Formulations

Abstract

There is provided an alkali metal salt of 4-chloro-N-[2-[(4-chlorophenyl)methyl]-3-oxo-1,2,4-thiadiazol-5-yl]benzamide and formulations thereof. This salt finds particular utility in the treatment or prevention of a disorder or condition ameliorated by the activation of AMPK.

Claims

1. An alkali metal salt of 4-chloro-N-[2-[(4-chlorophenyl)methyl]-3-oxo-1,2,4-thiadiazol-5-yl]benzamide.

2. The alkali metal salt of claim 1 wherein the alkali metal is sodium.

3. The alkali metal salt of claim 1 wherein the alkali metal salt is potassium.

4. A pharmaceutical formulation comprising the alkali metal salt of claim 1.

5. The pharmaceutical formulation according to claim 4, wherein said alkali metal salt has been milled.

6. The pharmaceutical formulation according to claim 4, wherein the formulation comprises particles containing the alkali metal salt, said particles having a particle size distribution defined by a D90 of less than 10 μm.

7. The pharmaceutical formulation according to claim 6, wherein the particles have a particle size distribution defined by a D90 of less than 9 μm; a D50 of less than 6 μm; a D50 of less than 5 μm; a D10 of less than 2 μm; or a D10 of less than 1.5 μm.

8. The pharmaceutical formulation according to claim 4, wherein the formulation further comprises an enteric coating.

9. The pharmaceutical formulation according to claim 8, wherein the enteric coating comprises beeswax, shellac, ethylcellulose polymers, carboxymethylethylcellulose, hydroxypropyl methylcellulose phthalate, acrylic acid and methacrylic acid copolymers, methacrylic acid copolymers, methyl methacrylate copolymers, ethoxyethyl methacrylates, cyanoethyl methacrylate, methyl methacrylate, copolymers, methacrylic acid copolymers, methyl methacrylate copolymers, methyl methacrylate copolymers, methacrylate copolymers, methacrylic acid copolymer, aminoalkyl methacrylate copolymer, methacrylic acid copolymers, methyl methacrylate copolymers, poly(acrylic acid), poly(methacrylic acid, methacrylic acid alkylamide copolymer, poly(methyl methacrylate), poly(methacrylic acid) (anhydride), methyl methacrylate, polymethacrylate, methyl methacrylate copolymer, poly(methyl methacrylate), poly(methyl methacrylate) copolymer, polyacrylamide, aminoalkyl methacrylate copolymer, poly(methacrylic acid anhydride), glycidyl methacrylate copolymers), cellulose acetate phthalate, or polyvinyl acetate phthalate.

10. The pharmaceutical formulation according to claim 4, wherein the formulation further comprises at least one pharmaceutically acceptable excipient.

11. The pharmaceutical formulation according to claim 4, wherein the formulation further comprises a lubricant, a binder, a filler, a surfactant, a diluent, an anti-adherent, a coating, a flavouring, a colourant, a glidant, a preservative, a sweetener, a disintegrant, an adsorbent, a buffering agent, an antioxidant, a chelating agent, a dissolution enhancer, a dissolution retardant, or a wetting agent, or any combination thereof.

12. The pharmaceutical formulation according to claim 4, wherein the formulation further comprises PVP K30, Na-docusate and mannitol.

13. The pharmaceutical formulation according to claim 4, wherein the formulation is provided in the form of a capsule or a tablet.

14. The pharmaceutical formulation according to claim 13, wherein an enteric coating is present on said capsule or tablet.

15. The pharmaceutical formulation according to claim 13, wherein the capsule or tablet contains particles comprising the alkali metal salt, and wherein each particle is coated with an enteric coating.

16. A pharmaceutical formulation according to claim 4, wherein the alkali metal salt has been milled so that particles containing the alkali metal salt have a particle size distribution defined by a D90 of less than 10 μm, and wherein the formulation further comprises an enteric coating.

17. A method of treatment of a disorder or condition ameliorated by the activation of AMPK comprising administering to a subject in need thereof the alkali metal salt of claim 1.

18. The method of treatment according to claim 17, where in the disorder or condition ameliorated by the activation of AMPK is type 2 diabetes.

19. The method of treatment according to claim 17, where in the disorder or condition ameliorated by the activation of AMPK is a condition associated with hyperinsulinemia selected from the group consisting of obesity and cardiovascular disease.

20. The method of treatment according to claim 17, where in the disorder or condition ameliorated by the activation of AMPK is cancer.

21. A process for preparing a pharmaceutical formulation of claim 6, wherein the process comprises milling the alkali metal salt to produce the particles having a particle size distribution defined by a D90 of less than 10 μm.

22. The process according to claim 21, wherein the process comprises dry milling.

23. The process according to claim 21, wherein the alkali metal salt is mixed with one or more pharmaceutically acceptable excipients after milling of the alkali metal salt.

24. The process according to claim 21, wherein the pharmaceutical formulation is provided in the form of a capsule or tablet and the process further comprises the step of coating said capsule or tablet with an enteric coating after the milled alkali metal salt is incorporated into said capsule or tablet.

25. The process according to claim 21, wherein the pharmaceutical formulation is provided in the form of a capsule or tablet and the process further comprises the step of applying an enteric coating to the milled alkali metal salt prior to incorporating said milled alkali metal salt into the capsule or tablet.

26. A pharmaceutical formulation comprising the alkali metal salt of claim 2; and at least one pharmaceutically acceptable excipient.

27. A pharmaceutical formulation comprising the alkali metal salt of claim 3; and at least one pharmaceutically acceptable excipient.

28. The pharmaceutical formulation according to claim 26, wherein said alkali metal salt has been milled.

29. The pharmaceutical formulation according to claim 26, wherein the formulation comprises particles containing the alkali metal salt, said particles having a particle size distribution defined by a D90 of less than 10 μm.

30. The pharmaceutical formulation according to claim 29, wherein the particles have a particle size distribution defined by a D90 of less than 9μm; a D50 of less than 6 μm; a D50 of less than 5 μm; a D10 of less than 2 μm; or a D10 of less than 1.5 μm.

31. The pharmaceutical formulation according to claim 26, wherein the formulation further comprises an enteric coating.

32. The pharmaceutical formulation according to claim 31, wherein the enteric coating comprises beeswax, shellac, ethylcellulose polymers, carboxymethylethylcellulose, hydroxypropyl methylcellulose phthalate, acrylic acid and methacrylic acid copolymers, methacrylic acid copolymers, methyl methacrylate copolymers, ethoxyethyl methacrylates, cyanoethyl methacrylate, methyl methacrylate, copolymers, methacrylic acid copolymers, methyl methacrylate copolymers, methyl methacrylate copolymers, methacrylate copolymers, methacrylic acid copolymer, aminoalkyl methacrylate copolymer, methacrylic acid copolymers, methyl methacrylate copolymers, poly(acrylic acid), poly(methacrylic acid, methacrylic acid alkylamide copolymer, poly(methyl methacrylate), poly(methacrylic acid) (anhydride), methyl methacrylate, polymethacrylate, methyl methacrylate copolymer, poly(methyl methacrylate), poly(methyl methacrylate) copolymer, polyacrylamide, aminoalkyl methacrylate copolymer, poly(methacrylic acid anhydride), glycidyl methacrylate copolymers), cellulose acetate phthalate, or polyvinyl acetate phthalate.

33. The pharmaceutical formulation according to claim 26, wherein the formulation further comprises a lubricant, a binder, a filler, a surfactant, a diluent, an anti-adherent, a coating, a flavouring, a colourant, a glidant, a preservative, a sweetener, a disintegrant, an adsorbent, a buffering agent, an antioxidant, a chelating agent, a dissolution enhancer, a dissolution retardant, or a wetting agent, or any combination thereof.

34. The pharmaceutical formulation according to claim 26, wherein the formulation further comprises PVP K30, Na-docusate and mannitol.

35. The pharmaceutical formulation according to claim 26, wherein the formulation is provided in the form of a capsule or a tablet.

36. The pharmaceutical formulation according to claim 35, wherein an enteric coating is present on said capsule or tablet.

37. The pharmaceutical formulation according to claim 35, wherein the capsule or tablet contains particles comprising the alkali metal salt, and wherein each particle is coated with an enteric coating.

38. A pharmaceutical formulation according to claim 26, wherein the alkali metal salt has been milled so that particles containing the alkali metal salt have a particle size distribution defined by a D90 of less than 10 μm, and wherein the formulation further comprises an enteric coating.

39. A method of treatment of a disorder or condition ameliorated by the activation of AMPK comprising administering to a subject in need thereof the alkali metal salt of claim 2.

40. The method of treatment according to claim 39, where in the disorder or condition ameliorated by the activation of AMPK is type 2 diabetes.

41. The method of treatment according to claim 39, where in the disorder or condition ameliorated by the activation of AMPK is a condition associated with hyperinsulinemia selected from the group consisting of obesity and cardiovascular disease.

42. The method of treatment according to claim 39, where in the disorder or condition ameliorated by the activation of AMPK is cancer.

43. A process for preparing a pharmaceutical formulation of claim 29, wherein the process comprises milling the alkali metal salt to produce the particles having a particle size distribution defined by a D90 of less than 10 μm.

44. The process according to claim 43, wherein the process comprises dry milling.

45. The process according to claim 43, wherein the alkali metal salt is mixed with one or more pharmaceutically acceptable excipients after milling of the alkali metal salt.

46. The process according to claim 43, wherein the pharmaceutical formulation is provided in the form of a capsule or tablet and the process further comprises the step of coating said capsule or tablet with an enteric coating after the milled alkali metal salt is incorporated into said capsule or tablet.

47. The process according to claim 43, wherein the pharmaceutical formulation is provided in the form of a capsule or tablet and the process further comprises the step of applying an enteric coating to the milled alkali metal salt prior to incorporating said milled alkali metal salt into the capsule or tablet.

Description

FIGURES

(1) The following drawings are provided to illustrate various aspects of the present inventive concept and are not intended to limit the scope of the present invention unless specified herein.

(2) FIG. 1 shows comparative results of oral pharmacokinetic studies using 4-chloro-N-[2-[(4-chlorophenyl)methyl]-3-oxo-1,2,4-thiadiazol-5-yl]benzamide (Compound 1) in a suspension and in non-coated capsules, and 4-chloro-N-[2-[(4-chlorophenyl)methyl]-3-oxo-1,2,4-thiadiazol-5-yl]benzamide sodium salt (Compound 2) and 4-chloro-N-[2-[(4-chlorophenyl)methyl]-3-oxo-1,2,4-thiadiazol-5-yl]benzamide potassium salt (Compound 3) separately in non-coated capsules.

(3) FIG. 2 shows comparative results of oral pharmacokinetic studies using Compound 1 in a suspension and in enteric coated capsules, and Compound 2 and Compound 3 separately in enteric coated capsules.

(4) FIGS. 3 and 4 show absolute and relative C.sub.max results for formulations of Compound 1 as a suspension and in non-coated capsules, and Compound 2 and Compound 3 separately in non-coated capsules.

(5) FIGS. 5 and 6 show absolute and relative AUC results for formulations of Compound 1 as a suspension and in non-coated capsules, and Compound 2 and Compound 3 separately in non-coated capsules.

(6) FIGS. 7 and 8 show absolute and relative C.sub.max results for formulations of Compound 1 as a suspension and in enteric coated capsules, and Compound 2 and Compound 3 separately in enteric coated capsules.

(7) FIGS. 9 and 10 show absolute and relative AUC results for formulations of Compound 1 as a suspension and in enteric coated capsules, and Compound 2 and Compound 3 separately in enteric coated capsules.

EXAMPLES

Abbreviations

(8) AUC.sub.0-t: Area under the concentration-time curve from time zero to last quantifiable concentration AUC.sub.0-∞: Area under the concentration-time curve from time zero to infinity b.w.: Body weight CE: Collision energy CL: Clearance C.sub.max: Peak plasma concentration. CXP: Collision exit potential DLS: Dynamic light scattering DP: Declustering potential EP Entrance potential h: Hours HPLC: High performance liquid chromatography ISD: Internal standard K.sub.2EDTA Dipotassium ethylene diamine tetra acetic acid LC: Liquid chromatography LC-MS/MS: Liquid chromatography—(tandem) mass spectrometry LS: Light scattering MRT: Mean residence time. min: Minutes PVP K30: Polyvinylpyrrolidone K 30 rpm: Revolutions per minute RT: Room temperature Tv.sub.1/2: Half-life T.sub.max: Time to reach the peak plasma concentration v/v: Volume by volume w/v: Weight per volume

(9) The present invention will be further described by reference to the following examples, which are not intended to limit the scope of the invention.

(10) Materials

(11) 4-Chloro-N-[2-[(4-chlorophenyl)methyl]-3-oxo-1,2,4-thiadiazol-5-yl]benzamide (compound 1), 4-chloro-N-[2-[(4-chlorophenyl)methyl]-3-oxo-1,2,4-thiadiazol-5-yl]benzamide sodium salt (compound 2) and 4-chloro-N-[2-[(4-chlorophenyl)methyl]-3-oxo-1,2,4-thiadiazol-5-yl]benzamide potassium salt (compound 3) were prepared by Anthem Biosciences.

(12) Sodium-docusate, PVP K30 and mannitol were supplied by Sigma-Aldrich.

Preparation 1—4-chloro-N-[2-[(4-chlorophenyl)methyl]-3-oxo-1,2,4-thiadiazol-5-yl]benzamide Sodium Salt (Compound 2)

(13) ##STR00005##

(14) Procedure:

(15) To a suspension of 4-chloro-N-[2-[(4-chlorophenyl)methyl]-3-oxo-1,2,4-thiadiazol-5-yl]benzamide (100 g, 0.2629 mol) in isopropanol (1.0 L) was added slowly a solution of sodium hydroxide (11.56 g, 0.2891 mol) in water (100 mL) at 25±5° C. The mass was stirred for 3 h at 25±5° C. and cooled to 5±5° C. The mass was stirred for 3 h at 5±5° C. and filtered to collect the solids. The solids were washed with isopropanol (300 mL) and dried for 8 h under reduced pressure at 35±5° C. The dried solids were micronized twice using an air jet mill with 4.0 kg/cm.sup.2 of primary pressure, 7.0 kg/cm.sup.2 of secondary pressure and screw feeder with 8 RPM to isolate the desired sodium salt as white solid (50 g, 48%).

Preparation 2—4-chloro-N-[2-(4-chlorophenyl)methyl-3-oxo-1,2,4-thiadiazol-5-yl]benzamide Potassium Salt (Compound 3)

(16) ##STR00006##

(17) Procedure:

(18) To a suspension of 4-chloro-N-[2-[(4-chlorophenyl)methyl]-3-oxo-1,2,4-thiadiazol-5-yl]benzamide (100 g, 0.2629 mol) in isopropanol (1.0 L) was added slowly a solution of potassium hydroxide (16.22 g, 0.2891 mol) in water (100 mL) at 25±5° C. The mass was stirred for 3 h at 25±5° C. and cooled to 5±5° C. The mass was stirred for 3 h at 5±5° C. and filtered to collect the solids. The solids were washed with isopropanol (300 mL) and dried for 8 h under reduced pressure at 35±5° C. The dried solids were micronized twice using an air jet mill with 4.0 kg/cm.sup.2 of primary pressure, 7.0 kg/cm.sup.2 of secondary pressure and screw feeder with 8 RPM to isolate the desired potassium salt as white solid (55 g, 50%).

Example 1—Milled Products

(19) Milling was performed for Examples 1a to 1c using an air jet mill (Equipment code: CP-AJM-01; Promas engineers) with the following parameters:

(20) TABLE-US-00001 Parameter Value Primary pressure (argon/nitrogen) 4.0 kg/cm.sup.2 Secondary pressure (argon/nitrogen) 7.0 kg/cm.sup.2 Screw feeder RPM 8

(21) Micronisation was repeated in all examples by performing the air jet milling for a second time using the same parameters. This allowed the particle size D90 to be reduced to less than 10 μm.

(22) Particle Size Analysis

(23) Reagents

(24) TABLE-US-00002 Name Grade Water HPLC Tween 20 Laboratory Reagent

(25) Instrument

(26) TABLE-US-00003 Balance Analytical balance Particle size Analyzer Shimadzu SALD-2300

(27) Method Parameter

(28) TABLE-US-00004 Dispersion method Flow Cell measurement technique with sampler Refractive Index 3.00-0.20i (nearly equal to Fraunhofer) Dispersion Agent Tween 20 Pump Speed 5.0 Time of sonication 10 Blank 4 drops of Tween 20 in 25 mL water

(29) Preparations

(30) Four drops of Tween 20 were added to 25 mL of water and the mixture was sonicated for 3 minutes to form the dispersant solution.

(31) 0.05 g of milled material was transferred into a 250 mL glass beaker. 25 mL of the dispersant solution (Tween 20/water mixture) was added to the beaker with continuous swirling for 2 to 3 minutes. The suspension was transferred into the measuring unit and the particle size distribution measurements were conducted in triplicate.

(32) TABLE-US-00005 TABLE 1 Average particle size results for dry-milled products Compound Example Particle size distribution (μm) No. No. Milling D10 D50 D90 D100 1 Example 1a Before 13.5 28.0 55.2 187.0 milling Example 1b After 1.sup.st 1.8 5.0 11.4 33.7 milling Example 1c After 2.sup.nd 1.2 3.7 8.3 21.4 milling 2 Example 1d Before 1.11 7.62 31.7 185 milling Example 1e After 1.sup.st 0.508 3.58 11.5 31.1 milling Example 1f After 2.sup.nd 0.414 2.70 8.29 24.0 milling 3 Example 1g Before 0.876 13.4 205 665 milling Example 1h After 1.sup.st 0.382 3.64 26.2 584 milling Example 1i After 2.sup.nd 0.289 1.57 11.7 35.3 milling

Examples 2 to 8—Single Dose Oral Pharmacokinetic Studies in Rabbits

(33) The studies detailed below (and referred to as Examples 2 to 8) were conducted to provide comparative single dose oral pharmacokinetics data for Compound 1, as well as sodium and potassium salts of that compound, using non-coated and enteric-coated capsules in male New Zealand White rabbits,

(34) Formulation Preparation

(35) 1. Capsules

(36) Ready to use enteric coated and non-coated capsules were used. Capsules were obtained from CapsulCN International Co., Ltd.

(37) Compound 1 was made using the process described in WO 2011/004162 and milled as described for Example 1c. The non-coated or enteric coated gelatin capsules were individually filled with 180 mg of dry-milled Compound 1 together with accompanying excipients as indicated. Both capsule types were filled with Compound 1 together with 1.8 mg of sodium docusate, 0.18 mg of PVP K30 and 9 mg of mannitol.

(38) Similarly, Compound 2, non-coated or enteric coated gelatin capsules were individually filled with 180 mg of dry-milled Compound 2 (as obtained in Example 1f) together with 1.8 mg of sodium docusate, 0.18 mg of PVP K30 and 9 mg of mannitol.

(39) For Compound 3, non-coated or enteric coated gelatin capsules were individually filled with 90 mg of dry-milled Compound 3 (as obtained in Example 1i) together with 0.90 mg of sodium docusate, 0.09 mg of PVP K30 and 4.50 mg of mannitol.

(40) Once prepared, the capsules were stored in a desiccator at between 19 and 25° C. prior to administration to the animals.

(41) Details of the formulations are summarized in Table 2.

(42) 2. Suspensions

(43) Compound 1 was made using the process described in WO 2011/004162.

(44) A 2% w/v methylcellulose solution in 4 mM phosphate buffer pH 7.4 was prepared.

(45) 40 mL of the 2% w/v methyl cellulose solution was added to a 250 mL conical flask together with 10 g of 2 mm glass beads, and vigorously stirred. Compound 1 (720 mg) was slowly added to the solution and the mixture was with continuously stirred for 1 h. The so homogenate was transferred to separate flask and its pH recorded. The suspension formulation was prepared before administration to the animals.

(46) Animal Husbandry

(47) Rabbits (New Zealand white; male) were housed under standard laboratory conditions, in environmentally monitored air-conditioned room with adequate fresh air supply (10-15 air changes per hour), room temperature (22±3° C.) and relative humidity 30 to 70%, with 12-hour light and 12-hour dark cycle. The temperature and relative humidity were recorded once daily.

(48) Each animal was housed in a standard stainless steel rabbit cage SS-304 (Size: L 24″×B 18″×H 18″) with stainless steel mesh and removable bottom tray for refuse disposal, food hopper for holding pelleted food, holder for drinking water bottle and siphon tube and label holder. Clean, sterilized corncob was provided as bedding material.

(49) The animals were fed ad libitum throughout the acclimatization and experimental periods, with Krishna Valley Agrotech rabbit feed.

(50) Water was provided ad libitum throughout the acclimatization and experimental periods Water from an Aqua guard water filter cum purifier was autoclaved and provided in polypropylene water bottles with stainless steel sipper tubes.

(51) Acclimatization

(52) The animals were acclimatized for a minimum period of 1 weeks (7 days) to facility room conditions and observed for clinical signs daily. Veterinary examination of all the animals were performed on the day of receipt, daily and on the day of randomization.

(53) Grouping

(54) Animal grouping was done by the method of body weight stratification and randomization. The animals selected for the study were weighed and grouped in to body weight ranges. These body weight stratified rabbits were distributed to all the study groups in equal numbers if possible, such that body weight variation of animals used does not exceed ±20% of the mean body weight. The grouping was done one day prior to the initiation of treatment.

(55) Study Design

(56) TABLE-US-00006 TABLE 2 Study Design No of animals Compound Dose Dose Concentration Example per group no. Vehicle (mg/kg b.w.) Volume of compound 2 3 1 Non-coated 90 1 capsule 180 mg capsules per animal per capsule 3 3 2 Non-coated 90 1 capsule 180 mg capsules per animal per capsule 4 3 3 Non-coated 90 2 capsules 90 mg capsules per animal per capsule 5 3 1 Enteric-coated 90 1 capsule 180 mg capsules per animal per capsule 6 3 2 Enteric-coated 90 1 capsule 180 mg capsules per animal par capsule 7 3 3 Enteric-coated 90 2 capsules 90 mg capsules per animal per capsule 8 3 1 Standard 90 5 mL/kg 18 mg/mL suspension b.w.

(57) Dose Administration

(58) Adult healthy male New Zealand white rabbits aged approximately 2 to 3 months old were used for experimentation after 7 days of acclimatization.

(59) 1. Capsules

(60) To dose the filled capsules, a soft plastic dosing tube was used. The filled capsule was inserted into the dosing tube so that the short end of the capsule protrudes slightly from the tip of the tube. The tip of the capsule was dipped in mineral oil to aid swallowing.

(61) The head was grasped firmly with one hand about the maxilla. The dosing tube containing the capsule was inserted behind the incisors. The dosing tube was slid straight into the back of the mouth. The capsule was ejected by pushing the plunger on the dosing tube. The dosing tube was removed, and the rabbit's mouth was closed. The neck was stroked gently to facilitate swallowing.

(62) 2. Suspension

(63) To dose the suspension, an infant feeding tube was used. The feeding tube was inserted through the mouth of the rabbit to the oesophagus and the stomach, and ascertained that it has not been placed in the trachea before dosing to the animals. The suspension of the Compound 1 was administered through the feeding tube. After the administration of the suspension, drinking water of approximately 2.0-2.5 mL was administered to flush out the contents in the feeding tube.

(64) Blood Sampling

(65) The animals were restrained in a rabbit restrainer and blood samples (400-500 μL/time point) were collected via the central ear artery at 0.16, 0.25, 0.50, 1.0, 2.0, 4.0, 6.0, 8.0, 24.0, 48.0 and 72 hours post-dosing. Collected blood specimens were centrifuged at 4000 or 6000 rpm, 4° C. for 10 minutes and plasma samples were separated and stored at ˜80° C. until analysis.

(66) Bioanalysis

(67) Concentrations of the analyte of Compound 1 in New Zealand white rabbits was determined using an API 3200 Q-trap LC-MS/MS system.

(68) Method Summary

(69) Chromatographic separation was achieved on Zorbax G18, 50×4.6 mm, 5 μm column with methanol-0.1% formic acid as mobile phase with gradient elution. The flow rate was set at 1.0 mL min.sup.−1. Detection was accomplished by a triple-quadrupole tandem mass f spectrometer in multiple-reaction monitoring (MRM) scanning via electrospray ionization source, applied in the positive mode. The optimised mass transition ion-pairs for quantitation were m/z 379.999.fwdarw.125.000 for the compound 1 and m/z 376.165.fwdarw.165.00 for the ISD (Haloperidol). Calibration plots were linear over the range of 11.062 to 20594.820 ng/mL.

(70) Buffer (0.1% Formic Acid)

(71) About 1.0 mL of formic acid was added to 999 mL of ultrapure water type-1 to make the buffer. This solution was stored at room temperature and used within two days from the date of preparation.

(72) Dilution Solvent (Methanol:Water. 80:20% v/v)

(73) Exactly 800 mL of methanol and 200 mL of ultrapure water type-1 were added to a reagent bottle, mixed well, and sonicated. This solution was stored at room temperature and used within seven days from the date of preparation.

(74) Rinsing Solvent (Methanol:Water, 50:50% v/v)

(75) Exactly 500 mL of methanol and 500 mL of ultrapure water type-1 were added to a reagent bottle, mixed well, and sonicated. This solution was stored at room temperature and used within three days from the date of preparation.

Preparation of Compound 1 Stock Solution

(76) 3.044 mg of compound 1 was weighed, transferred into a 5.0 mL volumetric flask and dissolved in 5 mL of methanol to obtain a 605.756 μg/mL stock solution. The final concentration of compound 1 was corrected according to the potency of the standard and the actual amount weighed.

(77) Internal Standard Stock Solution

(78) About 2.00 mg of Haloperidol (Sigma-Aldrich) was weighed, transferred into a 5.0 mL volumetric flask and dissolved in methanol to obtain a 400 μg/mL internal standard stock solution. The final concentration of Haloperidol was corrected according to the potency of the standard.

Preparation of Internal Standard (Haloperidol) Working Solution

(79) About 0.250 mL of the internal standard stock solution was diluted to 10 mL using a dilution solvent (methanol/water, 80:20) to obtain about approximately 10 μg/mL solution.

Preparation of Calibration Curve for Compound 1 in Plasma

(80) Calibration curve standards were prepared in a range between 11.062-20594.820 ng/mL (Prepared concentrations: 11.062, 20.112, 40.224, 80.449, 160.897, 321.794, 643.588, 1287.176, 2574.353, 5148.705, 10297.410 and 20594.820 ng/mL) in plasma by spiking blank plasma with aqueous analyte standards.

(81) Quality control samples LQC, MQC and HOC falling in the calibration curve range were prepared for Compound 1 (prepared concentrations: 40.224, 5148.705 and 10297.410 ng/mL) in plasma by spiking blank plasma with suitable aqueous analyte standards.

(82) Liquid-Liquid extraction method for plasma samples 10 μL of ISD working solution (approx. 10 μg/mL) was added to an RIA vial. Exactly 50 μL of rabbit plasma was added from a polypropylene capped tube/vial into the RIA vial and vortexed for 30-40 seconds. 2.5 mL of TBME was added to the RIA vial and then vortexed for 10 minutes at 2000 rpm using a vibramax shaker, The samples were centrifuged at 4000 rpm for 10 minutes at 4° C. 2 mL of organic layer was separated and evaporated to dryness for 20 minutes at 50° C. using a turbo evaporator. The sample residues were reconstituted with 200 μL of reconstitution solvent (methanol). The reconstituted samples were then transferred into auto sampler vials. 10 μL of the reconstituted sample was injected into the AR 3200 Q Trap LC-MS/MS system.

(83) Instrumentation Conditions LC Parameters Column: Zorbax C18, 50×4.6 mm, 5 μm Mobile phase: Methanol (A): 0.1% Formic acid (B) Separation mode: Gradient Mode Flow rate: 1.0 mL/min Injection volume: 10 μL Auto sampler temperature: 10° C. Column oven temperature: 40° C. LC-MS/MS API 3200 QTRAP Source Turbo Ion Spray Polarity Positive Scan type MRM m/z of analyte (Compound 1) [M+1] 379.999/125.000 m/z of internal standard (Haloperidol) 376.165/165.000 [M+1] Ion Spray Voltage (IS) 5500 Temperature (TEM) 500

(84) TABLE-US-00007 Compound name DP CE EP CXP Compound 1 66 25 4.5 16 Haloperidol 21 33 7 20.676

(85) Data Analysis

(86) The data of plasma concentration to respective time points for the analyte Compound 1 were used for the pharmacokinetic analysis. Pharmacokinetic analysis was performed using non-compartmental analysis (NCA) module of Phoenix WinNonlin 6.3 software to in determine the following pharmacokinetic parameters: C.sub.max T.sub.max AUC.sub.0-t AUC.sub.0-∞ T.sub.1/2: MRT

(87) Chromatograms of data acquired using the Analyst software version 1.6.1. were processed by the peak area ratio method. 1/×2 was used as the weighting factor. The concentration of the unknown is calculated from the following equation.
y=mx+c
Where, x=concentration of drug; m=slope of calibration curve; y=peak area ratio; and c=intercept of the calibration curve.

(88) Results

(89) The results for the single dose oral pharmacokinetic study in rabbits are tabulated in Tables 3 to 8 below and are shown graphically in FIGS. 1 to 10.

(90) The results show that there is an increase of between 50 and 100% in the systemic exposure of 4-chloro-N-[2-[(4-chlorophenyl)methyl]-3-oxo-1,2,4-thiadiazol-5-yl]benzamide when it is administered as a dry milled sodium salt (compound 2) compared to free base active ingredient (compound 1)—compare Examples 2 and 3, compare also Example 5 and 6; FIGS. 3 to 10. The dry milled potassium salt (compound 3) gave a more modest improvement compared with the milled free base active ingredient—compare Examples 2 and 4, compare also Examples 5 and 7; FIGS. 3 to 10.

(91) When compared to a standard suspension of the active ingredient (example 8), there was a surprising and substantial (up to six-fold) increase in the systemic exposure of 4-chloro-N-[2-[(4-chlorophenyl)methyl]-3-oxo-1,2,4-thiadiazol-5-yl]benzamide when the active ingredient was administered as a dry milled sodium salt (compound 2) in either non-coated or enteric-coated capsules

(92) Thus, the systemic exposure of 4-chloro-N-[2-[(4-chlorophenyl)methyl]-3-oxo-1,2,4-thiadiazol-5-yl]benzamide is increased by administration of the compound in the form of an alkali metal salt. The greatest improvements in systemic exposure were observed when the drug was administered in the form of the sodium salt.

(93) TABLE-US-00008 TABLE 3 Mean Plasma Pharmacokinetic Parameters For Compound 1 (4-chloro-N-[2-((4-chlorophenyl)methyl]-3- oxo-1,2,4-thiadiazol-5-yl]benzamide) Example 2 5 8 Compound No.  1  1  1 Vehicle Non-coated Enteric coated Suspension capsule capsule Dose (mg/kg b.w.) 90 90 90 C.sub.max (μg/mL) 63.70 ± 3.79  60.8 ± 15.7  31.2 ± 11.5 T.sub.max (h) 32.00 ± 13.86 40.00 ± 13.86 19 ± 9 AUC.sub.last (h*μg/mL)  3448 ± 405.1  2988 ± 665.8  952 ± 175 AUC.sub.inf (h*μg/mL) 7394 ± 3873 18639 ± 12808 .sup. 1123 ± 259.1 AUC.sub.extrap (%) 46.71 ± 19.06 82.44 ± 9.84  14 ± 9 T.sub.1/2 (h) 68.17 ± 43.03 252.2 ± 133.0 36 ± 8 MRT.sub.last (h) 36.86 ± 2.56  45.17 ± 2.54  25 ± 5

(94) TABLE-US-00009 TABLE 4 Plasma Concentration of Compound 1 (4-chloro-N-[2-[(4-chlorophenyl)methyl]-3- oxo-1,2,4-thiadiazol-5-yl]benzamide) Plasma concentration of dry milled compound 1 (ng/mL) Example 2 Example 5 Example 8 Time (h) Mean SD Mean SD Mean SD 0.00 0.000 0.000 0.000 0.000 0 0 0.16 0.000 0.000 0.000 0.000 1211 502 0.25 0.000 0.000 0.000 0.000 4421 3757 0.50 50.474 87.424 0.000 0.000 7250 3732 1.00 386.171 538.817 0.000 0.000 9387 4013 2.00 1530.898 1490.214 5.857 10.144 10099 4801 4.00 13407.634 5140.695 206.003 224.379 11642 3964 6.00 25336.370 5542.720 307.917 452.404 138880 3937 8.00 39160.355 4845.192 1505.092 1239.461 28718 13800 24.00 63648.538 3803.119 45705.076 7965.993 18423 10570 48.00 54789.036 11519.885 59671.800 16461.504 8831 5336 72.00 35657.688 11014.522 52298.238 22101.996 3032 1914 Lower limit of 11.062 ng/mL quantification

(95) TABLE-US-00010 TABLE 5 Mean Plasma Pharmacokinetic Parameters For Dry Milled Compound 2 (4-chloro-N-[2-[(4-chlorophenyl)methyl]-3- oxo-1,2,4-thiadiazol-5-yl]benzamide sodium salt) Example 3 6 Compound No.  2  2 Vehicle Non-coated Enteric- coated capsule capsule Dose (mg/kg b.w.) 90 90 C.sub.max (μg/mL) 98.97 ± 34.56 101.4 ± 17.04 T.sub.max (h) 32.00 ± 13.86 32.00 ± 13.86 AUC.sub.last (h*μg/mL) 5985 ± 2274 5708 ± 1142 AUC.sub.inf (h*μg/mL) 38101 ± 34673 55694 ± 22737 AUC.sub.extrap (%) 79.01 ± 8.87  88.81 ± 3.497 T.sub.1/2 (h) 226.7 ± 139.9 367.6 ± 109.2 MRT.sub.last (h) 38.79 ± 1.20  42.99 ± 1.59 

(96) TABLE-US-00011 TABLE 6 Plasma Concentration of Dry Milled Compound 2 (4-chloro- N-[2-[(4-chlorophenyl)methyl]-3-oxo-1,2,4-thiadiazol- 5-yl]benzamide sodium salt) Plasma concentration of dry milled compound 2 (ng/mL) Example 3 Example 6 Time (h) Mean SD Mean SD 0.00 0.000 0.000 0.000 0.000 0.16 0.000 0.000 0.000 0.000 0.25 51.798 64.697 0.000 0.000 0.50 206.386 140.625 0.000 0.000 1.00 594.725 272.012 9.027 15.636 2.00 1377.183 827.033 13.276 22.995 4.00 15776.855 19265.479 624.216 737.401 6.00 46144.063 18439.160 11866.638 20124.750 8.00 71215.612 27009.076 18413.782 30416.066 24.00 98809.233 34294.286 99879.409 15454.358 48.00 93029.388 38125.274 101178.612 17297.678 72.00 84071.553 36175.531 90966.319 16842.785 Lower limit of 11.062 ng/mL quantification

(97) TABLE-US-00012 TABLE 7 Mean Plasma Pharmacokinetic Parameters For Dry Milled Compound 3 (4-chloro-N-[2-[(4-chlorophenyl)methyl]-3- oxo-1,2,4-thiadiazol-5-yl]benzamide potassium salt) Example 4 7 Compound No.  3  3 Vehicle Non-coated capsule Enteric-coated capsule Dose (mg/kg b.w.) 90 90 C.sub.max (μg/mL) 81.90 ± 10.4  79.47 ± 5.43  T.sub.max (h) 40.00 ± 13.86 48.00 ± 0.00  AUC.sub.last (h*μg/mL)  4526 ± 589.4  3993 ± 43.27 AUC.sub.inf (h*μg/mL) 9370 ± 3377 18614 ± 13840 AUC.sub.extrap (%) 52.23 ± 10.63 69.48 ± 19.80 T.sub.1/2 (h) 71.56 ± 22.63 188.57 ± 158.39 MRT.sub.last (h) 39.563 ± 3.879  41.971 ± 0.696 

(98) TABLE-US-00013 TABLE 8 Plasma Concentration of Dry Milled Compound 3 (4-chloro- N-[2-[(4-chlorophenyl)methyl]-3-oxo-1,2,4-thiadiazol- 5-yl]benzamide potassium salt) Plasma concentration of dry milled compound 3 (ng/mL) Example 4 Example 7 Time (h) Mean SD Mean SD 0.00 0.000 0.000 0.000 0.000 0.16 169.000 264.220 0.000 0.000 0.25 255.336 379.795 0.000 0.000 0.50 457.796 516.389 0.000 0.000 1.00 2349.624 3185.747 4.721 8.177 2.00 4456.537 3014.162 93.881 84.157 4.00 16997.495 5642.942 2558.779 3400.345 6.00 21418.736 5907.019 7906.592 8461.297 8.00 39754.464 15506.499 16147.149 13934.820 24.00 75168.570 5463.233 66084.614 5140.470 48.00 77379.051 17862.078 79465.599 5432.733 72.00 60164.693 23815.560 49785.289 7425.826 Lower limit of 11.062 ng/mL quantification

Examples 9 and 10

(99) The following studies (referred to as Examples 9 and 10) provided comparative single dose oral pharmacokinetics data for Compound 2 (the sodium salt of Compound 1) using enteric-coated capsules in male New Zealand White Rabbits in the presence of a pH modifier (magnesium oxide). The studies were conducted using the methods described above in respect of Examples 2 to 8, except where indicated below.

(100) Enteric coated gelatin capsules were individually filled with the following:

(101) Ex. 9: Compound 2 (62.5 mg), sodium docusate (6.25 mg), PVP K30 (0.625 mg) and magnesium oxide (125 mg).

(102) Ex. 10: Compound 2 (62.5 mg), sodium docusate (6.25 mg), PVP K30 (0.625 mg) and mannitol (125 mg).

(103) Animals received 25 mg/kg b.w. of Compound 2 in a single dose.

(104) Results

(105) The results for the single dose oral pharmacokinetic study in rabbits are tabulated in Table 9 below.

(106) The results show that there is an increase in the systemic exposure of 4-chloro-N-[2-[(4-chlorophenyl)methyl]-3-oxo-1,2,4-thiadiazol-5-yl]benzamide when Compound 2 is formulated together with an alkaline excipient in comparison with formulation in the absence of an alkaline excipient.

(107) TABLE-US-00014 TABLE 9 Mean Plasma Pharmacokinetic Parameters For Compound 2 co-formulated with an alkaline excipient Example 9 10 Compound No.  2  2 Vehicle Enteric coated capsule Enteric coated capsule with MgO with mannitol Dose (mg/kg b.w.) 25 25 C.sub.max (μg/mL) 49.60 ± 4.80 47.43 ± 7.06 T.sub.max (h) 24.00 ± 0.00  32.00 ± 13.86 AUC.sub.last (h*μg/mL) 1703.93 ± 247.60 1472.50 ± 78.72  MRT.sub.last (h) 27.67 ± 0.11 29.98 ± 1.73

Example 11

(108) The following study is a single dose oral pharmacokinetics study for Compound 2 using enteric-coated capsules in male New Zealand White Rabbits in the presence of a pH modifier (magnesium oxide). The study was conducted using the methods described above in respect of Examples 2 to 8, except where indicated below.

(109) Enteric coated gelatin capsules were individually filled with Compound 2 (106 mg, as obtained in Example 1f), sodium docusate (10.6 mg), PVP K30 (1.1 mg) and magnesium oxide (100 mg).

(110) Animals received 53 mg/kg b.w. of Compound 2 (equivalent to 50 mg/kg Compound 1) in a single dose.

(111) Results

(112) The results for the single dose oral pharmacokinetic study in rabbits are tabulated in Table 10 below.

(113) The results show that there is a significant amount of systemic exposure of 4-chloro-N-[2-[(4-chlorophenyl)methyl]-3-oxo-1,2,4-thiadiazol-5-yl]benzamide when Compound 2 is formulated together with an alkaline excipient.

(114) TABLE-US-00015 TABLE 10 Mean Plasma Pharmacokinetic Parameters For Compound 2 co-formulated with an alkaline excipient Example 11 Compound No. 2 Vehicle Enteric coated capsule with MgO Dose (mg/kg b.w.) 53 mg/kg b.w. (equivalent to 50 mg/kg b.w of Compound 1) C.sub.max (μg/mL) 153.00 ± 24.33  T.sub.max (h) 48.00 ± 24.00 AUC.sub.last (h*μg/ml) 8925.87 ± 1186.00 MRT.sub.last (h) 40.93 ± 1.43 

Example 12

(115) The following illustrates representative pharmaceutical tablet dosage forms containing Compound 2 for therapeutic or prophylactic use in humans:

(116) TABLE-US-00016 Ingredient Quantity (mg) Quantity (%) Compound 2 106.06 30.30 Microcrystalline cellulose 66.15 18.9 Lactose monohydrate 132.29 37.8 Sodium starch glycollate 26.25 7.5 Colloidal silicon dioxide 8.75 2.5 Magnesium stearate 10.50 3 Total 350 100

(117) TABLE-US-00017 Ingredient Quantity (mg) Quantity (%) Compound 2 106.06 30.30 Microcrystalline cellulose 63.81 18.23 Lactose monohydrate 127.63 36.47 Sodium starch glycollate 26.25 7.5 Colloidal silicon dioxide 8.75 2.5 Sodium stearyl fumarate 17.50 5 Total 350 100

(118) These formulations may be obtained by conventional procedures well known in the pharmaceutical art. The tablets may also be enteric coated by conventional means, for example to provide a coating of polymethacrylate.