FIELD

The present invention relates to compositions comprising Fenofibrate for use in the treatment, prophylaxis and/or prevention of hypoglycaemia in type 1 diabetes or other related diseases in humans including Latent Autoimmune Diabetes of Adulthood (LADA). This treatment is particularly relevant for subjects that are genetically predisposed to these diseases. The daily dose ranges from 1-100 mg/kg bodyweight per day.

BACKGROUND

Hypoglycaemia symptoms rank from minor discomfort to seizures and death. Symptoms typically come on quickly. The average individual with type 1 diabetes experiences about two episodes of symptomatic hypoglycemia per week. Severe hypoglycemia (requiring help for recovery) has an annual prevalence of 30 - 40% and an annual incidence of 1.0 - 1.7 episodes per patient per year. A common cause of hypoglycemia in type 1 diabetes patients is insulin and sulfonylureas which are used to treat type 1 diabetes. The risk is greater in people with type 1 diabetes who have eaten less than usual, exercised more than usual, or have drunk alcohol. The glucose level that defines hypoglycemia is variable. In people with type 1 diabetes, levels below 3.9 mmol/L (70 mg/dL) is diagnostic. Other tests that may be useful in determining the cause include insulin and C peptide levels in the blood. Hyperglycemia (high blood sugar) is the opposite condition.

Hypoglycemia is for patients with type 1 diabetes prevented by matching food intake with the amount of exercise and the medications used. When people feel their blood sugar is low, testing with a glucose monitor is recommended. Some people have few initial symptoms of low blood sugar and frequent routine testing in this group is recommended. Treatment of hypoglycemia includes eating foods high in simple sugars. It is therefore recommended that individuals with type 1 diabetes also carry a source of sugar with them. If a person is not able to take food by mouth, an injection of glucose or glucagon may help.

Thus, due to the dramatic and recurrent fluctuations in glucose levels which typically comes quickly without any prior warning and results in seizures, or even death, there is an unmet need for providing compositions such as for example food supplements that is more reliable than eating foods high in simple sugars.

Growing evidence has suggested that the autonomic nervous system, in particular, the

sympathetic nerves, play an important role in the pathology of type 1 diabetes, with an observed loss of islet sympathetic nerves in newly diagnosed individuals with type 1 diabetes. The loss of pancreatic sympathetic nerves is a contributing factor for loss of glucagon release observed in individuals with type 1 diabetes and thereby the occurrence of hypoglycemia. EP0482498 relates to a method provided for preventing diabetes or preventing the onset of or reducing the risk of complications normally associated with diabetes. The term "complications normally associated with diabetes" in EP0482498 includes atherosclerosis (leading to myocardial infarction and cerebral ischemia), peripheral artery disease, nephropathy, retinopathy and neuropathy, and the solution provided is a therapeutically effective amount of a cholesterollowering drug alone such as Fenofibrate or in combination with an angiotensin converting enzyme inhibitor, which is administered systemically, such as orally or parenterally to a diabetic patient. EP0482498 does not disclose hypoglycaemia and does not provide any supporting data.

ClinicalTrials.gov Identifier: NCT01320345 is an ongoing clinical trial with Fenofibrate for evaluating the effects of Fenofibrate versus placebo on Macular Thickness and Volume (FAME 1 EYE) in adults with type 1 diabetes mellitus who are at high risk of eye damage. The use of Fenofibrate for reducing hypoglycemia in type 1 diabetes patients is not disclosed.

In Elijah I.E. et al.: "Role of the PPAR-a agonist Fenofibrate in severe pediatric burn"

JOURNAL OF THE INTERNATIONAL SOCIETY FOR BURN INJURIES, (2012),

Vol. 38(4), pages 481-486, Fenofibrate is described to prevent insulin-induced hypoglycemia in pediatric burn patients. Pediatric burn patients cannot be compared to type 1 diabetes or LADA, for various reasons including that type 1 diabetes and LADA is the results of reduced insulin production causing hyperglycemia while burn patients get hyperglycemia because of increased hepatic glucose production. Moreover, type 1 diabetes and LADA are autoimmune diseases, which is not the case for pediatric burn patients. The paper does not mention type 1 diabetes or LADA.

Hypoglycemia, despite advances in technology, remains the major limiting factor for individuals with type 1 diabetes in achieving optimal blood glucose control and hypoglycaemia is linked to psychological distress and increases in morbidity and mortality.

SUMMARY

Identification of new therapeutic approaches with a focus on improving sympathetic nerve function in pancreatic islets may be an improvement over current therapies focusing on continuous glucose homeostasis.

In the present application, the inventors treated the type 1 diabetes animal model NOD mice with Fenofibrate. Through a combination of mass spectrometry and stereology analysis, they found that Fenofibrate increased the amount of long-chain C24:1 sulfatide which is primarily found in nervous tissue and that this was associated with an increased volume of sympathetic nerves in pancreatic islets. Thus, in its broadest aspect, the present invention relates to a composition comprising Fenofibrate for use in the treatment, prophylaxis and/or prevention of hypoglycemia in a type 1 diabetes patient or other related diseases in humans including Latent Autoimmune Diabetes of Adulthood (LADA).

In the present application, the inventors further evaluated the effect of Fenofibrate on blood glucose homeostasis and looked at the effect on pancreas morphology. The inventors also present a human case report, wherein a type 1 diabetes patient treated with Fenofibrate had a reduced number of hypoglycaemia episodes.

DETAILED DESCRIPTION

In the present application, the inventors show how Fenofibrate has several beneficial effects on pancreatic islets in NOD mice. Fenofibrate increased the amount of the beneficial sphingolipid sulfatide in the pancreas, prevented loss of pancreatic sympathetic nerves and provided a more stable blood glucose, thus the application relates to a composition comprising Fenofibrate for use in the treatment, prophylaxis and/or prevention of hypoglycemia in patients with type 1 diabetes and/or LADA.

Thus, the present invention relates to a composition comprising Fenofibrate for use as a method for preventing hypoglycemia in a type 1 diabetic patient comprising administrating a daily dosage of 1 - 100 mg/kg bodyweight of Fenofibrate to a type 1 diabetic patient in need thereof.

Thus, the also present invention relates to a composition comprising Fenofibrate for use as a method for treatment, prophylaxis and/or prevention of LADA comprising administrating a daily dosage of 1 - 100 mg/kg bodyweight of Fenofibrate to a LADA patient in need thereof.

The invention also relates to a composition comprising Fenofibrate for use as a method for treatment, prophylaxis and/or prevention of hypoglycemia in a type 1 diabetic patient comprising administrating a daily dosage of 1 - 100 mg/kg bodyweight of Fenofibrate to a type 1 diabetic patient in need thereof.

Increased number of sympathetic neurons in islets following Fenofibrate treatment

As shown in Example 3, the total volume of sympathetic neurons was increased by Fenofibrate as evidenced by increased tyrosine hydroxylase (TH) expression in islets (Figure 10). There was an inverse correlation between islet inflammation (insulitis) score to islet sympathetic nerve fibers (p=0.047 r ² =0.34) and between insulitis score and TH expressing cells/islet volume (p=0.005 r ² =0.56) (Figure 1 1 & 12).

Thus, in one or more exemplary embodiments, the invention relates to a composition comprising Fenofibrate for use in increasing the number of sympathetic neurons in islets. Improving Blood glucose homeostasis

Loss of islet sympathetic neurons are known to reduce glucose tolerance in mice and so the present inventors wanted to investigate if the increased amount of islet sympathetic nerves would be reflected in improved blood glucose homeostasis. The present inventors show that the

Fenofibrate treated mice had a lower non-fasting glycemia than healthy NOD mice on a control diet. (5.3 mmol/l vs 6.1 mmol/l. Age 12 to 30 weeks Figure 13). Fenofibrate treatment furthermore resulted in a more stable glycemia as seen by the reduced standard deviation (Figure 14). The reduced non-fasting glycemia was associated with an increased fasting glycemia with controls (3.1 mmol/l) vs Fenofibrate (3.8mmol/l) (Figure 15).

A glucose tolerance test (GTT) showed that Fenofibrate treated mice had improved glucose tolerance at age 13 weeks (Figure 16). 4/12 control mice showed signs of fasting hyperinsulinemia, this was not the case in any of the Fenofibrate treated mice (0/12, c ² p=0.03) (Figure 17). The inventors furthermore found lower fasting glucagon levels in Fenofibrate treated mice (Figure 18).

HOMA-IR, which is a mathematical method used to quantify insulin resistance, was used to evaluate if these changes in glucose homeostasis were the result of altered insulin sensitivity. HOMA-IR revealed no difference in insulin sensitivity (Figure 19, p=0.48).

The inventors next used an insulin tolerance test to quantify insulin resistance and the mice ability to recover from insulin-induced hypoglycemia. Control and Fenofibrate-treated mice were injected with insulin and the following decline and return to normal glycemia was evaluated. There was no difference in the declining phase illustrating that Fenofibrate does not change insulin sensitivity. The Fenofibrate-treated mice had a quicker return to normal glycemia following the insulin-induced hypoglycemia (Figure 20, p=0.016)

The inventors also show a human case report in which a 19-year-old girl newly diagnosed with type 1 diabetes started taking Fenofibrate seven days after her diagnosis. The patient took her last dose of insulin 19 days later and has now been without insulin for 10 months with improved blood glucose homeostasis and a reduced number of hypoglycemia episodes (Figure 21 ).

Combined, the above-mentioned results show how Fenofibrate treatment improves blood glucose homeostasis and protects against insulin-induced hypoglycemia.

In one or more exemplary embodiments, the invention relates to a composition comprising Fenofibrate for use in preventing the loss of pancreatic sympathetic nerves in a LADA and/or type 1 diabetic patient. Fenofibrate

Fenofibrate, marketed as Tricor, Fenoglide, Lipofen and under several other brand names, is a drug of the fibrate class. The chemical formula is C20H21CIO4, having a molar mass of 360.831 g/mol. It is mainly used to reduce cholesterol levels in people at risk of cardiovascular disease. Like other fibrates, it is used in addition to diet, where it reduces both low-density lipoprotein (LDL) and very low-density lipoprotein (VLDL) levels, as well as increasing high-density lipoprotein (HDL) levels and reducing triglyceride levels. Fenofibrate has been used since 1975 and is one of the most commonly prescribed fibrates.

Type 1 diabetes patients

Type 1 diabetes, once known as diabetes mellitus type 1 , juvenile diabetes or insulin-dependent diabetes, is a chronic condition in which the pancreas produces little or no insulin. This results in high blood glucose levels. Despite active research, type 1 diabetes has no cure. Treatment focuses on managing blood sugar levels with insulin, diet, and lifestyle to prevent complications. The classical symptoms are frequent urination, increased thirst, increased hunger, and weight loss. Additional symptoms may include blurry vision, feeling tired, and poor wound healing. Symptoms typically develop over a short period of time.

The cause of type 1 diabetes is unknown. However, it is believed to involve a combination of genetic and environmental factors. Risk factors include having a family member with the condition. The underlying mechanism involves an autoimmune destruction of the insulin-producing beta cells in the pancreas. Diabetes is diagnosed by testing the level of sugar or A1C in the blood. Type 1 diabetes can be distinguished from type 2 by testing for the presence of autoantibodies.

The present invention relates to type 1 diabetes patients who typically have autoantibodies.

In one embodiment, the present invention relates to type 1 diabetes patients having dramatic and recurrent fluctuations in glucose levels. Some people with type 1 diabetes experience dramatic and recurrent fluctuations in glucose levels, often occurring for no apparent reason. This may result in a variety of symptoms including ranking from minor discomfort to loss of consciousness, seizures, or even death. Symptoms typically appear quickly.

Hypoglycemia

Hypoglycemia is a very common occurrence in people with diabetes, usually the result of a mismatch in the balance between insulin, food intake and physical activity.

Symptoms include excess sweating, excessive hunger, fainting, fatigue, lightheadedness, and shakiness. Mild cases are self-treated by eating or drinking something with a high amount of sugar. Severe cases can lead to unconsciousness and are treated with intravenous glucose or injections with glucagon. Continuous glucose monitors can alert patients to the presence of dangerously high or low blood sugar levels, but technical issues have limited the effect that these devices have had on clinical practice.

In the present context, hypoglycemia in a type 1 diabetes patient relates to a blood glucose of less than typically 70 mg/dl (3.9 mmol/l) in a said patient.

Latent Autoimmune Diabetes of Adulthood (LADA)

LADA is a form of type 1 diabetes that develops later into adulthood. LADA tends to develop more slowly than type 1 diabetes in childhood and, because LADA can sometimes appear similar to type 2 diabetes, doctors may mistakenly diagnose LADA as type 2 diabetes.

In one or more exemplary embodiments, the invention relates to a composition comprising Fenofibrate for use in the treatment of LADA.

In one embodiment, the LADA patients may have dramatic and recurrent fluctuations in glucose levels.

LADA is sometimes referred to as type 1.5 diabetes. This is not an official term, but it does illustrate the fact that LADA is a form of type 1 diabetes that shares some characteristics with type 2 diabetes.

As a form of type 1 diabetes, LADA is an autoimmune disease in which the body’s immune system attacks and kills off insulin-producing cells.

The reasons why LADA can often be mistaken for type 2 diabetes is it develops over a longer period of time than type 1 diabetes in children or younger adults.

Whereas type 1 diabetes in children tends to develop quickly, sometimes within the space of days, LADA develops more slowly, sometimes over a period of years.

The slower onset of diabetes symptoms being presented in people over 35 years may lead a GP to initially diagnose a case of LADA as type 2 diabetes.

Dosage regimen

A Fenofibrate dosage regimen range of 1 - 100 mg/kg bodyweight has already been suggested for use in treatment of Type 1 Diabetes Mellitus in a clinical trial. The daily dose range of the present invention contains Fenofibrate in an amount sufficient to administer to a subject a dosage from 0.1 mg to 100 mg per kg bodyweight per day.

In one embodiment, the composition contains Fenofibrate in an amount sufficient to administer to a subject a dosage from 1 mg to 100 mg per kg bodyweight per day.

In one embodiment, the composition contains Fenofibrate in an amount sufficient to administer to a subject a dosage from 0.1 mg to 1 mg per kg bodyweight per day.

In one embodiment, the composition contains Fenofibrate in an amount sufficient to administer to a subject a dosage from 1 mg to 3 mg per kg bodyweight per day.

In one embodiment, the composition contains Fenofibrate in an amount sufficient to administer to a subject a dosage from 3 mg to 5 mg per kg bodyweight per day.

In one embodiment, the composition contains Fenofibrate in an amount sufficient to administer to a subject a dosage from 5 mg to 7 mg per kg bodyweight per day

In one embodiment, the composition contains Fenofibrate in an amount sufficient to administer to a subject a dosage from 8 mg to 10 mg per kg bodyweight per day

In one embodiment, the composition contains Fenofibrate in an amount sufficient to administer to a subject a dosage from 10 mg to 14 mg per kg bodyweight per day

In one embodiment, the composition contains Fenofibrate in an amount sufficient to administer to a subject a dosage from 14 mg to 17 mg per kg bodyweight per day

In one embodiment, the composition contains Fenofibrate in an amount sufficient to administer to a subject a dosage from 17 mg to 20 mg per kg bodyweight per day

In one embodiment, the composition contains Fenofibrate in an amount sufficient to administer to a subject a dosage from 20 mg to 23 mg per kg bodyweight per day

In one embodiment, the composition contains Fenofibrate in an amount sufficient to administer to a subject a dosage from 23 mg to 26 mg per kg bodyweight per day

In one embodiment, the composition contains Fenofibrate in an amount sufficient to administer to a subject a dosage from 26 mg to 30 mg per kg bodyweight per day In one embodiment, the composition contains Fenofibrate in an amount sufficient to administer to a subject a dosage from 1 mg to 30 mg per kg bodyweight per day.

In one embodiment, the composition contains Fenofibrate in an amount sufficient to administer to a subject a dosage from 30 mg to 60 mg per kg body weight per day.

In one embodiment, the composition contains Fenofibrate in an amount sufficient to administer to a subject a dosage from 60 mg to 90 mg per kg bodyweight per day.

In one embodiment, the composition contains Fenofibrate in an amount sufficient to administer to a subject a dosage from 90 mg to 100 mg per kg bodyweight per day.

In one embodiment, the composition is administered to the subject in a dosage regime comprising an oral dose once daily.

In one embodiment, the composition is administered to the subject in a dosage regime comprising an oral dose twice-daily. Thus, in this embodiment, the composition contains Fenofibrate in an amount sufficient to administer to a subject a dosage from 0.05 mg to 50 mg per kg bodyweight twice-daily, such as but not limited to 1-50 mg per kg bodyweight twice-daily, 5-50 mg per kg bodyweight twice-daily, 10-50 mg per kg bodyweight twice-daily, 15-50 mg per kg bodyweight twice-daily, 20-50 mg per kg bodyweight twice-daily, 25-50 mg per kg bodyweight twice-daily, 30- 50 mg per kg bodyweight twice-daily, 35-50 mg per kg bodyweight twice-daily, 40-50 mg per kg bodyweight twice-daily, 45-50 mg per kg bodyweight twice-daily, twice-daily 10-20 mg per kg bodyweight twice-daily, 5-10 mg per kg bodyweight twice-daily, 5-40 mg per kg bodyweight twice- daily, 5-15 mg per kg bodyweight twice-daily, 5-45 mg per kg bodyweight twice-daily, 5-20 mg per kg bodyweight twice-daily, or 25-45 mg per kg bodyweight twice-daily.

The composition(s) according to the present invention must be administered for a time sufficient for the effect to occur.

In one embodiment, the composition is administered for at least 3 months.

In one embodiment, the composition is administered for at least 4 months.

In one embodiment, the composition is administered for at least 5 months.

In one embodiment, the composition is administered for at least 6months.

In one embodiment, the composition is administered for at least 7 months. In one embodiment, the composition is administered for at least 8 months.

In one embodiment, the composition is administered for at least 9 months.

In one embodiment, the composition is administered for at least 10 months.

In one embodiment, the composition is administered for at least 11 months.

In one embodiment, the composition is administered for at least 12 months.

Nutraceutical formulations

The present invention is further directed to provide the dietary supplement as a nutraceutical formulation aforementioned in the form of powders, tablets, capsules, soft gelatin capsules, controlled release capsules and tablets, lozenges and chewable preparations, liquid suspensions, suspensions in an edible supporting matrix or foodstuff and oral rehydration solutions. Nutraceutical formulation is a mean known to the skilled person and nutraceutical formulations enables consumption of an effective amount of the Fenofibrate to a human diagnosed with type 1 diabetes.

A nutraceutical is a pharmaceutical-grade and standardized nutrient, also known as dietary supplements or food additives by the FDA under the authority of the Federal Food, Drug, and Cosmetic Act.

The formulations of the present invention can be prepared and administered in a wide variety of oral dosage forms. It is obvious to those skilled in the art that the dosage forms may comprise Fenofibrate in combination with an effective amount of one or more dietary ingredients selected from the traditional group of minerals, fatty acids, amino acids and metabolites thereof, phytonutrients, vitamins and coenzymes.

For preparing the Fenofibrate compositions according to the present invention, nutraceutical compatible carriers and excipients may be either solid or liquid. Solid form preparations include powders, tablets, capsules, soft gelatin capsules, controlled release capsules and tablets, lozenges and chewable preparations, and suspensions in an edible supporting matrix or foodstuff. Liquid preparations include liquid suspensions and oral rehydration solutions. These preparations may contain, in addition to the active component, colorants, flavors, stabilizers, buffers, artificial and natural sweeteners, thickeners, solubilizing agents, dispersants, sorbants, glidants, disintegrants, and the like.

The nutraceutical compositions are preferably prepared in unit dosage form whereby the preparation is subdivided into unit doses containing appropriate quantities of the active component. The unit dosage form can be a packaged preparation containing discrete quantities of packaged tablets, lozenge, capsules, soft gelatin capsules, or controlled release capsules and tablets. Preferably, the controlled release period is suitable for once-daily (i.e., once within a 24-hour period) dosing of an effective dose of the nutraceutical compositions.

Optimally, the tablets, lozenge, capsules, soft gelatin capsules, or controlled release capsules and tablets may be coated with any pharmaceutically acceptable coating. The coatings may be applied for such purposes as product identification, printability of tablet, light protection, aesthetic appearance, and patient compliance. Numerous pharmaceutically acceptable coating formulations have been developed by the pharmaceutical industry and are well known to the pharmaceutical scientist and are also commercially available.

Food or beverage

The term "nutraceutical" as used herein denotes a usefulness in both the nutritional and pharmaceutical field of application. Thus, the novel nutraceutical compositions can find use as supplement to food and beverages, and as pharmaceutical formulations. As will be evident from the foregoing, the term nutraceutical composition also comprises food and beverages containing the above-specified active ingredients.

Thus, one embodiment relates to a composition according to the invention, wherein the nutraceutical is a food or beverage or a supplement composition for a food or beverage.

GENERAL

It should be understood that any feature and/or aspect discussed above in connections with the compounds according to the invention apply by analogy to the methods described herein.

The following figures and examples are provided below to illustrate the present invention. They are intended to be illustrative and are not to be construed as limiting in any way.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1

Sulfatide content in pancreas. 10pg tissue samples taken from 13-week-old mice were analysed by mass spectrometry, n=4. Lipid amount is shown as pmol/mg protein. Amount of the individual sulfatide species, white box control; black box Fenofibrate. Data is mean ± SEM, two-tailed unpaired Student’s f-test. ^* p<0.05; ^** p<0.01 ; ^*** p<0.001.

Figure 2

Sulfatide content in pancreas. 10pg tissue samples taken from 13-week-old mice were analysed by mass spectrometry, n=4. Lipid amount is shown as pmol/mg protein. Total sulfatide amount in pancreas, white box control; black box Fenofibrate. Data is mean ± SEM, two-tailed unpaired Student’s f-test. *p<0.05.

Figure 3

Pancreas weight in 13-week-old mice (n=12-13). Data shown as mean ± SEM. Unpaired two-tailed Student’s f-test.

Figure 4

Pancreas volume in 13-week-old mice as measured by stereology (n=11-13). Data shown as mean ± SEM. Unpaired two-tailed Student’s f-test.

Figure 5

Islet volume in 13-week-old mice as measured by stereology (n=11-13). Data shown as mean ± SEM. Unpaired two-tailed Student’s f-test.

Figure 6

Representative tyrosine hydroxylase staining in control mice, 13-week-old. Black arrow indicates nerve fiber and red arrow indicated tyrosine hydroxylase positive cell Scale bar 50pm.

Figure 7

Representative tyrosine hydroxylase staining in Fenofibrate-treated mice, 13-week-old.

Representative tyrosine hydroxylase staining in control mice, 13-week-old. Black arrow indicates nerve fiber and red arrow indicated tyrosine hydroxylase positive cell Scale bar 50pm.

Figure 8

Nerve fibre volume/islet volume in 13-week-old mice as measured by stereology (n=5-7). Data shown as mean ± SEM. Unpaired two-tailed Student’s f-test *p<0.05. Figure 9

Tyrosine hydroxylase (TH) positive cell volume/islet volume in 13-week-old mice as measured by stereology (n=5-7). Data shown as mean ± SEM. Unpaired two-tailed Student’s f-test *p<0.05.

Figure 10

Total tyrosine hydroxylase (TH) positive cell volume/islet volume in 13-week-old mice as measured by stereology (n=5-7). Data shown as mean ± SEM. Unpaired two-tailed Student’s ί-test *p<0.05.

^** p<0.01.

Figure 11

Correlation between insulitis score and nerve fibre volume/islet volume. Linear regression.

Figure 12

Correlation between insulitis score and tyrosine hydroxylase positive cell volume/islet volume. Linear regression.

Figure 13

Non-fasting glycemia in healthy NOD mice (n=15-16) from age 12 to 30 weeks. Unpaired two-tailed Student’s f-test. Data shown as mean ± SEM. ****p<0.0001.

Figure 14

Standard deviation as a measurement for the stability in blood glucose regulation (n=15-16).

Unpaired two-tailed Student’s f-test. Data shown as mean ± SEM. *p<0.05.

Figure 15

Fasting glycemia in healthy NOD mice (n=11-12) from age 12 to 30 weeks. Unpaired two-tailed Student’s f-test. Data shown as mean ± SEM. *p<0.05.

Figure 16

Glucose tolerance test on 13-week-old mice. Mice were injected with 1 M glucose 0.01 ml/g body weight and glycemia were measured at the indicated time (n=5-6). White squares represent control and black circles represent Fenofibrate treat mice, Results quantified by area under the curve calculation *p<0.05.

Figure 17

Fasting serum insulin levels, mice above the dashed line were defined as hyperinsulinemic n=12. Data shown as mean ± SEM.

Figure 18

Fasting serum glucagon levels. Unpaired two-tailed Student’s t-test. Data shown as mean ± SEM. ^* p<0.05. Figure 19

Homeostasis model assessment of insulin resistance (HOMA-IR). Unpaired two-tailed Student’s t- test. Data shown as mean ± SEM.

Figure 20

Insulin tolerance test was performed on healthy 13-week-old mice and blood glucose was measured at the indicated time. n=5. White squares represent control and black circles represent Fenofibrate treat mice. Results quantified by area under the curve calculation. For the declining phase, 0-60 minutes p=0.62 and for the increasing phase, time 60 to 150 minutes p=0.016. Data shown as mean ± SEM. Dashed line illustrates the change from decline to increasing phase.

Figure 21

Fenofibrate treatment eliminates demand for insulin therapy in a newly diagnosed type 1 diabetes patient. A 19-year-old girl diagnosed with type 1 diabetes began taking Fenofibrate 160mg/day 1 week after diagnosis. 2 units of insulin was taken at day 133 as a precaution when the patient was admitted to hospital in Sri Lanka with a high fever during her vacation. Graph shows her random mean blood glucose values and insulin intake.

EXAMPLES

Example 1 Experimental set ups

Animals

All animal experiments were conducted in agreement with the Directive 2010/63/EU of the European Parliament, the Council of 22 September 2010 on the protection of animals used for scientific purposes, and the Danish Animal Experimentation Act (LBK 474 15/05/2014). The Danish Animal Experiments Inspectorate approved the study (reference 2016-15-0201-00841 ) and the local ethical committee (EMED: P 15-383 and P 16-440). Female NOD mice (Taconic Biosciences, Hudson, USA) were housed at the Department of Experimental Medicine (University of

Copenhagen, Denmark) and kept in a Specific Pathogen Free (SPF) animal facility (temperature 22 C degrees, 12 h light cycle, air change 16 times per hour, and humidity 55 ±10%). 3-week-old mice were randomly distributed into two groups receiving either standard Altromin 1320 diet (Altromin, Lage, Germany) or a modified Altromin 1320 diet containing 0.01 % Fenofibrate (Sigma, St Louis, MO, USA). All mice had free access to diet and drinking water. There was no difference in body weight between the two groups at the start of the experiment. Mice were treated from the age of 3 weeks and until the experiment was terminated at age 13 weeks. Mice were weighed each week and food and water intake were measured weekly by weighing of the food racks or water flask, respectively. Glucose monitoring was performed using Freestyle Lite (Abbott Diabetes Care, Alameda, CA, USA). At the end of the experiment, animals were killed by cervical dislocation. Serum insulin and glucagon was measured using Mercodia mouse insulin or glucagon ELISA kit, respectively, (Mercodia, Uppsala, Sweden).

Insulin and glucose tolerance test

13-week-old mice were fasted as described above. Fasting blood glucose was measured from the tail tip and used as time point 0 minutes. Mice were then, for the glucose tolerance test, intraperitoneally injected with 0.01 ml 1 mol/l glucose/g body weight. Glucose concentrations were measured as described above at time 15, 30, 45, 60, 90, and 120 minutes. Due to the difference in fasted blood glucose between groups, the results are shown as the per cent increase in blood glucose compared to the fasting blood glucose concentration of each mouse. Area under the curve (AUC) was calculated using the fasting blood glucose level as baseline. For the insulin tolerance test mice were intraperitoneally injected with insulin (Actrapid, Novo Nordisk, Denmark) 0.75 units/kg body weight. Glucose concentrations were measured at time 15, 30, 45, 60, 90, 120, and 150 minutes. Results are shown as the per cent change in blood glucose compared to the fasting blood glucose concentration of each mouse. Glucose concentrations were declining for the first 60 minutes. An AUC for time 0-60 minutes was used as a measurement of insulin tolerance. Glucose concentrations during the 60-150-minute period were increasing and an AUC calculated with 60- minute glucose as 100% was used as a measurement of the mice’s response to hypoglycemia.

Homeostatic model assessment

For calculation of homeostasis model assessment of insulin resistance (HOMA-IR), the mice were fasted for six hours and blood glucose measured as described above. Mice were killed by cervical dislocation and blood was collected immediately by heart puncture. Insulin concentration was measured in blood serum as described above HOMA-IR was calculated as fasting serum insulin content mU/L x fasting blood glucose mM

Organ weight, insulitis, and stereology

Mice were killed, and organs carefully removed and weighed. The pancreas was fixed it in 10% neutral buffered formalin overnight until paraffin embedding. Pancreata was cut in 5pm sections in its entirety. For insulitis scoring, sections were stained with haematoxylin and eosin (H&E) and evaluated using an BX53 microscope (Olympus America, Inc., Melville, NY, USA). 30 islets from each mouse were scored blindly according to the following scale: 0, no infiltration; 1 , intact islets but with few mononuclear cells surrounding the islets; 2, peri-insulitis (multiple mononuclear cells surrounding the islets); 3, islet infiltration below 50%; 4, islet infiltration above 50%. For stereological evaluation of islet and pancreas volume, every 30th section was H&E stained scanned at 10X magnification using NanoZoomer- XR (Hamamatsu, Hamamatsu City, Japan). The resulting pictures were investigated using newCAST software (Visiopharm, Hoersholm, Denmark). A point-counting grid with 25 intersections and 1 encircled unit intersections was applied to the images and they were blindly counted for total number of intersections touching islets åP(islet) and total number of unit intersections touching pancreas åP(pancreas). The total islet volume (Visiet) and total pancreas volume (Vpancreas) were estimated using the following equation based on the Cavalieri method.

Where a/p designates the area per point of the grid tsectbn is the section thickness and Nsectbn(p-p) the number of sections between the sections used for counting.

For stereology analysis of sympathetic nerves, every 30th section was stained for tyrosine hydroxylase (ab112, Abeam, Cambridge, UK) and analysed by the same principles as for islet and pancreas volume. However, slides were scanned at 40X magnification and, a point-counting grid with 256 intersections was applied to the images upon counting.

Lipid measurement

For lipid measurement, 10mg samples were taken from the tale of the pancreas, snap frozen and kept at -80 C until analysis. Samples were homogenized at 4°C on TissueLyser II (QIAGEN, Hilden, Germany) in 155mM ammonium acetate. Total protein concentration was measured using Pierce BCA Protein Assay (Thermo Fisher Scientific, Waltham, MA, USA). Aliquots corresponding to 100pg protein were subjected to lipid extraction by a modified Bligh and Dyer protocol executed at room temperature. The sample aliquots were spiked with 10mI_ of 50nM, corresponding to 0.5pmol of SHexCer 30:1 :2 standard. Quantification of sulfatide species were performed on a (U)HPLC UltiMate 3000 RSLCnano System (Thermo Fisher Scientific) interfaced on-line to quadrupole-orbitrap mass spectrometer Q-Exactive (Thermo Fisher Scientific). We used a silica column 0.5 x 150mm (YMC-Pack Silica analytical column, 3pm particles). We used Lipid Xplorer (https://wiki.mpi-cbg.de/lipidx/Main_Page) to extract data, selecting a time range based on the eluted peaks of the sulfatide species.

Statistics

Statistical analysis was performed using Graphpad Prism version 6.01 (La Jolla, CA, USA) and data is shown as mean ± SEM unless otherwise noted. All data were assessed to ensure equal variance and normal distribution between groups. Data was log-transformed before analysis if not normally distributed. For comparisons between groups, a two-tailed unpaired Student’s f-test was used. Correlation between nervous tissue and insulitis score was performed with a linear regression. The percentage of mice with hyperinsulinemia was evaluated using a c ² test. A p-value of less than 0.05 was considered significant. ^* p<0.05; ^** p<0.01 ; ^*** p<0.001 ; ^**** p<0.0001.

Example 2 - Fenofibrate increases the amount of C24 sulfatide in islets

To evaluate the effect of Fenofibrate on sulfatide metabolism we employed a mass spectrometry approach. Female NOD mice were treated with Fenofibrate from age 3 weeks as to proceed the development of insulitis starting at age 4 weeks.10pg samples taken from the pancreatic tail of mice age 13 weeks. The mass spectrometry analysis showed a significant increase in the amount of long-chain C24:0 and C24:1 sulfatide as well as an increase in C20:1 (Figure 1 ). We also found an overall increase in the amount of all sulfatide species following Fenofibrate treatment (Figure 2).

Example 3 - Fenofibrate prevents loss of pancreatic nerves

Pancreatic C24:0 sulfatide is found in both islets and neuronal tissue while C24:1 is primarily found in only in neuronal tissue. So, the possibility exists that the increased amount of sulfatide might reflect a change in islet and/or neuronal volume as there was no difference in pancreatic mass at age 13 weeks (Figure 3). Pancreata from 13-week-old mice were therefore analysed by stereology. H&E staining revealed that Fenofibrate did not affect pancreas or islet volume (Figure 4 & 5). Next, we stained for TH as to examine the level of sympathetic pancreatic nerves (Figure 6 & 7). We find an increased volume of islet sympathetic nerve fibers in Fenofibrate treated mice. (Figure 8). We also noticed that Fenofibrate increases the volume of TH expressing cells in islets (Figure 9). The total volume of TH expression in islets was also increased by Fenofibrate (Figure 10). There was an inverse correlation between insulitis score to islet sympathetic nerve fibers (p=0.047 r ² =0.34) and between insulitis score and TH expressing cells/islet volume (p=0.005 r ² =0.56) (Figure 11 & 12).

Example 4 - Fenofibrate improves blood glucose homeostasis

Loss of pancreatic sympathetic neurons are known to reduce glucose tolerance in mice and so we wanted to investigate if the increased amount of pancreatic sympathetic nerves would be reflected in improved blood glucose homeostasis. Non-fasting glycemia was measured once a week from age 12 to 30-weeks and showed that Fenofibrate treated mice had a lower non-fasting glycemia than healthy NOD mice on a control diet. (5.3 mmol/l vs 6.1 mmol/l. Figure 13). Fenofibrate treatment furthermore resulted in a more stable glycemia as seen by the reduced standard deviation (Figure 14). The reduced non-fasting glycemia was associated with an increased fasting glycemia with controls (3.1 mmol/l) vs Fenofibrate (3.8mmol/l) (Figure 15). Next, a glucose tolerance test (GTT) showed that Fenofibrate treated mice had improved glucose tolerance at age 13 weeks (Figure 16). 4/12 control mice showed signs of fasting hyperinsulinemia, this was not the case in any of the Fenofibrate treated mice (0/12, c ² p=0.03) (Figure 17). We furthermore found lower fasted glucagon levels in Fenofibrate treated mice (Figure 18). HOMA-IR, which is a mathematical method used to quantify insulin resistance, to evaluate if these changes in glucose homeostasis were the result of altered insulin sensitivity. HOMA-IR revealed no difference in insulin sensitivity (Figure 19, p=0.48). We next used an insulin tolerance test to quantify insulin resistance and the mice ability to recover from insulin-induced hypoglycemia. Control and Fenofibrate-treated mice were injected with insulin and the following decline and return to normal glycemia was evaluated. There was no difference in the declining phase illustrating that Fenofibrate does not change insulin sensitivity (Figure 19, p=0.62). The Fenofibrate-treated mice had a quicker return to normal glycemia following the insulin-induced hypoglycemia. (Figure 20, p=0.016).