Therapy

Abstract

The present invention relates to a complex comprising a polypeptide having a sequence of a naturally occurring alpha-lactalbumin, or a functional variant thereof; or a peptide of up to 50 amino acids comprising an alpha-helical domain of said polypeptide; and a fatty acid or lipid or salt thereof for use in therapy for the treatment of metabolic-related disorders, particularly metabolic-related disorders that are modulated by, or otherwise affected by the leptin and / or adiponectin pathway.

Description

[0001]THERAPY TECHNICAL FIELD The present invention relates to a complex comprising a polypeptide having a sequence of a naturally occurring alpha-lactalbumin, or a functional variant thereof; or a peptide of up to 50 amino acids comprising an alpha-helical domain of said polypeptide; and a fatty acid or lipid or salt thereof for use in therapy for the treatment of metabolic-related disorders, particularly metabolic-related disorders that are modulated by, or otherwise affected by the leptin and / or adiponectin pathway. BACKGROUND The main function of adipose tissue is to store energy, in the form of fat, and it comprises 20-25% of the total body weight in healthy individuals. Adipose tissue cells (adipocytes) secrete bioactive molecules called adipocytokines (also called adipokines). Adiponectin, the most abundantly secreted adipokine, is a well-known homeostatic factor for regulating 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 like appetite and energy expenditure. It induces feelings of satiety, reducing food intake, and inhibits glucose uptake. Leptin is also an upstream regulator of adiponectin and serum levels of the two proteins exhibit an inversely proportional relationship. 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 adiposity-related dysfunctions. Leptin is involved in regulation of energy homeostasis and hunger by providing a sensation of satiety (fullness). Leptin does this by binding to the LEPR-gene encoded long isoform leptin receptor-B (LEPR-B), which activates downstream Janus kinase 2 (JAK2) - Signal Transducer and Activator of Transcription 3 (STAT3) signaling pathway in a subset of the hypothalamic neurons in the brain (Park HK, Ahima RS. Leptin signaling. F1000Prime Rep. 2014;6:73.). This drives production of anorexigenic peptides which lead to suppressed appetite and increased energy expenditure. In obesity, despite higher levels of circulating leptin as a result of increased adiposity, the feeling of satiety is inadequate due to dysregulation of the leptin signaling. As a cytokine, leptin promotes inflammatory response and upregulates proinflammatory cytokines such as interleukin 6 (IL-6) and Tumor Necrosis Factor alpha (TNF-⍺). Obesity predisposes to pro-inflammatory states by elevated IL-6 and TNF-⍺ levels, which in turn increase susceptibility to type 2 diabetes and cardiovascular diseases. HAMLET (human alpha-lactalbumin made lethal to tumor cells) is the first member of a family of tumoricidal unfolded protein-lipid complexes, consisting of partially unfolded α- lactalbumin and oleic acid. Initially isolated in the form of a fraction obtained by passing a casein containing fraction of human milk down an ion exchange column under high salt conditions (WO96 / 004929), it was found to be biologically active and in particular had an antibacterial activity. Subsequently, other methods for preparing active complexes have been derived including methods in which α-lactalbumin from various sources and oleic acid are heated together in solution. In addition, HAMLET and related complexes such as BAMLET, derived from bovine alpha-lactalbumin, have been found to kill transformed cells such as tumor cells or papilloma cells, as well as having antiviral activity. The inventors have further identified other effects of complexes and peptides related to HAMLET and BAMLET, particularly metabolic effects, especially those controlled or otherwise affected by the leptin and / or adiponectin pathways. The inventors have identified that the complexes bind to both leptin receptors and adiponectin receptors. As a result, the complexes can be used to modulate the activity of those receptors, and to treat conditions affected or controlled by the pathways associated with those receptors. SUMMARY OF THE INVENTION The invention provides a polypeptide having a sequence of a naturally occurring alpha- lactalbumin, or a functional variant thereof; or a peptide of up to 50 amino acids comprising an alpha-helical domain of said polypeptide, for use the treatment or prevention of metabolic-related conditions, particularly those modulated or controlled by the leptin pathway. The invention also provides a complex comprising a polypeptide having a sequence of a naturally occurring alpha-lactalbumin, or a functional variant thereof; or a peptide of up to 50 amino acids comprising an alpha-helical domain of said polypeptide; and a fatty acid or lipid or salt thereof, for use in the treatment or prevention of metabolic-related conditions, particularly those modulated or controlled by the leptin pathway. Optionally, the complex or the polypeptide is for use in modulating the leptin receptor. For example, the complex or the polypeptide may be for use in inhibiting the leptin receptor. Alternatively, it may be for use in activating the leptin receptor. The leptin receptor is well-known in the art. Leptin receptor activation is reliant on dimerization. Leptin binds to an Ig-like domain on a first leptin receptor and to the CHR2 domain on a second leptin receptor to induce dimerization. Without being bound by theory, the inventors understand that the complex or the polypeptide of the invention can bind to the Ig-like domain and / or to the CHR2 domain, preventing or reducing dimerization and activation, or preventing activation of the second leptin receptor. Alternatively, the complex or the polypeptide of the invention could be used to bring about dimerization or activation of the receptors. The invention also provides a method of modulating the leptin receptor comprising administering a polypeptide having a sequence of a naturally occurring alpha-lactalbumin, or a functional variant thereof; or a peptide of up to 50 amino acids comprising an alpha- helical domain of said polypeptide, to a subject in need thereof. The invention also provides a method of treating metabolic-related conditions comprising administering a polypeptide having a sequence of a naturally occurring alpha-lactalbumin, or a functional variant thereof; or a peptide of up to 50 amino acids comprising an alpha- helical domain of said polypeptide, to a subject in need thereof. The invention also provides a method of modulating the leptin receptor comprising administering a complex comprising a polypeptide having a sequence of a naturally occurring alpha-lactalbumin, or a functional variant thereof; or a peptide of up to 50 amino acids comprising an alpha-helical domain of said polypeptide; and a fatty acid or lipid or salt thereof, to a subject in need thereof. The invention also provides a method of treating metabolic-related conditions, particularly those modulated by the leptin receptor or leptin pathway, comprising administering a complex comprising a polypeptide having a sequence of a naturally occurring alpha- lactalbumin, or a functional variant thereof; or a peptide of up to 50 amino acids comprising an alpha-helical domain of said polypeptide; and a fatty acid or lipid or salt thereof, to a subject in need thereof. Metabolic-related conditions include conditions in which the metabolism is affected, such as insulin resistance, type II diabetes, metabolic syndrome, non-alcoholic fatty acid liver disease, cirrhosis, high blood pressure. The polypeptide or the complex may be used to modulate insulin tolerance or sensitivity, lipid metabolism and / or glucose metabolism and is therefore useful in the treatment of conditions arising from challenges with such processes. Conditions modulated by the leptin receptor include, but are not limited to hyperleptinemia, leptin resistance, lack of or reduced sensitivity to leptin, leptin-related weight gain and / or obesity, increased or reduced hunger stimulation, leptin-related increased or decreased metabolism, leptin-related metabolic syndrome, leptin-related non-alcoholic fatty liver disease, Rabson–Mendenhall syndrome, depression and food addiction, hypoleptinemia, hyperinsulinemia, fatty liver disease, dyslipidemia, hypogonadotropic hypogonadism, congenital or acquired generalized lipodystrophy (GL), type 1 diabetes, and hypothalamic amenorrhea. In one embodiment, the metabolic condition is one or more of obesity, hyperphagia, transient hyperglycemia, glucose intolerance, and elevated plasma insulin. The polypeptide or the complex may be for use in subjects with hyperleptinemia. For example, the compositions may be for use in female subjects with leptin levels above about 14, 14.5, 15, or 15.2 nanograms leptin per millilitre (ng / mL) or male subjects with leptin levels above about 11, 11.5, 12, or 12.5 ng / mL leptin. The polypeptide or the complex may be for use in subjects with hypoleptinemia. For example, the compositions may be for use in subjects with leptin levels below about 0.5 nanograms per millilitre (ng / mL). The polypeptide or the complex may be for use in subjects with increased leptin expression or increased leptin receptor expression, when compared to the expected expression levels, such as an increase of about 1%, 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60% or more. The polypeptide or the complex may be for use in subjects with decreased leptin expression or decreased leptin receptor expression, when compared to the expected expression levels, such as a decrease of about 1%, 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60% or more. The invention also provides a complex comprising a polypeptide having a sequence of a naturally occurring alpha-lactalbumin, or a functional variant thereof; or a peptide of up to 50 amino acids comprising an alpha-helical domain of said polypeptide; and a fatty acid or lipid or salt thereof, for use in the treatment or prevention of metabolic-related conditions, particularly those modulated or controlled by the adiponectin pathway. Optionally, the complex is for use in modulating the adiponectin receptor, either directly, for example by binding to the receptor, or indirectly. For example, the complex may be for use in inhibiting or reducing the activity of the adiponectin receptor. Alternatively, it may be for use in activating or increasing the activity of the adiponectin receptor. The adiponectin receptor is well-known in the art. Without being bound by theory, the inventors understand that the complexes of the invention can bind to the adiponectin receptor, inhibiting it, or activating it. The invention provides a polypeptide having a sequence of a naturally occurring alpha- lactalbumin, or a functional variant thereof; or a peptide of up to 50 amino acids comprising an alpha-helical domain of said polypeptide, for use the treatment or prevention of metabolic-related conditions, particularly those modulated or controlled by the adiponectin pathway. The invention also provides a complex comprising a polypeptide having a sequence of a naturally occurring alpha-lactalbumin, or a functional variant thereof; or a peptide of up to 50 amino acids comprising an alpha-helical domain of said polypeptide; and a fatty acid or lipid or salt thereof, for use in the treatment or prevention of metabolic-related conditions, particularly those modulated or controlled by the adiponectin pathway. The polypeptide or complex may be for use in subjects with increased adiponectin levels. For example, the polypeptide or complex may be for use in female subjects with adiponectin levels above about 35, 36 or 37 micrograms per millilitre (ug / mL) in subjects with a BMI below 25; above about 28, 29 or 30ug / mL in subjects with a BMI between 25 and 30; and above about 18, 19 or 20 in subjects with a BMI above 30. The polypeptide or complex may be for use in subjects with decreased adiponectin levels. For example, the polypeptide or complex may be for use in male subjects with adiponectin levels above about 35, 36 or 37 ug / mL in subjects with a BMI below 25; above about 28, 29 or 30 ug / mL in subjects with a BMI between 25 and 30; and above about 20, 21 or 22 ug / mL in subjects with a BMI above 30. The polypeptide or complex may be for use in female subjects with adiponectin levels below about 7, 6 or 5ug / mL in subjects with a BMI below 25; below about 7, 6 or 5 ug / mL in subjects with a BMI between 25 and 30; and below about 4, 3, or 2 ug / mL in subjects with a BMI above 30. The polypeptide or complex may be for use in male subjects with adiponectin levels below about 7, 6 or 5 ug / mL in subjects with a BMI below 25; below about 6, 5 or 4 ug / mL in subjects with a BMI between 25 and 30; and below about 6, 5 or 4 ug / mL in subjects with a BMI above 30. The polypeptide or complex may be for use in subjects with increased adiponectin expression or increased adiponectin receptor expression, when compared to the expected expression levels, such as an increase of about 1%, 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60% or more. The polypeptide or complex may be for use in subjects with decreased adiponectin expression or decreased adiponectin receptor expression, when compared to the expected expression levels, such as a decrease of about 1%, 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60% or more. Conditions modulated by the adiponectin receptor include, obesity, type 2 diabetes, metabolic syndrome, atherosclerosis, and cardiovascular disorders including myocardial infarctions. The invention further provides a method of modulating the leptin receptor and adiponectin receptor simultaneously or sequentially, comprising administering a complex as previously described. Without being bound by theory, the inventors understand that the fatty acid or salt of the complex can bind to the adiponectin receptor. When doing so, it is likely to increase the fluidity of the membrane in which the receptor is found. The complex can then recruit the leptin receptor, binding to it via the polypeptide. The polypeptide and the complex In one embodiment, the polypeptide has a sequence of a naturally occurring alpha- lactalbumin, preferably a human or bovine alpha-lactalbumin, more preferably a bovine alpha-lactalbumin. In one embodiment, the alpha-helical domain is the Alpha 1 (residues 1-39) or Alpha 2 (residues 81-123) domain of human alpha-lactalbumin, being of of SEQ ID NO 3 or SEQ ID NO 4; KQFTK XELSQLLKDIDGYGGIALPELI XTMFHTSGYDTQ (SEQ ID NO 3) LDDDITDDIM XAKKILDIKGIDYWLAHKALXTEKLEQWL XEKL (SEQ ID NO 4) where X is an amino acid residue other than cysteine. In an embodiment, the complex comprises a peptide of about or less than 45, 42, or 40 amino acids, in particular 39 amino acids, preferably corresponding to the Alpha 1 domain of human alpha-lactalbumin. In an embodiment, the polypeptide has less than 45, 42, or 40 amino acids, in particular 39 amino acids, preferably corresponding to the Alpha 1 domain of human alpha-lactalbumin. In one embodiment, the functional variant consists of a sequence lacking disulfide bonds. In one embodiment, the functional variant consists of a sequence in which cysteine residues in the native alpha-lactalbumin are changed to other amino acid residues, preferably alanine residues. In one embodiment, the fatty acid or lipid or salt thereof is a fatty acid or salt thereof. In one embodiment, the fatty acid or salt thereof is oleic acid or an oleate salt. In one embodiment, the polypeptide has the sequence of bovine alpha-lactalbumin and the fatty acid or salt thereof is oleic acid or an oleate salt. The polypeptide present in the complex may have the sequence of an α-lactalbumin or a variant thereof as described above. The complex may be referred to as a biologically active complex. As used herein, the term "biologically active" means that the complex has a biological activity, which is different from, or stronger than the individual components. In particular, the complex is able to induce cell death in particular selectively in tumor cells and / or has a bactericidal or antiviral effect not seen with the native protein including for example monomeric α-lactalbumin forms, although other therapeutic effects may be available. The expression "variant" refers to proteins or polypeptides having a similar biological function but in which the amino acid sequence differs from the base sequence from which it is derived in that one or more amino acids within the sequence are substituted for other amino acids. Amino acid substitutions may be regarded as "conservative" where an amino acid is replaced with a different amino acid with broadly similar properties. Non- conservative substitutions are where amino acids are replaced with amino acids of a different type. By "conservative substitution" is meant the substitution of an amino acid by another amino acid of the same class, in which the classes are defined as follows: Class Amino acid examples Nonpolar: A, V, L, I, P, M, F, W Uncharged polar: G, S, T, C, Y, N, Q Acidic: D, E Basic: K, R, H. As is well known to those skilled in the art, altering the primary structure of a peptide by a conservative substitution may not significantly alter the activity of that peptide because the side-chain of the amino acid which is inserted into the sequence may be able to form similar bonds and contacts as the side chain of the amino acid which has been substituted out. This is so even when the substitution is in a region which is critical in determining the peptide's conformation. Non-conservative substitutions are possible provided that these do not interrupt the function of the DNA binding domain polypeptides. Broadly speaking, fewer non-conservative substitutions will be possible without altering the biological activity of the polypeptides. Determination of the effect of any substitution (and, indeed, of any amino acid deletion or insertion) is wholly within the routine capabilities of the skilled person, who can readily determine whether a variant polypeptide retains the fundamental properties and activity of the basic protein. For example, when determining whether a variant of the polypeptide falls within the scope of the invention, the skilled person will determine whether complexes comprising the variant retain biological activity (e.g., tumor cell death) of complexes formed with unfolded forms of the native protein and the polypeptide has at least 60%, preferably at least 70%, more preferably at least 80%, yet more preferably 90%, 95%, 96%, 97%, 98%, 99% or 100% of the native protein. Variants of the polypeptide may comprise or consist essentially of an amino acid sequence with at least 70% identity, for example at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 96%, 97%, 98% or 99% identity to a native protein sequence such as an alpha- lactalbumin or lysozyme sequence. The level of sequence identity is suitably determined using the BLASTP computer program with the native protein sequences as the base sequence. This means that native protein sequences form the sequence against which the percentage identity is determined. The BLAST software is publicly available at http: / / blast.ncbi.nlm.nih.gov / Blast.cgi (accessible on 12 March 2009). In a particular embodiment, the polypeptide is an α-lactalbumin such as human, bovine or ovine α-lactalbumin. Whilst variants of these as described above may be useful in the invention, for nutraceutical use in particular, it may be preferable to utilize the native proteins in the products. A particular embodiment used human α-lactalbumin. In another embodiment, the α-lactalbumin is bovine α-lactalbumin. The sequence of a wide range of α- lactalbumins is known in the literature, for example as shown in Watanabe et al., J. Vet Med Sci, (2000) 62(11); 1217-1219. In another embodiment, the polypeptide comprises a recombinant protein having the sequence of α-lactalbumin or a fragment thereof, but which lacks intra-molecular disulfide bonds or cross-links. By ensuring that the recombinant protein lacks intra-molecular disulfide crosslinks, the molecule will be three-dimensionally non-native and completely inactive in terms of its original endogenous biological activity. This is achieved by changing cysteine residues in the native α-lactalbumin to other residues, in particular alanine residues. Preferably all cysteine residues will be changed to other residues, such as alanine residues. In particular the recombinant protein is based upon the sequence of human α- lactalbumin but α-lactalbumin from other sources, including bovine or ovine α-lactalbumin may be used to derive the recombinant protein. In a particular embodiment, the polypeptide is a recombinant protein having the sequence of native mature α-lactalbumin but which has all of the cysteines found at positions 6, 28, 61, 73, 77, 91, 111 and 120 in the full length sequence of mature human α-lactalbumin mutated to other amino acids, such as alanine, which do not give rise to disulphide bridges. Thus, a particular of a protein that may be utilised in accordance with the invention comprises a protein of SEQ ID NO 1. where the bold type indicates positions of mutations of cysteines in native human α- lactalbumin. As reported in WO2010079362, additional amino acid residues, for example up to 20 amino acids, may be attached at N and / or C terminal of the protein, if convenient, for example for expression purposes. Thus, in particular, a recombinant protein as shown in SEQ ID NO. 1 but with an additional methionine at the N-terminus (SEQ ID NO 2 shown below) has been used in the complex of the invention. The polypeptide used in the complex is suitably in pure form, and is suitably prepared using conventional methods of peptide synthesis or by recombinant expression. In particular, DNA encoding the required recombinant α-lactalbumin can be inserted into suitable expression vectors such as plasmids, which can then be employed to transform host cells, for example, prokaryotic cells such as E. coli or eukaryotic cells such as particular insect cells using conventional methods. Suitable fatty acids or lipids include those known to provide biologically active complexes. These include fatty acids, for example as described in WO2008058547. Where salts are used, these are suitably water soluble salt. Particular examples of suitable salts may include alkali or alkaline earth metal salts. In a particular embodiment, the salt is an alkali metal salt such as a sodium- or potassium salt. Where used in pharmaceuticals, the salts will be pharmaceutically acceptable. Particular examples of fatty acids or lipids used in the present invention are those having from 4-30, for example from 6 to 28, such as from 8 to 26 carbon atoms. In particular embodiments, the fatty acid or lipid has from 10 to 24, such as from 12 to 22, for example from 14 to 20 carbon atoms. In particular, the fatty acid or lipid will have 16, 17, 18 or 20 carbon atoms. The fatty acids may be saturated or unsaturated. In particular however, the complexes of the invention utilize fatty acids or salts of fatty acids having 18 carbon atoms. In one embodiment, the complexes of the invention utilize fatty acids or salts of fatty acids having 18 carbon atoms and wherein the fatty acid chain is unsaturated. In one embodiment, the fatty acid or salt of the fatty acid is a C18:1 fatty acid or salt thereof. A specific example is a C18:1 fatty acid or salt thereof of formula CH 3(CH 2) 7CH=CH(CH 2) 7COOH or CH 3(CH 2) 7CH=CH(CH 2) 7COO-. In one embodiment, the fatty acid or salt thereof is oleic acid or oleate salt. The complex may be prepared using methods similar to those described for example in WO99 / 26979, WO2008 / 138348, WO2010 / 131237, WO2014 / 023976, WO2018 / 210759, and WO2022 / 073982 the content of which is incorporated herein by reference. Not only has it been found that complexes can be prepared by contacting unfolded α-lactalbumin or derivatives thereof with co-factors in particular oleic acid or salts thereof under ion exchange conditions such as those found on an ion exchange column, but also incubation of solutions of α-lactalbumin or derivatives thereof with a co-factor at elevated temperatures, for example of from 50-80°C, for example from 50-70°C and in particular between 55-60°C will result in the production of suitable complexes for use in the invention. These methods however have generally focused on attempting to recreate the conditions in which the protein becomes unfolded and complexed with oleic ions. Such work has focused on using pure proteins including recombinant variant versions of the base proteins to facilitate the production of active complexes. Such starting materials however can also increase the cost of production. It is known that complexes obtained using α-lactalbumin from sources other than human milk, and in particular, BAMLET, obtained using bovine α-lactalbumin shows a qualitatively similar effects on cells and in particular on tumor cells as HAMLET (see for instance, Rammer et al. (2010) Mol. Cancer Ther. 9(1) 24-32). Therefore, effects demonstrated hereinafter using BAMLET would be similarly observed if HAMLET or compositions based upon HAMLET are used instead of BAMLET. Dosage The amount of complex administered to an individual will depend upon a variety of factors including the nature of the composition as well as the risk factor. However, as a general rule, when administered perorally, from 1mg to 20g / dose of the biologically active complex is used for each administration, which is suitably administered daily. The daily dose may be, for example, at least or about 1mg, 2mg, 5mg, 10mg, 15mg, 20mg, 25mg, 50mg, 75mg, 100mg, 200mg, 300mg, 400mg, 500mg, 750mg, 1g, 2g, 3g, 4g, 5g, 7.5g, 10g, 12.5g, 15g, or 17.5g. Alternatively, or additionally, the daily dose may be less than 25g, 22.5g, 20g, 17.5g, 15g, 10g, 7.5g, 5g, 4g, 3g, 2g, 1g, 750mg, 500mg, 400mg, 300mg, 200mg, 100mg, 75mg, 50mg, 25mg, 20mg, 15mg, 10mg or 5mg. Alternatively, the complex may be for administration in a dosage of 0.1g to 1g per kg of bodyweight, daily. It may be for administration in a dosage of at least or about 0.1g, 0.2g, 0.3g, 0.4g, 0.5g, 0.6g, 0.g, 0.8g, 0.9g or 1g per kg of bodyweight, daily. Alternatively, or additionally, it may be for administration in a dosage of less than 1.5g, 1g, 0.9g, 0.8g, 0.7g, 0.6g 0.5g, 0.4g, 0.3g, 0.2g, 0.1 per kg of bodyweight, daily. Food and beverage compositions The complex or pharmaceutical composition may be in the form of a beverage, particularly drinking water, or a foodstuff, such as baby-food, or as an additive or component for a beverage or foodstuff, such as a powder for mixing into a drink, for example, in the manner of a protein shake. Such food and beverage compositions may be produced using standard techniques. Similarly, the complex or pharmaceutical composition may be in the form of a composition for providing parental or, preferably, intravenous nutrition. The invention also provides a foodstuff, beverage, food additive or other nutritional composition comprising the complex as defined, particularly for use in treating cancer or a metabolic-related condition, as described in earlier aspects of the invention. Examples of such compositions include water-based or milk-based drinks, particularly drinking water; baby-food; nutritional compositions for parental or intravenous administration; food additives, for example powders for mixing into drinks or food; nutritional capsules, gels or tablets. General Throughout the description and claims of this specification, the words “comprise” and “contain” and variations of the words, for example “comprising” and “comprises”, mean “including but not limited to”, and do not exclude other components, integers or steps. Moreover, the singular encompasses the plural unless the context otherwise requires: in particular, where the indefinite article is used, the specification is to be understood as contemplating plurality as well as singularity, unless the context requires otherwise. Preferred features of each aspect of the invention may be as described in connection with any of the other aspects. Within the scope of this application, it is expressly intended that the various aspects, embodiments, examples and alternatives set out in the preceding paragraphs, in the claims and / or in the following description and drawings, and in particular the individual features thereof, may be taken independently or in any combination. That is, all embodiments and / or features of any embodiment can be combined in any way and / or combination, unless such features are incompatible. BRIEF DESCRIPTION OF THE DRAWINGS One or more embodiments of the invention will now be described, by way of example only, with reference to the accompanying drawings, in which: Figure 1 shows the leptin receptor (LepR) isoforms’ structure. Figure 2 shows the leptin receptor dimer structure and mechanism for leptin binding. Figure 3 shows the functional consequence of LepR and Alpha1 interaction. Figure 4 shows a prediction of interactions between Alpha1 and the Ig-like domain+CHR2 of LepR. Figure 5 shows a prediction of interactions between Alpha1 and the full extracellular region of LepR. Figure 6A shows the adiponectin receptor 2 structure including the binding pocket for oleic acid. Figure 6B the adiponectin receptor 2 structure showing intracellular cavity containing oleic acid. Figure 7A shows Alpha1 peptide sequence and AlphaFold predicted structure. Figure 7B shows in silico prediction of an Alpha1 binding pocket to in adiponectin receptors. Figure 8 shows the treatment schedule for Lepoband wildtype mice treated with BAMLET. Figure 9 shows the effect of BAMLET on weight gain and blood glucose levels in Lepoband wildtype mice. Figure 10 shows protective effect of peroral BAMLET treatment against a high fat diet (HFD) on systemic metabolism and adipocyte expansion in C57 / BL6 mice. a, Schematic representation of the experiment. C57BL / 6 mice, 8 weeks old were subjected to time- resolved analysis for 4 and 7 weeks of diet intervention, each time point including the following three groups: Chow: n = 8, HFD: n = 8, HFD+BAMLET: n = 9. The mice received 10 mg of BAMLET in 200 μl PBS (BAMLET) or 200 μl of PBS alone (placebo) in drinking water for four weeks and then received 20 mg of BAMLET in 200 μl PBS (BAMLET) or 200 μl of PBS alone (placebo) in drinking water for 3 three weeks. The mice were sacrificed at four weeks or seven weeks. b, Percent reduction change of body weight between 3 groups. c, Percent reduction change of Blood glucose between 3 groups. d, Different change of body weight and blood glucose between HFD+ BAMLET compared to placebo (HFD) group. e, Organ weight of: Adipose Epididymal tissue (eWAT), Adipose Inguinal tissue (IngWAT), Retro-peritoneal adipose tissue (ReWAT) and liver. Data are presented as the means ± S.E.M.s from two experiments and were analyzed by two-way ANOVA with Šídák's multiple comparisons test, the mice drinking BAMLET compared to the placebo group. For each organ, the first bar is Chow, the second bar is HFD + PBS and the third bar is HFD + BAMLET. f, % change in tissue weight between C57 / BL6 HFD+ BAMLET compared to placebo (HFD) group. Figure 11 shows regulation of metabolism genes and metabolic function. Figure 12 shows Alpha1 peptide interacts with adiponectin receptor 2 (AdipoR2) (residues 131-386) and Leptin Receptor isoform B (LEPR-B). a, Schematic illlustrating the reaction between the 39 amino acid long Alpha1 peptide and oleic acid molecules to form the Alpha1-oleate complex. (Brisuda, et al.) b, Previously reported structural studies of AdipoR2 (residues 100-386) by Vasiliauskaité-Brooks et al. show direct evidence of an oleic acid binding to the central cavity of AdipoR2, confirmed by crystallography. c, AlphaFold 3 prediction showing strong interaction between AdipoR2 and one oleic acid molecule (OLA), at this known oleic acid binding cavity. d, The Alpha1 peptide was predicted to weakly interact with the extracellular region of the truncated AdipoR2 protein. e, The Alpha1-oleate complex, comprising of an additional five OLA molecules, was predicted to interact with the truncated AdipoR2 with higher confidence. f, Magnified interaction of two oleic acid molecules of the alpha1-oleate complex situated inside the transmembrane cavity and at the opening between TM5 and TM6 of the tunnel-like cavity of AdipoR2 g-h, The surface- exposed extracellular part of LEPR-B was predicted to interact with the alpha1 peptide through its immunoglobulin like (Ig-like) domain. Alpha1 was predicted to interact with the LEPR-B segment containing Ig-like domain and CHR2 domain, at the Ig-like domain (g). This prediction was strengthened when the entire extracellular region of LEPR-B was used (h). i-j, Overlaying this prediction with a cryo-EM structure (PDB 8DH8) of a Leptin-bound LEPR complex suggested two possible effects. Alpha1 peptide can bind to the Ig-like domain of the first LEPR (LEPR I) (i) or the second LEPR (LEPR II) (j). Figure 13 shows the experiment using B6.Cg-Lepob / J mice. For each organ in e, the first bar is Chow + PBS, and the second bar is Chow + BAMLET. Figure 14 shows drinking BAMLET prevents activation of the top upregulated genes and metabolism related functions induced by a HFD in the intestines of B6.Cg-Lepob / J mice. a. Table listing the top 10 activated (first in the list) and 10 inhibited (second in the list) genes in response to HFD or HFD+BAMLET. Genes strongly activated by HFD were not longer regulated in mice eating HFD and drinking BAMLET. Changes in gene expression are measured as fold change (FC) compared to the Chow group (significance cutoff FC>2 and P<0.05). b. Histogram of metabolism related functions strongly regulated in response to HFD and compared to HFD+BAMLET. BAMLET drinking strongly reduced the regulation of diabetes related functions and abrogated the regulation of lipid metabolism and insulin resistance, compared to mice eating normal diet (Chow group) (green: HFD+BAMLET, red: HFD). DETAILED DESCRIPTION Example 1 Preparation of alpha1-oleate Alpha1 was synthesized using Fmoc solid phase chemistry (Mimotopes). The alpha1 sequence is: aa 1-39 Ac-KQFTKAELSQLLKDIDGYGGIA-LPELIATMFHTSGYDTQ-OH. A. The leptin receptor - Interaction of the alpha1 peptide with leptin receptors Leptin is a 16 kDa protein that exerts its effects by activating the leptin receptor (LepR), which exists in six isoforms (LepR-A, LepR-B, LepR-C, LepR-D, LepR-E and LepR-F). These isoforms share a homologous extracellular region and can be distinguished by their intracellular domains. Among these, isoform LepR-B, with the longest intracellular region, is the most signaling-competent and is mainly expressed in the hypothalamus. (Fig. 1) Structurally, the extracellular region of LepR is comprised of a short N-terminal helix, followed by a cytokine-homology region (CHR) 1, an Ig-like domain, CHR2, and a fibronectin type III region. Dimerization of two LepR’s, triggered by Leptin binding, is necessary for inducing the conformational changes required for receptor activation. Leptin binds to LepR-B in an asymmetrical architecture, interacting with a high-affinity site located 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. Structural insights into the mechanism of leptin receptor activation. Nature Communications. 2023;14(1):1797.). Both sites are required to induce the conformational change necessary for activation. (Fig. 2) This activation triggers the JAK-STAT cascade through JAK2 activation and STAT3 phosphorylation, which is involved in processes such as immunity, cell division, cell death, and tumor formation. An in silico approach was applied to investigate whether the Alpha1 peptide showed affinity for LepR. AlphaFold, a software which predicts protein structures and interactions, was used to screen for this potential interaction. LepR was found to interact with Alpha1 through its Ig-like domain. Initially, the inventors focused on analyzing the sequence of the Ig-like domain and CHR2, which are vital for leptin binding. Despite a relatively low ipTM score of 0.367, Alpha1 was predicted to bind to the Ig-like domain of LEPR at residues 369-373, 402-405 and 416-422. (Fig. 4) Upon expanding the analysis to include the entire extracellular region of LEPR, rather than just the Ig-like domain and CHR2, the ipTM score increased to 0.52. Remarkably, Alpha1 was still predicted to bind to the same residues as in the previous model, bolstering the credibility of the prediction. (Fig. 5) These findings suggest that Alpha1 prevents activation of LepR. Overlaying the highest- scoring predicted model with a cryo-EM structure of a leptin-bound LepR dimer (1) shows two possible mechanisms for LepR inhibition. If Alpha1 binds to the receptor that should bind to leptin through the Ig-like domain (LepR I), leptin binding would be completely inhibited (Fig. 3A). If Alpha1 binds to the receptor that should bind to leptin through CHR2 (LepR II), leptin binding may be possible, but the dimer would not be able to undergo the necessary conformational change to become competent (Fig. 3B) B. The adiponectin receptor - Interactions of the alpha1-oleate complex with the Adiponectin receptors APN in humans is 244 amino acids and 28kDa. Circulating APN oligomers exist as trimers (Low molecular weight), hexamers (Middle molecule weight) and multimer (High molecular weight) form, with the latter being the most biologically active form. APN also exists in very small quantities in the plasma in a globular form (as a result of proteolysis). APN in humans is 244 amino acids and 28kDa. Circulating APN oligomers exist as trimers (Low molecular weight), hexamers (Middle molecule weight) and multimer (High molecular weight) form, with the latter being the most biologically active form. APN also exists in very small quantities in the plasma in a globular form (as a result of proteolysis). All these metabolic processes are mediated via three receptors, AdipoR1, AdipoR2, and T- cadherin. The AMPK pathway and the PPAR pathway are activated via adiponectin binding 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, opposite to that of the classical G-Protein coupled receptors (GPCRs). Previously reported crystal structures of the AdipoR1 and AdipoR2 proteins interestingly show that the major cavity 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 suggest ceramidase 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 Applied Sciences. 2020;2(4):533.). AdipoR1 and AdipoR2 also have an adiponectin-independent function of 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 hu man cell types and independently of adiponectin. J Lipid Res. 2019;60(5):995-1004). We find this highly useful information in learning about Alpha1-H’s tumor-killing mechanism, given the fact that the complex has an oleic acid component and could therefore potentially interact with the AdipoR1 and AdipoR2 receptors at the membrane level. Furthermore, Alpha1-oleate is highly membrane active, suggesting that the combined effects of alpha1-oleate and the adiponectin receptors may be essential to initiate the extensive tumor cell to alpha1-oleate, that leads to tumor cell death. (Fig. 6). In silico modeling was used to predict if the alpha1 peptide shows affinity for the AdipoQ receptors, using the same approach as described for the LepR interactions. The predicted structures suggested a common binding pocket for Alpha1 (shown in green) near the intracellular N-terminal domain of both adiponectin receptors (shown in cyan and orange) (Fig. 7). This observation provides a context for further investigations into the AdipoQ receptors and their interactions with alpha1-oleate. Example 2 To exert its effects on the regulation of energy homeostasis and hunger, Leptin binds to the Leptin receptor (LepR), activating the JAK2-STAT3 signaling pathway, which is involved in processes such as immunity, cell division, cell death, and tumor formation. This cascade stimulates the production of anorexigenic peptides, reducing appetite and increasing energy expenditure. However, in obesity, despite elevated levels of circulating Leptin associated with increased adiposity, satiety is compromised due to Leptin signaling dysregulation. Lepobmice (commonly referred to as ob or ob / ob) have a nonsense point mutation in the gene that encodes Leptin, resulting in the absence of this hormone. As a result of the missing Leptin, Lepobmice exhibit obesity, hyperphagia, transient hyperglycemia, glucose intolerance, and elevated plasma insulin. As such, this strain is used to model phases I and II of diabetes type II and obesity. The inventors tested the effects of BAMLET on Lepobmice. BAMLET was administered to Lepobmice and to wildtype mice as shown in figure 8. Mice were fed either a high fat diet (HFD), normal chow, or HFD plus BAMLET. The effects on weight gain and blood glucose levels are shown in figure 9. Effects on weight gain.The Lepob mice were shown to gain weight during the three-week observation period,consistent with previous reports on the consequences of the mutation that depletes functional Leptin. In contrast, no significant increase in weight gain was observed in thee BAMLET treated mice, compared to the control which received regular chow. BL6 (wildtype) mice exhibited a similar trend, albeit with a less pronounced difference compared to high-fat diet- fed mice. Effects on blood glucose levels. BAMLET administration further led to slight decrease in blood glucose levels in obese mice, compared to those on a high-fat diet alone. Notably, blood glucose levels in all BL6 groups remained within the normal range, yielding non-biologically significant differences between the groups. This outcome is within expectations, considering that metabolic changes in wildtype mice typically evolve over a longer period of time. Example 3 This study proposes a new molecular concept for the treatment of obesity and diabetes based on potent metabolic effects of complexes formed by alpha-lactalbumin and oleic acid. The bovine protein complex BAMLET inhibited weight gain and reduced blood glucose levels, in healthy mice fed a high fat diet and ob / B6.Cg-Lepob / J mice but not in healthy mice on a normal diet. The related, peptide based complex alpha1-oleate regulated metabolism in human tissues, inhibiting ADIPOQ and LEP, which encode the lipid- and glucose- regulating proteins adiponectin and leptin. Treatment is further shown to inhibit genes in interconnected ADIPOQ and LEP gene networks, affecting glucose metabolism and lipid synthesis in treated tissues. Based on in silico modeling, a mechanism of direct receptor interaction is proposed, involving oleic acid binding pockets of the adiponectin receptor ADIPOR2 and extracellular peptide epitopes. Binding of the alpha1 peptide to the immunoglobulin-like domain of the leptin receptor is further predicted to potentially inhibit its dimerization and activation by leptin. BAMLET treatment in the drinking water inhibited the activation of cancer related gene expression in mice fed a high fat diet and caused a potent shift in metabolic gene expression, in intestinal and liver tissues, which was attenuated in ob / B6.Cg-Lepob / J mice. These findings provide a new molecular approach for targeting and inhibiting key regulators of metabolism, with independent effects on obesity, type II diabetes and cancer and suggest that these effects may target hosts with a metabolic dysfunction, caused by excessive fat intake or a leptin deficiency. INTRODUCTION Metabolic dysfunctions are increasingly recognized as a major health threat world- wide, associated with diabetes, metabolic syndromes, obesity and cancer. Classical, therapeutic approaches have focused on compensating the metabolic dysfunctions by administering insulin or modifying dietary habits. Recently, additional molecular mechanisms underlying metabolic disorders have been identified and major therapeutic advances include the design of drugs that target the metabolic syndrome, affecting both obesity and type 2 diabetes mellitus. In recent years, synthetic analogues of pancreatic incretin hormones glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP), such as semaglutide and tirzepatide, have been introduced to target key mechanisms underlying obesity and type 2 diabetes mellitus (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). Their broad effects include increasing insulin secretion in a glucose-dependent manner and suppressing glucagon secretion in the pancreas when glucose levels are high, as well as slowing gastric emptying and inducing satiety of hunger by acting on peripheral and central receptors of the gut-brain axis. These treatments are commonly associated with gastrointestinal adverse events, such as nausea, vomiting and diarrhea, limiting patient tolerability (Holst JJ. GLP-1 physiology in obesity and development of incretin-based drugs for chronic weight management. Nature Metabolism. 2024;6(10):1866-85). We have discovered a family of protein-oleic acid complexes that rapidly kill cancer cells, by an apoptosis like mechanism. The HAMLET complexes insert into lipid bilayers and are taken up by tumor cells and tissues. The second generation, peptide based alpha1-oleate complex, formed by a synthetic peptide comprising the N-terminal domain of alpha- lactalbumin and oleic acid, has shown therapeutic efficacy in animal models and in a placebo-controlled clinical study of non-muscle invasive bladder cancer (NMIBC), where dose-dependent effects on tumor number and tumor size were accompanied by marked changes in gene expression profiles in 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 alpha1‐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 alpha1‐oleate treatment in patients with bladder cancer. Cancer Medicine. 2024;13(17):e70149). Potent effects of peroral HAMLET treatment effects were reported in colon cancer prone APCMin+ / -mice, with a significant delay in tumor progression (Tran HT, Wan MLY, Ambite I, Cavalera M, Grossi M, Háček J, et al. BAMLET administration via drinking water inhibits intestinal tumor development and promotes long- term health. Scientific Reports. 2024;14(1):3838). Effects on metabolic pathways were also detected in the intestines of mice treated with BAMLET, a complex formed by bovine alpha- lactalbumin and oleic acid. This study investigated the metabolic effects of BAMLET treatment in greater detail. Effects on obesity and glucose metabolism were quantified after peroral BAMLET treatment in normal C57BL / 6 mice on high fat diet and in Lep deficient ob / ob mice. Metabolic responses were characterized by gene expression analysis, identifying potent effects on glucose and lipid metabolism in tissues of treated mice compared to placebo. Metabolic effects were also quantified locally in tissues from alpha1-oleate treated patients with bladder cancer. Identified targets regulated by these molecules include both Adiponectin and Leptin, key regulators of metabolic homeostasis, lipid metabolism, glucose levels and insulin sensitivity, via interactions with the Adiponectin and Leptin receptors. The results suggest a strong potential for therapeutic targeting of metabolic disorders using these molecules. RESULTS Metabolic effects of per oral BAMLET administration in the drinking water To investigate if the BAMLET complex affects metabolic end points, BAMLET was administered daily in the drinking water to healthy C57BL / 6 mice (Figure 10). The treatment group received 10 mg / day of BAMLET in phosphate buffered saline (PBS) during weeks 1- 4 and 20 mg / day of BAMLET during weeks 5-7. The placebo group received drinking water supplemented with PBS (Fig. 10a). The C57BL / 6 mice were fed a high fat diet (HFD) or a normal diet (chow) and body weights and blood glucose levels were measured weekly and tissues were collected at sacrifice after four or seven weeks of treatment. As expected, body weights and blood glucose levels increased more rapidly in C57BL / 6 mice fed a HFD than in mice fed chow (p values). Body weight gain was significantly reduced by BAMLET treatment in mice fed a HFD, compared to placebo, suggesting a potent effect on weight gain (Fig. 10b-d). A similar effect was observed for blood glucose levels, where BAMLET treatment significantly reduced blood glucose levels in mice fed a HFD, compared to placebo (Fig. 10c). The body weight was reduced by an average of 19% and blood glucose levels by 21% after seven weeks, in BAMLET treated mice on a HFD compared to placebo (Fig. 10d). There was no further increase in effects on body weight and blood glucose at higher doses of BAMLET (50 mg / day or 100 mg / day for seven weeks). BAMLET treatment reduced body weights and blood glucose to similar levels as the control group on a chow diet, suggesting a return to near healthy metabolic conditions. Side effects were not detected in the tissues of BAMLET treated control mice fed chow (Gross pathology scores). Importantly, BAMLET treatment did not affect weight gain or blood glucose levels in healthy C57BL / 6 mice fed chow, suggesting that the metabolic changes targeted by BAMLET are initiated by HFD consumption. Effects on adipose tissue growth Effects of Bamlet on adipose tissue growth were quantified by measuring the size and weight of epididymal white adipose tissue (eWAT), inguinal white adipose tissue (IngWAT) and retro-peritoneal adipose tissue (ReWAT), after 7 weeks of treatment, compared to placebo (Fig. 10e, f). As expected, the build up of epididymal (EPI) fat, inguinal (ING) fat and retro-peritoneal fat was significantly higher in mice fed HFD, compared to chow (Fig. 10e, f). This build-up of fat tissue was suppressed in BAMLET treated C57 / BL6 mice fed a HFD, as shown by a significant reduction in size and lower weight of white adipose tissue (eWAT, IngWAT and ReWAT), compared to the placebo group (Fig. 10e, f). Liver weights were not affected by BAMLET treatment but tissue analysis showed significantly less histologic evidence of “fatty liver” in BAMLET treated mice than in the placebo group. H&E-stained liver tissue sections showed centrilobular micro-vacuolar and macro-vacuolar steatosis and binucleated hepatocytes in C57 / BL6 mice fed a HFD. These changes were associated with the HFD as they were not seen in mice fed chow. Centrilobular micro-vacuolar and macro-vacuolar steatosis and binucleated hepatocytes were significantly less evident in BAMLET treated mice fed a HFD than in the placebo group. The results suggest a potent anti-obesity effect of BAMLET, preventing adipose tissue establishment, growth and liver steatosis. Furthermore, the effects on blood glucose levels suggested a ‘’return to health’’ in the BAMLET treated group, compared to the placebo group, fed a HFD. Regulation of metabolic genes and functions in human bladder cancer tissues treated with alpha1-oleate Effects on metabolism were further investigated using alpha1-oleate, a complex formed by the N-terminal peptide of human alpha-lactalbumin and oleic acid. Bladder tissue biopsies were obtained from patients with non-muscle invasive bladder cancer, who were treated with the alpha1-oleate complex in a placebo-controlled study (EudraCTNo:2016‐004269‐14, ClinicalTrials.gov NCT03560479). Each patient received six intravesical instillations ofalpha1-oleate or placebo during one month, and the molecular response to alpha1-oleate treatment was evaluated by sequencing of RNA from tissue biopsies obtained at transurethral resection after the end of treatment. The gene expression profiles were compared between patients receiving 8.5 mM of alpha1-oleate or placebo (PBS). RNA sequencing detected a significant treatment response to alpha1-oleate (8.5mM), 2894 total regulated genes compared to placebo, with a predominance of inhibition (2058 genes, 71%) (cut off FC ≥ 2, P < 0.05). Major regulated categories included genes related to only cancer (1751 genes), only metabolism (678 genes) and both cancer and metabolism (657 genes). Genes involved in glucose metabolism (261 genes), lipid metabolism (164) or both functions (195 genes) were affected by alpha1-oleate treatment (Fig. 11a). Functional analysis identified top regulated glucose metabolism functions as glucose metabolism disorder, glucose uptake, glucose synthesis, the metabolism of carbohydrates and glucose tolerance and top regulated lipid metabolism functions as the synthesis of lipid, fatty acid metabolism, concentration of fatty acids, synthesis of terpenoids and steroids (Fig. 11b). Network analysis of the 678 regulated metabolism genes confirmed the glucose metabolism disorder network as strongly regulated (Fig 11c), comprising 455 genes with a predominance of inhibition (341 out of 455 genes, 75%). A similar pattern was observed for the synthesis of lipid gene network, with a predominance of inhibition (99 out of 118 genes, 84%), confirming that alpha1-oleate treatment inhibits metabolism associated gene expression, compared to placebo (Fig 11d). Regulation of metabolism including Adiponectin and Leptin gene networks A potent tumor response to alpha1-oleate treatment was identified, with fold change (FC) values ranging from -26907 to 142. Top regulated genes in treated tumors included GC, encoding the vitamin D binding GC protein associated with tumor cell movement and migration (FC = -26907), ADIPOQ encoding Adiponectin (FC = -5353), CALB1S encoding the calbindin 1 protein, affecting cell survival (FC = -429), BAAT, encoding the bile acid- CoA:amino N-acyltransferase (FC = -273), CSF3 encoding colony stimulating factor 3 (FC = -171), EREG, encoding epiregulin (FC = -127), and KRT79, encoding Keratin 79 (FC = 142). Additionally, the magnitude (FC) of inhibition was greater than that of activation for the top regulated genes. Strikingly, the top regulated genes included metabolism-associated genes GC, ADIPOQ, CALB1, BAAT, CSF3 and EREG. Further analysis of the top regulated metabolism-associated gene subset identified a predominance of inhibition of genes, including also CIDEC, encoding Cell Death Inducing DFFA Like Effector C (FC = -79), TF, encoding Transferrin (FC = -64)and LEP, encoding leptin (FC = -60). (Fig. 11e)Adiponectin and Leptin are important metabolic hormones or adipokines, with broad effects on metabolism and health. As both molecules were strongly affected at the gene expression level in alpha1-oleate treated tumors, their gene networks and interdependence was investigated. In networks of ADIPOQ- and LEP- dependent genes, 66 / 78 and 55 / 70 genes were identified as down-regulated by alpha1-oleate treatment. Detailed analysis of genes in the ADIPOQ and LEP networks showed evidence of a strong interconnection, suggesting a combined effect on glucose metabolism disorder and synthesis of lipid functions (Fig. 11f). Moreover, upstream regulator analysis identified 14 genes regulating ADIPOQ, of which 13 were downregulated, including LEP as the top upstream regulator (Fig. 11g). Functional analysis confirmed the broad effect of alpha1-oleate treatment on the ADIPOQ gene network, predicted to broadly 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, phospholipid and membrane lipid metabolism. Similarly, the LEP dependent network regulating lipid metabolism was strongly downregulated, including the metabolism of membrane lipids, lipid oxidation, and stimulation of lipid synthesis and glucose metabolism disorder and carbohydrate transport functions were affected, but predicted to be less strongly regulated than by adiponectin. The network of the GC gene, which was the most strongly regulated (FC = -26907), comprised only 19 genes. Regulation of adiponectin protein levels The metabolic response to alpha1‐oleate was further investigated at the protein level, using a Luminex human adipokine panel (Adiponectin / Acrp30, C-Reactive Protein, Calbindin D, Cathepsin S, CXCL2 / GRO beta / MIP-2 / CINC-3, DPPIV / CD26, IGFBP-rp1 / IGFBP-7, Insulin, Leptin / OB, M-CSF, Myeloperoxidase (MPO), PBEF / Visfatin, Resistin, TIMP-1, TREM-1, Vitamin D BP (encoded by GC). Urine samples were collected from alpha1-oleate treated patients before the onset of treatment (Pre V1) and after each of the six intravesical alpha1-oleate instillations (Post V1 to V6). A significant increase in urine adiponectin levels was detected at the early time points (P = 0.008 for Post V1) and (P < 0.001 for Post V3), followed by a decline at the end of the treatment period, suggesting an initial adiponectin response to alpha1-oleate treatment. There was no change in urine adiponectin levels in the placebo group (P = 0.727 Post V1) and (P = 0.222 Post V6). A similar with an early increase in urine protein levels followed by a decrease was also seen for adipokines Interactions of alpha1-oleate with adiponectin and leptin receptors predicted by in silico modelling The 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 (TMs) of the AdipoR1 and AdipoR2 receptor proteins have identified a major cavity that can be occupied by oleic acid (9, 12) (Fig. 12b). Beginning in the upper half of the transmembrane AdipoR2 structure between TM5 and TM6, an uninterrupted tunnel-like cavity has been proposed to link the upper lipid bilayer of the plasma membrane to the oleic acid binding pocket and to continue further down to a cytoplasmic opening, potentially modulating entry and exit of molecules to or from the receptor. The alpha1-oleate complexes are formed by the structurally flexible alpha1 peptide and three to five oleic acid molecules that create a hydrophobic lipid core, as shown using nuclear magnetic resonance and computational simulations (Nat Comm. Brisuda 2021) (Fig. 12a). Given this, it is plausible that the complex interacts with the adiponectin receptors and that oleic acid, presented by the complex to the adiponectin receptors, may reach the oleic acid binding cavity of these receptors. To address this question, AlphaFold 3 (Abramson, J., et al., Nature, 2024) was used to explore structural interactions between the truncated AdipoR2 (residues 131-386) and the alpha1-oleate complex. The resulting structures underwent a sanity check in PyMOL and were ranked in terms of their interface predicted template modelling (iPTM) scores. First, a single oleic acid molecule (OLA) was predicted by the software to strongly interact with the truncated AdipoR2, at the known oleic acid binding site, confirming the oleic acid interaction with the truncated AdipoR2 (ipTM: 0.83) (Fig. 12c). Second, the truncated AdipoR2 was submitted to AlphaFold 3 together with the alpha1-oleate complex, comprising one alpha1 peptide residue and 5 oleic acid molecules (ipTM: 0.72) (Fig. 12d). The Alpha1 peptide component of the complex was predicted to interact with the extracellular segment of AdipoR2, predominantly with the loop between TM5 and TM6. One oleic acid was shown to occupy the oleic acid binding site of AdipoR2 while another was situated at the transmembrane opening of the tunnel-like cavity (Fig. 12d, e). The binding of the complex was compared to the binding of the alpha1 peptide, by submitting the truncated AdipoR2 with the alpha1 peptide to AlphaFold 3. The peptide was predicted to interact with the extracellular loop between TM5 and TM6, but with a weaker score (iPTM: 0.19) (Fig. 12f). The analysis suggests that the alpha1-oleate complex recognizes and binds to the extracellular domain of the AdipoR2 receptor, that oleic acid delivered by the complex reaches the oleic binding cavity and that the complex binds more efficiently than the alpha1 peptide alone. Surface exposed residues between the TM5 and TM6 (aa 302-375) in AdipoR2 were predicted to interact with the alpha1-peptide as well as the complex. Due to their proximity to the entrance of the tunnel-like cavity, it may be speculated that the alpha1-oleate complex facilitates the delivery of an oleic acid molecule to AdipoR2 via this site. The oleic acid molecule would then follow the tunnel, ultimately reaching the oleic acid binding site of AdipoR2 and the cytoplasm. in contrast for predicted interactions between the alpha1-oleate complex or the alpha1 peptide and the full-length AdipoR2, Alpha Fold 3 consistently positioned the Alpha1 peptide at an intracellular binding site defined by aa 84–128 on the N-terminal domain as well as aa residues 207–211 between the TM2 and TM3 domains A similar intraceullar binding site for the complex was detected in full-length AdipoR1. Interactions of alpha1-oleate with the Leptin receptors predicted by in silico modelling and protein binding assays Potential interactions between alpha1-oleate and Leptin receptor B (LEPR-B) were investigated using the AlphaFold 2 Multimer protocol. Structurally, the extracellular region of LepR is comprised of a short N-terminal helix, followed by a cytokine-homology region (CHR) 1, an Ig-like domain, CHR2, and a fibronectin type III region. Dimerization of LepR, triggered by Leptin binding, is essential for receptor activation, with Leptin binding to LepR- B in an asymmetrical architecture, interacting with a high-affinity site located within CHR2 and a low-affinity site within the Ig-like domain. Both sites are required to induce the conformational change necessary for activation which occurs after leptin binding. This activation leads to JAK2 activation and STAT3 phosphorylation, thus triggering the JAK- STAT cascade. Cryo-EM analysis studies have shown leptin binding forms an asymmetric dimeric or trimeric Leptin:LepR complex in 2:2 or 3:3 stoichiometries respectively, where Leptin binds at ‘Site 2’ with high-affinity to the cytokine receptor homology domain 2 domain (CHR2) of LEPR while binding on ‘Site 3’ with low-affinity to the immunoglobulin like domain (IgD) of the adjacent receptor in the complex. The ’Site 3’ leptin binding at the Ig-like domain of LEPR plays a crucial role in the rate-limiting step of leptin-LEPR binding. After leptin initially binds with high affinity at Site 2, the subsequent low-affinity interaction at Site 3 triggers the conformational changes necessary for dimerization and the formation of the signaling- competent 2:2 complex. The LEPR-B was predicted by the AlphaFold 2 multimer protocol to interact with the alpha1 peptide through its immunoglobulin like (Ig-like) domain of LEPR at residues 369-373, 402- 405 and 416-422 (ipTM score of 0.367), which are directly involved in dimerization (Fig. 12g). An increase in the ipTM score to 0.52 resulted from expanding the analysis to include the entire extracellular region of LEPR, rather than just the Ig-like domain and CHR2 (Fig. 12h). The analysis predicted binding of alpha1 to the same residues on the Ig-like domain as in the previous model. Predictions of interactions with the complex by AlphaFold 3 confirmed the binding of the peptide to the Ig like domain with a similar score as the peptide alone (iPTM score 0.32 versus 0. 39). Binding to the extracellular domain were predicted to be increased for the complex compared to the peptide. These findings suggest that the binding to LEPR of the alpha1 peptide and alpha1-oleate may prevent the activation of LepR by Leptin. Overlaying the highest-scoring predicted model with a cryo-EM structure of a Leptin-bound LEPR dimer suggested two possible effects. First, the binding of alpha1 to the Leptin receptor binding site on the Ig-like domain (LEPR I) would directly inhibit leptin binding (Fig. 12i). Second, alpha1 binding to the adjacent Ig-like domain (LEPR II) would prevent the conformational changes necessary for the formation of the dimeric Leptin:LEPR complex, thereby disrupting downstream signaling (Fig. 12i). Effect on weight gain and blood glucose levels in B6.Cg-Lepob / J mice We subsequently used B6.Cg-Lepob / J mice (ob or ob / ob) to investigate the involvement of Leptin and leptin receptors in the metabolic response to the BAMLET complexes, using the protocol described in Figure 13a. The B6.Cg-Lepob / J mice develop extreme obesity due to extreme fat tissue growth, hyperphagia, hyperglycemia, glucose intolerance and elevated plasma insulin levels and are used as a model of obesity and type II diabetes. Mice in the treatment group were continuously administered BAMLET in the drinking water (20 mg / day) and the placebo group received drinking water supplemented with PBS. As expected, the B6.Cg-Lepob / J mice showed more rapid weight gain and increase in blood glucose levels than the C57BL / 6 WT mice with a more potent effect in mice fed HFD than in those fed chow. To optimize the treatment effect, BAMLET treatment was started at four weeks of age, compared to eight weeks in the C57BL / 6 mice. The BAMLET treatment group showed a significant delay in weight gain compared to the placebo group after four weeks of treatment (p<0.01), and further reduced after seven weeks of treatment (p<0.001). A similar pattern was seen for blood glucose levels, which were significantly lower in the BAMLET treated group than in the placebo group after four weeks (p<0.01) and further reduced after seven weeks of treatment (p<0.001). The effect of BAMLET was more pronounced in B6.Cg-Lepob / J mice receiving chow than in those fed a HFD (Fig. 13). Gene expression in BAMLET treated Lep deficient mice To further investigate the response to BAMLET treatment, in Lep deficient ob / ob mice, intestinal RNA was subjected to genome-wide transcriptomic analysis. Changes in gene expression were quantified in mice fed a HFD compared to chow and Lep deficient ob / ob mice treated with BAMLET in the drinking water for four weeks were compared to the placebo group. Treatment was initiated at 8 weeks of age. A marked change in gene expression profile was detected in mice fed a HFD compared to chow (Fig. 14a). Identified top regulated genes (FC>2, p<0.05) were involved in metabolism and included LEP (FC = 122.92) and ADIPOQ (FC = 48.77), which were strongly activated. Activated genes further included SCD1 (sterol metabolism, FC = 50.8),TRARG1 (trafficking regulator of GLUT4 1, FC=32.4), and NPR3 (natriuretic peptide receptor3, FC = 47.2) (Fig. 14a). In contrast, these strongly activated metabolic genes were not regulated in BAMLET-treated Lep deficient ob / ob mice on a HFDLEP and AdipoQ expression was not activated in the BAMLET treated group (Fig. 14a). The genes that showed reduced expression in the HFD group compared to chow, were not strongly affected by BAMLET treatment. Functional analysis identified a number of diabetes- and lipid storage- related functions as significantly enriched in mice fed a HFD, but not in BAMLET treated mice. This included functional categories such as insulin dependent diabetes, glucose metabolism disorder, diabetes mellitus and insulin resistance, storage of lipids and uptake of fatty acids (Fig. 14b). Furthermore, a direct comparison showed strong downregulation of LEP (FC = -43.81) and ADIPOQ (FC = 16.65) in Lep deficient ob / ob mice on a HFD treated with BAMLET compared to placebo. Network analysis was further performed to evaluate the down-stream effects of BAMLET treatment. ADIPOQ and LEP dependent gene networks were activated in Lep deficient mice fed a HFD compared to chow, with activation of 38 and 52 genes, respectively (Fig 14c, d). The majority of these genes were no longer regulated in the BAMLET treated mice, suggesting that the treated mice maintain an expression level for the top-regulated genes and metabolic functions, comparable to the group fed chow, despite being fed a HFD (Fig. 14e). DISCUSSION This study identified a new molecular approach to the treatment of obesity and type II diabetes. Based on studies in animal models, the BAMLET complex was shown to reduce weight gain and blood glucose levels, specifically in mice fed a high fat diet or ob / ob mice, without affecting healthy mice on a normal diet, suggesting an effect specific for excessive fat consumption or metabolic abnormalities. The effects were not limited to lipid metabolism, however, as broad effects on glucose metabolism were detected, with a profile of strong inhibition. The lack of effect of BAMLET treatment in healthy mice is reassuring, as it suggests that aberrations of metabolism need to be present for BAMLET to exert its effects. Potent effects on metabolism were further detected in cancer tissues from patients treated with the alpha1-oleate complex for bladder cancer, supporting human relevance of these findings. The results further propose a molecular basis for these effects, involving direct interactions with the adiponectin and leptin receptors. Structural studies using AlphaFold identified both the AdipoR receptors and the LepR receptors as potential targets of this family of alpha- lactalbumin complexes. Previously reported crystal structures of the AdipoR1 and AdipoR2 receptors have identified a cavity suitable for occupation by oleic acid and fatty acids released from ceramide by ceramidases have been proposed as natural ligands. This study indicates that exogenous ligands may fit the oleic acid binding pocket in AdipoR1 and R2 and identifies the alpha 1-oleate complex as a candidate molecule for such interactions. In addition, the N-terminal alpha1 peptide of alpha-lactalbumin was shown to fit a surface exposed peptide sequence in TM5 and TM6, proximal to the transmembrane opening of the oleic acid binding pocket, suggesting the potential for active delivery of oleic acid by the complex through a combined effect. Interestingly, there was no evidence of AMPK or PPAR activation, suggesting that these interactions may be unrelated to the activation of intracellular signaling by Adiponectin, the natural ligand. Further studies will be required to validate the in silico predictions and understand the consequences of such interactions for the receptor bearing cell and tissue. The in silico analysis further predicted that the leptin receptor is targeted by alpha1-oleate, and that alpha1 may prevent the activation of LepR. Overlaying the highest-scoring predicted model with a cryo-EM structure of a Leptin-bound LepR dimer suggested two possible mechanisms for inhibition of receptor activation. Alpha1's predicted binding to the Ig-like domain of the LepR, could potentially inhibit leptin binding. Binding to the CRH2 domain of LepR II was not predicted to affect Leptin binding, but to inhibit the conformational change needed to form an active hetero-dimer. However, in cases where Leptin is absent (e.g. Lepobmice), alpha1 might also serve as an agonist for its receptor, counteracting leptin deficiency in these mice by binding to the same site within LepR and acting as a replacement for Leptin. The activation of LepR-B by alpha1 could explain how BAMLET administration is able to prevent the increase in weight and blood glucose levels seen in Leptin-deficient mice. In cases where Leptin is not fully absent but exists in low amounts, alpha1 could also bind to and activate the short or secretory isoforms of LepR, promoting the transport of Leptin to the hypothalamus, maximizing the effect of whatever low levels of Leptin remain. At a local level, in tumors or tissues with an overabundance of Leptin, activation of these low signaling competent isoforms of LepR by alpha1 could also lead to increased Leptin clearance, promoting the reinstatement of a healthy balance of hormone levels. Altered lipid metabolism and obesity are well-known risk factors, promoting the development of disease world-wide, 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 studies have identified mechanisms by which obesity drives the development of cancer, by promoting cancer cell proliferation, tumor growth and chronic inflammatory states. Identifying such key mechanisms would increase opportunities to specifically target the metabolic aspect of tumor development as well as other diseases arising from imbalances affecting metabolism. In this study, mice on a high fat diet showed a highly significant increase in cancer gene expression that was inhibited by BAMLET treatment. The results further demonstrate strong effects on metabolism in cancer patients treated with alpha1-oleate, a peptide-lipid complex comprising the N-terminal peptide of human alpha-lactalbumin and oleic acid. Gene expression analysis in tumors from alpha1- oleate treated NMIBC patients showed a profound inhibition of cancer associated as well as metabolism associated genes, resembling the response to BAMLET in mice on a high fat diet. Particularly, key molecules of glucose and lipid metabolism including ADIPOQ and LEP adipokines, showed a strong inhibition in alpha1-oleate treated patients. The study did not include information about patient obesity, but the response was general for all tumors examined, suggesting that the effect of alph1-oleate was cancer related. The ability to inhibit major metabolic functions observed in this study therefore suggest that BAMLET and alpha1-oleate treatment might be useful to prevent or target obesity as well as obesity driven cancer development. MATERIALS AND METHODS Animals and high-fat diet intervention B6.Cg-Lepob / J mice and C57BL / 6 were obtained from The Jackson Laboratory at approximately seven weeks of age. The mice were housed in cages (four to five animals per cage) with food and water ad libitum under a 12:12-hour light-dark cycle at 22°C−25°C. Mice were acclimated for approximately two weeks at the local animal facility at Biomedical Centre (BMC), Lund University, to reduce stress from transportation. Mice were fed chow diet or HFD (D12492 60 E% fat content; Research Diets, New Brunswick, NJ, USA). For the BAMLET therapeutic protocol, eight-week-old male mice (n = 8 - 10 per group) were provided daily with BAMLET (10 mg / day in 5 ml of PBS for 4 weeks and 20 mg / day in 5 ml of PBS for 3 weeks) in the drinking water for 7 weeks and sacrificed at 4 weeks and 7 weeks. Mice were sacrificed by isoflurane anesthesia overdose (Dechra, Cat# 200-129). All animal procedures were approved by the Malmö / Lund Committee for Animal Experiment Ethics, Lund, Sweden. BAMLET The BAMLET complex was made by mixing bovine alpha-lactalbumin (Agropur, Cat# BiPRO Alpha 9000; Sigma-Aldrich, Cat# L6010) with oleic acid (Sigma-Aldrich, Cat# O1008) in PBS (0.137 M Sodium chloride, 0.0027 M Potassium Chloride, 0.01 M Sodium Phosphate Dibasic, 0.0018 M Potassium Phosphate Monobasic, pH = 7.2–7.4) and vortexing for 30 s. The stoichiometry of protein to oleic acid was 1:5, which has been shown by NMR to result in a mixture where the fatty acid is bound to bovine alpha-lactalbumin, without an excess of free fatty acid. The complex was used immediately for experiments. Bovine alpha- 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. Clinical study The single center trial of alpha1‐oleate in patients with NMIBC (EudraCTNo:2016–004269‐14, ClinicalTrials.gov NCT 03560479) was amended with a dose‐finding part, using the sameeligibility criteria, schedule of visits, study treatment administration, primary and secondary end‐points and assessments as the first part of the study. The patients in the dose‐finding part received a five times higher dose (8.5 mM, n = 6) of alpha1‐oleate. All patients receiving 8.5 mM of alpha1‐oleate completed the treatment. Primary end points were safety, change in tumor size and tumor cell shedding into the urine. Secondary end points were histopathology evaluation of tumor biopsies, quantification of alpha1‐oleate uptake by the tumor, apoptosis induction and treatment effects on gene expression. RNA sequence analysis RNA samples were prepared by Illumina TruSeq Stranded mRNA Library Prep Kit (20020594), and libraries were multiplexed and sequenced using NextSeq 500 / 550 High Output Kits (v2.5 2x75 Cycles) with an average of 22 million reads per sample. Raw sequencing data was demultiplexed using bcl2fastq (version 2.18) and RSEM (1.3) was used for abundance estimation using the human genome release 37 / Ensemble 75. Quality control of the samples were visualized using dimensionality reduction (i.e PCA), MA-plots as well as RNA-seq intrinsic biases (such as GC bias, transcriptome complexity and alignment quality). Differential expression analysis was performed using R (version 3.4) and DESeq2 package. Fold change were calculated by comparing tumors in the treated to the placebo group. Relative expression levels were analyzed and genes with an absolute fold change > 2.0 and P < 0.05 were considered as differentially expressed. Differentially expressed genes were functionally characterized using the Ingenuity Pathway Analysis version 57662101 (IPA, Qiagen) software. AlphaFold Protein interactions were predicted using AlphaFold 3, a deep learning-based algorithm designed for high-accuracy protein structure and interaction determination. Full-length and truncated forms of the target protein, (ADIPOR2; UniProt ID: Q86V24 and Alpha1), were used as inputs. Parameters were set according to the default guidelines. The resulting structures were evaluated using the interface predicted Template Modeling (ipTM) scores to assess prediction confidence. The 5 top scoring predicted complexes for each search were visualized and analyzed using PyMOL (the PyMOL Molecular Graphics System, Version 3.1.3, Schrodinger, LLC.), a molecular visualization system. Structural alignment was performed to assess consistency between predicted and experimentally resolved structures in Protein Data Bank (PDB) (where available). Models with highest confidence ipTM scores were selected for further computational refinement and experimental validation, if applicable. Interacting residues were identified by selecting atoms within a distance of 4 Å between the target protein and the binding molecule or peptide). ADIPOR1 (UniProt accession number: Q96A54) and ADIPOR2 (UniProt accession number: Q86V24) sequences were sourced from the UniProt database and submitted together with the Alpha1 peptide sequence to ColabFold using 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 tree search. Nature Communications. 2022;13(1):6028.. Evaluation of the resulting complexes was conducted by visually assessing the top 5 scoring models using PyMOL (Open source). Complexes exhibiting evident conflicts, such as structural interpenetration, were filtered out during this process. Ethics declarations Clinical The study was approved by the State Institute for Drug Control (SUKL) in the Czech Republic; number 273799 / 17‐I and the Ethics Committee of the Motol University Hospital; number EK‐786 / 17. All patients were included in the study after informed consent. The dose escalation part of the study was an extension of the single center, placebo controlled, double blinded randomized Phase I / II interventional clinical trial of NMIBC (EudraCTNumber: 2016–004269‐14 and ClinicalTrials.gov NCT03560479). Demographic data,morbidity and health parameters as well as tumor characteristics were recorded by the study physicians in the electronic Case Report Form (eCRF) and closely monitored by an external monitor. Animal experiments were approved by the Malmö / Lund Animal Experimental Ethics Committee at the Lund District Court, Sweden (#01302-20). Animal care and animal experimental protocols followed institutional, national, and European Union guidelines and were governed by the European Parliament and Council Directive (2016 / 63, EU), the Swedish Animal Welfare Act (Djurskyddslagen 1988:534), the Swedish Welfare Ordinance (Djurskydssförordningen 1988:539) and Institutional Animal Care and Use Committee (IACUC) Guidelines. Results were reported in accordance with ARRIVE guidelines Statistical analyses The FDR or P-value for defining the differentially expression genes in IPA in the 8.5mM Alpha1H-treated tumors compared to placebo was set at <0.05. The following statistical analysis was performed using GraphPad Prism where statistical significance was defined as P < 0.05. The normality of the data was tested using the Shapiro-Wilk normality test, which revealed that the data was not normally distributed. The non-parametric Mann Whitney t-test was used to compare the median urinary adiponectin levels between 8.5mM AlphaH-treated and placebo-treated groups. The repeated measures two-way ANOVA test using Šidák’s multiple comparison test was used to examine the effects of time (visits) and treatment (8.5mM AlphaH and Placebo) on urinary adiponectin levels. The correlation between the number of cells shed post-instillation of either placebo or the Alpha1H complex and the amount of adiponectin was determined using the linear regression analysis, where R2= 0.307 with P = 0.001. The mean % cell death was shown with error bars representing the standard deviation. Again, the normality of the data was tested using the Shapiro-Wilk normality test, which revealed that the data normally distributed. Therefore, a one-way ANOVA followed by Tukey’s multiple comparison tests was used to compare the parametric data.

Claims

CLAIMS A polypeptide having a sequence of a naturally occurring alpha-lactalbumin, or a functional variant thereof; or a peptide of up to 50 amino acids comprising an alpha-helical domain of said polypeptide, for use the treatment or prevention of metabolic-related conditions. A complex comprising a polypeptide having a sequence of a naturally occurring alpha-lactalbumin, or a functional variant thereof; or a peptide of up to 50 amino acids comprising an alpha-helical domain of said polypeptide; and a fatty acid or lipid or salt thereof, for use in the treatment or prevention of metabolic-related conditions. A polypeptide for use according to claim 1 or a complex for use according to claim 2, wherein the metabolic-related condition is insulin resistance, type II diabetes, metabolic syndrome, non-alcoholic fatty acid liver disease, cirrhosis, or high blood pressure. A polypeptide for use according to claim 1 or a complex for use according to claim 2, wherein the metabolic-related conditions are modulated or controlled by the leptin pathway and / or the adiponectin pathway. A polypeptide or a complex for use according to claim 4, wherein the metabolic- related condition modulated or controlled by the leptin pathway is hyperleptinemia, leptin resistance, lack of or reduced sensitivity to leptin, leptin-related weight gain and / or obesity, increased or reduced hunger stimulation, leptin-related increased or decreased metabolism, leptin-related metabolic syndrome, leptin-related non-alcoholic fatty liver disease, Rabson–Mendenhall syndrome, depression and food addiction, hypoleptinemia, hyperinsulinemia, fatty liver disease, dyslipidemia, hypogonadotropic hypogonadism, congenital or acquired generalized lipodystrophy (GL), type 1 diabetes, or hypothalamic amenorrhea. A polypeptide or a complex for use according to claim 4, wherein the metabolic- related condition modulated or controlled by the leptin pathway is one or more of obesity, hyperphagia, transient hyperglycemia, glucose intolerance, and elevated plasma insulin. A polypeptide or a complex for use according to claim 4, wherein the metabolic- related condition modulated or controlled by the adiponectin pathway is obesity, type 2 diabetes, metabolic syndrome, atherosclerosis, and cardiovascular disorders including myocardial infarctions. A polypeptide having a sequence of a naturally occurring alpha-lactalbumin, or a functional variant thereof; or a peptide of up to 50 amino acids comprising an alpha-helical domain of said polypeptide, for use in modulating the leptin receptor and / or the adiponectin receptor.A complex comprising a polypeptide having a sequence of a naturally occurring alpha-lactalbumin, or a functional variant thereof; or a peptide of up to 50 amino acids comprising an alpha-helical domain of said polypeptide; and a fatty acid or lipid or salt thereof, for use in modulating the leptin receptor and / or the adiponectin receptor. A method of treating metabolic-related conditions comprising administering (1) a polypeptide having a sequence of a naturally occurring alpha-lactalbumin, or a functional variant thereof; or a peptide of up to 50 amino acids comprising an alpha-helical domain of said polypeptide, or (2) a complex comprising a polypeptide having a sequence of a naturally occurring alpha-lactalbumin, or a functional variant thereof; or a peptide of up to 50 amino acids comprising an alpha-helical domain of said polypeptide; and a fatty acid or lipid or salt thereof, to a subject in need thereof. A method according to claim 10, wherein the metabolic-related conditions are as defined in any one of claims 3-7. A method of modulating the leptin receptor and / or the adiponectin receptor comprising administering (1) a polypeptide having a sequence of a naturally occurring alpha-lactalbumin, or a functional variant thereof; or a peptide of up to 50 amino acids comprising an alpha-helical domain of said polypeptide, or (2) a complex comprising a polypeptide having a sequence of a naturally occurring alpha-lactalbumin, or a functional variant thereof; or a peptide of up to 50 amino acids comprising an alpha-helical domain of said polypeptide; and a fatty acid or lipid or salt thereof, to a subject in need thereof. A polypeptide for use of any one of claims 1 and 3-8, a complex for use of any one of claims 2, 3-7 or 9 or a method according to any one of claims 10-12, wherein the polypeptide has a sequence of a naturally occurring alpha-lactalbumin, a human or bovine alpha-lactalbumin or a bovine alpha-lactalbumin. A polypeptide for use of any one of claims 1 and 3-8, a complex for use of any one of claims 2, 3-7 or 9 or a method according to any one of claims 10-12, wherein the alpha- helical domain is KQFTK XELSQLLKDIDGYGGIALPELI XTMFHTSGYDTQ (SEQ ID NO 3) or LDDDITDDIM XAKKILDIKGIDYWLAHKALXTEKLEQWL XEKL (SEQ ID NO 4) where X is an amino acid residue other than cysteine. A complex for use of any one of claims 2, 3-7 or 9 or a method according to any one of claims 10-12, wherein the complex comprises a peptide of about or less than 45, 42, or 40 amino acids or a peptide of 39 amino acids. A polypeptide for use of any one of claims 1 and 3-8, a complex for use of any one of claims 2, 3-7 or 9 or a method according to any one of claims 10-12, wherein the functional variant (a) comprises or consists essentially of an amino acid sequence with at least 70%identity to the alpha-lactalbumin sequence, (b) consists of a sequence lacking disulfide bonds or (c) consists of a sequence in which cysteine residues in the native alpha- lactalbumin are changed to other amino acid residues. A complex for use of any one of claims 2, 3-7 or 9 or a method according to any one of claims 10-12, wherein the fatty acid or salt thereof is oleic acid or an oleate salt. A polypeptide for use of any one of claims 1 and 3-8, a complex for use of any one of claims 2, 3-7 or 9 or a method according to any one of claims 10-12, wherein the polypeptide is a recombinant protein having the sequence of SEQ ID NO: 1 or SEQ ID NO: 2.