CHOLINE KINASE INHIBITORS AS A THERAPEUTIC TREATMENT FOR OBESITY
TECHNICAL FIELD
5 The presently-disclosed subject matter relates to using choline kinase inhibitors to treat obesity, hyperglycemia, type 2 diabetes, supporting weight loss, increasing glucose uptake, and/or activating brown-adipose tissue thermogenesis in a subject in need thereof.
10 BACKGROUND
Obesity, defined by the World Health Organization (WHO) as abnormal or excessive fat accumulation that presents a risk to health, has become a national epidemic in the US. A body mass index (BMI) over 25 is considered overweight and a BMI over 30 is considered obese by the WHO. The onset of diabetes, which is
15 linked to obesity, has also reached epidemic proportions, with a disparate impact on African Americans, who are 60% more likely than European-Americans to be diagnosed with Type II diabetes mellitus (T2DM). Importantly, T2DM is one of the leading causes of premature deaths.
Being overweight or obese typically leads to an increase in non-esterified fatty
20 acids, hormones, cytokines, proinflammatory markers, and other molecules that lead to insulin resistance, generally via inflammation and the subsequent dysregulation of insulin receptors. Insulin resistance is a precursor to T2DM, and increased body mass is a one factor in the rising incidence of T2DM.
Weight loss, especially for obese patients, is an established strategy for
25 preventing the onset of diabetes. However, the best method of managing a patient’s weight is still debated. A wide variety of factors impact weight, including but not limited to inadequate amounts of physical exercise, high caloric intake, as well as individual metabolism. Doctors may recommend their patients decrease their caloric intake and/or increase their physical activity, but such goals can be difficult for
30 individuals.
SUMMARY
In one aspect, the present application discloses a method of enhancing weight loss comprising administering a choline kinase (“ChoK”) inhibitor to a subject
35 in need of weight loss. In one aspect, the present application discloses a method of enhancing weight loss comprising administering a flavonoid-containing compound having choline kinase inhibition activity to a subject in need of weight loss.
In one aspect, the present application discloses a method of enhancing weight loss comprising administering a compound selected from the group of RSM 932A, MN58b, Hemicholinium, EB-3D, EB-3P, ICL-CCIC-0019, and CK-37 to a subject in need of weight loss.
In one aspect, the present application discloses a method of treating hyperglycemia and/or type 2 diabetes in a subject, comprising administering to a subject in need of such treatment an effective amount of a choline kinase inhibitor.
In one aspect, the present application discloses a method of treating hyperglycemia and/or type 2 diabetes in a subject, comprising administering to a subject in need of such treatment an effective amount of a flavonoid-containing compound having choline kinase inhibition activity.
In one aspect, the present application discloses a method of treating hyperglycemia and/or type 2 diabetes in a subject, comprising administering to a subject in need of such treatment an effective amount of a compound selected from the group of RSM 932A, MN58b, Hemicholinium, EB-3D, EB-3P, ICL-CCIC-0019, and CK-37.
In yet another aspect the present application discloses a method of increasing glucose uptake in a subject in need of such uptake, comprising administering to said subject an effective amount of a choline kinase inhibitor.
In yet another aspect the present application discloses a method of increasing glucose uptake in a subject in need of such uptake, comprising administering to said subject an effective amount of an effective amount of a compound selected from the group of RSM 932A, MN58b, Hemicholinium, EB-3D, EB-3P, ICL-CCIC-0019, and CK-37.
In another aspect, the present application discloses a method of activating brown-adipose tissue (“BAT”) thermogenesis comprising administering an effective amount of a flavonoid-containing compound having choline kinase inhibition activity.
In yet another aspect the present application discloses a method of increasing glucose uptake in a subject in need of such uptake, comprising administering to said subject an effective amount of a choline kinase inhibitor.
In another aspect, the present application discloses a method of activating brown-adipose tissue (“BAT”) thermogenesis comprising administering an effective amount of a flavonoid-containing compound having choline kinase inhibition activity. In another aspect, the present application discloses a method of activating brown-adipose tissue (“BAT”) thermogenesis comprising administering effective amount of a compound selected from the group of RSM 932A, MN58b, Hemicholinium, EB-3D, EB-3P, ICL-CCIC-0019, and CK-37.
In another aspect, the present invention relates to a method for treating, preventing or ameliorating a fatty liver disease in a subject in need thereof comprising administering to said subject an effective amount of a choline kinase inhibitor.
In another aspect, the present invention relates to a method for treating, preventing or ameliorating a fatty liver disease in a subject in need thereof comprising administering to said subject an effective amount of a flavonoid-containing compound having choline kinase inhibitor.
In another aspect, the present invention relates to a method for treating, preventing or ameliorating a fatty liver disease in a subject in need thereof comprising administering to said subject an effective amount of a RSM 932A, MN58b, Hemicholinium, EB-3D, EB-3P, ICL-CCIC-0019, and CK-37
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 shows (A) Comparison of ChoKa expression in adipocyte- and preadipocyte- containing fraction/stromal vascular (SV) fraction from mice fed either standard chow or high fat diet (HF) (B) Expression of the PPAGy marker that is normally only expressed in adipocytes.
Figure 2 shows (A) microscopic images of 3T3-L1 Adipocytes treated with different concentrations of MN58b. Insets show adipocytes after addition of the RED-0 reagent on day 6 and (B) A graph of triacylglycerol (TAG) levels after treatment with different concentrations of MN58b.
Figure 3 shows (A) Microscopic images of 3T3-L1 Adipocytes collected on Day 6 of treatment with different concentrations of RSM-932A. Insets show adipocytes after addition of the RED-0 reagent. (B) A graph of TAG levels after treatment with different RSM-932A concentrations.
Figure 4 shows graphs of gene expression related to de novo lipogenesis (A), fatty acid (FA) uptake and esterification (B), and fatty acid oxidation (C) levels after treatment of 3T3-L1 cells with different concentrations of RSM-932A for 6 days.
Figure 5 shows graphs of gene expression related to de novo lipogenesis (A), fatty acid (FA) uptake and esterification (B), and fatty acid oxidation (C) levels after treatment of 3T3-L1 cells with different concentrations of MN58b for 6 days.
Figure 6 shows the effect in a obesity prevention test on (A) total body weight, (B) fat mass and (C) lean mass measured by Echo-MRI of mice fed a high fat diet for six weeks (n=5 per treatment) with administration of MN58b, Metformin or control arm (water).
Figure 7 Determination under an obesity prevention test of the effects of 6 weeks of HFD in mice tissues under control (water), ChoKa inhibition (MN58b) and metformin treatment (Metformin). The graphs show weight of gonadal white adipose tissue (gWAT), subcutaneous fat (SubQ), retroperitoneal fat (Retrofat.), Brown adipose tissue fat (BAT), liver, and skeletal muscle (Muscle).
Figure 8 Mice were fed with a HFD and simultaneously treated in the prevention study with control (water), ChoKa inhibitor (MN58b) and metformin (Metformin). (A) Glucose tolerance test (GTT) was performed at 6 weeks of indicated treatments; (B) Triacylglyride content (TG) in liver wet weight at week 6 was determined; (C) relative total liver weight versus total body weight; (D) Intake of food was determined relative to body weight under same conditions.
Figure 9 shows that weight gain (Figure 8A) and fat gain (Figure 8B) was attenuated under the treatment regime, while skeletal mass remained relatively unaffected (Figure 8C). Data was collected using Echo MRI over time for mice (n=5) in each treatment protocol arm (high fat diet for 15 weeks concomitant to treatment).
Figure 10 shows the effect of treatment of mice with the ChoKa inhibitor MN58b in different tissues under the treatment regime. Mice fed 20 weeks on HFD were treated as described and weight tissues were determined under control (water), ChoKa inhibitor (MN58b) and metformin treatment (Metformin). Masses of liver (liver), gonadal white adipose tissue (gWAT), subcutaneous fat (SubQ), retroperitoneal fat (RetroFat), brown adipose tissue (BAT) and skeletal muscle (Muscle) are shown.
Figure 11 shows a graph of glucose tolerance test (GTT, mg/dl) measurement over time for n=5 mice in the treatment study after 20 weeks of indicated treatments. (A) Glucose tolerance test; relative liver TG accumulation (B) and relative liver weight (C) versus total body weight.
Figure 12 Dose dependent treatment study total weight (A), fat mass (B) and lean mass (C) measurements.
Figure 13 shows a graph of (A) glucose tolerance test (mg/dl) and (B) insulin tolerance test (mg/dl) measurement over time for n=5 mice where MN58b was administered, compared to control and metformin.
Figure 14 shows (A) a graph of body weight after 8 weeks of administration of control (HFD), MN58b, semaglutide (SEMA) or MN58b and semaglutide (SEMA+MN58); (B) a graph of lean mass before treatment after 8 weeks of treatment under the same conditions, and; (C) a graph of fat mass before treatment and after 8 weeks of treatment under the same conditions.
DETAILED DESCRIPTION
The presently disclosed subject matter will now be described more fully. The presently disclosed subject matter can, however, be embodied in different forms and should not be construed as limited to the embodiments set forth herein below and in the accompanying Examples. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the embodiments to those skilled in the art.
All references listed herein, including but not limited to all patents, patent applications and publications thereof, and scientific journal articles, are incorporated herein by reference in their entireties to the extent that they supplement, explain, provide a background for, or teach methodology, techniques, and/or compositions employed herein.
Definitions
While the following terms are believed to be well understood by one of ordinary skill in the art, the following definitions are set forth to facilitate explanation of the presently disclosed subject matter.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which the presently disclosed subject matter belongs.
Following long-standing patent law convention, the terms “a” and “an” refer to “one or more” when used in this application, including the claims.
The term “and/or” when used in describing two or more items or conditions, refers to situations where all named items or conditions are present or applicable, or to situations wherein only one (or less than all) of the items or conditions is present or applicable.
The use of the term “or” in the claims is used to mean “and/or” unless explicitly indicated to refer to alternatives only or the alternatives are mutually exclusive, although the disclosure supports a definition that refers to only alternatives and “and/or.”
As used herein “another” can mean at least a second or more.
The term “comprising”, which is synonymous with “including,” “containing,” or “characterized by” is inclusive or open-ended and does not exclude additional, unrecited elements or method steps. “Comprising” is a term of art used in claim language which means that the named elements are essential, but other elements can be added and still form a construct within the scope of the claim.
As used herein, the phrase “consisting of” excludes any element, step, or ingredient not specified in the claim. When the phrase “consists of” appears in a clause of the body of a claim, rather than immediately following the preamble, it limits only the element set forth in that clause; other elements are not excluded from the claim as a whole.
As used herein, the phrase “consisting essentially of” limits the scope of a claim to the specified materials or steps, plus those that do not materially affect the basic and novel characteristic(s) of the claimed subject matter.
With respect to the terms “comprising”, “consisting of”, and “consisting essentially of”, where one of these three terms is used herein, the presently disclosed subject matter can include the use of either of the other two terms.
As used herein, the term “about”, when referring to a value is meant to encompass variations of in one example ±20% or ±10%, in another example ±5%, in another example ±1%, and in still another example ±0.1% from the specified amount, as such variations are appropriate to practice the disclosed documents.
In addition, all ranges disclosed herein are to be understood to encompass any and all subranges subsumed therein. For example, a stated range of “1.0 to 10.0” should be considered to include any and all subranges beginning with a minimum value of 1.0 or more and ending with a maximum value of 10.0 or less, e.g., 1.0 to 5.3, or 4.7 to 10.0, or 3.6 to 7.9.
All ranges disclosed herein are also to be considered to include the end points of the range, unless expressly stated otherwise. For example, a range of “between 5 and 10”, “from 5 to 10” or “5-10” should generally be considered to include the end points 5 and 10.
Further, when the phrase “up to” is used in connection with an amount or quantity, it is to be understood that the amount is at least a detectable amount or quantity. For example, a material present in an amount “up to” a specified amount can be present from a detectable amount and up to and including the specified amount.
As used herein, “thermogenesis” defined as the dissipation of energy through the production of heat and occurs in tissues including brown adipose tissue. As used herein, “obese” refers to a body mass index (“BMI”) at or over 30, where the BMI is calculated by dividing an individual’s body mass by the square of their height.
As used herein, “weight loss” refers to a reduction in bodyweight. Weight loss can comprise losing from 2% of an individual’s weight up to 25% of an individual’s weight or up to 20%, up to 15%, up to 10%, up to 5% or up to 2%. Analogously, an individual experiencing weight loss can lose between about 5% and about 20% of their weight, or between about 10% and about 15% of their weight.
The terms “choline kinase” and “ChoK” (also known as CK, CHK and choline phosphokinase) refer to an enzyme that catalyzes the first reaction in the CDP- choline pathway for phophatidylcholine (PC) biosynthesis (also known as the Kennedy pathway). ChoK phosphorylates choline to phosphocholine (PCho) in the presence of magnesium (Mg2+) using adenosine 5’-triphosphate (ATP) as a phosphate donor group.
At least 3 genes encoding for proteins with demonstrated choline kinase activity have been identified in the human genome, designated as ck-alpha, ck-beta, and HCEKV (USA patent US2003186241), and several genes encoded proteins of which are 30-65% homologous to those encoded by the ck genes, such as for example the genes CA116602, CHKL, CA116600, CA116599, CAH56371 , CA116603, BAA91793, CAI16598, and the genes CPT1 B, EKI2, SF1 , SHOX2, FHOD2, FLJ12242, KRT5, FBL, ARL61 P4, TOMM40, MLL, described in (http://www.ebi. ac.uk!cgi-bin/sumtab?tool=asdblast&jobid=blast-20050412- 18072127). A very relevant characteristic of the different choline kinase isoenzymes is that they have different biochemical properties, with important variations in their affinity for the choline substrate or for the ATP phosphate donor, and even in their active form, which can be presented as dimers or tetramers. Therefore it is necessary to define if there is a direct relationship between any of the different choline kinase isoenzymes identified and the attributed capacity to affect thermogenesis and adipogenesis and, as a consequence, obesity and T2DM in a subject.
On the other hand, choline kinase inhibition has been demonstrated to be a new and effective therapeutic approach for several human diseases in animal models including cancer, parasites infections, pathogenic bacteria, rheumatoid arthritis and inflammatory processes (9). However no relationship has been established of choline kinase activity and obesity or type 2 diabetes mellitus (T2DM).
As used herein, the term “Choline kinase inhibitor” relates to any compound capable of causing a decrease in the ChoK activity, including those compounds which prevent expression of the ChoK gene, leading to reduced ChoK mRNA or protein levels as well as compounds that inhibit ChoK causing a decrease in the activity of the enzyme.
Compounds leading to reduced ChoK mRNA levels can be identified using standard assays for determining mRNA expression levels such as RT-PCR, RNA protection analysis, Northern blot, in situ hybridization, microarray technology and the like.
Compounds leading to reduced ChoK protein levels can be identified using standard assays for determining protein expression levels such as Western-blot or Western transfer, ELISA (enzyme-linked immunosorbent assay), RIA (radioimmunoassay), competitive EIA (competitive enzyme immunoassay), DAS- ELISA (double antibody sandwich ELISA), immunocytochemical and immunohistochemical techniques, techniques based on the use of protein biochips or microarrays which include specific antibodies or assays based on colloidal precipitation in formats such as dipsticks.
The determination of the inhibitory capacity on the biological activity of choline kinase is detected using standard assays to measure the activity of choline kinase such as the methods based on the detection of the phosphorylation of [14C] labelled choline by ATP in the presence of purified recombinant choline kinase or a fraction enriched in choline kinase followed by detection of the phosphorylated choline using standard analytical techniques (e.g. TLC) as described in EP1710236.
In the case that the compounds are specific for choline kinase alpha (ChoKa), these can be identified by either detecting a decrease in ChoKa mRNA levels using probes specific for ChoKa and which do not hybridise under stringent conditions with other ChoK isoforms (e.g, ChoKP) or by detecting a decrease in ChoKa protein levels using antibodies specific for ChoKa and which do not bind other ChoK isoforms (e.g, ChoKP). Suitable reagents for the specific determination of ChoKa mRNA and ChoKa protein levels have been described in detail in W02006108905.
The term "disease associated with hyperglycemia" includes diseases such as diabetes, impaired glucose tolerance, impaired fasting glycemia, diabetic complications, obesity, hyperinsulinemia, hyperinsulinemic hypoglycemia, reactive hypoglycemia, hyperlipidemia, hypercholesterolemia, hypertriglyceridemia, lipid metabolism disorder, atherosclerosis, hypertension, congestive heart failure, edema, hyperuricemia and gout.
As used herein, “enhancing” refers to an increased or improved state relative to a control condition. For example, enhancing weight loss refers to a improvement or increase of the extent/degree weight loss with administration of the compounds of the present application compared to weight loss observed in the absence of the agent. For example, "enhancing" can refer to increase in the amount weight lost by a subject administered a compound of the present application by at least about 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120% 130%, 140% or 150% compared to the amount of weight lost by a subject not receiving a compound of the present application.
As used herein, "fatty liver disease" or "FLD" comprises a condition in which excess fat builds up in the liver. The two main types of fatty liver disease are alcoholic fatty liver disease and nonalcoholic fatty liver disease (NAFLD). As used herein, "alcoholic fatty liver disease" is a fatty liver disease related to heavy alcohol use. As used herein, "non-alcoholic fatty liver disease" or "NAFLD" is a range of fatty liver conditions which are not related to heavy alcohol use. Early stages of NAFLD can be symptomless but if not detected and managed, NAFLD can progress to serious liver damage. NAFLD can progress from a harmless fatty liver stage (steatosis) to nonalcoholic steatohepatitis (NASH), a more serious form of NAFLD, and eventually to fibrosis, cirrhosis, liver failure and liver cancer. As used herein, "non-alcoholic steatohepatitis" or "NASH" is a severe fatty liver condition, in which the liver is often inflamed.
As used herein, “inhibition” refers to a reduction, decrease or lessening relative to a control condition. For example, inhibition of choline kinase by an agent refers to a reduction, decrease or lessening of the extent/degree of choline kinase signalling in the absence of the agent, and/or in the presence of an appropriate control agent. For example, " inhibition " can refer to a reduction of about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100%.
The term “obesity” as used herein, is used to refer to an increase in adipose tissue, particularly of white adipose tissue. The abnormal increase of white adipose tissue can occur by increasing the existing adipocyte cell size or the differentiation of new adipocytes. Adipogenesis is the proliferation and differentiation of adipocyte precursor cells into mature adipocytes, which accumulate in adipose tissues distributed throughout the body. Adipogenesis related to the overall number of adipocytes in the body; therefore modulating adipogenesis is one approach for treating obesity.
The term "hyperglycemia" is used herein to refer to physiologically high blood glucose levels. Hyperglycemic refers to a state of physiologically high blood glucose levels. Blood glucose levels that are considered hyperglycemic will vary based on the species of the subject. For example, in rats, hyperglycemia refers to a blood glucose level > 200 mg/dL. In humans, hyperglycemia refers to a blood glucose level > 180 mg/dL.
The term “type 2 diabetes” or T2DM, also known as noninsulin dependent diabetes mellitus or adult-onset diabetes, refers to a condition in which blood glucose level is elevated i.e., hyperglycaemia or we can say, when production of insulin decrease than a body needs, body cells stop responding to the insulin. The term includes diet-treated type 2-diabetes, sulfonylurea-treated type 2-diabetes, far- advanced stage type 2-diabetes and long-term insulin-treated type 2-diabetes.
The terms "subject," "host," "patient," and "individual" are used interchangeably herein to refer to any mammalian subject for whom prognosis, diagnosis or therapy is desired, particularly humans. The subject may have previously suffered or is suffering from metabolic syndrome and/or its complications, may be suspected of suffering from metabolic syndrome and/or its complications or the subject may be a healthy individual with no previous signs or symptoms of metabolic syndrome and/or its complications.
Use of choline kinase inhibitors in the treatment of metabolic diseases
The authors of the present invention have found that administration of a ChoK inhibitor can lead to a decrease in BMI in obese patients.
Without being bound by theory, the ChoK inhibitors of the present application activate AMPK and/or BAT thermogenesis. ChoK inhibition has been shown to activate AMPK and lead to the uncoupling of the electron transport system in mitochondria (the latter event being an endpoint of the thermogenesis). AMPK is known to play a role in both BAT thermogenesis and adipogenesis; thus the present application identifies a role for ChoK in both BAT thermogenesis and adipocyte differentiation via the AMPK pathway. As shown herein, ChoK is an important target for the development of compounds in the treatment of obesity.
BAT thermogenic activity, particularly in overweight or obese patients, leads to reduced body weight, enhanced insulin sensitivity, and improved glucose metabolism. BAT thermogenesis is generally characterized by: (1) an upregulation of thermogenic genes (e.g., UCP-1); (2) an increase in energy expenditure (as measured by O2 consumption, an increase in temperature, and cold tolerance); (3) a decrease in ATP production and serum cholesterol and 4) a decrease in BMI. One strategy to reduce obesity and T2DM is to decrease BMI by increasing cellular energy expenditure. Energy expenditure can be elevated by activating BAT thermogenesis, which can lead to lipolysis and a lower BMI. BAT thermogenesis can be regulated by the energy sensor AMPK. Without being bound by theory, activating AMPK can lead to expression of the genes responsible for thermogenesis.
Accordingly, in one aspect, the present application discloses a method of enhancing weight loss comprising administering a choline kinase inhibitor (preferably a choline kinase a inhibitor) to a subject in need of weight loss. Alternatively, the invention relates to a choline kinase inhibitor (preferably a choline kinase a inhibitor) for use in a method of enhancing weight loss in a subject.
In another aspect the present application discloses a method of treating hyperglycemia and/or type 2 diabetes in a subject, comprising administering to a subject in need of such treatment an effective amount of a choline kinase inhibitor (preferably a choline kinase a inhibitor). Alternatively, the invention relates to a choline kinase inhibitor (preferably a choline kinase a inhibitor) for use in a method of treating hyperglycemia and/or type 2 diabetes in a subject.
In yet another aspect the present application discloses a method of increasing glucose uptake in a subject in need of such uptake, comprising administering to said subject an effective amount of a choline kinase inhibitor (preferably a choline kinase a inhibitor). Alternatively, the invention relates to a choline kinase inhibitor (preferably a choline kinase a inhibitor) for use in a method of increasing glucose uptake in a subject.
In yet another aspect the present application discloses a method of activating brown-adipose tissue thermogenesis comprising administering to a subject in need of such activating an effective amount of a choline kinase inhibitor (preferably a choline kinase a inhibitor). Alternatively, the invention relates to a choline kinase inhibitor (preferably a choline kinase a inhibitor) for use in a method of activating brown-adipose tissue thermogenesis.
In yet another aspect the present application discloses a method of activating 5' adenosine monophosphate-activated protein kinase comprising administering to a subject in need of such activating an effective amount of a choline kinase inhibitor (preferably a choline kinase a inhibitor). Alternatively, the invention relates to a choline kinase inhibitor (preferably a choline kinase a inhibitor) for use in a method of activating 5' adenosine monophosphate-activated protein kinase. There are two major types of adipocytes: white adipocytes and brown adipocytes. White adipocytes store energy as fat when energy intake overtakes energy expenditure. Brown/beige adipocyte tissue (BAT) carry out non-shivering thermogenesis upon cold exposure and adrenergic stimulation; thus BAT promotes energy expenditure. The imbalance of energy intake and expenditure leads to obesity. Activating the AMPK signaling pathway inhibits white adipogenesis, promotes brown adipogenesis, and promotes the "beiging" of white adipocytes. Both the inhibition of white adipocytes and the promotion of brown/beige adipocytes are therapeutic strategies for addressing obesity. In this way, the ChoK inhibitors that are shown herein to activate the AMPK pathway in adipose tissue can comprise a therapeutic for the treatment of obesity.
Treating obesity by enhancing weight loss can further address or ameliorate a number of weight-associated disorders, including but not limited to cardiovascular disease, joint inflammation, sleep apnea, respiratory disorder, and gallbladder disease. Accordingly, in yet another aspect the present application discloses a method of treating or ameliorating a weight-associated disorders, including but not limited to cardiovascular disease, joint inflammation, sleep apnea, respiratory disorder, and gallbladder disease comprising administering to a subject in need of such activating an effective amount of a choline kinase inhibitor (preferably a choline kinase a inhibitor). Alternatively, the invention relates to a choline kinase inhibitor (preferably a choline kinase a inhibitor) for use in a method of a method of treating or ameliorating a weight-associated disorders, including but not limited to cardiovascular disease, joint inflammation, sleep apnea, respiratory disorder, and gallbladder disease.
Exemplary choline kinase inhibitors that can be used in the present invention include compounds having a quaternary ammonium cationic core or tertiary amine susceptible to protonation to form quaternary cationic amines, with R groups selected from alkyl, aryl, and heterocyclic derivatives, coupled to alkylic or arylic chains, preferentially including heteroatoms able to form hydrogen bonds. In other words compounds that function as choline kinase inhibitors having the general formula
[R1R2R3R4N]+ wherein Ri, R2, R3 and R3 are independently selected from alkyl, aryl, and heterocyclic groups, or having the general formula
[R!R2R3N]+ where Ri, R2, and R3 are independently selected from alkyl, aryl, and heterocyclic groups, and the amine can be protonated to form a quaternary ammonium ion. Alkyl (e.g..methyl, ethyl, propyl), Aryl (e.g., phenyl, substituted phenyl) and heterocyclic groups (e.g., pyridine, morpholine, quinoline, etc.). Examples of choline kinase inhibitors that are quaternary ammonium ions with heterocyclic groups include HC3, MN58B, V-11-0711, EB3P, ICL-CCIC-0019, PL48, and G8V. Examples of choline kinase inhibitors that are quaternary ammonium ions with arylic groups are RSM- 932A and CK-37 and EB3P. Examples of choline kinase inhibitors that a quaternary ammonium ions with alky groups are RSM-932A and MN58b. Exemplary choline kinase inhibitors that can be used in the present invention are described under I to XIII in Table 1.
In an embodiment, the choline kinase inhibitor is a compound having the structure:
In another embodiment, the choline kinase inhibitor is a compounds capable of promoting an increase in the activity and/or expression of choline kinase beta (ChoKp) ora functionally equivalent variant thereof as described in international patent application WO201 0001369. ChoKp promotes a decrease in the growth of tumors caused by overexpression of ChoKa and thus, ChoKp as well as compositions which promote an increase in the activity and/or expression of ChoKp can also be used as ChoKa inhibitors in the compositions of the present invention. In a preferred embodiment, the compound capable of increasing the expression of ChoKp is a polynucleotide which comprises a nucleic acid sequence which encodes a ChoKp or a functionally variant thereof. The polynucleotide is of human origin and is defined by SEQ ID NO:1 (GenEMBL AB029886). In another preferred embodiment, the compound capable of increasing the expression of ChoKp is the ChoKp polypeptide as defined by SEQ ID NO:2 (UniProt accession Q9Y259) or a functionally equivalent variant thereof. The term “variants of ChoKp”, as used herein, is understood as a polypeptide which shows substantially the same properties of ChoKp in terms of (i) its capacity to prevent the increase in PCho caused by an increase in ChoKa activity; (ii) its capacity to prevente the oncogenic transformation of cell caused by an increase in the expression of ChoKa or (iii) its capacity to promote an increase in the activity of phosphatidyletanolamine methyl transferase (PEMT). Methods for the identification of variants of ChoK having the properties described herein are described in co-pending Spanish patent application P200802007.
Variants of the ChoKp suitable for use as ChoKa inhibitors in the compositions of the present invention preferably have a sequence identity with said ChoKp cytokines of at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%. The degree of identity between the variants and ChoKp is determined using computer algorithms and methods that are widely known for the persons skilled in the art. The identity between two amino acid sequences is preferably determined by using the BLASTP algorithm [BLASTManual, Altschul, S., et al., NCBI NLM NIH Bethesda, Md. 20894, Altschul, S., et a!., J. Mol. Biol. 21 5: 403-410 (1990)].
In another embodiment, the choline kinase inhibitor is an inhibitory antibody capable of specifically binding to an inhibiting the activity of choline kinase and, in particular, monoclonal antibodies that recognise the catalytic domain or the dimerization domain of ChoKa and therefore inhibit ChoKa activity. In a preferred embodiment, the inhibitory antibodies are monoclonal antibodies as defined in WO2007138143. In a still more preferred embodiment, the inhibitory antibodies are the antibodies AD3, AD8 and AD11 as defined in WO2007138143.
Further examples of ChoK inhibitors include any inhibitor of ChoKa activity or expression, and/or inhibitor of ChoKp activity or expression being an inhibitory nucleic acid that specifically inhibit expression and/or inhibits ChoKa activation and/or inhibits ChoKp activation. This includes any type of molecule, for example, a polynucleotide, a peptide, antisense oligonucleotide, antibody or antibody fragment, a peptidomimetic, peptoids such as vinylogous peptoids, a small organic molecule, or the like, and can act in any of various ways to reduce or inhibit choline kinase- mediated choline phosphorylation.
In various embodiments, the inhibitor of choline phosphorylation is an inhibitory nucleic acid that inhibits the expression of ChoKa or ChoKa. For example, the inhibitory nucleic acid can be siRNA, shRNA, oligonucleotides, antisense RNA, a ribozyme or a guide RNA (gRNA) that inhibit such activity or expression.
As used herein, an “inhibitory nucleic acid” means an RNA, DNA, or a combination thereof that interferes or interrupts the translation of mRNA. Inhibitory nucleic acids can be single or double stranded. The nucleotides of the inhibitory nucleic acid can be chemically modified, natural or artificial. The terms “short- inhibitory RNA” and “siRNA” interchangeably refer to short double-stranded RNA oligonucleotides that mediate RNA interference (also referred to as “RNA-mediated interference” or“RNAi”). The terms “small hairpin RNA” and “shRNA” interchangeably refer to an artificial RNA molecule with a tight hairpin turn that can be used to silence target gene expression via RNAi. RNAi is a highly conserved gene silencing event functioning through targeted destruction of individual mRNA by a homologous double-stranded small interfering RNA (siRNA) (Fire, A. et al., Nature 391 :806-811 (1998)). Methods for the design of siRNA or shRNA target sequences have been described including siRNA target sequences as well as sequence, structural and conformational properties (Henshel et al., Nucl. Acids Res., 32: 113-20 (2004); lli- Tei et al., Nucl. Acids Res., 3: 936-48 (2004); Sui et al., Proc. Natl. Acad. Sci. USA, 99: 5515-20 (2002); Elbashir et al., Methods, 26: 199-213 (2002); Duxbury and Whang, J. Surg. Res., 117: 339-44 (2004). Also relevant enzymatic requirements must be taken into consideration (Czaudema et al., Nucl. Acids Res., 31 : 2705-16 (2003)). Many computer programs are available to aid in the design of suitable siRNA and shRNAs for use in suppressing expression or inhibiting choline kinase-mediated phosphorylation.
In an embodiment, the ChoK inhibitor is the small hairpin RNA (shRNA) as defined in SEQ ID NO:1 or the siRNA defined by Glunde et al. (Cancer Res., 2005, 65:11034-11043).
As used herein, the term “antisense”, refers to a characteristic of an oligonucleotide or other nucleic acid having a base sequence complementary or substantially complementary to a target nucleic acid to which it is capable of hybridizing. In some embodiments, a target nucleic acid is a target gene mRNA. In some embodiments, hybridization is required for or results in at one activity, e.g., a decrease in the level, expression or activity of the target nucleic acid or a gene product thereof. The term "antisense oligonucleotide", as used herein, refers to an oligonucleotide complementary to a target nucleic acid. In some embodiments, an antisense oligonucleotide is capable of directing a decrease in the level, expression or activity of a target nucleic acid or a product thereof. In some embodiments, an antisense oligonucleotide is capable of directing a decrease in the level, expression or activity of the target nucleic acid or a product thereof, via a mechanism that involves RNA interference.
As used herein, the term “ribozyme” refers to RNA molecules that cleave mRNA at site-specific recognition sequences and that can be used to destroy target mRNAs, particularly through the use of hammerhead ribozymes. Hammerhead ribozymes cleave mRNAs at locations dictated by flanking regions that form complementary base pairs with the target mRNA. Preferably, the target mRNA has the following sequence of two bases: 5 -UG-3'. The construction and production of hammerhead ribozymes is well known in the art.
Gene targeting ribozymes may contain a hybridizing region complementary to two regions, each of at least 5 and preferably each of 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19 or 20 contiguous nucleotides in length of a target mRNA. In addition, ribozymes possess highly specific endoribonuclease activity, which autocatalytically cleaves the target sense mRNA.
With regard to antisense, siRNA or ribozyme oligonucleotides, phosphorothioate oligonucleotides can be used. Modifications of the phosphodiester linkage as well as of the heterocycle or the sugar may provide an increase in efficiency. Phophorothioate is used to modify the phosphodiester linkage. An N3 -P5' phosphoramidate linkage has been described as stabilizing oligonucleotides to nucleases and increasing the binding to RNA. Peptide nucleic acid (PNA) linkage is a complete replacement of the ribose and phosphodiester backbone and is stable to nucleases, increases the binding affinity to RNA, and does not allow cleavage by RNAse H. Its basic structure is also amenable to modifications that may allow its optimization as an antisense component. With respect to modifications of the heterocycle, certain heterocycle modifications have proven to augment antisense effects without interfering with RNAse H activity. An example of such modification is C-5 thiazole modification. Finally, modification of the sugar may also be considered. 2'-O-propyl and 2 '-meth oxy ethoxy ribose modifications stabilize oligonucleotides to nucleases in cell culture and in vivo.
Inhibitory nucleic acids, such as siRNA, shRNA, ribozymes, or antisense molecules, can be synthesized and introduced into cells using methods known in the art. Molecules can be synthesized chemically or enzymatically in vitro (Micura, Agnes Chem. Int. Ed. Emgl. 41 2265-9 (2002); Paddison et al., Proc. Natl. Acad. Sci. USA, 99:1443-8 2002) or endogenously expressed inside the cells in the form of shRNAs (Yu et al., Proc. Natl. Acad. Sci. USA, 99:6047-52 (2002); McManus et al., RNA 8, 842-50 (2002)). Plasmid-based expression systems using RNA polymerase III U6 or H1 , or RNA polymerase II U1 , small nuclear RNA promoters, have been used for endogenous expression of shRNAs (Brummelkamp et al., Science, 296: 550-3 (2002); Sui et al., Proc. Natl. Acad. Sci. USA, 99: 5515-20 (2002); Novarino et al., J. Neurosci., 24: 5322-30 (2004)). Synthetic siRNAs can be delivered by electroporation or by using lipophilic agents (McManus et al., RNA 8, 842-50 (2002); Kishida et al., J. Gene Med., 6: 105-10 (2004)). Alternatively, plasmid systems can be used to stably express small hairpin RNAs (shRNA) for the suppression of target genes (Dykxhoorn et al. , Nat. Rev. Mol. Biol., 4:457-67 (2003)). Various viral delivery systems have been developed to deliver shRNA-expressing cassettes into cells that are difficult to transfect (Brummelkamp et al., Cancer Cell, 2: 243-7 (2002); Rubinson et al., Nat. Genet., 33: 401-6 2003). Furthermore, siRNAs can also be delivered into live animals. (Hasuwa et al., FEBS Lett., 532, 227-30 (2002); Carmell et al., Nat. Struct. Biol., 10: 91-2 (2003); Kobayashi et al., J. Pharmacol. Exp. Ther., 308:688-93 (2004)). Inhibitory oligonucleotides can be delivered to a cell by direct transfection or transfection and expression via an expression vector. Appropriate expression vectors include mammalian expression vectors and viral vectors, into which has been cloned an inhibitory oligonucleotide with the appropriate regulatory sequences including a promoter to result in expression of the antisense RNA in a host cell. Suitable promoters can be constitutive or development-specific promoters. Transfection delivery can be achieved by liposomal transfection reagents, known in the art (e.g., Xtreme transfection reagent, Roche, Alameda, Calif.; Lipofectamine formulations, Invitrogen, Carlsbad, Calif.). Delivery mediated by cationic liposomes, by retroviral vectors and direct delivery are efficient. Another possible delivery mode is targeting using antibody to cell surface markers for the target cellsThe term “gRNA”, as used herein, refers to a RNA molecule that can be used to direct an endonuclease to a target site in the genome, thereby mediating CRISPR- based targeting of a target nucleic acid.
CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) is an acronym for DNA loci that contain multiple, short, direct repetitions of base sequences. The prokaryotic CRISPR/Cas system has been adapted for use as gene editing (silencing, enhancing or changing specific genes) for use in eukaryotes (see, for example, Cong, Science, 15:339(6121):819-823 (2013) and Jinek, et al., Science, 337(6096): 816-21 (2012)). By transfecting a cell with elements including a Cas gene and specifically designed CRISPRs, nucleic acid sequences can be cut and modified at any desired location. Methods of preparing compositions for use in genome editing using the CRISPR/Cas systems are described in detail in US Pub. No. 2016/0340661 , US Pub. No. 20160340662, US Pub. No. 2016/0354487, US Pub. No. 2016/0355796, US Pub. No. 20160355797, and WO 2014/018423, which are specifically incorporated by reference herein in their entireties.
Thus, as used herein, “CRISPR system” refers collectively to transcripts and other elements involved in the expression of or directing the activity of CRISPR- associated (“Cas”) genes, including sequences encoding a Cas gene, a tracr (transactivating CRISPR) sequence (e.g., tracrRNA or an active partial tracrRNA), a tracr- mate sequence (encompassing a “direct repeat” and a tracrRNA-processed partial direct repeat in the context of an endogenous CRISPR system), a guide sequence (also referred to as a “spacer”, “guide RNA” or “gRNA” in the context of an endogenous CRISPR system), or other sequences and transcripts from a CRISPR locus. One or more tracr mate sequences operably linked to a guide sequence (e.g., direct repeat-spacer-direct repeat) can also be referred to as “pre-crRNA” (pre- CRISPR RNA) before processing or crRNA after processing by a nucleaseThere are many resources available for helping practitioners determine suitable target sites once a desired DNA target sequence is identified. For example, numerous public resources, including a bioinformatically generated list of about 190,000 potential sgRNAs, targeting more than 40% of human exons, are available to aid practitioners in selecting target sites and designing the associate sgRNA to affect a nick or double strand break at the site. See also, crispr.u-psud.fr, a tool designed to help scientists find CRISPR targeting sites in a wide range of species and generate the appropriate crRNA sequencesln some embodiments, one or more vectors driving expression of one or more elements of a CRISPR system are introduced into a target cell such that expression of the elements of the CRISPR system direct formation of a CRISPR complex at one or more target sites. Accordingly, cleavage of DNA by the genome editing vector or composition can be used to delete nucleic acid material from a target DNA sequence by cleaving the target DNA sequence and allowing the cell to repair the sequence. As such, the compositions can be used to modify DNA in a sitespecific, i.e., “targeted” way, for example gene knock-out, gene knock-in, gene editing, gene tagging, etc., as used in, for example, gene therapyWhile the specifics can be varied in different engineered CRISPR systems, the overall methodology is similar. A practitioner interested in using CRISPR technology to target a DNA sequence can insert a short DNA fragment containing the target sequence into a guide RNA expression plasmid. The sgRNA expression plasmid contains the target sequence (about 20 nucleotides), a form of the tracrRNA sequence (the scaffold) as well as a suitable promoter and necessary elements for proper processing in eukaryotic cells. Such vectors are commercially available (see, for example, Addgene). Many of the systems rely on custom, complementary oligos that are annealed to form a double stranded DNA and then cloned into the sgRNA expression plasmid. Co-expression of the sgRNA and the appropriate Cas enzyme from the same or separate plasmids in transfected cells results in a single or double strand break (depending of the activity of the Cas enzyme) at the desired target site.
As described above, certain aspects of the present disclosure involve CRISPR- based targeting of the target nucleic acid, i.e. the choline kinase gene, which may involve use of a CRISPR-CAS9 targeting system. CRISPR-CAS9 systems may involve the use of a CRISPR RNA (crRNA), a trans- activating CRISPR RNA (tracrRNA), and a CAS9 protein. The crRNA and tracrRNA aid in directing the CAS9 protein to a target nucleic acid sequence, and these RNA molecules can be specifically engineered to target specific nucleic acid sequences. In particular, certain aspects of the present disclosure involve the use of a single guide RNA (gRNA) that reconstitutes the function of the crRNA and the tracrRNA. As disclosed herein, gRNA molecules may be used to direct a dCAS9 protein to a target nucleic acid sequence.
A gRNA as provided herein typically comprises a targeting domain and a binding domain. The targeting domain (also termed "targeting sequence") may comprise a nucleic acid sequence that binds to a target site, e.g., to a genomic nucleic acid molecule within a cell. The target site may be a double-stranded DNA sequence comprising a PAM sequence as well as the target sequence, which is located on the same strand as, and directly adjacent to, the PAM sequence. The targeting domain of the gRNA may comprise an RNA sequence that corresponds to the target sequence, i.e. , it resembles the sequence of the target domain, sometimes with one or more mismatches, but typically comprising an RNA sequence instead of a DNA sequence. The targeting domain of the gRNA thus may base pair (in full or partial complementarity) with the sequence of the double- stranded target site that is complementary to the target sequence, and thus with the strand complementary to the strand that comprises the PAM sequence. It will be understood that the targeting domain of the gRNA typically does not include a sequence that resembles the PAM sequence. It will further be understood that the location of the PAM may be 5’ or 3’ of the target sequence, depending on the nuclease employed. For example, the PAM is typically 3’ of the target sequence for Cas9 nucleases, and 5’ of the target sequence for Casl2a nucleases.
In some embodiments, the targeting domain sequence comprises between 17 and 30 nucleotides and corresponds fully to the target sequence (i.e., without any mismatch nucleotides). In some embodiments, however, the targeting domain sequence may comprise one or more, but typically not more than 4, mismatches, e.g., 1 , 2, 3, or 4 mismatches. As the targeting domain is part of gRNA, which is an RNA molecule, it will typically comprise ribonucleotides, while the DNA targeting domain will comprise deoxyribonucleotides.
Further, the agent can be administered in any way typical of an agent used to treat the particular type of above-mentioned diseases or under conditions that facilitate contact of the agent with the target diseased cells and, if appropriate, entry into the cells. Entry of a polynucleotide agent into a cell, for example, can be facilitated by incorporating the polynucleotide into a viral vector that can infect the cells. Thus, the inhibitory nucleic acid can be delivered in, for example, a lentiviral vector, a herpesvirus vector or an adenoviral vector. If a viral vector specific for the cell type is not available, the vector can be modified to express a receptor (or ligand) specific for a ligand (or receptor) expressed on the target cell, or can be encapsulated within a liposome, which also can be modified to include such a ligand (or receptor). A peptide agent can be introduced into a cell by various methods, including, for example, by engineering the peptide to contain a protein transduction domain such as the human immunodeficiency virus TAT protein transduction domain, which can facilitate translocation of the peptide into the cell.
Generally, the choline kinase inhibitor to be administered to a subject may be formulated in a composition (e.g., a pharmaceutical composition) suitable for such administration. Such formulated agents are useful as medicaments for treating a subject suffering from any of the above-mentioned diseases, in part, by elevated or abnormally elevated ChoKa or ChoKa expression or choline phosphorylation.
Pharmaceutically acceptable carriers useful for formulating an agent for administration to a subject are well known in the art and include, for example, aqueous solutions such as water or physiologically buffered saline or other solvents or vehicles such as glycols, glycerol, oils such as olive oil or injectable organic esters. A pharmaceutically acceptable carrier can contain physiologically acceptable compounds that act, for example, to stabilize or to increase the absorption of the conjugate. Such physiologically acceptable compounds include, for example, carbohydrates, such as glucose, sucrose or dextrans, antioxidants, such as ascorbic acid or glutathione, chelating agents, low molecular weight proteins or other stabilizers or excipients. One skilled in the art would know that the choice of a pharmaceutically acceptable carrier, including a physiologically acceptable compound, depends, for example, on the physico-chemical characteristics of the therapeutic agent and on the route of administration of the composition, which can be, for example, orally or parenterally such as intravenously, and by injection, intubation, or other such method known in the art. The pharmaceutical composition also can contain a second (or more) compound(s) such as a diagnostic reagent, nutritional substance, toxin, or therapeutic agent, for example, a cancer chemotherapeutic agent and/or vitamin(s).
In general, a suitable daily dose of a compound/inhibitor of the invention will be that amount of the compound/inhibitor that is the lowest dose effective to produce a therapeutic effect. Such an effective dose will generally depend upon the factors described above. Generally, intravenous, intracerebroventricular and subcutaneous doses of the compounds of this invention for a patient will range from about 0.0001 to about 100 mg per kilogram of body weight per day which can be administered in single or multiple doses.
In an embodiment of the invention, the choline kinase inhibitor is used in the treatment of type 2 diabetes mellitus, including diet-treated type 2-diabetes, sulfonylurea-treated type 2-diabetes, far-advanced stage type 2-diabetes and longterm insulin-treated type 2-diabetes.
In an embodiment of the invention, the choline kinase inhibitor is used for the treatment of “hyperglycemia” and, in particular, long-term hyperglycemia as well as any disease associated with long-term hyperglycemia such as diabetes, impaired glucose tolerance, impaired fasting glycemia, diabetic complications, obesity, hyperinsulinemia, hyperinsulinemic hypoglycemia, reactive hypoglycemia, hyperlipidemia, hypercholesterolemia, hypertriglyceridemia, lipid metabolism disorder, atherosclerosis, hypertension, congestive heart failure, edema, hyperuricemia and gout.
It will be understood that the subjects treated according to the methods of the present invention may be subjects which do not have history of metabolic disease or, alternatively, subjects that may be or may have been under a treatment, such as, but not limited to, diabetes, CVD, blood pressure or lipid modifying medication, or may not have had any previous treatment or medication.
The subject to be treated may have previously suffered or is suffering from cardiovascular disease and/or its complication(s) and may have been or may be undergoing statin treatment. The subject may have previously suffered or is suffering from type 2 diabetes and/or its complications and may have been or may be undergoing diabetes treatment.
In some embodiments of the aforementioned methods and uses of the present disclosure, the subject has a blood glucose level in the normal range. For example, in some embodiments, the blood glucose level of the subject includes a haemoglobin A1c level of about 5.7 percent or less, such as about 5.5 percent or less, such as about 5.0 percent or less. In some embodiments, the subject has a fasting blood sugar on awakening of about 100 mg/dL or less. In some embodiments, the subject has a before-meal glucose level of about 70 mg/dL to about about 99 mg/dL. In some embodiments, the subject has a glucose level of about 140 mg/dL or less about two hours after a meal. Typically, the subject has a fasting venous plasma glucose concentration of less than 6.1 mmol/L (110 mg/dL).
In some embodiments of the aforementioned methods and uses of the present disclosure, the subject has a blood pressure level in the normal range. In some embodiments of the aforementioned methods and uses of the present disclosure, the subject has a blood lipid levels, such as, but not limited to, triglyceride levels, high-density lipoprotein cholesterol levels and/or low-density lipoprotein cholesterol levels, in the normal range.
In some embodiments, the subject to be treated shows an alteration of one or more metabolic syndrome markers.
As used herein, "metabolic syndrome marker", "marker of metabolic syndrome", "metabolic syndrome biomarker", "biomarker of metabolic syndrome", "metabolic marker", "marker of the present disclosure", "biomarker of the present disclosure", or the similar, comprises at least one biomarker for metabolic syndrome and/or one or more of its complications comprising stearoylsphingosine and/or palmitoylsphingosine as disclosed on the present disclosure. "Metabolic syndrome marker", "marker of metabolic syndrome", "metabolic syndrome biomarker", "biomarker of metabolic syndrome", "metabolic marker", "marker of the present disclosure", "biomarker of the present disclosure", or the similar, may be used interchangeably in the present disclosure.
As used herein, a "combination of metabolic syndrome markers", a ’’metabolic syndrome marker combination", a "marker combination", a "metabolic marker combination", a "biomarker combination"or the similar, comprises a combination of at least one biomarker for metabolic syndrome and/or one or more of its complications and/or one or more other biomarkers, parameters or health data. As used herein, the combination may be e.g. a ratio, sum, difference, product, remainder, value, score, calculation, formula, equation, algorithm, or any combination thereof. In one embodiment, the combination comprises stearoylsphingosine and/or palmitoylsphingosine. In one embodiment, the combination comprises stearoylsphingosine/palmitoylsphingosine or palmitoylsphingosine/stearoylsphingosine. A "combination of metabolic syndrome markers", a ’’metabolic syndrome marker combination", a "marker combination", a "metabolic marker combination", a "biomarker combination"or the similar, may be used interchangeably in the present disclosure.
In certain embodiments, a metabolic syndrome marker and/or metabolic syndrome marker combination comprises, in addition to stearoylsphingosine and/or palmitoylsphingosine, one or more of other clinical parameters, such as, but not limited to, BMI, weight, height, age, gender, waist circumference, blood pressure, systolic blood pressure, diastolic blood pressure, glucose, triglycerides, high-density lipoprotein (HDL) cholesterol, HbA1c, low-density lipoprotein (LDL) cholesterol, insulin, CRP and/or any combination thereof.
In certain embodiments, a metabolic syndrome marker and/or metabolic syndrome marker combination further comprises one or more of other biomarkers or parameters, including, but not limited to, triglyseride (TG), total cholesterol (TC), low- density lipoprotein (LDL), low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein (HDL), high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein particle (LDL-P), high-density lipoprotein particle (HDL-P), Apolipoprotein B (ApoB), Apolipoprotein Al (ApoAl), Apolipoprotein All (ApoAII), Apolipoprotein C (ApoC), C-reactive protein (CRP), troponin T (TNT or TnT), troponin I (TNI or Tnl), B- type natriuretic peptide (BNP), N-terminal pro B-type natriuretic peptide (NT- proBNP), Cystatin C, creatine kinase (CK), glycated haemoglobin A1c (HbA1c), glucose, suppression of tumorigenicity 2 (St2), Galectin, trimethylamine-N-oxide (TMAO), lipoprotein-associated phospholipase A2 (Lp-PLA2), growth differentiation factor 15 (GDF15), lipoprotein (a) (Lp(a)), any other lipoprotein subgroup composition or particle number, and/or any combination thereof. In certain embodiments, a metabolic syndrome marker and/or metabolic syndrome marker combination further comprises one or more of other personal information or health data, including, but not limited to, sex, age, blood pressure, BMI, weight, height, smoking status, diabetes, lipid lowering treatment or other medication, ethnic background, genetic data, geographical location, history of CVD and/or CV events, family history of CVD and/or CV events, history of diabetes, family history of diabetes, history of other diseases, family history of other diseases, medical imaging data, e.g. from angiography or computed tomography (CT), and/or any combination thereof.
Combination therapy
The authors of the present invention have observed that the combined use of a choline kinase inhibitor and a GLP-1 analog resulted in a decrease in weight in overweight mice compared to mice being fed a high fat diet alone, and compared to mice being administered the choline kinase inhibitor or the GLP-1 agonist alone. .
Accordingly, in another aspect, the invention relates to a composition comprising a choline kinase inhibitor and a GLP-1 analog. The invention also relates to a choline kinase inhibitor for use in (a) treating, preventing or ameliorating a weight- associated disorder, (b) enhancing weight loss, (c) increasing glucose uptake, (d) activating brown-adipose tissue thermogenesis, (e) activating 5' adenosine monophosphate-activated protein kinase or (f) treating, preventing or ameliorating a fatty liver disease in a subject in need thereof when used in combination with a GLP- 1 analog.
The term "composition" refers to one or more compounds in various combinations according to alternative embodiments of this invention.
The term choline kinase inhibitor has been defined above. Choline kinase Inhibitors to be used in the combinations according to the present invention include any of the inhibitors defined above in the context of the medical uses.
In an embodiment, the choline kinase inhibitor forming part of the composition according to the invention is a choline kinase inhibitor specific for the choline kinase alpha isoform.
In an embodiment, the choline kinase inhibitor forming part of the composition according to the invention is a compound of Formula I: wherein:
Q' represents the conjugate base of a pharmaceutically suitable organic or inorganic acid;
Ri and R'i, represent, independently of each other, an aryl radical optionally substituted by halogen, trifluoromethyl, hydroxyl, Ci-e alkyl, amino or alkoxyl;
R2 and R'2, represent, independently of each other, an aryl radical optionally substituted by halogen, trifluoromethyl, hydroxyl, Ci-e, alkyl, amino or alkoxyl;
R3 and R'3, represent, independently of each other, either a radical selected from the group formed by H, halogen, trifluoromethyl, hydroxyl, amino, alkoxyl and Ci-e alkyl optionally substituted by trifluoromethyl, hydroxyl, amino or alkoxyl, or together with R4 and R'4, respectively, and independently of each other, a -CH=CH-CH=CH- radical optionally substituted by halogen, trifluormethyl, hydroxyl, Ci-e alkyl, amino or alkoxyl; R4 and R'4, represent, independently of each other, either a radical selected from the group formed by H and C1.6 alkyl optionally substituted by halogen, trifluoromethyl, hydroxyl, amino or alkoxyl, or together with R3 and R'3 respectively, and independently of each other, a -CH=CH-CH=CH- radical optionally substituted by halogen, trifluoromethyl, hydroxyl, Ci-e, alkyl, amino or alkoxyl and
- A represents a spacer group comprising any divalent organic structure acting as a joining link between the two pyridinium groups present in the structure defined by formula I In an embodiment, the choline kinase inhibitor forming part of the composition according to the invention is compound of Table 2.
In an embodiment, the choline kinase inhibitor forming part of the composition according to the invention is RSM932.
In an embodiment, the choline kinase inhibitor forming part of the composition according to the invention is a compound of Formula II: n is 0, 1 , 2 or 3
Z is any a structural group selected from the group of:
- wherein Y is selected from the group of -H, -CH3, -CH2-OH, -CO-CH3,
CN, -NH2, -N(CH3)2, pyrrolidine, piperidine, perhydroazepine, -OH, -O-CO- C15H31.
In an embodiment, the choline kinase inhibitor forming part of the composition according to the invention is Ta compound selected from the group consisting of RSM 932A, MN58b, Hemicholinium, EB-3D, EB-3P, ICL-CCIC-0019, CK-37, any of the compounds of Table 1 , 1 ,2-ethylene-p-(bis-benzyldimethyldiyl), symmetrical bis- and triscationic compounds, a compound of the bispyridinium cyclophane family, GRQF- JCR795b, GRQF-MN94b. The term “GLP-1 analog” is used herein interchangeably with “GLP1- receptor agonist” and includes, without limitation, GLP-1 agonists, GLP-1/GIP dual agonists, GLP-1/FGF21 dual agonists, GLP-1/GCGR dual agonists and GLP-1/GIP/GCGR triple agonists, and/or a THR-beta agonist. That is, in certain embodiments, the invention relates to the use of the ChoKa inhibitor in combination with a GLP-1 analog for the treatment of metabolic diseases or disorders. In a particular embodiment, the invention relates to the use of the ChoKa inhibitor in combination with a GLP-1 analog for the treatment of the treatment of fatty liver diseases, in particular NAFLD and/or NASH.
The term "GLP-1 agonist" as used herein refers to a compound, which fully or partially activates the human GLP-1 receptor. The term is thus equal to the term "GLP-1 receptor agonist" used in other documents. The term GLP-1 agonist as well as the specific GLP-1 agonists described herein also encompass salt forms thereof. It follows that the GLP-1 agonist should display "GLP-1 activity" which refers to the ability of the compound, i.e. a GLP-1 analogue or a compound comprising a GLP-1 analogue, to bind to the GLP-1 receptor and initiate a signal transduction pathway resulting in insulinotropic action or other physiological effects as is known in the art. In some embodiments the "GLP-1 agonist" binds to a GLP-1 receptor, e.g., with an affinity constant (KD) or activate the receptor with a potency (ECso) of below 1 mM, e.g. below 100 nM as measured by methods known in the art and exhibits insulinotropic activity, where insulinotropic activity may be measured in vivo or in vitro assays known to those of ordinary skill in the art. For example, the GLP-1 agonist may be administered to an animal with increased blood glucose (e.g. obtained using an Intravenous Glucose Tolerance Test (IVGTT). A person skilled in the art will be able to determine a suitable glucose dosage and a suitable blood sampling regime, e.g. depending on the species of the animal, for the IVGTT) and measure the plasma insulin concentration overtime. Suitable assays have been described in such as WO 2015/155151. The term half maximal effective concentration (EC50) generally refers to the concentration which induces a response halfway between the baseline and maximum, by reference to the dose response curve. EC50 is used as a measure of the potency of a compound and represents the concentration where 50 percent of its maximal effect is observed. The lower the EC50 value, the better the potency. In one embodiment, the potency (EC50) as determined (without HSA) is 5-1000 pM, such as 10-750 pM, 10-500 pM or 10-200 pM. In one embodiment the EC50 (without HSA) is at most 500 pM, such as at most 300 pM, such as at most 200 pM. In some embodiments the GLP-1 agonist is a GLP-1 analogue, optionally comprising "one substituent". The term "analogue" as used herein referring to a GLP- 1 peptide (hereafter "peptide") means a peptide wherein at least one amino acid residue of the peptide has been substituted with another amino acid residue and/or wherein at least one amino acid residue has been deleted from the peptide and/or wherein at least one amino acid residue has been added to the peptide and/or wherein at least one amino acid residue of the peptide has been modified. Such addition or deletion of amino acid residues may take place at the N- terminal of the peptide and/or at the C-terminal of the peptide. In some embodiments the term "GLP- 1 analogue" or "analogue of GLP-1" as used herein refers to a peptide, or a compound, which is a variant of the human Glucagon-Like Peptide-1 (GLP-1 (7-37)). GLP-1 (7-37) has the sequence HAEGTFTSDVSSYLEGQAAKEFIAWLVKGRG (SEQ ID NO: 2). In some embodiments the term "variant" refers to a compound which comprises one or more amino acid substitutions, deletions, additions and/or insertions. In one embodiment the GLP-1 agonist exhibits at least 60 percent, 65 percent, 70 percent, 80 percent or 90 percent sequence identity to GLP-1 (7-37) over the entire length of GLP-1 (7-37). In general, the term GLP-1 agonist is meant to encompass the GLP-1 agonist and any pharmaceutically acceptable salt, amide, or ester thereof. In some embodiments the composition comprises the GLP-1 agonist or a pharmaceutically acceptable salt, amide, or ester thereof. In some embodiments the composition comprises the GLP-1 agonist and one or more pharmaceutically acceptable counter ions. In certain embodiments, the ChoKa inhibitor may be administered with a GLP-1 agonist selected from one or more of the GLP-1 agonists disclosed in W093/19175, W096/29342, WO98/08871 , WO99/43707, WO99/43706, W099/43341 , WO99/43708, W02005/027978, W02005/058954, W02005/058958, W02006/005667, W02006/037810, W02006/037811 , W02006/097537,
W02006/097538, W02008/023050, W02009/030738, W02009/030771 ,
W02009/030774 and W02021/219710, which are incorporated herein by reference in their entirety. In certain embodiments, the ChoKa inhibitor may be administered with a GLP-1 agonist selected from the group consisting of: Dulaglutide (Trulicity(R)), Exenatide (Byetta(R)), Exenatide extended-release (Bydureon(R)), Liraglutide (Victoza(R)), Lixisenatide (Adlyxin(R)), Semaglutide injection (Ozempic(R)), and Semaglutide tablets (Rybelsus(R)). In some embodiments, the GLP-1 agonist is semaglutide having a formula of N- epsilon26-[2-(2- {2-[2-(2-{2-[(S)-4-carboxy-4-(17- carboxy-heptadecanoylamino)butyrylamino]ethoxy}ethoxy) acetylamino]ethoxy}ethoxy)acetyl] [Aib8,Arg34]GLP-1 (7-37). The term "GLP-1 agonist" as used herein also encompasses dual or triple agonists that can activate more than one receptor. These dual or triple agonists may be molecules that have the ability to activate the GLP-1 receptor and at least one further receptor. In some embodiments, the dual or triple agonist may be a chimeric molecule comprising a first portion that activates the GLP-1 receptor and further portions that activate further receptors. In certain embodiments, the GLP-1 agonist is a dual or triple agonist that, in addition to the GLP-1 receptor, further activates one or more of: a glucose-dependent insulinotropic polypeptide (GIP) receptor, a Fibroblast growth factor 21 (FGF21) receptor, and a glucagon receptor (GCGR). In certain embodiments, the inhibitor of the invention, in particular the siRNA molecules disclosed herein, may be combined with a GLP-1/GIP dual agonist for the treatment of metabolic diseases or disorders.
The term "GLP-1/GIP dual agonist" as used in the context of the present invention refers to a substance or ligand that can activate the GLP-1 receptor and the glucose-dependent insulinotropic polypeptide (GIP) receptor. GLP-1/GIP receptor co-agonists and their potential medical uses are described in several patent applications such as WO 2010/011439, WO 2013/164483, WO 2014/192284, WO 2015/067715, WO 2015/022420, WO 2015/086728, WO 2015/086729, WO 2016/111971 , WO 2020/023386, US 9745360, US 2014/162945, US 2014/0357552, WO 2021/150673, WO 2021/260530, WO 2022/018185, and WO 2022/079639, which are fully incorporated herein by reference. In certain embodiments, the inhibitor of the invention, in particular ChoKa inhibitor may be combined with the GLP-1/GIP dual agonist tirzepatide (Mounjaro(R)) for the treatment of metabolic diseases or disorders. In a particular embodiment, the ChoKa inhibitor may be combined with the GLP-1/GIP dual agonist tirzepatide (Mounjaro(R)) for the treatment of fatty liver diseases, in particular NAFLD and/or NASH. As used herein, "tirzepatide" means a GLP-1/GIP dual agonist peptide as described in U.S. Pat. No.9, 474, 780 and described by CAS Registry Number: 2023788-19-2. Tirzepatide is described in Example 1 of U.S. Pat. No.9,474, 780, with the following sequence: YX1EGTFTSDYSIX2LDKIAQKAFVQWLMGGPSSGAPPPS (SEQ ID NO: 3) wherein Xi is a-amino isobutyric acid (Aib); X2 is Aib; K at position 20 is chemically modified through conjugation to the epsilon-amino group of the K side-chain with (2-[2-(2- Amino- ethoxy)-ethoxy]-acetyl)2-(gammaGlu)1-CO-(CH2)18-CO2H; and the C- terminal amino acid is amidated as a C-terminal primary amide.
In certain embodiments, the ChoKa inhibitor may be combined with a GLP-1/ FGF21 dual agonist for the treatment of metabolic diseases or disorders. In a particular embodiment, the ChoKa inhibitor may be combined with a GLP-1/FGF21 dual agonist for the treatment of fatty liver diseases, in particular NAFLD and/or NASH. The term "GLP-1/FGF21 dual agonist" as used in the context of the present invention refers to a substance or ligand that can activate the GLP-1 receptor and the Fibroblast growth factor 21 (FGF21) receptor. Preferably, the GLP-1/FGF21 dual agonist is a chimeric molecule comprising a GLP-1 agonist as defined herein above and the molecule FGF21 or a functionally active variant or analogue thereof. In certain embodiments, the GLP-1/FGF21 dual agonist may further comprise an antibody Fc region. GLP-1/ FGF21 receptor co-agonists and their potential medical uses are described in several patent applications such as WO 2010/142665, WO2014/037373, WO 2018/115401 , WO 2018/166461 , WO 2019/243557, and WO 2022/002408, which are fully incorporated herein by reference.
In certain embodiments, the ChoKa inhibitor may be combined with a GLP-1/ GCGR dual agonist for the treatment of metabolic diseases or disorders. In a particular embodiment, the ChoKa inhibitor may be combined with a GLP-1/GCGR dual agonist for the treatment of fatty liver diseases, in particular NAFLD and/or NASH. The term "GLP-1/ GCGR dual agonist" as used in the context of the present invention refers to a substance or ligand that can activate the GLP-1 receptor and the glucagon receptor (GCGR). GLP-1/GCGR receptor co-agonists and their potential medical uses are described in several patent applications such as, WO 2008/101017, WO 2009/155258, WO 2011/075393, WO 2011/160630, WO 2014/056872, WO 2014/091316, WO 2015/086733, WO 2017/181452, WO 2018/100174, WO 2019/030268, WO 2019/060660 and WO 2023/006923, which are fully incorporated herein by reference. In certain embodiments, the ChoKa inhibitor may be combined with the GLP-1/ GCGR dual agonist Survodutide (Bl 456906) for the treatment of metabolic diseases or disorders. In a particular embodiment, the ChoKa inhibitor may be combined with the GLP-1/ GCGR dual agonist Survodutide (Bl 456906) for the treatment of fatty liver diseases, in particular NAFLD and/or NASH.
In certain embodiments, the choline kinase inhibitor may be combined with a GLP-1/GIP/GCGR triple agonist for the treatment of metabolic diseases or disorders. In a particular embodiment, the choline kinase inhibitor may be combined with a GLP- 1/GIP/GCGR triple agonist for the treatment of fatty liver diseases, in particular NAFLD and/or NASH. The term "GLP-1/GIP/GCGR triple agonist" as used in the context of the present invention refers to a substance or ligand that can activate the GLP-1 receptor, the GIP receptor and the glucagon receptor (GCGR). GLP- 1/GIP/GCGR receptor co-agonists and their potential medical uses are described in several patent applications such as, WO 2014/096150, WO 2015/067716, WO 2019/125292, WO 2022/090447, and WO 2022/268029, which are fully incorporated herein by reference. In certain embodiments, the choline kinase inhibitor may be combined with Retatrutid (LY-3437943) for the treatment of metabolic diseases or disorders. In a particular embodiment, the fatty liver diseases may be combined with Retatrutid (LY-3437943) for the treatment of fatty liver diseases, in particular NAFLD and/or NASH.
In an embodiment, the GLP-1 agonist forming part of the compositions according to the invention is selected from the group consisting of Dulaglutide, Exenatide, Liraglutide, Lixisenatide, Semaglutide, tirzepatide, and Survodutide.
In an embodiment, the GLP-1 agonist forming part of the compositions according to the invention is semaglutide.
In an embodiment, the compositions according to the invention contain MN58b and a GLP-1 agonist selected from the group consisting of Dulaglutide, Exenatide, Liraglutide, Lixisenatide, Semaglutide, tirzepatide, and Survodutide+ semaglutide.
In an embodiment, the compositions according to the invention contain a choline kinase inhibitor selected from the group consisting of RSM 932A, MN58b, Hemicholinium, EB-3D, EB-3P, ICL-CCIC-0019, CK-37, any of the compounds of Table 1 , 1 ,2-ethylene-p-(bis-benzyldimethyldiyl), symmetrical bis- and triscationic compounds, a compound of the bispyridinium cyclophane family, GRQF-JCR795b, GRQF-MN94ba and semaglutide.
In an embodiment, the compositions according to the invention contain MN58b and Semaglutide.
The compounds that form part of the compositions of the invention include not only the compounds as such but also pharmaceutically acceptable salts, solvates, prodrugs thereof. The term “pharmaceutically acceptable salts, solvates, prodrugs” refers to any pharmaceutically salt, ester, solvate or any other compound which when administered to a receptor is able to provide (directly or indirectly) a compound as described in the present document. However, it will be observed that pharmaceutically unacceptable salts are also within the scope of the invention because the latter can be useful in the preparation of pharmaceutically acceptable salts. The preparation of salts, prodrugs and derivatives can be carried out by means of methods known in the art. For example, pharmaceutically acceptable salts of compounds provided in the present document are synthesized by means of conventional chemical methods from an original compound containing a basic or acid residue. Such salts are generally prepared, for example, by reacting the free acid or base forms of the compounds with a stoichiometric amount of the suitable base or acid in water or an organic solvent or a mixture of both. Non-aqueous media such as DMSO (dimethylsulphoxide), ether, ethyl acetate, ethanol, isopropanol or acetonitrile are generally preferred. Examples of acid addition salts include mineral acid addition salts such as for example, hydrochloride, bromohydrate, iodohydrate, sulfate, nitrate, phosphate and organic acid addition salts such as for example, acetate, maleate, fumarate, citrate, oxalate, succinate, tartrate, malate, mandelate, methanesulfonate and p-toluenesulfonate. Examples of base addition salts include inorganic salts such as for example sodium, potassium, bromide, calcium, ammonium, magnesium, aluminium and lithium salts and organic base salts such as for example ethylenediamine, ethanolamine, N,N-dialkylenethanolamine, triethanolamine, glucamine and basic amino acid salts.
The particularly preferred derivatives or prodrugs are those increasing the bioavailability of the compounds of this invention when such compounds are administered to a patient (for example, by making a compound administered orally be absorbed more easily by blood), or enhancing the release of the original compound in a biological compartment (for example, the brain or the lymphatic system) in relation to the original species.
The invention also provides compositions wherein at least one of the compounds are found as prodrug. The term “prodrug” is used in its widest sense and includes those derivatives which are converted in vivo into the compounds of the invention. Such derivatives are evident for the persons skilled in the art and depending on the functional groups present in the molecule and without limitation, include the following derivatives of the present compounds: esters, amino acid esters, phosphate esters, metal salt sulfonate esters, carbamates and amides. Examples of methods for producing a prodrug of a given active compound are known by the person skilled in the art and can be found for example in Krogsgaard-Larsen et al. “Textbook of Drug design and Discovery” Taylor & Francis (April 2002).
The compounds of the invention can be in crystalline form as free compounds or as solvates and it is intended that both of them are within the scope of the present invention. The solvation methods are generally known in the art. The suitable solvates are pharmaceutically acceptable solvates. In a particular embodiment, the solvate is a hydrate.
The compounds forming the compositions of the invention can include enantiomers, depending on the presence of chiral centers on a C, or isomers, depending on the presence of multiple bonds (for example, Z, E). The individual isomers, enantiomers or diastereoisomers and the mixtures thereof are included within the scope of the present invention.
The different substituents selected for the different compounds of the invention provide a series of factors considerably affecting the values of log P. Thus, hydroxyl groups act as hydrogen bond donors and intra or intermolecular links can be established even in the case of phenols. The presence of carbonyl or carboxyl groups generates proton acceptor groups in the molecule. The presence of halogens generates very deficient carbons and considerably modifies the biological properties. The amino groups generate good nucleophiles on the molecule and in most cases significantly modify its polarity and polarizability and the presence of additional alkyl and/or aryl groups increases the lypophilicity of the molecules.
In another aspect, the invention provides pharmaceutical compositions comprising the compositions of the invention, their pharmaceutically acceptable salt, derivative, prodrug, solvate or stereoisomer thereof together with a pharmaceutically acceptable carrier, adjuvant or vehicle for the administration to a patient.
The phrase "pharmaceutically acceptable carrier" as used herein means a pharmaceutically acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material, involved in carrying or transporting the subject agents from one organ, or portion of the body, to another organ, or portion of the body. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation. Some examples of materials which can serve as pharmaceutically acceptable carriers include: (1) sugars, such as lactose, glucose and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients, such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerin, sorbitol, mannitol, solutol and polyethylene glycol; (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering agents, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20) phosphate buffer solutions; and (21) other non-toxic compatible substances employed in pharmaceutical formulations such as DMSO (dimethylsulphoxide) and its derivatives.
The pharmaceutical compositions can be administered by any suitable administration route, for example an oral, topical, rectal or parenteral route (including subcutaneous, intraperitoneal, intradermal, intramuscular and intravenous route).
Suitable pharmaceutical forms for oral administration include any solid composition (tablets, pastilles, capsules, granules, etc.) or liquid composition (solutions, suspensions, emulsions, syrups, etc.) and can contain conventional excipients known in the art, such as binding agents, for example syrup, acacia, gelatin, sorbitol, tragacanth, or polyvinylpyrrolidone; fillers, for example lactose, sugar, cornstarch, calcium fosfate, sorbitol or glycine; lubricants for the preparation of tablets, for example magnesium stearate, disintegrants, for example starch, polyvinylpirrolidone, sodium starch glycolate or microcrystalline cellulose; or pharmaceutically acceptable wetting agents such as sodium laurylsulfate.
Solid oral compositions can be prepared by means of conventional methods for mixing, filling or preparing tablets. The repeated mixing operations can be used to distribute the active ingredient through the entire compositions by using large amounts of filler agents. Such operations are conventional in the art. The tablets can be prepared, for example by means of wet or dry granulation and can be optionally coated according to methods well known in normal pharmaceutical practice, particularly with an enteric coating.
The pharmaceutical compositions can also be adapted for parenteral administration such as sterile solutions, suspensions or lyophilized products in a suitable unitary pharmaceutical form. Suitable excipients such as bulk agents, buffering agents or surfactants can be used. The mentioned formulations will be prepared using usual methods such as those described or referred to in Spanish Pharmacopoeia and the Pharmacopoeia of the United States and in similar reference texts.
The administration of the compounds or compositions used in the present invention can be by any suitable method, such as intravenous infusion, oral preparations and intraperitoneal and intravenous administration. Nevertheless, the preferred administration route will depend on the patient’s condition. Oral administration is preferred due to the comfort for the patient and the chronic character of the diseases which are to be treated. For their application in therapy, the compositions of the invention will preferably be found in pharmaceutically acceptable or substantially pure form, i.e. the compositions of the invention have a pharmaceutically acceptable purity level excluding the pharmaceutically acceptable excipients and not including material considered to be toxic at the normal dosage levels. The purity levels of the inhibitors of choline kinase or of the GLP-1 agonists compounds preferably exceed 50%, more preferably exceed 70%, more preferable exceed 90%. In a preferred embodiment, they exceed 95%.
The therapeutically effective amounts of the compounds that form part of the compositions according to the present invention will generally depend, among other factors, on the individual who is to be treated, on the severity of the disease said individual suffers from, on the administration form chosen etc. For this reason, the doses mentioned in this invention must be considered as guides for the person skilled in the art and the latter must adjust the doses according to the variables mentioned previously. Nevertheless, the compounds can be administered once or more times a day, for example, 1 , 2, 3 or 4 times a day in a typical daily total amount comprised between 1 and 200 mg/kg body mass/day, preferably 1-10 mg/kg body mass/day. In the same manner an inhibitor of choline kinase can be administered once or more times a day, for example, 1 , 2, 3 or 4 times a day in a typical daily total amount comprised between 1 and 200 mg/kg body mass/day, preferably 1-10 mg/kg body mass/day.
The compositions according to the present invention can be formulated as a single preparation or, alternatively, they may be provided as a product for the simultaneous, concurrent, separate or sequential administration.
The combinations disclosed herein may comprise the choline kinase inhibitor and the GLP-1 analog in one or more pharmaceutical compositions. For example, the choline kinase inhibitor and the GLP-1 agonist may be co-formulated in the same pharmaceutical composition, or they may each be formulated in separate pharmaceutical compositions. Pharmaceutical compositions comprising a choline kinase inhibitor and the GLP-1 analog can be prepared by any method known in the art of pharmacology. In general, such preparatory methods include the steps of bringing the choline kinase inhibitor and/or GLP-1 analog into association with one or more carriers, diluents, adjuvants, excipients or other accessory ingredients and then, if necessary and/or desirable, shaping and/or packaging the product into a desired single- or multi-dose unit. In certain embodiments, unit dosage compositions are those containing a daily dose or unit, daily sub-dose, as herein above described, or an appropriate fraction thereof, of the choline kinase inhibitor and/or GLP-1 analog. In some embodiments, the choline kinase inhibitor and the GLP-1 analog may be provided in a single unit dose (i.e., as a pharmaceutical composition), or they may be provided in two or more separate unit doses, which may be intended for simultaneous, separate or sequential administration. As used herein, a "unit dose" is discrete amount of the pharmaceutical composition comprising a predetermined amount of the active ingredient. The amount of the active ingredient (i.e., the choline kinase inhibitor or GLP-1 analog) is generally equal to the dosage of the active ingredient that would be administered to a subject and/or a convenient fraction of such a dosage such as, for example, one-half or one-third of such a dosage.
In certain embodiments, the administration of an effective amount of the choline kinase inhibitor and of the GLP- 1 analog results is carried out one or more times a day. The administration for a 70 kg adult human may comprise about 0.0001 mg to about 4000 mg, about 0.0001 mg to about 3000 mg, about 0.0001 mg to about 200 mg, about 0.001 mg to about 1500 mg, about 0.01 mg to about 1000 mg, about 0.1 mg to about 1000 mg, about 1 mg to about 1000 mg, about 1 mg to about 100 mg, about 10 mg to about 1000 mg, or about 100 mg to about 1000 mg, of the 4- epimerase inhibitor per unit dosage form. In certain embodiments, formulations of the compositions comprising a choline kinase inhibitor and a GLP-1 analog may be at dosage levels sufficient to deliver from about 0.001 mg/kg to about 100 mg/kg, from about 0.01 mg/kg to about 50 mg/kg, from about 0.1 mg/kg to about 40 mg/kg, from about 0.5 mg/kg to about 30 mg/kg, from about 0.01 mg/kg to about 10 mg/kg, from about 0.1 mg/kg to about 10 mg/kg, and from about 1 mg/kg to about 25 mg/kg, of subject body weight per day, one or more times a day, to obtain the desired therapeutic effect.
The compositions described in this invention, their pharmaceutically acceptable salts, prodrugs and/or solvates, as well as the pharmaceutical compositions containing them can be used together with other additional drugs to provide a combination therapy. Said additional drugs can form part of the same pharmaceutical composition or can alternatively be provided in the form of a separate composition for its simultaneous or non-simultaneous administration with the pharmaceutical composition according to the invention. The other drugs can form part of the same composition or be provided as a separate composition for its administration at the same time or at different times.
The compositions of the invention can be used in combination with other therapeutic agents such as metformin, sulfonylurea and sulfonylurea-like agents, thiazolidinediones, Peroxisome Proliferator-Activated Receptor (PPAR) gamma modulators, PPAR alpha modulators, Protein Tyrosine Phosphatase- IB inhibitors, Insulin Receptor Tyrosine Kinase activators, 11 p-hydroxysteroid dehydrogenase inhibitors, glycogen phosphorylase inhibitors, glucokinase activators, p-3 adrenergic agonists, and glucagon receptor agonists.
In addition, the compositions of the invention can be used in combination with one or more anti-obesity agents. Anti-obesity agents include, serotonin (5HT) transport inhibitors, including, but not limited to, paroxetine, fluoxetine, fenfluramine, fluvoxamine, sertraline, and imipramine.
Anti-obesity agents also include selective serotonin reuptake inhibitors, including, but not limited to dexfenfluramine, fluoxetine, sibutramine and combinations thereof.
Anti-obesity agents also include selective serotonin agonists and selective 5- HT2C receptor agonists, antagonists/inverse agonists of the central cannabinoid receptors (the CB-1 receptors), including, but not limited to, rimonabant (Sanofi Synthelabo), and SR-147778 (Sanofi Synthelabo),
Anti-obesity agents also include melanocortins and melanocortin agonists, metabotropic glutamate subtype 5 receptor (mGluR5) antagonists, topiramate, phentermine or combinations, neuropeptide Y1 (NPY1) antagonists, neuropeptide Y5 antagonists, melanin-concentrating hormone (MCH) antagonists including melaninconcentrating hormone 1 receptor (MCH1 R) antagonists, such as T-226296 (Takeda) and melanin-concentrating hormone 2 receptor (MCH2R) antagonists, nalmefene (REVEX(R)), 3-methoxynaltrexone naloxone, naltrexone, bupropion, naloxonazine, beta-funaltrexamine, deltal ([D-Ala2,Leu5,Cys6]-enkephalin (DALCE), naltrindole isothiocyanate, and nor-binaltorphamine or combinations thereof, orexin antagonists, neuropeptide Y2 (NPY2) agonists, neuropeptide Y4 (NPY4) agonists, histamine 3 (H3) antagonist/inverse agonists, cholecystokinin (CCK) and CCK agonists, ghrelin antagonists, obestatin and obestatin analogs and amylinomimetics (e.g., a amylin-calcitonin receptor co-agonists such as davalintide), The invention also relates to the use of compositions according to the invention for use in (a) treating, preventing or ameliorating a weight-associated disorder, (b) enhancing weight loss, (c) increasing glucose uptake, (d) activating brown-adipose tissue thermogenesis, (e) activating 5' adenosine monophosphate-activated protein kinase or (f) treating, preventing or ameliorating a fatty liver disease in a subject in need thereof. In an embodiment, the weight-associated disorder is selected from the group consisting of obesity, hyperglycemia, type 2 diabetes, a cardiovascular disease, joint inflammation, sleep apnea, a respiratory disorder, a gallbladder disease, elevated glucose levels, elevated insulin levels, non-alcoholic fatty liver disease and diabetic nephropathy.
In yet another embodiment, the fatty liver disease is non-alcoholic fatty liver disease (NAFLD) and non-alcoholic steatohepatitis (NASH).
Method for enhancing the therapeutic efficacy of a GLP-1 analog
The authors of the present invention have observed that the combined use of a choline kinase inhibitor and a GLP-1 analog resulted in a decrease in weight in overweight mice compared to mice being fed a high fat diet alone, and compared to mice being administered the choline kinase inhibitor or the GLP-1 agonist alone. .
Accordingly, in another aspect, the invention relates to a method for enhancing the therapeutic effectiveness of a GLP-1 analog comprising the administration of the GLP-1 analog with a choline kinase inhibitor.
The term “enhancing the therapeutic effectiveness of a GLP-1 analog” as used herein refers to the ability in increasing the therapeutic effect of the GLP-1 analog in one or more of the diseases for which these analogs are commonly used. In some embodiments, the choline kinase inhibitor enhances the effectiveness of the GLP-1 analog in treatment of a metabolic disorder. In some embodiments, the choline kinase inhibitor enhances the effectiveness of the GLP-1 analog in treatment of diabetes. In some embodiments, the choline kinase inhibitor enhances the effectiveness of the GLP-1 analog in treatment of obesity. In some embodiments, the choline kinase inhibitor enhances the effectiveness of the GLP-1 analog in treatment of metabolic syndrome.
In some embodiments, the choline kinase inhibitor enhances the effectiveness of the GLP-1 analog in treatment of diabetes (type I diabetes, type II diabetes), overweight and obesity, steatohepatitis (NASH, ASH), cardiovascular disease, fatty liver, cirrhosis, and non-alcoholic fatty liver disease , metabolic syndrome and various diabetic complications.
In an embodiment, the choline kinase inhibitor is used for enhancing the therapeutic effectiveness of the GLP-1 analog in treating, preventing or ameliorating a weight-associated disorder wherein the weight-associated disorder is selected from the group consisting of obesity, hyperglycemia, type 2 diabetes, a cardiovascular disease, joint inflammation, sleep apnea, a respiratory disorder, a gallbladder disease, elevated glucose levels, elevated insulin levels, non-alcoholic fatty liver disease and diabetic nephropathy.
In an embodiment, the choline kinase inhibitor is used for enhancing the therapeutic effectiveness of the GLP-1 analog in treating, preventing or ameliorating a fatty liver disease in a subject wherein the fatty liver disease is non-alcoholic fatty liver disease (NAFLD) and non-alcoholic steatohepatitis (NASH).
In some embodiments, the choline kinase inhibitor is capable of enhancing effectiveness of the GLP-1 analog by at least about 5 percent compared to the subject in which only the GLP-1 analog is administered. In other instances, the increase in effectiveness is of at least about 10 percent, of at least about 15 percent, 20 percent, 25 percent, 30 percent 40 percent, or 50 percent.
The terms “choline kinase inhibitor” and “GLP-1 analog” have been described above and are equally applicable to the instant method of the invention.
In an embodiment of the method for enhancing the therapeutic effectiveness of a GLP-1 analog, the choline kinase inhibitor is a choline kinase inhibitor specific for the choline kinase alpha isoform.
In an embodiment of the method for enhancing the therapeutic effectiveness of a GLP-1 analog, the choline kinase inhibitor is a compound of Formula I: wherein:
Q- represents the conjugate base of a pharmaceutically suitable organic or inorganic acid;
Ri and R'i, represent, independently of each other, an aryl radical optionally substituted by halogen, trifluoromethyl, hydroxyl, C 1-6 alkyl, amino or alkoxyl;
R2 and R'2, represent, independently of each other, an aryl radical optionally substituted by halogen, trifluoromethyl, hydroxyl, C1-6 alkyl, amino or alkoxyl; Rs and R'3, represent, independently of each other, either a radical selected from the group formed by H, halogen, trifluoromethyl, hydroxyl, amino, alkoxyl and C1-6 alkyl optionally substituted by trifluoromethyl, hydroxyl, amino or alkoxyl, or together with R4 and R'4, respectively, and independently of each other, a -CH=CH-CH=CH- radical optionally substituted by halogen, trifluormethyl, hydroxyl, C1-6 alkyl, amino or alkoxyl;
R4 and R'4, represent, independently of each other, either a radical selected from the group formed by H and C1-6 alkyl optionally substituted by halogen, trifluoromethyl, hydroxyl, amino or alkoxyl, or together with R3 and R'3 respectively, and independently of each other, a -CH=CH-CH=CH- radical optionally substituted by halogen, trifluoromethyl, hydroxyl C1-6 alkyl, amino or alkoxyl;
- A represents a spacer group comprising any divalent organic structure acting as a joining link between the two pyridinium groups present in the structure defined by formula I.
In an embodiment of the method for enhancing the therapeutic effectiveness of a GLP-1 analog, the choline kinase inhibitor is a compound of Table 2.
In an embodiment of the method for enhancing the therapeutic effectiveness of a GLP-1 analog, the choline kinase inhibitor is RSM932.
In an embodiment of the method for enhancing the therapeutic effectiveness of a GLP-1 analog, the choline kinase inhibitor is a compound of Formula II: n is 0, 1 , 2 or 3
Z is any a structural group selected from the group of: - wherein Y is selected from the group of -H, -CH3, -CH2-OH, -CO-CH3,
CN, -NH2, -N(CH3)2, pyrrolidine, piperidine, perhydroazepine, -OH, -O-CO- C15H31.
In an embodiment of the method for enhancing the therapeutic effectiveness of a GLP-1 analog, the choline kinase inhibitor is a compound selected from the group consisting of RSM 932A, MN58b, Hemicholinium, EB-3D, EB-3P, ICL-CCIC- 0019, CK-37, any of the compounds of Table 1 , 1 ,2-ethylene-p-(bis- benzyldimethyldiyl), symmetrical bis- and triscationic compounds, a compound of the bispyridinium cyclophane family, GRQF-JCR795b, GRQF-MN94b.
In an embodiment of the method for enhancing the therapeutic effectiveness of a GLP-1 analog, the GLP-1 analog is selected from the group consisting of Dulaglutide, Exenatide, Liraglutide, Lixisenatide, Semaglutide, tirzepatide, and Survodutide.
In an embodiment of the method for enhancing the effectiveness of a GLP-1 analog, the GLP-1 analog is semaglutide.
In an embodiment of the method for enhancing the therapeutic effectiveness of a GLP-1 analog, the choline kinase inhibitor is MN58b and the GLP-1 agonist selected from the group consisting of Dulaglutide, Exenatide, Liraglutide, Lixisenatide, Semaglutide, tirzepatide, and Survodutide+ semaglutide.
In an embodiment of the method for enhancing the therapeutic effectiveness of a GLP-1 analog, the choline kinase is inhibitor is selected from the group consisting of RSM 932A, MN58b, Hemicholinium, EB-3D, EB-3P, ICL-CCIC-0019, CK-37, any of the compounds of Table 1 , 1 ,2-ethylene-p-(bis-benzyldimethyldiyl), symmetrical bis- and triscationic compounds, a compound of the bispyridinium cyclophane family, GRQF-JCR795b, GRQF-MN94ba and the GLP-1 analog is semaglutide.
In an embodiment of the method for enhancing the therapeutic effectiveness of a GLP-1 analog, the choline kinase is inhibitor is MN58b and the GLP-1 analog is Semaglutide.
NUMBERED EMBODIMENTS
1. A method of enhancing weight loss comprising administering an effective amount of a choline kinase inhibitor to a subject in need of weight loss.
2. A method of treating hyperglycemia and/or type 2 diabetes in a subject, comprising administering to a subject in need of such treatment an effective amount of a choline kinase inhibitor. 3. A method of increasing glucose uptake in a subject in need of such uptake, comprising administering to said subject an effective amount of a choline kinase inhibitor.
4. A method of activating brown-adipose tissue thermogenesis comprising administering to a subject in need of such activating an effective amount of a choline kinase inhibitor.
5. A method of activating 5' adenosine monophosphate-activated protein kinase comprising administering to a subject in need of such activating an effective amount of a choline kinase inhibitor.
6. A method of treating or ameliorating a weight-associated disorders, including but not limited to cardiovascular disease, joint inflammation, sleep apnea, respiratory disorder, and gallbladder disease, wherein such method comprises administering an effective amount of a choline kinase inhibitor to a subject in need thereof.
7. A method of treatment or amelioration of a metabolic disorder, such as type 2 diabetes, elevated glucose levels, elevated insulin levels, obesity, nonalcoholic fatty liver disease, cardiovascular diseases or diabetic nephropathy comprising administering to a subject in need thereof an effective amount of a choline kinase inhibitor.
8. The method of any of embodiments 1 to 7, wherein said choline kinase inhibitor is a choline kinase inhibitor specific for the choline kinase alpha isoform.
9. The method of any of embodiments 1 to 7, wherein said choline kinase inhibitor is a compound having a flavonoid moiety.
10. The method of any one of embodiments 1 to 7, wherein said choline kinase inhibitor is a compound of formula I or of formula II wherein Formula I is wherein:
Q- represents the conjugate base of a pharmaceutically suitable organic or inorganic acid;
Ri and R'i, represent, independently of each other, an aryl radical optionally substituted by halogen, trifluoromethyl, hydroxyl, C 1-6 alkyl, amino or alkoxyl;
R2 and R'2, represent, independently of each other, an aryl radical optionally substituted by halogen, trifluoromethyl, hydroxyl, C1-6, alkyl, amino or alkoxyl;
R3 and R'3, represent, independently of each other, either a radical selected from the group formed by H, halogen, trifluoromethyl, hydroxyl, amino, alkoxyl and C1-6 alkyl optionally substituted by trifluoromethyl, hydroxyl, amino or alkoxyl, or together with R4 and R'4, respectively, and independently of each other, a -CH=CH-CH=CH- radical optionally substituted by halogen, trifluormethyl, hydroxyl, C 1-6 alkyl, amino or alkoxyl;
R4 and R'4, represent, independently of each other, either a radical selected from the group formed by H and C1-6 alkyl optionally substituted by halogen, trifluoromethyl, hydroxyl, amino or alkoxyl, or together with R3 and R'3 respectively, and independently of each other, a -CH=CH-CH=CH- radical optionally substituted by halogen, trifluoromethyl, hydroxyi C1-6, alky 4 amino or alkoxyl;
- A represents a spacer group comprising any divalent organic structure acting as a joining link between the two pyridinium groups present in the structure defined by formula I. and wherein Formula II is wherein n is 0, 1 , 2 or 3
Z is any a structural group selected from the group of:
- wherein Y is selected from the group of -H, -CH3, -CH2-OH, -CO-CH3, -CN, -NH2, -N(CH3)2, pyrrolidine, piperidine, perhydroazepine, -OH, -O-CO- C15H31. 11. The method of any one of embodiments 1 to 7, wherein said choline kinase inhibitor is a compound selected from Table 2.
12. The method of any one of embodiments 1 to 7, wherein said choline kinase inhibitor is a compound selected from the group consisting of RSM 932A, MN58b, Hemicholinium, EB-3D, EB-3P, ICL-CCIC-0019, CK-37, any of the compounds of Table 1 , 1 ,2-ethylene-p-(bis-benzyldimethyldiyl), symmetrical bis- and triscationic compounds, a compound of the bispyridinium cyclophane family, GRQF-JCR795b and GRQF-MN94b.
13. The method of any one of embodiments 1 to 7, wherein said choline kinase inhibitor is a compound selected from the group consisting of RSM 932A, MN58b, Hemicholinium, EB-3D, EB-3P, ICL-CCIC-0019 and CK-37.
14. The method of any one of embodiments 1 to 7, wherein said choline kinase inhibitor is RSM 932A or MN58b.
15. The method of any one of embodiments 1-15, further comprising administering a compound selected from the group consisting of semaglutide, orlistat, bupropion-naltrexone, tirzepatide, liraglutide, phenterminetopiramate, setmelanotide and bimagrumab. The invention is described below by way of the following examples which are to be construed as merely illustrative and not limiting the scope of the invention.
EXAMPLES
The following Examples have been included to provide guidance to one of ordinary skill in the art for practicing representative embodiments of the presently disclosed subject matter. Considering the present disclosure and the general level of skill in the art, those of skill can appreciate that the following Examples are intended to be exemplary only and that numerous changes, modifications, and alterations can be employed without departing from the scope of the presently disclosed subject matter.
EXAMPLE 1
Determination of ChoKa in cell models for adipocyte differentiation
To analyze a potential relationship of ChoKa function and adipocytes regulation, the levels of the enzyme were determined in adipocyte and preadipocyte containing fractions from mice fed a normal chow or a high fat diet.
Figure 1A shows that ChoKa was more highly expressed in both the preadipocytes and adipocytes of mice fed a high fat diet. As controls for the differentiation process under the experimental conditions, the levels of Ppagly was also determined in the same fractions as shown in Figure 1 B. The increased ChoKa expression in adipocytes correlated with a high fat diet suggests that ChoKa may play a role in adipogenesis.
Method
Generally following the method reported in Zebisch, K., Voigt, V., Wabitsch, M. and Brandsch, M., 2012. Protocol for effective differentiation of 3T3-L1 cells to adipocytes. Analytical biochemistry, 425(1), pp.88-90, wild type 3T3-L2 preadipocytes were treated with IBMX (3-isobutyl-1 -methylxanthine), dexamethasone and insulin to induce differentiation to adipocytes. The cells were also treated with MN58b or RSM932A to test the effects of these choline kinase inhibitors on differentiation as described in more detail below.
Specifically, on days 1-3 wild type 3T3-L1 pre-adipocytes were treated with 1 g/m! IBMX+ 0.25 pM Dexamethasone, inducing differentiation of the pre- adipocytes into adipocytes. In a variation from the referenced method, on days 4-6 the cells were further treated with 1 pg/m! insulin, supporting differentiation.
On each of days 1-6 the cells were treated with choline kinase inhibitors according to Table 1.
Table 1. Administration of a Choline Kinase a Inhibitor during pre-adipocyte differentiation to adipocytes
The treated cells were visualized by microscopy; lipid accumulation was detected using the RED-0 staining method and visualized by microscopy. Triacecylglycerol (TAG) levels were quantified by mass spectrometry; TAG is the lipid molecule that stores energy in adipocytes and is generally used as a measure of the lipid content in differentiated adipocytes.
Results
In each example, the addition of MN58b and RSM-932A blocked differentiation of the adipocytes in dose dependent manner. Treatment with vehicle alone (Control with no inhibitor) allowed full differentiation of 3T3-L1 cells at day 6, while administration of either inhibitor at 10 pM led to cell death, indicating the dose was toxic to the cells.
Figure 2A shows microscopic images of 3T3-L1 adipocytes treated with different concentrations of MN58b. Insets show adipocytes after addition of the RED- O reagent on day 6.
Lower doses (1 pM and 0.1 pM) of MN58b interfered with cell differentiation, and abrogated lipid accumulation. Figure 2A shows images collected from RED-O, wherein the RED-0 dye stains lipid deposits in the cells, thereby serving as an indicator of total lipid storage. Total lipid storage, is in turn, an indicator of the amount of lipogenesis that has occurred in the cells. The more intense and widespread the red color that results from the RED-0 dye, the greater the lipid deposits and therefore the greater the lipogenic activity in the cell. These results are consistent with lipogenesis being blocked by ChoKa inhibitor treatment, without being bound by theory, such blockage may occur by way of AMPK activation, as AMPK activation is known to block lipogenesis. On Day 6, the differentiated adipocytes treated with either 0.1 M or 1.0 M MN58b showed reduced concentration of TAG in a dose dependent manner (Figure 2B). TAG is the main lipid being stored in adipocytes and is therefore a measure of the production and storage of lipids in adipocytes. A reduced TAG concentration is consistent with the result seen with the RED-0 method: a reduction in lipogenesis, as evidenced by a smaller amount of lipids in the cells.
Similar results were observed after treatment with RSM-932A (Figure 3). Microscopic images of 3T3-L1 Adipocytes collected on Day 6 of treatment with different concentrations of RSM-932A (Figure 3A). Insets show adipocytes after addition of the RED-0 reagent. (Figure 3B) A graph of TAG concentrations of different RSM-932a treatments.
In this way, the use of MN58b and RSM-932A is correlated with absence of adipocyte fat storage cells, a property consistent with one path for a new therapeutic treating obesity.
The observed blocked differentiation of adipocytes shows that the AMPK pathway is affected by each of MN58b and RSM-932A. As discussed above, the AMPK pathway implicates thermogenesis, which is also related to weight loss.
The expression of adipocyte genes Ppary, Srebf, Acc1 , Fasn, CD36, Fabp 4, Lipinl , Dgat2, Ppara, Pgda, CPT1a, and CPT2 were all analyzed on Day 6 of treatment with 0.1 uM and 1.0 uM of RSM-932A or with treatment with with 0.1 uM and 1.0 uM of MN58b. Figure 4 shows graphs of gene expression related to (A) de novo lipogenesis, (B) fatty acid uptake and esterification, and (C) fatty acid oxidation with different RSM-932a treatments. The expression of the de novo lipogenesis genes Ppary, Srebf, Acc1 , and Fasn were reduced (as shown by qPCR methods), as were the gene expression of Fatty acid uptake genes CD36, Fabp 4, Lipinl , Dgat2, and the gene expression of fatty acid oxidation genes Ppara, Pgda, CPT1a, and CPT2. Figure 5 A,B,C show analogous results with treatment with MN58b. These genes are all expected to be modulated upon activation of the AMPK pathway, which leads to inhibition of lipogenesis, fatty acid uptake and fatty acid oxidation. This is consistent with AMPK and its downstream pathway being activated with ChoKa inhibitors.
EXAMPLE 2
Effect of ChoKa inhibitors in obesity prevention Method
Fifteen 6-week old C57/BL/6J make mice (Jackson Laboratories) were fed a 45% fat high fat diet (Research Diets, Inc New Brunswick, NJ) concurrently with one of three different high fat diet cohorts identified below by random assignment (n=5 for each cohort):
1) 45% High fat diet (“HFD” D12451 , Rodent Diet With 45 kcal% Fat) +water (oral gavage);
2) 45% HFD + 2.5 mg/kg MN58b (oral gavage)
3) 45% HFD + 250 mg/kg metformin(oral gavage).
Mice were weighed once a week throughout the study. Mice from each HFD cohort were analyzed using Echo MRI (magnetic resonance imaging) at the start of the study and once every two weeks for 6 weeks to evaluate fat mass , and lean mass. Metformin is used herein as a positive control.
Mice from each HFD cohort were sacrificed after 6 weeks of HFD and weights of liver, muscle and different types of fat were measured, following the method disclosed Bagchi DP, MacDougald OA. Identification and Dissection of Diverse Mouse Adipose Depots. J Vis Exp. 2019 Jul 11 ;(149): 10.3791/59499.
A glucose tolerance test were carried out on the MN58b treated mice on the 6th week of the prevention study with the following protocol:
1 . Mice tails were marked and mice weighed the night before the test.
2. Mice were clean fasted for 16 hours (overnight) before the test
3. The morning of the test:
A. A 20% glucose solution in saline (diluted commercial 50% glucose solution with saline) was prepared
B. Made a small tail snip to collect blood; mouse blood glucose was measured using a commercial glucose meter from Bayer
C. Mice were injected with 1 g/kg of the 20% glucose solution via intraperitoneal injection (I.P. injection)
D. Blood samples were collected 15, 30, 60, 90, and 120 minutes post glucose injection and blood glucose measurements recorded.
In addition, food intake of the mice were monitored.
Results
As shown in Figure 6, treatment with MN58b mirrored the effects of administering metformin in each of changes in body weight (Figure 6A), fat mass (Figure 6B) and lean mass (Figure 6C). Specifically, when administered to ‘normal weight’ mice, MN58b administered in conjunction with a high fat diet prevented weight gain and decreased overall fat mass compared to mice being fed a high fat diet alone, while lean mass was maintained.
The results show that after 6 weeks of HFD of sacrificial weights of liver, muscle and fat revealed that the mass of liver and of muscle were equivalent to control, while the fat deposits of each type were reduced compared to control with the administration of MN58b (Figure 7). The graphs in Figure 7 also show the weight of gonadal white adipose tissue (gWAT), subcutaneous fat (SubQ), retroperitoneal fat (Retrofat.), Brown adipose tissue fat (BAT).
Figure 8A shows a graph of blood glucose (mg/dl) measurement over time for n=5 mice administered MN58b compared to control and metformin compared to control. The glucose tolerance test revealed that treatment with MN58b improved glucose tolerance in the prevention model of mice fed a high fat diet compared to control. Figure 8D revealed that treatment with MN58b did not reduce food intake, which indicated that Mn58b does not function by reducing appetite, but rather by another mechanism.
EXAMPLE 3
Effect of ChoKa inhibitors in the treatment of obesity
Method
An experiment was performed to demonstrate the potential in vivo efficacy of treatment with the ChoKa inhibitor MN58b in an obese mice model. Fifteen 6-week old male C57/BL/6J mice (Jackson Laboratories) were fed a high fat diet (HFD, Research Diets, Inc New Brunswick, NJ) for 8 weeks followed by 12 weeks treatment except where noted with one of three different high fat diet cohorts identified below (n=5 for each cohort):
1) 45% HFD + water (oral gavage);
2) 45% HFD + 2.5 mg/kg MN58b (oral gavage) for 7 weeks, followed by treatment for 3 weeks with HFD + 5 mg/kg MN58b (oral gavage)
3) 45% HFD + 250 mg/kg metformin (oral gavage).
Mice from each cohort were weighed each week of the experiment. Mouse fat and lean mass were analyzed using Echo MRI (magnetic resonance imaging) at the start of treatment and once every two weeks after administration of the treatment started.
Mice fed 20 weeks on HFD were treated as described and weight of tissues were determined under control (water), ChoKa inhibitor (MN58b) and metformin treatment (Metformin). Masses of liver (liver), gonadal white adipose tissue (gWAT), subcutaneous fat (SubQ), retroperitoneal fat (RetroFat), brown adipose tissue (BAT) and skeletal muscle (Muscle) were measured.
A glucose tolerance test was performed as described above.
Results
Figure 9 demonstrates that weight gain (Figure 9A) and fat gain (Figure 9B) was attenuated under the treatment regime described here, while skeletal mass remained relatively unaffected (Figure 9C).
Consistent with the results of the prevention study, Figure 10 shows the effect of treatment of mice with the ChoKa inhibitor MN58b in different tissues. As in the prevention model, different types of fat tissue had their weight reduced with MN58b treatment with respect to water control, while the lean mass (muscle) remained unchanged with MN58b treatment.
Furthermore, these results indicate that treatment with MN58b improved glucose tolerance as in the prevention model described above (Figure 11A).
EXAMPLE 4
Dose dependence of ChoKa inhibitors in the treatment of obesity
Method
A second experiment was performed to demonstrate the potential in vivo efficacy of treatment in a dose dependent manner with the ChoKa inhibitor MN58b in an obese mice model. 50 6-week old male C57/BL/6J mice (Jackson Laboratories) were fed a high fat diet (HFD, Research Diets, Inc New Brunswick, NJ) for 8 weeks followed by 14 weeks treatment with with one of four different high fat diet cohorts identified below:
1) 45% HFD + water (oral gavage n=15);
2) 45% HFD + 5 mg/kg MN58b (oral gavage, n=15).
3) 45% HFD + 10 mg/kg MN58b (oral gavage, n=15).
4) 45% HFD + 250 mg/kg metformin (oral gavage, n=5).
As a control, an additional 5 mice were treated with a low fat chow diet for the entire 22 week duration of the study (n=5).
Mice from each cohort were weighed each week of the experiment. Mouse fat mass was analyzed using Echo MRI (magnetic resonance imaging) at the start of treatment and once every two weeks after administration of the treatment started.
A glucose tolerance test was performed as described above, at 10 weeks of treatment. In addition, in this second treatment test, an insulin tolerance test was carried out on the MN58b treated mice on the 14th week of the study with the following protocol:
1 . Mice tails were marked and mice weighed the night before the test.
2. Mice were clean fasted for 6 before the test
3. The morning of the test:
A. 1 unit/kg of insulin was injected intraperitoneally into the mice.
B. Blood samples were collected 15, 30, 60, 90, and 120 minutes post insulin injection and blood glucose measurements recorded, as done in the glucose tolerance tests.
Results
In Figure 12 we confirm that MN58b attenuates total weight (Figure 12A) and fat mass (Figure 12B) with respect to a high fat diet control and leaves lean mass within range of the low fat chow diet control (Figure 12C). In addition, the effects of MN58b were found to be dose dependent, with a stronger effect on weight and fat mass with MN58b at 10 mg/kg compared to 5 mg/kg. Likewise, Figure 13A revealed a higher glucose tolerance under a treatment regime with MN58b in comparison to the control, confirming the results from the prevention test described in Figure 8A, and the results of the glucose tolerance test from the first treatment test, as seen in Figure 11A.
The insulin tolerance test revealed that mice treated with MN58b were more sensitive to insulin than control mice treated with water (Figure 13B).
EXAMPLE 5
Synergistic effects between ChoKa inhibitors and GLP-1 analogs
Method
Semaglutide is a known approved obesity treatment. The synergism between semiglutide and ChoK inhibitors was studied here.
Twenty 8-week old male C57/BL/6J mice (Jackson Laboratories) were fed a high fat diet (HFD) for 6 weeks and then were randomized into 4 groups of 5 mice each followed by 8 weeks treatment with one the following schedules:
(1) Control: 8 weeks Control (HFD);
(2) Treatment (SEMA): 8 weeks HFD + 0.01 mg/kg semaglutide (subcutaneous injection, 7 days a week)
(3) Treatment (MN): 8 weeks HFD + 5 mg/kg MN58b (oral gavage, 5 days a week) (4) Treatment Synergism: 8 weeks HFD + 0.01 mg/kg semaglutide + 5 mg/kg MN58b.
Mice were weighed twice a week throughout the study.
Results
As shown in Figure 14A MN58b + semaglutide caused the body weight to decrease when administered to ‘overweight’ mice compared to mice being fed a high fat diet alone, and synergistically compared to mice being administered MN58b or semaglutide alone. Administration of 0.01 mg/kg semaglutide caused weight loss. Thus, co-administration of MN58b and semaglutide led to increased weight loss with respect to administration of semaglutide or MN58 alone. Meanwhile, lean mass did not vary over time among treatments (Figure 14B). Fat mass increase over the length of the study was highly attenuated in the semaglutide + MN58b condition (Figure 14C) with respect to all other conditions and the control, which is consistent with the synergistic behavior displayed in the total weight, and suggests that this combination of drugs is potent in the prevention of fat gain.
EXAMPLE 6
Effect of ChoKa inhibitors in reducing fat deposition in liver
Methods
Mice from the first prevention test (Example 2) and first treatment test (Example 3) had their liver triglycerides analyzed. To measure triglycerides in liver, mouse livers were harvested and triglyceride levels were measured per gram wet weight of liver using the Wako Diagnostics L-Type TG M kit.
Results
Liver tissue showed a marked and significant reduction in triglycerides accumulation with both MN58b and metformin, with respect to water controls, in both the prevention (Figure 8B) and treatment (Figure 11 B) regimes. Meanwhile, there was no significant effect on liver weight with MN58b treatment with respect to control in the prevention study (Figure 8C) with a slight significant difference in liver weight with respect to control in the first treatment study (Figure 11C).
It will be understood that various details of the presently disclosed subject matter may be changed without departing from the scope of the presently disclosed subject matter. Furthermore, the foregoing description is for the purpose of illustration only, and not for the purpose of limitation. Any and all compositions, uses, and/or methods shown and/or described expressly or by implication in the information provided herewith, including but not limited to features that may be apparent and/or understood by those of skill in the art.
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