Calcitonin Analogues for Treating Diseases and Disorders

Abstract

Calcitonin analogues as a medicament for producing a decrease in liver triglycerides or for reducing fat accumulation in the liver of a subject are provided.

Claims

1. A calcitonin analogue as a medicament for producing a decrease in liver triglycerides or for reducing fat accumulation in the liver of a subject, wherein the calcitonin analogue is in accordance with SEQ ID NO: 1 or SEQ ID NO: 2.

2. The calcitonin analogue as claimed in claim 1, wherein the wherein the calcitonin analogue is in accordance with SEQ ID NO: 3.

3. The calcitonin analogue as claimed in claim 1, wherein the wherein the calcitonin analogue is in accordance with SEQ ID NO: 4.

4. The calcitonin analogue as claimed in claim 1, wherein the wherein the calcitonin analogue is in accordance with SEQ ID NO: 5.

5. The calcitonin analogue as claimed in claim 1, wherein the wherein the calcitonin analogue is in accordance with and one of SEQ ID NOS: 6-74.

6. A calcitonin analogue as a medicament for producing a decrease in liver triglycerides/reducing fat accumulation in the liver of a subject, wherein the calcitonin analogue is in accordance with any one of SEQ ID NOS: 54, 55, 57 or 58.

7. A method of producing a decrease in liver triglycerides/reducing fat accumulation in the liver of a subject mammal in need thereof which comprises administering an effective amount of a calcitonin analogue as claimed in claim 1.

8. The method as claimed in claim 7, wherein the amount administered is from 0.001 to 50 g/kg/day.

9. The method as claimed in claim 7, wherein the amount administered is from 0.01 to 5 g/kg/day.

10. The method as claimed in claim 7 or claim 8, wherein the subject is a human.

Description

[0072] The invention will be further illustrated and explained by the following non-limiting example which makes reference to the accompanying drawings in which:

[0073] FIGS. 1A-1C show food intake, bodyweight and weight of the epididymal adipose tissue depot at termination in a 16-day study.

[0074] FIG. 2 shows liver triacylglycerols and arachidonic acid following 16-days of treatment in obese rats.

[0075] FIG. 3 shows liver free fatty acids following 16-days of treatment in obese rats.

[0076] FIG. 4 shows bodyweight as a function of treatment over 60 days presented as baseline normalized bodyweight as a function of time in five dose groups and a vehicle arm.

[0077] FIG. 5 shows absolute bodyweight at termination of the study. Lean represents a non-obese control group, and the vehicle is the obese control group. The pair-fed groups to the right illustrate groups where the food of the control group has been restricted to match the level of the corresponding active treatment group, and thereby shows the weight loss introduced specifically by the reduction in food intake.

[0078] FIG. 6 shows measurement of the weight of the perirenal fat depot at termination.

[0079] FIG. 7 shows results of extraction of triacylglycerols from the liver collected at termination of the experiment.

[0080] FIGS. 8A-8F show the results of body weight reduction using KBP-042.

[0081] FIG. 9 summarises all of the results from the tests performed in Example 2.

[0082] FIGS. 10A-10F show the results of Adipose Tissue reduction using KBP-042.

[0083] FIGS. 11A-11F show Oil red 0 staining of frozen liver sections (magnification 40): (FIG. 11A) Lean; (FIG. 11B) Vehicle; (FIG. 11C) 2.5 g/kg KBP-042; (FIG. 11D) 2.5 g/kg KBP-089; (FIG. 11E) Pair-fed KBP-089; (FIG. 11F) Quantification.

[0084] FIGS. 12A-12L shows blood glucose and insulin levels during oral glucose tolerance test (OGTT) performed in animals treated with KBP-042 or vehicle.

[0085] FIGS. 13A-13B show the effect of KBP-042 on insulin sensitivity.

EXAMPLE 1

[0086] Two studies were conducted, and in both studies the obese rats used were generated as follows:

[0087] To obtain obese rats, male Sprague-Dawley rats aged 12 weeks were placed on a diet consisting of regular rodent chow and a 60 kcal % high-fat diet (no. D12495; Research Diets) for a total of 12 wk. Following the 12 weeks on high fat diet the rats were randomized into treatment groups based on weight.

[0088] The initial study was a 16-day treatment study, in which three groups were studied: Lean (same age but no high fat diet), vehicle (obese control group), and KBP-042 at 7.5 g/kg/day. Throughout the experiment the bodyweight and food intake were monitored, and at termination liver and fat depots were collected for weighing and potential analysis of triglyceride, free fatty acids and arachidonic acid content.

[0089] The second study consisted of the following groups: vehicle, KBP-042 0.625 g/kg/day, KBP-042 1.25 g/kg/day, KBP-042 2.5 g/kg/day, KBP-042 5 g/kg/day, KBP-042 10 g/kg/day, pair-fed (calorie restriction) matching the 5 g/kg/day and pair-fed (calorie restriction) matching the 10 g/kg/day, to allow assessment of the effect of KBP-042 on bodyweight independent of its effect on food intake.

[0090] Throughout the experiment the bodyweight and food intake were monitored, and at termination liver and fat depots were collected for weighing and potential analysis of triglyceride content.

Tissue Lipid Analysis

Preparation of Internal Standard

[0091] As the quantification procedure contains several steps an internal standard was added to each sample extraction, which enables the results to be normalized and correcting for any loss. The internal standard was prepared as a solution containing 3.66 mg/mL C19:0 TAG (Sigma-Aldrich, product number: T4632), 2.73 mg/mL C17:0 Phospholipid (PL), 0.4 mg/mL C19:0 FFA (Sigma-Aldrich, product number: N-8263), 0.096 mg/mL C15:0 Diacylglycerol (DAG), 0.06 mg/mL d31-tagged Ceramide, 0.03 mg/mL d7-tagged Sphingosine, 1.036 mg/mL Sitostanol, 0.04 mg/mL d31-tagged Sphingomyelin and 1.596 mg/mL C15:0 Cholesterolester in Chloroform:Methanol 2:1 (v/v).

Tissue Lipid Extraction

[0092] The method is based on Folch et al [89][90, 91]. At the time of extraction the samples were portioned at 500 mg individually and transferred to glass tubes, followed by the addition of 10 mL 100 g/mL butylated hydroxytoluene (BHT) in chloroform:methanol 2:1 (v:v), and placed on ice. Each sample was added 150 L of the internal standard solution. Homogenization was performed using a rotor-stator submersion blender (IKA Ultra-Turrax T25) with the sample tubes submerged in ice-water. The samples were each homogenized for 310 seconds with 50 second intervals, where after the homogenate is transferred to 35 mL screw-cap centrifuge tubes. The aggregate was cleaned in 5 mL chloroform:methanol 2:1 in the original glass tube, which was then transferred quantitatively to the homogenate. The homogenate was added 3 mL (0.24 times the total homogenate volume) of 0.73% (w/v) NaCl in MiliQ H.sub.2O and mixed, followed by centrifugation at 2800.Math.g for 5 minutes at 4 C., to create a 2-phase system. The lower phase was transferred to a clean 12.5 mL centrifugation tube. The homogenate (the upper phase) was then added 3 mL chloroform:methanol 85:15 and mixed, followed by another centrifugation step. The new lower phase was then isolated and added to the first extract and dried down under nitrogen in a 40.0 water bath, followed by resolubilization in 300 L chloroform:methanol 2:1. At this point the lipid extracts can be stored at 20 C.

[0093] Lipid Fractionation

[0094] The lipid extracts were fractionated on amino-propyl columns (Phenomenex Strata NH2, product number: 8B-5009-HBJ). The columns were pre-washed with 22 mL chloroform:methanol 23:1 (v:v) and left to dry. During fractionation the columns were not allowed to dry. The columns were primed with 21 mL hexane followed by application of the lipid extracts. Fraction 1 containing cholesterol esters was eluted with 3 mL Hexane into a clean tube. Fraction 2 containing TAG was eluted using 3 mL hexane:chloroform:ethylacetate 100:5:5 (v:v:v) into a clean tube. Fraction 3 containing DAG, Cholesterol and Ceramide was eluted using 32 mL chloroform:methanol 23:1 (v:v) into a new glass. Fraction 4 containing FFA was eluted using 5 mL 2% acetic acid in diethyl ether into a new glass. Fraction 5 containing PL was eluted using 4 mL methanol into a new glass. Fractions 1 and 2 were dried down under nitrogen at 40.0 and re-dissolved in 300 L chloroform:methanol 95:5 and stored at 20 C. Fraction 4 and half of fraction 5 (fraction 5A) were dried down under nitrogen at 40.0 and re-dissolved in 300 L chloroform:methanol 2:1 and stored at 20 C. Fraction 3 was equally dried down and re-dissolved in 135 L chloroform and 67.5 L isopropanol, transferred to HPLC vials and stored at 20 C. The other half of fraction (fraction 5B) was dried down under nitrogen and re-dissolved in 200 L chloroform:isopropanol 1:1 (v:v), transferred to HPLC vials and stored at 20 C.

Fatty Acid Methylation

[0095] To allow gas chromatographic analysis of the fatty acids they must be converted to the more volatile Fatty Acid Methyl Ester (FAME) form. This is performed using a boron trifluoride catalyzed methylation in methanol. For the methylation step triacylglycerols must be hydrolysed, which is performed under alkaline conditions in the first step of the procedure. This is followed by methylation and extraction of the FAME products. Fractions were dried down under nitrogen at 40 C., followed by the addition of 1 mL of 0.5 M NaOH in methanol. The sample tubes tightly closed with screw caps were allowed to reflux for 5 minutes at 80 C. in a heating block to mediate hydrolysis. After the hydrolysis step the samples were allowed to cool to room temperature before the addition of 1 mL 20% BF in methanol and 0.5 mL 0.1% hydroquinone in methanol. The samples were then allowed to reflux at 80 C. for 2 minutes to mediate the methylation, followed by cooling. 2 mL 0.73% NaCl in Milli-Q was added to the samples followed by mixing for 10 seconds. This increases the polarity of the methanol phase by increasing the content of water. The samples were then added 0.5 mL heptane followed by mixing for 10 seconds, to extract the FAMEs, and centrifuged at 2800.Math.g for 1 minute. The top phase (heptane) was transferred to a clean 3 mL centrifuge tube. An additional 0.5 mL heptane was added to the methylated sample, mixed, centrifuged and transferred to the first extract. The remaining solution was discarded. The top phases (heptane extract) were added 1 mL saturated alkaline NaCl solution, mixed for 10 seconds and centrifuged at 2800.Math.g for 1 minute. The top phase was then transferred to a new 3 mL tube. The TAG fraction is ready to use after methylation and were transferred to GC vials with low-volume inserts.

Lipid Analysis Using GC-FID

[0096] The methylated lipid fractions were analysed using an Agilent 6890N gas chromatograph with a fused silica capillary column (Sigma-Aldrich; Supelco SP-2380, product number: 24111) and a Flame Ionization Detector (FID).

[0097] Data Processing

[0098] Total triacylglycerol and arachidonic acid were calculated as the ratio of the total peak area (Areatot) (subtracted the area of the internal standard) to the area of the internal standard Areain-std), multiplied by the mass of the internal standard(min-std). This is detailed in equation (1). To achieve the final concentration, the total content was normalised by sample weight.

[00001]$\begin{array}{cc}\mathrm{Total}.Math..Math.\mathrm{Content}=\frac{\left({\mathrm{Area}}_{\mathrm{tot}}-{\mathrm{Area}}_{i.Math..Math.n-\mathrm{std}}\right)}{{\mathrm{Area}}_{i.Math..Math.n-\mathrm{std}}}.Math.m_{i.Math..Math.n-\mathrm{std}}& \mathrm{Equation}.Math..Math.\left(1\right)\end{array}$

The content of individual fatty acids were identified and calculated as the ratio of the individual fatty acid peak area (AreaF A) to total identified fatty acid area (AreaID) subtracted the peak area of the internal standard (Areain-std) and expressed in percent. This is detailed in equation (2).

[00002]$\begin{array}{cc}\mathrm{Area}.Math..Math.{\%}_{\mathrm{FA}}=\frac{{\mathrm{Area}}_{\mathrm{FA}}}{{\mathrm{Area}}_{\mathrm{ID}}-{\mathrm{Area}}_{i.Math..Math.n-\mathrm{std}}}.Math.100& \mathrm{Equation}.Math..Math.\left(2\right)\end{array}$

Results

[0099] Results of the 16 day initial study are shown in FIGS. 1A-1C and 2-3. As seen in FIGS. 1A-1C, the peptide

TABLE-US-00012 (SEQIDNO:54) AcCSNLSTCVLGKLSQELHKLQTYPRTDVGANAP-NH.sub.2(KBP-042)
reduced food intake (A) body weight gain (B) and visceral fat (epididymal)(C) within 16-days of treatment when compared to saline.

[0100] As seen in FIG. 2, the KBP-peptide produced an improvement in triacylglycerols and arachidonic acid (a pro-inflammatory mediator) content in extracted liver tissue.

[0101] As seen in FIG. 3, the KBP-peptide produced an improvement in Free Fatty Acids content in extracted liver tissue.

[0102] The 16-day study thus demonstrated that KBP-042 as expected produced a pronounced reduction in food intake early, which led to a marked weight reduction. Furthermore, fats depots were reduced. Importantly, analysis of the liver fatty acid composition showed that KBP-042 reduced triacylglycerols and free fatty acid accumulation, indicating a benefit on fatty liver. Finally, KBP-042 reduced the levels of the fatty acid arachidonic acid in the liver, and arachidonic acid is a known pro-inflammatory mediator, and therefore reduction of the levels of this molecule should be beneficial in terms of preventing fatty liver and steatosis in the liver.

[0103] The results of the 8-week study are seen in FIGS. 4-7, confirmed all of these initial study findings.

[0104] As seen in FIG. 4, the KBP-peptide produced a marked weight reduction even when compared to the calorie restricted groups (pair-fed), showing a substantial weight loss independent of the regulation of food intake. The 8-week study also included a pair-fed control (calorie restriction), and as such also confirmed the beneficial effects of KBP-042 on bodyweight and fatty liver independent of the reduction in food intake.

[0105] As seen in FIG. 5, the KBP peptide reduced perirenal fat depot measurements compared to vehicle, even in a pair fed experiment.

[0106] As seen in FIG. 6, the KBP peptide reduced triglyceride content of the liver compared to vehicle, even in a pair fed experiment.

[0107] As seen in FIG. 7, the KBP-peptide produced an improvement in triacylglycerols after 8 weeks of treatment.

EXAMPLE 2BODY WEIGHT REDUCTION (KBP-042)

[0108] A normal diet group (ND) was included as a reference for all parameters studied in high fat diet (HFD) rats. Endpoint data from the ND group appear in FIG. 8 and all results from the performed tests are summarized in FIG. 9 and are compared to HFD-Vehicle.

[0109] FIGS. 8A-8F shows: FIG. 8A) Absolute body weight progression during the study from randomization at day 0 to last day of treatment, day 56; FIG. 8B) Vehicle-corrected body weights; FIG. 8C) Endpoint body weights; FIG. 8D) Food intake of all treatment groups during the entire study. Food intake was monitored every day for the first 6 days followed by weekly monitoring. Pair-fed groups were fed the same as the average for their corresponding treatment group (5 g/kg or 10 g/kg). n=10 for all groups except vehicle (n=12). Statistical analysis between groups for FIG. 8C), FIG. 8E) and FIG. 8F) were performed as a One-way ANOVA followed by Tukey's post-hoc test with the following annotations: ###P<0.001 vs. ND-control *P<0.05, **P<0.01, ***P<0.001 vs. HFD-Vehicle, P<0.01, P<0.001 vs. Pair-fed 5 g/kg, P<0.01, P<0.001 vs. Pair-fed 10 g/kg, Data are expressed as meanSEM.

[0110] In general the HFD-Vehicles had impaired glucose tolerance (higher total area under the curve) in the oral and intravenous glucose tolerance tests, with higher insulin levels in both tests. All data showing that HFD rats were obese and pre-diabetic at the initiation of treatment were as expected.

[0111] After treatment with KBP-042 for 8 weeks, there was a dose-dependent and sustained reduction of body weight. A large weight loss was observed in the initial phase of the study (FIGS. 8A and 8B) in the three highest treatment groups (2.5 g/kg, 5 g/kg and 10 g/kg), as well as the two corresponding pair-fed groups (Pair-fed 5 g/kg and Pair-fed 10 g/kg). This corresponds well with the large reduction in food intake in the first 6 days of treatment (FIG. 8D). However, after this transient large reduction in feeding, food intake normalised during the course of the study. The pair-fed groups gained weight again after food intake increased, whereas the KBP-042 treatment sustained the initial weight reduction throughout the 56 days of treatment. At day 56 animals were weighed and treatment with KBP-042 was evaluated. Body weight was significantly lower for the 2.5 g/kg, 5 g/kg and 10 g/kg groups compared to the HFD-vehicle. The reduced food intake corresponds well with the body weight change for the three highest treatment groups (2.5 g/kg, 5 g/kg and 10 g/kg) (FIG. 8E), although the pair-fed groups which received the same amount of food as their corresponding treatment-group, did not have significantly different weight than the HFD-vehicle animals.

[0112] On the basis of food intake and body weight change the food efficiency was calculated (FIG. 8F), and as expected KBP-042 treatment with 2.5 g/kg, 5 g/kg and 10 g/kg resulted in a drastic reduction in food efficiency, which was significantly different from the pair-fed controls, possibly indicating increased energy expenditure.

[0113] In conclusion, KBP-042 mediated a large reduction in body weight and maintained weight loss for 8 weeks.

EXAMPLE 3REDUCTION IN ADIPOSE TISSUE (KBP-042)

[0114] At the end of the study of Example 2 three different adipose tissues were isolated.

[0115] The results are shown in FIGS. 10A-10F: FIGS. 10A-10CWeight of isolated epididymal, inguinal and perirenal white adipose tissue respectively after 56 days of treatment; FIG. 10DTotal triacylglyceride content extracted from liver tissue after treatment with KBP-042 or saline for 56 days; FIGS. 10E-10FPlasma Adiponectin and Leptin levels respectively after 56 days of treatment. n=10 for all groups except vehicle (n=12). Statistical analysis between groups were performed as a One-way ANOVA followed by Tukey's post-hoc test with the following annotations: ##P<0.01, ###P<0.001 vs. ND-control. *P<0.05, **P<0.01, ***P<0.001 vs. HFD-Vehicle. P<0.05, P<0.01 vs. Pair-fed 5 g/kg. P<0.05 vs. Pair-fed 10 g/kg, Data are expressed as meanSEM

[0116] As seen in FIGS. 10A-10C, the weights of isolated epididymal and perirenal white adipose tissues were significantly reduced after treatment with 10 g/kg of KBP-042. For the perirenal adipose tissue even the 2.5, 5 and 10 g/kg groups showed a significant reduction in size. The same reduction was not seen in the pair-fed controls. There was a trend towards reducing inguinal white adipose tissue.

[0117] To assess lipid accumulation in liver, triacylglycerols (TAG) were extracted from the liver and analyzed (FIG. 10D). The HFD-Vehicle group had dramatically higher TAG levels compared to ND group, as expected. This accumulation was significantly reduced after treatment with 10 g/kg KBP-042, while the corresponding pair-fed control group did not show a significant reduction in liver TAG. The large variances in the TAG levels make it difficult to achieve statistical significance however the trends indicated a dose-dependent effect. In order to assess whether the treatment altered fatty acid metabolism in selective ways (e.g. metabolism of saturated vs. monounsaturated vs. polyunsaturated), we also analysed the fatty acid composition of hepatic TAG. The results show that there were no differences in the relative distribution, i.e. the treatment caused a general reduction in TAG without effects on metabolism of specific fatty acid types (FIG. 9).

[0118] Finally, the two adipokines adiponectin and leptin were measured after 56 days of treatment (FIGS. 10E and 10F). Adiponectin levels were significantly increased in response to treatment with doses of 1.25 g/kg, 2.5 g/kg, 5 g/kg and 10 g/kg KBP-042. With respect to leptin, there was trend towards reduced levels of plasma leptin, which reached statistical significance when comparing 10 g/kg KBP-042 to the corresponding pair-fed control.

[0119] In summary, fat depots, lipid, and adipokine data support a strongly improved metabolic status as a function of KBP-042 therapy. Adipose tissues and ectopic lipid accumulation were reduced by KBP-042.

EXAMPLE 4REDUCED LEVELS OF LIVER FAT (KBP-042 AND KBP-089)

[0120] At termination of the study of Example 2 the rat livers were embedded using snap freezing in OCT, and then sectioned using a cryomicrotome. Sections were prepared from four groups, KBP-042 in HFD rats, KBP-089 in HFD rats, control HFD rats, and a lean rat comparison.

[0121] The sections were stained using Oil-Red-O staining. Staining of liver sections from HFD-fed rats (FIG. 11B) showed a significant 57.1% increase (p<0.01) in lipid accumulation compared to lean rats (FIG. 11A). Lipid accumulation in liver from rats daily treated with 2.5 g/kg KBP-042 (FIG. 11C) decreased HFD-induced lipid accumulation significant with 78.3% (p<0.05) compared to vehicle and was similar to lean rats (p=0.8173). Lipid accumulation in liver from rats treated with 2.5 g/kg KBP-089 (FIG. 11D) reduced lipid accumulation significant by 155.3% (p<0.001) compared to vehicle and was furthermore similar to lean rats (p=0.9976). The lipid accumulation in pair-fed KBP-089 liver exceeded KBP-089 treated rats with 66.5% (p<0.001) and was not significant different from vehicle (0.6711). Statistical test performed was one-way ANOVA with Dunnet's post test for multiple comparisons. ** p<0.01, *** p<0.001.

[0122] Thus, as seen in FIGS. 11C and 11D, KBP-042 and KBP-089 treatment led to a substantial reduction in lipids present in the liver sections all the way down to the level observed in the lean rats. When quantifying the intensity of the staining (FIG. 11F), these data confirmed that KBP-042 and KBP-089 induced a significant reduction of liver lipid accumulation.

[0123] The results indicate that HFD-induced lipid accumulation in liver tissue can be decreased by KBP-treatment but not by calorie restriction.

EXAMPLE 5IMPROVED GLUCOSE TOLERANCE (KBP-042)

[0124] To assess whether the weight and liver fat reductions manifested in improved glucose tolerance, an oral glucose tolerance test was performed both in treatment naive animals (after a single injection), following 3 weeks and again after 7 weeks of treatment.

[0125] Animals were challenged with an oral glucose bolus (2 g/kg) at time=0, and dosed with either KBP-042 or saline at t=30. FIGS. 12A, 12B and 12C show blood glucose levels during acute OGTT, OGTT after 3 weeks, and OGTT after 7 weeks respectively. FIGS. 12D, 12E and 12F show the area under the curve for acute OGTT, OGTT after 3 weeks, and OGTT after 7 weeks respectively. FIGS. 12G, 12H and 12I show insulin levels during acute OGTT, OGTT after 3 weeks, and OGTT after 7 weeks respectively. FIGS. 12J, 12K and 12L show insulin levels during acute OGTT, OGTT after 3 weeks, and OGTT after 7 weeks respectively expressed as area under the curve. n=10 for all groups except vehicle (n=12). Statistical analysis between groups were performed as a One-way ANOVA followed by Tukey's post-hoc test with the following annotations: *P<0.05, **P<0.01, ***P<0.001 vs. HFD-Vehicle. P<0.01, P<0.001 vs. Pair-fed 5 g/kg. P<0.01, P<0.001 vs. Pair-fed 10 g/kg, Data are expressed as meanSEM.

[0126] The OGTT performed after an acute dose showed a slightly impaired glucose tolerance for the 10 g/kg group compared to HFD-Vehicle (FIG. 12A). A hyperglycemic effect was observed 30 minutes after the subcutaneous administration of KBP-042 (at t=0). The total area under the curve (tAUC) was significantly increased with injection of 10 g/kg KBP-042 (FIG. 12D). However, the insulin levels during the first 60 minutes after glucose administration were reduced in animals dosed with KBP-042 (FIGS. 12G and 12J).

[0127] After 3 weeks of treatment with KBP-042 or saline the three highest doses of KBP-042 (2.5 g/kg, 5 g/kg and 10 g/kg) resulted in a significantly lowered tAUC (FIGS. 12B and 12E). Interestingly, the insulin levels were lowered for all treatment groups except the 0.625 g/kg KBP-042 group (FIGS. 12H and 12K). The pair-fed 10 g/kg group also had a reduced insulin response (FIG. 12K).

[0128] When the OGTT was performed at week 7 of treatment (FIG. 12C) only the two highest dose groups (5 g/kg and 10 g/kg) showed improved glucose tolerance when tAUC was considered (FIG. 12F). Interestingly, the three highest dose groups had increased glucose tolerance (or maintained for the 2.5 g/kg group), while drastically reduced insulin levels were observed within the first 60 minutes after glucose administration (FIGS. 12I and 12L). No changes in glucose tolerance or insulin levels were observed in the pair-fed groups compared to HFD-Vehicle.

[0129] In conclusion, treatment with KBP-042 improved glucose tolerance with reduced insulin levels after chronic treatment.

EXAMPLE 6EFFECT OF KBP-042 ON INSULIN SENSITIVITY

[0130] As liver fat is known to decrease insulin sensitivity, the effect of KBP-042 on insulin sensitivity was considered using the glucose infusion rate (GIR) in the hyperinsulinemic-euglycemic clamp. For this study ND rats were compared to insulin resistant HFD rats and 5 g/kg KBP-042 treated HFD rats.

[0131] The results are shown in FIGS. 13A-13B: FIG. 13AGlucose infusion rate (GIR) at steady state during hyperinsulinemic-euglycemic clamp when blood glucose was clamped at basal levels after 21 days treatment; FIG. 13BBody weight at hyperinsulinemic-euglycemic clamp experiment day after 21 days of treatment. Statistical analysis between groups was performed as a One-way ANOVA followed by Tukey's post-hoc test with the following annotations: *P<0.05, **P<0.01, ***P<0.001. Data are expressed as meanSEM.

[0132] FIG. 13A shows GIR reduced by 30% (p=0.057) in the HFD group compared to ND. The treatment with KBP-042 led to a significant increase in GIR (82%, p<0.001) compared to HFD-Vehicle. When KBP-042 treatment is compared to ND GIR is increased with 27% (p<0.05). As expected the body weight was increased after HFD for 10 weeks as compared to ND (FIG. 13B). FIG. 13B shows that the treatment with KBP-042 for 21 days reduced the body weight by 18% and the body weight was not significantly different from the ND rats at the end of the study.

[0133] Thus, KBP-042 improved whole-body insulin sensitivity in the hyperinsulinemic-euglycemic clamp

[0134] In summary, we here present a novel possibility for reduction of fatty liver, a disease which has become prominent within the last decades due to the increasing occurrence of obesity.

[0135] In this specification, unless expressly otherwise indicated, the word or is used in the sense of an operator that returns a true value when either or both of the stated conditions is met, as opposed to the operator exclusive or which requires that only one of the conditions is met. The word comprising is used in the sense of including rather than in to mean consisting of. All prior teachings acknowledged above are hereby incorporated by reference. No acknowledgement of any prior published document herein should be taken to be an admission or representation that the teaching thereof was common general knowledge in Australia or elsewhere at the date hereof.