treatment

Abstract

This invention relates to a complex comprising a polypeptide having a naturally occurring α-lactalbumin sequence or a functional variant thereof; or a peptide comprising up to 50 amino acids of the α-helical domain of said polypeptide; and a fatty acid or lipid or a salt thereof, for the treatment of metabolic-related conditions, particularly those regulated by or otherwise affected by the leptin and / or adiponectin pathway.

Description

Technical Field

[0001] This invention relates to a complex comprising a polypeptide having a naturally occurring α-lactalbumin sequence or a functional variant thereof; or a peptide comprising up to 50 amino acids of the α-helical domain of said polypeptide; and a fatty acid or lipid or a salt thereof, for the treatment of metabolic-related conditions, particularly those regulated by or otherwise affected by the leptin and / or adiponectin pathway. Background Technology

[0002] The primary function of adipose tissue is to store energy in the form of fat, accounting for 20-25% of the total body weight of a healthy individual. Adipose tissue cells (adipocytes) secrete bioactive molecules called adipokines (also known as adipokines). Adiponectin, the most abundant secreted adipokine, is a well-known homeostatic factor that regulates glucose levels, lipid metabolism, and insulin sensitivity through its anti-inflammatory, anti-fibrotic, and antioxidant effects. Leptin, a peptide hormone primarily released by adipose tissue, plays a crucial role in regulating metabolic functions such as appetite and energy expenditure. It induces satiety, reduces food intake, and inhibits glucose uptake. Leptin is also an upstream regulator of adiponectin; serum levels of these two proteins are inversely related. Adiponectin and leptin are associated with risk factors for metabolic disorders, including obesity, diabetes, and cardiometabolic diseases. The leptin / adiponectin ratio has been proposed as a promising biomarker for obesity-related functional impairments.

[0003] Leptin participates in the regulation of energy homeostasis and hunger by providing a feeling of satiety. Leptin achieves this by binding to the long subtype of leptin receptor-B (LEPR-B) encoded by the LEPR gene, which activates the Janus kinase 2 (JAK2) signal transducer and activator of transcription 3 (STAT3) signaling pathway in a downstream subset of hypothalamic neurons (Park HK, Ahima RS. Leptin signaling. F1000Prime Rep. 2014;6:73.). This promotes the production of anorexia peptides, thereby suppressing appetite and increasing energy expenditure. In obesity, although circulating leptin levels are elevated due to increased fat mass, satiety is insufficient due to dysregulation of leptin signaling. As a cytokine, leptin promotes inflammatory responses and upregulates pro-inflammatory cytokines such as interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α). Obesity predisposes to elevated levels of IL-6 and TNF-α, thereby increasing susceptibility to type 2 diabetes and cardiovascular disease.

[0004] HAMLET (human α-lactalbumin, a tumor cell lethal protein) is the first member of a family of unfolded protein-lipid complexes that kill tumor cells. It consists of partially unfolded α-lactalbumin and oleic acid. Initially isolated as a fraction obtained by passing casein-containing breast milk fractions through an ion-exchange column under high-salt conditions (WO96 / 004929), it was found to possess biological activity, particularly antibacterial activity. Subsequently, other methods for preparing the active complex have been developed, including heating α-lactalbumin and oleic acid together in solution. Furthermore, HAMLET and related complexes, such as BAMLET, derived from bovine α-lactalbumin, have been found to kill transformed cells, such as tumor cells or papilloma cells, and possess antiviral activity.

[0005] The inventors further identified additional effects of the complexes and peptides associated with HAMLET and BAMLET, particularly metabolic effects, especially those controlled or otherwise influenced by the leptin and / or adiponectin pathways. The inventors have determined that the complexes bind to leptin and adiponectin receptors. Therefore, these complexes can be used to modulate the activity of these receptors and to treat conditions affected or controlled by pathways associated with these receptors. Summary of the Invention

[0006] This invention provides a polypeptide having a naturally occurring α-lactalbumin sequence, or a functional variant thereof; or a peptide comprising up to 50 amino acids of the α-helical domain of said polypeptide, for the treatment or prevention of metabolic-related conditions, particularly those regulated or controlled by the leptin pathway.

[0007] The present invention also provides a complex comprising a polypeptide having a naturally occurring α-lactalbumin sequence or a functional variant thereof; or a peptide comprising up to 50 amino acids containing an α-helical domain of said polypeptide; and a fatty acid or lipid or a salt thereof, for the treatment or prevention of metabolic-related conditions, particularly those regulated or controlled by the leptin pathway.

[0008] Optionally, the complex or peptide is used to modulate the leptin receptor. For example, the complex or peptide can be used to inhibit the leptin receptor. Alternatively, it can be used to activate the leptin receptor.

[0009] Leptin receptors are well known in the art. Activation of the leptin receptor depends on dimerization. Leptin binds to the Ig-like domain on the first leptin receptor and to the CHR2 domain on the second leptin receptor to induce dimerization. Without being bound by theory, the inventors understand that the complexes or peptides of the present invention can bind to the Ig-like domain and / or the CHR2 domain, thereby preventing or reducing dimerization and activation, or preventing activation of the second leptin receptor.

[0010] Alternatively, the complexes or peptides of the present invention can be used to dimerize or activate receptors.

[0011] The present invention also provides a method for modulating leptin receptors, comprising administering to a subject in need a polypeptide having a naturally occurring α-lactalbumin sequence or a functional variant thereof; or a peptide comprising up to 50 amino acids of the α-helical domain of said polypeptide.

[0012] The present invention also provides a method for treating metabolic-related conditions, comprising administering to a subject in need a polypeptide having a naturally occurring α-lactalbumin sequence or a functional variant thereof; or a peptide comprising up to 50 amino acids of the α-helical domain of said polypeptide.

[0013] The present invention also provides a method for modulating leptin receptors, comprising administering a complex to a subject in need, the complex comprising a polypeptide having a naturally occurring α-lactalbumin sequence or a functional variant thereof; or a peptide comprising up to 50 amino acids containing an α-helical domain of said polypeptide; and a fatty acid or a lipid or salt thereof.

[0014] The present invention also provides a method for treating metabolic-related conditions, particularly those regulated by leptin receptors or the leptin pathway, comprising administering a complex to a subject in need, the complex comprising a polypeptide having a naturally occurring α-lactalbumin sequence or a functional variant thereof; or a peptide comprising up to 50 amino acids containing an α-helical domain of said polypeptide; and a fatty acid or a lipid or salt thereof.

[0015] Metabolic disorders include those affecting metabolism, such as insulin resistance, type 2 diabetes, metabolic syndrome, non-alcoholic fatty liver disease, cirrhosis, and hypertension. Peptides or complexes can be used to regulate insulin resistance or sensitivity, lipid metabolism, and / or glucose metabolism, and therefore can be used to treat conditions arising from these processes.

[0016] Leptin receptor-regulated disorders include, but are not limited to, hyperleptinemia, leptin resistance, leptin deficiency or decreased sensitivity, leptin-related weight gain and / or obesity, increased or decreased hunger stimulation, increased or decreased leptin-related metabolism, leptin-related metabolic syndrome, leptin-related non-alcoholic fatty liver disease, Rabson-Mendenhall syndrome, depression and food addiction, hypoleptinemia, hyperinsulinemia, fatty liver, dyslipidemia, hypogonadotropic hypogonadism, congenital or acquired systemic lipodystrophy (GL), type 1 diabetes mellitus, and hypothalamic amenorrhea.

[0017] In one implementation, metabolic disorders are one or more of obesity, hyperphagia, transient hyperglycemia, glucose intolerance, and elevated plasma insulin.

[0018] The peptide or complex can be used in subjects with hyperleptinemia. For example, the composition can be used in female subjects with leptin levels above about 14, 14.5, 15, or 15.2 ng / mL, or in male subjects with leptin levels above about 11, 11.5, 12, or 12.5 ng / mL.

[0019] The peptide or complex can be used in subjects with hypoleptinemia. For example, this composition can be used in subjects with leptin levels below about 0.5 nanograms per milliliter (ng / mL).

[0020] The peptide or complex can be used to increase leptin expression or leptin receptor expression in subjects compared to the expected expression level, for example, by about 1%, 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60% or more.

[0021] The peptide or complex can be used in subjects with reduced leptin expression or reduced leptin receptor expression compared to the expected expression level, for example, a reduction of about 1%, 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60% or more.

[0022] The present invention also provides a complex comprising a polypeptide having a naturally occurring α-lactalbumin sequence or a functional variant thereof; or a peptide comprising up to 50 amino acids of the α-helical domain of said polypeptide; and a fatty acid or lipid or a salt thereof, for the treatment or prevention of metabolic-related conditions, particularly those regulated or controlled by the adiponectin pathway.

[0023] Optionally, the complex is used to directly (e.g., by binding to the receptor) or indirectly regulate the adiponectin receptor. For example, the complex can be used to inhibit or reduce the activity of the adiponectin receptor. Alternatively, it can be used to activate or increase the activity of the adiponectin receptor.

[0024] Adiponectin receptors are well known in the art. Without being bound by theory, the inventors understand that the complexes of the present invention can bind to adiponectin receptors, inhibiting or activating them.

[0025] The present invention provides a polypeptide having a naturally occurring α-lactalbumin sequence or a functional variant thereof; or a peptide comprising up to 50 amino acids of the α-helical domain of said polypeptide, for the treatment or prevention of metabolic-related conditions, particularly those regulated or controlled by the adiponectin pathway.

[0026] The present invention also provides a complex comprising a polypeptide having a naturally occurring α-lactalbumin sequence or a functional variant thereof; or a peptide comprising up to 50 amino acids containing an α-helical domain of said polypeptide; and a fatty acid or lipid or a salt thereof, for the treatment or prevention of metabolic-related conditions, particularly those regulated or controlled by the adiponectin pathway.

[0027] The peptide or complex can be used in subjects with elevated adiponectin levels. For example, the peptide or complex can be used in female subjects with a BMI below 25 and adiponectin levels above approximately 35, 36, or 37 μg / mL; subjects with a BMI between 25 and 30 and adiponectin levels above approximately 28, 29, or 30 μg / mL; and subjects with a BMI above 30 and adiponectin levels above approximately 18, 19, or 20.

[0028] The peptide or complex can be used in subjects with reduced adiponectin levels. For example, the peptide or complex can be used in male subjects with a BMI below 25 and adiponectin levels above approximately 35, 36, or 37 μg / mL; in subjects with a BMI between 25 and 30 and adiponectin levels above approximately 28, 29, or 30 μg / mL; and in subjects with a BMI above 30 and adiponectin levels above approximately 20, 21, or 22 μg / mL.

[0029] This peptide or complex can be used in female subjects with a BMI below 25 and adiponectin levels below approximately 7, 6, or 5 μg / mL; subjects with a BMI between 25 and 30 and adiponectin levels below approximately 7, 6, or 5 μg / mL; and subjects with a BMI above 30 and adiponectin levels below approximately 4, 3, or 2 μg / mL.

[0030] The peptide or complex may be used in male subjects with a BMI below 25 and adiponectin levels below approximately 7, 6, or 5 μg / mL; in subjects with a BMI between 25 and 30 and adiponectin levels below approximately 6, 5, or 4 μg / mL; and in subjects with a BMI above 30 and adiponectin levels below approximately 6, 5, or 4 μg / mL.

[0031] This peptide or complex can be used in subjects with increased adiponectin expression or adiponectin receptor expression compared to the expected expression level, for example, an increase of approximately 1%, 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, or more.

[0032] This peptide or complex can be used in subjects whose adiponectin expression or adiponectin receptor expression is reduced compared to the expected expression level, for example, by approximately 1%, 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, or more.

[0033] Adiponectin receptor-mediated disorders include obesity, type 2 diabetes, metabolic syndrome, atherosclerosis, and cardiovascular diseases, including myocardial infarction.

[0034] The present invention also provides a method for simultaneously or sequentially modulating leptin receptors and adiponectin receptors, comprising administering the complex as described above.

[0035] Unbound by theory, the inventors understood that the fatty acids or salts of the complex could bind to adiponectin receptors. This could increase the fluidity of the receptor's membrane. The complex could then recruit and bind to leptin receptors via peptides.

[0036] peptides and complexes

[0037] In one embodiment, the polypeptide has the sequence of a naturally occurring α-lactalbumin, preferably human or bovine α-lactalbumin, more preferably bovine α-lactalbumin.

[0038] In one embodiment, the α-helical domain is the α1 (residues 1-39) or α2 (residues 81-123) domain of human α-lactalbumin, as shown in SEQ ID NO:3 or SEQ ID NO:4; KQFTKXELSQLLKDIDGYGGIALPELIXTMFHTSGYDTQ (SEQ ID NO:3)LDDDITDDIMXAKKILDIKGIDYWLAHKALXTEKLEQWLXEKL (SEQ ID NO:4), where X is an amino acid residue other than cysteine.

[0039] In one embodiment, the complex comprises about or less than 45, 42 or 40 amino acids, particularly a peptide of 39 amino acids, preferably corresponding to the α1 domain of human α-lactalbumin.

[0040] In one embodiment, the polypeptide has fewer than 45, 42, or 40 amino acids, particularly 39 amino acids, preferably corresponding to the α1 domain of human α-lactalbumin.

[0041] In one embodiment, the functional variant consists of a sequence lacking disulfide bonds. In another embodiment, the functional variant consists of a sequence in which cysteine ​​residues in native α-lactalbumin are replaced by other amino acid residues, preferably alanine residues.

[0042] In one embodiment, the fatty acid or lipid or its salt is a fatty acid or its salt. In one embodiment, the fatty acid or its salt is oleic acid or oleate.

[0043] In one embodiment, the polypeptide has the sequence of bovine α-lactalbumin, and the fatty acid or its salt is oleic acid or oleate.

[0044] The polypeptides present in the complex may have the sequence of α-lactalbumin or its variants as described above.

[0045] This complex may be referred to as a bioactive complex. As used herein, the term "bioactive" means that the complex possesses biological activity that is different from or stronger than that of the individual components. In particular, the complex is capable of inducing cell death, especially selectively inducing tumor cell death, and / or possesses bactericidal or antiviral activity not found in natural proteins, including monomeric α-lactalbumin forms, although other therapeutic effects may be possible.

[0046] Expression "variants" refer to proteins or polypeptides that have similar biological functions but whose amino acid sequences differ from those from those from which they are derived; that is, one or more amino acids in the sequence are replaced by other amino acids. Amino acid substitutions are considered "conserved" substitutions when an amino acid is replaced by a different amino acid with broadly similar properties. Non-conserved substitutions occur when an amino acid is replaced by an amino acid of a different type.

[0047] "Conservative substitution" refers to the substitution of an amino acid by another amino acid of the same class, where the categories are defined as follows:

[0048] As is well known to those skilled in the art, altering the primary structure of a peptide through conservative substitution may not significantly change its activity because the side chains of the inserted amino acids may be able to form bonds and contacts similar to those of the substituted amino acid side chains. This is true even when the substitution is located in a critical region that determines the peptide conformation.

[0049] Non-conserved substitutions are possible, provided they do not disrupt the function of the DNA-binding domain peptide. Overall, the number of non-conserved substitutions that do not alter the peptide's biological activity will be relatively small.

[0050] Determining the effect of any substitution (and virtually any amino acid deletion or insertion) is entirely within the ordinary capabilities of those skilled in the art, who can readily determine whether the variant peptide retains the basic properties and activity of the underlying protein. For example, when determining whether a variant of a peptide falls within the scope of this invention, those skilled in the art will determine whether a complex formed from the unfolded form of the native protein and a complex containing the variant formed from the peptide retains biological activity (such as tumor cell death), and whether the peptide has at least 60%, preferably at least 70%, more preferably at least 80%, more preferably 90%, 95%, 96%, 97%, 98%, 99%, or 100% of the native protein activity.

[0051] Variants of polypeptides may comprise or consist substantially of an amino acid sequence that is at least 70% identical to a natural protein sequence such as α-lactalbumin or lysozyme, for example, at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 96%, 97%, 98%, or 99% identical.

[0052] Using the BLASTP computer program, the level of sequence identity is appropriately determined using the natural protein sequence as the base sequence. This means that the natural protein sequence forms a sequence with a defined percentage of identity. The BLAST software is publicly available at the following website. http: / / blast.ncbi.nlm.nih.gov / Blast.cgi (Available for review on March 12, 2009).

[0053] In one particular embodiment, the polypeptide is an α-lactalbumin, such as human, bovine, or sheep α-lactalbumin. While these variants as described above can be used in the present invention, particularly for nutritional supplements, natural proteins are preferred. One particular embodiment uses human α-lactalbumin. In another embodiment, the α-lactalbumin is bovine α-lactalbumin. Sequences of various α-lactalbumins are known in the literature, for example as shown in Watanabe et al. J. Vet Med Sci, (2000) 62(11); 1217-1219.

[0054] In another embodiment, the polypeptide comprises a recombinant protein having an α-lactalbumin sequence or a fragment thereof, but lacking intramolecular disulfide bonds or cross-links. By ensuring that the recombinant protein lacks intramolecular disulfide cross-links, the molecule is unnatural in three dimensions and completely loses its original endogenous biological activity. This is achieved by altering cysteine ​​residues in natural α-lactalbumin to other amino acid residues (especially alanine residues). Preferably, all cysteine ​​residues are substituted with other residues, such as alanine residues. In particular, the recombinant protein is based on the human α-lactalbumin sequence, but α-lactalbumin from other sources (including bovine or sheep α-lactalbumin) can also be used to derive the recombinant protein.

[0055] In one particular embodiment, the polypeptide is a recombinant protein having the sequence of native mature α-lactalbumin, but with all cysteine ​​residues at positions 6, 28, 61, 73, 77, 91, 111, and 120 of the full-length mature human α-lactalbumin sequence mutated to other amino acids (such as alanine) that do not form disulfide bridges. Therefore, according to the present invention, a specific protein comprising the protein of SEQ ID NO: 1 can be used.

[0056]

[0057] The bold text indicates the location of cysteine ​​mutations in natural human α-lactalbumin.

[0058] As described in WO2010079362, for convenience (e.g., for expression purposes), additional amino acid residues, such as up to 20 amino acids, can be linked to the N-terminus and / or C-terminus of a protein. Therefore, the recombinant protein shown in SEQ ID NO: 1, but with an additional methionine added to the N-terminus (as shown in SEQ ID NO: 2 below), has been used in the complexes of this invention.

[0059]

[0060] The polypeptides used in the complex are suitably in pure form and are prepared using conventional peptide synthesis methods or by recombinant expression. In particular, the DNA encoding the desired recombinant α-lactalbumin can be inserted into a suitable expression vector, such as a plasmid, and then used to transform host cells, such as prokaryotic cells like E. coli or eukaryotic cells like specific insect cells.

[0061] Suitable fatty acids or lipids include those known to provide bioactive complexes. These include fatty acids, such as those described in WO2008058547. When using salts, these salts are suitable water-soluble salts. Specific examples of suitable salts may include alkali metal salts or alkaline earth metal salts. In a particular embodiment, the salt is an alkali metal salt, such as a sodium or potassium salt. When used in pharmaceutical manufacturing, these salts are pharmaceutically acceptable.

[0062] Specific examples of fatty acids or lipids used in this invention are those having 4 to 30, for example 6 to 28, for example 8 to 26 carbon atoms. In certain embodiments, the fatty acids or lipids have 10 to 24, such as 12 to 22, for example 14 to 20 carbon atoms. In particular, the fatty acids or lipids will have 16, 17, 18, or 20 carbon atoms. The fatty acids can be saturated or unsaturated.

[0063] However, specifically, the complex of the present invention utilizes a fatty acid or fatty acid salt having 18 carbon atoms. In one embodiment, the complex of the present invention utilizes a fatty acid or fatty acid salt having 18 carbon atoms and an unsaturated fatty acid chain. In one embodiment, the fatty acid or fatty acid salt is a C18:1 fatty acid or a salt thereof. A specific example is the formula CH3(CH2)7CH=CH(CH2)7COOH or CH3(CH2)7CH=CH(CH2)7COO. - In one embodiment, the fatty acid or its salt is oleic acid or oleate.

[0064] This complex can be prepared using methods similar to those described in, for example, WO99 / 26979, WO2008 / 138348, WO2010 / 131237, WO2014 / 023976, WO2018 / 210759 and WO2022 / 073982, the contents of which are incorporated herein by reference. It has been found that the complex can be prepared not only by contacting unfolded α-lactalbumin or its derivatives with a cofactor, particularly oleic acid or its salts, under ion-exchange conditions, such as those found on ion-exchange columns, but also by incubating a solution of α-lactalbumin or its derivatives with a cofactor at high temperatures (e.g., 50-80°C, e.g., 50-70°C, particularly 55-60°C), thereby producing a complex suitable for the present invention.

[0065] However, these methods typically focus on attempting to reconstruct the conditions under which proteins unfold and complex with oleic acid ions. Such work emphasizes the use of pure proteins, including recombinant variants of the basic protein, to facilitate the production of the active complex. However, this starting material also increases production costs.

[0066] Complexes obtained using α-lactalbumin from sources other than human milk, particularly BAMLET obtained using bovine α-lactalbumin, are known to exhibit qualitatively similar effects on cells, especially tumor cells, to those of HAMLET (see, for example, Rammer et al. (2010) Mol. Cancer Ther. 9(1) 24-32). Therefore, similar effects demonstrated below with the use of BAMLET can be observed if HAMLET or HAMLET-based compositions are used instead of BAMLET.

[0067] dose

[0068] The amount of the complex administered to an individual will depend on a variety of factors, including the nature of the composition and risk factors. However, as a general rule, when administered orally, a dose of 1 mg to 20 g of the bioactive complex is suitable for daily administration. Daily doses can be, for example, at least or about 1 mg, 2 mg, 5 mg, 10 mg, 15 mg, 20 mg, 25 mg, 50 mg, 75 mg, 100 mg, 200 mg, 300 mg, 400 mg, 500 mg, 750 mg, 1 g, 2 g, 3 g, 4 g, 5 g, 7.5 g, 10 g, 12.5 g, 15 g, or 17.5 g. Alternatively, the daily dose may be less than 25 g, 22.5 g, 20 g, 17.5 g, 15 g, 10 g, 7.5 g, 5 g, 4 g, 3 g, 2 g, 1 g, 750 mg, 500 mg, 400 mg, 300 mg, 200 mg, 100 mg, 75 mg, 50 mg, 25 mg, 20 mg, 15 mg, 10 mg, or 5 mg. Alternatively, the complex may be administered daily at a dose of 0.1 g to 1 g per kilogram of body weight. It may be administered at a dose of at least or about 0.1 g, 0.2 g, 0.3 g, 0.4 g, 0.5 g, 0.6 g, 0.7 g, 0.8 g, 0.9 g, or 1 g per kilogram of body weight per day. As an alternative or supplement, it can be administered at doses of less than 1.5 g, 1 g, 0.9 g, 0.8 g, 0.7 g, 0.6 g, 0.5 g, 0.4 g, 0.3 g, 0.2 g, or 0.1 g per kilogram of body weight per day.

[0069] Food and beverage compositions

[0070] Complex or pharmaceutical compositions may be in the form of beverages, particularly drinking water, or in the form of foods such as infant formula, or as additives or components of beverages or foods, such as powders for mixing into beverages, for example, in the form of protein shakes. Such food and beverage compositions may be produced using standard techniques. Similarly, complex or pharmaceutical compositions may be in the form of compositions for providing parenteral or preferably intravenous nutrition.

[0071] The present invention also provides food, beverage, food additive or other nutritional compositions comprising the defined complex, particularly for the treatment of cancer or metabolic-related conditions, as described above.

[0072] Examples of such compositions include water-based or milk-based beverages, particularly drinking water; infant foods; nutritional compositions for parenteral or intravenous administration; food additives, such as powders for mixing into beverages or foods; and nutritional capsules, gels, or tablets.

[0073] General

[0074] In the description and claims of this specification, the words “comprise” and “contain,” as well as variations of these words such as “comprising” or “comprises,” mean “including, but not limited to,” and do not exclude other components, integers, or steps. Furthermore, unless the context requires otherwise, the singular includes the plural: particularly in the case of the use of the indefinite article, unless the context requires otherwise, the specification should be understood to consider both the plural and the singular.

[0075] Preferred features of each aspect of the invention may be as described in conjunction with any other aspect. Within the scope of this application, it is expressly intended that the various aspects, embodiments, examples, and alternatives listed in the foregoing paragraphs, claims, and / or the following description and drawings, particularly their respective features, may be employed independently or in any combination. That is, features of all and / or any embodiments may be combined in any manner and / or combination unless these features are incompatible. Attached Figure Description

[0076] One or more embodiments of the invention will now be described by way of example only with reference to the accompanying drawings, wherein:

[0077] Figure 1 The structure of the leptin receptor (LepR) isoform is shown.

[0078] Figure 2 The structure of the leptin receptor dimer and the mechanism of leptin binding were shown.

[0079] Figure 3 shows the functional results of the interaction between LepR and α1.

[0080] Figure 4 The predicted interaction between the Ig-like domains +CHR2 of α1 and LepR is shown.

[0081] Figure 5 The predicted interactions between α1 and LepR throughout the extracellular region are shown.

[0082] Figure 6A shows the structure of adiponectin receptor 2, including the oleic acid binding pocket.

[0083] Figure 6B shows the structure of adiponectin receptor 2, revealing the intracellular lumen containing oleic acid.

[0084] Figure 7A shows the α1 peptide sequence and the AlphaFold predicted structure.

[0085] Figure 7B shows a computer simulation of the binding of the α1 binding pocket to the adiponectin receptor. in silico )predict.

[0086] Figure 8 Lep treated with BAMLET was shown ob Treatment regimens for wild-type mice.

[0087] Figure 9 Showing BAMLET to Lep ob Effects on weight gain and blood glucose levels in wild-type mice.

[0088] Figure 10 shows the protective effect of oral BAMLET treatment against high-fat diet (HFD) on systemic metabolism and adipocyte expansion in C57 / BL6 mice. a. Schematic diagram of the experiment. Time-resolved analyses of dietary interventions were performed on 8-week-old C57BL / 6 mice at 4 and 7 weeks. Each time point included the following three groups: Chow (normal diet): n=8, HFD: n=8, HFD+BAMLET: n=9. Mice received 10 mg BAMLET in 200 μl PBS (BAMLET) or 200 μl PBS alone (placebo) in drinking water for 4 weeks, and then received 20 mg BAMLET dissolved in 200 μl PBS (BAMLET group) or 200 μl PBS (placebo group) in drinking water for 3 weeks. Mice were sacrificed at four or seven weeks. b. Percentage change in body weight among the three groups. c. Percentage change in blood glucose in the three groups. d. The changes in body weight and blood glucose differed between the HFD+BAMLET group and the placebo (HFD) group. e. Organ weights: epididymal adipose tissue (eWAT), inguinal adipose tissue (IngWAT), perirenal adipose tissue (ReWAT), and liver. Data are expressed as mean ± standard error of two experiments and were analyzed using two-way ANOVA and Šídák multiple comparison tests, comparing mice treated with BAMLET to the placebo group. For each organ, the first column is Chow, the second column is HFD+PBS, and the third column is HFD+BAMLET. f. Percentage change in tissue weight in the C57 / BL6 HFD+BAMLET group compared to the placebo (HFD) group.

[0089] Figure 11 shows the regulation of metabolic genes and metabolic functions.

[0090] Figure 12 shows the interactions of the α1 peptide with adiponectin receptor 2 (AdipoR2) (residues 131-386) and leptin receptor subtype B (LEPR-B). a) Schematic diagram showing the reaction between the 39-amino acid-long α1 peptide and an oleic acid molecule to form an α1-oleate complex (Brisuda et al.). b) Previous structural studies of AdipoR2 (residues 100-386) (Vasiliauskaité-Brooks et al.) provided direct evidence of oleic acid binding to the central cavity of AdipoR2, which was confirmed by crystallography. c) AlphaFold 3 predictions show a strong interaction between AdipoR2 and an oleic acid molecule (OLA) at the known oleic acid-binding cavity. d) A weak interaction is predicted between the α1 peptide and the extracellular region of the truncated AdipoR2 protein. e) An α1-oleate complex composed of five additional OLA molecules is predicted to interact with the truncated AdipoR2 with higher confidence. f. Magnified interaction of two oleic acid molecules in the α1-oleic acid complex located within the transmembrane lumen of AdipoR2 and at the opening between the tunnel-like cavities TM5 and TM6. gh. Predicted interaction of the surface-exposed extracellular portion of the complex with the α1 peptide via its immunoglobulin-like (Ig-like) domain. α1 is predicted to interact with the LEPR-B fragment containing both the Ig-like and CHR2 domains, at the Ig-like domain (g). This prediction is reinforced when the entire extracellular region of LEPR-B is used (h). ij. Superimposed with the cryo-electron microscopy structure of the leptin-binding LEPR complex (PDB 8DH8), suggesting two possible effects. The α1 peptide may bind to the Ig-like domain of either the first LEPR (LEPR I) (i) or the second LEPR (LEPR II) (j).

[0091] Figure 13 shows the use of B6.Cg-Lep ob Experiments with J mice. For each organ in e, the first strip is Chow + PBS, and the second strip is Chow + BAMLET.

[0092] Figure 14 shows that drinking BAMLET can prevent B6.Cg-Lep obThe top-ranked upregulated genes and activation of metabolic-related functions induced by HFD in the gut of / J mice. a. A table listing the top 10 activated (first in the list) and 10 repressed (second in the list) genes in response to HFD or HFD+BAMLET. Genes strongly activated by HFD were no longer regulated in mice fed HFD and drinking BAMLET. Changes in gene expression compared to the Chow group were measured as fold changes (FC) (significance threshold FC>2, P<0.05). b. Histogram of strong regulation of metabolic-related functions in response to HFD and compared to HFD+BAMLET. Compared to mice on a normal diet (Chow group), drinking BAMLET significantly reduced the regulation of diabetes-related functions and eliminated the regulation of lipid metabolism and insulin resistance (green: HFD+BAMLET, red: HFD). Detailed Implementation

[0093] Example 1

[0094] method

[0095] Preparation of α1-oleate

[0096] α1 was synthesized using Fmoc solid-phase chemistry (Mimotopes). The α1 sequence is: aa 1-39 Ac-KQFTKAELSQLLKDIDGYGGIA-LPELIATMFHTSGYDTQ-OH.

[0097] A. Interaction between leptin receptor-α1 peptide and leptin receptor

[0098] Leptin is a 16 kDa protein that functions by activating the leptin receptor (LepR). LepR exists in six isoforms (LepR-a, LepR-B, LepR-C, LepR-D, LepR-E, and LepR-F). These isoforms share homologous extracellular regions and can be distinguished by their intracellular domains. Among them, the LepR-B isoform, with the longest intracellular region, has the strongest signal transduction capacity and is mainly expressed in the hypothalamus. Figure 1 ).

[0099] Structurally, the extracellular region of LepR consists of a short N-terminal helix followed by the cytokine homology region (CHR) 1, an Ig-like domain, CHR2, and a fibronectin type III region. Dimerization of the two LepRs induced by leptin binding is necessary for the conformational changes required to induce receptor activation. Leptin binds to LepR-B in an asymmetric structure, interacting with a high-affinity site within CHR2 and a low-affinity site within the Ig-like domain (Saxton RA, Caveney NA, Moya-Garzon MD, Householder KD, Rodriguez GE, Burdsall KA et al., Structuralinsights into the mechanism of leptin receptor activation. Nature Communications. 2023;14(1):1797). Both sites require conformational changes to induce activation. Figure 2 This activation triggers the JAK-STAT cascade through JAK2 activation and STAT3 phosphorylation. STAT3 is involved in processes such as immunity, cell division, cell death, and tumor formation.

[0100] Computer simulations were used to investigate whether the α1 peptide exhibits affinity for LepR. The software AlphaFold, which predicts protein structure and interactions, was used to screen for potential interactions. Leptin was found to interact with α1 via its Ig-like domain. Initially, the inventors focused on analyzing the Ig-like domain and the CHR2 sequence, which are crucial for leptin binding. Although the ipTM score was relatively low at 0.367, α1 was predicted to bind to the Ig-like domain of LEPR at residues 369–373, 402–405, and 416–422. Figure 4 Extending the analysis to include the entire extracellular region of LEPR, not just the Ig-like domain and CHR2, the ipTM score increased to 0.52. Notably, α1 was still predicted to bind to residues in the previous model, thus enhancing the reliability of the prediction. Figure 5 ).

[0101] These findings suggest that α1 can prevent LepR activation. Superimposing the highest-scoring prediction model with the frozen EM structure of the leptin-binding LepR dimer (1) reveals two possible mechanisms of LepR inhibition. If α1 binds to the receptor that should bind to leptin via the Ig-like domain (LepRI), leptin binding is completely inhibited (Fig. 3A). If α1 binds to the receptor that should bind to leptin via CHR2 (LepR II), leptin binding is possible, but the dimer will not undergo the necessary conformational change to acquire activity (Fig. 3B).

[0102] B. Interaction between adiponectin receptor-α1-oleic acid complex and adiponectin receptor

[0103] Human APN consists of 244 amino acids and has a molecular weight of 28 kDa. Circulating APN oligomers exist as trimers (low molecular weight), hexamers (medium molecular weight), and polymers (high molecular weight), the latter being the most biologically active form. APN also exists in plasma in globular form (due to protein hydrolysis).

[0104] All these metabolic processes are mediated by three receptors: AdipoR1, AdipoR2, and T-cadherin. The AMPK and PPAR pathways are activated by the binding of adiponectin to AdipoR1 and AdipoR2, respectively. AdipoR1 and AdipoR2 contain seven transmembrane domains with high structural homology, as well as an intracellular N-terminus and an extracellular C-terminus, unlike classic G protein-coupled receptors (GPCRs).

[0105] The previously reported crystal structures of AdipoR1 and AdipoR2 proteins interestingly suggest that major cavities in the protein structure can be occupied by oleic acid (Vasiliauskaité-Brooks I, Sounier R, Rochaix P, Bellot G, Fortier M, Hoh F et al., Structural insights into adiponectin receptors suggestceramidase activity. Nature. 2017;544(7648):120-3. Tanabe H, Fujii Y, Okada-Iwabu M, Iwabu M, Nakamura Y, Hosaka T et al. Crystal structures of the human adiponectin receptors. Nature. 2015;520(7547):312-6. Muratore M, Komai AM. Theoretical study of the adiponectin receptors: binding site characterization and molecular dynamics of possible ligands for drug design. SN AppliedSciences. 2020;2(4):533). AdipoR1 and AdipoR2 also have adiponectin-independent functions in maintaining membrane fluidity in many types of human cells (Ruiz M, Ståhlman M, Borén J, Pilon M. AdipoR1 and AdipoR2 maintain membrane fluidity in most human cell types and independently of adiponectin. J Lipid Res. 2019;60(5):995-1004). Considering that the complex has an oleic acid component and may therefore interact with AdipoR1 and AdipoR2 receptors at the membrane level, we found this information to be very useful for understanding the tumor-killing mechanism of α1-H. In addition, α1-oleate has high membrane activity, suggesting that the combined effect of α1-oleate and adiponectin receptor may be crucial to initiating the widespread conversion of tumor cells to α1-oleate (leading to tumor cell death) (Figure 6).

[0106] Using the same approach as with LepR interactions, computer simulations were employed to predict whether the α1 peptide exhibits affinity for the AdipoQ receptor. The predicted structures revealed a common binding pocket (shown in green) for α1 near the intracellular N-terminal domains of both adiponectin receptors (Figure 7). This observation provides context for further investigation of the AdipoQ receptor and its interaction with α1-oleate.

[0107] Example 2

[0108] To exert its regulatory effects on energy homeostasis and hunger, leptin binds to its receptor (LepR), activating the JAK2-STAT3 signaling pathway, which is involved in processes such as immunity, cell division, cell death, and tumorigenesis. This cascade stimulates the production of anorexia peptides, reducing appetite and increasing energy expenditure. However, in obesity, although elevated circulating leptin levels are associated with increased obesity, satiety is diminished due to dysregulation of leptin signaling.

[0109] Lep ob Mice (commonly known as ob or ob / ob) have a nonsense point mutation in the gene encoding leptin, resulting in the absence of this hormone. Due to the lack of leptin, Lep... ob Mice exhibited obesity, overeating, transient hyperglycemia, glucose intolerance, and elevated plasma insulin levels. Therefore, this strain was used to simulate phase I and II of type 2 diabetes and obesity.

[0110] The inventors tested BAMLET on Lep ob Effects on mice. For example... Figure 8 As shown, for Lep ob BAMLET was administered to mice and wild-type mice. Mice were fed a high-fat diet (HFD), normal diet, or HFD plus BAMLET. Effects on weight gain and blood glucose levels are described in [link to relevant documentation]. Figure 9 .

[0111] Effects on weight gain .

[0112] Lep ob Mice gained weight during the three-week observation period, consistent with previous reports on the consequences of functional leptin deficiency mutations. In contrast, mice treated with BAMLET did not show a significant increase in weight gain compared to the control group receiving a standard diet. BL6 (wild-type) mice exhibited a similar trend, although the difference was less pronounced compared to mice fed a high-fat diet.

[0113] Effects on blood glucose levels .

[0114] BAMLET administration also resulted in a slight decrease in blood glucose levels in obese mice compared to the high-fat diet-only group. Notably, blood glucose levels remained within the normal range in all BL6 groups, and the differences between groups were not biologically significant. This result is expected, given that metabolic changes in wild-type mice typically evolve over a longer period.

[0115] Example 3

[0116] This study proposes a novel molecular concept for the treatment of obesity and diabetes based on the potential metabolic effects of a complex formed from α-lactalbumin and oleic acid. In healthy mice fed a high-fat diet and ob / B6.Ag-Lep ob In / J mice, the bovine protein complex BAMLET inhibited weight gain and reduced blood glucose levels, but had no effect on healthy mice on a normal diet. The associated peptide-based complex α1-oleate regulates metabolism in human tissues, inhibiting ADIPOQ and LEP, two proteins encoding lipid and glucose-regulating proteins adiponectin and leptin. Treatment further showed inhibition of genes in the interconnected ADIPOQ and LEP gene network, affecting glucose metabolism and lipid synthesis in treated tissues. Based on computer simulations, a direct receptor interaction mechanism was proposed involving the oleic acid-binding pocket and extracellular peptide epitopes of the adiponectin receptor ADIPOR2. It was further predicted that the binding of the α1-peptide to the immunoglobulin-like domain of the leptin receptor may inhibit leptin dimerization and activation. Treatment with BAMLET in drinking water inhibited the activation of cancer-related gene expression in mice fed a high-fat diet and induced a potent metabolic shift in gene expression in the gut and liver tissues, a shift observed in ob / B6.Cg-Lep. ob The effects were attenuated in J mice. These findings provide new molecular approaches to targeting and inhibiting key metabolic regulators that have independent effects on obesity, type 2 diabetes, and cancer, and suggest that these effects may target hosts with metabolic dysfunction caused by excessive fat intake or leptin deficiency.

[0117] introduction

[0118] Metabolic dysfunction is increasingly recognized as a major health threat worldwide, associated with diabetes, metabolic syndrome, obesity, and cancer. Traditional treatments have focused on compensating for metabolic dysfunction through insulin injections or dietary changes. Recently, other molecular mechanisms of metabolic diseases have been identified, and major therapeutic advances include the design of drugs targeting metabolic syndrome, which affects obesity and type 2 diabetes. In recent years, synthetic analogues of the pancreatic incretin hormone glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP), such as smegglutide and tirzepatide, have been introduced to target key mechanisms of obesity and type 2 diabetes (Fisman EZ, Tenenbaum A. The dual glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like peptide-1 (GLP-1) receptor agonist tirzepatide: a novel cardiometabolic therapeutic prospect. Cardiovascular diabetology. 2021;20(1):22). Its broad effects include increasing insulin secretion in a glucose-dependent manner when blood glucose is high, inhibiting glucagon secretion from the pancreas, delaying gastric emptying, and inducing hunger satiety by acting on peripheral and central receptors in the gut-brain axis. These treatments commonly cause gastrointestinal adverse events, such as nausea, vomiting, and diarrhea, limiting patient tolerance (Holst JJ. GLP-1 physiology in obesity and development of incretin-based drugs for chronic weight management. Nature Metabolism. 2024;6(10):1866-85).

[0119] We have discovered a family of protein-oleic acid complexes that rapidly kill cancer cells through an apoptosis-like mechanism. The HAMLET complex inserts into the lipid bilayer and is absorbed by tumor cells and tissues. The second-generation peptide-based α1-oleate complex, formed by a synthetic peptide containing the N-terminal domain of α-lactalbumin and oleic acid, has shown therapeutic efficacy in animal models and dose-dependent tumor number and size effects in a placebo-controlled clinical study of non-muscle-invasive bladder cancer (NMIBC). It also showed significant changes in the gene expression profile of the treated tumors compared to the placebo group (Hien TT, Ambite I, Butler D, Wan MLY, Tran TH, Höglund U, et al. Bladder cancer therapy without toxicity—a dose-escalation study of α1-oleate. International journal of cancer. 2020;147(9):2479-92 and Haq F, Sabari S, Háček J, Brisuda A, Ambite I, Cavalera M, et al. Clinical and molecular response to α1-oleate treatment inpatients with bladder cancer. Cancer Medicine. 2024;13(17):e70149). Oral HAMLET treatment in colorectal cancer-susceptible APC Min+ / - Potential effects in mice have also been reported, with a significant delay in tumor progression (Tran HT, WanMLY, Ambite I, Cavalera M, Grossi M, Háček J, et al. BAMLET administered via drinking water inhibits intestinal tumor development and promotes long-term health. Scientific Reports. 2024;14(1):3838). Effects on metabolic pathways have also been detected in the intestines of mice treated with BAMLET (a complex formed by bovine α-lactalbumin and oleic acid).

[0120] This study investigated the metabolic effects of BAMLET treatment in more detail. The effects of oral BAMLET treatment on obesity and glucose metabolism were quantified in normal C57BL / 6 mice on a high-fat diet and Lep-deficient ob / ob mice. Metabolic responses were characterized by gene expression analysis, and the potent effects on glucose and lipid metabolism in treated mouse tissues were determined compared to placebo. Metabolic effects were also locally quantified in tissues of cancerous bladder patients treated with α1-oleic acid. These identified molecular regulatory targets include adiponectin and leptin—key regulators of metabolic homeostasis, lipid metabolism, blood glucose levels, and insulin sensitivity—which function through interactions with adiponectin and leptin receptors. These results demonstrate the strong potential of using these molecular therapies to target metabolic disorders.

[0121] result

[0122] Effects of BAMLET in drinking water on metabolism

[0123] To investigate whether the BAMLET complex affected metabolic endpoints, BAMLET was administered daily to the drinking water of healthy C57BL / 6 mice (Figure 10). The treatment group received 10 mg / day of phosphate-buffered saline (PBS) BAMLET from weeks 1 to 4 and 20 mg / day of BAMLET from weeks 5 to 7. The placebo group received drinking water supplemented with PBS (Figure 10a). C57BL / 6 mice were fed either a high-fat diet (HFD) or a normal diet (chow), and body weight and blood glucose levels were measured weekly. Tissue was collected at sacrifice four to seven weeks after treatment. As expected, body weight and blood glucose levels increased more rapidly in the HFD-fed C57BL / 6 mice than in the cow group (p-value).

[0124] Compared with placebo, BAMLET treatment significantly reduced body weight gain in mice fed HFD, indicating a significant effect on body weight gain (Figure 10b-d). A similar effect was observed in blood glucose levels, with BAMLET treatment significantly reducing blood glucose levels in mice fed HFD compared with placebo (Figure 10c).

[0125] Compared with placebo, HFD mice treated with BAMLET experienced an average weight loss of 19% and a 21% reduction in blood glucose levels after seven weeks (Figure 10d). Higher doses of BAMLET (50 mg / day or 100 mg / day for 7 weeks) did not further increase the effects on weight and blood glucose.

[0126] BAMLET treatment reduced body weight and blood glucose to levels similar to those in the normal diet control group, indicating near-healthy metabolic status. No side effects were detected in the tissues of BAMLET-treated control mice fed with chow (gross pathology score). Importantly, BAMLET treatment did not affect body weight gain or blood glucose levels in chow-fed healthy C57BL / 6 mice, suggesting that the metabolic changes targeted by BAMLET are initiated by HFD intake.

[0127] Effects on adipose tissue growth

[0128] The effect of Bamlet on adipose tissue growth was quantified by measuring the size and weight of epididymal white adipose tissue (eWAT), inguinal white adipose tissue (IngWAT), and perirenal adipose tissue (ReWAT) after seven weeks of treatment, compared with placebo (Fig. 10e, f). As expected, HFD-fed mice showed significantly greater accumulation of epididymal (EDI), inguinal (ING), and perirenal adipose tissue compared with the normal diet group (Fig. 10e, f). This adipose tissue accumulation was inhibited in HFD-fed BAMLET-treated C57 / BL6 mice, as evidenced by a significant reduction in the size and weight of white adipose tissue (eWAT, IngWAT, and ReWAT) compared with the placebo group (Fig. 10e, f).

[0129] BAMLET treatment did not affect liver weight, but histological analysis showed significantly less histological evidence of "fatty liver" in BAMLET-treated mice than in the placebo group. H&E-stained liver sections revealed centrilobular microvacuolar and macrovacuolar steatosis and binucleated hepatocytes in C57 / BL6 mice fed with HFD. These changes were associated with HFD because they were not observed in mice fed a normal diet. Centrilobular microvacuolar and macrovacuolar steatosis and binucleated hepatocytes were significantly less pronounced in BAMLET-treated mice fed with HFD compared to the placebo group.

[0130] The results showed that BAMLET has a potent anti-obesity effect, preventing the formation, growth, and hepatic steatosis of adipose tissue. Furthermore, compared to the placebo group taking HFD, the BAMLET treatment group demonstrated a “restoration to health” effect on blood glucose levels.

[0131] Regulation of metabolic genes and functions in human bladder cancer tissue by α1-oleate

[0132] The effects on metabolism were further investigated using α1-oleate, a complex formed from the N-terminal peptide of human α-lactalbumin and oleic acid. Bladder tissue biopsies were obtained from patients with non-muscle-invasive cancer who received α1-oleate complex treatment in a placebo-controlled study (EudraCT No: 2016-004269-14). ClinicalTrials.gov NCT03560479 Each patient received six intravesical instillations of α1-oleate or placebo over one month, and the molecular response to α1-oleate treatment was assessed by RNA sequencing of tissue biopsies obtained via transurethral resection at the end of treatment. Gene expression profiles were compared between patients receiving 8.5 mM α1-oleate or placebo (PBS).

[0133] RNA sequencing detected a significant therapeutic response to α1-oleate (8.5 mM), with 2894 regulated genes compared to placebo, predominantly repressed (2058 genes, 71%) (cutoff FC≥2, P<0.05). The main regulated categories included genes solely associated with cancer (1751 genes), genes solely associated with metabolism (678 genes), and genes associated with both cancer and metabolism. Genes involved in glucose metabolism (261 genes), lipid metabolism (164 genes), or both (195 genes) were affected by α1-oleate treatment (Figure 11a). Functional analysis identified the top-ranked regulated glucose metabolism functions as glucose metabolism disorders, glucose uptake, glucose synthesis, carbohydrate metabolism, and glucose tolerance, and the top-ranked regulated lipid metabolism functions as lipid synthesis, fatty acid metabolism, fatty acid concentration, terpene compound synthesis, and steroid synthesis (Figure 11b).

[0134] Network analysis of 678 regulated metabolic genes confirmed that the glucose metabolism disorder network was strongly regulated (Fig. 11c), consisting of 455 predominantly repressive genes (341 out of 455 genes, 75%). A similar pattern was observed in the synthesis of lipid genes, predominantly repressive (99 out of 118 genes, 84%), confirming that α1-oleate treatment suppressed the expression of metabolism-related genes compared to placebo (Fig. 11d).

[0135] Metabolic regulation, including adiponectin and leptin gene networks

[0136] A strong tumor response to α1-oleate treatment was observed, with fold change (FC) values ​​ranging from -26907 to 142. The top-ranking regulatory genes involved in tumor treatment included GC, encoding a vitamin D-binding GC protein associated with tumor cell motility and migration (FC = -26907); ADIPOQ, encoding adiponectin (FC = -5353); CALB1S, encoding cadherin 1 protein, affecting cell survival (FC = -429); BAAT, encoding bile acid coenzyme A:amino N-acyltransferase (FC = -273); CSF3, encoding colony-stimulating factor 3 (FC = -171); EREG, encoding an epidermal regulatory protein (FC = -127); and KRT79, encoding keratin 79 (FC ​​= 142). Furthermore, the magnitude of inhibition (FC) was greater than the magnitude of activation of the top-ranking regulated genes.

[0137] Notably, the top-ranked regulated genes included metabolism-related genes GC, ADIPOQ, CALB1, BAAT, CSF3, and EREG. Further analysis of the top-ranked subset of regulated metabolism-related genes identified gene repression dominance, which also included CIDEC (encoding Cell Death Inducing DFFA Like Effector C, FC = -79), TF (encoding transferrin, FC = -64), and LEP (encoding leptin, FC = -60) (Figure 11e).

[0138] Adiponectin and leptin are important metabolic hormones or adipokines with broad effects on metabolism and health. Since the gene expression levels of both molecules are strongly affected in tumors treated with α1-oleate, their gene networks and interdependencies were investigated. In the ADIPOQ- and LEP-dependent gene networks, 66 / 78 and 55 / 70 genes were identified as downregulated by α1-oleate treatment. Detailed analysis of genes in the ADIPOQ and LEP networks revealed evidence of strong interrelationships, suggesting their shared role in glucose metabolism disorders and lipid synthesis (Figure 11f). Furthermore, upstream regulatory factor analysis identified 14 genes regulating ADIPOQ, 13 of which were downregulated, including LEP as a leading upstream regulator (Figure 11g).

[0139] Functional analysis confirmed the broad effects of α1-oleate treatment on the ADIPOQ gene network, which is expected to extensively affect glucose metabolism disorders, glucose tolerance, glucose synthesis, transport, and uptake. Lipid metabolism functions were also strongly downregulated in the ADIPOQ-dependent network, including fatty acid oxidation, lipid release, phospholipids, and membrane lipid metabolism. Similarly, the LEP-dependent network regulating lipid metabolism was strongly downregulated, including membrane lipid metabolism, lipid oxidation, and stimulation of lipid synthesis; glucose metabolism disorders and carbohydrate transport functions were affected, but the degree of regulation was expected to be less than that of adiponectin. The GC gene network showed the strongest regulation (FC=-26907), consisting of only 19 genes.

[0140] Regulation of adiponectin levels

[0141] The metabolic response to α1-oleate was further investigated at the protein level using the Luminex human adipokines group (adiponectin / Acrp30, C-reactive protein, cadherin D, cathepsin S, CXCL2 / GROβ / MIP-2 / CINC-3, DPPIV / CD26, IGFBP-rp1 / IGFBP-7, insulin, leptin / OB, M-CSF, myeloperoxidase (MPO), PBEF / endothelin, resistin, TIMP-1, TREM-1, and vitamin D BP (encoded by GC)). Urine samples were collected from patients treated with α1-oleate before treatment initiation (before V1) and after six intravesical infusions of α1-oleate (after V1 to V6).

[0142] Significantly elevated urinary adiponectin levels were detected at early time points (P=0.008 after V1 and P<0.001 after V3), followed by a decrease at the end of treatment, indicating an initial adiponectin response to α1-oleate therapy. Urinary adiponectin levels remained unchanged in the placebo group (P=0.727 after V1 and P=0.222 after V6). A similar pattern of early elevation followed by a decrease in adipokines was observed.

[0143] Computer simulation modeling predicts the interaction between α1-oleate and adiponectin and leptin receptors.

[0144] Adiponectin receptors AdipoR1 and AdipoR2 are defined by seven transmembrane helices, resembling inverted G protein-coupled receptors, and an intracellular domain involved in metabolic signaling. Previously reported crystal structures of the transmembrane domains (TM) of the AdipoR1 and AdipoR2 receptor proteins have identified a major cavity that can be occupied by oleic acid (9,12) (Fig. 12b). Starting from the upper half of the transmembrane AdipoR2 structure between TM5 and TM6, studies have proposed a continuous channel-like cavity connecting the upper lipid bilayer of the plasma membrane to the oleic acid-binding pocket and extending downwards to the cytoplasmic opening, potentially regulating molecule entry and exit from the receptor. As shown by NMR and computational simulations (Nat Comm. Brisuda 2021) (Fig. 12a), the α1-oleate complex consists of a hydrophobic lipid core formed by a structurally flexible α1-peptide and three to five oleic acid molecules. Given this, it is reasonable that the complex may interact with adiponectin receptors, and that the oleic acid presented by the complex to the adiponectin receptors may reach the oleic acid binding cavities of these receptors.

[0145] To address this issue, we used AlphaFold 3 (Abramson, J. et al., Nature, 2024) to explore the structural interactions between truncated AdipoR2 (residues 131-386) and the α1-oleate complex. The resulting structures were subjected to plausibility checks in PyMOL and ranked according to their Interface Prediction Template Modeling (iPTM) scores. First, the software predicted a strong interaction between a single oleic acid molecule (OLA) and truncated AdipoR2 at known oleic acid binding sites, confirming the interaction between oleic acid and truncated AdipoR2 (ipTM: 0.83) (Fig. 12c). Second, truncated AdipoR2 was submitted to AlphaFold 3 along with the α1-oleate complex consisting of one α1-peptide residue and five oleic acid molecules (ipTM: 0.72) (Fig. 12d). The α1-peptide component of this complex is expected to interact with the extracellular segment of AdipoR2, primarily through loop interactions between TM5 and TM6. One type of oleic acid was shown to occupy the oleic acid binding site of AdipoR2, while another oleic acid was located at the transmembrane opening of the tunnel-like lumen (Fig. 12d, e). The binding of the complex was compared to that of the α1-peptide by submitting the truncated AdipoR2 along with the α1-peptide to AlphaFold 3. The peptide was expected to interact with the extracellular loop between TM5 and TM6, but scored low (iPTM: 0.19) (Fig. 12f). Analysis showed that the α1-oleate complex recognizes and binds to the extracellular domain of the AdipoR2 receptor, the oleic acid delivered by the complex reaches the oleic acid binding lumen, and the complex binds more efficiently than the α1-peptide alone.

[0146] The exposed surface residues between TM5 and TM6 in AdipoR2 (aa 302-375) are expected to interact with the α1 peptide and the complex. Given their proximity to the entrance of the channel-like cavity, it can be inferred that the α1-oleate complex facilitates the transport of oleic acid molecules to AdipoR2 through this site. The oleic acid molecules then travel along the channel, eventually reaching the oleic acid binding site and cytoplasm of AdipoR2.

[0147] In contrast to the predicted interaction between the α1 oleate complex or α1 peptide and full-length AdipoR2, AlphaFold 3 consistently localizes the α1 peptide to the intracellular binding site defined by aa residues 84-128 on the N-terminal domain and aa residues 207-211 between the TM2 and TM3 domains. A similar intracellular binding site for this complex was detected in full-length AdipoR1.

[0148] Computer simulation modeling and protein binding analysis predict the interaction between α1-oleate and leptin receptor.

[0149] The potential interaction between α1-oleate and leptin receptor B (LEPR-B) was investigated using the AlphaFold 2 multimer protocol. Structurally, the extracellular region of LepR consists of a short N-terminal helix followed by the cytokine homology region (CHR)1, an Ig-like domain, CHR2, and a fibronectin type III region. LepR dimerization induced by leptin binding is crucial for receptor activation. Leptin binds to LepR-B in an asymmetric structure, interacting with both a high-affinity site within CHR2 and a low-affinity site within the Ig-like domain. Both sites require the conformational changes necessary to induce activation following leptin binding. This activation leads to JAK2 activation and STAT3 phosphorylation, thereby triggering the JAK-STAT cascade.

[0150] Cryo-electron microscopy analysis revealed that leptin binds in stoichiometry at either a 2:2 or 3:3 ratio to form asymmetric dimer or trimer leptin:LepR complexes. Leptin binds with high affinity to the cytokine receptor homology domain 2 (CHR2) of LepR at "site 2," while binding with low affinity to the immunoglobulin-like domain (IgD) of the adjacent receptor in the complex at "site 3." The 'site 3' leptin binding at the IgD domain plays a crucial role in the rate-limiting step of leptin-LEPR binding. After initially binding with high affinity at site 2, the subsequent low-affinity interaction at site 3 triggers the conformational change and signal transduction capacity required for dimerization, leading to the formation of the 2:2 complex.

[0151] The AlphaFold 2 multimerization scheme predicted that LEPR-B interacts with the α1 peptide through its immunoglobulin-like (Ig-like) domains at residues 369-373, 402-405, and 416-422 of LEPR (ipTM score of 0.367), which are directly involved in dimerization (Fig. 12g). Expanding the analysis to include the complete extracellular region of LEPR (rather than just the Ig-like domain and CHR2) improved the ipTM score to 0.52 (Fig. 12h). The analysis predicted that α1 binds to the same residues in the Ig-like domain as in the previous model. AlphaFold 3's predictions of complex interactions confirmed the binding of the peptide to the Ig-like domain, with scores similar to those of the peptide alone (ipTM scores of 0.32 vs. 0.39). The predicted binding of the complex to the extracellular domain was increased compared to the peptide.

[0152] These findings suggest that the binding of α1 peptide and α1 oleate to LEPR may prevent leptin from activating LEPR. Superimposing the highest-scoring predictive model with the cryogenic EM structure of the leptin-bound LEPR dimer revealed two possible effects. First, binding of α1 to the leptin receptor binding site (LEPR I) on the Ig-like domain directly inhibits leptin binding (Fig. 12i). Second, binding of α1 to the adjacent Ig-like domain (LEPR II) prevents the conformational change required for the formation of the dimer leptin:LEPR complex, thereby disrupting downstream signaling (Fig. 12i).

[0153] For B6.Cg-Lep ob Effects of body weight gain and blood glucose levels in J mice

[0154] Subsequently, we used B6.Cg-Lep ob / J mice (ob or ob / ob) were used, and the protocol described in Figure 13a was used to investigate the roles of leptin and leptin receptor in the metabolic response of the BAMLET complex. B6.Cg-Lep ob / J mice developed extreme obesity due to extreme adipose tissue growth, overeating, hyperglycemia, glucose intolerance, and elevated plasma insulin levels, and were used as a model of obesity and type 2 diabetes. Mice in the treatment group received BAMLET (20 mg / day) continuously in their drinking water, while the placebo group received drinking water supplemented with PBS. As expected, B6.Cg-Lep... ob / J mice showed faster weight gain and elevated blood glucose levels than C57BL / 6 WT mice, and the effects were stronger on mice fed HFD than on mice fed a normal diet.

[0155] To optimize treatment efficacy, BAMLET treatment was initiated at 4 weeks of age, while C57BL / 6 mice were 8 weeks old. Compared to the placebo group, the BAMLET treatment group showed a significantly delayed increase in body weight after four weeks of treatment (p<0.01), which further decreased after seven weeks (p<0.001). A similar pattern was observed in blood glucose levels, with significantly lower levels in the BAMLET treatment group than in the placebo group after four weeks (p<0.01), and a further decrease after seven weeks (p<0.001). BAMLET was administered to mice receiving a normal diet and B6.Cg-Lep... ob The effect was more pronounced in / J mice than in mice fed HFD (Figure 13).

[0156] BAMLET treatment Lep Gene expression in defective mice

[0157] To further investigate the response to BAMLET treatment, Lep Defect type ob / ob In mice, whole-genome transcriptome analysis of intestinal RNA was performed. Gene expression changes in HFD-fed mice compared to a normal diet group were quantified, and mice treated with BAMLET in their drinking water for four weeks were also analyzed. Lep defect ob / ob Mice were compared with the placebo group. Treatment began at 8 weeks of age.

[0158] Significant changes in gene expression profiles were detected in HFD mice compared to the normal diet group. Figure 14a The top-ranked regulated genes identified (FC>2, p<0.05) involved in metabolism, including LEP (FC = 122.92) and ADIPOQ (FC = 48.77), were strongly activated. Further activated genes included SCD1 (sterol metabolism, FC = 50.8), TRARG1 (a transport regulator of GLUT4 1, FC = 32.4), and NPR3 (natriuretic peptide receptor 3, FC = 47.2). Figure 14a In contrast, these strongly activated metabolic genes are less active in BAMLET treatment. Lep Defect type ob / ob In mice (HFD diet), LEP and AdipoQ expression were not regulated, and in the BAMLET treatment group, LEP and AdipoQ expression were not activated. Figure 14a Genes with reduced expression in the HFD group compared to the normal diet group were not strongly affected by BAMLET treatment.

[0159] Functional analysis revealed that many functions associated with diabetes and lipid storage were significantly enhanced in mice fed HFD, but not in mice treated with BAMLET. These included functional categories such as insulin-dependent diabetes, glucose metabolism disorders, diabetes and insulin resistance, lipid storage, and fatty acid uptake. Figure 14b Furthermore, direct comparisons showed that HFD patients treated with BAMLET had significantly better outcomes compared to placebo. Lep Defect type ob / ob LEP (FC=-43.81) and ADIPOQ (FC=16.65) in mice were significantly downregulated.

[0160] Further network analysis was performed to evaluate the downstream effects of BAMLET treatment. Compared with the normal diet group, those fed HFD... Lep In defective mice, ADIPOQ and LEP-dependent gene networks were activated, activating 38 and 52 genes, respectively. Figure 14c ,d). Most of these genes were no longer regulated in BAMLET-treated mice, indicating that despite being fed HFD, the treated mice maintained expression levels of the highest regulatory genes and metabolic functions comparable to the normal diet group (Fig. 14e).

[0161] discuss

[0162] This study identifies a novel molecular approach to treating obesity and type 2 diabetes. Based on animal models, the BAMLET complex was shown to reduce weight gain and blood glucose levels, particularly in mice fed a high-fat diet or obese / obese mice, without affecting healthy mice on a normal diet, suggesting a specific effect on excessive fat intake or metabolic abnormalities. However, these effects are not limited to lipid metabolism, as a broad and strong inhibitory effect on glucose metabolism was detected. BAMLET treatment was ineffective and relatively safe in healthy mice, indicating that metabolic abnormalities are required for BAMLET to be effective. Further potential metabolic effects were detected in cancer tissues from cancer patients treated with the α1-oleic acid complex, supporting the relevance of these findings to humans.

[0163] These results further elucidate the molecular basis of these effects, involving direct interactions with adiponectin and leptin receptors. Structural studies using AlphaFold have shown that both the AdipoR and LepR receptors are potential targets of the α-lactalbumin complex family. Previously reported crystal structures of the AdipoR1 and AdipoR2 receptors have identified cavities suitable for oleic acid occupancy, and fatty acids released from ceramides by ceramidinase have been proposed as natural ligands. This study suggests that exogenous adiponectin may fit into the oleic acid-binding pockets in AdipoR1 and R2, and identifies the α1-oleic acid complex as a candidate molecule for this interaction. Furthermore, the N-terminal α1 peptide of α-lactalbumin was shown to conform to the surface-exposed peptide sequences near the transmembrane opening of the oleic acid-binding pocket in TM5 and TM6, indicating that the complex may actively deliver oleic acid through co-action. Interestingly, there was no evidence of activation of AMPK or PPAR, suggesting that these interactions may be independent of the activation of intracellular signaling by the natural ligand adiponectin. Further research is needed to validate the computational predictions and understand the effects of this interaction on receptor-carrying cells and tissues.

[0164] Computer simulations further predicted that the leptin receptor is a target of α1 oleate, and α1 may prevent LepR activation. Superimposing the highest-scoring prediction model with the frozen EM structure of the leptin-binding LepR dimer revealed two possible mechanisms for inhibiting receptor activation. α1-predicted binding to the Ig-like domain of LepR may inhibit leptin binding. Binding to the CRH2 domain of LepR II is expected not to affect leptin binding, but to inhibit the conformational change required to form the active heterodimer. However, in the absence of leptin (e.g., LepR II),... ob In mice, α1 may act as an agonist of the leptin receptor by binding to the same site on its receptor and serving as a substitute for leptin. α1 activation of LepR-B may explain how BAMLET administration prevents increases in body weight and blood glucose levels in leptin-deficient mice. In cases where leptin is not completely absent but present at low levels, α1 can also bind to and activate short- or secretory LepR isoforms, promoting leptin transport to the hypothalamus and maximizing the effect of remaining low-level leptin. At local levels, in tumors or tissues with excess leptin, α1 activation of these low-signaling-activity LepR isoforms can also lead to increased leptin clearance, promoting the restoration of a healthy hormonal balance.

[0165] Altered lipid metabolism and obesity are well-known risk factors that promote the development of diseases worldwide, including cancer (Mossberg AK, Wullt B, Gustafsson L, Mansson W, Ljunggren E, Svanborg C. Bladder cancers respond to intravesical instillation of HAMLET (human alpha-lactalbumin made lethal to tumor cells). Int J Cancer. 2007;121(6):1352-9). Extensive research has identified mechanisms by which obesity drives cancer development by promoting cancer cell proliferation, tumor growth, and a chronic inflammatory state. Identifying these key mechanisms will increase the chances of developing metabolic aspects specifically targeting tumor development, as well as other diseases caused by imbalances affecting metabolism. In this study, mice on a high-fat diet showed a highly significant increase in cancer gene expression, which was suppressed by BAMLET treatment. These results further demonstrate the strong metabolic impact of alpha-1-oleic acid (ALA), a peptide-lipid complex comprising the N-terminal peptide of human alpha-lactalbumin and oleic acid, in cancer patients treated with ALA. Gene expression analysis in tumors of NMIBC patients treated with α1-oleic acid showed significant suppression of cancer-related and metabolism-related genes, similar to the response of mice on a high-fat diet to BAMLET. In particular, key molecules of glucose and lipid metabolism, including ADIPOQ and LEP adipokines, exhibited strong inhibitory effects in patients treated with α1-oleic acid. This study did not include information on patient obesity, but the response to all examined tumors was generalized, suggesting that the role of α1-oleic acid is related to cancer. Therefore, the ability to suppress major metabolic functions observed in this study suggests that BAMLET and α1-oleic acid treatment may help prevent or target obesity and obesity-driven cancer development.

[0166] Materials and Methods

[0167] Animals and high-fat diet intervention

[0168] B6.Cg-Lep ob / J mice and C57BL / 6 mice were purchased from the Jackson Laboratory and were approximately seven weeks old. Mice were housed in cages (four to five animals per cage) at 22°C–25°C with a 12:12 hour light-dark cycle and free access to food and water. Mice were acclimatized for approximately two weeks at the local animal facility at Lund University's Biomedical Center (BMC) to reduce transport stress. Mice were fed a normal diet or HFD (D12492, 60% E fat content; Research Diets, New Brunswick, NJ, USA).

[0169] For the BAMLET treatment regimen, 8-week-old male mice (n=8-10 per group) were given BAMLET in their drinking water daily (10 mg / day in 5 ml PBS for 4 weeks, and 20 mg / day in 5 ml PBS for 3 weeks) for 7 weeks, and were sacrificed at weeks 4 and 7. Mice were sacrificed by overdose anesthesia with isoflurane (Dechra, Cat# 200-129). All animal procedures were approved by the Lund-Malmö / Lund Animal Ethics Committee in Sweden.

[0170] BAMLET

[0171] The BAMLET complex was prepared by incubation in PBS (0.137 M sodium chloride, 0.0027 M potassium chloride, 0.01 M disodium hydrogen phosphate, 0.0018 M potassium dihydrogen phosphate, pH = 7.2–7.4) and vortexing for 30 seconds. The stoichiometric ratio of protein to oleic acid was 1:5, and NMR analysis showed that this resulted in a mixture of fatty acids bound to bovine α-lactalbumin without excess free fatty acids. The complex was immediately used in experiments. Bovine α-lactalbumin and oleic acid were used as controls. The BAMLET complex has been extensively characterized in previous studies, including the stoichiometry of protein and oleic acid.

[0172] Clinical research

[0173] Using the same eligibility criteria, schedule of visits, study treatment management, primary and secondary endpoints, and assessments as in Part I of the study, a single-center trial of α1-oleate in patients with NMIBC (EudraCT No: 2016–004269‐14) was conducted. ClinicalTrials.gov NCT 03560479 The dose discovery section was revised. Patients in the dose discovery section received a five-fold higher dose (8.5 mM, n = 6) of α1-oleate. All patients completed treatment with 8.5 mM α1-oleate. The primary endpoints were safety, changes in tumor size, and tumor cell shedding into the urine. Secondary endpoints were histopathological evaluation of tumor biopsy, quantification of tumor uptake of α1-oleate, apoptosis induction, and the effect of treatment on gene expression.

[0174] RNA sequence analysis

[0175] RNA samples were prepared using the Illumina TruSeq Stranded mRNA Library Prep Kit (20020594). Libraries were reused and sequenced using the NextSeq 500 / 550 high-output kit (v2.5 2x75 cycles), with an average read count of 22 million per sample. Raw sequencing data were demultiplexed using bcl2fastq (version 2.18), and abundance was estimated using RSEM (1.3) with Human Genome Version 37 / Ensemble 75. Sample quality control was visualized using dimensionality reduction (i.e., PCA), MA plots, and intrinsic RNA-seq biases (such as GC bias, transcriptome complexity, and alignment quality). Differential expression analysis was performed using R (version 3.4) and the DESeq2 software package. Fold changes were calculated by comparing tumors in the treatment and placebo groups. Relative expression levels were analyzed; genes with an absolute fold change >2.0 and P < 0.05 were considered differentially expressed. Differentially expressed genes were functionally characterized using Ingenuity Pathway Analysis version 57662101 (IPA, Qiagen) software.

[0176] AlphaFold

[0177] Protein interactions were predicted using AlphaFold 3, a deep learning algorithm designed for high-precision protein structure and interaction determination. The target protein (ADIPOR2, UniProt ID: Q86V24) and α1 in both full-length and truncated forms were used as input. Parameters were set according to the default guidelines. The resulting structures were evaluated for prediction confidence using Interface Prediction Template Modeling (ipTM) scores. The five highest-scoring predicted complexes from each search were visualized and analyzed using PyMOL (PyMOL Molecular Graphics System, version 3.1.3, Schrodinger, LLC.), a molecular visualization system. Structural alignments were performed to assess the consistency (if any) between the predicted structures and experimentally resolved structures in the Protein Data Bank (PDB). The model with the highest ipTM score was selected for further computational refinement and experimental validation (if applicable). Interacting residues were identified by selecting atoms within 4 Å of the target protein and the binding molecule or peptide.

[0178] The sequences ADIPOR1 (UniProt accession number: Q96A54) and ADIPOR2 (Uni-Prot accession number: Q86V24) were obtained from the UniProt database and submitted to ColabFold along with the α1 peptide sequence using the AlphaFold2 multimer (Mirdita M, Schütze K, Moriwaki Y, Heo L, Ovchinnikov S, Steinegger M. ColabFold:making protein folding accessible to all. Nature Methods. 2022;19(6):679-82 and Bryant P, Pozzati G, Zhu W, Shenoy A, Kundrotas P, Elofsson A. Predicting the structure of large protein complexes using AlphaFold and Monte Carlo treesearch. Nature Communications. 2022;13(1):6028). The obtained complexes were evaluated using the top 5 scoring models in PyMOL (open source) for visual assessment. During this process, complexes exhibiting significant conflicts (such as structural interpenetration) are filtered out.

[0179] Ethical Statement

[0180] clinical

[0181] This study was approved by the Czech National Medicines Regulatory Agency (SUKL), under approval number 273799 / 17-I, and by the Ethics Committee of Motor University Hospital, under approval number EK-786 / 17. All patients were enrolled after giving informed consent. The dose-escalation portion was a single-center, placebo-controlled, double-blind, randomized phase I / II interventional clinical trial of NMIBC (EudraCT number: 2016-004269-14 and...). ClinicalTrials.gov NCT03560479 This is an extension of the existing data. Demographic data, prevalence and health parameters, as well as tumor characteristics, are recorded by research physicians in electronic case report forms (eCRFs) and closely monitored by external monitors.

[0182] Animal experiments were approved by the Malmö / Lund Animal Experimentation Ethics Committee of the Lund Regional Court in Sweden (#01302-20). Animal care and experimental protocols followed institutional, national, and EU guidelines and were governed by European Parliament and Council directives (2016 / 63, EU), the Swedish Animal Welfare Act (Djurskyddslagen 1988:534), the Swedish Welfare Ordinance (Djurskyddsförordningen 1988:539), and the guidelines of the Committee on the Management and Use of Laboratory Animals (IACUC). Results were in accordance with the ARRIVE guidelines (…). https: / / arriveguidelines.org )Report.

[0183] Statistical analysis

[0184] Compared with placebo, the FDR or P value for defining differentially expressed IPA genes in tumors treated with 8.5 mM α1H was set at <0.05.

[0185] The following statistical analysis was performed using GraphPad Prism, where statistical significance was defined as P < 0.05.

[0186] The normality of the data was tested using the Shapiro-Wilk normality test, which showed that the data were not normally distributed. A nonparametric Mann-Whitney t-test was used to compare the median urinary adiponectin level between the 8.5 mM AlphaH treatment group and the placebo treatment group.

[0187] Repeated measures two-way ANOVA combined with Šídák's multiple comparison test was used to examine the effects of time (number of visits) and treatment (8.5 mM AlphaH vs. placebo) on urinary adiponectin levels. Linear regression analysis was used to determine the correlation between the number of exfoliated cells and adiponectin levels after perfusion of placebo or α1H complex, R0. 2 = 0.307, P = 0.001. The mean percentage of cell death is expressed as standard deviation in error bars. The normality of the data was tested again using the Shapiro-Wilk normality test, and the results showed that the data were normally distributed. Therefore, one-way ANOVA combined with the Tukey multiple comparison test was used to compare the parametric data.

Claims

1. A polypeptide having a naturally occurring α-lactalbumin sequence, or a functional variant thereof; or a peptide comprising up to 50 amino acids of an α-helical domain of said polypeptide, for the treatment or prevention of metabolic-related conditions.

2. A complex comprising a polypeptide having a naturally occurring α-lactalbumin sequence or a functional variant thereof; or a peptide comprising up to 50 amino acids including an α-helical domain of said polypeptide; and a fatty acid or lipid or a salt thereof, for the treatment or prevention of metabolic-related conditions.

3. The polypeptide of claim 1 or the complex of claim 2, wherein the metabolic-related condition is insulin resistance, type 2 diabetes, metabolic syndrome, non-alcoholic fatty liver disease, cirrhosis, or hypertension.

4. The polypeptide of claim 1 or the complex of claim 2, wherein the metabolic-related condition is regulated or controlled by the leptin pathway and / or the adiponectin pathway.

5. The polypeptide or complex for the use of claim 4, wherein the metabolic-related disorders regulated or controlled by the leptin pathway are hyperleptinemia, leptin resistance, leptin deficiency or decreased sensitivity, leptin-related weight gain and / or obesity, increased or decreased hunger stimulation, increased or decreased leptin-related metabolism, leptin-related metabolic syndrome, leptin-related non-alcoholic fatty liver disease, Rabson-Mendenhall syndrome, depression and food addiction, hypoleptinemia, hyperinsulinemia, fatty liver, dyslipidemia, hypogonadotropic hypogonadism, congenital or acquired systemic lipodystrophy (GL), type 1 diabetes, or hypothalamic amenorrhea.

6. The polypeptide or complex for the use of claim 4, wherein the metabolic-related condition regulated or controlled by the leptin pathway is one or more of obesity, overeating, transient hyperglycemia, glucose intolerance, and elevated plasma insulin.

7. The polypeptide or complex for the use of claim 4, wherein the metabolic-related conditions regulated or controlled by the adiponectin pathway are obesity, type 2 diabetes, metabolic syndrome, atherosclerosis, and cardiovascular diseases including myocardial infarction.

8. A polypeptide having a naturally occurring α-lactalbumin sequence or a functional variant thereof; or a peptide comprising up to 50 amino acids of the α-helical domain of said polypeptide for regulating leptin receptors and / or adiponectin receptors.

9. A complex comprising a polypeptide having a naturally occurring α-lactalbumin sequence or a functional variant thereof; or a peptide comprising an α-helical domain of said polypeptide of up to 50 amino acids; and a fatty acid or lipid or a salt thereof for regulating leptin receptors and / or adiponectin receptors.

10. A method of treating a metabolic-related condition, comprising administering to a subject in need (1) a polypeptide having a naturally occurring α-lactalbumin sequence or a functional variant thereof; or a peptide comprising up to 50 amino acids of an α-helical domain of said polypeptide; or (2) a complex comprising a polypeptide having a naturally occurring α-lactalbumin sequence or a functional variant thereof; or a peptide comprising up to 50 amino acids of an α-helical domain of said polypeptide; and a fatty acid or lipid or a salt thereof.

11. The method of claim 10, wherein the metabolic-related condition is as defined in any one of claims 3-7.

12. A method of modulating leptin receptors and / or adiponectin receptors, comprising administering to a subject in need (1) a polypeptide having a naturally occurring α-lactalbumin sequence or a functional variant thereof; or a peptide comprising up to 50 amino acids of an α-helical domain of said polypeptide, or (2) a complex comprising a polypeptide having a naturally occurring α-lactalbumin sequence or a functional variant thereof; or a peptide comprising up to 50 amino acids of an α-helical domain of said polypeptide; and a fatty acid or lipid or a salt thereof.

13. A polypeptide for use according to any one of claims 1 and 3-8, a complex for use according to any one of claims 2, 3-7 or 9, or the method according to any one of claims 10-12, wherein the polypeptide has the sequence of naturally occurring α-lactalbumin, human or bovine α-lactalbumin or bovine α-lactalbumin.

14. A polypeptide for use according to any one of claims 1 and 3-8, a complex for use according to any one of claims 2, 3-7 or 9, or the method according to any one of claims 10-12, wherein the α-helix domain is KQFTKXELSQLLKDIDGYGGIALPELI XTMFHTSGYDTQ (SEQ ID NO: 3) or LDDITDDIMXAKKILDIKGIDYWLAHKALXTEKLEQWL XEKL (SEQ ID NO: 4), wherein X is an amino acid residue other than cysteine.

15. The complex for use according to any one of claims 2, 3-7 or 9, or the method according to any one of claims 10-12, wherein the complex comprises a peptide of about 45, 42 or 40 amino acids or a peptide of 39 amino acids.

16. A polypeptide for use according to any one of claims 1 and 3-8, a complex for use according to any one of claims 2, 3-7 or 9, or the method according to any one of claims 10-12, wherein the functional variant (A) comprises or is substantially composed of an amino acid sequence having at least 70% identity with the α-lactalbumin sequence, (b) comprises a sequence lacking disulfide bonds, or (c) comprises a sequence in which cysteine ​​residues in natural α-lactalbumin are replaced with other amino acid residues.

17. The complex for use according to any one of claims 2, 3-7 or 9, or the method according to any one of claims 10-12, wherein the fatty acid or its salt is oleic acid or oleate.

18. A polypeptide for use according to any one of claims 1 and 3-8, a complex for use according to any one of claims 2, 3-7 or 9, or the method according to any one of claims 10-12, wherein the polypeptide is a recombinant protein having the sequence SEQ ID NO: 1 or SEQ ID NO: 2.

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