Patent Application
20200069774
The present disclosure relates to methods for controlling body weight and/or appetite in a subject. In one particular application, the method involves administering to an overweight or obese subject an effective amount of a first agent comprising MIC-1 and/or a MIC-1 agonist and a second agent comprising leptin and/or a leptin agonist, for the purpose of achieving weight loss.
The present application claims priority from Australian Provisional Patent Application No 2016905018 titled “Treatment of obesity and eating disorders” filed on 6 Dec. 2016, the content of which is hereby incorporated by reference in its entirety.
The following application(s) or publication(s) are referred to in the present application and their contents are hereby incorporated by reference in their entirety:
Amirah E-E A et al., Expert Opin Drug Deliv 12(12): 1923-1941 (2015);
International Patent Application No PCT/GB1996/001094 (WO 96/034885) titled “Biologically active peptide fragments of OB protein”;
International Patent Application No PCT/AU1996/000386 (WO 97/000958) titled “Novel TGF-b like cytokine”;
International Patent Application No PCT/US2009/001231 (WO 2009/108340) titled “Leptin agonist and methods of use”;
International Patent Application No PCT/US2013/023465 (WO 2013/113008) titled “Growth differentiation factor 15 (GDF-15) polypeptides”;
International Patent Application No PCT/EP2015/063596 (WO 2015/197446) titled “MIC-1 fusion proteins and uses thereof”;
International Patent Application No PCT/EP2017/050695 (WO 2017/121865) titled “MIC-1 receptor and uses thereof”;
International Patent Application No PCT/EP2017/062583 (WO 2017/202936) titled “MIC-1 compounds and use thereof”; and
U.S. Pat. No. 5,225,539 titled “Recombinant altered antibodies and methods of making altered antibodies”.
The control of body weight is a complex process that is multifactorial and influenced by a number of factors including appetite, food ingestion and excretion, energy utilisation and expenditure. A number of soluble mediators are known to be involved in regulating various aspects of this process and include systemic- and/or nervous system-derived hormones, cytokines and other mediators such as ghrelin, glucagon-like peptide 1 (GLP-1), amylin, insulin, cholecystokinin (CCK), proopiomelanocortin (POMC), agouti-related peptide (AgRP), and neuropeptide Y (NPY). Normal weight control is important to good health and overweight, and especially obesity, may greatly increase morbidity and mortality in individuals. On the other hand, having a lower than average weight can also be problematic, and in developed societies, where sufficient food is available, this is more frequently due to diseases including some chronic inflammatory disorders, chronic renal and cardiac failure, liver cirrhosis, chronic obstructive pulmonary disease (COPD), eating disorders such as anorexia nervosa, and cancer. Especially in the late stages of cancer, anorexia/cachexia is common (occurring in most terminally ill cancer patients), and is responsible for about a quarter of all cancer-related deaths.
Some years ago, the present Applicant cloned and characterised a novel human TGF-b superfamily cytokine that was named macrophage inhibitory cytokine-1 (MIC-1)1-7, but has since also become known, amongst others, as prostate derived factor (PDF), placental bone morphogenetic protein (PLAB), and growth/differentiation factor-15 (GDF-15)7. Under resting conditions, placenta is the only tissue expressing large amounts of MIC-17, but epithelial cells in a wide variety of other organs also normally express smaller amounts of MIC-1 mRNA such that MIC-1 circulates in all individuals with a broad normal range of 0.15-1.15 ng/ml serum8. These serum levels increase with age and a number of physiological variants such as body mass index (BMI), but are also increased in many different disease processes, especially those associated with malignancy, inflammation and injury7, 9-12. In some of these, especially malignancies, such as those of the breast, prostate, pancreas, ovary and colon10-12, 14, these serum levels can rise dramatically. In addition, in a study of several hundred patients with colonic polyps or colon cancer15, the present Applicant showed that elevation of serum levels of MIC-1 occurs in a progressive stepwise manner, reflecting colon cancer pathogenesis, with progression from normal to benign and then to dysplastic colonic polyps and finally colon cancer. However, whilst MIC-1 serum levels increase in disease such as these cancers, its overall biological effect, in most instances, is likely to be beneficial, as animal studies have indicated that MIC-1 over-expression mitigates disease16-19. Most importantly, a recent study demonstrated that transgenic mice over-expressing MIC-1, whether on a chow or high fat diet (HFD), have a markedly prolonged life expectancy20. This suggests that disease associated elevation of serum MIC-1 levels may represent an incompletely effective attempt to control the pathological process in question.
The present Applicant has previously found that mice with tumour xenografts, engineered to over-express human MIC-1, became anorexic/cachectic21, 22. Further, it was observed that the serum levels of the tumour-derived MIC-1 in the mice were proportional to the degree of weight loss, with more marked effects occurring in mice with serum levels greater than about 8-10 ng/ml. Importantly, these serum levels are well within the range of that commonly seen in patients with diseases such as advanced cancer (ie cancer, which has typically metastasized, and which is not considered curable but responds to treatment (eg by disease-directed therapy) to prolong life). Moreover, mice with tumours over-expressing MIC-1 were found to eat less and lose a substantial amount of fat and muscle, which could be reversed, without modifying tumour progression, by administering an antagonistic anti-MIC-1 monoclonal antibody21, 22. In addition, in a comparative study, it was found that the administration of recombinant MIC-1 to mice reproduced these findings21 and were consistent with studies of humans correlating serum MIC-1 levels with reduced BMI or anorexia/cachexia syndromes21, 23-25. Also, the studies with MIC-1 over-expressing transgenic mice found that these mice were lean, protected from the development of obesity and showed an improved level of glucose tolerance when placed on an obesogenic diet21, 26, 27. It was also considered that these transgenic mice may have increased thermogenesis20, 28. Further, it has been found that mice bearing a germline gene deletion of MIC-1 are obese, eat more and weigh more throughout their life than syngeneic mice. These findings suggest that MIC-1 plays a role in the physiological regulation of energy homeostasis and that this pathway becomes subverted in disease states leading to an anorexia/cachexia syndrome.
As mentioned above, mice with MIC-1 over-expressing tumours become anorexic/cachectic and have a decreased food intake. This is mediated by actions on the hypothalamus and brainstem, which is a major mechanism for the observed weight loss21. This is supported by the finding that surgical lesioning of the brainstem area postrema (AP) and the medial (m) nucleus of solitary tract (NTS) of mice prevents the anorexic actions of recombinant MIC-129. However, it is suspected that there is much more involved and, notably, the studies described above did not exclude metabolic actions of MIC-1 outside of the central nervous system (CNS) or its possible actions on different CNS centres. Moreover, the present Applicant considers that the metabolic actions of MIC-1 may interact with other agents involved in appetite control and/or energy homeostasis (eg some adipokines and peptide hormones such as ghrelin and proopiomelanocortin). This led to the investigation of whether there is interaction between the effects of MIC-1 and the adipokine, leptin.
In a first aspect, the present disclosure relates to a method of controlling body weight and/or appetite in a subject, said method comprising administering to said subject an effective amount of:
Preferably, the first agent comprises MIC-1 and/or a MIC-1 agonist.
The subject may be an overweight or obese subject and, in one embodiment, the subject may be an overweight or obese subject with a low serum leptin level.
In a second aspect, the present disclosure relates to a pharmaceutical composition comprising:
Preferably, the first agent comprises MIC-1 and/or a MIC-1 agonist.
The pharmaceutical composition may be suitable for, for example, oral, buccal, nasal, intramuscular and intravenous administration.
In a third aspect, the present disclosure relates to a method of controlling body weight and/or appetite in a subject, said method comprising the steps of:
determining whether said subject has a low, normal or high serum level of leptin; and
administering to said subject, where determined as having a low or normal serum level of leptin, an effective amount of:
administering to said subject, where determined as having a high serum level of leptin, an effective amount of:
In a fourth aspect, the present disclosure relates to a kit comprising first and second containers (eg vials), wherein the first container contains a first agent comprising MIC-1, a MIC-1 agonist and/or a MIC-1-inducing agent, and the second container contains a second agent comprising leptin and/or a leptin agonist; optionally packaged with instructions for the use of the kit in the method of the first or third aspects.
In a fifth aspect, the present disclosure relates to a method of increasing body weight and/or appetite in a subject, said method comprising administering to said subject an effective amount of:
The subject may be a subject suffering from anorexia or cachexia.
In a sixth aspect, the disclosure relates to a pharmaceutical composition comprising:
In a seventh aspect, the present disclosure relates to a kit comprising first and second containers (eg vials), wherein the first container contains a MIC-1-inhibiting agent and the second container contains a leptin-inhibiting agent; optionally packaged with instructions for the use of the kit in the method of the fifth aspect.
An investigation into a possible interaction between MIC-1 and the adipokine, leptin, using an obese mouse model, determined that treatment with MIC-1 and leptin caused significantly greater weight loss than that observed with MIC-1 or leptin alone (see Examples hereinafter).
In a first aspect, this disclosure relates to a method of controlling body weight (eg achieving weight loss) and/or appetite in a subject, said method comprising administering to said subject an effective amount of:
The method of the first aspect may be conducted to achieve appetite control and/or body weight control in a subject. For example, for body weight control, the method may be conducted to achieve weight loss in a subject or, at least, to prevent further gain of body weight in a subject. The subject may be a subject in need of body weight control such as, for example, an overweight or obese subject (for example, a subject with diet-induced obesity (DIO)) and/or a reduction in appetite. For the purposes of the present disclosure, an overweight subject is considered to be a subject with a body mass index (BMI) of 25 to 30, and an obese subject is considered to be a subject with a BMI of 30 or more33, 61.
Preferably, the method of the first aspect is a method of decreasing body weight (ie achieving weight loss) or preventing body weight gain.
In one particular embodiment of the method of the first aspect, the subject is an overweight or obese subject with a lower than normal serum leptin level (ie a serum leptin level that is lower than the typical range for a normal subject) or, otherwise, a relatively low serum leptin level for an obese subject or relatively low serum level given the degree of adiposity present in the subject.
For normal (ie non-obese) human subjects, the normal serum level range of leptin is broad; for example, about 3.5 to 14.0 ng/ml for adult women and 4.5 to 25.5 ng/ml for adult men31 after overnight fasting. A lower than normal serum leptin level may be regarded as a level at the lower end of these ranges (eg less than about 6 ng/ml for adult women and less than about 12 ng/ml for adult men) or lower than these ranges.
For most obese human subjects, their serum leptin level (following overnight fasting) will be high (nb. it has been found that obese humans have (on average) leptin levels four times higher than non-obese individuals60) and correlates with BMI68. For example, it has been reported that the mean serum leptin level after overnight fasting for obese subjects is 31.3 ng/ml60 (nb. in this study, obese subjects were considered as having a BMI of ≥27.3 for men and ≥27.8 for women). Elsewhere, it has been reported that the mean serum leptin level for obese adult women is 38.2 and that the mean serum leptin level for obese adult men is 27.061. Given such knowledge, those skilled in the art can readily determine serum leptins levels which would be regarded as being relatively low for an obese subject. However, in one embodiment of the method of the first aspect, a relatively low serum leptin level for an obese subject (after overnight fasting) may be, for an adult woman, less than about 30 ng/ml or, more preferably, less than 25 or less than 20 ng/ml, and for an adult man, less than about 20 ng/ml or, more preferably, less than 15 ng/ml.
Population studies have suggested that about 10% of obese individuals have low serum leptin levels59. Lower than normal serum leptin levels or levels which are relatively low for an obese subject may be caused by, for example, impairment of leptin regulation (especially, reduced leptin production by adipose tissue67) which may or may not be associated with certain diseases and conditions such as congenital leptin deficiency34, intra-uterine growth retardation (IUGR)32 and other factors. Subjects with a lower than normal serum leptin level or relatively low serum leptin level (for an obese subject) can be identified by measuring serum leptin levels in accordance with any suitable assay method known to those skilled in the art including standard immunoassays (eg a leptin ELISA). Thus, in a particular embodiment, the method may be a method of decreasing body weight (ie achieving weight loss) or preventing body weight gain in an overweight or obese subject with a lower than normal serum leptin level or a relatively low serum leptin level for an obese subject. It is considered that in accordance with the results achieved in the investigation described hereinafter, administering to such a subject an agent comprising, for example, MIC-1 and/or a MIC-1 agonist and an agent comprising leptin and/or a leptin agonist will be more effective for body weight control than the use of MIC-1 and/or a MIC-1 agonist alone.
The first agent comprising MIC-1, a MIC-1 agonist and/or a MIC-1-inducing agent may, for example, be administered to the subject concurrently (eg in combination) with the second agent comprising leptin and/or leptin agonists, or otherwise the first agent comprising MIC-1, a MIC-1 agonist and/or a MIC-1-inducing agent and the second agent comprising leptin and/or leptin agonist may be administered to the subject consecutively and in either order. As used herein, it is to be understood that when administered consecutively, the respective agents may be administered one after another with practically no time interval (ie one is administered effectively immediately after the other) or, otherwise, after an interval of 1 to 5 minutes or more (eg 10 minutes, 30 minutes, 60 minutes, 4 hours or 12 hours). Where the first and second agents are administered consecutively, they may each be formulated for administration in a pharmaceutical composition form including a pharmacologically acceptable carrier and/or excipient, which may be the same or different. The pharmaceutical compositions may optionally comprise other substances such as absorption enhancers including surfactants (eg sodium lauryl sulphate, laureth-9, sodium dodecylsulphate, sodium taurodihydrofusidate and poly oxyethylene ethers) and chelating agents (eg EDTA, citric acid and salicylates). The pharmaceutical compositions may be suitable for, for example, oral, buccal, nasal, intramuscular and intravenous administration. In some embodiments, the pharmaceutical compositions are particularly suitable for nasal administration (ie intranasal delivery), which has been previously shown as an efficacious route for the administration of TGF-b superfamily members (GDF-5 and GDNF) and other proteins and peptides (recently reviewed by Amirah E-E A and B L Waszczak56; the entire disclosure of which is incorporated herein by reference). Suitable pharmaceutical compositions for nasal administration include, but are not limited to, liposomal compositions57 and lipid microemulsions58.
Typically, the pharmaceutical compositions will be administered to the subject in an amount which is effective for controlling body weight and/or appetite. The composition comprising the first agent may be administered to provide, for example, an amount of the agent comprising MIC-1, a MIC-1 agonist and/or a MIC-1-inducing agent that is between about 0.01 and about 100 mg/kg body weight per day, or between about 0.05 and 25 mg/kg body weight per day of the agent comprising MIC-1, a MIC-1 agonist and/or a MIC-1-inducing agent. The composition comprising the second agent may be administered to provide, for example, an amount of the agent comprising leptin and/or leptin agonist that is between about 0.01 and about 5000 mg/kg body weight per day, between about 10 and 1000 mg/kg body weight per day of the agent comprising leptin and/or leptin agonist, or between about 100 and 500 mg/kg body weight per day of the agent comprising leptin and/or leptin agonist. The pharmaceutical compositions may be intended for single daily administration, multiple daily administration, or controlled or sustained release, as needed to achieve the most effective results.
Preferably, the first agent comprising MIC-1, a MIC-1 agonist and/or a MIC-1-inducing agent and the second agent comprising leptin and/or leptin agonist are formulated in combination for administration in a single pharmaceutical composition. The combination composition may further comprise, for example, a pharmacologically acceptable carrier and/or excipient, optionally together with others substances such as absorption enhancers (including surfactants and chelating agents) and protease inhibitors (such as aprotinin (trypsin/chymotrypsin inhibitor), amastatin, bestatin, boroleucine, and puromycin (aminopeptidase inhibitors)) for improving bioavailability, especially oral bioavailability. Such a combination pharmaceutical composition may be suitable for, for example, oral, buccal, nasal, intramuscular and intravenous administration. In some embodiments, the pharmaceutical composition is particularly suitable for nasal administration (ie intranasal delivery).
Typically, a combination pharmaceutical composition will be administered to the subject in an amount which is effective for controlling body weight and/or appetite. Thus, the combination composition may be administered to provide, for example, an amount of the agent comprising MIC-1, a MIC-1 agonist and/or a MIC-1-inducing agent that is between about 0.01 and about 100 mg/kg body weight per day or between about 0.05 and 25 mg/kg body weight per day, and an amount of the agent comprising leptin and/or leptin agonist that is between about 0.01 and about 5000 mg/kg body weight per day, between about 100 and 1000 mg/kg body weight per day, or between about 100 and 500 mg/kg body weight per day of the agent comprising leptin and/or leptin agonist. The combination pharmaceutical composition may be intended for single daily administration, multiple daily administration, or controlled or sustained release, as needed to achieve the most effective results. However, notwithstanding the above, it will be understood by those skilled in the art that the administered amount of the combination composition, and the frequency of administration for any particular subject, may vary and depend upon a variety of factors including the activity of the active agents (eg the first and second agents), the metabolic stability and length of action of the active agents, the age, body weight, sex, mode and time of administration, and the rate of excretion of the active agents.
The method may involve administering a pharmaceutical composition or combination pharmaceutical composition comprising a first agent that comprises a MIC-1 agent such as a recombinant MIC-1 or synthetic MIC-1 (ie MIC-1 produced by protein synthesis techniques) comprising, preferably, a dimer of a polypeptide comprising a native MIC-1 amino acid sequence (eg the native amino acid sequence of a mature (processed) MIC-1 protein) or a variant MIC-1 amino acid sequence. Suitable variants of MIC-1 amino acid sequences may include a naturally occurring or non-natural variant amino acid sequence of a native amino acid sequence that may include one or more minor sequence variations which, preferably, do not substantially alter the function of the polypeptide (eg despite the variation(s), the polypeptide maintains the ability of binding to and activating the MIC-1 receptor, known as the GDNF family receptor α-like GFRAL)30, 60, 70). The native human MIC-1 amino acid sequence was originally published by the present Applicant in International patent publication No WO 97/000958 (the entire disclosure of which is incorporated herein by reference, wherein MIC-1 is referred to as CL13). An example of a naturally occurring variant of that sequence, which is suitable for use in the method of the first aspect, comprises a His→Asp substitution at position 6 (ie H6D) of the mature MIC-1 amino acid sequence. Other variants of MIC-1 amino acid sequences may include one or more conservative amino acid substitutions such as: G, A, V, I, L, M; D, E; N, Q; S, T; K, R, H; F, Y, W, H; and P, Nα-alkylamino acids. Other substitutions may include the substitution of one or more L-amino acid(s) with a D-amino acid(s). Preferably, any amino acid substitution comprises a substitution with an amino acid selected from the twenty (20) standard amino acids encoded by the genetic code (ie the canonical amino acids). Some suitable examples of amino acid substitutions have been described in International patent publication no WO 2017/202936 (the entire disclosure of which is incorporated herein by reference) such as N3E, P11E, H18E, R21E, A30E, A47E, R53E, A54E, M57E, M57L, R67E, L68E, A75E, A81E, P85E, M86L, L105E and K107E, which may improve solubility, improve stability and/or prevent oxidation of the MIC-1. However, amino acid substitutions with non-canonical amino acids such as, for example, certain Nα-alkylamino acids (eg N-methyl glycine (sarcosine) and N-methyl alanine), and other amino acids such as 2-aminobutyric acid (Abu), naphthylalanine (Nal), amino isobutyric acid, 3-aminoadipic acid (Aad), ornithine, citruline, amino-oxyserine, homo-arginine, norleucine (Nle), aminosuberic acid and β-2- and β-3-napthylalanine, ring-substituted phenylalanine (Phe) derivatives (eg 2,3,4,5,6-pentafluoro-phenylalanine, 4-chloro-phenylalanine, methyl-phenylalanine and phosphono-phenylalanine), phospho-tyrosine (pTyr), selenocysteine and selenomethionine, are also contemplated. Other sequence variations that may be present include one or more amino acid deletion (such as, for example, the deletion of Asn and position 3 (des-N3)) or addition (eg insertion). Other additions that may be made to, for example, the N- or C-terminal sequence may comprise the addition of a single amino acid (eg Ala), short amino acid sequences (eg 2 to 10 amino acids in length) or long amino acid sequences (eg 11 or more amino acids) which may confer various additional functionalities or properties, such as improved bioavailability, extended circulating half life, improved solubility and/or stability, and improved protein recovery or expression (eg a fusion partner). Numerous examples of MIC-1 agents comprising an additional amino acid sequence (of 3 to 36 amino acids in length or more) at the N-terminal (ie an N-terminal extension) have been described in WO 2017/202936 and are considered as suitable for use in the method of the first aspect. MIC-1 agents comprising an additional long amino acid amino sequence (eg 11 or more amino acids) at the N- or C-terminal may be considered as a MIC-1 conjugate. MIC-1 conjugates suitable for use in the method of the first aspect may comprise, for example, a conjugate of a MIC-1 with a conjugate partner selected from the group comprising fusion partners to improve protein recovery or expression (eg Human serum albumin (HSA), Glutathione S-transferase (GST), the pro-region of another member of the TGF-β superfamily, and various affinity-tags such as a polyhistidine tag (His-tag) or FLAG-tag), and Fc polypeptides and other polypeptides capable of dimerising. The MIC-1 molecule of a MIC-1 conjugate may optionally be conjugated to the conjugate partner via a linker sequence as is well known to those skilled in the art.
Some particular examples of suitable MIC-1 conjugates are described in International patent publication No WO 2015/197446 (the entire disclosure of which is incorporated herein by reference). These conjugates comprise the fusion partner HSA (or a functional variant thereof) at the N-terminus conjugated via a linker sequence to a MIC-1 comprising a native MIC-1 or variant MIC-1 amino acid sequence at the C-terminus of the conjugate. The peptide linker is 10 to 50 amino acids in length and comprises the amino acid sequence [X-Ym]n, wherein X is Asp or Glu, Y is Ala, m is from 2 to 4, and n is at least 2.
In some embodiments, the first agent comprises a MIC-1 conjugate that is a conjugate of MIC-1 and one or more Fc polypeptides. The term “Fc polypeptide” will be well understood by those skilled in the art as referring to the non-antigen binding portion or “crystallisable fragment” of an immunoglobulin or antibody, or fragment thereof, through which an antibody is able to mediate effector functions, such as binding to a cellular receptor and inducing immune responses. The Fc conjugate partner may enable the formation of a dimer of the MIC-1 conjugate by dimerisation between Fc polypeptides. Some particular examples of MIC-1-Fc polypeptide conjugates are described in International patent publication No WO 2013/113008 (the entire disclosure of which is incorporated herein by reference).
Where the method involves administering a pharmaceutical composition or combination pharmaceutical composition comprising a first agent that comprises recombinant MIC-1, preferably the recombinant MIC-1 is recombinant human MIC-1 (rhMIC-1), preferably in dimeric form, or a conjugate of rhMIC-1 (eg a conjugate of rhMIC-1 with an Fc polypeptide in dimeric form).
The method may involve administering a pharmaceutical composition or combination pharmaceutical composition comprising a first agent that comprises a MIC-1 agonist. As used herein, the term “MIC-1 agonist” is to be understood as referring to any agent that binds to and activates a MIC-1 receptor30, 69, 70 to produce, at least partially, a biological response or activity of MIC-1 (eg agents which mimic an activity of MIC-1). Suitable MIC-1 agonists may, for example, stimulate glycosaminoglycan and collagen production in simple assays such as those described in WO 97/000958 supra (where an observed increase in the production of these matrix proteins indicates MIC-1 agonist activity). Additionally or alternatively, suitable MIC-1 agonists may be identified by, for example, bioassay (eg by subcutaneously administering the agent to mice and monitoring body weight and food intake to confirm that they are decreased). Examples of such bioassays are described in WO 2015/197446 supra. Another approach to identifying suitable MIC-1 agonists involves the incubation of the agent with a cell line stably expressing the MIC-1 receptor and co-receptor RET and detecting an increase in phosphorylation levels of any one or more of RET, extracellular signal-regulated kinase (ERK) and the serine/threonine-specific protein kinase AKT69. Further bioassays that are useful in identifying and/or assessing suitable MIC-1 agonists are described in International patent publication No WO 2017/121865 (the entire disclosure of which is incorporated herein by reference).
As such, the term “MIC-1 agonist” encompasses agents comprising an active MIC-1 fragment, analogues and peptide mimetics of the active domains of MIC-1, and small organic molecules which mimic MIC-1 activity. Active MIC-1 fragments may be conjugated with a conjugate partner selected from the group comprising fusion partners to improve protein recovery or expression, and Fc polypeptides and other polypeptides capable of dimerising.
In some embodiments, the method involves administering a pharmaceutical composition or combination pharmaceutical composition comprising a first agent that comprises an active MIC-1 fragment. Suitable active MIC-1 fragments preferably comprise a fragment of MIC-1 that retains the characteristic and highly conserved seven-cysteine domain of members of the TGF-β superfamily (which spans about 80 amino acids and encompasses most of the mature form of the respective proteins) but which lacks one or more of the N-terminal sequence amino acids of the mature MIC-1 protein (eg amino acid (aa) 1-13); that is, an N-terminal truncation. Particular examples include (Δ1-13)MIC-1 (where all of the 13 N-terminal amino acids have been deleted), (Δ1-10)MIC-1, (Δ1-5)MIC-1, (Δ1-3)MIC-1 (specifically described in the abovementioned WO 2017/202936) and (Δ1)MIC-1 (where only the N-terminal amino acid of the mature MIC-1 protein (alanine (Ala) in mature human MIC-1) is deleted). An active MIC-1 fragment may comprise an active variant MIC-1 amino acid sequence as described above, but preferably, comprises an active fragment of a MIC-1 comprising a native MIC-1 amino acid sequence. Active MIC-1 fragments suitable for use in the method of the first aspect can be readily produced by those skilled in the art by using, for example, standard recombinant or protein synthesis techniques. A first agent comprising an active MIC-1 fragment may further comprise a conjugate partner selected from the group comprising fusion partners to improve protein recovery or expression, and Fc polypeptides and other polypeptides capable of dimerising.
The method may involve administering a pharmaceutical composition or combination pharmaceutical composition comprising a first agent that comprises a MIC-1-inducing agent. As used herein, the term “MIC-1-inducing agent” is to be understood as referring to any agent that induces, directly or indirectly, expression of endogenous MIC-1 in a subject. Suitable MIC-1-inducing agents may, for example, be identified by simple MIC-1 expression assays such as those described in WO 97/000958 supra involving the culture of macrophages (eg peripheral blood derived macrophages), monocytoid cell lines (eg U937) and/or human neonatal fibroblast cell lines (eg CCD34Lu) with the agent and determining increased expression of MIC-1 mRNA and/or MIC-1 protein.
As such, the term “MIC-1-inducing agent” encompasses agents which enhance transcription or translation of the MIC-1 gene such as the p53 transcription factor (which is often seen in elevated levels in diseases associated with MIC-1 over-expression), factors which enhance p53 expression or activity (eg nutlin62), non-steroidal anti-inflammatory drugs (NSAIDS) known to induce MIC-1 expression (eg sulindac sulfide and other NSAIDS that induce expression of the EGR-1 transcription factor63-65), and various anti-tumourigenic compounds (eg resveratrol, genistein, diallyl disulfide, retinoid 6-[3-adamantyl)-4-hydroxyphenyl]-2-naphthalene carboxylic acid, 2-(4-amino-3-methylphenyl)-5-fluorobenzothiazole, and peroxisome proliferator-activated receptor-γ ligands63).
As will be well known to those skilled in the art, a first agent comprising MIC-1, a MIC-1 agonist and/or a MIC-1-inducing agent as described above (particularly, a first agent that comprises an active MIC-1 fragment) may also comprise other compounds, complexing agents and substances which may be covalently or non-covalently linked, which may confer various additional functionalities or properties, such as improved bioavailability. For example, a first agent may comprise MIC-1, a MIC-1 agonist and/or a MIC-1-inducing agent that is covalently or non-covalently linked to polyethylene glycol polymer chains (eg PEGylation) and various well known absorption enhancers (particularly for improving oral bioavailability) including, for example, fatty acids and their derivatives (eg oleic acid, linoleic acid, caprylic acid, capric acid, mono and di-glycerides, and acylcarnitines such as lauroyl-L-carnitine chloride and palmitoylcarnitine chloride), cationic polymers (eg chitosan and its derivatives) and anionic polymers (eg carbopol and polyacrylic acid derivatives, and N-acetyl cysteine), and monomers for the formation of hydrogels (eg 2-hydroxyethyl methacrylate, ethylene glycol dimethacrylate, N-isopropyl acrylamide, acrylic acid and methacrylic acid).
Notwithstanding the above, a first agent comprising MIC-1 and/or a MIC-1 agonist suitable for use in the method of the first aspect may additionally or alternatively, comprise amino acid sequences that have been modified either by natural processes, such as post-translational processing, or by chemical modification techniques such as those well known to those skilled in the art. Such modifications can occur anywhere in the molecule, including for example, within the peptide backbone, the amino acid side-chains and/or the C-terminal. It will also be appreciated that the same types of modifications may be present in the same or at varying degrees at several sites in the molecule. One particular example of chemical modification is C-terminal amidation. Accordingly, the MIC-1 and/or MIC-1 agonist of the first agent may have an amide group at the C-terminal. Methods for the amidation of the C-terminal of a polypeptide (eg α-amidation) are well known to those skilled in the art. The C-terminal amide may avoid carboxypeptidase degradation of the MIC-1 or MIC-1 agonist.
The method may involve administering a pharmaceutical composition or combination pharmaceutical composition comprising a second agent that comprises a leptin agent such as recombinant leptin or synthetic leptin (ie leptin produced by protein synthesis techniques) comprising a native leptin amino acid sequence (eg the native amino acid sequence of the 146 aa mature human leptin protein) or a variant leptin amino acid sequence. Suitable variant leptin amino acid sequences may include a naturally occurring or non-natural variant amino acid sequence of a native amino acid sequence that may include one or more minor sequence variations which, preferably, do not substantially alter the function of the polypeptide (eg despite the variation(s), the polypeptide maintains the ability of binding to and activating a leptin receptor (LEP-R, but also known as OB-R)). One example of a non-natural variant of leptin is the leptin W100E variant (comprising a Trp→Glu substitution at position 100)51. Other variants of leptin amino acid sequences may include one or more conservative amino acid substitutions such as: G, A, V, I, L, M; D, E; N, Q; S, T; K, R, H; F, Y, W, H; and P, Nα-alkylamino acids. Other substitutions may include the substitution of one or more L-amino acid(s) with a D-amino acid(s). Preferably, any amino acid substitution comprises a substitution with an amino acid selected from the twenty (20) standard amino acids encoded by the genetic code (ie the canonical amino acids). However, amino acid substitutions with non-canonical amino acids such as those mentioned above at paragraph [0047], are also contemplated. Other sequence variations that may be present include one or more amino acid deletion or addition (eg insertion). Other additions that may be made to, for example, the N- or C-terminal sequence may comprise the addition of a single amino acid (eg Ala or Met; nb. an N-methionyl residue is included in one well known analogue of human leptin, namely N-methionyl leptin (metrepletin) which is considered to be suitable for use in the method of the first aspect), short amino acid sequences (eg 2 to 10 amino acids in length) or long amino acid sequences (eg 11 or more amino acids) which may confer various additional functionalities or properties, such as improved bioavailability, protein recovery or expression (eg a fusion partner). Variant leptin agents comprising an additional long amino acid amino sequence (eg 11 or more amino acids) at the N- or C-terminal may be considered as a leptin conjugate. Leptin conjugates suitable for use in the method of the first aspect may comprise, for example, a conjugate of a leptin with a conjugate partner selected from the group comprising fusion partners to improve protein recovery or expression (eg Human serum albumin (HSA), Glutathione S-transferase (GST), and various affinity-tags such as a polyhistidine tag (His-tag) or FLAG-tag). The leptin molecule of a leptin conjugate may optionally be conjugated to the conjugate partner via a linker sequence as is well known to those skilled in the art.
Where the method involves administering a pharmaceutical composition or combination pharmaceutical composition comprising a second agent that comprises recombinant leptin, preferably the recombinant leptin is recombinant human leptin (rhLeptin) or a conjugate of rhLeptin (eg a conjugate of rhLeptin with HSA).
The method may involve administering a pharmaceutical composition or combination pharmaceutical composition comprising a second agent that comprises a leptin agonist. As used herein, the term “leptin agonist” is to be understood as referring to any agent that binds to and activates a leptin receptor to produce, at least partially, a biological response or activity of leptin (eg agents which mimic an activity of leptin). Suitable leptin agonists may be readily assayed to determine their ability to bind to and activate a leptin receptor (LEP-R) using a cellular assay as described in, for example, International patent publication No WO 2009/108340 (the entire disclosure of which is incorporated herein by reference), wherein a cell line expressing LEP-R will be stimulated to grow as a result of treatment with an agent with leptin agonist activity. Alternatively or additionally, suitable leptin agonists may be identified by bioassay involving determining their ability to substitute for leptin in leptin-deficient animals.
As such, the term “leptin agonist” encompasses agents comprising an active leptin fragment, analogues and peptide mimetics of the active domains of leptin such as the 12-residue glycosylated leptin-based peptidomimetic E1/6-amino-hexanoic acid (Aca)52 and other molecules based upon the third receptor-binding region (“site III”) of leptin, and small organic molecules which mimic leptin activity. Active leptin fragments may be conjugated with a conjugate partner selected from the group comprising fusion partners to improve protein recovery or expression.
In some embodiments, the method involves administering a pharmaceutical composition or combination pharmaceutical composition comprising a second agent that comprises an active leptin fragment. Suitable leptin fragments may include leptin 22-56, leptin 57-9235, leptin 93-12243 and leptin 116-13036. Further examples of active leptin fragments which may be suitable for use in the method of the first aspect have been described in International patent publication No WO 96/34885 (the entire disclosure of which is incorporated herein by reference) and include fragments of leptin comprising amino acids 26 to 39 (ie leptin 26-39), 74 to 88, 93 to 113 or 142 to 161. An active leptin fragment may comprise an active variant leptin amino acid sequence as described above, but preferably, comprises an active fragment of a leptin comprising a native leptin amino acid sequence. Active leptin fragments suitable for use in the method of the first aspect can be readily produced by those skilled in the art by using, for example, standard recombinant or protein synthesis techniques. A second agent comprising an active leptin fragment may further comprise a conjugate partner selected from the group comprising fusion partners to improve protein recovery or expression.
As will be well known to those skilled in the art, a second agent comprising leptin and/or a leptin agonist as described above (particularly, a second agent that comprises an active leptin fragment) may also comprise other compounds, complexing agents and substances which may be covalently or non-covalently linked, which may confer various additional functionalities or properties, such as improved bioavailability. For example, a second agent may comprise leptin and/or a leptin agonist that is covalently or non-covalently linked to polyethylene glycol polymer chains (eg PEGylation) and various well known absorption enhancers (particularly for improving oral bioavailability) including, for example, fatty acids and their derivatives, cationic polymers and anionic polymers, and monomers for the formation of hydrogels. In addition, and notwithstanding the above, a second agent comprising leptin and/or a leptin agonist suitable for use in the method of the first aspect may additionally or alternatively, comprise amino acid sequences that have been modified either by natural processes, such as post-translational processing, or by chemical modification techniques such as those well known to those skilled in the art. Such modifications can occur anywhere in the molecule, including for example, within the peptide backbone, the amino acid side-chains and/or the C-terminal. It will also be appreciated that the same types of modifications may be present in the same or at varying degrees at several sites in the molecule. One particular example of chemical modification is C-terminal amidation (ie to provide the leptin or leptin agonist with a C-terminal amide group) to prevent carboxypeptidase degradation.
In one particular embodiment, the method of the first aspect involves administering a first pharmaceutical composition comprising a first agent that comprises MIC-1 or a MIC-1 conjugate, and a second pharmaceutical composition comprising a second agent that comprises leptin or a leptin conjugate. The first and second pharmaceutical compositions may be administered concurrently or consecutively in either order.
In another particular embodiment, the method of the first aspect involves administering a combination pharmaceutical composition comprising a first agent that comprises MIC-1 or a MIC-1 conjugate, and a second agent that comprises leptin or a leptin conjugate.
As mentioned above, the method of the first aspect may be conducted to achieve body weight control in an overweight or obese subject. However, the method may also be useful for a subject who might otherwise desire weight loss for reasons of well-being or vanity.
The method of the first aspect may be particularly useful for an overweight or obese subject who is suffering Type 2 diabetes (T2D). As is well known, T2D is a major complication of obesity and several epidemiological studies have linked MIC-1 to diabetes and insulin resistance. For example, increased serum MIC-1 levels have been found to independently predict the presence of insulin resistance28, 39 as well as progress to Type 2 diabetes40-42. Thus, it is expected that the administration of a first agent comprising MIC-1, a MIC-1 agonist and/or a MIC-1-inducing agent along with a second agent comprising leptin and/or a leptin agonist, will provide a useful therapy for diabetes and/or one or more complications of T2D, obesity or overweight (eg non-alcoholic steatohepatitis (NASH) or glucose intolerance) both because of body weight reduction and the direct effects of MIC-1 (or the MIC-1 agonist) on glucose tolerance and homeostasis.
Typically, the subject will be a human. However, the method of the first aspect may also be applicable to non-human subjects such as, for example, livestock (eg cattle, sheep and horses), exotic animals (eg tigers, lions, elephants and the like) and companion animals (such as dogs and cats).
The first aspect of the present disclosure also relates to the use of:
In a second aspect, the disclosure relates to a pharmaceutical composition comprising:
The pharmaceutical composition may be suitable for, for example, oral, buccal, nasal, intramuscular and intravenous administration. The composition may further comprise, for example, a pharmacologically acceptable carrier and/or excipient, optionally together with others substances such as absorption enhancers and protease inhibitors for improving bioavailability, especially oral bioavailability. The composition may be intended for single daily administration, multiple daily administration, or controlled or sustained release, as needed to achieve the most effective results.
The determination by the present Applicant that treatment with MIC-1 and leptin caused significantly greater weight loss in an obese mouse model than was observed with MIC-1 or leptin alone (see Examples 2 and 3 hereinafter), suggests that where a subject (eg an overweight or obese subject) has a higher than normal serum leptin level (ie a serum leptin level that is typical of most obese subjects and is therefore higher than the typical range for a normal non-obese subject), then it may be sufficient, in a method of controlling body weight and/or appetite in a subject, to administer only the first agent comprising MIC-1, a MIC-1 agonist and/or MIC-1-inducing agent (that is, there may be no need to administer the second agent comprising leptin and/or a leptin agonist).
Thus, in a third aspect, the present disclosure relates to a method of controlling body weight (eg achieving weight loss) and/or appetite in a subject, said method comprising the steps of:
determining whether said subject has a low, normal or high serum level of leptin; and
administering to said subject, where determined as having a low or normal serum level of leptin, an effective amount of:
administering to said subject, where determined as having a high serum level of leptin, an effective amount of:
As mentioned above, for normal (ie non-obese) human subjects, the normal serum level range of leptin is broad; for example, about 3.5 to 14.0 ng/ml for adult women and 4.5 to 25.5 ng/ml for adult men after overnight fasting. Thus, a relatively high serum leptin level for a normal human subject may be regarded as a level at the higher end of these ranges (eg more than about 10 ng/ml for adult women and more than about 18 ng/ml for adult men). Such higher than normal serum leptin levels may be caused by, for example, impairment of leptin regulation associated with certain diseases and conditions such as chronic inflammatory diseases (such as inflammatory bowel disease, inflammatory nephritis, pelvic endometriosis, non-alcoholic hepatitis, chronic pulmonary inflammation, Behcet's disease and Graves' disease') and some cancers (such as breast cancer38). On the other hand, for an obese human subject, the serum leptin level would normally be expected to be high (ie as described above at paragraph [0041]), and a relatively low serum leptin level for an obese human subject (after overnight fasting) may therefore be, for example, for an adult woman, less than about 30 ng/ml or, more preferably, less than 25 or less than 20 ng/ml, and for an adult man, less than about 20 ng/ml or, more preferably, less than 15 ng/ml.
The step of determining the relative level of serum leptin may comprise measuring serum leptin levels in accordance with any suitable assay method known to those skilled in the art including standard immunoassays (eg a leptin ELISA).
The step of administering the first and second agents may be as described above in relation to the method of the first aspect.
The third aspect of the present disclosure also relates to the use of:
In a fourth aspect, the present disclosure relates to a kit comprising first and second containers (eg vials), wherein the first container contains a first agent comprising MIC-1, a MIC-1 agonist and/or a MIC-1-inducing agent, and the second container contains a second agent comprising leptin and/or a leptin agonist; optionally packaged with instructions for the use of the kit in the method of the first or third aspects.
The first and second agents provided in the containers of the kit may be as described above in relation to the method of the first aspect. The kit may further comprise materials and agents for assaying MIC-1 and/or leptin as described above.
The present Applicant has previously described methods for treating anorexia or cachexia by administering to a subject suffering from such condition a MIC-1-inhibiting agent such as an antagonistic anti-MIC-1 monoclonal antibody21, 22. It is considered that the finding described hereinafter in the Examples, that treatment with MIC-1 and leptin caused significantly greater weight loss in an obese mouse model than was observed with MIC-1 or leptin alone, is also of relevance to the treatment of anorexia and cachexia. Further, it was found that when administered to normal (non obese) chow fed mice, the combination of MIC-1 and leptin caused a much greater loss of lean mass which is considered especially important in the pathogenesis of anorexia/cachexia syndromes. Thus, it is expected that treatment of an anorexic/cachectic subject with agents to inhibit both MIC-1 and leptin, will achieve significantly improved results (ie in the form of weight gain or increase in fat and lean mass, or at least, a reduction in any further loss of body weight or lean or fat mass) than that achieved by treatment with a MIC-1-inhibiting agent alone.
Thus, in a fifth aspect, the present disclosure relates to a method of increasing body weight and/or appetite in a subject, said method comprising administering to said subject an effective amount of:
The method of the fifth aspect may be conducted to achieve an increase in body weight and/or appetite or, at least, control of body weight and/or appetite in a subject. For example, the method may be conducted to achieve body weight gain in a subject or, at least, a reduction in any loss of body weight (eg to prevent further weight loss in an anorexic/cachectic subject). The subject may be, for example, a subject suffering from anorexia or cachexia. Cachectic subjects may be subjects suffering from decreased appetite and/or weight loss associated with inflammatory disease (eg rheumatoid arthritis), chronic renal and/or cardiac failure, and/or cancer (particularly, an epithelial cancer such as breast, prostate, colonic, rectal, bladder, lung and pancreatic cancer). The method may, however, also be useful for the treatment of decreased appetite and/or weight loss associated with any other disease, condition or treatment wherein MIC-1 is over-expressed (eg injury, inflammation, stress, and radiotherapy and chemotherapy). Subjects suitable for treatment with the method of the fifth aspect may therefore show MIC-1 over-expression or, at least, a serum level of MIC-1 that is consistently at the high end of the normal serum level of 0.15-1.15 ng/ml. Such subjects can be determined by the detection of a relatively high serum MIC-1 level (eg from a whole blood or serum sample) using any suitable assay for MIC-1 known to those skilled in the art including standard immunoassays (eg a MIC-1 ELISA4). Additionally, subjects suitable for treatment with the method of the fifth aspect may show a relatively high serum level of leptin. For example, a serum level of leptin at the higher end of the normal ranges mentioned above (eg more than about 10 ng/ml for adult women and less than about 18 ng/ml for adult men). As mentioned above, the relative level of serum leptin may be determined by measuring serum leptin levels in accordance with any suitable assay method known to those skilled in the art including standard immunoassays (eg a leptin ELISA).
In one particular embodiment of the method of the fifth aspect, the subject is a cachectic subject suffering from decreased body weight and/or appetite associated with advanced cancer, where a high total tumour mass often leads to a high serum level of MIC-1. Leptin is also commonly over-expressed in cancerous tissues, especially those of the pancreas, colon, lung, breast, ovary and prostate (nb. the searchable database, The Human Protein Atlas, reveals that leptin is over-expressed at both the mRNA and protein levels in a significant proportion of patients with malignant tumours in these organs). Also, as mentioned above, some cancers (eg breast cancer) are known to cause higher than normal serum leptin levels; however, since leptin is prominently or mainly expressed in adipose tissue (ie such that leptin levels may, at least in part, reflect the fat mass of a subject) which is typically rapidly lost in cachexia, the effect of a leptin-expressing tumour on the serum leptin level may be greatly offset by the reduced level of leptin expression from adipose tissue (ie due to loss of adipose tissue). Thus, a cachectic subject with advanced cancer may or may not also show a high serum level of leptin, but typically, will at least show a higher serum leptin level than that of other age-, height- and gender-matched subjects with a comparable degree of adiposity.
The MIC-1-inhibiting agent may decrease the amount of MIC-1 (ie endogenous MIC-1) in the subject (particularly, the serum level of MIC-1) and/or decrease the activity of MIC-1 in the subject.
The leptin-inhibiting agent may decrease the amount of leptin (ie endogenous leptin) in the subject (particularly, the serum level of leptin) and/or decrease the activity of leptin in the subject.
The MIC-1-inhibiting agent may, for example, be administered to the subject concurrently (eg in combination) with the leptin-inhibiting agent, or otherwise the MIC-1-inhibiting agent and the leptin-inhibiting agent may be administered to the subject consecutively and in either order. As such, when administered consecutively, the respective agents may be administered one after another with practically no time interval (ie one is administered effectively immediately after the other) or, otherwise, after an interval of, for example, 1 to 5 minutes, 10 minutes, 30 minutes, 60 minutes, 4 hours or 12 hours or more. Where the MIC-1-inhibiting agent and leptin-inhibiting agent are administered consecutively, they may each be formulated for administration in a pharmaceutical composition form including a pharmacologically acceptable carrier and/or excipient, which may be the same or different. The pharmaceutical compositions may be suitable for, for example, oral, buccal, nasal, intramuscular and intravenous administration. The pharmaceutical compositions may optionally comprise other substances such as absorption enhancers (including surfactants) and chelating agents. Typically, the pharmaceutical compositions will be administered to the subject in an amount which is effective for increasing body weight and/or appetite. The composition comprising the MIC-1-inhibiting agent may be administered to provide, for example, an amount of the agent that is between about 0.01 and about 100 mg/kg body weight per day, or between about 0.05 and 25 mg/kg body weight per day of the agent. The composition comprising the leptin-inhibiting agent may be administered to provide, for example, an amount of the agent that is between about 0.01 and about 1000 mg/kg body weight per day, or between about 1 and 750 mg/kg body weight per day of the agent. However, it will be understood by those skilled in the art that the administered amount of the pharmaceutical compositions, and the frequency of administration for any particular subject, may vary and depend upon a variety of factors including the activity of the active agents (eg the MIC-1-inhibiting agent and the leptin-inhibiting agent), the metabolic stability and length of action of the active agents, the age, body weight, sex, mode and time of administration, and the rate of excretion of the active agents. The pharmaceutical compositions may be intended for single daily administration, multiple daily administration, or controlled or sustained release, as needed to achieve the most effective results.
The MIC-1-inhibiting agent may comprise an antagonistic anti-MIC-1 antibody (ie a neutralising MIC-1 antibody) or a fragment thereof (eg MIC-1-binding fragments including those selected from Fab fragments, recombinant scFv fragments44 and single-domain (sdAb) antibodies), catalytic or inhibitory oligonucleotide molecule targeted against the MIC-1 gene (eg a ribozyme, DNAzyme, antisense RNA, or small inhibitory RNA (siRNA)), inhibitor of MIC-1 transcription or translation, a soluble extra-cytoplasmic receptor domain of the MIC-1 receptor30, 69, 70, or a peptide or small organic molecule which inhibits, for example, MIC-1 binding to its receptor, MIC-1 receptor phosphorylation, or MIC-1 receptor signalling. One example of a soluble extra-cytoplasmic receptor domain of the MIC-1 receptor that may be suitable for use as a MIC-1-inhibiting agent is a protein expressed by an alternative murine mRNA splice variant denoted GRAL-B72, or a human ortholog thereof; which lack a transmembrane and cytoplasmic domain. Suitable MIC-1-inhibiting agents may be assayed by one or more simple assays relevant to the intended mechanism of inhibition. For example, antagonistic anti-MIC-1 antibodies may be assessed for their ability to bind to MIC-1 using standard immunoassays and/or inhibit (antagonise) MIC-1 activities observed in the assays and bioassays described above at paragraph [0051]. For some other MIC-1-inhibiting agents it may be more relevant to determine inhibition of expression of MIC-1 mRNA or MIC-1 protein in the cellular assays described in paragraph [0054] above.
In some embodiments, the MIC-1-inhibiting agent comprises an antagonistic anti-MIC-1 antibody or a fragment thereof. Preferably, the MIC-1-inhibiting agent comprises a human anti-MIC-1 antibody (such as may be produced by well known techniques using transgenic mice or phage display45, 46) or a chimeric or humanised anti-MIC-1 antibody (such as may be produced by well known techniques described in, for example, U.S. Pat. No. 5,225,539 (the entire disclosure of which is incorporated herein by reference), by specificity determining residue (SDR) grafting47, by affinity maturation using phage display48, or using heavy chain complementarity-determining region 3 grafting coupled with in vitro somatic hypermutation49).
The leptin-inhibiting agent may comprise an antagonistic anti-leptin antibody (ie a neutralising leptin antibody) or a fragment thereof (eg leptin-binding fragments including those selected from Fab fragments, recombinant scFv fragments and single-domain (sdAb) antibodies), a catalytic or inhibitory oligonucleotide molecule targeted against the leptin (ob or lep) gene (eg a ribozyme, DNAzyme, antisense RNA, or small inhibitory RNA (siRNA)), an inhibitor of ob transcription or translation (eg silibinin and curcumin50, a soluble extra-cytoplasmic receptor domain of the leptin receptor (eg the LEP-R:Fc fusion protein described by De Rosa et al.55), an antagonistic anti-leptin receptor antibody (ie a neutralising anti-LEP-R antibody54) or a fragment thereof (eg leptin receptor-binding fragments including those selected from Fab fragments, recombinant scFv fragments and single-domain (sdAb) antibodies), or a leptin mutant, peptide or small organic molecule which inhibits, for example, leptin binding to its receptor, leptin receptor phosphorylation, or leptin receptor signalling (eg by binding, but not activating, the leptin receptor). One suitable example of a leptin mutant includes an Arg→Glu substitution at position 128 that abolishes biological activity without affecting receptor binding53. Another suitable example of a leptin mutant is a dominant negative form of leptin66, 74. Suitable leptin-inhibiting agents may be assayed by one or more simple assays relevant to the intended mechanism of inhibition. For example, antagonistic anti-leptin antibodies may be assessed for their ability to bind to leptin using standard immunoassays and/or inhibit (antagonise) leptin activities observed in the assays and bioassays described above at paragraph [0060]. For some other leptin-inhibiting agents it may be more relevant to determine inhibition of expression of leptin mRNA or leptin protein in suitable cellular assays.
In some embodiments, the leptin-inhibiting agent comprises an antagonistic anti-leptin antibody or a fragment thereof. Preferably, the leptin-inhibiting agent comprises a human anti-leptin antibody, or a chimeric or humanised anti-leptin antibody. Such antibodies may be produced using, for example, any of the well known methods described in paragraph [0088] above.
The fifth aspect of the present disclosure also relates to the use of:
In a sixth aspect, the disclosure relates to a pharmaceutical composition comprising:
The pharmaceutical composition of the sixth aspect may be suitable for, for example, oral, buccal, nasal, intramuscular and intravenous administration. The composition may further comprise, for example, a pharmacologically acceptable carrier and/or excipient, optionally together with others substances such as absorption enhancers and protease inhibitors for improving bioavailability, especially oral bioavailability. The composition may be intended for single daily administration, multiple daily administration, or controlled or sustained release, as needed to achieve the most effective results.
In a seventh aspect, the present disclosure relates to a kit comprising first and second containers (eg vials), wherein the first container contains a MIC-1-inhibiting agent and the second container contains a leptin-inhibiting agent; optionally packaged with instructions for the use of the kit in the method of the fifth aspect.
The MIC-1-inhibiting agent and leptin-inhibiting agent provided in the containers of the kit of the seventh aspect may be as described above in relation to the method of the fifth aspect. The kit may further comprise materials and agents for assaying MIC-1 and/or leptin as described above.
The disclosure is hereinafter expanded by way of the following non-limiting examples and accompanying figures.
A study was conducted using groups of ten (10) male 22 week old C57BL6 mice, CHOW fed or with diet-induced obesity (DIO) from 12 weeks of a high fat diet (HFD). The mice were infused via osmotic minipump (ag Alzet® model 2002; Durect Corporation, Cupertino, Calif., United States of America) with either vehicle (CHOWc or DIOc) or recombinant murine MIC-1 (0.5 μg/gBW/day; CHOWm or DIOm) produced in accordance with a previously described method21. It was found that MIC-1 induced sustained weight loss, accompanied by a decrease in food intake (
The data obtained in this study therefore indicates that MIC-1 has very favourable characteristics for its use as an anti-obesity agent. In particular, the study found that MIC-1 is much more effective in obese than lean mice and, in further support of this, also found that the rate of weight loss in the obese mice becomes progressively less as those mice lost weight and it causes insignificant loss of lean mass. In similar experiments72 conducted over a period of 34 days (ie prolonged infusion with recombinant MIC-1 for 34 days) with mice fed on normal chow or HFD for 16 weeks in order to induce DIO, it was also observed that MIC-1 decreased food intake and body weight of DIO mice and chow fed mice compared with vehicle treated control mice. In the DIO mice, these effects were also accompanied by a greater reduction in fat mass. Further, whilst MIC-1 treated chow fed mice lost lean as well as fat-mass, the MIC-1 treated DIO mice lost fat mass alone. The reduction in body weight and adiposity was due largely to reduced food intake, but MIC-1 treated DIO mice also displayed increased energy expenditure that may be due to increased thermogenesis. In addition, the MIC-1 treated DIO mice showed higher circulating levels of adiponectin and lower tissue expression and circulating levels of leptin and inflammatory mediators associated with insulin resistance, and displayed marked improvement in the steatohepatitis of obesity. Peripheral insulin and glucose intolerance were improved in both MIC-1 treated DIO and normal chow fed mice compared to vehicle-treated control mice.
A study was conducted using chow fed 11 week old C57BL6 mice continuously infused (using a standard osmotic minipump) with either vehicle, recombinant murine MIC-1 (0.5 μg/g body weight/24 hrs), leptin (0.5 μg/g body weight/24 hrs), or MIC-1 plus leptin (0.5 μg/g body weight MIC-1+0.5 μg/g body weight leptin/24 hrs) for six (6) days. The leptin was recombinant murine leptin sourced from Creative Biomart (Shirley, N.Y., United States of America).
The results are shown in
The data obtained in this study also indicates that MIC-1 has very favourable characteristics for its use as an anti-obesity agent, particularly when used in combination with leptin. It is considered that prolonged use of MIC-1 and/or leptin will lead to sustained weight loss and correct the metabolic consequences of diet-induced obesity (DIO).
Vehicle, murine leptin (0.5 μg/g/day) and recombinant murine MIC-1 (0.5 μg/g/day) were infused in binary combinations into normal chow fed male mice and male mice rendered obese (mice with diet-induced obesity (DIO)) and leptin-resistant after 24 weeks on a high fat diet. The results are shown in
The results confirm that HFD mice are more responsive to MIC-1-induced weight loss than chow fed mice. In particular, it was observed in this study that the HFD mice lost proportionately more weight and continued to lose weight whilst the chow fed mice weight loss plateaued by about 11 days. In addition, the results showed that when using doses of leptin that are equipotent with MIC-1 in inducing weight loss in chow fed mice (see
Further experiments were conducted to demonstrate and characterise an interaction between MIC-1 and leptin in mice with diet induced obesity (DIO) mice and normal chow fed mice, using 7 mice per group. The methods used were similar to those described in relation to Examples 1-3 and/or were as previously described71. Each mouse was implanted with two osmotic minipumps (Alzet model 2002; Durect Corporation). One pump contained either vehicle or MIC-1 (recombinant mature domain of murine MIC-1 expressed and secreted from P. pastoris grown in minimal media and highly purified in a 3 step procedure, then lyophilised and stored at −80° C. until use, at which time it was reconstituted in 4 mM HCL then diluted to the appropriate concentration in PBS vehicle) and the second pump contained either vehicle or leptin (recombinant murine leptin produced in E. coli as a single, non-glycosylated, polypeptide chain consisting of 147 aa's, with a molecular weight of 16.1 kDa). Vehicle or MIC-1 was infused via the minipump at 0.5 μg/gBW/day for the duration of the experiment, while vehicle or leptin was infused at 0.2 μg/gBW/day for the duration of the experiment. Body weight (BW) was monitored for the first 13 days during which mice were largely undisturbed. Whole body fat-mass and lean-mass were measured during treatment using dual-energy X-ray absorptiometry (DEXA; Lunar PIXImus2 mouse densitometer; GE Healthcare, Waukesha, Wis., United States of America). Investigations such as DEXA and food intake measurements, which substantially disrupt mouse behaviours, were undertaken between days 15 and 19, over which period body weight was not monitored. Effects on normal liver and liver from DIO were investigated using tissue from dissected livers fixed, sectioned and stained with haematoxylin and eosin. Standard glucose tolerance tests (GTT) and insulin sensitivity tests (ITT) were also performed (at day 14) on the treated mice.
Results are shown in
As it had been previously demonstrated, it was found that DIO mice treated in these experiments with MIC-1 lost a substantial amount of body weight. On the other hand, DIO mice treated with leptin alone (ie leptin+vehicle) displayed unaltered body weight, which is consistent with well known leptin resistance of severely obese mice and humans. However, when combined with MIC-1, MIC-1+leptin treated mice lost further body weight over mice treated only with MIC-1 (ie MIC-1+vehicle), suggesting a synergistic relationship between MIC-1 and leptin (
It was observed that the body weight lost by the DIO mice treated with MIC-1 or the combination of MIC-1+leptin was largely fat mass (see
Using liver sections prepared as described above, histology clearly demonstrated the presence of steatohepatitis in vehicle treated DIO mice (data not shown) compared to the normal mouse liver. Hepatic steatosis is a major cause of morbidity in obesity and can lead to non-alcoholic steatohepatitis (NASH) and sometimes cirrhosis and liver failure. It was found that the observed steatohepatitis improved over the treatment period in the mice infused with MIC-1 alone (ie MIC-1+vehicle), and returned essentially to normal with infusion of the combination of MIC-1 and leptin treatment, indicating that these agents offer considerable promise as a combination therapy to treat or prevent steatohepatitis and thereby reduce the risk of development of complicating cirrhosis and liver failure.
With the IGT and ITT, it was found that the combination treatment with leptin potentiated a previously recognised ability of MIC-1 to improve glucose tolerance and insulin sensitivity of obese mice (see
Ob/Ob mice have a mutant inactive form of leptin and thus these mice have a functionally inactive leptin pathway, which leads to severe, rapidly progressive obesity and its consequences on a normal diet. The mice do, however, have an intact leptin receptor and signalling pathway and can thus respond to the administration of leptin despite not responding to their endogenous mutant leptin. The mice were used in the experimentation here, in order to further examine the interaction between MIC-1 and leptin, in an obesity model without elevated background levels of normal leptin (cf. as occurs in DIO mice). The methods used in the experiments were as described in relation to Examples 1-3 and/or as previously described71.
Ob/ob genetically leptin deficient mice have no bioactive circulating leptin and are severely obese even on a normal chow diet. They experience some weight reduction following treatment with MIC-1 alone although this is less than that seen with mice that are obese because of a high fat diet (see
The results observed in the previous examples, particularly Example 4, suggested that the anorexia/cachexia-inducing activity of over-expressed MIC-1 could be reduced by inhibiting the leptin pathway. In this study, mice were administered with a leptin antagonist (mouse super leptin antagonist (SMLA); Protein Laboratories Rehevot Ltd, Rehevot, Israel) by continuous infusion. SMLA is a mutant mouse leptin molecule with high affinity for the leptin receptor but which that does not activate the leptin receptor and thus acts as a dominant negative competitive inhibitor of leptin action74. The mice were subcutaneously implanted with two osmotic minipumps; one minipump infusing MIC-1 or vehicle (0.5 μg/gBW/day) and the other infusing SMLA or vehicle (1.5 μ/gBW/day). The methods used were as described in relation to Examples 1-3 and/or as previously described72.
Results are shown in
Throughout the specification and the claims that follow, unless the context requires otherwise, the words “comprise” and “include” and variations such as “comprising” and “including” will be understood to imply the inclusion of a stated integer or group of integers, but not the exclusion of any other integer or group of integers.
The reference to any prior art in this specification is not, and should not be taken as, an acknowledgement of any form of suggestion that such prior art forms part of the common general knowledge.
It will be appreciated by those skilled in the art that the subject matter described herein is not restricted in its use to the particular application described. Neither is the subject matter restricted in its preferred embodiment with regard to the particular elements and/or features described or depicted herein, but is capable of numerous rearrangements, modifications and substitutions without departing from the scope as set forth and defined by the following claims.
| Number | Date | Country | Kind |
|---|---|---|---|
| 2016905018 | Dec 2016 | AU | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/AU2017/000262 | 12/6/2017 | WO | 00 |
