodiments of the present invention, the half-life The extended protein contains HSA or a functional variant thereof in which the cysteine residue at position 34 of HSA is replaced by serine or alanine. It contains HSA or a functional variant thereof.

[0029] In certain embodiments, the fusion protein of the present invention contains a half-life extended protein having an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-3. It contains a half-life extended protein having an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-3.

[0030] Any suitable linker can be used in the fusion protein according to the embodiments of the present invention. As used herein, the term "linker" refers to a linking moiety including a peptide linker. Any suitable linker can be used in the fusion protein according to the embodiments of the present invention. As used herein, the term "linker" refers to a linking moiety including a peptide linker. The linker preferably ensures proper folding, minimizes steric hindrance, and does not significantly interfere with the structure of each functional element within the fusion protein. The linker preferably ensures proper folding, minimizes steric hindrance, and does not significantly interfere with the structure of each functional element within the fusion protein. In certain embodiments of the present invention, the peptide linker contains 2 to 120 amino acids. For example, the peptide linker can be 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, 4 0, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53 0, 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, 8 0, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 1 05, 106, 107, 108, 109, 110, 111, 112, 113, 114, 1 05, 106, 107, 108, 109, 110, 111, 112, 113, 114, 1 It contains 15, 116, 117, 118, 119, or 120 amino acids.

[0031] In embodiments of the present invention, the linker increases the flexibility of the fusion protein element. In certain specific embodiments of the present invention, the linker contains, but is not limited to, GS-(GGGGS)n or AS-(GGGGS )n-GT, a flexible linker containing the sequence (GGGGS)n (where n is 2 to 2 0, for example 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 ).

[0032] In other embodiments of the present invention, the linker is structured. In certain specific embodiments of the present invention, the linker contains, but is not limited to, AS-(AP)n-GT or AS-(EAAAK)n-GT and includes a structured linker containing the sequence (AP)n or (EAAAK)n (where n is 2 to 2 0, for example 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 ). In other embodiments of the present invention, the linker contains the sequence (GGGGA) n , (PGGGS) n , (AGGGS) n or GGS-(EGK SSGSGSESKST) n -GGS (n is 2 to 20).

[0033] In embodiments of the present invention, the fusion protein has at least 90% sequence identity with the amino acid sequences of SEQ ID NO: 5, 25-30, 36-37, 4 0, 48, 55-56, 59-60, or 64-75. In certain specific embodiments of the present invention, the fusion protein consists of SEQ ID NO: 5 , 25-30, 36-37, 40, 48, 55-56, 59-60, and 64-75 . It includes an amino acid sequence selected from the group consisting of. In a more specific embodiment of the present invention, the fusion tan protein includes an amino acid sequence selected from the group consisting of SEQ ID NO: 5, 25-30, 40, 55-56, 55-56, 59-60, and 7 0. In a still more specific embodiment of the present invention the fusion protein includes the amino acid sequence of SEQ ID NO: 92, SEQ ID NO: 60, or SEQ ID NO: 26 . The fusion protein may have a small extension such as a tag that facilitates purification, such as a polyhistidine tag, an antigenic epitope, or a binding domain, at the amino terminus or carboxyl terminus of the protein.

[0034] The fusion proteins disclosed herein include, but are not limited to, the effect on food intake, oral glucose tolerance test, measurement of blood glucose level, analysis of insulin resistance, weight change , pharmacokinetic analysis, toxicokinetic analysis, immunoassay and mass spectrometry of the level and stability of the full-length fusion protein, and ex vivo stability analysis in human plasma, and the biological activity of GDF15 can be characterized or evaluated.

[0035] The present invention also provides an isolated nucleic acid molecule encoding the fusion protein of the present invention. In an embodiment of the present invention the isolated nucleic acid molecule encodes a fusion protein 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 including an amino acid sequence selected from the group consisting of SEQ ID NO: 5, 25-31, 36-37, 40, 48, 55-56, 59-60, 64-75 . In a particular embodiment, the isolated nucleic acid molecule encodes a fusion protein including an amino acid sequence selected from the group consisting of SEQ ID NO: 5, 25-31, 36-37, 40, 48, 55-56, 59-60, 64-75 , and 92. In more specific embodiments, the isolated nucleic acid molecule encodes a fusion protein comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 5, 25-30, 40, 55-56 , 59-60, 70, and 92. In more specific embodiments, the isolated nucleic acid molecule comprises the nucleotide sequences of SEQ ID NO: 76-91, 9 5, and 110.

[0036] According to other embodiments of the present invention, the nucleic acid molecule encoding the fusion protein can be present within an expression vector . Examples of expression vectors include, but are not limited to, vectors for expressing recombinant proteins, and vectors for delivering nucleic acids into the body of a subject and expressing them in the tissues of the subject, such as viral vectors . Examples of viral vectors suitable for use with the present invention include, but are not limited to adenoviral vectors, adeno-associated viral vectors, lentiviral vectors . The vector may be a non-viral vector. Examples of non-viral vectors include, but are not limited to plasmids, bacterial artificial chromosomes, yeast artificial chromosomes, bacteriophages, etc. The vector can include any element for establishing the conventional functions of an expression vector, such as a promoter, ribosome binding element , terminator, enhancer, selection marker, or origin of replication . .

[0037] According to other embodiments of the present invention, the nucleic acid molecule encoding the fusion protein is known in the art in view of the present disclosure to improve recombinant expression from desired host cells such as human embryonic kidney (HEK) cells or Chinese hamster ovary (CHO) cells . It can be codon-optimized using technology.

[0038] The present invention also provides a host cell comprising a nucleic acid molecule encoding the fusion protein of the present invention. Examples of host cells include, but are not limited to, host cells for expressing recombinant proteins, and host cells for delivering a nucleic acid into a subject's body for expression in the subject's tissue. Examples of host cells suitable for use with the present invention include, but are not limited to, HEK or CHO cells.

[0039] In another general aspect, the present invention relates to a method for obtaining the fusion protein of the present invention. In a general aspect, such a method comprises: (1) culturing a host cell comprising a nucleic acid molecule encoding the fusion protein under conditions under which the fusion protein is produced; and (2) recovering the fusion protein produced by the host cell. The fusion protein can be further purified using methods well known in the art.

[0040] In certain embodiments, the fusion protein is expressed in a host cell and purified from the host cell using one or a combination of standard purification methods including, but not limited to, affinity chromatography, size exclusion chromatography, ultrafiltration, and dialysis. Preferably, the fusion protein is purified to be protease-free.

[0041] The present invention also provides a pharmaceutical composition comprising the fusion protein of the present invention and a pharmaceutically acceptable carrier.

[0042] The present invention also provides a nucleic acid molecule encoding the fusion protein of the present invention and a pharmaceutically acceptable carrier. and further provides a composition comprising the same. A composition comprising a nucleic acid molecule encoding the fusion protein of the present invention may comprise a delivery vehicle for introducing the nucleic acid molecule into a cell for expressing the fusion protein. Examples of nucleic acid delivery vehicles include liposomes, biocompatible polymers including natural polymers and synthetic polymers, lipoproteins, polypeptides, polysaccharides, lipopolysaccharides, artificial virus envelopes, metal particles, and viruses such as bacteria, baculoviruses, adenoviruses and retroviruses, bacteriophages, cosmids, plasmids, fungal vectors, and other recombinant vectors commonly used in the art described for expression in various eukaryotic hosts. The present invention also relates to a kit comprising the pharmaceutical composition of the present invention. Such a kit may comprise a first container having the dried fusion protein of the present invention and a second container having an aqueous solution to be mixed with the dried fusion protein prior to administration

[0043] to a subject, or may comprise a single container containing the liquid pharmaceutical composition of the present invention. Such a kit may comprise a single-dose unit or a multi-dose unit of the pharmaceutical composition of the present invention. Such a kit may comprise one or more pre-filled syringes (e.g., liquid syringes and lyosyringes). The kit may also comprise instructions for use. Such instructions for use may describe the use and properties of the materials provided in the kit and may be adapted to the specific metabolic disorder being treated. The present invention relates to metabolic disorders such as type 2 diabetes, elevated blood glucose levels, elevated insulin levels, obesity, dyslipidemia, diabetic nephropathy, myocardial ischemic disorders, congestive heart failure, or rheumatoid arthritis, and can be made to be suitable for the exact metabolic disorder being treated. The instructions for use may describe the use and properties of the materials provided in the kit and may be adapted to the specific metabolic disorder being treated.

[0044] The present invention also relates to the use of the fusion protein or the pharmaceutical composition of the present invention for the prevention or treatment of metabolic disorders, disorders, Also relates to the use of the pharmaceutical composition described herein for treating or preventing harm or a condition . According to an embodiment of the present invention, a method of treating or preventing a metabolic disease, disorder, or condition in a subject in need of treatment comprises administering to the subject a therapeutically or prophylactically effective amount of the pharmaceutical composition of the present invention . Any of the pharmaceutical compositions described herein, including a pharmaceutical composition comprising the fusion protein of the present invention or a pharmaceutical composition comprising a nucleic acid encoding the fusion protein can be used in the method of the present invention .

[0045] Disclosed herein is a method of reducing the body weight of a subject, comprising administering a composition comprising a fusion protein having SEQ ID NO: 92 and at least one pharmaceutically acceptable carrier or diluent , wherein the fusion protein is administered at a dose in the range of about 0.8 mg to about 90 mg and the subject has a body weight of 80 kg or more . .

[0046] According to a particular embodiment, the subject is overweight. In a particular embodiment of the present invention, the subject has a BMI of 25 kg / m or more, and in a particular embodiment, the subject has a BMI in the range of 25 kg / m 2 to 29.9 kg / m 2 2 .

[0047] According to a particular embodiment, the fusion protein is administered at a dose selected from the group consisting of 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 a particular embodiment, 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 In an embodiment, 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 m g. In other embodiments, the fusion protein is administered at a dose of about 90 mg .

[0048] 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 certain such embodiments, the fusion protein is 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 a dose selected from the group consisting of about 1.08 mg / kg . In certain 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 / k g. 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 about 1.08 mg / kg .

[0049] According to a particular embodiment of the invention, the fusion protein is administered by subcutaneous injection.

[0050] According to a particular embodiment of the invention, the fusion protein is administered to the subject once a week.

[0051] A method for reducing the food intake of a subject, comprising administering a composition comprising a fusion protein having 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 about 0.8 mg to about 90 mg, and the subject weighs 80 kg or more. A method for reducing the food intake of a subject, comprising administering a composition comprising a fusion protein having 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 about 0.8 mg to about 90 mg, and the subject weighs 80 kg or more. A method for reducing the food intake of a subject, comprising administering a composition comprising a fusion protein having 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 about 0.8 mg to about 90 mg, and the subject weighs 80 kg or more. A method for reducing the food intake of a subject, comprising administering a composition comprising a fusion protein having 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 about 0.8 mg to about 90 mg, and the subject weighs 80 kg or more.

[0052] According to certain embodiments, the subject is overweight. In certain embodiments of the invention, the subject has a BMI of 25 kg / m 25 kg / m 2 or more, and in certain embodiments, the subject has a BMI in the range of 25 kg / m 2 to 29.9 kg / m 2 In certain embodiments, the subject has a BMI in the range of 25 kg / m to 29.9 kg / m.

[0053] According to certain embodiments, the fusion protein is administered at a dose selected from the group consisting of 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 certain 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. According to certain embodiments, the fusion protein is administered at a dose selected from the group consisting of 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 certain 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. According to certain embodiments, the fusion protein is administered at a dose selected from the group consisting of 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 certain 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. According to certain embodiments, the fusion protein is administered at a dose selected from the group consisting of 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 certain 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. According to certain embodiments, the fusion protein is administered at a dose selected from the group consisting of 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 certain 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. According to certain embodiments, the fusion protein is administered at a dose selected from the group consisting of 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 certain 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. According to certain embodiments, the fusion protein is administered at a dose selected from the group consisting of 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 certain 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. According to certain embodiments, the fusion protein is administered at a dose selected from the group consisting of 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 certain 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. According to certain embodiments, the fusion protein is administered at a dose selected from the group consisting of 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 certain 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.

[0054] According to certain embodiments, the fusion protein is administered at a dose of about 0.01 mg / kg to about 1.08 mg / kg. It is administered in a dosage range of / kg. In certain such embodiments, the fusion protein is about 0. 01 mg / kg, about 0.03 mg / kg, about 0.09 mg / kg, about 0.18 mg / kg from the group consisting of about 0.36 mg / kg, about 0.72 mg / kg, and about 1.08 mg / kg and is administered at a dosage selected therefrom. In certain embodiments, the fusion protein is about 0.01 mg / kg. In other embodiments, the fusion protein is about 0.03 mg / kg. In other embodiments, the fusion protein is about 0.09 mg / k g. In other embodiments, the fusion protein is about 0.18 mg / kg and is administered at a dosage of. In other embodiments, the fusion protein is about 0.36 mg / kg at a dosage and is administered. In other embodiments, the fusion protein is about 0.72 mg / kg at a dosage and is administered. In other embodiments, the fusion protein is about 1.08 mg / kg at a dosage and is administered.

[0055] According to certain embodiments of the invention, the fusion protein is administered by subcutaneous injection.

[0056] According to certain embodiments of the invention, the fusion protein is administered to the subject once a week.

[0057] When referring to numerical ranges, cutoffs, or specific values, the term "about" is used to indicate that the recited value can vary by up to 10% from the recited value. Thus the term "about" encompasses 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 defined value. is used to include.

[0058] As used herein, "subject" means any animal, particularly a mammal, and most particularly a human, to be treated or having been treated by the method according to an embodiment of the present invention. It also includes any animal that is being or has been treated by the methods of the present invention, particularly mammals, and most particularly humans. As used herein, the term "mammal" includes all mammals. 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, and more particularly humans.

[0059] As used herein, "overweight" refers to excessive body weight. Using various parameters such as the subject's age, height, sex, and health status, it is determined whether the subject is overweight compared to a reference healthy individual. For example, by dividing the weight of the subject in kilograms by the square of the height of the subject in meters, the body mass index (BMI) of the subject is calculated, and the subject may be considered overweight or obese. An adult with a BMI in the range of 18.5 - 24.9 kg / m 2 is considered to have a normal weight, an adult with a BMI of 25 - 29.9 kg / m 2 may be considered overweight (pre-obese), and an adult with a BMI of 3 0 kg / m 2 or higher may be considered obese. Increased appetite often contributes to excessive body weight.

[0060] "Metabolic disease, disorder or condition" refers to any disorder associated with abnormal metabolism. Examples of metabolic diseases, disorders or conditions that can be treated according to the methods of the present invention include, but are not limited to, type 2 diabetes, elevated blood glucose levels, elevated insulin levels, obesity, overweight conditions, and the like. Body weight, dyslipidemia, diabetic nephropathy, myocardial ischemic disorder, congestive heart failure, or chronic arthritis Rheumatism.

[0061] As used herein, the terms "treating", "being treated", and "treatment" refer to administering a composition to a subject to obtain a desired therapeutic or clinical outcome in the subject. In one embodiment the terms "treating", "being treated", and "treatment" refer to reducing, alleviating, or delaying the progression or development of a metabolic disease such as type 2 diabetes, elevated blood glucose levels, elevated insulin levels, obesity, dyslipidemia, diabetic nephropathy, myocardial ischemic disorder, congestive heart failure, or chronic arthritis by administering the pharmaceutical composition of the present invention.

[0062] According to an embodiment of the present invention, the pharmaceutical composition of the present invention can be administered to a subject by any method well known to those skilled in the art in view of the present disclosure, such as intramuscular, subcutaneous, oral, intravenous, cutaneous, intra-mucosal (e.g., intra-intestinal), intranasal, or intraperitoneal administration routes. In certain embodiments the pharmaceutical composition of the present invention is administered to a subject by intravenous injection or subcutaneous injection.

[0063] As used herein, administration "once a week" is carried out within one day. Preferably, administration "once a week" is carried out in a single step such as a single injection.

[0064] In certain embodiments, the present invention provides a dosage of a clinically proven and clinically effective GDF15 fusion protein having the sequence of SEQ ID NO: 92 for use in a method of reducing the body weight of a subject, and the clinically proven and clinically effective dosage ranges from 0.8 mg to 90 mg for a subject weighing 80 kg or more ​​​​​​​​​is administered as a single subcutaneous (SC) injection at a dose of

[0065] In certain embodiments, the present invention provides a method for reducing food intake in a subject. A clinically proven safe and clinically proven effective vaccine having a sequence including SEQ ID NO:92. The present invention provides a method for administering a GDF15 fusion protein having a dose that has been clinically proven to be safe and The clinically proven effective dose is 0.8 mg to 90 mg for subjects weighing 80 kg or more. The drug is administered as a single subcutaneous (SC) injection at doses ranging from 0.1 mg / kg to 100 mg / kg.

[0066] According to the present invention as defined herein, the term "clinically proven to be safe" or treatment with a GDF15 fusion protein having a sequence comprising SEQ ID NO:92. It refers to a favorable risk:benefit ratio with respect to adverse vital signs (heart rate, contractility, and diastolic blood pressure, body temperature), adverse standard laboratory tests (hematology, clinical chemistry, urinalysis, lipids, clotting), allergic reactions / hypersensitivity, adverse local injection site reactions, or adverse EKG The frequency or severity of adverse events is relatively low or decreasing, and / or It is declining.

[0067] According to the present invention as defined herein, "clinically proven" or "clinically The term "proven effective against GDF15 fusion proteins having a sequence comprising SEQ ID NO:92" refers to Regarding the dosage, administration regimen, or treatment with protein, This refers to a decrease in appetite, a decrease in food preference assessed using questionnaires, or a loss of body weight.

[0068] As used herein, a reduction in body weight is at least 3%, at least 4%, at least Also 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 a decrease of any numerical value between these values.

[0069] As used herein, a decrease in food intake is 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 2 9%, at least 30%, or a decrease of any numerical value between these values. Food intake is , the grams of each food item consumed, and the calories estimated based on its nutritional content can be measured by measurement.

[0070] As used herein, unless otherwise specified, the term "clinically proven" (used independently or used to modify the terms "safety" and / or "efficacy") means proven by clinical trials that meet the standards of the US Food and Drug Administration, EMEA, or the corresponding national regulatory agency. For example, a clinical trial is a randomized double-blind trial of appropriate size used to clinically prove the effect of a drug. It may be. In some embodiments, "clinically proven" means proven by a clinical trial that meets the standards of the US Food and Drug Administration, EMEA, or the corresponding national regulatory agency for Phase I clinical trials. A method of reducing the weight of a subject, comprising administering a composition comprising a fusion protein having SEQ ID NO: 92 and at least one pharmaceutically acceptable carrier or diluent, wherein the fusion protein is administered at a dose in the range of 0.8 mg to 90 mg, and the weight of the subject is 80 kg or more.

[0071] Embodiment 1. A method of reducing the weight of a subject, comprising administering a composition comprising a fusion protein having SEQ ID NO: 92 and at least one pharmaceutically acceptable carrier or diluent, wherein the fusion protein is administered at a dose in the range of 0.8 mg to 90 mg, and the weight of the subject is 80 kg or more. 2. The method according to embodiment 1, wherein the subject is overweight. 3. The method according to embodiment 2, wherein the subject has a BMI of 25 kg / m2 or more. 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. 5. The method according to embodiment 1, wherein the fusion protein is administered at a dose selected from the group consisting of 0.8 mg, 2.5 mg, 7.5 mg, 15 mg, 30 mg, 60 mg, and 90 mg. 6. The method according to embodiment 5, wherein the fusion protein is administered at a dose of 0.8 mg. 7. The method according to embodiment 5, wherein the fusion protein is administered at a dose of 2.5 mg. 8. The method according to embodiment 5, wherein the fusion protein is administered at a dose of 7.5 mg. 9. The method according to embodiment 5, wherein the fusion protein is administered at a dose of 15 mg. 10. The method according to embodiment 5, wherein the fusion protein is administered at a dose of 30 mg. 11. The method according to embodiment 5, wherein the fusion protein is administered at a dose of 60 mg. 12. The method according to embodiment 5, wherein the fusion protein is administered at a dose of 90 mg. 13. The method according to any one of embodiments 1 to 12, wherein the composition is administered by subcutaneous injection. 14. The method according to any one of embodiments 1 to 12, wherein the composition is administered by intravenous injection. 15. The method according to any one of embodiments 1 to 12, wherein the composition is administered by intramuscular injection. 16. The method according to any one of embodiments 1 to 12, wherein the composition is administered orally. 13. The method according to embodiment 1, wherein the fusion protein is administered by subcutaneous injection. 14. A method for reducing the body weight of a subject, comprising administering a composition comprising a fusion protein having SEQ ID NO: 92 and , at least one pharmaceutically acceptable carrier or diluent, wherein the fusion protein is administered at a dose in the range of 0.01 mg / kg to 1.08 mg / kg. 15. The method according to embodiment 14, wherein the fusion protein is administered at a dose of 0.01 mg / kg, 0.03 mg / kg, 0.09 m g / kg, 0.18 mg / kg, 0.36 mg / kg, 0.72 mg / kg, and 1.0 8 mg / kg, selected from the group consisting of. 16. The method according to embodiment 15, wherein the fusion protein is administered at a dose of 0.01 mg / kg. 17. The method according to embodiment 15, wherein the fusion protein is administered at a dose of 0.03 mg / kg. 18. The method according to embodiment 15, wherein the fusion protein is administered at a dose of 0.09 mg / kg. 19. The method according to embodiment 15, wherein the fusion protein is administered at a dose of 0.18 mg / kg. 20. The method according to embodiment 15, wherein the fusion protein is administered at a dose of 0.36 mg / kg. 21. The method according to embodiment 15, wherein the fusion protein is administered at a dose of 0.72 mg / kg. 22. The method according to embodiment 15, wherein the fusion protein is administered at a dose of 1.08 mg / kg. 23. The method according to embodiment 14, wherein the fusion protein is administered by subcutaneous injection. 24. The method according to embodiment 14, wherein the composition is administered to the subject once a week. 25. The method according to embodiment 1, wherein the composition is administered to the subject once a week. 1A. A method for reducing food intake in a subject, the method comprising administering a composition comprising a fusion protein having SEQ ID NO: 92 and at least one pharmaceutically acceptable carrier or diluent, wherein the fusion protein is administered at a dose in the range of 0.8 mg to 90 mg and the subject has a body weight of 80 kg or more. 2A. The method according to embodiment 1A, wherein the subject is overweight. 3A. The method according to embodiment 2A, wherein the subject has a BMI of 25 kg / m2 or more. 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. 5A. The method according to embodiment 1A, wherein the fusion protein is administered at a dose selected from the group consisting of 0.8 mg, 2.5 mg, 7.5 mg, 15 mg, 30 mg, 60 mg, and 90 mg. 6A. The method according to embodiment 5A, wherein the fusion protein is administered at a dose of 0.8 mg. 7A. The method according to embodiment 5A, wherein the fusion protein is administered at a dose of 2.5 mg. 8A. The method according to embodiment 5A, wherein the fusion protein is administered at a dose of 7.5 mg. 9A. The method according to embodiment 5A, wherein the fusion protein is administered at a dose of 15 mg. 10A. The method according to embodiment 5A, wherein the fusion protein is administered at a dose of 30 mg. 11A. The method according to embodiment 5A, wherein the fusion protein is administered at a dose of 60 mg. 12A. The method according to embodiment 5A, wherein the fusion protein is administered at a dose of 90 mg. 13A. The method according to embodiment 1A, wherein the fusion protein is administered by subcutaneous injection. 14A. A method for reducing food intake of a subject, the method comprising administering a composition comprising a fusion protein having SEQ ID NO: 92 and at least one pharmaceutically acceptable carrier or diluent, wherein the fusion protein is administered at a dosage in the range of 0.01 mg / kg to 1.08 mg / kg. 15A. The method according to embodiment 14A, wherein the fusion protein is administered at a dosage selected from the group consisting of 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. 16A. The method according to embodiment 15A, wherein the fusion protein is administered at a dosage of 0.01 mg / kg. 17A. The method according to embodiment 15A, wherein the fusion protein is administered at a dosage of 0.03 mg / kg. 18A. The method according to embodiment 15A, wherein the fusion protein is administered at a dosage of 0.09 mg / kg. 19A. The method according to embodiment 15A, wherein the fusion protein is administered at a dosage of 0.18 mg / kg. 20A. The method according to embodiment 15A, wherein the fusion protein is administered at a dosage of 0.36 mg / kg. 21A. The method according to embodiment 15A, wherein the fusion protein is administered at a dosage of 0.72 mg / kg. 22A. The method according to embodiment 15A, wherein the fusion protein is administered at a dosage of 1.08 mg / kg. 23A. The method according to embodiment 15A, wherein the fusion protein is administered by subcutaneous injection. ​​​​​​​​​​​​​24A. The method according to embodiment 14A, wherein the composition is administered to the subject once a week. 25A. The method according to embodiment 1A, wherein the composition is administered to the subject once a week. 1B. A method for reducing the body weight of a subject, comprising administering to the subject once a week a composition comprising a fusion protein having SEQ ID NO: 92 and at least one pharmaceutically acceptable carrier or diluent, wherein the fusion protein is administered at a dose in the range of 0.8 mg to 90 mg, and the subject has a body weight of 80 kg or more. 2B. The method according to embodiment 1B, wherein the subject is overweight. 3B. The method according to embodiment 2B, wherein the subject has a BMI of 25 kg / m2 or more. 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. 5B. The method according to embodiment 1B, wherein the fusion protein is administered at a dose selected from the group consisting of 0.8 mg, 2.5 mg, 7.5 mg, 15 mg, 30 mg, 60 mg, and 90 mg. 6B. The method according to embodiment 5B, wherein the fusion protein is administered at a dose of 0.8 mg. 7B. The method according to embodiment 5B, wherein the fusion protein is administered at a dose of 2.5 mg. 8B. The method according to embodiment 5B, wherein the fusion protein is administered at a dose of 7.5 mg. 9B. The method according to embodiment 5B, wherein the fusion protein is administered at a dose of 15 mg. 10B. The method according to embodiment 5B, wherein the fusion protein is administered at a dose of 30 mg. 11B. The method according to embodiment 5B, wherein the fusion protein is administered at a dose of 60 mg. 12B. The method according to embodiment 5B, wherein the fusion protein is administered at a dose of 90 mg. . 13B. The method according to Embodiment 1B, wherein the fusion protein is administered by subcutaneous injection. 14B. A method for reducing the weight of a subject, comprising a fusion protein having SEQ ID NO: 92 and at least one pharmaceutically acceptable carrier or diluent, and administering the composition once a week to the subject such that the fusion protein is administered at a dose in the range of 0.01 mg / kg to 1.08 mg / k g. 15B. The method according to Embodiment 14B, wherein the fusion protein is administered at a dose selected from the group consisting of 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. 16B. The method according to Embodiment 15B, wherein the fusion protein is administered at a dose of 0.01 mg / kg . 17B. The method according to Embodiment 15B, wherein the fusion protein is administered at a dose of 0.03 mg / kg . 18B. The method according to Embodiment 15B, wherein the fusion protein is administered at a dose of 0.09 mg / kg . 19B. The method according to Embodiment 15B, wherein the fusion protein is administered at a dose of 0.18 mg / kg . 20B. The method according to Embodiment 15B, wherein the fusion protein is administered at a dose of 0.36 mg / kg . 21B. The method according to Embodiment 15B, wherein the fusion protein is administered at a dose of 0.72 mg / kg . 22B. The method according to Embodiment 15B, wherein the fusion protein is administered at a dose of 1.08 mg / kg . 23B. The method according to Embodiment 22B, wherein the fusion protein is administered by subcutaneous injection . 1C. A method of reducing the food intake of a subject, comprising administering to the subject a composition comprising a fusion protein having SEQ ID NO: 92 and at least one pharmaceutically acceptable carrier or diluent, administering once a week, wherein the fusion protein is administered at a dose in the range of 0.8 mg to 90 mg, and the subject weighs 80 kg or more. 2C. The method according to embodiment 1C, wherein the subject is overweight. 3C. The method according to embodiment 2C, wherein the subject has a BMI of 25 kg / m2 or more. 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. 5C. The method according to embodiment 1C, wherein the fusion protein is administered at a dose selected from the group consisting of 0.8 mg, 2.5 mg, 7.5 mg, 15 mg, 30 mg, 60 mg, and 90 mg. 6C. The method according to embodiment 5C, wherein the fusion protein is administered at a dose of 0.8 mg. 7C. The method according to embodiment 5C, wherein the fusion protein is administered at a dose of 2.5 mg. 8C. The method according to embodiment 5C, wherein the fusion protein is administered at a dose of 7.5 mg. 9C. The method according to embodiment 5C, wherein the fusion protein is administered at a dose of 15 mg. 10C. The method according to embodiment 5C, wherein the fusion protein is administered at a dose of 30 mg. 11C. The method according to embodiment 5C, wherein the fusion protein is administered at a dose of 60 mg. 12C. The method according to embodiment 5C, wherein the fusion protein is administered at a dose of 90 mg. 13C. The method according to embodiment 1C, wherein the fusion protein is administered by subcutaneous injection. ​​​​​​​​​14C. A method for reducing the food intake of a subject, comprising administering to the subject once a week a composition comprising the fusion protein having SEQ ID NO: 92 and at least one pharmaceutically acceptable carrier or diluent, wherein the fusion protein is administered at a dose in the range of 0.01 mg / kg to 1.08 mg / kg. 15C. The method according to embodiment 14C, wherein the fusion protein is administered at a dose selected from the group consisting of 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. 16C. The method according to embodiment 15C, wherein the fusion protein is administered at a dose of 0.01 mg / kg. 17C. The method according to embodiment 15C, wherein the fusion protein is administered at a dose of 0.03 mg. 18C. The method according to embodiment 15C, wherein the fusion protein is administered at a dose of 0.09 mg / kg. 19C. The method according to embodiment 15C, wherein the fusion protein is administered at a dose of 0.18 mg. 20C. The method according to embodiment 15C, wherein the fusion protein is administered at a dose of 0.36 mg / kg. 21C. The method according to embodiment 15C, wherein the fusion protein is administered at a dose of 0.72 mg. 22C. The method according to embodiment 15C, wherein the fusion protein is administered at a dose of 1.08 mg / kg. 23C. The method according to embodiment 22C, wherein the fusion protein is administered by subcutaneous injection.

Examples

[0072] ​​​​​​​​​​​​​​The following examples of the present invention are for further explaining the essence of the present invention. Those skilled in the art should be able to manufacture and use the present invention and implement the method claimed by using the above description and the following exemplary examples. It should be understood that the following examples do not limit the present invention, and the scope of the present invention is defined by the appended claims.

[0073] Example 1: Design of a fusion molecule containing GDF15 (influence of shortening of GDF15) Similar to other members of the TGFβ family, GDF15 forms a dimer in the endoplasmic reticulum, is cleaved in a furin-like manner to produce secreted mature GDF15 (amino acids 197 - 308), and is synthesized as a preproprotein. The secreted mature GDF15 homodimer is approximately 25 kDa, and each monomer can form up to four intramolecular disulfide bonds, and the components of the homodimer are linked by one intermolecular disulfide bond.

[0074] In the present invention, the crystal structure of GDF15 was determined and is shown in FIGS. 1A and 1B. This crystal structure shows that the C-terminus of mature GDF15 is embedded in the dimer interface, while the N-terminus is exposed. This exposed terminus enables the ligation of fusion proteins such as a protein with an extended half-life for the N-terminus of GDF15.

[0075] This crystal structure also shows a novel disulfide pairing pattern of cysteine residues in GDF15. TGFβ1 has pairings of C1 - C3 and C2 - C7 (i.e., the pairing between its first and third cysteine residues and the pairing between its second and seventh cysteine residues), ​​​​​​​​​whereas GDF15 has pairings of C1-C2 and C3-C7, with respect to having pairings between residues) having pairings of (refer to Figures 1A and 1B). This unique disulfide pairing results in a loop formed by the C1-C2 pairing located away from the cysteine knot containing other disulfide bonds at the N-terminus of the protein. Due to this structure it is predicted that the N-terminus of GDF15 may not be important for dimer formation or overall protein folding, and that GDF15 and its N-terminal fusion proteins may not be affected even by N-terminal deletions that delete residues within the C1 and C2, residues within the C1-C2 loop, or even residues on the C-terminal side of C2.

[0076] Example 2: Design of fusion molecules containing GDF15 (effect of linker) Different linkers between the HSA molecule and the GDF15 molecule were evaluated. Both a flexible linker containing the sequence (GGG GS)n and a structured linker containing the sequence (AP)n or (EAAAK)n (where n is from 2 to 20) were evaluated.

[0077] Fusion proteins containing different linkers were compared for their biophysical properties, their effects on food intake in lean mice, their pharmacokinetic (PK) values in those mice, and their ex vivo stability in human blood. The results of the linker mutants tested are shown in Table 1. The molecule containing SEQ ID NO: 31 and containing the (EAAAK) linker showed aggregation by HPLC. The remaining 7 linker mutants in Table 1 did not show aggregation. 8

[0078]

Table 1

[0079] Even in these variants, the stability of the linker was evaluated by in vivo tests in mice and by ex vivo stability tests in human whole blood and plasma samples. The results from these tests were analyzed using two detection modalities. The presence of both molecules on both sides of the linker was measured using an immunoassay with a pair of an anti-GDF15 capture antibody and an anti-HSA detection antibody to evaluate how complete the linker was. The overall integrity of the molecule was roughly characterized using different surrogate peptide sequences from HSA and GDF15 by liquid chromatography / mass spectrometry (LC-MS). The immunoassay showed a stable PK profile for all linker variants, and no disappearance of the spike in sample concentration in plasma was observed over 48 hours for any linker variant. The LC-MS results were consistent with the immunoassay and showed that the surrogate peptides from different parts of the HSA and GDF15 molecules were complete. The PK profiles of the linker variants analyzed by LC-MS using surrogate peptides showed a similar trend for different linker variants, and all linker variants showed detectable levels on day 7. All variants in Table 1, except for SEQ ID NO: 31, showed the desired biophysical properties and PK values Even in these variants, the stability of the linker was evaluated by in vivo tests in mice and by ex vivo stability tests in human whole blood and plasma samples. The results from these tests were analyzed using two detection modalities. The presence of both molecules on both sides of the linker was measured using an immunoassay with a pair of an anti-GDF15 capture antibody and an anti-HSA detection antibody to evaluate how complete the linker was. The overall integrity of the molecule was roughly characterized using different surrogate peptide sequences from HSA and GDF15 by liquid chromatography / mass spectrometry (LC-MS). The immunoassay showed a stable PK profile for all linker variants, and no disappearance of the spike in sample concentration in plasma was observed over 48 hours for any linker variant. The LC-MS results were consistent with the immunoassay and showed that the surrogate peptides from different parts of the HSA and GDF15 molecules were complete. The PK profiles of the linker variants analyzed by LC-MS using surrogate peptides showed a similar trend for different linker variants, and all linker variants showed detectable levels on day 7. All variants in Table 1, except for SEQ ID NO: 31, showed the desired biophysical properties and PK values and plasma samples. The results from these tests were analyzed using two detection modalities. The presence of both molecules on both sides of the linker was measured using an immunoassay with a pair of an anti-GDF15 capture antibody and an anti-HSA detection antibody to evaluate how complete the linker was. The overall integrity of the molecule was roughly characterized using different surrogate peptide sequences from HSA and GDF15 by liquid chromatography / mass spectrometry (LC-MS). The immunoassay showed a stable PK profile for all linker variants, and no disappearance of the spike in sample concentration in plasma was observed over 48 hours for any linker variant. The LC-MS results were consistent with the immunoassay and showed that the surrogate peptides from different parts of the HSA and GDF15 molecules were complete. The PK profiles of the linker variants analyzed by LC-MS using surrogate peptides showed a similar trend for different linker variants, and all linker variants showed detectable levels on day 7. All variants in Table 1, except for SEQ ID NO: 31, showed the desired biophysical properties and PK values and plasma samples. The results from these tests were analyzed using two detection modalities. The presence of both molecules on both sides of the linker was measured using an immunoassay with a pair of an anti-GDF15 capture antibody and an anti-HSA detection antibody to evaluate how complete the linker was. The overall integrity of the molecule was roughly characterized using different surrogate peptide sequences from HSA and GDF15 by liquid chromatography / mass spectrometry (LC-MS). The immunoassay showed a stable PK profile for all linker variants, and no disappearance of the spike in sample concentration in plasma was observed over 48 hours for any linker variant. The LC-MS results were consistent with the immunoassay and showed that the surrogate peptides from different parts of the HSA and GDF15 molecules were complete. The PK profiles of the linker variants analyzed by LC-MS using surrogate peptides showed a similar trend for different linker variants, and all linker variants showed detectable levels on day 7. All variants in Table 1, except for SEQ ID NO: 31, showed the desired biophysical properties and PK values and plasma samples. The results from these tests were analyzed using two detection modalities. The presence of both molecules on both sides of the linker was measured using an immunoassay with a pair of an anti-GDF15 capture antibody and an anti-HSA detection antibody to evaluate how complete the linker was. The overall integrity of the molecule was roughly characterized using different surrogate peptide sequences from HSA and GDF15 by liquid chromatography / mass spectrometry (LC-MS). The immunoassay showed a stable PK profile for all linker variants, and no disappearance of the spike in sample concentration in plasma was observed over 48 hours for any linker variant. The LC-MS results were consistent with the immunoassay and showed that the surrogate peptides from different parts of the HSA and GDF15 molecules were complete. The PK profiles of the linker variants analyzed by LC-MS using surrogate peptides showed a similar trend for different linker variants, and all linker variants showed detectable levels on day 7. All variants in Table 1, except for SEQ ID NO: 31, showed the desired biophysical properties and PK values and plasma samples. The results from these tests were analyzed using two detection modalities. The presence of both molecules on both sides of the linker was measured using an immunoassay with a pair of an anti-GDF15 capture antibody and an anti-HSA detection antibody to evaluate how complete the linker was. The overall integrity of the molecule was roughly characterized using different surrogate peptide sequences from HSA and GDF15 by liquid chromatography / mass spectrometry (LC-MS). The immunoassay showed a stable PK profile for all linker variants, and no disappearance of the spike in sample concentration in plasma was observed over 48 hours for any linker variant. The LC-MS results were consistent with the immunoassay and showed that the surrogate peptides from different parts of the HSA and GDF15 molecules were complete. The PK profiles of the linker variants analyzed by LC-MS using surrogate peptides showed a similar trend for different linker variants, and all linker variants showed detectable levels on day 7. All variants in Table 1, except for SEQ ID NO: 31, showed the desired biophysical properties and PK values and plasma samples. The results from these tests were analyzed using two detection modalities. The presence of both molecules on both sides of the linker was measured using an immunoassay with a pair of an anti-GDF15 capture antibody and an anti-HSA detection antibody to evaluate how complete the linker was. The overall integrity of the molecule was roughly characterized using different surrogate peptide sequences from HSA and GDF15 by liquid chromatography / mass spectrometry (LC-MS). The immunoassay showed a stable PK profile for all linker variants, and no disappearance of the spike in sample concentration in plasma was observed over 48 hours for any linker variant. The LC-MS results were consistent with the immunoassay and showed that the surrogate peptides from different parts of the HSA and GDF15 molecules were complete. The PK profiles of the linker variants analyzed by LC-MS using surrogate peptides showed a similar trend for different linker variants, and all linker variants showed detectable levels on day 7. All variants in Table 1, except for SEQ ID NO: 31, showed the desired biophysical properties and PK values and plasma samples. The results from these tests were analyzed using two detection modalities. The presence of both molecules on both sides of the linker was measured using an immunoassay with a pair of an anti-GDF15 capture antibody and an anti-HSA detection antibody to evaluate how complete the linker was. The overall integrity of the molecule was roughly characterized using different surrogate peptide sequences from HSA and GDF15 by liquid chromatography / mass spectrometry (LC-MS). The immunoassay showed a stable PK profile for all linker variants, and no disappearance of the spike in sample concentration in plasma was observed over 48 hours for any linker variant. The LC-MS results were consistent with the immunoassay and showed that the surrogate peptides from different parts of the HSA and GDF15 molecules were complete. The PK profiles of the linker variants analyzed by LC-MS using surrogate peptides showed a similar trend for different linker variants, and all linker variants showed detectable levels on day 7. All variants in Table 1, except for SEQ ID NO: 31, showed the desired biophysical properties and PK values and plasma samples. The results from these tests were analyzed using two detection modalities. The presence of both molecules on both sides of the linker was measured using an immunoassay with a pair of an anti-GDF15 capture antibody and an anti-HSA detection antibody to evaluate how complete the linker was. The overall integrity of the molecule was roughly characterized using different surrogate peptide sequences from HSA and GDF15 by liquid chromatography / mass spectrometry (LC-MS). The immunoassay showed a stable PK profile for all linker variants, and no disappearance of the spike in sample concentration in plasma was observed over 48 hours for any linker variant. The LC-MS results were consistent with the immunoassay and showed that the surrogate peptides from different parts of the HSA and GDF15 molecules were complete. The PK profiles of the linker variants analyzed by LC-MS using surrogate peptides showed a similar trend for different linker variants, and all linker variants showed detectable levels on day 7. All variants in Table 1, except for SEQ ID NO: 31, showed the desired biophysical properties and PK values and plasma samples. The results from these tests were analyzed using two detection modalities. The presence of both molecules on both sides of the linker was measured using an immunoassay with a pair of an anti-GDF15 capture antibody and an anti-HSA detection antibody to evaluate how complete the linker was. The overall integrity of the molecule was roughly characterized using different surrogate peptide sequences from HSA and GDF15 by liquid chromatography / mass spectrometry (LC-MS). The immunoassay showed a stable PK profile for all linker variants, and no disappearance of the spike in sample concentration in plasma was observed over 48 hours for any linker variant. The LC-MS results were consistent with the immunoassay and showed that the surrogate peptides from different parts of the HSA and GDF15 molecules were complete. The PK profiles of the linker variants analyzed by LC-MS using surrogate peptides showed a similar trend for different linker variants, and all linker variants showed detectable levels on day 7. All variants in Table 1, except for SEQ ID NO: 31, showed the desired biophysical properties and PK values and plasma samples. The results from these tests were analyzed using two detection modalities. The presence of both molecules on both sides of the linker was measured using an immunoassay with a pair of an anti-GDF15 capture antibody and an anti-HSA detection antibody to evaluate how complete the linker was. The overall integrity of the molecule was roughly characterized using different surrogate peptide sequences from HSA and GDF15 by liquid chromatography / mass spectrometry (LC-MS). The immunoassay showed a stable PK profile for all linker variants, and no disappearance of the spike in sample concentration in plasma was observed over 48 hours for any linker variant. The LC-MS results were consistent with the immunoassay and showed that the surrogate peptides from different parts of the HSA and GDF15 molecules were complete. The PK profiles of the linker variants analyzed by LC-MS using surrogate peptides showed a similar trend for different linker variants, and all linker variants showed detectable levels on day 7. All variants in Table 1, except for SEQ ID NO: 31, showed the desired biophysical properties and PK values and plasma samples. The results from these tests were analyzed using two detection modalities. The presence of both molecules on both sides of the linker was measured using an immunoassay with a pair of an anti-GDF15 capture antibody and an anti-HSA detection antibody to evaluate how complete the linker was. The overall integrity of the molecule was roughly characterized using different surrogate peptide sequences from HSA and GDF15 by liquid chromatography / mass spectrometry (LC-MS). The immunoassay showed a stable PK profile for all linker variants, and no disappearance of the spike in sample concentration in plasma was observed over 48 hours for any linker variant. The LC-MS results were consistent with the immunoassay and showed that the surrogate peptides from different parts of the HSA and GDF15 molecules were complete. The PK profiles of the linker variants analyzed by LC-MS using surrogate peptides showed a similar trend for different linker variants, and all linker variants showed detectable levels on day 7. All variants in Table 1, except for SEQ ID NO: 31, showed the desired biophysical properties and PK values

[0080] Each linker variant was evaluated for its in vivo activity in lean mice by performing a food intake test Table 2 shows the effect of the linker variants on the effect of the fusion protein on the decrease in food intake Table 2 shows the effect of the linker variants on the effect of the fusion protein on the decrease in food intake For the flexible (GGGGS)n linker, increasing the linker length to 2, 4, and 8 residues significantly increased the effectiveness of the fusion protein. For the more rigid (AP)n linker, this trend was less pronounced, suggesting that the degree of freedom of GDF15 molecules within the fusion protein plays an important role in its effect.

[0081]

Table 2

[0082] Example 3: Design of a fusion molecule containing GDF15 (effect of HSA mutations) A recombinant protein was designed in which human serum albumin as a half-life extended protein was fused to the N-terminus of GDF15 by a linker. According to this design, since the interface for dimerization of GDF15 is not destabilized, natural intermolecular chain disulfide bonds are formed, and GDF15 homodimers with fused HSA proteins extending from each GDF15 arm are obtained. According to this approach, only one gene is required to produce the HSA-GDF15 homodimer.

[0083] Natural human serum albumin protein contains 35 cysteine (Cys, C) residues that form 17 disulfide bonds, and the Cys-34 residue is the only free cysteine in the molecule. This free Cys-34 has been shown to function as a free radical scavenger by trapping multiple reactive oxygen species (ROS) and reactive nitrogen species (RNS). Therefore, this free Cys was mutated to minimize the risk of heterogeneity due to oxidation.

[0084] The free cysteine at position 34 of HSA was mutated to serine or alanine, and GDF15 fusion molecules with these HSA( C34S) or HSA(C34A) mutations were analyzed. All of these molecules were purified using a three-step purification method of (i) ion exchange chromatography, (ii) hydrophobic interaction chromato graphy, and (iii) size exclusion chromatography. At the time these molecules were first produced, HPLC analysis showed that both molecules were pure and showed no aggregation (Table 3).

[0085] However, two weeks after production, the fusion protein with the HSA(C34S) mutation (including SEQ ID NO: 48) showed aggregation by HPLC, while the protein with the HSA(C34A) mutation (SEQ ID NO: 40) remained free of aggregation even after four weeks.

[0086]

[0087] Example 4: Protease cleavage tendency of GDF15 The inventors observed that the arginine residue (R198) at amino acid position 198 of GDF15 is susceptible to protease degradation within the HSA -GDF15 fusion molecule. Such degradation results in a heterogeneous population, which is undesirable as a therapeutic composition. Such cleavage can be prevented by a cocktail of protease inhibitors. Each purification method was examined for protease removal. Table 4 shows two types of HSA affinity columns tested for the purification of HSA-GDF15 fusion proteins as measured by HPLC. At the time of purification ​​​​At the point where, regardless of the method used for purification, the purity of the HSA-GDF15 fusion protein was 100 %, and it was complete. At low concentrations (2 - 5 mg / ml), the protein purified by any method was completely retained throughout the 4-week test period. However, at high concentrations (40 - 50 mg / ml), only the antibody-based HSA resin (CaptureSele ct) produced a protease-free protein, which was completely retained throughout the 4-week test period. The protein obtained from the HSA-ligand-based resin (Albupure) was initially complete, but decomposed over time when stored at high concentrations. By adding a protease inhibitor cocktail (PI) and EDTA, the degradation of the high-concentration HSA-GDF15 fusion protein purified using Alb upure's resin was completely stopped. Therefore, the purification method plays an important role in generating a stable therapeutic composition. The corresponding degradation was not observed either in vivo or ex vivo, indicating that once the therapeutic composition is prepared as protease-free, the degradation of the fusion protein is not a problem in vivo. Therefore, a purification method that can effectively remove potential proteases during production, such as the method using CaptureSelect's resin, is essential for effectively manufacturing a homogeneous, complete, and stable GDF15 therapeutic drug. protein was initially complete but decomposed over time and with storage at high concentrations. By adding a protease inhibitor cocktail (PI) and EDTA, the degradation of the high-concentration HSA-GDF15 fusion protein purified using Alb upure's resin was completely stopped. Therefore, the purification method plays an important role in generating a stable therapeutic composition. The corresponding degradation was not observed either in vivo or ex vivo, indicating that once the therapeutic composition is prepared as protease-free, the degradation of the fusion protein is not a problem in vivo. Therefore, a purification method that can effectively remove potential proteases during production, such as the method using CaptureSelect's resin, is essential for effectively manufacturing a homogeneous, complete, and stable GDF15 therapeutic drug. role. Such corresponding degradation was not observed either in vivo or ex vivo, indicating that once the therapeutic composition is prepared as protease-free, the degradation of the fusion protein is not a problem in vivo. Therefore, a purification method that can effectively remove potential proteases during production, such as the method using CaptureSelect's resin, is essential for effectively manufacturing a homogeneous, complete, and stable GDF15 therapeutic drug. drug. drug. drug. drug. drug.

[0088]

Table 4

[0089] Example 5: N-terminal deletion mutants of GDF15 ​According to the GDF15 crystal structure shown in FIGS. 1A and 1B, GD involved in the deletion mutant The N-terminus of F15 is predicted to be unimportant for dimer formation and overall protein folding. According to this crystal structure, it is also predicted that such N-terminal deletions do not affect any potential receptor interactions. Various N-terminal deletions of GDF15 were tested for in vivo activity using HSA-GDF15 fusion proteins containing them.

[0090] An N-terminal deletion mutant of GDF15 with the protease cleavage site (R198) of GDF15 removed was designed. Immediately after the R198 residue, there is a potential deamination site for residues N199 - G200, but substrate deamination is still not preferred in therapeutic compositions. The N-terminal deletion of GDF15 can remove both the proteolytic cleavage site and the deamination site. The resulting GDF15 deletion mutants incorporated into the fusion protein with HSA were GDF15 (201 - 308; SEQ ID NO: 8), GDF15 (202 - 308; SEQ ID NO: 9), and G DF15 (211 - 308; SEQ ID NO: 11). In vivo experiments in mice showed that the N-terminal deletion mutants of GDF15 were still active in reducing food intake (FIG. 17). From the results of the tests, it was confirmed that these N-terminal deletion mutants of GDF15 were properly expressed, formed appropriate dimers, and were active in vivo. (201 - 308; SEQ ID NO: 8), GDF15 (202 - 308; SEQ ID NO: 9), and G DF15 (211 - 308; SEQ ID NO: 11). In vivo experiments in mice showed that the N-terminal deletion mutants of GDF15 were still active in reducing food intake (FIG. 17). From the results of the tests, it was confirmed that these N-terminal deletion mutants of GDF15 were properly expressed, formed appropriate dimers, and were active in vivo. confirmed.

[0091] Example 6: Inactive mutants of GDF15 Table 5 shows 12 mutants of GDF15 created to abolish the in vivo activity of GDF15 and identify the functional epitopes of GDF15. These mutations The variants include five types of single mutants, two types of double mutants, and five types of triple mutants. HSA-GDF15 fusion proteins containing these mutations were characterized for their biophysical properties and activities (Table 5). Of the 12 mutants, one showed no expression, and four formed aggregates over time, indicating that these mutations interfere with protein folding and biophysical properties. Of the remaining seven mutants, four had single mutations in GDF15, and these mutants were tested for their ability to reduce food intake compared to the wild type in mice. Three single mutants (I89R, I89W, and W32A) lost their in vivo activity, while the remaining mutant (Q60W) showed activity similar to the wild type. These results suggest that the I89R, I89W, and W32A mutations interfere with the interaction of GDF15 with its receptor / coreceptor, indicating that functional epitopes of GDF15 are located around residues I89 and W32. The numbering of the mutations is based on mature GDF15 present 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 the mature GDF15 protein.

[0092] [Table 5] * For purification purposes, a 6xHis tag was attached to the N-terminus.

[0093] Example 7: Expression and Purification Methods Expression To express more than 20 ml, it was grown in Expi293™ expression medium. Expression was performed using HEK Expi293 (trademark) cells. The cells were grown at 37 °C with shaking at 125 RPM under 8% CO2. The Expi293 (trademark) Expression Kit was used to transfect the cells at 2.5 × 106 cells per mL. For every liter of transfected cells, 1 mg of total DNA was diluted in 25 mL of Opti-MEM, and 2.6 mL of Expi293 (trademark) reagent was diluted in 25 mL of Opti-MEM and incubated at room temperature for 5 minutes. The diluted DNA and the diluted Expi293 reagent were combined and incubated at room temperature for 20 minutes. 6 Next, this DNA complex was added to the cells. The cells were placed in a shaking incubator overnight. On the day after transfection, 5 mL of the kit's Enhancer1 was diluted in 50 mL of the kit's Enhancer2, and the total volume of the two Enhancers was added to the cells. The transfected cells were incubated in the incubator for another 4 days and then harvested. The cells were concentrated by centrifugation at 6000 g for 30 minutes and then filtered through a 0.2 μm filter prior to the purification process.

[0094] Expression was also performed in CHO cells. The plasmid was purified and characterized. Prior to transfection, one aliquot of 20 μg of plasmid DNA containing the coding region of HSA-GDF15 was linearized by restriction enzyme digestion with Acl I. This digestion with the restriction endonuclease ensures the removal of the ampicillin resistance gene. Two 15 μg aliquots of linearized DNA were transfected into two 1 × 106 CHO cells using a BTX ECM 830 Electro Cell Manipulator (Harvard Apparatus, Holliston, Massachusetts). 7Cells (transfection pools A and B as described) were transfected. The cells were electroporated three times at 250 V with a pulse length of 15 milliseconds and a pulse interval of 5 seconds in a cuvette with an electrode gap of 4 mm. The transfected cells were transferred to MACH-1 + L-glutamine in a shaking flask and incubated for 1 day . Transfection pools A and B were centrifuged, resuspended in MACH-1 + MSX, transferred to a shaking flask and incubated for 6 days. The transfected HSA fusion protein-producing cells from transfection pools A and B were pooled and seeded in methylcellulose on the 8th day after electroporation . Purification Two-step purification using CaptureSelect resin and size exclusion chromatography was used. The cell supernatant from transiently transfected Expi293TM cells was loaded onto a pre-equilibrated (PBS, pH

[0095] 7.2) HSA CaptureSelect column (CaptureSelect human albumin affinity matrix sold by ThermoFisher Scientific) at a suitable volume of 10 mg of protein per 1 ml of resin. After loading, unbound proteins were removed by washing the column with 10 column volumes ( CV) of PBS (pH 7.2). HSA-GDF15 bound to the column was eluted with 10 CV of 2 M MgCl 2 in 20 mM Tris (pH 7.0). The peak fractions were pooled, filtered (0.2 µm), and dialyzed against PBS (pH 7.2) at 4 °C. After dialysis, the protein was filtered again (0.2 µm) and applied to size exclusion chromatography. The eluted fractions were collected and concentrated using Amicon Ultra centrifugal filter units (Millipore). The concentrated protein was then 2 filtered again (0.2 µm) and stored at -80 °C. ​​​​​ After concentrating to the appropriate volume, it was loaded onto a 26 / 60 Superdex 200 column (GE Healthcare). Proteins eluted by size exclusion chromatography (SEC) with high purity (measured by SDS-PAGE) were pooled. Protein concentration was measured by absorbance at 280 nm using a BioTek Synergy HTTM spectrophotometer. The quality of the purified protein was evaluated by SDS-PAGE and analytical size exclusion HPLC (SE-HPLC, Dionex HPLC system). Endotoxin levels were measured using the LAL assay (Pyrotell®-T, Associates of Cape Cod). thcare). After concentrating to the appropriate volume, it was loaded onto a 26 / 60 Superdex 200 column (GE Healthcare). Proteins eluted by size exclusion chromatography (SEC) with high purity (measured by SDS-PAGE) were pooled. Protein concentration was measured by absorbance at 280 nm using a BioTek Synergy HTTM spectrophotometer. The quality of the purified protein was evaluated by SDS-PAGE and analytical size exclusion HPLC (SE-HPLC, Dionex HPLC system). Endotoxin levels were measured using the LAL assay (Pyrotell®-T, Associates of Cape Cod). After concentrating to the appropriate volume, it was loaded onto a 26 / 60 Superdex 200 column (GE Healthcare). Proteins eluted by size exclusion chromatography (SEC) with high purity (measured by SDS-PAGE) were pooled. Protein concentration was measured by absorbance at 280 nm using a BioTek Synergy HTTM spectrophotometer. The quality of the purified protein was evaluated by SDS-PAGE and analytical size exclusion HPLC (SE-HPLC, Dionex HPLC system). Endotoxin levels were measured using the LAL assay (Pyrotell®-T, Associates of Cape Cod). After concentrating to the appropriate volume, it was loaded onto a 26 / 60 Superdex 200 column (GE Healthcare). Proteins eluted by size exclusion chromatography (SEC) with high purity (measured by SDS-PAGE) were pooled. Protein concentration was measured by absorbance at 280 nm using a BioTek Synergy HTTM spectrophotometer. The quality of the purified protein was evaluated by SDS-PAGE and analytical size exclusion HPLC (SE-HPLC, Dionex HPLC system). Endotoxin levels were measured using the LAL assay (Pyrotell®-T, Associates of Cape Cod). After concentrating to the appropriate volume, it was loaded onto a 26 / 60 Superdex 200 column (GE Healthcare). Proteins eluted by size exclusion chromatography (SEC) with high purity (measured by SDS-PAGE) were pooled. Protein concentration was measured by absorbance at 280 nm using a BioTek Synergy HTTM spectrophotometer. The quality of the purified protein was evaluated by SDS-PAGE and analytical size exclusion HPLC (SE-HPLC, Dionex HPLC system). Endotoxin levels were measured using the LAL assay (Pyrotell®-T, Associates of Cape Cod). After concentrating to the appropriate volume, it was loaded onto a 26 / 60 Superdex 200 column (GE Healthcare). Proteins eluted by size exclusion chromatography (SEC) with high purity (measured by SDS-PAGE) were pooled. Protein concentration was measured by absorbance at 280 nm using a BioTek Synergy HTTM spectrophotometer. The quality of the purified protein was evaluated by SDS-PAGE and analytical size exclusion HPLC (SE-HPLC, Dionex HPLC system). Endotoxin levels were measured using the LAL assay (Pyrotell®-T, Associates of Cape Cod). After concentrating to the appropriate volume, it was loaded onto a 26 / 60 Superdex 200 column (GE Healthcare). Proteins eluted by size exclusion chromatography (SEC) with high purity (measured by SDS-PAGE) were pooled. Protein concentration was measured by absorbance at 280 nm using a BioTek Synergy HTTM spectrophotometer. The quality of the purified protein was evaluated by SDS-PAGE and analytical size exclusion HPLC (SE-HPLC, Dionex HPLC system). Endotoxin levels were measured using the LAL assay (Pyrotell®-T, Associates of Cape Cod).

[0096] Two-step purification using Albupure resin and SEC was used. The HSA-GDF15 fusion protein was purified at room temperature using Albupure resin (ProMetic BioSciences) with a synthetic triazine ligand immobilized to selectively bind to HSA. The expression supernatant was passed through the Albupure resin. Next, the resin was washed first with 4 CV of PBS (pH 7.2), and then with 4 CV of 50 mM Tris (pH 8.0), 150 mM NaCl buffer. The HSA-GDF15 bound to the column was eluted with 4 CV of PBS buffer (pH 7.2) containing 100 mM sodium octanoate. The fraction containing the protein was concentrated to 10 mL using a spin concentrator (Amicon) with a cut-off value of 30,000 kDa molecular weight, and then passed through a 26 / 60 Superdex S200pg column (GE) equilibrated with PBS buffer (pH 7.2). Two-step purification using Albupure resin and SEC was used. The HSA-GDF15 fusion protein was purified at room temperature using Albupure resin (ProMetic BioSciences) with a synthetic triazine ligand immobilized to selectively bind to HSA. The expression supernatant was passed through the Albupure resin. Next, the resin was washed first with 4 CV of PBS (pH 7.2), and then with 4 CV of 50 mM Tris (pH 8.0), 150 mM NaCl buffer. The HSA-GDF15 bound to the column was eluted with 4 CV of PBS buffer (pH 7.2) containing 100 mM sodium octanoate. The fraction containing the protein was concentrated to 10 mL using a spin concentrator (Amicon) with a cut-off value of 30,000 kDa molecular weight, and then passed through a 26 / 60 Superdex S200pg column (GE) equilibrated with PBS buffer (pH 7.2). Two-step purification using Albupure resin and SEC was used. The HSA-GDF15 fusion protein was purified at room temperature using Albupure resin (ProMetic BioSciences) with a synthetic triazine ligand immobilized to selectively bind to HSA. The expression supernatant was passed through the Albupure resin. Next, the resin was washed first with 4 CV of PBS (pH 7.2), and then with 4 CV of 50 mM Tris (pH 8.0), 150 mM NaCl buffer. The HSA-GDF15 bound to the column was eluted with 4 CV of PBS buffer (pH 7.2) containing 100 mM sodium octanoate. The fraction containing the protein was concentrated to 10 mL using a spin concentrator (Amicon) with a cut-off value of 30,000 kDa molecular weight, and then passed through a 26 / 60 Superdex S200pg column (GE) equilibrated with PBS buffer (pH 7.2). Two-step purification using Albupure resin and SEC was used. The HSA-GDF15 fusion protein was purified at room temperature using Albupure resin (ProMetic BioSciences) with a synthetic triazine ligand immobilized to selectively bind to HSA. The expression supernatant was passed through the Albupure resin. Next, the resin was washed first with 4 CV of PBS (pH 7.2), and then with 4 CV of 50 mM Tris (pH 8.0), 150 mM NaCl buffer. The HSA-GDF15 bound to the column was eluted with 4 CV of PBS buffer (pH 7.2) containing 100 mM sodium octanoate. The fraction containing the protein was concentrated to 10 mL using a spin concentrator (Amicon) with a cut-off value of 30,000 kDa molecular weight, and then passed through a 26 / 60 Superdex S200pg column (GE) equilibrated with PBS buffer (pH 7.2). Two-step purification using Albupure resin and SEC was used. The HSA-GDF15 fusion protein was purified at room temperature using Albupure resin (ProMetic BioSciences) with a synthetic triazine ligand immobilized to selectively bind to HSA. The expression supernatant was passed through the Albupure resin. Next, the resin was washed first with 4 CV of PBS (pH 7.2), and then with 4 CV of 50 mM Tris (pH 8.0), 150 mM NaCl buffer. The HSA-GDF15 bound to the column was eluted with 4 CV of PBS buffer (pH 7.2) containing 100 mM sodium octanoate. The fraction containing the protein was concentrated to 10 mL using a spin concentrator (Amicon) with a cut-off value of 30,000 kDa molecular weight, and then passed through a 26 / 60 Superdex S200pg column (GE) equilibrated with PBS buffer (pH 7.2). Two-step purification using Albupure resin and SEC was used. The HSA-GDF15 fusion protein was purified at room temperature using Albupure resin (ProMetic BioSciences) with a synthetic triazine ligand immobilized to selectively bind to HSA. The expression supernatant was passed through the Albupure resin. Next, the resin was washed first with 4 CV of PBS (pH 7.2), and then with 4 CV of 50 mM Tris (pH 8.0), 150 mM NaCl buffer. The HSA-GDF15 bound to the column was eluted with 4 CV of PBS buffer (pH 7.2) containing 100 mM sodium octanoate. The fraction containing the protein was concentrated to 10 mL using a spin concentrator (Amicon) with a cut-off value of 30,000 kDa molecular weight, and then passed through a 26 / 60 Superdex S200pg column (GE) equilibrated with PBS buffer (pH 7.2). Two-step purification using Albupure resin and SEC was used. The HSA-GDF15 fusion protein was purified at room temperature using Albupure resin (ProMetic BioSciences) with a synthetic triazine ligand immobilized to selectively bind to HSA. The expression supernatant was passed through the Albupure resin. Next, the resin was washed first with 4 CV of PBS (pH 7.2), and then with 4 CV of 50 mM Tris (pH 8.0), 150 mM NaCl buffer. The HSA-GDF15 bound to the column was eluted with 4 CV of PBS buffer (pH 7.2) containing 100 mM sodium octanoate. The fraction containing the protein was concentrated to 10 mL using a spin concentrator (Amicon) with a cut-off value of 30,000 kDa molecular weight, and then passed through a 26 / 60 Superdex S200pg column (GE) equilibrated with PBS buffer (pH 7.2). Two-step purification using Albupure resin and SEC was used. The HSA-GDF15 fusion protein was purified at room temperature using Albupure resin (ProMetic BioSciences) with a synthetic triazine ligand immobilized to selectively bind to HSA. The expression supernatant was passed through the Albupure resin. Next, the resin was washed first with 4 CV of PBS (pH 7.2), and then with 4 CV of 50 mM Tris (pH 8.0), 150 mM NaCl buffer. The HSA-GDF15 bound to the column was eluted with 4 CV of PBS buffer (pH 7.2) containing 100 mM sodium octanoate. The fraction containing the protein was concentrated to 10 mL using a spin concentrator (Amicon) with a cut-off value of 30,000 kDa molecular weight, and then passed through a 26 / 60 Superdex S200pg column (GE) equilibrated with PBS buffer (pH 7.2). Two-step purification using Albupure resin and SEC was used. The HSA-GDF15 fusion protein was purified at room temperature using Albupure resin (ProMetic BioSciences) with a synthetic triazine ligand immobilized to selectively bind to HSA. The expression supernatant was passed through the Albupure resin. Next, the resin was washed first with 4 CV of PBS (pH 7.2), and then with 4 CV of 50 mM Tris (pH 8.0), 150 mM NaCl buffer. The HSA-GDF15 bound to the column was eluted with 4 CV of PBS buffer (pH 7.2) containing 100 mM sodium octanoate. The fraction containing the protein was concentrated to 10 mL using a spin concentrator (Amicon) with a cut-off value of 30,000 kDa molecular weight, and then passed through a 26 / 60 Superdex S200pg column (GE) equilibrated with PBS buffer (pH 7.2). Two-step purification using Albupure resin and SEC was used. The HSA-GDF15 fusion protein was purified at room temperature using Albupure resin (ProMetic BioSciences) with a synthetic triazine ligand immobilized to selectively bind to HSA. The expression supernatant was passed through the Albupure resin. Next, the resin was washed first with 4 CV of PBS (pH 7.2), and then with 4 CV of 50 mM Tris (pH 8.0), 150 mM NaCl buffer. The HSA-GDF15 bound to the column was eluted with 4 CV of PBS buffer (pH 7.2) containing 100 mM sodium octanoate. The fraction containing the protein was concentrated to 10 mL using a spin concentrator (Amicon) with a cut-off value of 30,000 kDa molecular weight, and then passed through a 26 / 60 Superdex S200pg column (GE) equilibrated with PBS buffer (pH 7.2). Performed. The SEC fraction containing the HSA-GDF15 dimer was identified by SDS-PAGE and pooled for analysis. The protein purity was evaluated by SDS-PAGE and SE-HPLC. In Examples 8 to 14 and Example 19, the characteristics of the exemplary fusion protein of the present invention having the amino acid sequence of SEQ ID NO: 60 were evaluated. This fusion protein is a complete recombinant protein existing as a homodimer of a fusion of mature human GDF15 with HSA via a 42-amino acid linker GS-(GGGGS) consisting of glycine and serine residues. The predicted molecular weight of this fusion protein is 162,696 Da, and one natural free cysteine at position 34 of HSA is mutated to serine. This specific HSA-GDF15 fusion protein will be simply referred to as "FP1" in the following examples for simplicity. The 6xHis-tagged variant of FP1 containing the AS-(GGGGS)x8-GT linker (6xHis-FP1, SEQ ID NO: 26) was used for comparison in some of the following examples.

[0097] Example 8: Effect of FP1 on food intake in C57B1 / 6 mice The purpose of this study was to demonstrate the dose-response effect of FP1 on the inhibition of food intake in C57B1 / 6 mice. Male C57Bl / 6 mice were acclimated in BioDAQ cages for at least 72 hours. 8 Subsequently, the mice were grouped into 6 groups of 8 animals each based on their food intake 24 hours in advance. Between 4:00 and 5:00 pm, the body weights of the animals were measured and they were administered with a solvent or a solution containing FP1. The predicted molecular weight of this fusion protein is 162,696 Da, and one natural free cysteine at position 34 of HSA is mutated to serine. This specific HSA-GDF 15 fusion protein will be simply referred to as "FP1" in the following examples for simplicity. The 6xHis-tagged variant of FP1 containing the AS-(GGGGS)x8-GT linker (6xHis-FP1, SEQ ID NO: 26) was used for comparison in some of the following examples. 15 fusion protein will be simply referred to as "FP1" in the following examples for simplicity. The 6xHis-tagged variant of FP1 containing the AS-(GGGGS)x8-GT linker (6xHis-FP1, SEQ ID NO: 26) was used for comparison in some of the following examples. The 6xHis-tagged variant of FP1 containing the AS-(GGGGS)x8-GT linker (6xHis-FP1, SEQ ID NO: 26) was used for comparison in some of the following examples. The 6xHis-tagged variant of FP1 containing the AS-(GGGGS)x8-GT linker (6xHis-FP1, SEQ ID NO: 26) was used for comparison in some of the following examples. Between 4:00 and 5:00 pm, the body weights of the animals were measured and they were administered with a solvent or a solution containing FP1.

[0098] Example 8: Effect of FP1 on food intake in C57B1 / 6 mice The purpose of this study was to demonstrate the dose-response effect of FP1 on the inhibition of food intake in C57B1 / 6 mice. Male C57Bl / 6 mice were acclimated in BioDAQ cages for at least 72 hours.

[0099] Male C57Bl / 6 mice were acclimated in BioDAQ cages for at least 72 hours. Subsequently, the mice were grouped into 6 groups of 8 animals each based on their food intake 24 hours in advance. Between 4:00 and 5:00 pm, the body weights of the animals were measured and they were administered with a solvent or a solution containing FP1. The composition was administered by subcutaneous injection. For each cage, changes in food intake were continuously recorded over 48 hours after injection using the BioDAQ system. In this study, 6xHis-FP1 was used as a comparison.

[0100] The results (Figure 2 and Table 6) were presented as the average of cumulative food intake at predetermined time intervals. The results showed that subcutaneous administration of FP1 to C57Bl / 6 mice significantly inhibited food intake compared to vehicle-treated animals at all doses and time points tested. 6xHis-FP1 reduced food intake at a dose of 8 nmol / kg.

[0101]

Table 6

[0102] Example 9: Effect of FP1 on food intake in Sprague Dawley rats The purpose of this study was to demonstrate the dose-response effect of FP1 on the inhibition of food intake in Sprague Dawley rats.

[0103] Male Sprague-Dawley rats were acclimated to the BioDAQ cages for a minimum of 72 hours. Subsequently, the rats were pre-grouped into 6 groups of 8 animals each based on food intake over a prior 24-hour period. Between 4:00 and 5:00 PM, the animals were weighed, and vehicle or ​​​​​​​A composition containing the fusion protein was administered by subcutaneous injection. For each cage, the change in food intake was continuously recorded by the BioDAQ system over 48 hours after injection. In this test, 6xHis-FP1 was used for comparison.

[0104] The results are shown in Figures 3 and 7. Subcutaneous administration of FP1 inhibited food intake at doses of 2.5 nmol / kg and 10 nmol / kg compared to vehicle-treated animals. The inhibition rate reached a significant difference only at the highest dose tested (10 nmol / kg) 24 and 48 hours after administration. FP1 decreased food intake at a dose of 8 nmol / kg, and the effect was significant at 24 and 48 hours.

[0105]

Table 7

[0106] Example 10: Effect of FP1 on glucose homeostasis and body weight in diet-induced obesity (DIO) mice The purpose of this study was to evaluate the effect of FP1 on food intake, body weight, and glucose homeostasis over the entire 2-week period in which DIO C57Bl / 6 mice were treated.

[0107] The body weights of male DIO mice were measured, and FP1 was administered subcutaneously at 2 mL / kg every 3 days (q3d) on days 0, 3, 6, 9, and 12. Similarly in the vehicle-treated group and the rosiglitazone-treated group. (q3d) ​​​​​PBS was administered according to the regimen. Control rosiglitazone was given at 0.015% in the diet freely. Mouse and diet weights were recorded daily. A blood glucose meter (One Touch® Ultra®, Lifescan, Milpitas, CA) was used to measure blood glucose levels. Body fat mass and fat-free mass were quantified in awake mice using a Bruker Mini-Spec LF110 by time domain NMR (TD-NMR). In an oral glucose tolerance test (OGTT), mice were fasted for 4 h. Glucose (2 g / kg) was administered by forced oral gavage at 10 mL / kg, and blood glucose levels were measured by tail snip at 0, 30, 60, 90, and 120 min. Insulin was measured at 0, 30, and 90 min after glucose administration.

[0108] At the end of the study, mice were euthanized by CO2 inhalation and terminal blood samples were collected. Serum was placed into 96-well plates on wet ice and stored at -80 °C. The liver was excised, and the fat content relative to the total liver slice mass was evaluated by TD-NMR using a Bruker MiniSpec mq60 according to the manufacturer's instructions.

[0109] The homeostasis model assessment of insulin resistance (HOMA-IR) was calculated by dividing the product of the fasting blood glucose level (mg / dL) and insulin level (mU / L) by a factor of 405.

[0110] Treatment of DIO mice with 1 nmol / kg and 10 nmol / kg of FP1 q3d decreased body weight (Table 8) and food intake (Table 9). The decreases reached statistical significance at specific timepoints described below.

[0111] FP1 reduced body weight in DIO mice at 1 (days 2 - 14) and 10 nmol / kg (days 1 - 14 ). A significant decrease in food intake was observed on days 1 and 2 of the test at a dose of 1 nmol / kg and on days 1, 8, and 9 at a dose of 10 nmol / kg (Table 9).

[0112]

Table 8

[0113]

Table 9

[0114] In the OGTT performed on day 14 of the test, FP1 significantly decreased blood glucose levels compared to vehicle-treated animals at all three doses tested at all time points after time point 0 (Table 1 0). When further quantified as area under the curve (AUC) and ΔAUC, all three doses tested were significantly lower compared to the vehicle (Table 10 and Figures 5A and 5B).

[0115]

Table 10

[0116] Fasting blood glucose levels were measured at the start of the test (day 0), day 7, and day 13 (Table 11, and Figure 6). FP1 significantly decreased blood glucose levels statistically at doses of 1 nmol / kg and 10 nmol / kg on day 13 of the test .

[0117]

Table 11

[0118] Plasma insulin levels during the OGTT were 0.1 nmol / k at 30 min in FP1. The 1 and 10 nmol / kg doses were significantly higher than the corresponding vehicle group at the same time point. The insulin excursion during the OGTT as measured by total AUC was: The 0.1 nmol / kg dose of FP1 was higher than that of the vehicle group (Table 12), and the 1 and 10 nmol / kg doses were In both cases, the difference was significant only at the lowest dose. At 90 min, mice treated with 1 and 10 nmol / kg FP1 showed lower IgE levels. The effect was not statistically significant. HOMA-IR, which was used as a marker, was measured on the 14th day of the study. At this point, FP1 was At 10 nmol / kg, it reduced HOMA-IR and improved insulin sensitivity (Table 13 and Figure 7).

[0119] [Table 12] Data are expressed as mean ± SEM. n=8 / group * p<0.05 compared with the vehicle-treated group

[0120] [Table 13] Data are expressed as mean±SEM. n=8 / group * p<0.05 compared with the vehicle-treated group

[0121] The magnitude of weight loss achieved by day 13 was not related to absolute body fat mass or body mass at any dose. There was no measurable change in percent fat (Table 14). A significant decrease in absolute fat-free mass was observed. This decrease was not observed when expressed as the fat-free rate (%). On the 15th day of the test, a final autopsy was performed to measure liver weight ( Table 15). FP1 decreased the absolute liver weight and the liver weight as a percentage of body weight (%) at a dose of 10 nmol / kg. A decrease was observed at a dose of 1 nmol / kg, but this did not reach statistical significance for either parameter. Liver fat was measured by NMR in a liver biopsy (Table 1 6). The FP1 fusion protein decreased the liver fat content, expressed as a percentage of liver biopsy weight (%), at doses of 1 and 10 nmol / kg. This decrease was significant at the higher dose .

Table 14

[0122]

Table 15

[0123]

Table 16

[0124]

Table 16

[0125] Example 11: Effect of FP1 on blood glucose level and body weight in ob / ob mice The purpose of this study was to evaluate the effect of an 8-day treatment with It was aimed to evaluate the effect of FP1 on body weight and blood glucose level over the course of the treatment.

[0126] The body weights of male ob / ob mice were measured, and FP1 was subcutaneously administered at 2 mL / kg every three days (q3 d) on days 0, 3, and 6. The weights of the mice and the diet were recorded daily. The blood glucose level was measured daily using a blood glucose meter. At the end of the test, the mice were euthanized and the final blood samples were collected.

[0127] At a dose of 1 nmol / kg, FP1 significantly reduced the body weight in ob / ob mice compared to the vehicle-treated mice from day 2 to day 8 (expressed as a percentage of the starting body weight). At a dose of 10 nmol / kg, FP1 reduced the body weight in ob / ob mice compared to the vehicle-treated mice from day 1 to day 8 (expressed as a percentage of the starting body weight) (Table 17 and Figure 8).

[0128]

Table 17

[0129] At a dose of 10 nmol / kg, FP1 reduced the fasting blood glucose level in ob / ob mice compared to the vehicle-treated mice on days 1 and 2 and from day 4 to day 8 of the test. A decrease in blood glucose level was observed at 1 nmol / kg, but this effect did not reach statistical significance (Table 18 and Figure 9).

[0130]

Table 18

[0131] ​​Example 12: Pharmacokinetics in Multiple Species Pharmacokinetics in Mice FP1 was administered to female C57Bl / 6 mice at a dose of 2 mg / kg in PBS (pH 7) by intravenous and subcutaneous routes. After both administration routes, blood samples were collected and serum was processed to measure the drug concentration over 7 days. The concentration of FP1 was measured by immunoassay . The drug concentration / time profiles in serum are summarized in Tables 19 and 20 and shown in Figure 10 .

[0132]

Table 19

[0133]

Table 20

[0134] Pharmacokinetic analysis showed that the terminal half-lives of FP1 in C57Bl / 6 mice after subcutaneous and intravenous administrations were 1.67 and 1.57 days, respectively (Table 21). FP1 showed an average bioavailability of approximately 71% after subcutaneous administration.

[0135]

Table 21

[0136] Pharmacokinetics in Rats FP1 was administered to female Sprague Dawley rats at a dose of 2 mg / kg in PBS (pH 7) by intravenous and subcutaneous routes. After both administration routes, blood samples were collected and serum was processed to measure the drug concentration over 7 days. The concentration of FP1 was measured by immunoassay ​​It was measured. The drug concentration / time profiles in serum are summarized in Tables 22 and 23 and are shown in Figure 11.

[0137]

Table 22

[0138]

Table 23

[0139] Pharmacokinetic analysis showed that the terminal half-lives of FP1 were 1.34 and 1.51 days in Sprague Dawley rats after subcutaneous and intravenous administration, respectively (Table 24). FP1 showed an average bioavailability of approximately 23% after subcutaneous administration.

[0140]

Table 24

[0141] Pharmacokinetics in monkeys FP1 was intravenously and subcutaneously administered to naive male cynomolgus monkeys (Macaca fascicularis) at a dose of 1 mg / kg in PBS (pH 7 ). After both routes of administration, blood samples were collected, serum was processed, and drug concentrations were measured for 21 days using immunoassay bioanalysis. The drug concentration / time profiles in serum are summarized in Tables 25 and 26 and are shown in Figure 12.

[0142]

Table 25

[0143]

Table 26

[0144] Pharmacokinetic analysis showed terminal half-lives of 8. 5 and 9.2 days for FP1 in cynomolgus monkeys after subcutaneous and intravenous administration, respectively, and an average bioavailability of approximately 88% after subcutaneous administration (Table 27).

[0145]

Table 27

[0146] The concentrations of the intact dimer present in the serum of cynomolgus monkeys after intravenous and subcutaneous administration were quantified using immunoaffinity capture LCMS analysis (Tables 28 and 29, and Figures 13 and 14). The concentrations measured by this method were similar to those measured by immunoassay (IA), indicating that FP1 circulates as an intact dimer and that there is no detectable metabolic instability in cynomolgus monkeys.

[0147]

Table 28

[0148]

Table 29

[0149] The concentrations of the analyte in the serum of cynomolgus monkeys after intravenous and subcutaneous administration were also measured by immunoaffinity capture trypsin digestion LC-MS / MS analysis (Tables 30 and 31). Near the N-terminus of the HSA region within FP1, near the N-terminus of GDF15, and near the C-terminus of GDF15, respectively, are located ​​​The tryptic digestion peptides to be used, namely, ALV (ALVLIAFAQYLQQSPFE DHVK), ASL (ASLEDLGWADWVLSPR), and TDT (TDTGVS LQTYDDLLAK) were selected. These peptides were observed as surrogate peptides for FP1. All concentrations of the surrogate peptides were comparable to each other and to the concentration measured by immunoassay, indicating that the GDF15 sequence within FP1 was fully preserved and linked to the complete HSA sequence in vivo.

[0150]

Table 30

[0151]

Table 31

[0152] Human plasma stability assay The purpose of this study was to analyze the ex vivo stability of FP1 in human plasma. Fresh, non-frozen human plasma was prepared from heparinized blood of two subjects (one male and one female) by centrifugation. FP1 was incubated in this substrate at 37°C for 0, 4, 24 and 48 hours while gently mixing. The concentration of FP1 was measured by immunoassay. The average difference (%) from the starting concentration (0 hours) was in the range of -4.1 to -12.9, and no increase over time was observed, indicating that FP1 was stable in ex vivo human plasma for up to 48 hours (Table 32 and Figure 15).

[0153]

Table 32

[0154] The concentration of the complete dimer present after incubation in human plasma was quantified using immunoaffinity capture LCMS. The concentrations measured by this method were stable over time (0, 4, 24, and 48 hours), indicating that FP1 was retained as the complete dimer in ex vivo human plasma for up to 48 hours (Table 33 and Figure 16).

[0155]

Table 33

[0156] In Examples 15 - 19, the properties of the exemplary fusion protein of the present invention described in Example 5, which has the amino acid sequence of SEQ ID NO: 92 (encoded by the nucleotide sequences of SEQ ID NO: 95 (codon optimization 1 ) and SEQ ID NO: 110 (codon optimization 2)), were evaluated. This fusion protein is a complete recombinant protein that exists as a homodimer of a fusion of the mature human GDF15 deletion mutant (201 - 3 08, SEQ ID NO: 8) with HSA(C34S) via a 42 - amino - acid linker GS-(GGG GS) consisting of glycine and serine residues. One natural free cysteine at position 34 of HSA has been mutated to serine. For simplicity, this specific 8 HSA - GDF15 fusion protein will be referred to as "FP2" in the following examples.

[0157] SEQ ID NO: 92: DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQSPFEDHV KLVNEVTEFAKTCVADESAENCDKSLHTLFGDKLCTVATL RETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEV ​​​​​​DVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKR YKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKC ASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTK VHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCE KPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYA EAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKC CAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGE YKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKH PEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTES LVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKE RQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCK ADDKETCFAEEGKKLVAASQAALGLGSGGGGSGGGGSGGG GSGGGGSGGGGSGGGGSGGGGSGGGGSDHCPLGPGRCCRL HTVRASLEDLGWADWVLSPREVQVTMCIGACPSQFRAANM HAQIKTSLHRLKPDTVPAPCCVPASYNPMVLIQKTDTGVS LQTYDDLLAKDCHCI

[0158] Example 13: In vitro agonist potency of FP2 The in vitro agonist potency of FP2 was determined using a cell-based pAKT assay with SK-N-AS cells stably overexpressing human GDF15 receptor (GFRAL). using a cell-based pAKT assay with SK-N-AS cells stably overexpressing human GDF15 receptor (GFRAL). It was evaluated. GFRAL activity was determined by measuring the level of phosphorylated AKT ( Ser473) in stable transfected SK-N-AS human neuroblastoma cells (ATCC) overexpressing human GFRAL. Phosphorylation of AKT after treating GFRAL-expressing cells with test articles at various concentrations was measured using the Phosp ho-AKT(Ser473) Assay kit (Cisbio, Bedford, MA) according to the manufacturer's instructions. Using the obtained data, the EC value was calculated using Prism statistical software (GraphPad Software San Diego). F P2 activated pAKT at a half-maximal effect concentration (EC ) of 2.908 ± 0.239 nM (N = 3). Native GDF15 functioned as a control for the assay and showed agonist activity at an EC 50 of 0.153 ± 0.00 8 nM (N = 3). 50 50

[0159] Example 14: Effect of FP2 on food intake in C57Bl / 6 mice FP2 was evaluated for its property of reducing food intake in male C57Bl / 6 mice after a single administration. Male C57Bl / 6N mice (10 - 12 weeks old) obtained from Taconic Biosciences (Hudson, NY) were used in the study. The mice were housed individually in a temperature-controlled room with a 12-hour light / dark cycle (6:00 AM / 6:00 PM) and allowed free access to water and food. Male C57Bl / 6 mice were acclimated to the environment in BioDAQ cages for at least 72 hours. Then, the mice were grouped into 6 groups of 8 animals each based on their food intake in the last 24 hours. Between 4:00 - 5:00 PM, the body weight of the animals was ​​​​​​​​​Measured, and a solvent or a compound was administered by subcutaneous injection. For each cage, the change in food intake was continuously recorded by a BioDAQ system over 48 hours after administration of the compound. In this test, 6xHis-FP1 was used for comparison. FP2 showed a significant effect of reducing food intake at 12, 24, and 48 hours after administration at all the tested dose levels (Table 34). A decrease in the rate of change (%) of food intake was observed for all time points and all dose levels in mice compared to PBS (Table 35).

[0160] FP2 showed a significant effect of reducing food intake at 12, 24, and 48 hours after administration at all the tested dose levels (Table 34). A decrease in the rate of change (%) of food intake was observed for all time points and all dose levels in mice compared to PBS (Table 35).

Table 34

[0161]

Table 34

[0162]

Table 35

[0163] Example 15: Effect of FP2 on food intake in Sprague Dawley rats FP2 was administered once daily to male Sprague-Dawley rats to evaluate food intake and body weight. Animals weighing 200-225 g were fed Obtained from Arles River Labs (Wilmington, MA), delivery within 1 week The animals were kept in an Alpha dome in a temperature-controlled room with a 12-hour light / dark cycle. Rats were housed individually in cages containing rye bedding and plastic tubes for concentration. The mice were allowed free access to water and fed a test rodent diet; Irradiated Certif ied PicoLab® Rodent Diet 20,5K75 * (Pu rina Mills,St.Louis,MO via ASAP Quakerto The rats were weighed and administered 100 mg of ... The results were determined and recorded.

[0164] The animals were allowed to acclimate in BioDAQ cages for at least 72 hours. Based on the intake, the animals were divided into 6 groups of 8 each during the last 24 hours. Between 0 and 5:00, the animals were weighed and administered the solvent or compound by subcutaneous injection. The change in food intake for each cage was recorded by the BioDAQ system after compound administration. Recorded continuously over 48 hours. In this test, 6XHis-FP1 was used as a comparison for reference.

[0165] The dose-dependent decrease in food intake was tested after a single administration of FP2. At a dose of 0.3 nmol / kg, no significant difference was observed in food intake. At 1 nmol / kg, a significant effect of decreasing food intake was observed at 12 hours, but not at 24 or 48 hours. At dose levels of 3 and 10 nmol / kg, a significant decrease in food intake was observed at all time points (Table 36, Figure 19). A decrease in the rate of change (%) in food intake was observed for all time points and all dose levels compared to PBS (Table 37). Although there was a significant effect on reducing food intake at 12 hours, it was not observed at 24 or 48 hours At dose levels of 3 and 10 nmol / kg, a significant decrease in food intake was observed at all time points (Table 36, Figure 19). A decrease in the rate of change (%) in food intake was observed for all time points and all dose levels compared to PBS (Table 37).

Table 36

[0166] The data are presented as mean ± SEM. Each value is compared with PBS * p ≤ 0.05 ** p ≤ 0.01 **** p ≤ 0.001 The statistical analysis methods used were ANOVA and Dunnett's multiple comparison test. n = 8 / group

[0167]

Table 37

[0168] Example 16: Effect of FP2 on food intake, body weight, and glucose homeostasis in diet-induced obese (DIO) C57Bl / 6 mice Male DIO C57Bl / 6 mice were repeatedly administered FP2 for 8 days, and their ability to reduce food intake and body weight and improve glucose homeostasis was evaluated. Male DIO C57Bl / 6 mice (21 weeks old, fed a high-fat diet for 15 weeks) obtained from Taconic Biosciences (Hudson, NY) were used in the study. The mice were housed individually in a temperature-controlled room with a 12-hour light / dark cycle (6:00 AM / 6:00 PM), allowed free access to water, and fed Research Diets D12492 (Research Diets, Inc., New Brunswick, NJ). The mice were acclimatized to the mouse facility for at least one week before the start of the study. The endpoints of the study were measurements of food intake, body weight, body composition, and blood glucose endpoints (OGTT, blood glucose levels). One day before dosing, the body weight of the animals was measured and the animals were grouped by body weight (BW). Administration was by subcutaneous injection. Animals receiving FP2 were given this compound on days 0, 3, 6, 9, and 12. Sterile PBS was similarly administered to the vehicle and rosiglitazone groups on these days. Rosiglitazone was given freely in the diet at 0.015 wt / wt%. Body weight and food intake were recorded daily for 15 days. Blood glucose levels were measured on days 0, 7, and 13. An oral glucose tolerance test (OGTT) was performed on day 14. O During the GTT, insulin levels were measured at the selected time points. On day 15, the mice were euthanized with CO 2 and the final blood samples for exposure were collected by cardiac puncture. A total of 15 mice in three mice per dosing group were used for separate PK arms. For the exposure-response (E-R) analysis of FP2 in DIO mice Most of the animals in the pharmacodynamics (PD) (effect) arm had undetectable drug concentrations on the last test day when PK samples were obtained, presumably due to immunogenicity. Therefore, instead of individual PK from the PD arm, the mean PK profile from the PK arm was used

[0169] to perform exposure-response (on days 3, 6, and 9, respectively) for the rate of change in body weight (%) from baseline in the PD arm at the corresponding dose levels. In this method, it is assumed that the PK arm exhibits similar behavior to the PD arm with respect to drug exposure. For the exposure-response (E-R) analysis of FP2 in DIO mice Most of the animals in the pharmacodynamics (PD) (effect) arm had undetectable drug concentrations on the last test day when PK samples were obtained, presumably due to immunogenicity. Therefore, instead of individual PK from the PD arm, the mean PK profile from the PK arm was used to perform exposure-response (on days 3, 6, and 9, respectively) for the rate of change in body weight (%) from baseline in the PD arm at the corresponding dose levels. In this method, it is assumed that the PK arm exhibits similar behavior to the PD arm with respect to drug exposure. to perform exposure-response (on days 3, 6, and 9, respectively) for the rate of change in body weight (%) from baseline in the PD arm at the corresponding dose levels. In this method, it is assumed that the PK arm exhibits similar behavior to the PD arm with respect to drug exposure. An E model (GraphPad Prism 6, log of agonist value vs. response) was used to correlate the exposure data with the response data (log values were converted to drug concentrations). The Hill

[0170] E max model (GraphPad Prism 6, log of agonist value vs. response) was used to correlate the exposure data with the response data (log values were converted to drug concentrations). The Hill Slope was set to 1. The model fitted to the EC values had EC 50 values with EC 10 fitted to them, although the E max estimates were different (E max = -4.26%, -8.18%, and -9. 85%, respectively), were within a factor of 2 at days 3, 6, and 9. On day 9, some animals also showed a loss of drug exposure, presumably due to the formation of potential ADA, so the E-R parameters based on the day 9 data require caution in interpretation.

[0171] The effect of 2-week F on food intake, body weight, glucose homeostasis, and hepatic fat content The effect of exposure to P2 was evaluated in diet-induced obese male C57Bl / 6 mice. 0.3 nM ol / kg treatment group of 1.7 - 3.3 nM of FP2, 1.0 nmol / kg treatment group of 7.1 ~14 nM, 3.0 nmol / kg treatment group of 20.8 - 41.6 nM, and 10 nmol / kg treatment group of 28.5 - 112.9 nM of FP2 trough exposure values were maintained until day 9 in the PK arm of the study (n = 2 or 3, Table 49). After day 9, despite continuing q3d administration, a decrease in circulating values was observed in the majority of animals (Table 49). Consistent with this accelerated clearance lance, the majority of animals in the PD arm of the study had undetectable circulating FP2 values on day 15 (Table 50).

[0172] Treatment of DIO mice with q3d of FP2 decreased food intake (Table 38), body weight (Tables 39, 40, and Figure 20), and fed blood glucose levels compared to vehicle treatment (Tables 43 and Figure 23). A significant decrease in food intake was observed at 0.3 nmol / kg on days 2, 5, and 8, at 1.0 nmol / kg throughout days 1 - 7, at 3.0 nmol / kg on days 1 2, 4 - 6, and 8, and at 10.0 nmol / kg on days 1, 3 - 6, 8, and 9. The rate of change in body weight (%) was significant at 0.3 nmol / kg from day 5 to day 13, at 1.0 nmol / kg and 10.0 nmol / kg from day 3 It was intentional. The decrease in postprandial blood glucose was significant on day 7 in animals at the dose level of 3.0 nmol / kg and on day 13 in animals at the dose levels of 3.0 and 10.0 nmol / kg. It was significant.

[0173] DIO mice treated with FP2 at q3d showed improved glucose tolerance on day 14 in an oral glucose challenge compared to the solvent-treated group (Table 41, Figures 21A and 21B). Blood glucose levels were significantly lower at 30 minutes in the 0.3 nmol / kg group, at 60 minutes and 120 minutes in the 1.0 nmol / kg group, at 120 minutes in the 3.0 nmol / kg group, and at 3 0, 90, and 120 minutes in the 10.0 nmol / kg group. The total area under the curve was significant in all dosing groups. Insulin levels in the glucose challenge were significantly lower at 30 minutes in the 0.3 and 10.0 nmol / kg groups (Table 42, Figures 22A and 22B). Furthermore, compared with solvent-treated animals, DIO mice after q3d treatment with 10.0 nmol / kg of FP2 for 14 days showed a significant decrease in the calculated fasting HOMA-IR value, indicating improved insulin sensitivity ([[]]END]] Table 44 and Figure 24).

[0174] Body composition was measured by MRI on day -1 and day 13 before the start of the study (Tables 47 and 4 8). DIO mice treated with 1.0 nmol / kg and 10.0 nmol / kg of FP2 showed a significant decrease in body fat mass on day 13, while no change in fat-free mass was observed in any treatment group. On day 13, the 10.0 nmol / kg treatment group showed a significant increase in fat-free rate (%) and a significant decrease in body fat rate (%) compared to the solvent-treated group. From day -1 to day 13, the changes in fat-free mass were 0.3 nmol / kg, 1.0 nmol / kg, for and was significant in the 10.0 nmol / kg treatment group. For the fat removal rate (%), 1.0, 3. 0, and 10.0 nmol / kg treatment groups were significant. The changes from day - 1 to day 13 were significant for body fat mass and fat removal rate (%) in all treatment groups compared to the solvent.

[0175] There was no significant difference in the serum level of endogenous mouse GDF15 between solvent - treated animals and mice treated with FP2 at q3d for 15 days (Table 46).

[0176] Conclusion: These results indicate that high drug exposure generally is associated with a greater rate of change in body weight (%) from baseline at the population level in all the dosing groups tested on days 3, 6, and 9.

[0177] Exposure to FP2 for 2 weeks led to a decrease in food intake, body weight loss, blood glucose reduction, improved glucose tolerance and insulin sensitivity in DIO mice. A significant decrease in food intake was obtained over multiple days at 1.0, 3.0, and 10.0 nmol / kg of q3d. Body weight decreased significantly starting from 3 to 5 days after the start of the test. The fasting blood glucose level on day 13 decreased significantly after q3d administration of 3.0 and 10.0 nmol / kg of FP2. Insulin sensitivity, indicated by a significant decrease in fasting HOMA - IR was obtained 14 days after q3d administration of 10.0 nmol / kg of FP2. On day 13, in DIO mice treated with 10.0 nmol / kg of FP2 at q3d a significant increase in fat removal rate (%) and a significant decrease in body fat rate (%) were observed.

[0178]

Table 38

[0179]

Table 39

[0180]

Table 40

[0181]

Table 41

[0182]

Table 42

[0183]

Table 43

[0184]

Table 44

[0185]

Table 45

[0186]

Table 46

[0187]

Table 47

[0188]

Table 48

[0189]

Table 49

[0190]

Table 50

[0191] Example 17: Pharmacokinetics and immune response in multiple species of FP2 Pharmacokinetics in mice The pharmacokinetic characteristics of FP2 were evaluated when it was administered subcutaneously to female C57Bl / 6 mice. FP2 was administered to female C57Bl / 6 mice (Sage Laboratories, St. Louis, Missouri) at a dose level of 2.0 mg / kg in PBS (pH 7.3 - 7.5) by subcutaneous (n = 5 samples at each time point) and intravenous (n = 5 samples at each time point) administration. Samples were collected at the last time point by terminal bleeding. Blood samples were collected, serum was processed, and the drug concentration was measured up to 168 hours. The concentration of FP2 was measured by immunoassay . The drug concentration profiles in plasma are summarized in Tables 51 and 52 and shown in Figure 25.

[0192] Pharmacokinetic analysis of FP2 in C57Bl / 6 mice showed that the terminal half - lives after intravenous and subcutaneous administration were approximately 1.51 days and 1.76 days, respectively, and the average bioavailability after subcutaneous administration was shown to be approximately 61%.

[0193]

Table 51

[0194]

Table 52

[0195]

Table 53

[0196] Drug kinetics in rats FP2 was administered subcutaneously (n = 5 samples per time point) and intravenously (n = 5 samples per time point) to Sprague - Dawley rats (Sage Laboratories Inc., St. Louis, Missouri) in PBS (pH 7.3 - 7.5) at a dose level of 2.0 mg / kg Samples were collected at the last time point by terminal bleeding. Blood samples were collected, serum was processed, and drug concentration was measured up to 168 hours. The concentration of FP2 was measured by immunoassay The drug concentration profiles in plasma are summarized in Tables 54 and 55 and shown in Figures 2 and 6. The pharmacokinetic parameters calculated from these data are summarized in Table 56

[0197] Pharmacokinetic analysis of FP2 in Sprague Dawley rats showed that the terminal half - lives after intravenous and subcutaneous administration were approximately 1.46 days and 1.37 days, respectively, and the average bioavailability after subcutaneous administration was approximately 28%

[0198]

Table 54

[0199]

Table 55

[0200]

Table 56

[0201] Drug kinetics in monkeys ​​FP2 was administered subcutaneously at 1 mg / kg and intravenously at 1 mg / kg to three male cynomolgus monkeys in PBS (pH 7.0 - 7.6) each. Blood samples were collected and plasma was processed , and the drug concentration was measured up to day 21.

[0202] The pharmacokinetics (PK) of FP2 were characterized after single intravenous (IV) (1.0 mg / kg ) and subcutaneous (SC) (1.0 mg / kg) administrations to cynomolgus monkeys. The drug concentration - time profiles in plasma after SC administration are shown in Tables 57 and 58 for immunoassay and LCMS analysis, respectively, and the drug concentration - time profiles in plasma after IV administration are shown in Tables 59 and 60 for immunoassay and LCMS analysis, respectively. The immunoassay data are graphed in Figure 27, and the LCMS data are shown in Figure 28. Using the results of immunoassay analysis, the terminal half - life (t

[0203] 1 / 2) of FP2 based on the average NCA was approximately 7.05 days and approximately 8.51 days after IV and SC administrations, respectively. The average PK parameters after IV and SC administrations are shown in Table 61. Using the results of immunoassay bioanalysis, the terminal half - life (t1 / 2 ) of FP2 estimated by the average non - compartmental model was approximately 7.05 days and approximately 8.51 days after IV and SC administrations, respectively. The average bioavailability (F%) of FP2 was estimated to be approximately 98.5% based on AUC in cynomolgus monkeys after SC administration and approximately 109.2% based on AUC . 0~最終 0~無限 .

[0204]

Table 57

[0205]

Table 58

[0206]

Table 59

[0207]

Table 60

[0208]

Table 61

[0209] Human plasma stability assay The ex vivo stability of FP2 was examined in fresh heparinized plasma at 37 °C up to 48 hours. Fresh non-frozen human plasma was prepared from heparinized blood of two subjects (one male and one female) by centrifugation. FP2 was incubated in this substrate at 37 °C for 0, 4, 24 and 48 hours with gentle mixing. The concentration of FP2 was measured by immunoassay. LCMS was performed after independent immune affinity capture to The concentration of the complete dimer present in the matrix was quantified.

[0210] In the immunoassay method, the recovery rate from the starting concentration was in the range of 104.8 - 94.1, and no decrease over time was observed, indicating that FP2 was stable in ex vivo human plasma for up to 48 hours (Figure 29 and Table 62). In the LCMS method, the concentration was stable over time and it was shown that FP2 was maintained as a complete dimer in ex vivo human plasma for up to 48 hours (Figure 30 and Table 63).

[0211] [Table 62]

[0212] [Table 63]

[0213] Example 18: Effects of single-dose FP1 and FP2 in cynomolgus monkeys The effects of FP1 and FP 2 on food intake and body weight in naïve cynomolgus monkeys after single-dose administration were evaluated.

[0214] FP1 was administered subcutaneously to a cohort of naïve cynomolgus monkeys at three dose levels of 1, 3, and 10 nmol / kg. A vehicle-treated group was also included. The animals were treated blindly. The study was conducted for a total of 6 weeks consisting of 2 weeks of baseline food intake measurement and data collection, and 4 weeks of data collection after single-dose administration of the compound. Plasma drug exposure was measured at 1, 7, 14, 21, and 28 days post-administration.

[0215] Treatment of cynomolgus monkeys with single-dose FP1 resulted in no significant differences in food intake and body ​Reduced weight (Figs. 31-32). A significant decrease in daily food intake was observed on days 4, 5, 6, and 8-12 at a dose level of 10 nmol / kg (Fig. 31). The weekly average value of daily food intake significantly decreased during the second week after administration at a dose level of 10 nmol / kg. At a dose level of 3 nmol / kg, a significant decrease rate (%) from the average weekly food intake was shown before the administration at the second week after administration, and at a dose level of 10 nmol / kg, a significant decrease rate (%) from the average weekly food intake was shown before the administrations at the first and second weeks after administration. A significant decrease in the weight change rate (%) from day 0 was observed on day 28 at a dose level of 3 nmol / kg and on days 14, 21, and 28 at a dose level of 10 nmol / kg (Fig. 32). FP2 was subcutaneously administered to a cohort of naive cynomolgus monkeys at three dose levels of 1, 3, and 10 nmol / kg. A vehicle-treated group was also included. The animals were treated in a blinded manner. The study was conducted for a total of 11 weeks, including a 5-week baseline food intake measurement and data collection, a 1-week treatment, and a 5-week washout period with data collection. Plasma drug exposure was measured on days 1, 7, 14, 21, 28, 35, and 42 after administration. Treatment of cynomolgus monkeys with a single dose of FP2 reduced food intake and body weight compared to vehicle treatment (Figs. 33-34). A significant decrease in daily food intake was observed on days 3, 5-8, 10, and 12 at a dose level of 3 nmol / kg, on days 3-38, and 40 at a dose level of 10 nmol / kg (Fig. 33). The weekly average value of daily food intake significantly decreased during the first week after administration at a dose level of 3 nmol / kg and at a dose level of 10 nmol / kg

[0216]

[0217] ​​​​​​​​​​​​​​​Significantly decreased in the 1st to 6th week. At a dosage level of 3 nmol / kg, at the 2nd week after administration, a significant decrease rate (%) was observed from the week before administration in the weekly average daily food intake, and at a dosage level of 10 nmol / kg, significant decrease rates (%) were observed from the week before administration in the weekly average daily food intake at the 1st and 6th weeks after administration. A significant decrease in the body weight change rate (%) from day 0 was observed at the dosage level of 1 nmol / kg from day 21 to 42, at the dosage level of 3 nmol / kg from day 14 to 42, and at the dosage level of 10 nmol / kg from day 7 to 42 (Figure 33).

[0218] Example 19: Effect of multiple administrations of FP2 in cynomolgus monkeys The effect of FP2 was evaluated by weekly subcutaneous injection at three dosing levels of 0.3, 1, and 10 nmol / kg to a cohort of naïve spontaneously overweight cynomolgus monkeys (aged 8 - 20 years, body weight 8.0 - 11.9 kg). Food intake was measured daily, body weight was measured weekly, and the general condition of the animals was evaluated daily. Treatment of overweight cynomolgus monkeys with FP2 once a week for 12 weeks reduced food intake (Figure 35) and body weight (Figure 36) compared to vehicle treatment. Circulating FP2 concentration was measured by immunoassay (Figure 37). Loss of exposure to FP2, presumably due to the production of anti-drug antibodies (ADA), was observed in some animals at a later time point . The figure shows data collected up to the point before the loss of exposure (defined as a decrease of more than 40% of the trough serum drug concentration from the previous measurement for the same animal). No treatment-related toxic effects were observed throughout the study.

[0219] Example 20: Thermal stability of the linker ​​​The thermal stability of different linkers linking HSA and GDF15 was examined. Fragmentation and aggregation properties were evaluated by diluting HSA-GDF15 fusion proteins with different linkers to 10 mg / ml. After adding 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 by size exclusion high performance liquid chromatography (SE-HPLC). The percentage of intact protein as well as aggregates and fragments (%) was quantified for these proteins. Table 64 shows that the HSA-GDF15 protein with a linker consisting of repeats of AP is the most stable against fragmentation under heat stress .

[0220] To evaluate whether these linkers affect the interaction between GDF15 and its receptor, a GFRAL-FC fusion protein was coated on plates and an immunoassay for detection with anti-GDF15 or anti-HS A was performed using monoclonal antibodies against GDF15 (Janssen) and HSA (K erafast, Inc., Boston, Massachusetts). This assay showed that all of these linkers have similar binding properties to the receptor, as shown in Table 66 .

[0221] [Table 64]

[0222] Example 21: Clinical trial protocol The safety, tolerability, pharmacokinetics (including absolute bioavailability), and immunogenicity of FP2 administered subcutaneously to healthy subjects except for being overweight were examined in a double-blind, placebo -controlled trial ​​​Placebo-controlled, randomized, single-dose escalating administration study

[0223] Protocol 64739090EDI1001; Phase 1

[0224] EudraCT number: 2018-000324-34

[0225] [Table 65-1]

[0226] [Table 65-2]

[0227] [Table 65-3]

[0228] [Table 65-4]

[0229] [Table 65-5]

[0230] [Table 66-1]

[0231] [Table 66-2]

[0232] [Table 66-3] a - The first part is double-blind and consists of a maximum of seven dosing groups. The second part is non-blind and consists of a single-dose group. The b-screening procedure must be conducted within 4 weeks (28 days) before the administration of the investigational drug on Day 1. It is required to be carried out. c-Informed consent - It is necessary to obtain it before starting any test-related procedures. d-Inclusion / exclusion criteria - The minimum criteria for the availability of documents supporting the eligibility criteria are described in "Section 4 Eligibility of the Subject Population" and are confirmed after verification of the pre-dose baseline assessment. It is confirmed after verification. e-For the list of clinical tests to be obtained, please refer to "Example 21 Section 9.6.2 Clinical Tests". Subjects need to fast for at least 10 hours before blood sampling (i.e., do not consume food or beverages (except water)). As a result for verification before randomization and administration on Day 1, baseline clinical tests may be obtained on Day -2 or Day -1. The baseline clinical tests are provided as such for verification before randomization and administration on Day 1. They may be obtained on Day -2 or Day -1. f-It is obtained only from all female subjects. g-Randomization is carried out on Day 1 after all evaluations on Day -2 and Day -1 have been conducted, reviewed, verified, and it is confirmed that the subjects meet all inclusion criteria and do not meet the exclusion criteria (e.g., test results, ECG, etc.). After confirmation, it is carried out on Day 1. h-Part 1 Dose escalation / subcutaneous (SC) administration: A single dose of FP2 or placebo (maximum volume 2 mL) is administered to the subjects. Part 2: A single dose of FP2 is administered to the subjects by IV infusion (constant rate) over 30 minutes (constant rate). All subjects in Part 1 and Part 2 need to fast overnight (at least 10 hours) from pre-dose to 3 hours after administration. Time 0 in Part 1 is the injection time of the investigational drug by SC, and time 0 in Part 2 is the start time of the IV infusion of the investigational drug. A single dose of FP2 or placebo (maximum volume 2 mL) is administered to the subjects by SC. Part 2: A single dose of FP2 is administered to the subjects by IV infusion (constant rate) over 30 minutes (constant rate). All subjects in Part 1 and Part 2 need to fast overnight (at least 10 hours) from pre-dose to 3 hours after administration. Time 0 in Part 1 is the injection time of the investigational drug by SC, and time 0 in Part 2 is the start time of the IV infusion of the investigational drug. Time 0 in Part 1 is the injection time of the investigational drug by SC, and time 0 in Part 2 is the start time of the IV infusion of the investigational drug. i-Vital signs need to be measured after 5 minutes of rest in the supine position, with the resting heart rate The number (HR) and blood pressure (BP) need to be included. If blood sampling or vital sign measurements are scheduled at the same time as the ECG recording, each procedure must be performed in the order of vital signs, ECG, PK, blood sampling for safety, and exploratory biomarker analysis. The measurements are taken with a fully automated sphygmomanometer. At all time points, a single blood pressure and time are measured and recorded. j - Continuous II - lead ECG monitoring is only performed in Part 2, starting 30 minutes before the start of the IV infusion on Day 1 and ending 2 hours after the end of the infusion. The principal investigator may, at their discretion, extend the duration of cardiac monitoring. k - 12 - lead ECG: Except for the screening ECG, all ECGs are measured three times. The subjects need to avoid distractions (e.g., TV, mobile phone), rest in the supine position in a quiet situation for at least 5 minutes, and refrain from talking or moving their limbs. The three ECGs need to be obtained individually within 2 minutes at each time point. If the ECG is performed at the same time as the PK sample for the same test, the PK sample needs to be collected immediately after the ECG. - The 12 - lead ECG on Day 1 needs to be synchronized (simultaneous) with the 12 - lead ECGs scheduled on Day 1 (i.e., before dosing, 1, 2, 4, 8, 12, and 24 hours after dosing). m - Body weight: Before breakfast and after urination on Days - 1, 2, 3, 4, and 5, and after urination on Day 1 before dosing. The body weight needs to be measured twice. The subjects need to be weighed on a calibrated scale without shoes and wearing a gown. n - The pre - dosing procedure needs to be obtained within 30 minutes before the test drug administration. o - For a detailed description of the timing of the 24 - hour food intake assessment and the Refer to "Example 21, Section 9.3, Pharmacodynamic Evaluation", "Time and Subject Schedule of Diet and VAS Questionnaire". p - At screening and serum pregnancy test are required for all women. Urine pregnancy test can be obtained at all other time points. Refer to the lab manual for the description of q - sample collection procedures and processes. r - Pharmacokinetic evaluation: All PK blood samplings should be carried out as close as possible to the scheduled time points. If an ECG is performed at the same time point, the PK specimens should be collected immediately after the end of the ECG. In Part 2 (IV infusion), all sampling time points are relative to the end of the infusion. The timing of pharmacokinetic sample collection may be changed if indicated by preliminary PK data from the previous dose (however, no additional samples are collected). s - Time point 0.5 (= t 0 (at the end of injection) is only applicable to IV administration in Part 2. t - Pharmacogenomic (DNA) samples need to be collected at the designated time points, but if necessary, they may be collected at later time points without deviating from the protocol. u - Adverse events and concomitant medications are recorded from the time of signing the informed consent until the final test procedures at the hospital visit at the end of the test. Additionally, for adverse events, queries are made at designated time points throughout the test (using open - ended questions). v - For guidelines on reporting the toxicity of local injection site reactions, refer to Table 70 in "Example 21, Section 9.6.8 Local Injection Site Reactions". w - For guidelines on the management of allergic reactions and / or hypersensitivity reactions, refer to "Example 21, Section 9.6.7 Allergic Reactions / General Hypersensitivity". ​​​​​​​​​​​​

[0233]

Table 67

[0234]

Table 68

[0235] 1. Introduction Growth differentiation factor 15 (GDF15) exists as a 25 kDa dimer in human plasma It is a circulating protein factor. Published data and internal data mainly support its role in the regulation of energy balance (i.e., food intake) that affects energy metabolism.

[0236] Subcutaneous (SC) administration of FP2 results in a decrease in food intake and subsequent body weight (BW) loss in rodents and non-human primates. Furthermore, SC treatment with FP2 probably improves glucose homeostasis and insulin resistance in diet-induced obesity (DIO) mice, likely due to body weight loss. FP2 acts mainly by binding to GDNF family receptor α-like (GFRAL), a recently identified GDF15 receptor expressed in the area postrema of the central nervous system (CNS). 5,17,24,15 FP2 reduces food intake, which results in body weight loss in obese subjects and is assumed to lead to improvement of obesity-related comorbidities.

[0237] 1.1 Background 1.1.1. Non-clinical trials Pharmacological profile The in vitro agonist potency of FP2 was evaluated using a cell-based pAKT assay with SK-N-AS cells stably overexpressing the human GFRAL receptor. FP2 activated pAKT at a half-maximal effective concentration (EC 50 ) of 2.908 ± 0.239 nM (N = 3) (see Example 14). Native GDF15 functioned as a control for the assay and had an EC of 0.153 ± 0.008 nM (N = 3) (EC 50showed agonist activity.

[0238] FP2 was evaluated for its ability to reduce food intake in multiple species. The single SC administration of FP2 strongly inhibited food intake in male C57Bl / 6 mice (see Example 14) and Sprague-D awley (SD) rats (see Example 15). A single SC administration of FP2 to naive spontaneously overweight cynomolgus monkeys resulted in a reduction in food intake and, as a result, a significant weight loss compared to vehicle-treated animals up to 4 weeks after administration (Example 18).

[0239] The repeated administration of FP2 every 3 days for 2 weeks decreased food intake and body weight in DIO mice and improved glucose tolerance and insulin sensitivity measured by the homeostasis model assessment (insulin resistance) (see Example 16). Weekly administration of FP2 to a cohort of naive spontaneously overweight cynomolgus monkeys for 12 weeks significantly decreased food intake and body weight compared to vehicle treatment (see Example 19). Loss of exposure at later time points was observed in some animals, presumably due to the production of anti-drug antibodies ( ADA). No treatment-related toxic effects were observed throughout the study.

[0240] Safety pharmacology Safety pharmacology endpoints (cardiovascular [CV], respiratory, and central nervous system [CNS function) were evaluated in cynomolgus monkeys (study number 8372593) and SD rats (study number 8371098) in accordance with the International Conference on Harmonization of Technical Requirements for Pharmaceuticals for Human Use (ICH) S6(R1) guidelines as part of a 4-week repeated-dose toxicity study conducted according to Good Laboratory Practice (GLP) standards for pharmaceutical safety testing. Performed. Furthermore, a stand-alone CV safety pharmacology study (study number T-2017-044) was conducted in cynomolgus monkeys equipped with a telemetry device.

[0241] Overall, IV and SC administrations of FP2 up to the maximum dose had no effect on CV endpoints, core body temperature, respiratory rate, neurological or behavioral endpoints.

[0242] Toxicity Non-clinical safety studies (see Table 65) were conducted in countries that are members of the mutual acceptance of chemical safety data of GLP, 21 CFR, Part 58, and / or the Organization for Economic Cooperation and Development (OECD) in accordance with the principles of OECD-GLP, including appropriate documentation. The FP2 test substance (batch number CVC_PCM01) used in the non-clinical safety studies is considered representative of the clinical trial substance.

[0243]

Table 69

[0244] Repeated administration of FP2 in cynomolgus monkeys and SD rats was generally well tolerated. There were no deaths and no obvious signs. Some findings (e.g., decreased food intake and body weight ) are considered the result of the proposed mechanism of action and are not regarded as toxic effects.

[0245] Selection of relevant species Since FP2 is a fully recombinant fusion protein with both human GDF15 and the HSA domain linked via a short peptide consisting of natural amino acids, the toxicity program was ​​Primarily in accordance with ICH guideline S6(R1) "Preclinical Safety E valuation of Biotechnology-Derived Pharm aceuticals" (Preclinical safety evaluation of biotechnology-derived pharmaceuticals). It is designed accordingly.

[0246] Regarding the determination of relevant animal species, for the GDF15 moiety of FP2 being the biologically active ingredient while the HSA moiety functions mainly through interaction with the neonatal Fc receptor (FcRn) to extend the half-life, thereby increasing the exposure of FP2. The GDF15 receptor (GFRAL) and the GFRAL signaling coreceptor (RET) have only recently been identified 1 7,24,5,15 .

[0247] Analysis of the amino acid sequence homology in silico of the biologically active ingredient (GDF15), its receptors (GFRAL and RET), its half-life extending ingredient (HSA), and the albumin receptor (FcRn) in different species revealed the highest similarity (95 - 100%) between humans and monkeys (i.e., cynomolgus monkeys), and a correspondingly high similarity (7 8 - 100%) between humans and rats (Table 66).

[0248]

Table 70

[0249] ​​In vitro binding assays demonstrated binding of FP2 to recombinant GFRAL proteins from human, rat, and cynomolgus monkey, with affinities within a 2-fold range between human and cynomolgus monkey and within a 5-fold range between human and rat. Furthermore, tissue expression analysis of GFRAL receptors in different species revealed comparable expression patterns (mainly in the area postrema of the hindbrain) in rat, monkey, and human. 24 .

[0250] In vivo pharmacodynamic (PD) studies demonstrated the putative PD effects of FP2 (e.g., reduction in food intake and body weight) in both cynomolgus monkey and rat.

[0251] However, single-dose PK studies also showed some significant differences in PK between cynomolgus monkey (T : approximately 7 - 9 days) and Sprague-Dawley rat (T : approximately 1 - 2 days) due to lower affinity of human HSA for rat FcRn receptor and similar affinity of human HSA for cynomolgus monkey FcRn. 1 / 2 1 / 2

[0252] Therefore, cynomolgus monkey was considered the most relevant / predictive animal species and was selected as the non-rodent species for conducting non-clinical safety studies to enable first-in-human (FIH). Rat was selected as the rodent toxicology test species with some limitations in PK.

[0253] Pharmacokinetic profile The PK and toxicokinetics (TK) of FP2 were characterized in rodents and lean cynomolgus monkeys after single-dose and up to 4 weeks of chronic dosing. Time to reach maximum concentration (T max ) ​​​​​​​​​​​​The median among them was estimated to be 1 day, 1 day, and 1.67 days in mice, rats, and cynomolgus monkeys, respectively. The clearance of FP2 (about 25 and 5 mL / day / kg in rodents and monkeys, respectively) and the elimination half-life (about 1.5 and 7.1 days in rodents and monkeys, respectively) are likely to be significantly different between monkeys and rodents due to the difference in the affinity of HSA for FcRn in rodents or monkeys (i.e., HSA binds to rodent FcRn with a lower affinity than human FcRn, whereas the affinity of HSA for monkey FcRn is similar to that for humans). Therefore, monkeys are considered to be a more predictive species for the PK of FP2 in humans than rodents. In a 90-kg human, the predicted elimination half-life is about 12 - 17 days.

[0254] FP2 has been shown to be stable in human plasma ex vivo for up to 48 hours and in cynomolgus monkeys in vivo after SC and IV administration as a complete dimer. The metabolism of intact FP2 is expected to proceed through standard proteolytic pathways.

[0255] 1.1.2. Clinical Trials Since this is the first administration of FP2 in humans, there is no clinical experience.

[0256] Pharmacokinetics and Immunogenicity in Humans To date, no human PK studies have been conducted with FP2.

[0257] Efficacy / Safety Trials To date, no clinical trials have been conducted with FP2.

[0258] 2. Objectives and Hypotheses 2.1 Objectives 2.1.1. First Part: Dose Escalation Single Administration​​​​ Overweight (BMI 25 to 29.9 kg / m 2 The subjects were otherwise healthy. After single ascending dose SC administration of FP2:

[0259] Main purpose To evaluate the safety and tolerability of FP2 administered subcutaneously (SC).

[0260] Secondary Objectives -Evaluate the penalty kicks in FP2. To evaluate the immunogenicity of FP2 in terms of potential ADA formation and to assess the effect of endogenous GDF To evaluate the possibility of antibody formation against 15. -Assess pharmacodynamic (PD) endpoints such as body weight and food intake.

[0261] exploratory purpose Administration of FP2 was assessed using a visual analogue scale (VAS) questionnaire. To determine whether the change in appetite was associated with changes in PD endpoints such as food preference and appetite assessment. evaluate. To evaluate whether endogenous levels of GDF15 are associated with PD endpoints . To evaluate whether PK in FP2 is associated with PD endpoints.

[0262] 2.1.2. Part 2: Absolute bioavailability Overweight (BMI 25 to 29.9 kg / m 2 The subjects were otherwise healthy. After a single intravenous (IV) dose of FP2:

[0263] Main purpose Age, sex, and weight matched (one of the dose-escalating SC arms listed above in Part 1) subjects were given a 30-minute short-term IV infusion (constant rate) The absolute SC bioavailability of FP2 is estimated by single dose administration.

[0264] Secondary Objectives To evaluate the safety and tolerability of FP2 administered IV

[0265] 2.2. Hypothesis Given that the primary objectives were safety and tolerability, no formal statistical hypotheses were established in this study. No tests of hypotheses were planned. All other analyses are exploratory.

[0266] 3. Study Design and Rationale Study design overview This is the first-in-human (FIH) test for FP2. The study was in two parts and was conducted at a single center in overweight but otherwise healthy subjects. Part 1 will evaluate the safety, tolerability, and PK of single ascending doses of FP2 administered SC. The second part was a 30-minute randomized, double-blind, placebo-controlled study to determine whether Systemic exposure and PK of FP2 administered as a single dose short-term IV infusion (fixed rate) in a 10-mL bolus This is an open-label, single-arm study to evaluate

[0267] Approximately 62 people were overweight (BMI 25 to 29.9 kg / m 2 (The following) Otherwise healthy male and female (non-reproductive potential) subjects are planned to participate in this study. Approximately 56 subjects will be randomly selected into Part 1 of this study. and approximately 6 subjects will be assigned to part 2.

[0268] Subjects will be screened for eligibility on Days -28 through -3. Eligible subjects will Subjects were admitted to the clinical trial unit (CRU) on day -2 and had baseline safety assessments. Have them receive the price. On the first and third days before the study, have the subjects undergo a 24-hour measurement of food intake and fill in a VAS questionnaire for appetite evaluation and food preference evaluation. Administer the test drug to the subjects on the first day, and have them continue to stay in the CRU until the morning of the fifth day for safety, tolerance, PK / ADA, and PD evaluations. They may be discharged when the test evaluations are completed. Request that the subjects return to the CRU at the outpatient clinic visits during the first week (seventh day), second week (fourteenth day), third week (twenty-first day), fourth week (twenty-eighth day), sixth week (forty-second day), eighth week (fifty-sixth day), tenth week (seventieth day), twelfth week (eighty-fourth day), and at the outpatient clinic visit after the end of the study (7 to 10 days later). The total test duration for each subject is approximately 17 weeks.

[0269] Figure 38 shows an overview of the test plan.

[0270] 3.1.1. Part 1: Single ascending dose Test sequentially in a maximum of seven dose groups (DGs) of healthy subjects who are not overweight (8 subjects per DG). Within each DG, randomize 6 subjects to FP2 and 2 subjects to placebo-matched. Therefore, the ratio of active drug to placebo is 3:1 at each dose level (see Table 67). Enroll 4 male and 4 female subjects (randomize 3 subjects to the active substance and 1 subject to the placebo in each sex group) in the first DG of Part 1 of the study where an undiluted test drug (i.e., a 50 mg / mL undiluted formulation) administration is planned so that each subject can be matched to the corresponding IV administration group in Part 2 of the study.

[0271] The planned dosing escalation scheme for FP2 is described in Table 67 below. The doses administered during the trial are calculated for an 80 kg individual based on the flat dosing approach specified in the column "FP2 (mg SC)".

[0272]

Table 71

[0273] The treatment is double-blind and randomized at each dose level.

[0274] At each dose level, subjects are divided into four subgroups (n = maximum 2 / subgroup) and dosed on different days. First, two sentinel subjects are dosed simultaneously on the same day (one with placebo, one with FP2), and after completing a 72-hour safety monitoring period, dosing of the next subject in that DG can commence. After verifying the safety data, up to two additional subjects per day (at approximately 2-hour intervals) can be dosed until all subjects have completed dosing. At least 10 days are placed between the last subject in the previous group and the first subject in the subsequent DG.

[0275] After completion of each dose level, preliminary safety and PK data are verified by the sponsor and the principal investigator (PI) of the trial to determine the next planned dosing level. Each dosing escalation decision is based on the blinded preliminary safety, tolerability, and PK data collected in all subjects in a particular DG at least 72 hours after dosing. The minimum number of evaluable subjects (i.e., subjects who have completed the study procedure at least 72 hours after dosing) required for verification of dosing escalation is N = 7 per DG.

[0276] The predicted mean serum exposure of FP2 (C max or AUC​​0~48時間 ) is the most relevant dose that exceeds the lowest no- observed adverse effect level (NOAEL) obtained from a 1-month GLP toxicity study in the most relevant species (i.e., cynomolgus monkey) will not be administered.

[0277] 3.1.2. Part 2: Absolute Bioavailability Part 2 is an open-label single-arm study to evaluate the systemic exposure and PK of a single administration of FP2 as a 30-minute IV infusion (constant rate) in healthy overweight (BMI 25 or more to 29.9 kg / m 2 2 or less) male and female subjects. The absolute bioavailability of the SC dosage form of FP2 will be measured using the PK data from Part 2. In Part 2, 6 healthy male (n = 3) and female (n = 3) subjects who are overweight (BMI 25 or more to 29.9 kg / m 2 or less) but otherwise healthy will be enrolled. The subjects will be matched for sex, age (±5 years), and weight (±5 kg) with subjects in the SC dosing group from Part 1 (presumably DG5; the first dosing group of 30 mg where an undiluted FP2 formulation of 50 mg / mL was used). Part 2 can be initiated before the completion of Part 1 of the study after the blinded pre-safety and tolerability data from DG before Part 1 of the study have been verified by the sponsor of the clinical trial and the principal investigator (PI) of the clinical trial.

[0278] In Part 2, each eligible subject will receive a single IV dose of FP2 administered as a constant rate short-term infusion over 30 minutes via an appropriate indwelling catheter in the antecubital vein. The IV dose in Part 2 will be selected based on the pre-safety and PK data in Part 1. 2 except that they are overweight (BMI 25 or more to 29.9 kg / m 2 or less). Subjects will be matched for sex, age (±5 years), and weight (±5 kg) with subjects in the SC dosing group from Part 1 (presumably DG5; the first dosing group of 30 mg where an undiluted FP2 formulation of 50 mg / mL was used). Part 2 can be initiated before the completion of Part 1 of the study after the blinded pre-safety and tolerability data from DG before Part 1 of the study have been verified by the sponsor of the clinical trial and the principal investigator (PI) of the clinical trial. Part 2 can be initiated before the completion of Part 1 of the study after the blinded pre-safety and tolerability data from DG before Part 1 of the study have been verified by the sponsor of the clinical trial and the principal investigator (PI) of the clinical trial. In Part 2, each eligible subject will receive a single IV dose of FP2 administered as a constant rate short-term infusion over 30 minutes via an appropriate indwelling catheter in the antecubital vein. The IV dose in Part 2 will be selected based on the pre-safety and PK data in Part 1. In Part 2, each eligible subject will receive a single IV dose of FP2 administered as a constant rate short-term infusion over 30 minutes via an appropriate indwelling catheter in the antecubital vein. The IV dose in Part 2 will be selected based on the pre-safety and PK data in Part 1. It can be done.

[0279] Each eligible subject in Part 2 will receive a single IV dose of FP2 administered as a constant rate short-term infusion over 30 minutes via an appropriate indwelling catheter in the antecubital vein. The IV dose in Part 2 will be selected based on the pre-safety and PK data in Part 1. The IV dose in Part 2 will be selected based on the pre-safety and PK data in Part 1. . The selected IV dose should not exceed one-third of the dose already evaluated as being fully acceptable in part 1, taking into account the expected differences in maximum exposure levels upon IV administration and, where appropriate, the incomplete bioavailability of the SC formulation. For details, see section 3.5 of Example 21, part 2 of dose selection. For safety monitoring, administer to one sentinel subject first, and after completing a 72-hour safety monitoring period, dosing to the next subject can be carried out. The remaining 5 subjects are further divided into subgroups (administered at least 24-hour intervals), and dosing is not carried out to more than 2 subjects per day (at approximately 2-hour intervals). and, where appropriate, in part 1 to account for the incomplete bioavailability of the SC formulation. not exceed one-third of the dose already evaluated as being fully acceptable. For details, see section 3.5 of Example 21, part 2 of dose selection. See section 3.5 of Example 21, part 2 of dose selection. For safety monitoring, administer to one sentinel subject first, and after completing a 72-hour safety monitoring period, dosing to the next subject can be carried out. The remaining 5 subjects are further divided into subgroups (administered at least 24-hour intervals), and dosing is not carried out to more than 2 subjects per day (at approximately 2-hour intervals). further divided (administered at least 24-hour intervals), and dosing is not carried out to more than 2 subjects per day (at approximately 2-hour intervals).

[0280] 3.2. Theoretical basis of the study design 3.2.1. Considerations in the overall study design The proposed study is a first-in-human (FIH), double-blind, randomized, placebo-controlled, single-ascending-dose (SAD) study in healthy adult subjects (except overweight) to evaluate the safety, tolerability, PK, immunogenicity, and PD (i.e., food intake, body weight, appetite assessment, and food preference) of FP2, which is a fully recombinant homodimer of GDF15 fused to HSA. This study is designed in accordance with relevant regulatory guidelines for first-in-human and other early clinical development studies (EMA Guidance EMEA / CHMP / SWP / 28367 / 07 Rev.1, 2017; FDA Guidance for Industry, 2005). Based on the available non-clinical data and pharmacological properties regarding the expected systemic safety,

[0281] This study is designed in accordance with relevant regulatory guidelines for first-in-human and other early clinical development studies (EMA Guidance EMEA / CHMP / SWP / 28367 / 07 Rev.1, 2017; FDA Guidance for Industry, 2005). EMA Guidance EMEA / CHMP / SWP / 28367 / 07 Rev.1, 2017; FDA Guidance for Industry , 2005).

[0282] Regarding the expected systemic safety, based on the available non-clinical data and pharmacological properties,​​​​ , FP2 is not considered to be a "high-risk" novel biological entity (NBE) according to the criteria outlined in the EMA's "Guideline on strategies to id entify and mitigate risks for first-in-h uman clinical trials with investigationa l medicinal products" (Guideline on strategies for identifying and reducing risks in first-in-human trials with investigational medicinal products). It is not considered an " 4 .

[0283] Important trial design elements such as the determination of the starting safety dose (based on estimated minimum pharmacological effect level [MABEL], pharmacologically active dose [PAD], and NOAEL data), the dose escalation strategy, and the definition of stopping criteria meet the current scientific, medical, and ethical standards and requirements (Sections 3.3, 3.4, 3.5, 3.6 of Example 21) and are consistent with the design of current FIH trials for comparable products with similar objectives. .3, 3.4, 3.5, 3.6) and are consistent with the design of current FIH trials for comparable products with similar objectives when considering other equivalent products with similar objectives.

[0284] The targeted patient population is well-defined and carefully selected based on a comprehensive set of applicable inclusion and exclusion criteria (see Section 4, Subject Population, of the protocol). . All subjects will be monitored with regular safety follow-up for 13 weeks after dosing. .

[0285] This trial will be designed and conducted at a specialized CRU under medical monitoring conditions that ensure, if necessary, the early detection of adverse events with a high probability and appropriate therapeutic intervention.

[0286] 3.2.2. Blinding, Control, Phase / Period of the Trial, Treatment Groups​​​ Part 1 A double-blind, placebo-controlled, randomized trial design allows for the best practical evaluation of the safety and tolerability profile of FP2 by minimizing potential biases during data collection and for the evaluation of clinical endpoints. To evaluate the frequency and magnitude of changes in clinical endpoints that occur when active treatment is not given, a placebo control is used in Part 1. Randomization is used to minimize bias in the assignment of subjects to treatment groups and to increase the likelihood that known and unknown subject attributes (e.g., demographic characteristics and baseline characteristics) are evenly balanced between treatment groups.

[0287] Part 2 Part 2 is a non-blind, single-arm trial design that provides PK data for the IV independent of the formulation for the disappearance of FP2, which cannot be obtained by other methods, and is used to estimate the absolute bioavailability (BA) of the SC FP2 dosage form.

[0288] 3.2.3. Test Population The rationale for enrolling healthy subjects, except for those who are overweight, is as follows.

[0289] FP2 is administered via the SC route, and drug absorption characteristics from SC tissue (i.e., absorption rate and absorption amount) tend to differ among different subjects (e.g., men and women) and target populations (i.e., lean subjects, overweight subjects, and obese subjects). Therefore, this study aims to determine the initial human PK in the relevant population to enable a reliable prediction of PK and dose selection for repeated dosing in a population closer to the target population before exposing overweight or obese subjects to longer-term studies. ​​​​​​​​​​​​​

[0290] The subject risk of healthy subjects other than being overweight is considered equivalent to that of lean healthy subjects, since subjects with clinically significant conditions (e.g., , type 2 diabetes, hypertension), which are known to be more frequently seen in overweight individuals according to the screening criteria, are excluded.

[0291] The enrollment of healthy subjects other than being overweight enables the preliminary evaluation of the safety and PD effects (such as food intake, body weight, appetite assessment, and food preference) of FP2 in a subject population similar to / equivalent to the test population expected to be enrolled in the second phase.

[0292] 3.2.4. Pharmacokinetics and Pharmacodynamics The timing and duration of PK sampling in this study are based on non-clinical PK data including allometric model predictions. By using this information, the complete characterization of the PK profile is enabled by a frequent blood sampling schedule, and the data required to define the major PK parameters necessary to support further clinical development is provided.

[0293] The 24-hour assessment of food intake (before dosing and at the predicted T max ), and the monitoring of body weight throughout the study enable the evaluation of the possible reduction in food intake (i.e., reduction in calorie intake, and the expected weight loss) that may occur upon single administration of FP2. By frequently filling out the VAS quality questionnaire, the characterization of the changes in appetite behavior that may be associated with the reduction in food intake and weight loss during treatment with single administration of FP2 can be performed.

[0294] 3.2.5. Safety and Tolerability The major findings recorded in a 1 - month GLP toxicity study in rats and monkeys using FP2 were considered secondary to the observed significant decreases in food intake and body weight, which was the hypothesized target pharmacokinetics for this class of drugs. Overall, FP2 was well tolerated in the toxicity study and no findings were noted that required special monitoring.

[0295] Therefore, safety monitoring in this study will consist of a series of standard safety evaluations including monitoring of vital signs (heart rate, systolic and diastolic blood pressure, body temperature), standard clinical tests (hematology, clinical chemistry, urinalysis, lipids, coagulation), physical examinations, monitoring of signs and symptoms / harmful events [TEAE] manifested by treatment including allergic reactions / hypersensitivity and local injection site reactions, and documentation of a series of standard 12 - lead ECGs. Continuous 12 - lead ECG monitoring will also be performed in Part 2.

[0296] 3.2.6. Immunogenicity Since the immunogenic potential of NBE is part of its overall safety profile, the potential immunogenicity of FP2 will be monitored by a series of quantifications of ADA and screening for antibodies that may be formed against endogenous GDF15.

[0297] 3.2.9. IV Administration (Part 2) Part 2 of the study was a single - dose IV infusion (constant rate) of FP2 over 30 minutes administered to healthy subjects, matched for age, sex and body weight to an appropriate SC reference group (Group 1 of Part 1 where the undiluted study drug was administered), except for being overweight, to assess systemic exposure and PK. ​​This is an open-label single-arm trial to evaluate and provides PK data for IV independent of the formulation regarding the disappearance of FP2 that cannot be obtained by other methods, and is used to estimate the absolute bioavailability (BA) of the SC dosage form of FP2. The six subjects who received FP2 by IV administration are typical in the estimation of absolute BA and function as a benchmark for the evaluation of the pharmaceutical quality attributes of the SC dosage form.

[0298] 3.3. Theoretical basis for dose selection and escalation in Part 1 At three dose levels tested in a repeated-dose study in overweight cynomolgus monkeys (see Example 19 for reference), to simultaneously characterize the PK and PD of FP2 (change rates (%) of food intake and BW compared to baseline), an indirect response PK / PD model combining the PK of FP2 with the physiological manifestation of the relationship between food intake and body weight (BW) was developed. In this study, the decrease in food intake was greatest between Weeks 2 and 3 and showed a dose-dependent attenuation with continuous treatment, while body weight decreased continuously until Week 4 (1 nmol / kg group) or Week 7 (10 nmol / kg group) and then reached a plateau. To characterize these observations, a novel physiological function-based PK / PD model was developed that describes the treatment-induced changes in both food intake (FI) and the resulting body weight (BW) by including terms that describe the compensatory changes in food intake and energy consumption that occur in response to changes in body weight. The change in body weight was described as the combined effect of the long-term effect of changes in food intake over time and changes in energy consumption. This PK / PD model can describe the trajectories of food intake and body weight during a 12-week study and overweight food intake (FI) and the resulting body weight (BW) by including terms that describe the compensatory changes in food intake and energy consumption that occur in response to changes in body weight. The change in body weight was described as the combined effect of the long-term effect of changes in food intake over time and changes in energy consumption. This PK / PD model can describe the trajectories of food intake and body weight during a 12-week study and overweight cynomolgus monkeys. Provided the exposure-response relationship of FP2 in overweight cynomolgus monkeys. The weight loss-dependent compensatory food intake term in this model maintains the parameters of the drug's effect on food intake constant over time for a specific exposure. This semi-mechanistic model developed in cynomolgus monkeys enables further translatable modeling, and the results indicate that the percentage of weight loss in humans is greater than that in cynomolgus monkeys for a specific rate of decrease in energy intake (%). This modeling result also quantitatively supports the mechanism of action of FP2, and weight loss is mainly brought about by the drug-induced decrease in food intake in overweight cynomolgus monkeys. Using this modeling approach, PAD and effective clinical doses / exposures in humans were determined. Based on the weight loss-dependent compensatory food intake term, the parameters of the drug's effect on food intake are maintained constant over time for a specific exposure. This semi-mechanistic model developed in cynomolgus monkeys enables further translatable modeling based on the known relationship between energy intake and weight changes in humans. and the results show that the percentage of weight loss in humans is greater than that in cynomolgus monkeys for a specific rate of decrease in energy intake (%). This modeling result also quantitatively supports the mechanism of action of FP2, and weight loss is mainly brought about by the drug-induced decrease in food intake in overweight cynomolgus monkeys. Using this modeling approach, PAD and effective clinical doses / exposures in humans were determined. Assuming that the PK / PD relationship of FP2, as well as physiologically relevant parameters and SC bioavailability, are translatable between overweight cynomolgus monkeys and humans, the predicted weekly SC dose in humans that would result in a 20% decrease in food intake at week 12 is approximately 0.08 mg / kg (approximately 0.5 nmol / kg). Based on the open literature and model simulations, the dose that would result in a 20% decrease in food intake at week 12 after a single weekly SC administration could correspond to a weight loss of more than 10% after one year of treatment.

[0299] 3.3.1. Reasons for the Justifiability of the Starting Dose The starting dose of this trial was based on toxicity (NOAEL) and pharmacological data (PAD). open literature 13、12、11 and model simulations, the dose that would result in a 20% decrease in food intake at week 12 after a single weekly SC administration could correspond to a weight loss of more than 10% after one year of treatment. 12 weeks after a single weekly SC administration could correspond to a weight loss of more than 10% after one year of treatment.

[0300] 3.3.1. Reasons for the Justifiability of the Starting Dose The starting dose of this trial was based on toxicity (NOAEL) and pharmacological data (PAD). , selected in accordance with the regulatory guidelines related to the First-in-Human trial 3,6 as per .

[0301] In the 1-month toxicity study, the NOAEL doses for rats and cynomolgus monkeys were 1 00 mg / kg for rats and 50 mg / kg for cynomolgus monkeys. The human equivalent dose (HED) was calculated by normalizing the dose with respect to body surface area. For the calculation of the maximum recommended starting dose (MRSD ), a safety margin of 10 was used as the default safety factor to protect human subjects receiving the initial 6 clinical dose . As reflected in the exposure ratio calculations described below, when a safety factor of 10 was used, the MRSD for FP2 was calculated to be 1.6 mg / kg BW. For a human with a body weight of 80 kg, the MRSD dose was calculated

[0302] to be 128 mg . Based on its pharmacological action, FP2 is expected to function like endogenous GDF15 to reduce food intake and cause weight loss. The mechanism of action and non-clinical safety profile of FP2 do not suggest that FP2 meets the criteria for being considered a high-risk pharmaceutical

[0303] . The results of the repeated-dose cynomolgus monkey study showed pharmacological activity (i.e., reduced food intake) at significantly lower doses, so the MRSD needs to be derived using a PAD-based approach . For this purpose, the human PK parameters of FP2 were predicted by allometric scaling of model-derived PK parameters fixed to the body weight after a single SC administration in overweight cynomolgus monkeys .

[0304] On this basis, PK and PD data across the three dose levels tested in cynomolgus monkeys were A PK / PD model for FP2 was developed using data from the 0.05 mg / kg A single SC dose of 0.3 nmol / kg (approximately 0.3 nmol / kg) was determined to be the threshold for significant pharmacological activity. It was estimated that 0.05 mg / kg / day would result in a reduction in maximum food intake of approximately 10%. The predicted level of reduced food intake with kg was not considered significant to the safety of the subjects. Since no other known significant safety PD effects were observed, a safety factor of 5 (default) was used. (instead of a safety factor of 10 for the 0.05 mg / kg dose estimated from the model) This safety factor selection was also performed for the human and cynomolgus monkey recombinant GFRAL fusion proteins. The in vitro binding affinity of FP2 to proteins was within a two-fold range, and the binding affinity of FP2 in monkeys and mice was within a two-fold range. Taking into account the comparable tissue expression pattern of GFRAL receptors in humans, This study uses a flat dosing approach for a body weight of 80 kg. Therefore, the MRSD based on the PAD is 0.8 mg (0.01 mg / kg × 80 kg), which is the MRSD based on the PAD and the MRS based on the NOAEL. This indicates that the concentration is approximately 160 times lower than that of D.

[0305] The MRSD based on a PAD of 0.01 mg / kg corresponds to a maximum serum drug concentration of approximately 0.6 nM. This is predicted to result in normal range levels of endogenous GDF15 (i.e., approximately 0.046nM or 1.15ng / mL) 13-fold higher 2 , and uncomplicated pregnancies. The median endogenous GDF15 level found in women with 5 times lower than ,000pg / mL20 . The value of this concentration, when corrected for the differences in 1) the binding affinity of human GFRAL receptor (11 - fold lower binding affinity of FP2 compared to endogenous GDF15), and 2) the difference in the potency of in vitro functional assays (about 19 - fold lower EC value of FP2 compared to endogenous GDF15 in the pAKT functional assay using rhGFRAL - expressing SK - N - AS cells), gives a prediction that the expected maximum concentration of FP2 in humans after a single SC administration of 0.01 mg / kg is within 1.2 - fold of the normal upper limit of endogenous GDF15 in lean individuals. fold lower EC 50 value) is such that, after a single SC administration of 0.01 mg / kg, the predicted maximum concentration of FP2 in humans is within 1.2 - fold of the normal upper limit of endogenous GDF15 in lean individuals. Moreover, the C

[0306] from an initial dose of 0.01 mg / kg is predicted to be lower than the EC max of the read - out value of the pAKT functional assay using rhGFRAL - expressing cells. of the read - out value of the pAKT functional assay using rhGFRAL - expressing cells. 10 is expected to be lower.

[0307] 3.3.2. Maximum Dose The NOAEL doses in 4 - week GLP rat or cynomolgus monkey toxicity studies were, respectively, , mean C values of 415 μg / mL (days 1 - 4, male and female) and 1117 μg / mL (days 22 - 29, max male and female), and mean AUC values of 883 μg·day / mL (days 1 - 4, male and female) and 6341 μg·day / mL (days 22 - 29, male and female). No significant differences in mean drug exposure (evaluated by C and AUC) and other TK parameters were observed between male max and female cynomolgus monkeys. Drug exposure (evaluated by Cmax and AUC after the first dose) in female rats tended to be slightly higher than that in male rats. The planned dose of 1.08 mg / kg (BW) in Part 1 (evaluated by Cmax and AUC 1~4日目 after the first dose) in female rats tended to be slightly higher than that in male rats. The planned dose of 1.08 mg / kg (BW) in Part 1 in Part 1 The maximum dose is about the average C in the NOAEL dose of cynomolgus monkeys max and about 97 to 21 times lower average C max and AUC exposure are expected to be given. In the first part of this study As it progresses, the PK data from each successive DG is used for simulations to predict the total body exposure of FP2 to further improve these exposure estimates. Subsequent doses can be adjusted based on verification of new safety properties, tolerability, and PK data, but ensure that the planned maximum dose is not exceeded.

[0308] The CV safety study in cynomolgus monkeys equipped with devices showed no significant findings up to the highest SC dose tested at 50 mg / kg, so it was not considered to limit the planned maximum exposures summarized above. exposure.

[0309] 3.4. Dose escalation The planned dose range in the first part is such that it enables characterization of the doses expected to provide a safety margin (≥10-fold) from the anticipated repeated-dose exposures of the therapeutically effective doses that will likely be investigated in future studies in obese subjects and to account for increased potential exposures in special populations (e.g., subjects with renal and hepatic impairment) and situations (e.g., drug-drug interaction studies, thorough QT / QTc studies, etc.). .

[0310] The proposed dose escalation strategy is that if the previous dose level has been shown to be safe and the exposure levels ( Cmax and AUC 0~72時間 ) exhibit generally dose-proportional linear PK (or a non-proportional increase in exposure amount), from the first two dose escalation steps of the study to the third dose level The concept of approximately 3-fold dose increments is followed. Under the same provisions, the first doses to dose levels 4, 5, and 6 are The third, fourth, and fifth dose escalation steps consisted of approximately two-fold dose escalations, whereas all Subsequent dose escalation steps (if any) are planned at approximately 50% dose increments (Table 3).

[0311] Preliminary safety and PK data will be reviewed by sponsor and PI upon completion of each dose level. Each dose escalation decision is made after at least Blinded preliminary safety, tolerability, and P data collected in all subjects in a given dose group at 72 hours Based on K data and preliminary PK data obtained at least 72 hours after dosing. Required evaluable subjects (i.e., those who have completed study procedures for a minimum of 72 hours post-dose) The minimum number of subjects enrolled in the study is N=7 per treatment group. Any further clinically relevant information will be reported by the Investigator at the review meeting. As the study progresses, cumulative safety data from previous dose arms will also be collected as part of each titration meeting. The planned doses will be evaluated periodically based on preliminary PK, safety, and / or tolerability data from previous doses. may be modified, reduced or repeated if supported by validity data. However, significant amendments to the study protocol have been issued and the legally competent health authorities (HHAs) have A) and an Independent Ethics Committee (IEC) will not be able to increase it unless it is submitted to them.

[0312] At least 10 days must elapse between the last subject in the previous treatment group and the first subject in the subsequent DG. Ku.

[0313] Using the NOAEL dose in 4-week GLP rat or cynomolgus monkey toxicity studies, The upper exposure limit targeted in this study is derived (for details, see Example 21, Section 3.3.2).

[0314] 3.5. Dose Selection Part 2 The dose intensity of Part 2 is selected based on the preliminary safety and PK data in Part 1, such that it does not exceed one-third of the dose evaluated as being fully tolerated in Part 1, while providing a safety margin of approximately three-fold lower in the case where the BA by SC of FP2 in overweight human subjects is significantly lower than that determined in lean cynomolgus monkeys (absolute BA at a single IV dose of 1.0 mg / kg is 99% ). The C value observed at IV administration in the 4-week cynomolgus monkey TK study (50 mg / kg (BW)) was only approximately two-fold higher than that observed at SC administration of the same dose , such that a three-fold reduction in the well-tolerated SC dose is expected to provide an appropriate margin with respect to the safe maximum exposure (C max ) predicted by a constant-rate IV infusion of FP2 over 30 minutes. For example, if a SC dose of 30 mg administered to dosing group 5 is considered safe and well-tolerated, the IV dose for Part 2 can be selected based on this D G and would be one-third of 30 mg, i.e., 10 mg max . This is based on the assumption that the absolute BA at SC administration in overweight subjects is as low as approximately 33%, such that at an IV dose intensity of 10 mg, the AUC at IV administration does not reliably exceed the AUC obtained after SC administration of 30 mg. BA is calculated from the AUC (C not) and the assumption of 33% BA is conservative, such that an IV dose intensity of one-third of the SC dose is expected to result in a lower C than the IV dose intensity of 30 mg SC. max not) and the assumption of 33% BA is conservative, such that an IV dose intensity of one-third of the SC dose is expected to result in a lower C than the IV dose intensity of 30 mg SC. max(i.e., at the end of injection) is highly likely to occur.

[0315] 3.6. Termination Criteria 3.6.1. Individual Termination Criteria - Part 1 In the first part of the test, only a single SC administration of FP2 is carried out. Therefore, individual termination criteria (such as interruption of administration) of the test drug cannot be applied. However, for randomized subjects, the test drug will not be administered if the test is terminated (see Example 21, Section 10.2).

[0316] 3.6.2. Individual Termination Criteria - Part 2 IV infusion will be interrupted in the case of a medically important AE, which means potential risk to the subject as judged by the principal investigator of the clinical trial. Such medically important AEs include, but are not limited to, the following findings.

[0317] The subject has an absolute QT corrected according to Fridericia's formula (QTcF) of 500 milliseconds or more, or an increase in QTcF from the baseline in two consecutive measurements (15-minute interval) after the first occurrence is greater than 60 milliseconds.

[0318] Based on continuous heart rate monitoring, a subject has tachycardia defined as a resting supine heart rate of more than 100 bpm for at least 15 minutes after the first occurrence.

[0319] Based on continuous heart rate monitoring, a subject has bradycardia defined as a resting supine heart rate of less than 45 bpm for at least 15 minutes after the first occurrence.

[0320] Defined as a resting systolic blood pressure (SBP) of more than 180 mmHg, and at least Subjects who developed hypertension lasting for at least 15 minutes.

[0321] The subject has a severe or serious adverse event, and the principal investigator of the clinical trial believes that for safety reasons, it is in the best interest of the subject to discontinue the intravenous injection.

[0322] At the time of discontinuation for one of these reasons, the administration of the investigational drug is considered definitively terminated (i.e., not restarted). The reason for discontinuation of the investigational drug is documented.

[0323] 3.6.3. Trial discontinuation criteria The progress of the trial (progress within the yet-to-be-completed DG or to a higher dose level) is put on hold at any time in the following cases. That is, - If the trial is discontinued (regardless of the treatment group or duration) due to any medically important or serious adverse event (SAE) that has been evaluated by the principal investigator of the clinical trial as possibly, probably, or highly likely related to the investigational drug in two subjects, or - If one subject has an SAE that has been evaluated by the principal investigator of the clinical trial as possibly, probably, or highly likely related to the investigational drug.

[0324] An independent internal DRC can be convened, independent of the clinical trial team (see Section 11.8). The purpose of the DRC is to verify all unblinded safety data. At the end of this detailed safety verification, one of the following recommendations is made. That is, Continue the planned study (i.e., there are no major safety concerns). Continue the trial by repeating the current dose in more subjects.

[0325] ​​​​​​​​​

[0326] The dose between the current dose and the next planned dose, or between the current dose and a lower previous dose Continue the test at this dose.

[0327] End the test.

[0328] 4. Subject population Screening for eligible subjects is performed within 28 days prior to the administration of the test drug.

[0329] For both Part 1 and Part 2, admit at least two additional preliminary subjects to the hospital on day - 2 of the inpatient period and conduct all evaluations until dosing to ensure that all dosing groups are properly randomized. If replacement is necessary, additional subjects can be enrolled. Each replacement subject completes all tests for the subject being replaced according to the randomization schedule (see Section 5 of the protocol, Assignment and Blinding of the Test Drug). No subject participates in more than one dosing group or only a part of this test. Re - screening can be performed for preliminary subjects to participate in another dosing group of the test.

[0330]

[0331] In Part 1, register 4 males and 4 females (randomize 3 in each gender group to the active substance and 1 to the placebo) to the first dosing group for which administration of the undiluted test drug (i.e., the undiluted formulation of 50 mg / mL) is planned so that each subject can be matched to the corresponding IV dosing group in Part 2 of the test.

[0332] The inclusion and exclusion criteria for enrolling subjects in this test are the following two sub - sets ​​​​​​​​​It is described in the protocol. If there are any questions regarding the following inclusion or exclusion criteria, the clinical trial The responsible physician shall consult with the appropriate sponsor representative and resolve all questions before registering the subject in the trial. Deviation cases are not permitted.

[0333] For considerations on statistical considerations in subject selection, refer to "Section 11.2 Sample Size Determination" in Example 21.

[0334] 4.1 Inclusion Criteria Each potential subject must meet all of the following criteria to be registered in this trial. That is , Male or female 18 to 45 years old (including the boundary values).

[0335] The body mass index (BMI) is 25.0 to 29.9 kg / m 2 (including the boundary values), and the weight is 80 kg or more.

[0336] Healthy based on physical examination, medical history, vital signs, clinical tests, and 12-lead ECG performed at screening and baseline (-2 days and / or -1 day). If any of the results are abnormal, the subject can be included only if the principal investigator of the clinical trial determines that the deviation from normal or abnormal is not clinically significant. This decision is recorded in the subject's original sample and signed by the principal investigator of the clinical trial.

[0337] The subject understands the purpose of the trial and the procedures required therefor and signs an informed consent form (ICF) indicating the intention to participate in the trial.

[0338] Women are women with no possibility of pregnancy, defined as any of the following. That is ,​​​ After menopause The postmenopausal state is defined as having no menstruation for at least 12 months without other medical causes and with follicle-stimulating hormone (FSH) levels at screening being in the postmenopausal range (> 40 IU / L or mIU / mL). However, if the subject's amenorrhea period is less than 12 months, two FSH measurements (one of which may be obtained from the subject's medical record) are required to confirm the postmenopausal state. All women must have a negative serum β-human chorionic gonadotropin (hCG) pregnancy test at screening and a negative urine pregnancy test at admission on day - 2 . Permanent infertility Permanent infertility methods include hysterectomy, bilateral salpingectomy, bilateral tubal occlusion / ligation, and bilateral oophorectomy, as documented in the medical record, or other forms of pregnancy incapability. All women must have a negative serum hCG pregnancy test at screening and a negative urine pregnancy test at admission on day - 2

[0339] The resting heart rate (after the subject has been in the supine position for 5 minutes) is 50 - 90 beats per minute (bpm ). If the heart rate is outside the range, a maximum of two repeated evaluations are permitted. The blood pressure (after the subject has been in the supine position for 5 minutes) is 90 - 140 mmHg in systolic (including the boundary value) and 90 mmHg or less in diastolic. If the blood pressure is outside the range, a maximum of two repeated evaluations are permitted.

[0340] Men (including men who have had vasectomies) are included if their partner is pregnant

[0341]

[0342]

[0342] ​​​​Also, consent to the use of a condom (to prevent the fetus from being exposed to the test drug by vaginal absorption ), and consent not to provide sperm during the test period and for three months after the end of the test. Male subjects should encourage their female partners to use an effective method of contraception (e.g., prescription oral contraceptives , contraceptive injections, intrauterine devices, double-barrier methods, and contraceptive patches) in addition to the condom used by the male subject.

[0343] Willingness to comply with the prohibitions and restrictions specified in the test protocol.

[0344] Subjects should like the food items provided for the 24-hour food intake assessment (at least one of the main dish and one of the side dishes from the lunch and dinner menus), typically consume them, and have a habitual meal pattern of three meals a day (breakfast, lunch, and dinner).

[0345] If consent is given to provide any DNA samples for research, sign a separate informed consent form. Refusal to consent to the provision of any DNA research samples does not exclude the subject from participating in this test.

[0346] 4.2. Exclusion Criteria Potential subjects who meet any of the following criteria will be excluded from participating in the test. That is, specifically, CV diseases (including arrhythmia, myocardial infarction, stroke, peripheral vascular disease), endocrine or metabolic diseases (e.g., diabetes, hyperthyroidism / hypothyroidism, severe hypertriglyceridemia [>400 mg / dL]), blood diseases (e.g., von Willebrand disease or other hemorrhagic diseases), respiratory organ diseases, liver or digestive diseases, nerve or mental diseases, eye diseases (retinal disorders or white (including cataracts), neoplastic diseases, skin diseases, kidney diseases, or other diseases that the investigator-in-charge should exclude the subject or may interfere with the interpretation of the test results (however, limited to these determined), history of serious diseases or medical disorders, or current activity.

[0347] Previous bariatric surgery, or dietary therapy including a commercial weight loss program, or Recent weight change (≧5%) due to drug treatment within 6 months of screening.

[0348] A lifetime history of any eating disorder, or a high risk of eating disorder (using the Questionnaire 5 [QEWP-5] related to eating and weight patterns) 25 Please refer to Addendum 1.

[0349] A lifetime history of malignant tumors, or a family history of susceptibility to malignant tumors defined as the same type of cancer in at least 2 close relatives on the same side of the family (defined as parents, siblings, children, grandparents, aunts, uncles, nephews, nieces), or multiple types of cancer in one close relative who is a close relative, or Close relatives who developed cancer at a young age (under 50 years old), or cancer that occurred in both organs of a pair (e.g., both kidneys), or multiple childhood cancers in siblings, or breast cancer in male close relatives, or close relatives who have cancers occurring in multiple generations (e.g., grandfather, father, son) are also excluded.

[0350] History of abnormal or positive results in routine cancer screening tests (e.g., prostate-specific antigen [PSA] in men, Papanicolaou [PAP] smear, or mammogram in women).

[0351] Genetic syndromes predisposing to cancer (e.g., BRCA1 and BRCA2, Lynch syndrome, familial adenomatous polyposis syndrome, Li-Fraumeni syndrome, and multiple endocrine neoplasia syndrome).

[0352] Aspartate aminotransferase (AST) and alanine aminotransferase (ALT) above the upper limit of normal (ULN ) of the clinical laboratory reference range at screening or on day - 2 of hospitalization.

[0353] Total bilirubin above 1.5 times the ULN (i.e., 1.5 × ULN), suggesting Gilbert syndrome (benign congenital non - hemolytic hypobilirubinemia due to UGT1A1 polymorphism).

[0354] Hemoglobin, hematocrit, or red blood cell count below the lower limit of normal of the clinical laboratory reference range at screening or on day - 2 of hospitalization.

[0355] Abnormal fasting blood glucose at screening or on day 2 (i.e., >125 mg / dL or > 6.9 mmol / L; matrix plasma from venous blood sample) and / or hemoglobin A1 c (HbA 1c )(i.e., >6.4% [high - performance liquid chromatography] or >42 mmol / mol (Hb). If non - compliance with the required overnight fasting period is suspected, blood glucose measurements can be repeated during the screening period.

[0356] Serum creatinine above the ULN of the clinical laboratory reference range at screening or on day - 2 of hospitalization.

[0357] Thyroid - stimulating hormone ( TSH) levels outside the reference limits of the clinical laboratory reference range at screening.​

[0358] Any unauthorized treatment, preliminary test, and concomitant treatment was conducted up to 30 days before the maximum of the first dose of the planned investigational drug.

[0359] History of drug or alcohol abuse according to the criteria of the "Diagnostic and Statistical Manual of Mental Disorders" (5th Edition) (D SM-V) within 2 years before screening, or drug or alcohol abuse at the time of screening or - on Day - 2 (barbiturates, opioids, cocaine, ca nnabinoids, amphetamines, and benzodiazepines, including but not limited to these) had a positive test result.

[0360] Known allergy, hypersensitivity, or intolerance to any of the excipients of FP2 (see "IB, Section 2.3 Formulation Information").

[0361] Donated blood or blood products (approx. 450 mL) within 2 months before the first administration of the investigational drug, or had a large amount of blood loss.

[0362] Received an investigational drug (including an investigational vaccine) or used an invasive investigational medical device within 1 month before the planned first administration of the investigational drug or within a period less than 10 times the half-life of the drug, whichever is longer.

[0363] Was pregnant, breastfeeding, or planning to become pregnant during the registration of this trial or within 12 weeks after the last administration of the investigational drug.

[0364] Was planning to have a child during the registration of this trial or within 12 weeks after the last administration of the investigational drug.

[0365] History of hepatitis B surface antigen (HBsAg) or hepatitis C antibody (anti-HCV) positive, or other clinically active liver disease, or positive HBsAg or anti-HCV test at the time of screening. Positive history of human immunodeficiency virus (HIV) antibody, or positive HIV test at the time of screening.

[0366] Positive history of human immunodeficiency virus (HIV) antibody, or positive HIV test at the time of screening. Positive.

[0367] Received major surgery (e.g., requiring general anesthesia) within 6 months prior to screening, or not fully recovered from surgery, or surgery is scheduled during the period when the subject is expected to participate in the study or within 12 weeks after the last dose of the study drug. Note: Subjects with scheduled surgical procedures under local anesthesia may participate if approved by the principal investigator of the clinical trial. Or not fully recovered from surgery, or surgery is scheduled during the period when the subject is expected to participate in the study or within 12 weeks after the last dose of the study drug. Note: Subjects with scheduled surgical procedures under local anesthesia may participate if approved by the principal investigator of the clinical trial. Or surgery is scheduled during the period when the subject is expected to participate in the study or within 12 weeks after the last dose of the study drug. Note: Subjects with scheduled surgical procedures under local anesthesia may participate if approved by the principal investigator of the clinical trial. Or surgery is scheduled during the period when the subject is expected to participate in the study or within 12 weeks after the last dose of the study drug. Note: Subjects with scheduled surgical procedures under local anesthesia may participate if approved by the principal investigator of the clinical trial. Or surgery is scheduled during the period when the subject is expected to participate in the study or within 12 weeks after the last dose of the study drug. Note: Subjects with scheduled surgical procedures under local anesthesia may participate if approved by the principal investigator of the clinical trial.

[0368] Subject smoked tobacco (or equivalent) and / or used nicotine-containing products 3 months prior to study drug administration, or positive cotinine test at the time of screening or admission on day - 2. Subject smoked tobacco (or equivalent) and / or used nicotine-containing products 3 months prior to study drug administration, or positive cotinine test at the time of screening or admission on day - 2. Subject smoked tobacco (or equivalent) and / or used nicotine-containing products 3 months prior to study drug administration, or positive cotinine test at the time of screening or admission on day - 2.

[0369] Drinks more than 1200 mL per day (i.e., 5 cups, total combined volume) of tea / coffee / cocoa / coke / caffeinated beverages (e.g., energy drinks) on average. Drinks more than 1200 mL per day (i.e., 5 cups, total combined volume) of tea / coffee / cocoa / coke / caffeinated beverages (e.g., energy drinks) on average. Drinks more than 1200 mL per day (i.e., 5 cups, total combined volume) of tea / coffee / cocoa / coke / caffeinated beverages (e.g., energy drinks) on average.

[0370] Subjects who are total vegetarians or vegetarians have food allergies or food intolerances. Subjects who are total vegetarians or vegetarians have food allergies or food intolerances.

[0371] Psychological and / or emotional problems that invalidate informed consent or limit the ability of the subject to comply with the requirements of the study. Psychological and / or emotional problems that invalidate informed consent or limit the ability of the subject to comply with the requirements of the study.

[0372] if the subject is unable or unwilling to undergo multiple venous punctures due to low tolerance or difficult access to veins or

[0373] According to the opinion of the principal investigator of the clinical trial, participation is not in the best interest of the subject (e.g., may compromise soundness) or there are any conditions that may interfere with, limit, or confound the evaluations specified in the protocol

[0374] a person who is an applicant for the clinical trial, an employee of the principal investigator of the clinical trial or the clinical trial facility, who is directly involved in the proposed test or other tests proposed based on the instructions of the principal investigator of the clinical trial or the clinical trial facility or a family member of such an employee, principal investigator of the clinical trial, or applicant for the clinical trial

[0375] the subject lives in a facility by order of a court or agency

[0376] is randomized in a previous dosing group of this trial

[0377] 4.3. Prohibited and Restricted Matters Potential subjects must be willing and required to comply with the following prohibited and restricted matters in order to be eligible to participate during the trial period

[0378] Agree to comply with all requirements to be met during the trial as described in the inclusion and exclusion criteria (e.g., contraception requirements)

[0379] Strenuous exercise may affect the evaluations and safety test results specified by the trial. Therefore, strenuous exercise (e.g., running long distances of 5 km / day, weightlifting or any physical activity that the subject is not accustomed to) shall be started 3 days before the trial and during the trial ​​​​​Throughout, it is necessary to avoid until the completion of the visit at the end of the trial.

[0380] Subjects are instructed to avoid donating blood for at least 3 months after the end of the trial (i.e., the visit at the end of the trial). They are so instructed.

[0381] Alcohol consumption or alcohol-containing products are not permitted starting at least 24 hours before screening and admission to the CRU on Day - 2 until the end of the stay on Day 5, and also not permitted for at least 24 hours before any other outpatient visit. During the remaining days of the trial, the alcohol consumption amount should be restricted to a maximum of 24 grams per day for men (i.e., 0.5 L of beer per day or 0.25 L of wine per day, or 3 glasses [2 cL per glass] of distilled spirits per day), and 12 grams per day for women (i.e., 0.25 L of beer per day or 0.125 L of wine per day, or 1.5 glasses [2 cL per glass] of distilled spirits per day). They are not permitted starting at least 24 hours before screening and admission to the CRU on Day - 2 until the end of the stay on Day 5, and also not permitted for at least 24 hours before any other outpatient visit. During the remaining days of the trial, the alcohol consumption amount should be restricted to a maximum of 24 grams per day for men (i.e., 0.5 L of beer per day or 0.25 L of wine per day, or 3 glasses [2 cL per glass] of distilled spirits per day), and 12 grams per day for women (i.e., 0.25 L of beer per day or 0.125 L of wine per day, or 1.5 glasses [2 cL per glass] of distilled spirits per day). For the remaining days of the trial, the alcohol consumption amount should be restricted to a maximum of 24 grams per day for men (i.e., 0.5 L of beer per day or 0.25 L of wine per day, or 3 glasses [2 cL per glass] of distilled spirits per day), and 12 grams per day for women (i.e., 0.25 L of beer per day or 0.125 L of wine per day, or 1.5 glasses [2 cL per glass] of distilled spirits per day). For the remaining days of the trial, the alcohol consumption amount should be restricted to a maximum of 24 grams per day for men (i.e., 0.5 L of beer per day or 0.25 L of wine per day, or 3 glasses [2 cL per glass] of distilled spirits per day), and 12 grams per day for women (i.e., 0.25 L of beer per day or 0.125 L of wine per day, or 1.5 glasses [2 cL per glass] of distilled spirits per day). For the remaining days of the trial, the alcohol consumption amount should be restricted to a maximum of 24 grams per day for men (i.e., 0.5 L of beer per day or 0.25 L of wine per day, or 3 glasses [2 cL per glass] of distilled spirits per day), and 12 grams per day for women (i.e., 0.25 L of beer per day or 0.125 L of wine per day, or 1.5 glasses [2 cL per glass] of distilled spirits per day). For the remaining days of the trial, the alcohol consumption amount should be restricted to a maximum of 24 grams per day for men (i.e., 0.5 L of beer per day or 0.25 L of wine per day, or 3 glasses [2 cL per glass] of distilled spirits per day), and 12 grams per day for women (i.e., 0.25 L of beer per day or 0.125 L of wine per day, or 1.5 glasses [2 cL per glass] of distilled spirits per day). For the remaining days of the trial, the alcohol consumption amount should be restricted to a maximum of 24 grams per day for men (i.e., 0.5 L of beer per day or 0.25 L of wine per day, or 3 glasses [2 cL per glass] of distilled spirits per day), and 12 grams per day for women (i.e., 0.25 L of beer per day or 0.125 L of wine per day, or 1.5 glasses [2 cL per glass] of distilled spirits per day). For the remaining days of the trial, the alcohol consumption amount should be restricted to a maximum of 24 grams per day for men (i.e., 0.5 L of beer per day or 0.25 L of wine per day, or 3 glasses [2 cL per glass] of distilled spirits per day), and 12 grams per day for women (i.e., 0.25 L of beer per day or 0.125 L of wine per day, or 1.5 glasses [2 cL per glass] of distilled spirits per day).

[0382] Subjects should avoid consuming any food or beverage containing grapefruit juice, Seville orange (including orange marmalade), or quinine (e.g., tonic water) from 48 hours before Day 1 until the end of the stay on Day 5. Subjects should avoid consuming any food or beverage containing grapefruit juice, Seville orange (including orange marmalade), or quinine (e.g., tonic water) from 48 hours before Day 1 until the end of the stay on Day 5. Subjects should avoid consuming any food or beverage containing grapefruit juice, Seville orange (including orange marmalade), or quinine (e.g., tonic water) from 48 hours before Day 1 until the end of the stay on Day 5.

[0383] Subjects should refrain from using any methylxanthine-containing products (e.g., chocolate bars or beverages, coffee, tea, cola, or energy drinks) from 48 hours before the administration of the study drug on Day 1 until Day 5. On the other days between screening and follow - up visits, subjects should, on average, consume no more than 1200 mL of tea / coffee / cocoa / coke per day. Subjects should refrain from using any methylxanthine-containing products (e.g., chocolate bars or beverages, coffee, tea, cola, or energy drinks) from 48 hours before the administration of the study drug on Day 1 until Day 5. On the other days between screening and follow - up visits, subjects should, on average, consume no more than 1200 mL of tea / coffee / cocoa / coke per day. Subjects should refrain from using any methylxanthine-containing products (e.g., chocolate bars or beverages, coffee, tea, cola, or energy drinks) from 48 hours before the administration of the study drug on Day 1 until Day 5. On the other days between screening and follow - up visits, subjects should, on average, consume no more than 1200 mL of tea / coffee / cocoa / coke per day. Subjects should refrain from using any methylxanthine-containing products (e.g., chocolate bars or beverages, coffee, tea, cola, or energy drinks) from 48 hours before the administration of the study drug on Day 1 until Day 5. On the other days between screening and follow - up visits, subjects should, on average, consume no more than 1200 mL of tea / coffee / cocoa / coke per day. Subjects are instructed not to drink the ーラ (5 cups, total combined volume).

[0384] Since subjects may interfere with drug screening, they are instructed to refrain from taking poppy seeds at least 72 hours before admission to the CRU.

[0385] Smoking of tobacco (or equivalents) and / or use of nicotine-containing products are not permitted from 3 months before administration of the study drug until the completion of the last visit at the end of the study.

[0386] Subjects do not consume any food or beverages other than those provided by the study site staff during the hospitalization phase.

[0387] Agree to comply with the contraception requirements described in the inclusion criteria. There is no information regarding the effect of FP2 on sperm or sperm production in the body, nor is there any information regarding its effect on fetal development. It is important that the subject and the subject's partner do not become pregnant during the study period or for up to 3 months after the study. If the subject's partner becomes pregnant during the study period or within 3 months after the end of the study (i.e., the last visit at the end of the study), the subject must report this to the principal investigator.

[0388] Section 1 All subjects eligible to participate in the study are randomized prior to administration of the study drug on Day 1. A computer-generated randomization schedule is provided by the sponsor and maintained at the pharmacy at the CRU.

[0389] Within each DG, subjects are assigned to active drug treatment (FP2) or placebo based on a computer-generated randomization schedule created prior to the study by the sponsor or under its supervision. is randomly assigned. Randomization is balanced by using randomly shuffled blocks. Six subjects in each DG are administered FP2, and two subjects are administered placebo. For the DG in Part 1 that is matched with Part 2, four females and four males are randomized (3 FP2 and 1 placebo) in a ratio of 3:1 for each sex group and enrolled. Four females and four males are randomized (3 FP2 and 1 placebo) in a ratio of 3:1 for each sex group and enrolled. Four females and four males are randomized (3 FP2 and 1 placebo) in a ratio of 3:1 for each sex group and enrolled.

[0390] Subjects in each DG are divided into four subgroups (n = up to 2 / subgroup) and administered on different days. On the first day of each DG, the first subgroup (sentinel group) consisting of two subjects is randomly assigned to either FP2 or placebo in a 1:1 ratio, and administered at approximately the same time on the same day to evaluate safety and tolerability up to 72 hours. Any AEs reported / observed in the subjects administered within the first subgroup that could affect the administration of the remaining subjects in the DG are reported to the sponsor before further randomization and administration of subjects. The remaining six subjects (1 placebo, 5 FP2) are randomly assigned to either FP2 or placebo in a ratio of 5:1 (5 FP2 and 1 placebo). After the sentinel subjects are administered, in each DG, the remaining six subjects are administered in groups of two (administration interval of approximately 2 hours) over approximately 3 days (at least 24 - hour intervals). are randomly assigned to either FP2 or placebo in a ratio of 5:1 (5 FP2 and 1 placebo). After the sentinel subjects are administered, in each DG, the remaining six subjects are randomly assigned to either FP2 or placebo in a ratio of 5:1 (5 FP2 and 1 placebo). After the sentinel subjects are administered, in each DG, the remaining six subjects are randomly assigned to either FP2 or placebo in a ratio of 5:1 (5 FP2 and 1 placebo). After the sentinel subjects are administered, in each DG, the remaining six subjects are administered in groups of two (administration interval of approximately 2 hours) over approximately 3 days (at least 24 - hour intervals). ) are administered.

[0391] The unblinded pharmacist at the CRU prepares the test drug doses for individual subjects according to the randomization schedule, masks the syringes by attaching blinded labels before dispensing to prevent accidental unblinding by the color of the solution. Administration of the test drug is at any stage of the trial to prevent accidental unblinding by the color of the solution. Administration of the test drug is at any stage of the trial It is conducted by clinical trial personnel who are not involved in the overall assessment. The principal investigator of the clinical trial is provided with a sealed randomization code for each subject, which includes the coded details of the investigational drug. These sealed codes are stored together in a restricted access area that is accessible 24 hours a day. All randomization codes are retrieved after the subject has completed participation in the trial, whether they have been opened or remain sealed. Data that could potentially unblind the assignment of the investigational drug (i.e., serum concentration of the investigational drug, data on anti-drug antibodies against FP2, data on investigational drug preparation / accountability, treatment assignment) is handled with special care to maintain the integrity of the blindness and minimize the potential for bias. This may include special provisions such as ensuring that the principal investigator of the clinical trial, the clinical team, or others cannot view the data until the database is locked and unblinding occurs, as necessary. Any access to PK data by a participating site or the trial team is anonymized (i.e., only group-level data and / or dummy subject numbers to which individual subject data is assigned). Under normal circumstances, the blindness should not be broken until all subjects have completed the trial and the database is finalized. Otherwise, the blindness of individual subjects may need to be broken only if specific emergency measures / sequences of actions are designated based on knowing the subject's treatment status. In such cases, the principal investigator of the clinical trial can determine what the investigational drug is by opening the sealed code as an emergency measure. The principal investigator of the clinical trial shall break the blindness of individual subjects only when specific emergency measures / sequences of actions are designated based on knowing the subject's treatment status. In such cases, the principal investigator of the clinical trial can determine what the investigational drug is by opening the sealed code as an emergency measure.

[0392] Data that could potentially unblind the assignment of the investigational drug (i.e., serum concentration of the investigational drug, data on anti-drug antibodies against FP2, data on investigational drug preparation / accountability, treatment assignment) is handled with special care to maintain the integrity of the blindness and minimize the potential for bias. This may include special provisions such as ensuring that the principal investigator of the clinical trial, the clinical team, or others cannot view the data until the database is locked and unblinding occurs, as necessary. Any access to PK data by a participating site or the trial team is anonymized (i.e., only group-level data and / or dummy subject numbers to which individual subject data is assigned). Under normal circumstances, the blindness should not be broken until all subjects have completed the trial and the database is finalized. Otherwise, the blindness of individual subjects may need to be broken only if specific emergency measures / sequences of actions are designated based on knowing the subject's treatment status. In such cases, the principal investigator of the clinical trial can determine what the investigational drug is by opening the sealed code as an emergency measure. Data that could potentially unblind the assignment of the investigational drug (i.e., serum concentration of the investigational drug, data on anti-drug antibodies against FP2, data on investigational drug preparation / accountability, treatment assignment) is handled with special care to maintain the integrity of the blindness and minimize the potential for bias. This may include special provisions such as ensuring that the principal investigator of the clinical trial, the clinical team, or others cannot view the data until the database is locked and unblinding occurs, as necessary.

[0393] Any access to PK data by a participating site or the trial team is anonymized (i.e., only group-level data and / or dummy subject numbers to which individual subject data is assigned). Under normal circumstances, the blindness should not be broken until all subjects have completed the trial and the database is finalized. Otherwise, the blindness of individual subjects may need to be broken only if specific emergency measures / sequences of actions are designated based on knowing the subject's treatment status. In such cases, the principal investigator of the clinical trial can determine what the investigational drug is by opening the sealed code as an emergency measure. The principal investigator of the clinical trial shall break the blindness of individual subjects only when specific emergency measures / sequences of actions are designated based on knowing the subject's treatment status. Before unblinding, if possible, contact the sponsor of the clinical trial or their designated representative to discuss that specific situation. Telephone contact with the sponsor of the clinical trial or their designated representative can be made 24 hours a day, 7 days a week. If the blind is unblinded, the sponsor of the clinical trial must be notified as soon as possible. The date, time, and reason for unblinding are documented in the source materials.

[0394] Subjects whose allocation of investigational medicinal product has been unblinded must continue to undergo the scheduled evaluations.

[0395] Generally, the randomization code is fully disclosed only when the trial is completed and the clinical database is locked. However, for verification by the unblinded DRC, the randomization code and, if necessary, the treatment and placebo groups to which the randomization codes have been assigned are disclosed to authorized persons.

[0396] Part 2 Part 2 is an unblinded, single-dose treatment in which the same IV dose of FP2 is administered to all subjects, so no special provisions for randomization or treatment allocation are required. .

[0397] Subjects are selected to match the individual subjects in the control SC dosing group in Part 1 of the trial for sex, age (±5 years), and weight (±5 kg).

[0398] Parts 1 and 2 For both Parts 1 and 2, and for each DG, at least two additional prospective subjects are admitted to the hospital on day - 2 and all evaluations up to dosing are performed to ensure that all dosing groups are truly randomized.

[0399] Start assigning randomization numbers from 1001 in the first part and from 3001 in the second part to eligible subjects in sequence. By registering additional subjects as replacement subjects, at least 7 subjects per dosing group complete the test procedure for at least 72 hours after dosing in the first part, and in the second part, 6 subjects complete the test procedure over a period of at least twice the half-life of FP2 (determined based on PK data from the dosing group before the first part). It is also possible to do so. Replacement subjects are given the same treatment as the subjects being replaced, and are assigned a new randomization number that is the same as the randomization number of the subject being replaced, but with the first digit changed to "2" in the first part and "4" in the second part. For example, in the first part, subject 100 4 is replaced by subject 2004, and in the second part, subject 3006 is replaced by subject 4006. In the second part, FP2 is administered to all subjects in an open-label format.

[0400] 6. Dosage and Administration 6.1 Test Drug FP2 is supplied as a sterile solution for injection, stored at -40°C, and protected from light. This solution has a yellowish-brown appearance, a pH of 6.5, and a concentration of 50 mg / mL of FP2 in 10 mM sodium phosphate, 8% sucrose, and 0.04% polysorbate 20. FP2 is provided in a frozen state in an R2 glass vial with a filling volume of 1.2 mL (Table 68).

[0401] The formulation buffer used in the FP2 formulation is also used as a placebo formulation in this test, and is also used as a diluent in the preparation of the first SC dose of FP2 (DG1 - DG4). ​​​​​​​​​It is supplied for use. This formulated buffer solution is a sterile, clear solution consisting of 10 mM sodium phosphate, 8.0% sucrose and 0.04% polysorbate 20. The formulated buffer solution is used to prepare the placebo injection solution. The formulated buffer solution is provided in a frozen state in an R2 glass vial with a filling volume of 1.2 mL.

[0402]

Table 72

[0403] Since the FP2 drug product (tan solution) and the placebo (clear, colorless solution) differ in appearance, unblinded pharmacists maintain the blindness of the test in Part 1 and prevent the test personnel and subjects from visually identifying the product according to the instructions in the "Investigational Medicinal Product (IMP) Handling Manual".

[0404] Detailed instructions regarding dosage preparation, administration procedures, and storage conditions are provided separately to the clinical trial implementation facilities as additional guidance materials.

[0405] Part 1 Administration: The investigational drug is administered by designated, trained, and qualified implementation facility personnel who are independent of the test team and not involved in any other aspects of the conduct of the clinical trial. After at least 10 hours of overnight fasting, the investigational drug (FP2 or placebo) is administered as a single SC dose (maximum volume 2 mL) as 1.0 mL using an insulin syringe (DG1, DG2, and DG5) or 2.0 mL syringe (DG3, DG4, DG6, and DG7) into the right lower quadrant of the abdomen on Day 1. The subjects in each treatment group are divided into four subgroups (n = maximum 2 / subgroup). ​It is divided and administered on different days. First, two sentinel subjects are administered at approximately the same time on the same day (one with placebo and one with FP2). After completing the 72-hour safety monitoring period, it can be administered to the next subject in that DG. After verification of the blinded safety data, up to two additional subjects per day can be administered alternately until all subjects in the dosing group have completed the administration (at approximately 2-hour intervals, one subject is administered around 7 am and one subject is administered around 9 am).

[0406] Time zero (0) is the time of injection of the test drug.

[0407] All SC injections are performed on the anterior abdominal wall, avoiding the area within 2 inches (approx. 5 cm) around the umbilicus. Before injection, the responsible investigator at the implementing facility checks / palpates the planned injection site. Injections should not be performed in areas of the abdominal wall evaluated as abnormal.

[0408] Emergency medical care and emergency equipment (including drugs for immediate use in the treatment of anaphylaxis) are available, and trained physicians are on-site in the dosing room at all times during the administration of the test drug to provide immediate medical care.

[0409] Part 2 Administration: An automatic infusion device (such as Braun Perfusor® Compact S or equivalent) is used, and FP2 is administered as a single dose as a constant-rate infusion over 30 minutes via an indwelling catheter into a suitable forearm vein through a separate line using an administration set equipped with a filter. The administration is performed at approximately the same time each day, but alternately (one subject around 7 am and one subject around 9 am per day). For the two sentinel subjects, Administer initially, and then administer to the next two subjects at least 24 hours after administration to the two sentinel subjects, and treat the last two subjects at least 24 hours after that. Administer to one sentinel subject first, and after completing the 72-hour safety monitoring period, administer to the next subject. The remaining five subjects can be further divided into subgroups (administered at intervals of at least 24 hours), and ensure that no more than two subjects are administered per day (at intervals of approximately 2 hours). Administer to one sentinel subject first, and after completing the 72-hour safety monitoring period, administer to the next subject. The remaining five subjects can be further divided into subgroups (administered at intervals of at least 24 hours), and ensure that no more than two subjects are administered per day (at intervals of approximately 2 hours). The remaining five subjects can be further divided into subgroups (administered at intervals of at least 24 hours), and ensure that no more than two subjects are administered per day (at intervals of approximately 2 hours). Time zero (0) is the start time of the IV infusion of the test drug.

[0410] Since placebo comparison is not planned for the IV administration of FP2, measures to blind the test drug are not necessary.

[0411] Since placebo comparison is not planned for the IV administration of FP2, measures to blind the test drug are not necessary. Since placebo comparison is not planned for the IV administration of FP2, measures to blind the test drug are not necessary.

[0412] Physicians with experience and training in emergency medicine and emergency equipment (including medications for immediate use in the treatment of anaphylaxis) should be immediately available in the dosing room for medical treatment at all times during the administration of the test drug. Physicians with experience and training in emergency medicine and emergency equipment (including medications for immediate use in the treatment of anaphylaxis) should be immediately available in the dosing room for medical treatment at all times during the administration of the test drug. Physicians with experience and training in emergency medicine and emergency equipment (including medications for immediate use in the treatment of anaphylaxis) should be immediately available in the dosing room for medical treatment at all times during the administration of the test drug.

[0413] Technical problems that require temporary interruption of the administration of the test drug (such as catheter failure and the need to place a new catheter) may occur. In this case, the infusion should be resumed as soon as possible, and the interruption time and the reasons for each should be documented. Technical problems that require temporary interruption of the administration of the test drug (such as catheter failure and the need to place a new catheter) may occur. In this case, the infusion should be resumed as soon as possible, and the interruption time and the reasons for each should be documented. Technical problems that require temporary interruption of the administration of the test drug (such as catheter failure and the need to place a new catheter) may occur. In this case, the infusion should be resumed as soon as possible, and the interruption time and the reasons for each should be documented. Technical problems that require temporary interruption of the administration of the test drug (such as catheter failure and the need to place a new catheter) may occur. In this case, the infusion should be resumed as soon as possible, and the interruption time and the reasons for each should be documented.

[0414] 7. Treatment Compliance The test drug is administered by a qualified test facility staff member as an SC injection (Part 1) or an IV infusion (Part 2), and the details of each administration are recorded in the electronic data collection system as applicable [Part 1 SC: injection date, injection time, injection volume, injection site; The test drug is administered by a qualified test facility staff member as an SC injection (Part 1) or an IV infusion (Part 2), and the details of each administration are recorded in the electronic data collection system as applicable [Part 1 SC: injection date, injection time, injection volume, injection site; The test drug is administered by a qualified test facility staff member as an SC injection (Part 1) or an IV infusion (Part 2), and the details of each administration are recorded in the electronic data collection system as applicable [Part 1 SC: injection date, injection time, injection volume, injection site; Part 2 IV: Start and stop times of IV injection, and injection volume

[0415] 8. Pretreatment and concomitant therapy Pretreatment administered up to 30 days before the first dose of the investigational drug is recorded. Throughout the study, no treatment is permitted within 30 days before the planned first dose of the investigational drug and during the study, except for paracetamol. Prescription or over-the-counter drugs (including prescription drugs or commercial drugs containing vaccines, vitamins, mineral supplements, nutritional supplements, herbal supplements [including St. John's wort, garlic extract, and herbal tea]) are not permitted. If a subject requires administration of a prescription or over-the-counter drug during the study, the subject can be registered for or continue in the study after obtaining the consent and approval of the study requester (or designated person) and the responsible investigator.

[0416] If administration of any concomitant therapy is required, it is reported in the appropriate section of the electronic case report form (eCRF). The information recorded includes the drug description, treatment period, dosing regimen, administration route, and its indication.

[0417] The use of paracetamol is permitted up to 3 days before administration of the investigational drug. Throughout the study, a maximum of three 500 mg doses per day and 3 g or less of paracetamol per week are permitted for the treatment of headache or other pain.

[0418] Concomitant therapy is recorded throughout the study from the start of the first dose of the investigational drug until the last visit at the end of the study. Concomitant therapy needs to be recorded only after the last visit at the end of the study if it is associated with new or worsening adverse events and serious adverse events that meet the criteria.

[0419] 9. Evaluation of the study​​​​​​​ 9.1. Test Procedure 9.1.1. General In the "Schedule of Time and Events", the PK frequency and timing applicable to this test , immunogenicity, PD, exploratory biomarkers, pharmacogenomics, and safety measurements are summarized.

[0420] The timing of meals and VAS questionnaires is specified in the "Schedule of Meal and VAS Questionnaire Time and Events".

[0421] If multiple evaluations are scheduled at the same time point and / or one or more evaluations are scheduled simultaneously with a meal, the procedure should be carried out in the order of vital signs, ECG, PK, blood sampling, VAS questionnaire for appetite assessment, meal, and VAS questionnaire for food preference (after the first bite of food). Blood sampling for PK evaluation should be kept as close as possible to the specified time. If the ECG is performed at the same time point as the PK, the PK specimen should be collected immediately after the end of the ECG. If necessary, other measurements can be performed prior to the specified time point. The order of multiple evaluations at the same time point should be the same throughout the test. The actual evaluation date and time should be recorded in the source document and eCRF.

[0422] If possible, vital signs (i.e., blood pressure [BP], heart rate [HR]) should be recorded from the arm opposite to the arm from which the blood sample is taken (except during the IV infusion time).

[0423] All women should undergo pregnancy tests at screening and throughout the test. Serum pregnancy tests should be performed at screening, and urine pregnancy tests should be obtained at all other time points in the "Schedule of Time and Events". ​​​​​​​​​​​

[0424]

Table 73

[0425] These volumes can be adjusted in the final test manual (allowing for changes in the diameter or availability of blood collection tubes) as long as the maximum amount of blood collected from each subject in this study does not exceed 500 mL. Additional blood samples can be taken as needed for further safety, immunogenicity, or PK evaluation based on the data obtained, but the total amount of blood collected from an individual subject in this study shall not exceed the amount described in the protocol of this study without prior approval from an independent ethics committee (IEC) and the health authorities. Repeat samples or samples outside the schedule can be taken for safety reasons or due to technical problems with the sample, and prior approval from an independent ethics committee (IEC) and the health authorities is not required. exceeding. changing but in this study, the total amount of blood collected from an individual subject shall not exceed the amount described in the protocol of this study without prior approval from an independent ethics committee (IEC) and the health authorities. . Repeat samples or samples outside the schedule can be taken for safety reasons or due to technical problems with the sample and prior approval from an independent ethics committee (IEC) and the health authorities is not required.​​

[0426] For each subject, the maximum amount of blood collected in this trial shall not exceed 500 mL. The total blood volume collected from each subject is approximately 315 mL.

[0427] 9.1.2. Screening Period (Parts 1 and 2) Potential subjects shall visit the screening clinic within 28 days before the administration of the investigational drug on Day 1 for a medical examination to determine their eligibility to participate in the trial. If the subject meets the enrollment criteria, they will be admitted to the CRU on Day - 2.

[0428] Before conducting any of the trial procedures, the PI (or the designated study personnel) shall review and explain the written ICF to each subject. No trial procedures (including fasting for the clinical examinations of the trial) can be carried out until the subject signs the ICF. At the screening clinic visit, all evaluations reported by the subject must be performed prior to any trial, procedure, or consultation to exclude subjects who do not meet any of these inclusion criteria.

[0429] The principal investigator of the clinical trial (or the designated study personnel) shall also review and explain the ICF described for any genetic research samples prior to pharmacogenomic blood sampling.

[0430] All adverse events, whether severe or not, shall be reported from the time the signed and dated informed consent form is obtained until the final trial procedure at the last visit at the end of the trial, and also reported by direct questioning at specific time points. (See "Schedule of Time and Events").

[0431] ​​​​​​​​​​The re-examination of outliers that may lead to exclusions is allowed only once. The re-examination can be conducted during a visit outside of the schedule. If any screening test is repeated, the test results must meet the eligibility requirements before admission to the CRU (e.g., on day -2) and are provided for verification by the principal investigator of the clinical trial.

[0432] 9.1.3. Treatment Period for Inpatients (Part 1 and Part 2) Day -2 and Day -1 (Baseline) Eligible subjects are admitted to the CRU on day -2 as specified in the "Schedule of Time and Events", and undergo baseline safety assessment (on day -2) and baseline ECG collection on day -1 (synchronized with the ECG on day 1). On day -1, the subjects also perform a 24-hour measurement of food intake and fill out a VAS questionnaire for appetite evaluation and food preference assessment.

[0433] Day 1 / Randomization and Administration After confirming that all registration criteria have been met, eligible subjects are randomized prior to the administration of the study drug on day 1.

[0434] The study drug is administered under the supervision of the PI or their designated person. For details and timing of the study procedure, please refer to the "Schedule of Time and Events".

[0435] For each dose level in Part 1 and Part 2, the subjects are divided into subgroups so that no more than 2 subjects per day are administered, and the administrations are carried out on different days. First, 2 sentinel subjects in Part 1 (1 placebo and 1 FP2) and 1 sentinel subject in Part 2 (FP2) are administered. After completing the 72-hour safety monitoring period, the subsequent subjects in the treatment group (FP2) are administered, and after completing the 72-hour safety monitoring period, the subsequent subjects in the treatment group ​​​​​​​​​​After verifying safety data, all subjects will complete the treatment. Up to two additional subjects may be dosed alternately per day (approximately 2 hours apart). at intervals).

[0436] Days 3 to 5 On the third day, measurements of 24-hour food intake and administration of the VAS questionnaire are repeated.

[0437] Subjects were withdrawn to the CRU until the morning of Day 5 for safety, tolerability, PK, and PD evaluations. Patients will remain in the hospital for the duration of the stay and may be discharged upon completion of study evaluations.

[0438] 9.1.4. Outpatient Period (Parts 1 and 2) Subjects were returned to the CRU and placed in a stable condition as detailed in the "Time and Event Schedule". Patients were fasted (for at least 10 h) for safety, tolerability, PK, PD, and immunogenicity assessments. Completion of the End of Study Visit will constitute the termination of the subject's participation in the study. A reasonable range for outpatient visits to occur at scheduled times (i.e., the specific days of each visit). Every effort should be made within the window of ±1 day for the fourth week. (day 28), with a ±3-day window allowed for the remainder of the study until the end-of-study visit. All subsequent visits will be allowed for the previously rescheduled patient visit. Must be scheduled relative to the date of first study drug administration (Day 1), not the date of hospitalization. There is.

[0439] Early Discontinuation If a subject discontinues the study for any reason before the end of the outpatient period, You need to get an evaluation.

[0440] 9.2. Pharmacokinetics and Immunogenicity Collect venous blood samples over time as shown in "Schedule of Time and Events". P The PK sampling time can be adjusted based on the preliminary PK data from the previous dosing group (for example, if the serum concentration of FP2 is below the lower limit of quantification [(LLOQ)], subsequent sampling times can be omitted). The actual dates and times of collection of each PK and immunogenicity blood sample (ADA) are recorded on the eCRF within the electronic data collection system. Subjects who discontinue their participation in the study must collect a final evaluation sample at the end. (for example, if the serum concentration of FP2 is below the lower limit of quantification [(LLOQ)], subsequent sampling times can be omitted). The actual dates and times of collection of each PK and immunogenicity blood sample (ADA) are recorded on the eCRF within the electronic data collection system. Subjects who discontinue their participation in the study must collect a final evaluation sample at the end.

[0441] 9.2.1. Evaluation Using the samples collected for the analysis of the serum concentration of FP2 and antibodies against FP2, further evaluation of the additional characteristics of immunogenicity or evaluation of related biomarkers, or evaluation of aspects of safety or efficacy to address concerns arising during or after the study period can be carried out. The confidentiality of the subjects is maintained. Using the samples collected for the analysis of the serum concentration of FP2 and antibodies against FP2, further evaluation of the additional characteristics of immunogenicity or evaluation of related biomarkers, or evaluation of aspects of safety or efficacy to address concerns arising during or after the study period can be carried out. The confidentiality of the subjects is maintained.

[0442] 9.2.2. Analytical Procedures Pharmacokinetics The sponsor of the clinical trial, or under its supervision, analyzes serum samples and determines the concentration of FP2 using a specific and sensitive immunoassay method that has been validated. The sponsor of the clinical trial, or under its supervision, analyzes serum samples and determines the concentration of FP2 using a specific and sensitive immunoassay method that has been validated.

[0443] Immunogenicity The sponsor of the clinical trial, or under its supervision, detects and characterizes anti-FP2 antibodies and potential antibodies against endogenous GDF15 in serum using an assay method that has been validated. The sponsor of the clinical trial, or under its supervision, detects and characterizes anti-FP2 antibodies and potential antibodies against endogenous GDF15 in serum using an assay method that has been validated. All samples collected for the detection of ADA are also evaluated for the FP2 serum concentration, enabling the interpretation of antibody data. All samples collected for the detection of ADA are also evaluated for the FP2 serum concentration, enabling the interpretation of antibody data.

[0444] 9.2.3. Pharmacokinetic Parameters The pharmacokinetic parameters of FP2 were calculated from the serum concentration-time profiles using non-compartmental analysis. The pharmacokinetic parameters after single administration of FP2 include, but are not limited to, the following. Although not limited thereto, the following can be mentioned. C max : Maximum observed serum concentration

[0445] T max : Time to reach the maximum observed serum concentration.

[0446] AUC inf : Area under the serum concentration-time curve from time 0 to infinity with extrapolation of the terminal elimination phase.

[0447] AUC last : Area under the serum concentration-time curve from time 0 to the time corresponding to the last quantifiable concentration.

[0448] T 1 / 2 = Terminal elimination half-life.

[0449] CL: Total systemic clearance (IV only).

[0450] CL / F: Apparent total systemic clearance after extravascular administration (SC only).

[0451] Vz: Volume of distribution based on the terminal elimination phase (IV only).

[0452] Vz / F: Apparent volume of distribution based on the terminal elimination phase after extravascular administration (SC only).

[0453] F (%): Absolute SC bioavailability calculated as the percentage of the systemically available dose. The absolute bioavailability is calculated using the following formula.

[0454]

Equation

[0455] 9.2.4. Evaluation of Immunogenicity In accordance with the "Schedule of Time and Events", anti-FP2 antibodies are evaluated in the blood collected from all subjects. Furthermore, blood samples must also be collected at the time of the subjects' visit at the end of the trial for those who discontinued the trial. These samples will be tested by the sponsor of the clinical trial or the designated person in charge of the sponsor of the clinical trial.

[0456] Serum samples are screened for antibodies that bind to FP2, and the titers of the confirmed positive samples are reported. Other analyses can be performed to further characterize the immunogenicity of FP2.

[0457] 9.3. Pharmacodynamic Evaluation Body weight is measured twice, before breakfast in the morning and after urination, as detailed in the "Schedule of Time and Events". Subjects are weighed on a calibrated scale while not wearing shoes and wearing a gown or light indoor clothing.

[0458] Twenty-four-hour food intake is evaluated by providing four meals (breakfast, lunch, snack, and dinner) that are served simultaneously on both - Day 1 and Day 3. The meals are standardized between days (the same meals on - Day 1 and Day 3). The meals are also standardized between subjects so that the composition and serving size per person are the same for all subjects, and are based on regional preferences and guidelines evaluated by a study dietitian. Each item of each meal is weighed before and after consumption, and the number of grams consumed of each meal item is calculated and recorded. The calories consumed are estimated based on the number of grams consumed of each meal item and the amount of its nutrients. ​​​​​​Evaluate the change in calorie intake from the first day to the third day. Food intake is not recorded On days (-2, 1, 2, 4, the first and fifth days), all meals (including breakfast and snacks) must be different from the meals provided on days -1 and 3.

[0459] A VAS questionnaire for evaluat...

Claims

1. 1. A method for reducing weight in a subject, comprising administering to a subject a fusion protein having SEQ ID NO:92 and at least one and at least one pharma- ceutically acceptable carrier or diluent. wherein the fusion protein is administered at a dose ranging from 0.8 mg to 90 mg, and is 80 kg or more.

2. The method of claim 1 , wherein the subject is overweight.

3. 3. The method of claim 2, wherein the subject has a BMI of 25 kg / m2 or greater.

4. 3. The subject having a BMI in the range of 25 kg / m2 to 29.9 kg / m2. The method according to

5. The fusion protein is present in an amount of 0.8 mg, 2.5 mg, 7.5 mg, 15 mg, 30 mg, 10. The method of claim 1, wherein the compound is administered at a dose selected from the group consisting of 60 mg, 60 mg, and 90 mg. method.

6. 6. The method of claim 5, wherein the fusion protein is administered at a dose of 0.8 mg.

7. 6. The method of claim 5, wherein the fusion protein is administered in a dose of 2.5 mg.

8. 6. The method of claim 5, wherein the fusion protein is administered at a dose of 7.5 mg.

9. 6. The method of claim 5, wherein the fusion protein is administered in a dose of 15 mg.

10. 6. The method of claim 5, wherein the fusion protein is administered in a dose of 30 mg.

11. 6. The method of claim 5, wherein the fusion protein is administered in a dose of 60 mg.

12. 6. The method of claim 5, wherein the fusion protein is administered in a dose of 90 mg.

13. The method of claim 1 , wherein the fusion protein is administered by subcutaneous injection.

14. 10. The method of claim 1, wherein the composition is administered to the subject once a week.

15. 1. A method for reducing food intake in a subject, comprising administering to a subject a fusion protein having SEQ ID NO:92 and and at least one pharma- ceutically acceptable carrier or diluent. wherein the fusion protein is administered at a dose ranging from 0.8 mg to 90 mg, and the subject The body weight is 80 kg or more.

16. 16. The method of claim 15, wherein the subject is overweight.

17. 17. The method of claim 16, wherein the subject has a BMI of 25 kg / m2 or greater.

18. 10. The method of claim 1, wherein the subject has a BMI in the range of 25 kg / m2 to 29.9 kg / m2.

7. The method according to claim 7.

19. The fusion protein is administered at a dose selected from the group consisting of 0.8 mg, 2.5 mg, 7.5 mg, 15 mg, 30 mg, 60 mg, and 90 mg, the method according to claim 15, wherein.

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

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

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

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

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

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

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

27. The method according to claim 15, wherein the fusion protein is administered by subcutaneous injection.

28. The method according to claim 15, wherein the composition is administered to the subject once a week.

29. A method of reducing the body weight of a subject, comprising administering a composition comprising a fusion protein having 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. Method.

30. The method according to claim 29, wherein the composition is administered at a dose selected from the group consisting of 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. Method according to claim 29.

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

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

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

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

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

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

37. 30. The method of claim 29, wherein the composition is administered at a dose of 1.08 mg / kg.

38. 30. The method of claim 29, wherein the fusion protein is administered by subcutaneous injection.

39. 30. The method of claim 29, wherein the composition is administered to the subject once a week.

40. 1. A method for reducing food intake in a subject, comprising administering to a subject a fusion protein having SEQ ID NO:92 and and at least one pharma- ceutically acceptable carrier or diluent. wherein the composition is administered at a dose ranging from 0.01 mg / kg to 1.08 mg / kg. How to do it.

41. The composition is administered at 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 41. The method of claim 40, wherein the dose is selected from the group consisting of:

42. 42. The method of claim 41, wherein the composition is administered at a dose of 0.01 mg / kg.

43. 42. The method of claim 41, wherein the composition is administered at a dose of 0.03 mg / kg.

44. 42. The method of claim 41, wherein the composition is administered at a dose of 0.09 mg / kg.

45. 42. The method of claim 41, wherein the composition is administered at a dose of 0.18 mg / kg.

46. 42. The method of claim 41, wherein the composition is administered at a dose of 0.36 mg / kg.

47. 42. The method of claim 41, wherein the composition is administered at a dose of 0.72 mg / kg.

48. 42. The method of claim 41, wherein the composition is administered at a dose of 1.08 mg / kg.

49. 42. The method of claim 41, wherein the fusion protein is administered by subcutaneous injection.

50. 42. The method of claim 41, wherein the composition is administered to the subject once a week.

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