Compositions and compounds for simultaneous delivery of uridine and ketroleucine - Patent Application 20070229933

Abstract

Compounds and compositions that deliver both uridine and ketroucine are useful in treating disorders characterized by muscle weakness or loss of lean muscle mass. One such compound is 5'-O-ketroucyl-2',3'-di-O-acetyluridine.

Description

[Background technology]

[0001] Age-related neuromuscular dysfunction underlies several major disease states, including sarcopenia and muscle disuse atrophy. Both of these conditions involve impaired energy metabolism in the muscle and nervous system, as well as reduced muscle mass. A satisfactory treatment for these conditions should address both pathogenic factors, defective energy metabolism and reduced muscle protein.

[0002] Oral delivery of uridine for therapeutic purposes is limited by its poor bioavailability, approximately 7% in both humans and mice. Although ester prodrugs of uridine have been found to improve its bioavailability, only one, 2',3',5,-tri-O-acetyluridine (or uridine triacetate), has been found to be suitable for delivering sufficient uridine for some clinical purposes. The bioavailability of oral uridine triacetate has been measured to be approximately 50% (Ashour 1996). Summary of the Invention

[0003] The present invention provides the compound 5'-O-ketroucyl-2',3'-di-O-acetyluridine, also known as 2',3'-di-O-acetyl-5'-O-(α-ketoisocaproyl)uridine. The present invention also provides compositions comprising a therapeutically effective amount of a combination of a ketroucine compound and a uridine prodrug.

[0004] The present invention provides compositions, compounds and methods for treating or preventing sarcopenia (and other muscle wasting conditions such as cachexia), dynapenia (pathological loss of muscle strength), exercise intolerance (pathological loss of endurance during vigorous activity), and muscle disuse atrophy.Conditions associated with high risk of muscle wasting and exercise intolerance are also included, including chronic kidney disease, chronic obstructive pulmonary disease (COPD) and chronic heart failure.Specifically, compositions and compounds are provided that deliver therapeutic amounts of both uridine and ketroleucine. [Brief description of the drawings]

[0005] [Figure 1] Survival in mice treated with 3-NP, UTA and NaKL. Group 1: 4% UTA diet, NaCl drinking water; Group 2: 4% UTA diet, NaKL drinking water; Group 3: chow diet, normal drinking water, UTA / NaCl gavage; Group 4: chow diet, normal drinking water, UTA / NaKL gavage; Group 5: chow diet, NaKL drinking water, UTA gavage [Diagram 2] Percentage of weight loss in mice treated with 3-NP, UTA and NaKL. Group 1: 4% UTA diet, NaCl drinking water; Group 2: 4% UTA diet, NaKL drinking water; Group 3: chow diet, normal drinking water, UTA / NaCl gavage; Group 4: chow diet, normal drinking water, UTA / NaKL gavage; Group 5: chow diet, NaKL drinking water, UTA gavage DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0006] Ketroucine (alpha-ketoisocaproic acid, 4-methyl-2-oxovalerate, KL) is a deaminated derivative of the essential branched-chain amino acid leucine. Leucine is not only a structural component of proteins, including those in skeletal muscle, but also acts as a regulator of muscle protein maintenance. Leucine metabolites, including ketroucine and β-hydroxy-β-methylbutyrate, can activate or maintain muscle protein synthesis.

[0007] Ketroleucine is also a pathogenic factor in certain metabolic disorders. Maple Syrup Urine Disease (MSUD, so named because of the characteristic odor of urine, similar to that of maple syrup) is a rare genetic disorder caused by a deficiency of the enzyme complex, branched-chain alpha-ketoacid dehydrogenase. This results in impaired catabolism of branched-chain amino acids and their deamination products, causing their accumulation in brain and other tissues, and is responsible for the odor of urine and other body fluids. MSUD is characterized by progressive neurological dysfunction that begins with lethargy, irritability, and anorexia, followed by abnormal movements, increased spasticity, and eventually neurological signs such as seizures and deep coma. If untreated, progressive brain damage is inevitable and death usually occurs within weeks or months. Ketroleucine accumulation impairs brain energy metabolism, in part, by inhibiting creatine kinase, a key enzyme for the relay of energy derived from mitochondrial ATP to cytosolic sites of energy utilization.

[0008] It is therefore unexpected that the combination of oral ketroleucine with oral uridine triacetate provided greater protection against mortality in a model of severe mitochondrial energy failure than that conferred by uridine triacetate or ketroleucine alone.

[0009] Ketroucine is also a short chain keto acid. The present disclosure also encompasses prodrugs of uridine that include ketroucine as a 5'-ester substituent, with optional 2' and / or 3' substituents including acetate or other 2 or 3 carbon atom carboxylic acids. The ketroucine substituent at the 5' position of the ribose moiety is combined with acetate substituents at the 2' and 3' positions to provide novel compounds that deliver uridine into the circulation while also delivering prodrug substituents that provide additional or complementary therapeutic benefits beyond enhanced uridine delivery.

[0010] The main clinical indication for either the prodrug of uridine with ketoleucine substitution or the combination of uridine triacetate + ketoleucine is for the prevention and treatment of skeletal muscle wasting disorders, including but not limited to muscle disuse atrophy (e.g., muscle mass and strength loss caused by exercise restraint and lack of exercise during long-term hospitalization) and sarcopenia (loss of lean muscle mass) or dynopenia (loss of muscle strength with or without loss of muscle mass).Higher risk and incidence of sarcopenia and dynopenia are found in people with comorbidities, including chronic kidney disease, COPD and chronic heart failure, and the compounds and compositions of the present disclosure are suitable for the treatment and prevention of these conditions.Cancer-related cachexia, pathological loss of body weight and muscle mass can also be treated or prevented using the agents of the present disclosure.

[0011] Suitable doses of the compounds and compositions of the present invention deliver the equivalent of 1-5 grams of uridine per dose to a human subject, advantageously 2-4 grams per dose, given orally 1-3 times per day. In the combination of uridine triacetate + ketroucine (generally in the form of sodium ketroucine or other salt), the ketroucine is present in the combined formulation in a molar equivalent ranging from 1 up to 4 times the molar amount of uridine. The molecular weight of ketroucine is approximately 130 daltons, compared to 370 for uridine triacetate.

[0012] Example 1: Synthesis of 2',3'-di-O-acetyl-5'-O-(α-ketoisocaproyl)uridine Preparation of 5'-O-(tert-butyldimethylsilyl)uridine [ka] Imidazole (32.68 g, 480 mmole) and tert-butyl(chloro)dimethylsilane (36.2 g, 240 mmole) were added sequentially to a solution of uridine (48.84 g, 200 mmole) in DMF (300 mL) cooled to 0° C. The mixture was stirred at 0° C. for 4 h and at room temperature for 17 h. TLC (silica gel, 10% MeOH / DCM) showed the reaction was complete. DMF was removed using a rotary evaporator under high vacuum. The crude product was dissolved in ethyl acetate and washed with water (3×500 mL). The organic layer was dried over anhydrous MgSO4 and concentrated to give 88% yield of monosilylated uridine as a crystalline compound containing trace amounts of silyl impurities, which was 1 Confirmed by H NMR (400 MHz, CDCl3) δ 0.11 (s, 6H), 0.92 (s, 9H), 3.84 (dd, 1H, J = 1.8, 11.7 Hz), 4.02 (dd, 1H, J = 2.2, 11.7 Hz), 4.10-4.30 (m, 3H), 5.68 (d, 1H, J = 8.1 Hz), 5.88 (d, 1H, J = 2.2 Hz), 8.07 (d, 1H, J = 8.0 Hz).

[0013] Preparation of 5'-O-(tert-butyldimethylsilyl)-2',3'-di-O-acetyluridine [ka] 5'-O-(tert-butyldimethylsilyl)uridine (52.0 g, 145 mmole) was dissolved in DCM (700 mL) and DMAP (40 g, 328 mmole) and acetic anhydride (35 mL, 371 mmole) were added sequentially. The reaction mixture was stirred at room temperature for 4 h under argon. TLC (silica gel, 50% ethyl acetate / hexanes) showed the reaction was complete. The reaction mixture was transferred into a separatory funnel and washed with water (2 x 500 mL). The organic layer was dried over anhydrous MgSO4, concentrated, and purified using flash chromatography (silica gel, 50% ethyl acetate / hexanes) to give 66.0 g (100%) of product. 1H NMR (400MHz, CDCl3) δ0.11(s, 6H), 0.92(s, 9H), 2.05(s, 3H), 2.11(s, 3H), 3.81(m, 1H), 3.90(m, 1H), 4.19~4.20( m, 1H), 5.27~5.33(m, 2H), 5.72(dd, 1H, J=1.8, 8.0Hz), 6.28(d, 1H, J=6.6Hz), 7.84(d, 1H, J=8.1Hz), 8.78(s, 1H), 13 C NMR (100MHz, CDCl3) δ -5.48, -5.45, 18.45, 20.54, 20.83, 25.99, 63.34, 71. 88, 73.52, 83.94, 85.47, 103.41, 139.64, 150.88, 163.24, 169.77, 170.07.

[0014] Preparation of 2',3'-di-O-acetyluridine [ka] 4 grams of para-toluenesulfonic acid was added to a solution of 5'-O-(tert-butyldimethylsilyl)-2',3'-di-O-acetyluridine (66.0 g, 0.149 mole) in dry MeOH (100 mL) and dry DCM (250 mL) cooled to 0°C. The reaction mixture was stirred at 0°C for 3 hours and then at room temperature overnight. TLC (silica gel, 50% ethyl acetate / hexanes) showed consumption of starting material. The reaction mixture was quenched with triethylamine and evaporated to dryness. The crude product was chromatographed using silica gel and hexanes / ethyl acetate (1:1) to give 39.5 g (81%) of product. 1 H NMR (400MHz, CDCl3) δ2.08(s, 3H), 2.13(s, 3H), 2.73(s, 1H), 3.85(dd, 1H, J=2.2, 12.1Hz), 3.94(dd, 1H, J=2.2, 12.1Hz), 4.21(q, 1H, J=2.2Hz), 5.45~5.49(AB, 2H), 5.78(dd, 1H, J=1.9, 8.1Hz), 6.06(m, 1H), 7.75(d, 1H, J=8.1Hz), 8.90(s, 1H), 13C NMR (100MHz, CDCl3) δ20.64, 20.85, 62.03, 71.41, 73.19, 83.69, 87.86, 103.45, 140.93, 150.71, 163.28, 169.97, 170.28.

[0015] Preparation of 2',3'-di-O-acetyl-5'-O-(α-ketoisocaproyl)uridine [ka] Dichloromethyl methyl ether (5.00 mL, 56.5 mmol) is added dropwise to α-ketoisocaproic acid (5.0 g, 38.5 mmol) during which gaseous HCl is evolved. After the addition is complete, the mixture is heated at 48-52 °C for 30 min. The mixture is then rapidly cooled in an ice bath. The mixture is slowly added via syringe to a mixture of 2',3'-di-O-acetyluridine (12.6 g, 38.4 mmol) and pyridine (9.0 mL, 112 mmol) in 180 mL of DCM cooled by an ice bath. The mixture is allowed to warm to room temperature overnight. 5 mL of water is added and the solvent is evaporated. The residue is partitioned between ethyl acetate (3 x 250 mL) and 1 M HCl (2 x 200 mL), 0.1 M HCl (100 mL), and brine (200 mL). The combined organic phases are dried over anhydrous MgSO4 and the solvent is evaporated. Purification by flash chromatography (60% ethyl acetate / hexanes) gave 14.2 g of the product as a white foamy solid (84%). 1 H NMR (400MHz, CDCl3) δ0.97~0.99(m, 6H), 2.10(s, 3H), 2.14(s, 3H), 2.18~2.24(m, 1H), 2.72~2.85(m, 2H), 4.37~4.41( m, 2H), 4.48~4.55(m, 1H), 5.35~5.40(m, 2H), 5.86(dd, 1H, J=2, 8Hz), 6.25(d, 1H, J=6Hz), 7.77(d, 1H, J=8Hz), 9.14(br s, 1H), 13C NMR (100MHz, CDCl3) δ20.35, 20.51, 22.35, 22.39, 24.14, 47.45, 64.59, 70.66, 72.3 5, 79.87, 86.30, 103.74, 139.88, 150.51, 160.02, 162.77, 169.43, 169.73, 193.08.

[0016] Example 2: Protective Effects of Oral Uridine Triacetate and Ketroecine in a Mouse Model of Mitochondrial Dysfunction The combination of uridine triacetate and ketoleucine was evaluated in a model of progressive and lethal dysfunction of mitochondrial oxidative phosphorylation. 3-nitropropionic acid (3-NP), an irreversible inhibitor of complex II of the mitochondrial electron transport system. Daily administration of 3-NP leads to a progressive decrease in mitochondrial energy reserve capacity until a threshold for baseline energy requirements for survival is breached, resulting in death from both cardiac failure and central nervous system dysfunction.

[0017] In this study, uridine triacetate was administered either by gavage as an oral bolus or by incorporation into rodent chow at a concentration of 4% w / w. Similarly, ketoleucine (in the form of its sodium salt, NaKL) was administered in different groups by gavage or by dissolving it in drinking water. Sodium chloride (NaCl) was used as a control substance in some groups to account for the sodium load from ketoleucine salt. Chemicals: Hydroxypropyl methylcellulose (HPMC), Catalog number H3785, Lot number SLBS5701, CAS9004-65-3, SIGMA-Aldrich. Uridine triacetate (UTA) for gavage, item, D000156, lot number Q000001095, Almac Sciences; Uridine triacetate (UTA) in diet, item and lot number, D000302, 2148-070, project number 1314A0071A, R-104 lot Q000003578, recovery of materials from Almac Sciences; Sodium chloride (NaCl), VWR, catalog number BDH9286-500G, lot number 1976C500, CAS 7647-14-5; 4-Methyl-2-oxovaleric acid sodium salt (Ketoleucine: NaKL), catalog number M326425, lot number 1-AWT-36-1, CAS 4502-00-5, Toronto Research Chemicals, and 3-nitropropionic acid (3NP), catalog number N5636, lot number 11101616, CAS504-88-1, SIGMA-Aldrich. Vehicle: UTA, NaKL, or NaCl in suspension was prepared in 0.75% HPMC. NaCL or NaKL in drinking water was prepared by dissolving in water. 3-NP solution was prepared in water. Dosage Formulation: Preparation: UTA was suspended in 0.75% HPMC at 50 mg / ml. NaKL was suspended in 0.75% HPMC at 100 mg / ml or included in the drinking water at 15.8 mg / ml. Groups not receiving NaKL received an amount of NaCL equimolar to the sodium contributed by NaKL, either by gavage (38.4 mg / ml) or in the drinking water (6.1 mg / ml). NaKL in the drinking water was filter sterilized after preparation. Drinking water containing NaKL was changed every 4 days. 3NP was weighed, dissolved in water, neutralized to pH 7 with 1N NaOH, and sterile filtered to a final concentration of 6.5 mg / ml. Dosing: Some groups of mice were gavaged with a UTA suspension containing 50 mg / ml UTA (0.02 ml / g bw, dose: 1,000 mg / Kg), or a similar UTA suspension also containing 100 mg / ml NaKL (0.02 ml / g bw, dose: 2,000 mg / Kg), or 38.42 mg / ml NaCl (0.02 ml / g bw, dose: 768 mg / Kg). Some mice will have NaKL (15.6 mg / ml, 104 mM) or an equimolar amount of NaCL (6.1 mg / ml) in their drinking water. Mice were injected ip with 6.5 mg / ml of 3-NP for a dose of 65 mg / Kg (0.01 ml / g bw). Animals: Female CD-1 mice. [Table 1] General Experimental Design: For the experiment, 50 mice were divided into 5 groups of 10 animals each: Group 1, Group 2, Group 3, Group 4, and Group 5. Group 1 was fed a diet of 4% UTA and had access to water containing an equimolar amount of NaCl (104 mM, 6.1 mg NaCl / ml) to that contributed by NaKL in the drinking water of the other groups. Mice were injected intraperitoneally with 6.5 mg / ml of 3-NP (0.01 ml / g bw) at 5 PM. Group 2 was fed a diet of 4% UTA and had access to water containing 15.6 mg NaKL / ml, 104 mM NaKL.) Mice were injected intraperitoneally with 6.5 mg / ml 3-NP (0.01 ml / g bw) at PM5. Group 3 was gavaged with 1000 mg / Kg UTA and 768 mg / Kg NaCl at 7 AM and 4 PM, and mice were injected intraperitoneally with 6.5 mg / ml 3-NP (0.01 ml / g bw) at 5 PM. Group 4 was gavaged with 1000mg / Kg UTA+2000mg / Kg NaKL at 7AM and 4PM and injected intraperitoneally with 6.5mg / ml 3-NP (0.01ml / g bw) at 5PM. Group 5 was gavaged with 1000 mg / Kg UTA at 7 AM and 4 PM and had access to water containing 15.6 mg / ml NaKL. Mice were injected intraperitoneally with 6.5 mg / ml 3-NP (0.01 ml / g bw) at 5 PM. Mice in groups 1 and 2 began the 4% UTA diet on Monday 5-6-19. Mice in groups 1, 2, and 5 began their respective drinking water treatments on Monday 5-6-19. This was to avoid the opportunity for the mice to associate any side effects of the 3NP injections with the change in drinking water or diet, thereby creating an aversion to necessary nutrition and hydration. Mice 3, 4, and 5 began gavage treatments on Wednesday 5-8-19 at 7 AM and 4 PM. All mice began 3NP treatments at 5 PM on Wednesday 5-8-19. Body weights were assessed when the mice were organized into groups on Monday 5-6 before being placed on food and drinking water treatments. Body weights were assessed again on Tuesday 5-7. Weight loss and mortality were assessed daily mornings beginning on Wednesday 5-8 before treatment began, and treatment was scheduled to continue for at least 14 days depending on the occurrence and extent of mortality. [Table 2]

[0018] The combination of uridine triacetate and NaKL, where both drugs were given by gavage, was significantly more effective in preventing mortality than either drug alone, given by gavage or in chow, and more effective than both uridine triacetate and NaKL given together in chow and drinking water, respectively. This may be due to the greater effectiveness of the higher peak concentrations of plasma uridine and ketroleucine obtained after oral bolus dosing versus the smaller but more prolonged rise in total body uridine and NaKL (and its metabolites) after administration in chow or drinking water. Uridine triacetate given by oral gavage and NaKL in drinking water (Group 5) was more effective against mortality than the other groups, except for oral gavage bolus administration of uridine triacetate and NaKL (Group 3).

[0019] Bolus (gavage) uridine triacetate with or without NaKL (Groups 3-5) provided better protection against weight loss than 4% (w / w) uridine triacetate in rodent chow (Groups 1 and 2) despite the larger total daily uridine triacetate dose (approximately 8000 mg / kg / day) in animals that received it in their chow. Bolus UTA ​​produced higher peak plasma uridine concentrations than UTA in the chow, indicating that protective mechanisms are better activated by periodic high-peak uridine versus constant lower-level systemic exposure. [Table 3]

Claims

1. The compound, 5'-O-ketroeusyl-2',3'-di-O-acetyluridine.

2. 10. A compound according to claim 1 for use in the treatment of a disorder characterized by muscle weakness or loss of lean muscle mass in a mammalian subject.

3. The compound of claim 2, wherein the disorder is selected from the group consisting of muscle wasting states, dynapenia, muscle disuse atrophy, and muscle wasting and exercise tolerance associated with chronic kidney disease, chronic obstructive pulmonary disease, and chronic heart failure.

4. The compound of claim 3, wherein the muscle wasting condition is selected from the group consisting of sarcopenia and cachexia.

5. The compound according to any one of claims 2 to 4, wherein the mammalian subject is a human subject.

6. A pharmaceutical composition comprising a therapeutically effective amount of an orally bioavailable uridine prodrug and a ketroucine compound.

7. The pharmaceutical composition of claim 6 , wherein the uridine prodrug is uridine triacetate.

8. 7. The pharmaceutical composition of claim 6, wherein the ketroucine compound is selected from the group consisting of 5'-O-ketroucyl-2',3'-di-O-acetyluridine, keutleucine, and therapeutically effective salts thereof.

9. A pharmaceutical composition according to any one of claims 6 to 8 for use in the treatment of a disorder characterised by muscle weakness or loss of lean muscle mass in a mammalian subject.

10. The pharmaceutical composition of claim 9, wherein the mammalian subject is a human subject.

11. 10. The pharmaceutical composition of claim 9, wherein the disorder is selected from the group consisting of muscle wasting states, dynapenia, muscle disuse atrophy, and muscle wasting and exercise tolerance associated with chronic kidney disease, chronic obstructive pulmonary disease, and chronic heart failure.

12. The pharmaceutical composition of claim 11, wherein the muscle wasting condition is selected from the group consisting of sarcopenia and cachexia.

13. The pharmaceutical composition of claim 11 or 12, wherein the mammalian subject is a human subject.

14. A method for producing 5'-O-ketroucyl-2',3'-di-O-acetyluridine, comprising the steps of: (i) mixing uridine with imidazole and tert-butyl(chloro)dimethylsilane to produce 5'-O-(tert-butyldimethylsilyl)uridine; (ii) mixing 5'-O-(tert-butyldimethylsilyl)uridine with 4-dimethylaminopyridine (DMAP) and acetic anhydride to produce 5'-O-(tert-butyldimethylsilyl)uridine-2',3'-di-O-acetyluridine; (iii) mixing 5'-O-(tert-butyldimethylsilyl)uridine-2',3'-di-O-acetyluridine with para-toluenesulfonic acid in a state to produce 2',3'-di-O-acetyluridine; (iv) mixing dichloromethyl methyl ether and α-ketoisocaproic acid in a manner to form a first mixture; and (v) adding the first mixture to a second mixture comprising the 2',3'-di-O-acetyluridine and pyridine under conditions to produce 5'-O-ketroeucyl-2',3'-di-O-acetyluridine; method.

15. The method of claim 14, wherein in step (i), prior to the addition of the imidazole and tert-butyl(chloro)dimethylsilane, the uridine is dissolved in dimethylformamide (DMF), and optionally further comprising, in step (i), mixing the uridine, imidazole, and tert-butyl(chloro)dimethylsilane at 0° C. for 4 hours and then at room temperature for 17 hours.

16. The method of claim 14 or 15, wherein in step (ii), prior to the addition of the DMAP and acetic anhydride, the 5'-O-(tert-butyldimethylsilyl)uridine is dissolved in dichloromethane (DCM), and optionally further, in step (ii), the 5'-O-(tert-butyldimethylsilyl)uridine, DMAP, and acetic anhydride are mixed at room temperature for 4 hours.

17. The method of claim 14, wherein in step (iii), prior to the addition of para-toluenesulfonic acid, the 5'-O-(tert-butyldimethylsilyl)uridine-2',3'-di-O-acetyluridine is dissolved in methanol (MeOH) and DCM, and optionally further, in step (iii), the 5'-O-(tert-butyldimethylsilyl)uridine-2',3'-di-O-acetyluridine and para-toluenesulfonic acid are mixed at 0°C for 3 hours and then at room temperature overnight.

18. The method of claim 14, wherein in step (iv), the first mixture is heated at 48-52° C. for 30 minutes and then cooled in an ice bath.

19. The method of any one of claims 14 to 18, wherein in step (v), the 2',3'-di-O-acetyluridine and pyridine are dissolved together in ice-bath cooled DCM to form the second mixture, and prior to addition of the first mixture in (iv), and optionally further, in step (v), the first mixture and the second mixture are warmed at room temperature overnight.

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