WO2011120576A1

WO2011120576A1 - Antidiabetic enolic glucoside of phenylpyruvic acid

Info

Publication number: WO2011120576A1
Application number: PCT/EP2010/054331
Authority: WO
Inventors: Stephen John Fey; Elizabeth Joubert; Johan Louw
Original assignee: ZADEC
Current assignee: ZADEC
Priority date: 2010-03-31
Filing date: 2010-03-31
Publication date: 2011-10-06
Anticipated expiration: 2012-09-30

Abstract

There is provided an antidiabetic enolic glucoside of phenylpyruvic acid for use as a medicament, especially a normoglycemic agent, i.e. for lowering blood glucose levels to normal levels in mammals that are obese, pre-diabetic or have diabetes, obesity and/or syndrome X. Hence the compound of the present invention help to manage blood sugar levels, i.e. helping the body by balancing the blood sugar levels; helping to keep balanced blood glucose levels, particularly in humans with diabetes; aiding by enhancing the glucose uptake by the cells and by reducing sugar levels, thus improving or restoring the glucose tolerance; optimizing the glycaemic response; normalizing the glucose tolerance.

Description

Antidiabetic enolic glucoside of phenylpyruvic acid

FIELD OF THE INVENTION

The present invention relates to a compound for use as a medicament, especially a normoglycemic agent, i.e. for lowering blood glucose levels to normal levels in mammals that are obese, pre-diabetic or have diabetes, obesity and/or syndrome X (metabolic syndrome).

BACKGROUND OF THE INVENTION

Diabetes mellitus defines a complex of metabolic diseases derived from multiple causative factors and is characterized by impaired glucose metabolism, usually associated with impaired protein and fat metabolism. This results in elevated fasting and postprandial serum glucose levels that leads to complications if left untreated.

Four different forms of diabetes mellitus are known, (1 ) type 1 diabetes mellitus (T1 D), (2) type 2 diabetes mellitus (T2D), (3) the so-called gestational diabetes mellitus, which begins or is recognized for the first time during pregnancy, and (4) some other forms which are mainly based on genetic defects. The two major forms of diabetes mellitus are the type 1 and type 2 diabetes mellitus, of which T2D is the most prevailing form. There are many theories for explaining the impairment of insulin production by the pancreas that leads to type 1 diabetic condition. Reference is made to two papers. The first is entitled "Possible toxic effects of normal and diabetic patient serum on pancreatic B-cells" by Lernmark A, Sehlin J, Taljedal IB, Kromann H, Nerup J. published in Diabetologia. 1978 Jan 14;14(1 ):25-31 . The second is "Autoimmune Imbalance and Double Negative T Cells Associated with Resistant, Prone and Diabetic Animals", Hosszufalusi, N., Chan, E., Granger, G., and Charles, M.; J Autoimmun, 5: 305-18 (1992). These papers show that inflammation of the pancreatic Islets interrupts insulin production. Specifically, the insulin producing beta cells in the pancreatic islets are destroyed by immune attack. Such beta cell destruction is recognized as being due to attack by several types of immune cells including NK (natural killer) cells and double negative T-Lymphocytes. The identification of antibodies against certain proteins (e.g. GAD65, insulin etc.) is used as one of the diagnostic parameters to detect T1 D. Even so this autoimmune attack is considered a secondary event following changes in the islets themselves and these changes probably set in many years before the clinical onset of diabetes.

T2D is associated with hyperglycaemia, hypercholesterolemia and hyperlipidemia. The insensitivity to insulin in T2D leads to a decrease in glucose utilization by the liver, muscle a nd th e ad i pose tissu e a nd to a n i n creased blood gl u cose level . U n control led hyperglycaemia is associated with the dysfunction and failure of various organs such as the eyes, heart, blood vessels, kidney and nerves thus leading to increased and premature mortality due to an increased risk for microvascular and macrovascular diseases, including nephropathy, neuropathy, retinopathy, ulceration of the legs and feet, fatty liver disease, hypertension, cardiovascular diseases, and cerebrovascular diseases (stroke), the so-called diabetic complications. Recent evidence showed that tight glycemic control is a major factor in the prevention of these complications in T2D. Therefore, optimal glycemic control by drugs or therapeutic regimens is an important approach for the treatment of T2D.

T2D is the form of diabetes mellitus which occurs predominantly in adults, in whom adequate production of insulin is available for use in the early stage of the diseases, yet a defect exists in insulin action especially insulin-mediated utilization and metabolism of glucose in peripheral tissues. The changes in various tissues associated with T2D also exist many years before clinical symptoms are detected.

T2D is diagnosed by raised levels of plasma glucose. Following diagnosis of diabetes by raised blood glucose levels, therapies such as diet and exercise and/or available medication can result in a temporary improvement in plasma glucose levels but cannot halt the progression of the disease. The rate of failure of these therapies is associated with the rate of continuing beta-cell decline. The incidence of T2D is increasing worldwide. Although genetic factors may play a role, the increase is normally attributed to life-style changes such as the adoption of a Western diet, high in fat, leads to obesity which can be a factor contributing to the increase of this disease. Life-style factors, such as increased fat intake and reduced exercise, have been shown to be associated with obesity and insulin resistance. In rats, high fat feeding induces a state of insulin resistance associated with diminished insulin-stimulated glycolysis and glycogen synthesis. This disease is a result of the peripheral insulin-responsive tissues, such as muscle and adipose tissue, displaying a significant decrease in response to insulin resulting in an increase in circulating glucose and fatty acids in the blood. The low response to insulin results in a decrease in glycolysis which in turn initiates gluconeogenesis and glycogenolysis in the liver, both of which are "switched off" by insulin under normal conditions.

Pancreatic cells are able to cope with the initial insulin resistant phase by producing an excess of i n s u l i n a n d i n creasi ng th e a mount of insulin secreted. The resulting hyperinsulinaemia to maintain normoglycaemia eventually brings about cell dysfunction leading to full blown diabetes. It is evident that T2D is dependent on insults occurring both at peripheral as well as the cell level.

Diabetes is considered to be insidious, since there is no cure known at this time. Various treatments, however, have been used to ameliorate diabetes.

At present, T1 D patients are treated with insulin. Unfortunately, the use of insulin currently requires multiple (often daily) doses, normal ly admi nistered by self-injection, with determination of the proper dosage of insulin requiring frequent estimations of the sugar in urine or blood, performed either by the patient or the administering physician. The unintended administration of an excess dose of insulin can result in hypoglycaemia, with adverse effects ranging from mild abnormalities in blood glucose to coma, or even death.

Therapy of T2D initially involves dietary and lifestyle changes (including increased exercise). When these measures fail to maintain adequate glycemic control, the patients are treated with oral hypoglycemic agents and/or exogenous insulin. The current oral pharmacological agents for the treatment of T2D include those that potentiate insulin secretion (sulphonylurea agents), those that improve the action of insulin in the liver (biguanide agents), insulin sensitizing agents (thiazolidinediones) and agents which act to inhibit the uptake of glucose in the gastrointestinal tract (oglucosidase inhibitors). Biguanides, such as metformin, became available for treatment of type 2 diabetes in the late 1950s, and have been effective hypoglycaemic agents ever since. As an insulin sensitizer, metformin acts predominantly on the liver, where it suppresses glucose release. Metformin has also been shown to inhibit the enzymatic activity of complex I of the respiratory chain and thereby impairs both mitochondrial function and cell respiration, and in so doing decreasing the ATP/ADP ratio which activates AMP activated protein kinases causing catabolic responses on the short term and insulin sensitization on the long term. This drug has been proven effective in both monotherapy and in combination with sulfonylureas or insulin. However, currently available agents generally fail to maintain adequate glycemic control in the long term due to progressive deterioration in hyperglycaemia, resulting from progressive loss of pancreatic cell function. The proportion of patients able to maintain target glycemic levels decreases markedly overtime necessitating the administration of additional/alternative pharmacological agents. Furthermore, the drugs may have unwanted side effects and are associated with high primary and secondary failure rates.

Therefore, there is a need for compounds with minimal side effects for the prevention, control and/or treatment of diabetes mellitus and for the prevention of the physical complications associated with it as mentioned above. Many patients are interested in alternative therapies which could minimize the side effects associated with high-dose of drugs and yield additive clinical benefits. Diabetes mellitus is a progressive and chronic disease, which usually is not recognized until significant damage has occurred to the pancreatic cells responsible for producing insulin and to the cardiovascular system. Therefore, there is also an increasing interest in the development of novel treatments of diabetes mellitus in people at risk especially in elderly persons, but also in obese children,

(who are at high risk for developing T1 D or T2D). Since T2D is often associated with symptoms from syndrome X ("metabolic syndrome"), such as hypertriglyceridemia or dyslipidemia, the compound according to the present invention is also useful for the treatment or prevention of syndrome X.

SUMMARY OF THE INVENTION

The present invention relates to the compound of formula I for use as a medicament:

(I)

In a preferred embodiment the present invention relates to an optical isomer of formula I, namely the compound of formula II for use as a medicament

When reference is made to the compound of formula (I), the isomer of formula (I I) is the most preferred optical isomer. Concerning further isomers of the Z-form of compound (I) or (II) is preferred, however, the E-form is also active.

The compound of the present invention is particularly useful for the treatment of T1 D, T2D, obesity and/or syndrome X. The present invention is also directed to dietary and pharmaceutical compositions containing this compound and to a method for the treatment of T1 D, T2D, obesity and/or syndrome X in animals including humans, said method comprising the step of administering an effective dose of a compound of the formula I to animals including humans which are in need thereof.

In the context of this invention "treatment" also encompasses co-treatment as well as prevention, and control. Animals in the context of the present invention may be mammals including humans. Preferred examples of mammals beside humans are dogs, cats, guinea pigs, Gack) rabbits, hares, ferrets, horses, monkeys, apes and ruminants (cattle, sheep and goats).

The present inventors have found that the compound of formula I is a particularly effective agent in the prevention, control and/or treatment of non-autoimmune T2D, obesity and/or syndrome X, in animals including humans, especially in mammals including humans. The present invention is also directed to the use of the compound of the formula I as defined above for

• helping to manage blood sugar levels, i.e. helping the body by balancing the blood sugar levels; helping to keep balanced blood glucose levels, particularly in humans with diabetes; aiding by enhancing the glucose uptake by the cells and by reducing sugar levels, thus improving or restoring the glucose tolerance; lowering the blood glucose level; optimizing the glycaemic response; normalizing the glucose tolerance; i.e. the compound of the formula I may be a a-glucosidase inhibitors, hyperglycaemia treating and/or controlling agents and blood glucose lowering agents; and amelioration of T1 D;

• reducing sweetness cravings;

• reducing appetite; · preserving or improving the pancreatic β-cell function, thus promoting a healthy pancreatic function; i.e. the compound of the formula I is an pancreatic β-cell function improver;

• treating or controlling the insulin sensitivity by e.g. helping to restore/enhance the insulin sensitivity; i.e. the compound of the formula I may be an insulin sensitizing agent;

• delaying, preventing or controlling non-autoimmune T2D and thus preventing also the diabetes accompanying disorders/complications such as the ones mentioned above, i.e. the compound of the formula I may be a T2D preventing agent. The compound of the present invention is particularly intended for the prevention of non- autoimmune T2D in those individuals in high risk to develop this disease, such as individuals with pre-diabetes, impaired glucose tolerance (IGT), or obesity.

The invention also relates to the use of the compound according to the invention, including the physiologically acceptable salts thereof, as pharmaceutical compositions. This invention also relates to pharmaceutical compositions, containing the compound according to the invention or a physiologically acceptable salt thereof, optionally together with one or more inert carriers and/or diluents.

This invention also relates to the use of at least one compound according to the invention or one of the physiological ly acceptable salts of such a com pou nd for preparing a pharmaceutical composition which is suitable for the treatment or prevention of diseases or conditions, such as diabetes.

This invention also relates to the use of the compound according to the invention for preparing a pharmaceutical composition which is suitable for the treatment of metabolic disorders.

The invention further relates to a process for preparing a pharmaceutical composition according to the invention, characterised in that a compound according to the invention is incorporated in one or more inert carriers and/or diluents by a non-chemical method.

The compound according to the invention is particularly suitable for the prevention or treatment of diseases, particularly metabolic disorders, or conditions such as T1 D and T2D, complications of diabetes (such as e.g. retinopathy, nephropathy or neuropathies, diabetic foot, ulcers, macroangiopathies), metabolic acidosis or ketosis, reactive hypoglycaemia, hyperinsulinaemia, glucose metabolic disorder, insulin resistance, metabolic syndrome, dyslipidaemias of different origins, atherosclerosis and related diseases, obesity, high blood pressure, chronic heart failure, oedema and hyperuricaemia. These substances are also suitable for preventing beta-cell degeneration such as e.g. apoptosis or necrosis of pancreatic beta cells. The substances are also suitable for improving or restoring the functionality of pancreatic cells, and also for increasing the number and size of pancreatic beta cells. The compound according to the invention may also be used as a diuretic or antihypertensive and is suitable for the prevention and treatment of acute renal failure.

I n particular, the compound according to the invention , includ ing the physiologically acceptable salts thereof, is suitable for the prevention or treatment of diabetes, particularly T1 D and T2D, and/or diabetic complications.

The dosage required to achieve the corresponding activity for treatment or prevention usually depends on the compound which is to be administered, the patient, the nature and gravity of the illness or condition and the method and frequency of administration and is for the patient's doctor to decide. For a skilled practitioner this would depend on inter alia efficiency of absorption, rate of metabolism, and excretion. Additionally, the gut environment may influence the uptake and stability of the compound of the invention. For this purpose, the compound of form ula I prepared according to the invention may be formulated, optionally together with other active substances, together with one or more inert conventional carriers and/or diluents, e.g. with corn starch, lactose, glucose, microcrystalline cellulose, magnesium stearate, polyvinylpyrrolidone, citric acid , tartaric acid , water, water/ethanol, water/glycerol, water/sorbitol, water/polyethylene glycol, propylene glycol, cetylstearyl alcohol, carboxymethylcellulose or fatty substances such as hard fat or suitable mixtures thereof, to produce conventional galenic preparations such as plain or coated tablets, capsules, powders, solutions, suspensions or suppositories.

The use of the compound according to the invention, or a physiologically acceptable salt thereof, in combination with another active substance may take place simultaneously or at staggered times, but particularly within a short space of time. If they are administered simultaneously, the two active substances are given to the patient together; while if they are used at staggered times the two active substances are given to the patient one after the other within a period of less than or equal to 12 hours, but particularly less than or equal to 6 hours. Consequently, in another aspect, this invention relates to a pharmaceutical composition which comprises a compound according to the invention or a physiologically acceptable salt of such a compound and at least one of the active substances described above as combination partners, optionally together with one or more inert carriers and/or diluents. DETAILED DESCRIPTION OF THE INVENTION

The term "diabetes" means a disease or condition that is generally characterized by metabolic defects in production and utilization of glucose which result in the failure to maintain appropriate blood sugar levels in the body. The result of these defects is elevated blood glucose, referred to as "hyperglycaemia." Two major forms of diabetes are Type 1 diabetes and Type 2 diabetes. As described above, Type 1 diabetes is generally the result of an absolute deficiency of insulin, the hormone which regulates glucose utilization. Type 2 diabetes often occurs in the face of normal, or even elevated levels of insulin and can result from the inability of tissues to respond appropriately to insulin. Most Type 2 diabetic patients are insulin resistant and have a relative deficiency of insulin, in that insulin secretion can not compensate for the resistance of peripheral tissues to respond to insulin. In addition, many Type 2 diabetics are obese. Other types of disorders of glucose homeostasis include impaired glucose tolerance, which is a metabolic stage intermediate between normal glucose homeostasis and diabetes. The guidelines for diagnosis for Type 2 diabetes and impaired glucose tolerance have been outlined by the American Diabetes Association (see, e.g. , The Expert Committee on the Diagnosis and Classification of Diabetes Mellitus, Diabetes Care, (1999) Vol 2 (Suppl 1 ): S5-19). The term "pharmaceutically acceptable salt" refers to salts of the compound of the present invention obtained when treating the compound with inorganic or organic acids, such as HCI, HBr, HI , H₂S0₄, H₃P0₄, acetic acid, propionic acid, glycollic acid, maleic acid, malonic acid, succinic acid, tartaric acid, citric acid, benzoic acid, ascorbic acid, etc. When reference is made to the compound of the present invention it means the isolated compound.

The present invention is also directed to the compound of the formula I as defined above for use as inhibitors of glucose uptake such as oglucosidase inhibitors, as hyperglycaemia treating and/or controlling agents, as blood glucose lowering agents, as blood lipids lowering agents, as insulin sensitizing agents, as glucose transporter activators, as pancreatic β-cell function improvers, as inhibitors of hepatic glucose production, as insulin mimetics and/or as enhancers of insulin release. The present invention is further directed to the use of a compound of the formula I as defined above for the manufacture of a composition for the treatment of diabetes, obesity and/or syndrome X. The composition is preferably used as inhibitor of glucose uptake such as oglucosidase inhibitor, as hyperglycaemia treating and/or controlling agent, as blood glucose lowering agent, as blood lipid lowering agent, as insulin sensitizing agent, as glucose transporter activator, as pancreatic β-cell function improver, as inhibitor of hepatic glucose production, as insulin mimetic and/or as enhancer of insulin release.

A further object of the present invention is a dietary composition containing at least a compound of the formula I as defined and with the preferences given as above.

The term "dietary compositions" comprises any type of (fortified) food, (fortified) (animal) feed and beverages including also clinical nutrition, and also dietary supplements as well as the corresponding additives: food additives, beverage additives, feed additives. Also encompassed is functional food/feed i.e. a food/feed that has been enhanced with vitamins or pharmaceuticals to provide further specific health benefits, as well as a nutraceutical, i.e. a pill or other pharmaceutical product that has nutritional value.

The dietary compositions according to the present invention may further contain protective hydrocolloids (such as gums, proteins, modified starches), binders, film forming agents, encapsulating agents/materials, wall/shell materia l s , m atrix compounds, coatings, emulsifiers, surface active agents, solubilizing agents (oils, fats, waxes, lecithins etc.), adsorbents, carriers, fillers, co-compounds, dispersing agents, wetting agents, processing aids (solvents), flowing agents, taste masking agents, weighting agents, jellyfying agents, gel forming agents, antioxidants and antimicrobials.

Another object of the present invention is a pharmaceutical composition containing at least one compound of the formula I as defined and with the preferences given as above and a conventional pharmaceutical carrier.

Beside a pharmaceutically acceptable carrier and at least one compound of the formula I the pharmaceutical compositions according to the present invention may further contain conventional pharmaceutical additives and adjuvants, excipients or diluents, including, but not limited to, water, gelatin of any origin, vegetable gums, lignin sulfonate, talc, sugars, starch, gum arabic, vegetable oils, polyalkylene glycols, flavoring agents, preservatives, stabilizers, emulsifying agents, buffers, lubricants, colorants, wetting agents, fillers, and the like. The carrier material can be organic or inorganic inert carrier material suitable for oral/parenteral/injectable administration. The dietary and pharmaceutical compositions according to the present invention may be in any galenic form that is suitable for administrating to the animal body including the human body, especially in any form that is conventional for oral administration, e.g. in solid form such as (additives/supplements for) food or feed, food or feed premix, fortified food or feed, tablets, pills, granules, dragees, capsules, and effervescent formulations such as powders and tablets, or in liquid form such as solutions, emulsions or suspensions as e.g. beverages, pastes and oily suspensions. The pastes may be filled into hard or soft shell capsules, whereby the capsules feature e.g. a matrix of (fish, swine, poultry, cow) gelatin, plant proteins or lignin sulfonate. Examples for other application forms are forms for sublingual, transdermal , parenteral or injectable administration . The dietary and pharmaceutical compositions may be in the form of controlled (delayed) release formulations. Furthermore, it has been demonstrated that by binding the compound of the present invention to secondary molecules, such as certain peptides, increased is stability prolonging the active period is ach ieved . The present invention also encompasses pro-drugs which are metabolised into more active entities.

Beverages encompass non-alcoholic and alcoholic drinks as well as liquid preparations to be added to drinking water and liquid food. Non-alcoholic drinks are e.g. soft drinks, sport drinks, fruit juices, lemonades, near-water drinks (i.e. water-based drinks with a low calorie content), teas and milk based drinks. Liquid food is e.g. soups and dairy products.

The invention also relates to a method for the treatment of a disease, including diabetes, such as T1 D and T2D, obesity and/or syndrome X in animals including humans, said method comprising the step of administering an effective dose of the compound of the formula I as defined above to animals including humans which are in need thereof.

Animals in the context of the present invention may be mammals including humans. Preferred examples of mammals beside humans are other primates, dogs, cats, guinea pigs, rabbits, hares, ferrets, horses, monkeys, apes and ruminants (cattle, sheep and goats). For humans a suitable daily dosage of a compound of the formula I may be within the range from 0.00003 mg per kg body weight to 60 mg per kg body weight per day. More preferred may be a daily dosage of 0.0003 to 6 mg per kg body weight, preferred may be a daily dosage of 0.0003 to 3 mg per kg body weight per day, especially preferred may be a daily dosage of 0.003 to 0.3 mg per kg body weight per day, most preferred may be a daily dosage of 0.015 to 0.06 mg per kg body weight per day.

The compound according to the invention may be obtained using methods of synthesis known in principle.

The invention is further illustrated by the following examples.

Example 1

3-Phenyl-2-(3,4,5-trihvdroxy-6-hvdroxy^ acid (RX1 )

The enolic glucoside of phenylpyruvic acid (here RX1 ) was isolated by solvent extraction followed by SPE and semi-preparative HPLC from a batch of rooibos (Aspalathus linearis). Alternatively, the compound can be isolated as described by Marais et al (Tetrahedron Letters, 1996).

As shown in Figure 1 RX1 is able to reduce the blood sugar of monkeys for prolonged periods of time. Figure 1 shows reduction in plasma glucose level of a diabetic primate M1081 (baseline glucose 6.3 mmol/L) over 6 h after a single dose of RX1 (tested at ca. 70.5 ug/6.78 kg animal = 10.4 ug/kg BW).

Example 2

Glucose stimulated insulin secretion rate AUC values of untreated and RX-1 treated prediabetic monkeys

Prediabetic vervet monkeys (fasting plasma glucose levels between 4.0 and 5.5 mM) were treated with 1 0 ug/kg RX-1 3 times daily with meals for 7 days. Blood samples were collected following 1.75g/kg oral glucose stimulation at 0, 5, 10, 15, 30, 60, 90, 120 and 180 minutes. AUC values calculated mean glucose stimulated insulin secretion values over the time interval 0 - 120 min. Four monkeys were used in each group (untreated, RX1 -treated).

As appears from Figure 2 RX-1 treatment decreased insulin secretion by 46% while achieving a better glycaemic control.

Example 3

Glucose uptake in a transformed 3T3 -L1 adipocytes

3T3-L1 cells were transformed in culture using modified DMEM differentiation media supplemented with insulin, dexametasone and isobutylmethylxanthine and cultured for 3 days. The transformed 3T3-L1 adipocytes were then cultured for a further 5 days in modified DMEM supplemented with 10% FCS before being exposed to insulin, metformin and the compound of the present invention (see Table 1 ). Glucose uptake over a three (3) hour period was determined after the 5 days of treatment using a colourometric glucose oxidase method (Biovision Inc, USA).

Table 1 shows the glucose uptake data of 3T3-L1 adipose cells following two (2) days of pre-sensitization with the relevant extracts, followed by a three (3) hour glucose uptake assay with media containing 8 mM glucose. The glucose concentration column represents the glucose concentration remaining in the media following three (3) hour exposure to the cells. The glucose uptake column represents glucose uptake from the media after a 3 hour exposure. SD represents the standard deviation. The percentage increases calculated from the relevant solvent vehicle and the P= values are reflected in the last two columns respectively.

The 3T3-L1 adipose cell glucose uptake assay showed that 3-Phenyl-2-(3,4,5-trihydroxy-6- hydroxymethyl-tetrahydro-pyran-2-yloxy)-a c ry l i c a c i d ( R X-1 ), (2R,3R,4S,5R,6S)-2- (Acetoxymethyl)-6-((Z)-3-methoxy-3-oxo-1 -phenylprop-1 -en-2-yloxy)tetrahydro-2H-pyran-

3,4,5-triyl triacetate (RX-1 -triacetate), and (Z)-Methyl 3-phenyl-2-((2S,3R,4S,5S,6R)-3,4,5- trihydroxy-6-(hydroxymethyl)tetra hydro-2H-pyran-2-yloxy)acrylate (RX-1 a e ry I a t e) significantly increased the glucose uptake over a 3 hour culture period.

Table 1

3T3-L1 Adipose Cell: Glucose Uptake Data

Example 4

Analysis of glucose-lowering properties of Rx-1 in in obese, insulin resistant Wistar rats The aim of the study was to determine whether the glucose-lowering properties of Rx-1 is related to the expression of genes involved in glucose uptake, insulin signalling, fatty acid oxidation, cytokine signalling and the glucagon receptor in liver and muscle, and the expression of genes involved in glucagon processing, insulin expression and transcription factors important for β-cell development in the pancreas.

Methods & Results

Three week old male rats were fed a high fat diet for 24 weeks to induce obesity and insulin resistance. Thereafter, rats were treated with 0.3 mg/kg Rx-1 daily for two weeks, and then with 3 mg/kg Rx-1 daily for seven days. Fasting glucose concentrations were measured before treatment, after two weeks treatment with 0.3 mg/kg Rx-1 and then again after seven days treatment with 3 mg/kg Rx-1 . After treatment with 3 mg/kg Rx-1 rats were terminated and liver, muscle and pancreas biopsies were taken. Quantitative real-time PCR was used to measure the expression of 12 genes in liver and muscle samples, and ten genes in pancreas samples.

Rats were housed at the Primate Unit (Medical Research Council, South Africa). Rat management including feeding, glucose measurements and terminations, were done according to standard operating procedures (Diabetes Discovery Platform , Medical Research Council). Briefly, three week old rats were fed a high fat diet for 24 weeks to induce T2D. The study group consisted of thirteen rats, eight rats were treated by daily gavage with 0.3 mg/kg Rx-1 for two weeks, and then with 3 mg/kg Rx-1 for seven days. Five rats were used as controls and were treated with water only for three weeks. Rats were terminated after treatment and liver, muscle and pancreas tissue harvested and stored in RNa/aier (Ambion) as recommended by the manufacturer. The study was approved by the ethics committee of the Medical Research Council of South Africa. -RNA extraction from liver tissue

Tissue was removed from RNA/aier, weighed (80-100 mg), and placed in a 2 ml microfuge tube containing 1 ml of Trizol (Invitrogen) and a stainless steel bead (Qiagen). Tissue was homogenised in the TissueLyser (Qiagen) at 25 Hz for 6 min, centrifuged at 12,000 g for 10 min at 4°C, and the supernatant removed and incubated at room temperature for 5 min. Thereafter, 0.2 ml of chloroform (Sigma) was added, shaken vigorously for 15 sec, and then incubated at room temperature for 3 min with occasional mixing. Samples were centrifuged 12,000 g for 15 min at 4°C and the aqueous phase was transferred to a new tube. RNA was precipitated by adding 0.5 ml isopropanol, mixed well for 30 sec, and placed at -20°C overnight. The following day, tubes were centrifuged at 12,000 g for 20 min at 4°C. The pellet was washed with 1 ml of 75 % ethanol and centrifuged at 12,000 g for 15 min at 4°C.

The wash step was repeated. After the second wash, the pellet was air dried by placing tubes with their lids open (on ice) in a PCR cabinet for 2 hours. Excess liquid was removed by blotting tubes on paper towel occasionally during this incubation. The pellet was resuspended by adding 100 μΙ RNase-free water and incubating at 55°C for 10 min. RNA concentrations were determined using a spectrophotometer (Nanodrop Technologies).

Thereafter, RNA was purified with the RNeasy Mini Kit according to the manufacturer's instructions (Qiagen) and concentrations again determined with the Nanodrop. Genomic DNA was removed by treating RNA with Turbo DNA-free DNase (Ambion) and incubating at 37°C for 90 min according to the manufacturer's instructions, but using 1 .5x the units of DNase and incubation time recommended by the kit. In brief, 20 μg RNA was incubated with

1 .5 μΙ (3 units) DNase, 5 μΙ DNase buffer, and nuclease-free water in a final reaction volume of 50 μΙ for 45 min at 37°C, thereafter, another 3 units of DNase was added and incubated for a further 45 min. DNase was inactivated by adding V₅ volume (10 μΙ) of the DNase inactivation reagent supplied with the kit. Reactions were incubated at room temperature for 2 min, and centrifuged at 14,000 rpm for 1 .5 min. The supernatant was removed and RNA concentrations were measured using the Nanodrop. The quality of the DNase-treated RNA was determined with the RNA 6000 Nano kit using the 2100 Bioanalyser Lab-on-a-Chip system as recommended by the manufacturers (Agilent technologies). -Reverse transcription

RNA extracted from liver, muscle and pancreas tissue was converted to cDNA using the High Capacity Reverse Transcription kit as recommended by the manufacturers (Applied Biosystems). In brief, 2 μg of DNase-treated RNA was added to nuclease-free water in a volume of 10 μΙ. Thereafter, 2 μΙ reaction buffer, 0.8 μΙ dNTPs, 2 μΙ random primers, 1 μΙ RNase-inhibitor, 1 μΙ reverse transcriptase, and 3.2 μΙ nuclease-free water were added. The same reaction without the reverse transcription enzyme (minus RT reaction) was set-up to investigate genomic DNA contamination. Reactions were incubated at 25°C for 1 0 min , 37°C for 120 min, and 85°C for 5 s to inactivate the reverse transcriptase. cDNA samples were stored at -20°C until expression analysis. -Quantitative real-time PCR, data collection and evaluation

The extent of genomic DNA contamination was investigated by performing qRT-PCR of the RT reactions. Undiluted cDNA (plus and minus RT reactions) prepared from liver, muscle, and pancreas were mixed with 12.5 μΙ SYBR Green mix (Applied Biosystems), 2.25 μΙ 10 μΜ Gapdh Forward Primer (900 nM), 2.25 μΙ 10 μΜ Gapdh Reverse Primer (900 nM), and H₂0 in a final volume of 25 μΙ. After all the reagents had been added, the PCR tubes were briefly spun to ensure that all solutions were at the bottom of the tubes. The PCR reactions were cond ucted on the ABI 7500 Seq uence Detection System I nstrument (Applied Biosystems) using the Absolute Quantification (AQ) Software (SDS V1.4), and labelling all samples as unknowns. Universal cycling conditions; 50°C for 2 min and 95° for 10 min, followed by 40 cycles of 95°C for 15 s and 60°C for 1 min were used. A dissociation curve was added. Data was acquired during the extension step (60°C for 1 min). After the run, default settings for the threshold cycle (C_T) and baseline were used and Ct values were exported to Excel for analysis.

For analysis of gene expression, 25 ng of cDNA prepared from liver, muscle and pancreas was mixed with 12.5 μΙ Taqman universal PCR master mix (Applied Biosystems), 1 .25 μΙ gene-specific primer and probe mixtures (predeveloped Taqman gene expression assays, Applied Biosystems), and H₂0 in a final volume of 25 μΙ. The Taqman assays that were used are listed in Table 1 . The suffix _m represents an assay whose probe spans an exon- exon junction of the associated gene and therefore will not detect genomic DNA, while the suffix _s represents an assay whose primers and probes are designed within a single exon, such assays will detect genomic DNA.

The PCR reactions were conducted on the AB I 7500 Sequence Detection System Instrument (Applied Biosystems) using universal cycling conditions as described before. All samples were run in duplicate. Data generated on the ABI 7500 Instrument were analysed with the ABI Relative Quantitation (RQ) software (SDS V1 .4) using a Ct threshold of 0.1 . Relative expression levels were determined by using the 2^"AACt method, where AACt = (Ct_gene studied-Ct ousekeeping gene)treated " (Ctg_ene studied' -Cthousekee ing gene)controi. The gene expression was normalised to housekeeping genes to correct for differences in cDNA loading. Two gene expression assays, β-actin (ActB) and glyceraldehyde-3-phosphate dehydrogenase (Gapdh) (Table 1 ) were used as endogenous controls. Relative gene expression data generated by the RQ software for each of the two endogenous controls individually or the data normalised to the average of the two endogenous controls were imported into Microsoft Excel and analysed.

-Statistical Analysis

Statistical analysis of normalised gene expression data before and after treatment was performed using two-tailed unpaired t tests (GraphPad Prism version 3.02 Software, San Diego, California, USA). Statistical significance was indicated by a P value < 0.05.

The aim of this study was to determine whether treatment with Rx-1 affected expression levels of genes involved in glucose uptake, insulin signalling, fatty acid oxidation, cytokine signalling and carbohydrate metabolism in the liver and muscle of OB/IR Wistar rats. The affect of Rx-1 treatment on the expression of genes involved in glucagon processing, insulin expression and transcription factors were analysed in the pancreas. Analysis of gene expression profiles after treatment may give insight into the mechanisms of action of Rx-1 .

Gene expression levels in Rx-1 treated and control rats were normalised to ActB, Gapdh or the average of ActB and Gapdh. Although gene expression varied according to the endogenous control used, generally, Rx-1 treatment upregulated genes involved in glucose uptake (Glutl and Glut2), insulin signalling (I R and I RS2), fatty acid oxidation (PPARa), cytokine signalling (SOCS3) and carbohydrate metabolism (GcgR) in the liver. Rx-1 treatment did not affect the expression of these genes in muscle samples. In the pancreas, Rx-1 treatment increased the expression of genes involved in glucagon processing, GLP- 1 R, Gcg and GcgR, the genes encoding insulin, Ins1 and Ins2 and the transcription factors I si 1 and Pdx1. None of the changes observed in the pancreas were statistically significant. The expression of Pcsk2 and nestin was unaffected by Rx-1 treatment. Neuro3 could not be detected in this study.

Rx-1 treatment increased Gck gene expression in the liver of OB/I R rats. However, the increase was not statistically significant. Gck is an enzyme predominantly expressed in the liver where it senses glucose and converts it to glucose-6-phosphate, the first step of glycolysis (Agius, 2008). A number of factors, including insulin (lynedjian et al. 1988) and phenolic compounds (Valentova et al. 2007) have been reported to upregulate Gck gene expression in the liver. Rx-1 treatment decreased Gck m RNA levels in muscle. It has previously been reported that muscle is not a major source of Gck activity. This study showed increased expression of Glutl and Glut2 in the liver of OB/IR Wistar rats treated with Rx-1 . Glut4 mRNA levels in the muscle of these animals were unaffected by treatment. Glucose is important for cellular metabolism and the synthesis of ATP through glycolysis and the citric acid cycle. Facilitative glucose transport into cells is mediated by members of the GLUT protein family that belong to a much larger superfamily of 12 transmembrane segment transporters. At present, thirteen mammalian glucose transporter isoforms have been identified (Joost et al. 2002). These proteins are expressed in a tissue- and cell-specific manner. GLUT1 is a widely expressed and mediates glucose transport into red cells and throughout the blood brain barrier, and provides most cells with their basal glucose requirement. It also plays a role in transporting glucose across epithelial and endothelial barrier tissues. Makni et al. (2008) reported that Glutl polymorphisms are associated with T2D in the Tunisian population.

GLUT2 is a high-Km isoform expressed in hepatocytes, pancreatic beta cells, and the basolateral mem branes of i ntestinal and renal epithelial cells. Single nucleotide polymorphisms (SNPs) in the Glut2 gene of Finnish subjects with impaired glucose tolerance were associated with a threefold risk for developing T2D (Laukkanen et al. 2005).

GLUT4 is expressed exclusively in the insulin-sensitive tissues, fat and muscle. It is responsible for increased glucose disposal in these tissues in the postprandial state and is important in whole-body glucose homeostasis. Insulin stimulation results in GLUT4 translocation from intracellular vesicles within a cell to the plasma membrane and increased glucose uptake. Failure of GLUT4 translocation results in insulin resistance and T2D. Glut4 gene expression and function is decreased during insulin resistance, T2D, obesity, and aging (Karnieli et al. 2008).

IR mRNA levels was increased in the liver of treated rats, whereas levels were unchanged in the muscle of these animals. The IR is a transmembrane protein that consists of an extracellular domain to which insulin binds and an intracellular domain with tyrosine kinase activity. Following insulin binding, the substrate tyrosine kinase activity of the I R initiates a cascade of cellular phosphorylation reactions where it phosphorylates a number of substrates including I RS1 and I RS2. These phosphorylated substrates then serve as docking molecules that bind to and activate cellular kinases, such as Pi3k, leading to glucose uptake, cell growth and protein synthesis (Youngren, 2007). Impaired IR function and signaling is associated with insulin resistance and T2D.

Rx-1 treatment increased I RS2 gene expression in the liver of treated rats. I RS1 mRNA levels were u nchanged in the l iver, whi le both I RS 1 and I RS2 m RNA levels were unchanged in the muscle of these animals. Four isoforms of insulin receptor substrate (IRS) proteins have been identified (Thirone et al. 2006), with I RS1 and I RS2 being the most important. There are tissue-specific differences in the roles of the IRS proteins, with I RS1 playing a prominent role in skeletal muscle, while IRS2 is more important in the liver (White, 2002).

Pi3k was upregulated in the liver only. However, the upregulation was not significant. Pi3k plays a key role in insulin signalling and has been shown to be blunted in tissues of patients with T2D. A number of studies have provided evidence suggesting that insulin resistance, the main cause of T2D can potentially be treated by targeting Pi3k itself or its up and downstream modulators (Jiang and Zhang, 2002).

PPARa was sign ificantly u pregu lated in the liver after Rx-1 treatment. PPARa is predominantly expressed in the liver, and to a lesser extent in muscle, where it controls lipid metabolism and glucose homeostasis (Lefebvre et al. 2006). PPARa agonists have been used to treat obesity, insulin resistance and T2D. One of the mechanisms whereby PPARa improves insulin resistance is by upregulating the genes for fatty acid metabolism.

The expression of SOCS1 and SOCS3 mRNA was increased in the liver and muscle of Rx- 1 treated rats. Only the upregulation of SOCS3 in the liver was statistically significant.

SOCS1 and SOCS3 are two of a family of eight proteins that are thought to regulate cellular responses to cytokines in a negative feedback manner (Yasukawa et al. 2000). Studies have shown that SOCS1 and SOCS3 expression is increased in the liver of OB/IR mice (Ueki et al. 2005). Antisense-mediated knockdown of liver SOCS1 or 3 expression reverses insulin resistance in obese, diabetic mice, strongly supporting a role for SOCS proteins in obesity related insulin resistance (Ueki et al. 2005). The contradictory results obtained in this study highlights the complex gene networks involved in cytokine signalling, insulin resistance and T2D. The main function of the SOCS proteins are as negative regulators of cytokine signalling, therefore, increased expression of these genes may result in decreased cytokine signalling which is beneficial during insulin resistance and T2D (Krebs and Hilton, 2001 ).

Rx-1 treatment increased the expression of the GcgR gene in the liver and muscle after treatment. The upregulation of GcgR was not statistically significant. Charbonneau reported that high fat diet feeding of rats decreased total hepatic GcgR by about 55% (Charbonneau et al. 2007). Our data therefore suggests that Rx-1 treatment reverses the diet-induced downregulation of the GcgR. GLP1 R gene expression was increased in the pancreas after Rx-1 treatment. The incretin hormones, glucagon like peptide 1 (GLP1 ) and glucose-dependent insulinotropic peptide or also known as gastric inhibitory peptide (GIP) stimulate insulin release after the ingestion of carbohydrates and fats, maintaining glucose homeostasis (Kieffer and Habener, 1999). Disruption of the gene encoding the GLP1 R results in glucose intolerance and the inability to secrete insulin in response to glucose (Scrocchi et al. 1996). Activation of the GLP1 R induces β-cell neogenesis and proliferation (Xu et al. 1999), while inhibiting apoptosis (Li et al. 2003).

Rx-1 treatment increased Pdx1 , Ins1 and Ins2 gene expression in the pancreas. Previous studies have reported that GLP1 treatment increases mRNA and protein levels of the transcription factor Pdx-1 (also known as I DX-1 , STF1 and I U F 1 ), and of insulin in the pancreas (Doyle and Egan, 2007). Other studies in our laboratory showed that circulating GLP1 levels were increased in the blood of Rx-1 treated OB/IR rats (Louw et al. 2008). Pdx1 activates insulin gene expression by binding to its promoter and also prolongs the half-life of insulin mRNA (Poitout et al. 2006). In vitro and in vivo studies in rodents have shown that insulin gene expression is greatly reduced under circumstances of chronically elevated levels of glucose and fatty acids (Poitout et al. 2006). Insulin is encoded by the genes, insulin 1 (Ins1 ) and insulin 2 (Ins2). It is speculated that in rodents Ins1 arose from Ins2 due to an RNA mediated duplication-transposition process. Humans only have one insulin gene, with homology to the highly conserved rodent Ins2 (Madadi et al. 2008).

Gcg, the GcgR and I si 1 mRNA levels were increased in the pancreas of OB/IR rats after Rx- 1 treatment. Glucagon is a hormone expressed in the liver where it stimulates glucose production. Isl1 has a critical role in the embryonic development of pancreatic endocrine cells (Ahlgren et al. 1997). In 2008, Koya et al. reported that treatment of streptozotocin- induced diabetic mice with recombinant Pdx-1 enhances β-cell regeneration and liver cell differentiation, restoring normoglycaemia. They further showed that I si 1 and Gcg mRNA levels in the liver and pancreas of these mice were upregulated after recombinant Pdx-1 treatment. Charbonneau et al. (2007) showed that total hepatic GcgR protein content was decreased in rats fed a high fat diet and that GcgR protein levels were increased slightly after exercise.

Nestin is a marker of pancreatic islet stem cells and it has been suggested that nestin- positive cells represent a multipotent pancreatic stem cell population, which could be used in future cell replacement therapies to cure diabetes (Lumelsky et al. 2001 ). In contrast,

Delacour et al. (2004) showed that nestin is expressed in adult pancreatic exocrine cells, and suggests that nestin is not a specific marker of islet endocrine cells. In our study, nestin mRNA levels were unaffected by Rx-1 treatment. Neurogenin 3 was not detected in the untreated or treated rats. Neurogenin-3 is a transcription factor expressed in endocrine progenitor cells and is required for endocrine-cell development in the pancreas (Habener et al. 2005). Lee et al. (2006) reported that Neurogenin-3 is not expressed in adult mouse pancreatic tissue. These results are in agreement with others (Dor et al. 2004) who have reported that replication of existing β-cells is the primary mechanism of β-cell regeneration in adult mice.

Pcsk2 or proconvertase 2 (PC2) mRNA levels were unaffected by Rx-1 treatment. In a-cells PC2 cleaves proglucagon to produce glucagon (Wideman et al. 2006). In summary, this study showed upregulation of the genes involved in glucose uptake, insulin signalling, fatty acid metabolism and cytokine signalling in the liver of Rx-1 treated rats. The expression of genes encoding the hormones insulin and glucagon were increased in the pancreas of these rats, while the transcription factors Pdx1 and I si 1 were also upregulated. GcgR mRNA levels were increased in both the liver and pancreas of Rx-1 treated rats. Taken together, these results suggest that Rx-1 treatment may reverse insulin resistance and increase fatty acid oxidation in OB/IR rats. Genes involved in glucose uptake (Glutl and Glut2), insulin signalling (IR and IRS2), fatty acid oxidation (PPARa), cytokine signalling (SOCS 1 and SOCS3) and the glucagon receptor were upregulated in the liver of Rx-1 treated rats. Only the glucagon receptor was upregulated in the muscle. The expression of the other genes was essentially unchanged.

Genes involved in glucagon processing (GLP1 R, Gcg and GcgR), insulin expression (Ins1 and Ins2) and the transcription factors (Isl1 and Pdx1 ) were upregulated in the pancreas of Rx-1 treated rats. The expression of Pcsk2 and nestin was unaffected by Rx-1 treatment, while neuro3 could not be detected.

Conclusion

Gene expression analysis is a useful technique that may give insight into the glucose- lowering mechanism of action of Rx-1 . Results from this study suggest that Rx-1 acts in the liver where it stimulates glucose uptake, insulin signalling and fatty acid oxidation. I n addition, Rx-1 seems to inhibit cytokine signalling, a hallmark of insulin resistance and type two diabetes. In the pancreas, Rx-1 treatment increased the expression of genes encoding insulin, the transcription factors, Isl1 and Pdx1 , and GLP1 R. Interestingly, GLP1 levels were also increased in the blood of these rats. Taken together, our results suggest that Rx-1 may reverse insulin resistance and increase glucose uptake and fatty acid oxidation in obese, insulin resistant rats.

Claims

1 . Compound of formula I or a physiologically acceptable salts thereof

for use as a medicament.

2. Compound of formula II or a physiologically acceptable salts thereof:

for use as a medicament.

3. Compound of formula I or II as defined in claim 1 or 2 for the treatment of diabetes, such as type 1 diabetes (T1 D) or type 2 diabetes (T2D), obesity and/or syndrome X.

4. A pharmaceutical composition containing a compound of formula I or II as defined in claim 1 or 2 and a conventional pharmaceutical carrier.

5. A method for the treatment of a metabolic disease, including diabetes, obesity and/or syndrome X in animals including humans, said method comprising the step of administering an effective dose of a compound of formula I or II as defined in claim 1 or 2 to animals including humans which are in need thereof.