Pharmaceutical composition comprising mineralocorticoid receptor antagonist and use thereof

Abstract

Provided are a pharmaceutical composition comprising a mineralocorticoid receptor antagonist and use thereof. When the pharmaceutical composition is orally administered to a patient having chronic kidney disease in need thereof, the effective and safe AUC ranges from 188 ng*h/mL to 3173 ng*h/mL, with bioavailability of 50% or more in mammals. When the pharmaceutical composition is orally administered at a daily dose of 0.1 to 1.0 mg to treat chronic kidney disease, the AUC is controlled at a safe and effective level.

Claims

1. A pharmaceutical composition, comprising: Compound I, 2-chloro-4-[(3S,3aR)-3-cyclopentyl-7-(4-hydroxylpiperidine-1-carbonyl)-3,3a,4,5-tetrahydro-2H-pyrazolo[3,4-f]quinolin-2-yl]benzonitrile: ##STR00003## one or more surfactants selected from the group consisting of benzalkonium chloride, sodium lauryl sulfonate, sodium dodecyl sulfate, glycerol, cholic acid, poloxamer, polyvinyl alcohol, Polysorbate 80, PVP K.sub.30 and polyethylene glycol, and one or more additional pharmaceutically acceptable carriers; wherein the pharmaceutical composition is a unit dose formulation comprising 0.1 to 1.0 mg of Compound I; the weight ratio of Compound I to the surfactant is between 1:0.1 to 1:20; and the pharmaceutical composition is a tablet or a capsule.

2. The pharmaceutical composition according to claim 1, wherein the unit dose formulation comprises 0.1 to 0.5 mg of Compound I.

3. The pharmaceutical composition according to claim 2, wherein the daily dose is from unit dose formulation comprises 0.2 to 0.5 mg of Compound I.

4. The pharmaceutical composition according to claim 1, wherein the D.sub.90 of Compound I is 25 μm or less.

5. The pharmaceutical composition according to claim 4, wherein the D.sub.90 of Compound I is 10 μm or less.

6. The pharmaceutical composition according to claim 1, wherein the pharmaceutical composition comprises one or more surfactants selected from the group consisting of benzalkonium chloride, sodium lauryl sulfonate, and sodium dodecyl sulfate.

7. The pharmaceutical composition according to claim 6, wherein the pharmaceutical composition comprises one surfactant selected from the group consisting of benzalkonium chloride, sodium lauryl sulfonate, and sodium dodecyl sulfate.

8. The pharmaceutical composition according to claim 1, wherein the weight ratio is between 1:1 to 1:20.

9. The pharmaceutical composition according to claim 1, wherein the unit dose formulation comprises 0.1 mg of Compound I.

10. The pharmaceutical composition according to claim 1, wherein the unit dose formulation comprises 0.15 mg of Compound I.

11. The pharmaceutical composition according to claim 1, wherein the unit dose formulation comprises 0.2 mg of Compound I.

12. The pharmaceutical composition according to claim 1, wherein the unit dose formulation comprises 0.25 mg of Compound I.

13. The pharmaceutical composition according to claim 1, wherein the unit dose formulation comprises 0.3 mg of Compound I.

14. The pharmaceutical composition according to claim 1, wherein the unit dose formulation comprises 0.35 mg of Compound I.

15. The pharmaceutical composition according to claim 1, wherein the unit dose formulation comprises 0.4 mg of Compound I.

16. The pharmaceutical composition according to claim 1, wherein the unit dose formulation comprises 0.45 mg of Compound I.

17. The pharmaceutical composition according to claim 1, wherein the unit dose formulation comprises 0.5 mg of Compound I.

18. The pharmaceutical composition according to claim 1, wherein the unit dose formulation comprises 0.6 mg of Compound I.

19. The pharmaceutical composition according to claim 1, wherein the unit dose formulation comprises 0.7 mg of Compound I.

20. The pharmaceutical composition according to claim 1, wherein the unit dose formulation comprises 0.8 mg of Compound I.

21. The pharmaceutical composition according to claim 1, wherein the unit dose formulation comprises 0.9 mg of Compound I.

22. The pharmaceutical composition according to claim 1, wherein the unit dose formulation comprises 1.0 mg of Compound I.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 is a curve showing the correlation between SBP variation based on baselines and AUC.sub.0-24 in a DSS rat model with hypertension and renal injury induced by a high content of salts.

(2) FIG. 2 is a curve showing the correlation between UACR variation based on baselines and AUC.sub.0-24 in a DSS rat model with hypertension and renal injury induced by a high content of salts.

DETAILED DESCRIPTION OF THE INVENTION

(3) The present invention includes, but not limited to, the following examples. Other embodiments for carrying out the technical solution of the present invention fall within the scopes as claimed in the present invention.

Example 1. Preparation of Compound I

(4) 2-chloro-4-[(3S,3aR)-3-cyclopentyl-7-(4-hydroxylpiperidine-1-carbonyl)-3,3a,4,5-tetrahydro-2H-pyrazolo[3,4-f]quinolin-2-yl]benzonitrile (herein referred to as “Compound I”), with the structure as shown below.

(5) ##STR00002##

(6) Compound I may be prepared by the method described in WO2012022121A1 or WO2014094664A1, or other synthesis methods.

Example 2. Exemplary Formulations of the Pharmaceutical Composition of the Present Invention

(7) The examples of the present invention present part of the formulations of the pharmaceutical composition, which are used for the administration of the pharmaceutical composition of the present invention. It should be noted that the formulation herein is not limited to the following ones, other formulations having bioavailability of 50% or more fall within the scopes of the present invention as claimed.

(8) Exemplary tablet formulations are shown below.

(9) Formulation 1. Each tablet contained 0.5 mg of Compound I.

(10) TABLE-US-00001 Compound I having D.sub.90 of 538 nm 0.5 g of C.sub.28H.sub.30ClN.sub.5O.sub.2 Microcrystalline cellulose PH101 30 g Mannitol 70 g Croscarmellose Sodium 6 g Polyvinyl pyrrolidone K30 2.15 g Magnesium stearate 2 g Silicon dioxide 2 g Formulated into 1000 tablets

(11) Manufacturing Process:

(12) (1) Polyvinylpyrrolidone K30 was formulated into an aqueous solution with a concentration of 5% (w/w), to be used as the binder;

(13) (2) Microcrystalline cellulose PH101, mannitol, and Croscarmellose Sodium were weighed in amounts as described above and sufficiently mixed for 15 minutes to obtain Mixture 1;

(14) (3) Suspension containing 0.5 g of nanosized Compound I was weighed, and then added into and mixed with Mixture 1; the resultant mixture was added with the aqueous solution of Polyvinylpyrrolidone K30, the binder, to obtain soft material, which was treated by a 24-mesh screen to make wet granules;
(4) The wet granules were dried at 55±5° C. for 2 to 3 hours, the granules' water content being controlled at a level below 2.5%;
(5) The dried granules were screened by the 24-mesh screen again;
(6) Magnesium stearate and Silicon dioxide were added to the granules at amounts described above and then mixed for 15 minutes;
(7) Tablet compression was done, with tablet hardness controlled at 6 to 10 kg;
(8) Tablets were coated;
(9) Tablets were packaged and warehoused.

(15) Formulation 2. Each tablet contained 0.5 mg of Compound I.

(16) TABLE-US-00002 Compound I having D.sub.90 of 4.0 μm 0.5 g of C.sub.28H.sub.30ClN.sub.5O.sub.2 Sodium dodecyl sulfate 5 g Microcrystalline cellulose PH101 40 g Lactose monohydrate 60 g Croscarmellose Sodium 3.5 g Hydroxypropyl cellulose 5 g Magnesium stearate 2.0 g Silicon dioxide 1.0 g Formulated into 1000 tablets

(17) Formulation 3. Each tablet contained 2.5 mg of Compound I.

(18) TABLE-US-00003 Compound I having D.sub.90 of 4.0 μm 2.5 g of C.sub.28H.sub.30ClN.sub.5O.sub.2 Sodium dodecyl sulfate 25 g Microcrystalline cellulose PH101 40 g Lactose monohydrate 60 g Croscarmellose Sodium 3.5 g Hydroxypropyl cellulose 5 g Magnesium stearate 2.0 g Silicon dioxide 1.0 g Formulated into 1000 tablets

(19) Manufacturing Process:

(20) (1) The starting materials were screened by a 30-mesh screen and stored for further use;

(21) (2) Compound I, sodium dodecyl sulfate, microcrystalline cellulose PH101, lactose monohydrate, Croscarmellose Sodium and hydroxypropyl cellulose were sufficiently mixed for 15 minutes;

(22) (3) Water was added into the mixture to obtain soft material which was treated by a 24-mesh screen to make wet granules;

(23) (4) The wet granules were dried at 55±5° C. for 2 to 3 hours, the granules' water content being controlled at a level below 2.5%;

(24) (5) The dried granules were screened by the 24-mesh screen again;

(25) (6) Magnesium stearate and silicon dioxide were added to the granules at amounts described above and then mixed for 15 minutes;

(26) (7) Tablet compression was done, with tablet hardness controlled at 6 to 10 kg;

(27) (8) Tablets were coated;

(28) (9) Tablets were packaged and warehoused.

(29) Exemplary capsule formulations are shown below.

(30) Formulation 1. Each capsule contained 0.1 mg of Compound I.

(31) TABLE-US-00004 Compound I having D.sub.90 of 5 μm 0.1 g of C.sub.28H.sub.30ClN.sub.5O.sub.2 Microcrystalline cellulose PH101 30.0 g Mannitol 70.0 g Croscarmellose Sodium 6.0 g Polyvinylpyrrolidone K30 2.15 g Magnesium stearate 2.0 g Silicon dioxide 2.0 g Formulated into 1000 capsules

(32) Formulation 2. Each capsule contained 0.25 mg of Compound I.

(33) TABLE-US-00005 Compound I having D.sub.90 of 5 μm 0.25 g of C.sub.28H.sub.30ClN.sub.5O.sub.2 Microcrystalline cellulose PH101 30.0 g Mannitol 70.0 g Croscarmellose Sodium 6.0 g Polyvinylpyrrolidone K30 2.15 g Magnesium stearate 2.0 g Silicon dioxide 2.0 g Formulated into 1000 capsules

(34) Manufacturing Process:

(35) (1) Polyvinylpyrrolidone K30 was formulated into an aqueous solution with a concentration of 5% (w/w), to be used as the binder;

(36) (2) Compound I, microcrystalline cellulose PH101, mannitol, and Croscarmellose Sodium were weighed in amounts described above and sufficiently mixed for 15 minutes; water content of the obtained mixture was measured;

(37) (3) The resultant mixture was added with the aqueous solution of Polyvinylpyrrolidone K.sub.30 as the binder to obtain soft material, which was treated by a 24-mesh screen to make wet granules; (4) The wet granules were dried at 55±5° C. for 2 to 3 hours, the granules' water content being controlled at a level below 2.5%;
(5) The dried granules were screened by the 24-mesh screen again;
(6) Magnesium stearate and Silicon dioxide were added to the granules at amounts described above and then mixed for 15 minutes;
(7) The amount of the obtained mixture to be inserted into the capsule was calculated and the mixture was weighed at this amount and put into the capsule;
(8) Capsules were put into bottles made of high-density polyethylene for oral solid drugs, and the bottles were sealed;
(9) The bottles were warehoused.

(38) Formulation 3. Each capsule contained 0.5 mg of Compound I.

(39) TABLE-US-00006 Compound I having D.sub.90 of 5 μm 0.5 g of C.sub.28H.sub.30ClN.sub.5O.sub.2 Microcrystalline cellulose PH101 30.0 g Mannitol 70.0 g Croscarmellose Sodium 6.0 g Polyvinylpyrrolidone K30 2.15 g Magnesium stearate 2.0 g Silicon dioxide 2.0 g Formulated into 1000 capsules

(40) Formulation 4. Each capsule contained 2.5 mg of Compound I.

(41) TABLE-US-00007 Compound I having D.sub.90 of 5 μm 2.5 g of C.sub.28H.sub.30ClN.sub.5O.sub.2 Microcrystalline cellulose PH101 30.0 g Mannitol 70.0 g Croscarmellose Sodium 6.0 g Polyvinylpyrrolidone K30 2.15 g Magnesium stearate 2.0 g Silicon dioxide 2.0 g Formulated into 1000 capsules

(42) Formulation 5. Each capsule contained 10 mg of Compound I.

(43) TABLE-US-00008 Compound I having D.sub.90 of 5 μm 10 g of C.sub.28H.sub.30ClN.sub.5O.sub.2 Microcrystalline cellulose PH101 30.0 g Mannitol 70.0 g Croscarmellose Sodium 6.0 g Polyvinylpyrrolidone K30 2.15 g Magnesium stearate 2.0 g Silicon dioxide 2.0 g Formulated into 1000 capsules

(44) Manufacturing Process:

(45) (1) Preparation of the binder solution: Polyvinylpyrrolidone K30 was formulated into an aqueous solution with a concentration of 3 to 5% (w/w), to be used as the binder;

(46) (2) Mixing: Microcrystalline cellulose PH101, mannitol, and Croscarmellose Sodium were passed through a 30-mesh screen and then transferred with Compound I to a high-shear granulation machine where they were mixed for 2 to 3 minutes at an agitating speed of 180 to 220 r/min and a cutting speed of 475 to 525 r/min;
(3) Granulation: The binder solution was added to the mixture obtained in step (2) within 5 to 7 minutes after the completion of step (2), and the mixture was subject to granulation at an agitating speed of 180 to 220 r/min and a cutting speed of 475 to 525 r/min, followed by granulation for 0.5 to 1.5 minutes at an agitating speed of 180 to 220 r/min and a cutting speed of 1800 to 2200 r/min;
(4) Drying: The wet granules were dried at 55±5, the granules' water content being controlled at a level below 2.5%;
(5) Breaking: The dried granules were put into Fitz Mill and forced through Screen #0033 at a rotating speed of 840 to 960 r/min; the breaking step can be performed using other methods or devices as long as the same effect can be produced;
(6) Mixing: Magnesium stearate and Silicon dioxide were screened, respectively, using the 30-mesh screen or some other devices; the silicone dioxide was then put into a V-shaped mixer and mixed for 12 minutes at 20 r/min; magnesium stearate was added to and mixed with the silicone dioxide for 3 minutes at 20 r/min;
(7) Capsule filling: The mixed powder was filled into capsules using a capsule filling machine (MF-30);
(8) Polishing: The capsules were polished using a capsule polishing machine;
(9) Packing and labeling: The capsules were put into HDPE bottles whose caps were screwed tight and sealed using an electromagnetic sealing machine; bottles were labeled later.

(47) Formulation 6. Each capsule contained 2.5 mg of Compound I.

(48) TABLE-US-00009 Compound I having D.sub.90 of 21.7 μm 2.5 g of C.sub.28H.sub.30ClN.sub.5O.sub.2 Microcrystalline cellulose PH101 40.0 g Lactose monohydrate 60.0 g Croscarmellose Sodium 3.0 g Magnesium stearate 2.0 g Silicon dioxide 2.0 g Formulated into 1000 capsules

(49) Manufacturing Process:

(50) (1) The starting materials were passed through a 30-mesh screen and stored for future use;

(51) (2) Mixing: Compound I, microcrystalline cellulose PH101, lactose monohydrate, Croscarmellose Sodium, magnesium stearate and silicon dioxide were evenly mixed for 15 minutes;

(52) (3) The amount to be inserted into the capsule was calculated, and the mixture at the amount was put into the capsules;

(53) (4) The capsules were put into bottles made of high-density polyethylene for oral solid drugs, and the bottles were sealed;

(54) (5) The bottles were warehoused.

(55) The exemplary self-emulsifying drug delivery system (SEDDS) is shown below.

(56) SEDDS Formulation

(57) CompoundI:ethanol:Kolliphor EL:Miglyol 812N=10 mg:1 g:5 g:4 g

(58) Manufacturing Process:

(59) Compound I was dissolved in ethanol, which was added and mixed with Kolliphor EL and Miglyol 812N to obtain an emulsified mixture.

(60) Exemplary soft capsule formulations are shown below.

(61) Formulation 1. Each soft capsule contained 0.25 mg of Compound I.

(62) TABLE-US-00010 Compound I 0.25 g of C.sub.28H.sub.30ClN.sub.5O.sub.2 Polyethylene glycol 400 240 g Propylene glycol 30 g Formulated into 1000 capsules

(63) Formulation 2. Each soft capsule contained 0.5 mg of Compound I.

(64) TABLE-US-00011 Compound I 0.5 g of C.sub.28H.sub.30ClN.sub.5O.sub.2 Polyethylene glycol 400 240 g Propylene glycol 30 g Formulated into 1000 capsules

(65) Formulation 3. Each soft capsule contained 1.0 mg of Compound I.

(66) TABLE-US-00012 Compound I 1.0 g of C.sub.28H.sub.30ClN.sub.5O.sub.2 Polyethylene glycol 400 240 g Propylene glycol 30 g Formulated into 1000 capsules

(67) Manufacturing Process:

(68) (1) Compound I was mixed with and dissolved in polyethylene glycol 400 and propylene glycol;

(69) (2) The mixture was pressed into soft capsules;

(70) (3) The soft capsules were packaged and warehoused.

(71) Exemplary suspension formulations are shown below.

(72) Formulation 1. The suspension contained 0.25 mg of Compound I per milliliter.

(73) TABLE-US-00013 Compound I 0.25 g of C.sub.28H.sub.30ClN.sub.5O.sub.2 Methyl cellulose 5 g Water added to 1000 mL Formulated into 1000 mL

(74) Manufacturing Process:

(75) (1) Methyl cellulose was dissolved in water whose amount was about 80% of that totally used; the solution was kept for future use;

(76) (2) Compound I was weighed and put into the solution; after sufficient mixing, Compound I was suspended in the solution;

(77) (3) Water was added to a total volume of 1000 mL, and sufficient mixing was done.

(78) Formulation 2 The suspension contained 0.5 mg of Compound I per milliliter.

(79) TABLE-US-00014 Compound I 0.5 g of C.sub.28H.sub.30ClN.sub.5O.sub.2 Methyl cellulose 5 g Water added to 1000 mL Formulated into 1000 mL

(80) Formulation 3. The suspension contained 1.0 mg of Compound I per milliliter.

(81) TABLE-US-00015 Compound I 1.0 g of C.sub.28H.sub.30ClN.sub.5O.sub.2 Methyl cellulose 5 g Water added to 1000 mL Formulated into 1000 mL

(82) Exemplary emulsion formulations are shown below.

(83) Formulation 1. The emulsion contained 0.25 mg of Compound I per milliliter.

(84) TABLE-US-00016 Compound I 0.25 g of C.sub.28H.sub.30ClN.sub.5O.sub.2 Polyethylene glycol 400 50 g Span 80 (Sorbitan oleate) 3 g Tween 80 6 g Soybean oil 50 g Glyceryl Monostearate 10 g Water added to 1000 mL Formulated into 1000 mL

(85) Manufacturing Process:

(86) (1) Water phase preparation: Compound I and polyethylene glycol 400 were heated to 60° C. so that Compound I was dissolved; the solution was kept warm for future use;

(87) (2) Oil phase preparation: Glyceryl Monostearate and soybean oil were heated to 60° C. and dissolved; the solution was kept warm for future use;

(88) (3) The water phase was added to and mixed at 60° C. with the oil phase, and the resultant mixture was added with and mixed at 60° C. with Span 80 and Tween 80; water was added to a total volume of 1000 mL; the obtained mixture was ground into uniform size using a colloid mill.

(89) Formulation 2. The emulsion contained 0.5 mg of Compound I per milliliter.

(90) TABLE-US-00017 Compound I 0.5 g of C.sub.28H.sub.30ClN.sub.5O.sub.2 Polyethylene glycol 400 50 g Span 80 3 g Tween 80 6 g Soybean oil 50 g Glyceryl Monostearate 10 g Water added to 1000 mL Formulated into 1000 mL

(91) Formulation 3. The emulsion contained 1.0 mg of Compound I per milliliter.

(92) TABLE-US-00018 Compound I 1.0 g of C.sub.28H.sub.30ClN.sub.5O.sub.2 Polyethylene glycol 400 50 g Span 80 3 g Tween 80 6 g Soybean oil 5 0 g Glyceryl Monostearate 10 g Water added to 1000 mL Formulated into 1000 mL

(93) Exemplary dripping pill formulations are shown below.

(94) Formulation 1. Each pill contained 0.1 mg of Compound I.

(95) TABLE-US-00019 Compound I having D.sub.90 of 5 μm 0.1 g of C.sub.28H.sub.30ClN.sub.5O.sub.2 Polyethylene glycol 4000 60.0 g Polyethylene glycol 6000 30.0 g Formulated into 1000 pills

(96) Formulation 2. Each pill contained 0.25 mg of Compound I.

(97) TABLE-US-00020 Compound I having D.sub.90 of 5 μm 0.25 g of C.sub.28H.sub.30ClN.sub.5O.sub.2 Polyethylene glycol 4000 60.0 g Polyethylene glycol 6000 30.0 g Formulated into 1000 pills

(98) Formulation 3. Each pill contained 0.5 mg of Compound I.

(99) TABLE-US-00021 Compound I having D.sub.90 of 5 μm 0.5 g of C.sub.28H.sub.30ClN.sub.5O.sub.2 Polyethylene glycol 4000 60.0 g Polyethylene glycol 6000 30.0 g Formulated into 1000 pills

(100) Formulation 4. Each pill contained 1.0 mg of Compound I.

(101) TABLE-US-00022 Compound I having D.sub.90 of 5 μm 1.0 g of C.sub.28H.sub.30ClN.sub.5O.sub.2 Polyethylene glycol 4000 60.0 g Polyethylene glycol 6000 30.0 g Formulated into 1000 pills

(102) Manufacturing Process:

(103) (1) Compound I, polyethylene glycol 4000 and polyethylene glycol 6000 were weighted at the amounts set forth above for future use;

(104) (2) Polyethylene glycol 4000 and polyethylene glycol 6000 were melted with a water bath at 80° C., and then added with Compound I which was dissolved with stirring; the mixture was kept at 80° C. for future use;

(105) (3) At the temperature of 80° C., the mixture was added dropwise into condensed dimethicone where the mixture was condensed as solid pills;

(106) (4) The pills were fetched out and drained dry, and then put into polyethylene bottles which were properly sealed.

(107) Exemplary oral melt tablet formulations are shown below.

(108) Formulation 1. Each oral melt tablet contained 0.25 mg of Compound I.

(109) TABLE-US-00023 Compound I 0.25 g of C.sub.28H.sub.30ClN.sub.5O.sub.2 Microcrystalline cellulose PH101 10 g Mannitol 80 g Croscarmellose Sodium 10 g Water q.s. Magnesium stearate 1.0 g Silicon dioxide 1.0 g Formulated into 1000 tablets

(110) Manufacturing Process:

(111) (1) Compound I, microcrystalline cellulose PH101, mannitol and Croscarmellose Sodium were evenly mixed;

(112) (2) Water was added to the mixture to perform granulation, and the obtained granules were dried;

(113) (3) Magnesium stearate and silicon dioxide were added to be uniformly mixed with granules;

(114) (4) Pelleting was done;

(115) (5) The obtained tablets were coated and then packaged.

(116) Formulation 2. Each oral melt tablet contained 0.5 mg of Compound I.

(117) TABLE-US-00024 Compound I 0.5 g of C.sub.28H.sub.30ClN.sub.5O.sub.2 Microcrystalline cellulose PH101 10 g Mannitol 80 g Croscarmellose Sodium 10 g Water q.s. Magnesium stearate 1.0 g Silicon dioxide 1.0 g Formulated into 1000 tablets

(118) Formulation 3. Each oral melt tablet contained 1.0 mg of Compound I.

(119) TABLE-US-00025 Compound I 1.0 g of C.sub.28H.sub.30ClN.sub.5O.sub.2 Microcrystalline cellulose PH101 10 g Mannitol 80 g Croscarmellose Sodium 10 g Water q.s. Magnesium stearate 1.0 g Silicon dioxide 1.0 g Formulated into 1000 tablets

(120) Exemplary sustained release tablet formulations are shown below.

(121) Formulation 1. Each sustained release tablet contained 0.25 mg of Compound I.

(122) TABLE-US-00026 Compound I 0.25 g of C.sub.28H.sub.30ClN.sub.5O.sub.2 Microcrystalline cellulose PH101 20 g Lactose 70 g Hydroxypropyl methylcellulose 40 g Polyvinylpyrrolidone K30 6 g Magnesium stearate 1.0 g Silicon dioxide 1.0 g Formulated into 1000 tablets

(123) Manufacturing Process:

(124) (1) Compound I and the additives were weighed;

(125) (2) They were sufficiently mixed for 15 minutes;

(126) (3) Pelleting was done;

(127) (4) The obtained tablets were coated and then packaged.

(128) Formulation 2. Each sustained release tablet contained 0.5 mg of Compound I.

(129) TABLE-US-00027 Compound I 0.5 g of C.sub.28H.sub.30ClN.sub.5O.sub.2 Microcrystalline cellulose PH101 20 g Lactose 70 g Hydroxypropyl methyl cellulose 40 g Polyvinylpyrrolidone K30 6 g Magnesium stearate 1.0 g Silicon dioxide 1.0 g Formulated into 1000 tablets

(130) Formulation 3. Each sustained release tablet contained 1.0 mg of Compound I.

(131) TABLE-US-00028 Compound I 1.0 g of C.sub.28H.sub.30ClN.sub.5O.sub.2 Microcrystalline cellulose PH101 20 g Lactose 70 g Hydroxypropyl methyl cellulose 40 g Polyvinylpyrrolidone K30 6 g Magnesium stearate 1.0 g Silicon dioxide 1.0 g Formulated into 1000 tablets

(132) Exemplary micro-capsule formulations are shown below.

(133) Formulation 1. Each gram of micro-capsules contained 0.25 mg of Compound I.

(134) TABLE-US-00029 Compound I 0.25 g of C.sub.28H.sub.30ClN.sub.5O.sub.2 Stearic acid 900 g 10% ethyl cellulose in ethanol 1000 mL 95% ethanol q.s. Formulated into 1000 g

(135) Manufacturing Process:

(136) (1) Stearic acid was weighed and melted in a water bath;

(137) (2) Compound I was weighed and then stirred with and dissolved in 10% ethyl cellulose in ethanol;

(138) (3) Ethanol (q.s.) was added to the mixture obtained in step (2), and the resultant mixture was added to the melt stearic acid; the mixture was kept heated in the water bath until uniform liquid was obtained;

(139) (4) The liquid were sprayed and cooled, with micro-capsules settled out;

(140) (5) The micro-capsules were collected.

(141) Formulation 2. Each gram of micro-capsules contained 0.5 mg of Compound I.

(142) TABLE-US-00030 Compound I 0.5 g of C.sub.28H.sub.30ClN.sub.5O.sub.2 Stearic acid 900 g 10% ethyl cellulose in ethanol 1000 mL 95% ethanol q.s. Formulated into 1000 g

(143) Formulation 3. Each gram of micro-capsules contained 1.0 mg of Compound I.

(144) TABLE-US-00031 Compound I 1.0 g of C.sub.28H.sub.30ClN.sub.5O.sub.2 Stearic acid 900 g 10% ethyl cellulose in ethanol 1000 mL 95% ethanol q.s. Formulated into 1000 g

(145) Exemplary liposome formulation is shown below.

(146) Formulation 1. Each milliliter of liposomes contained 0.25 mg of Compound I.

(147) TABLE-US-00032 Compound I 0.25 g of C.sub.28H.sub.30ClN.sub.5O.sub.2 Soybean lecithin 20 g Cholesterol 5 g Polyethylene glycol 4000 100 mL Ethanol q.s. Formulated into 1000 mL

(148) Manufacturing Process:

(149) (1) Soybean lecithin and cholesterol were weighed and dissolved in ethanol (q.s.);

(150) (2) Compound I was weighed and ultrasonically dissolved in polyethylene glycol 4000; water was added to the solution to a total volume of 800 mL;

(151) (3) The solution obtained in step (1) was slowed introduced into the solution obtained in step (2), and the resultant mixture was stirred at 55° C.; ethanol was completely removed, and water was added to a total volume of 1000 mL.

(152) Exemplary micelle formulation is shown below.

(153) Formulation 1. Each milliliter of micelle contained 0.25 mg of Compound I.

(154) TABLE-US-00033 Compound I 0.25 g of C.sub.28H.sub.30ClN.sub.5O.sub.2 Soybean lecithin 10 g Ethanol 100 g Polyethylene glycol 4000 50 g Water added to 1000 mL Formulated into 1000 mL

(155) Manufacturing Process:

(156) (1) Compound I, soybean lecithin and polyethylene glycol 4000 were weighed and dissolved in ethanol;

(157) (2) Water was added to a total volume of 1000 mL to dilute the obtained mixture.

(158) Exemplary solution formulations are shown below.

(159) Formulation 1. Each 5 milliliter of the solution contained 0.1 mg of Compound I.

(160) TABLE-US-00034 Compound I having D.sub.90 of 5 μm 0.1 g of C.sub.28H.sub.30ClN.sub.5O.sub.2 Sodium dodecyl sulfate 1.0 g Edetate disodium 1.0 g Citric acid 2.0 g Aspartame 5.0 g Aquae pro injectione Added to 5000 mL Formulated into 1000 ampoules

(161) Formulation 2. Each 5 milliliter of the solution contained 0.25 mg of Compound I.

(162) TABLE-US-00035 Compound I having D.sub.90 of 5 μm 0.25 g of C.sub.28H.sub.30ClN.sub.5O.sub.2 Sodium dodecyl sulfate 2.5 g Edetate disodium 1.0 g Citric acid 2.0 g Aspartame 5.0 g Aquae pro injectione Added to 5000 mL Formulated into 1000 ampoules

(163) Formulation 3. Each 5 milliliter of the solution contained 0.5 mg of Compound I.

(164) TABLE-US-00036 Compound I having D.sub.90 of 5 μm 0.5 g of C.sub.28H.sub.30ClN.sub.5O.sub.2 Sodium dodecyl sulfate 5.0 g Edetate disodium 1.0 g Citric acid 2.0 g Aspartame 5.0 g Aquae pro injectione Added to 5000 mL Formulated into 1000 ampoules

(165) Formulation 4. Each 5 milliliter of the solution contained 1.0 mg of Compound I.

(166) TABLE-US-00037 Compound I having D.sub.90 of 5 μm 1.0 g of C.sub.28H.sub.30ClN.sub.5O.sub.2 Sodium dodecyl sulfate 10.0 g Edetate disodium 1.0 g Citric acid 2.0 g Aspartame 5.0 g Aquae pro injectione Added to 5000 mL Formulated into 1000 ampoules

(167) Manufacturing Process:

(168) (1) Compound I and the additives were weighed;

(169) (2) Sodium dodecyl sulfate was dissolved in the Aquae pro injection (q.s.) and then added with Compound I which was stirred to dissolve; then edetate disodium, citric acid and aspartame were added into and dissolved in the resultant solution;

(170) (3) The obtained solution was subject to filtration and sterilization;

(171) (4) The solution was filled in ampoules, 5 mL for each;

(172) (5) The ampoules were packaged.

Example 3. Effect of Particle Size on Absorption

Example 3-1. Effect of Particle Size on Absorption

(173) Compound I with different formulations and/or different D.sub.90 values was compared for absorptions in male SD rats.

(174) Samples to test: Compound I was prepared by the method as described in Example 1 of WO2014094664A1, and then pulverized to provide Compound I samples having D.sub.90 of 538 nm, 3.8 μm, 21.7 μm, 41.5 μm and 71.6 μm, respectively; immediately before the tests, these samples were suspended in 0.5% MC to obtain 1 mg/mL suspensions.

(175) Solutions: 5% DMSO+95% (solution containing 6% HP-β-CD)

(176) Solid dispersions: Compound I: Polyvinylpyrrolidone K30=1:8 (w/w)

(177) The SD rats were intragastrically given a single dose of the test samples at a dose of 1.0 mg/kg, and blood was collected prior to the administration and 30 min, 1 h, 2 h, 4 h, 6 h, 8 h, 10 h, and 24 h after the administration.

(178) The formulation for intravenous injection was a solution where Compound I was dissolved in 5% DMSO+95% (6% HP-β-CD solution), and SD rats were administered at a dose of 2 mg/kg. The rats' blood samples were collected prior to the administration and 5 min, 15 min, 30 min, 1 h, 2 h, 4 h, 6 h, 8 h, and 24 h after the administration.

(179) Blood collection: The animals were fixed and had their tails warmed in a water bath 10 min prior to blood collection; about 100 μL of blood was collected through the tail vein for each animal and put into an anticoagulation tube containing hepatin; the blood samples were subject to centrifugation at 8000 rpm for 6 min at 4° C. to obtain plasma samples, which had to be obtained within 30 minutes after blood was collected; the plasma samples were stored in a freezer at −80° C. for future tests.

(180) Sample analysis: 50 μL of each plasma sample from the freezer was transferred to a centrifugal tube; 100 μL of water and 400 μL of MTBE standard solution (50 ng/mL) were added into the tube and well mixed with the plasma; the obtained mixture was mixed for 10 minutes using a vortex mixer and then subject to centrifugation for 10 minutes (4000 r/min); 300 μL of the supernatant was transferred to another centrifugal tube and blown dry with nitrogen gas; the resultant substance was dissolved in 200 μL of a solution consisting of methanol and water at a ratio of 1:1, of which 204 was used for the LC-MS/MS test.

(181) Data processing: The compound concentration was output by Analyst 1.6.1 (AB Sciex); the means, standard deviations, and variation coefficients were calculated using Microsoft Excel (no calculation needed if these parameters were directly output by Analyst 1.6.1), and pharmacokinetic parameters were determined using the NCA analysis object in Pharsight Phoenix 6.3.

(182) Results and Discussion

(183) TABLE-US-00038 TABLE 1 Absorption of Compound I of different particle sizes in male SD rats Test sample AUC.sub.INF (ng * h/mL) BA % Solution 1276 81.6 Solid dispersion 1202 76.9 Compound I with D.sub.90 of 538 nm 1287 82.3 Compound I with D.sub.90 of 3.8 μm 1045 66.9 Compound I with D.sub.90 of 21.7 μm 854 54.6 Compound I with D.sub.90 of 41.5 μm 545 34.8 Compound I with D.sub.90 of 72.0 μm 207 13.2 Note: The solution, when administered by intravenous injection at a dose of 2.0 mg/kg, resulted in an AUC.sub.INF of 3126 ng * h/mL.

Conclusion

(184) It can be seen from the Table above that, the lower the particle size of Compound I was, the higher bioavailability would be. When Compound I had a particle size of 21.7 μm, bioavailability reached 54.6%, meeting the clinic requirements.

Example 3-2. Effect of Particle Size on Absorption

(185) Sample to test: Compound I having D.sub.90 of 5 μm was suspended in 0.5% MC immediately before tests, to prepare 0.25 mg/mL suspension.

(186) Animals: Three Beagle Dogs.

(187) Method

(188) Drug Administration and Blood Collection:

(189) (1) All animals fasted for 12 h or longer before drug administration, and food was supplied 4 h after drug administration, with ad libitum access to water all the time. Animals were intragastrically given the test samples in a single dose at a dose of 0.5 mg/kg, and 200 μL of blood was collected for each animal, via the small saphenous vein, immediately prior to the administration (t=0) and 10 min, 30 min, 45 min, 1 h, 2 h, 4 h, 6 h, 8 h, 12 h, 24 h, 30 h, 48 h and 72 h after the administration. The collected blood was stored in a dry tube with heparin (normal saline containing 0.1% heparin sodium).
(2) Plasma preparation: The whole blood samples collected in step (1) were subject to centrifugation at a low speed (8000 r/min, 6 min, 4° C.) to separate the plasma (the whole blood was stored in a mobile freezer at about 0 to 4° C., and plasma had to be separated within 30 minutes after the blood collection), and the plasma was kept in dark in a freezer at −70° C. (or lower) for future analysis.

(190) Sample analysis: 50 μL of each plasma sample from the freezer was transferred to a centrifugal tube; 100 μL of water and 400 μL of MTBE standard solution (50 ng/mL) were added into the tube and well mixed with the plasma; the obtained mixture was mixed for 10 minutes using a vortex mixer and then subject to centrifugation for 5 minutes (12000 r/min); 300 μL of the supernatant was transferred to another centrifugal tube and blown dry with nitrogen gas; the resultant substance was dissolved in 200 μL of a solution consisting of methanol and water at a ratio of 1:1, of which 20 μL was used for the LC-MS/MS measurement.

(191) Data processing: The compound concentration was output by Analyst 1.6.1 (AB Sciex); the means, standard deviations, and variation coefficients were calculated using Microsoft Excel (no calculation needed if these parameters were directed output by Analyst 1.6.1), and pharmacokinetic parameters were determined using the NCA analysis object in Pharsight Phoenix 6.3.

(192) Results:

(193) TABLE-US-00039 TABLE 2 Absorption in Beagle dogs Test sample Dose (mg/kg) AUC.sub.0-∞ (ng * h/mL) BA % Compound I having 0.5 506 57.4 D.sub.90 of 5 μm Note: The solution, when administered by intravenous injection at a dose of 1.0 mg/kg, resulted in an AUC.sub.INF of 1762 ng * h/mL.

Example 3-3. Additives had No Effect on Absorption

(194) With the methods as described in Example 3-1, the capsule of Capsule formulation 6 as prepared in Example 2 was added in water to prepare a suspension with a concentration of 0.1 mg/ml, which was intragastrically given to SD rats in a single dose at a dose of 10 ml/kg (with an actual dose of 0.95 mg/kg).

(195) The AUC.sub.INF was proved to be 821 ng*h/mL (the solution, when administered by intravenous injection at a dose of 2.0 mg/kg, resulted in an AUC.sub.INF of 3126 ng*h/mL), with bioavailability of 52.5%.

Example 3-4. Effect of Particle Size on Absorption

(196) Samples to Test:

(197) Capsule of Capsule formulation 5 as prepared in Example 2, 10 mg of Compound I per capsule.

(198) Solutions: 5% DMSO+95% (6% HP-β-CD solution)

(199) Animals: Four Beagle Dogs.

(200) Method

(201) Drug Administration and Blood Collection:

(202) (1) Administration of capsules. All animals fasted for 12 h or longer before drug administration, and food was supplied 4 h after drug administration, with always ad libitum access to water. Beagle dogs were orally given the capsule of Capsule formulation 5 as prepared in Example 2 (two capsules for each Beagle, 10 mg of Compound I per capsule) in a single dose, and 200 μL of blood was collected for each animal, via the small saphenous vein, immediately prior to the administration (0 h) and 0.17 h, 0.50 h, 0.75 h, 1.0 h, 2.0 h, 4.0 h, 6.0 h, 8.0 h, 12 h and 24 h after the administration. The collected blood was stored in a dry tube with heparin (normal saline containing 0.1% heparin sodium).
(2) Plasma preparation. The whole blood samples collected in step (1) were subject to centrifugation at a low speed (8000 r/min, 6 min, 4° C.) to separate the plasma (the whole blood was stored in a mobile freezer at about 0 to 4° C., and plasma had to be separated within 30 minutes after the blood collection), and the plasma was kept in dark in a freezer at −70° C. (or lower) for future analysis.

(203) Sample analysis: 50 μL of each plasma sample from the freezer was transferred to a centrifugal tube; 100 μL of water and 400 μL of MTBE standard solution (50 ng/mL) were added into the tube and well mixed with the plasma; the obtained mixture was mixed for 10 minutes using a vortex mixer and then subject to centrifugation for 5 minutes (12000 r/min); 300 μL of the supernatant was transferred to another centrifugal tube and blown dry with nitrogen gas; the resultant substance was dissolved in 200 μL of a solution consisting of methanol and water at a ratio of 1:1, of which 20 μL was used for the LC-MS/MS measurement.

(204) Data processing: the compound concentration was output by Analyst 1.6.1 (AB Sciex); the means, standard deviations, and variation coefficients were calculated using Microsoft Excel (no calculation needed if these parameters were directed output by Analyst 1.6.1), and pharmacokinetic parameters were determined using the NCA analysis object in Pharsight Phoenix 6.3.

(205) Results:

(206) TABLE-US-00040 TABLE 3 Absorption of capsules in Beagle dogs Dose AUC.sub.0-∞ Test sample (mg/kg) (ng * h/mL) BA % Capsule of Capsule formulation 5 2.67 2361 50.2 Capsule of Capsule formulation 5 2.63 2611 56.3 Capsule of Capsule formulation 5 2.63 2314 49.9 Capsule of Capsule formulation 5 2.78 3349 68.4 Mean 2.67 2659 56.5 Note: The solutions, when administered by intravenous injection at a dose of 1.0 mg/kg, resulted in an AUC.sub.INF of 1762 ng * h/mL.

Example 3-5. Effect of Particle Size on Absorption

(207) Sample to test: Capsule of Capsule formulation 6 as prepared in Example 2, 2.5 mg of Compound I per capsule.

(208) Animals: Four Beagle Dogs.

(209) The Beagle dog were orally administered with the capsule of Capsule formulation 6 as prepared in Example 2, one capsule for each Beagle dog, 2.5 mg of Compound I per capsule. Experiments were performed using the methods described in Example 3-3.

(210) TABLE-US-00041 TABLE 4 Absorption of capsules in Beagle dogs Test sample Dose (mg/kg) AUC.sub.0-∞ (ng * h/mL) BA % Capsule of Capsule 0.23 239 59.0 formulation 6 Note: The solutions, when administered by intravenous injection at a dose of 1.0 mg/kg, resulted in an AUC.sub.INF of 1762 ng * h/mL.

Example 4. Effect of Surfactants on Absorption

Example 4-1. Effect of Surfactants on Absorption

(211) Samples to test: 1 mg/mL suspensions were formulated by mixing Compound I and a surfactant at ratios of 1:5, 1:10 and 1:20, respectively.

(212) SD rats were intragastrically given the test samples in a single dose at a dose of 1.0 mg/kg, and blood was collected prior to the administration and 30 min, 1 h, 2 h, 4 h, 6 h, 8 h, 10 h, and 24 h after the administration.

(213) Blood collection: The animals were fixed and had their tails warmed in a water bath 10 min prior to blood collection; about 100 μL of blood was collected through the tail vein for each animal and put into an anticoagulation tube containing hepatin; the blood samples were subject to centrifugation at 8000 rpm for 6 min at 4° C. to obtain plasma samples, which had to be obtained within 30 minutes after blood was collected; the plasma samples were stored in a freezer at −80° C. for future tests.

(214) Sample analysis: 50 μL of each plasma sample from the freezer was transferred to a centrifugal tube; 100 μL of water and 400 μL of MTBE standard solution (50 ng/mL) were added into the tube and well mixed with the plasma; the obtained mixture was mixed for 10 minutes using a vortex mixer and then subject to centrifugation for 5 minutes (12000 r/min); 300 μL of the supernatant was transferred to another centrifugal tube and blown dry with nitrogen gas; the resultant substance was dissolved in 200 μL of a solution consisting of methanol and water at a ratio of 1:1, of which 20 μL was used for the LC-MS/MS measurement.

(215) Data processing: the compound concentration was output by Analyst 1.6.1 (AB Sciex); the means, standard deviations, and variation coefficients were calculated using Microsoft Excel (no calculation needed if these parameters were directed output by Analyst 1.6.1), and pharmacokinetic parameters were determined using the NCA analysis object in Pharsight Phoenix 6.3.

(216) Results

(217) TABLE-US-00042 TABLE 5 Effect of the surfactant on Absorption (Compound I:the surfactant = 1:10) AUC.sub.INF Surfactant Dose (mg/kg) (ng * h/mL) BA % Glycerol 0.87 657 48.3 Propylene glycol 0.89 457 32.9 Polyvinyl alcohol 0.87 603 44.3 Polyethylene glycol 400 0.97 613 40.0 Sodium dodecyl sulfonate 0.99 831 53.7 Sodium dodecyl sulfate 1.01 964 61.1 Polyoxyl(40) stearate 0.88 427 31.0 PVP K.sub.30 0.94 608 41.4 Carbomer 0.91 289 20.1 Polysorbate 80 0.9 654 46.5 Poloxamer 188 0.97 712 47.0 Hydroxypropyl cellulose 0.92 535 37.2 Hydroxypropyl methyl cellulose 0.91 483 33.6 Kolliphor HS15 0.99 536 34.6 Cholic acid 0.96 701 46.7 Benzalkonium chloride 0.83 768 59.2 hydroxypropyl-β-cyclodextrin 0.91 350 24.6 Note: The solution, when administered by intravenous injection at a dose of 2.0 mg/kg, resulted in an AUC.sub.INF of 3126 ng * h/mL.

(218) TABLE-US-00043 TABLE 6 Effect of the surfactant on Absorption (Compound I:the surfactant = 1:20) Surfactant Dose (mg/kg) AUC.sub.INF (ng * h/mL) BA % Benzalkonium chloride 0.93 1402 96.5 Sodium dodecyl 0.98 953 62.2 sulfonate Sodium dodecyl sulfate 0.93 1297 89.2 Note: The solution, when administered by intravenous injection at a dose of 2.0 mg/kg, resulted in an AUC.sub.INF of 3126 ng * h/mL.

(219) TABLE-US-00044 TABLE 7 Effect of the surfactant on Absorption (Compound I:the surfactant = 1:5) Dose AUC.sub.INF Ratio Surfactant (mg/kg) (ng * h/mL) BA % 1:5 Benzalkonium chloride 0.80 855 68.4 1:5 Sodium dodecyl sulfonate 0.96 968 64.5 Note: The solution, when administered by intravenous injection at a dose of 2.0 mg/kg, resulted in an AUC.sub.INF of 3126 ng * h/mL.

Example 4-2. Effect of Surfactants on Absorption

(220) Samples to test: Tablet of Tablet formulation 3 prepared in Example 2, 2.5 mg of Compound I per tablet.

(221) Solutions: 5% DMSO+95% (6% HP-β-CD solution), intravenous (IV) administration.

(222) Animals: Eight Beagle Dogs, Four for Each Group.

(223) Method

(224) Drug Administration and Blood Collection:

(225) (1) All animals fasted for 12 h or longer before drug administration, and food was supplied 4 h after drug administration, with always ad libitum access to water. Beagle dogs were orally given the tablets prepared in Example 2, two tablets for each Beagle dog, 2.5 mg of Compound I per tablet, and 200 μL of blood was collected for each animal, via the small saphenous vein, 30 min, 45 min, 1 h, 2 h, 4 h, 6 h, 8 h, 12 h, and 24 h after the administration. The collected blood samples were stored in a dry tube with K2EDTA.
For the group with intravenous (IV) administration, blood was collected 5 min, 15 min, 30 min, 1 h, 2 h, 4 h, 6 h, 8 h, 12 h, and 24 h after the administration.
(2) Plasma preparation: The whole blood samples collected in step (1) were subject to centrifugation at a low speed (8000 r/min, 6 min, 4° C.) to separate the plasma (the whole blood was stored in a mobile freezer at about 0 to 4° C., and plasma had to be separated within 30 minutes after the blood collection), and the plasma was kept in dark in a freezer at −70° C. (or lower) for future analysis.

(226) Sample analysis: 50 μL of each plasma sample from the freezer was transferred to a centrifugal tube; 100 μL of water and 400 μL of MTBE standard solution (50 ng/mL) were added into the tube and well mixed with the plasma; the obtained mixture was mixed for 10 minutes using a vortex mixer and then subject to centrifugation for 5 minutes (12000 r/min); 300 μL of the supernatant was transferred to another centrifugal tube and blown dry with nitrogen gas; the resultant substance was dissolved in 200 μL of a solution consisting of methanol and water at a ratio of 1:1, of which 20 μL was used for the LC-MS/MS measurement.

(227) Data processing: the compound concentration was output by Analyst 1.6.1 (AB Sciex); the means, standard deviations, and variation coefficients were calculated using Microsoft Excel (no calculation needed if these parameters were directed output by Analyst 1.6.1), and pharmacokinetic parameters were determined using the NCA analysis object in Pharsight Phoenix 6.3.

(228) Results

(229) TABLE-US-00045 TABLE 8 Absorption in Beagle dogs Formulation Dose (mg/kg) AUC.sub.INF (ng * h/mL) F % Tablet formulation 3 0.59 1100 77.4 IV group 0.6 1445 NA

Example 4-3. Effect of Surfactants on Absorption

(230) Sample to Test:

(231) Self-emulsifying drug delivery system (SEDDS): CompoundI:ethanol:Kolliphor EL:Miglyol 812N=10 mg: 1 g: 5 g: 4 g.

(232) Solutions: 5% DMSO+95% (6% HP-β-CD solution)

(233) SD rats were intragastrically given the test samples in a single dose at a dose of 1.0 mg/kg, and blood was collected prior to the administration and 30 min, 1 h, 2 h, 4 h, 6 h, 8 h, 10 h, and 24 h after the administration.

(234) The formulation for intravenous injection was a solution where Compound I was dissolved in 5% DMSO+95% (6% HP-β-CD solution), and SD rats were administered at a dose of 2 mg/kg. The rats' blood samples were collected prior to the administration and 5 min, 15 min, 30 min, 1 h, 2 h, 4 h, 6 h, 8 h, and 24 h after the administration.

(235) Blood collection: The animals were fixed and had their tails warmed in a water bath 10 min prior to blood collection; about 100 μL of blood was collected through the tail vein for each animal and put into an anticoagulation tube containing hepatin; the blood samples were subject to centrifugation at 8000 rpm for 6 min at 4° C. to obtain plasma samples, which had to be obtained within 30 minutes after blood was collected; the plasma samples were stored in a freezer at −80° C. for future tests.

(236) Sample analysis: 50 μL of each plasma sample from the freezer was transferred to a centrifugal tube; 100 μL of water and 400 μL of MTBE standard solution (50 ng/mL) were added into the tube and well mixed with the plasma; the obtained mixture was mixed for 10 minutes using a vortex mixer and then subject to centrifugation for 5 minutes (12000 r/min); 300 μL of the supernatant was transferred to another centrifugal tube and blown dry with nitrogen gas; the resultant substance was dissolved in 200 μL of a solution consisting of methanol and water at a ratio of 1:1, of which 20 μL was used for the LC-MS/MS measurement.

(237) Data processing: the compound concentration was output by Analyst 1.6.1 (AB Sciex); the means, standard deviations, and variation coefficients were calculated using Microsoft Excel (no calculation needed if these parameters were directed output by Analyst 1.6.1), and pharmacokinetic parameters were determined using the NCA analysis object in Pharsight Phoenix 6.3.

Results and Conclusions

(238) The rats orally administered with the SEDDS drug had AUC.sub.INF of 1297 ng*h/mL (for the solution intravenously given at a dose of 2.0 mg/kg, AUC.sub.INF of 3126 ng*h/mL was generated), with bioavailability being 83.0%.

Example 5. Toxicity and Pharmacokinetic Studies in SD Rats Intragastrically Administered with Compound I for 13 Weeks and Recovered for 4 Weeks

(239) Sample to test: Solid dispersion of Compound I (Compound I:PVPK30=1:8 (w/w))

(240) Animals: SD Rats, SPF Level.

(241) Method:

(242) Two hundred and twenty SD rats were used in the studies. Three groups, i.e., high dose group, intermediate dose group and low dose group, were administered with Compound I with following doses, 20 males and 20 females for each group. In specific, male animals were given the suspension at the doses of 3, 9 and 30 mg/kg/day, respectively, while the females were administered at the doses of 1, 3, and 10 mg/kg/day, respectively. The animals in the control group, 20 males and 20 females, were given purified water. All animals fasted overnight before blood collections and anatomical examinations.

(243) In addition, for the pharmacokinetic study, 9 males and 9 females were assigned into the administration group, while 3 males and 3 females were for the control group.

Results and Conclusions

(244) TABLE-US-00046 TABLE 9 Serum potassium (K.sup.+) and AUC.sub.0-24 h levels in SD rats intragastrically administered with Compound I for 13 weeks Dose K.sup.+(mmol/L) Gender (mg/kg) (Mean ± SD) AUC.sub.0-24 h (ng * h/mL) Male 0 5.1 ± 0.3 / 3 5.0 ± 0.3  5430 9 5.0 ± 0.3 16500 30 5.1 ± 0.2 49900 Female 0 4.2 ± 0.3 / 1 4.4 ± 0.4  4080 3 4.4 ± 0.3 15500 10 4.5 ± 0.2 43600 Note: “/” means n.a. (not applicable).

(245) Based on results above, the NOAEL (no observed adverse effect level) was 30 mg/kg/day in male animals and 10 mg/kg/day in females, with the corresponding AUC.sub.0-24h being 49900 ng*h/mL and 43600 ng*h/mL, respectively, under which AUC level no obvious abnormal serum potassium level was observed.

Example 6. Efficacy Test in Salt-Sensitive Rats with Salt Induced Renal Injury

(246) Animals: Male Dahl/Ss Rats

(247) Test Article and Formulation:

(248) Solid dispersions of Compound I (Compound I:PVPK30=1:8 (w/w)): Solid dispersions of Compound I was formulated with suitable amount of sterile water for injection into suspensions having concentrations of 0.03, 0.10, 0.30 and 1.00 mg/mL, respectively. The suspensions were prepared immediately before use.

(249) The Dahl/ss rats were randomly divided into 6 groups based on the blood pressures tested before drug administration, i.e., Control group 1 with normal animals (n=10), Control group 2 with animals having induced renal injury (4% NaCl, n=12), Treatment groups with doses at 0.3 mg/kg/day (n=11), 1 mg/kg/day (n=11), 3 mg/kg/day (n=11) and 10 mg/kg/day (n=11), respectively, wherein n represents number of animals.

(250) Method

(251) The Dahl/ss rats were fed Research diet AIN-93G containing 4% NaCl to induce hypertensive nephropathy, so as to test the in vivo pharmacodynamics activity of Compound I.

(252) One week before the experiment, the blood pressures were measured twice in rats by the tail-cuff blood pressure measurement so that rats could accommodate to the blood pressure monitoring operation. Then, immediately before the test, the blood pressure was determined and used as the baseline value. According to the baseline blood pressures, the rats were randomly divided into groups. On the next day, the animals in Control group 2 and the treatment groups were fed Research diet AIN-93G containing 4% NaCl and water, ad libitum, to induce the renal injury model. The model build took 42 days. The animals in Control group 1 was given the normal AIN-93G diet.

(253) The animals in the treatment groups were intragastrically given Compound I twice a day at doses of 0.3, 1, 3 and 10 mg/kg/day, respectively, with the suspensions of Compound I being administered in a volume of 5 mL/kg per dose. The two control groups were given an equal amount of sterile water for injection.

(254) Blood pressure (systolic blood pressure, SBP) measurement: Blood pressure was measured for 6 weeks, once a week, and the blood pressure changes in these groups were analyzed.

(255) Pathological examination for the kidney and heart: At the end of the study, rats were sacrificed painlessly, from which bilateral kidneys and the heart were collected for histopathological analysis. The kidneys were stained using Hematoxylin-Eosin (HE) to semi-quantitatively evaluate renal injuries. The thickness of the left ventricular wall was measured for each heart to analyze the heart injury.

(256) Further, blood samples at 0 h, 1 h, 2 h, 4 h, 6 h, 8 h, 10 h, and 24 h post the last administration were collected from the tail vein of rats before the end of the study. Blood samples were put into a low-temperature high-speed centrifuge and centrifuged for 6 min at 4° C., 8000 rpm. Plasma samples were collected and stored in a freezer at −80° C. until plasma drug concentration were measured.

Results and Conclusions

(257) According to the test results, Blood pressure (SBP) or Urine albumin-to-creatinine ratio (UACR) vs. AUC.sub.0-24 curve was plotted (see FIG. 1 and FIG. 2).

(258) The SBP or UACR vs. AUC.sub.0-24 curve showed an undulation point at 100 h*ng/mL of AUC after which the curve tended to be flat and SBP and UACR were significantly decreased. It was indicated that Compound I had good efficacy and took effect when AUC reached 100 h*ng/mL.

Example 7. Safety, Tolerability, and Pharmacokinetics Studies in Healthy Subjects with a Single Dose

(259) Five groups of healthy subjects were given capsules at doses of 0.5 mg (1 capsule, 0.5 mg of Compound I per capsule), 1.0 mg (2 capsules, 0.5 mg of Compound I per capsule), 2.5 mg (1 capsule, 2.5 mg of Compound I per capsule), 10 mg (1 capsule, 10 mg of Compound I per capsule), and 30 mg (3 capsules, 10 mg of Compound I per capsule), each group containing 8 subjects. The drug administration was done only once.

(260) The capsules mentioned above were the ones prepared in Example 2, i.e., the capsules of Capsule formulation 3 (0.5 mg of Compound I per capsule), Capsule formulation 4 (2.5 mg of Compound I per capsule), and Capsule formulation 5 (10 mg of Compound I per capsule).

(261) Plasma samples were collected to measure plasma drug concentration, and the pharmacokinetic parameters were analyzed using the non-compartment model. Serum samples were collected to measure serum potassium levels.

(262) Results

(263) In the healthy subjects administered at the dose of 0.5 to 30 mg/day, AUC.sub.0-24 of Compound I was 162.5 to 5016 ng*h/mL, and T.sub.1/2 was about 60 hours, with no elevated serum potassium level observed.

(264) TABLE-US-00047 TABLE 10 Pharmacodynamics results Pharmacodynamics Dose parameters 0.5 mg 1.0 mg 2.5 mg 10 mg 30 mg AUC.sub.0-24 (h * ng/mL) 162.5 396.7 639.8 3077 5016 T.sub.1/2 (h) 61.5 60.9 53.3 52.8 73.9

Example 8. Safety, Tolerability, and Pharmacokinetics Studies in Healthy Subjects with Multiple Doses

(265) Two groups of healthy subjects were given capsules once a day at daily doses of 2.5 mg (1 capsule for each administration, 2.5 mg of Compound I per capsule), and 5.0 mg (2 capsules for each administration, 2.5 mg of Compound I per capsule), respectively, each group containing 6 subjects. The administration continued for 14 consecutive days. The capsules used in this study were of Capsule formulation 4 prepared in Example 2.

(266) Plasma samples were collected to measure plasma drug concentration, and the pharmacokinetic parameters were analyzed using the non-compartment model. Serum samples were collected to measure serum potassium levels.

(267) Results

(268) In the healthy subjects administered in multiple doses at the daily dose of 2.5 mg/day, the average steady-state AUC.sub.0-24 was 2865±821 ng*h/mL, with no elevated serum potassium level observed.

(269) In the healthy subjects administered in multiple doses at the daily dose of 5.0 mg/day, the average steady-state AUC.sub.0-24 was 6376±1028 ng*h/mL, with transient elevated serum potassium level observed in three subjects.

Example 9. Safety, Tolerability, and Pharmacokinetics Studies in Patients Having Chronic Kidney Disease

(270) The patients in this example were those having chronic kidney disease with symptoms of renal failure, proteinuria, acute kidney injury, glomerular nephritis, renal cyst, urinary frequency, renal calculus, obstructive uropathy, and etc. These patients also had other diseases or implications such as diabetes mellitus, hyperlipidemia, hypercholesterolemia, hypertension, peripheral vascular diseases, coronary artery disease, and etc.

(271) These patients continued to take the medications that they had prior to the present clinical trial, the medications being, for example, angiotensin converting enzyme inhibitors, such as Lisinopril, Benazepril, Enalapril, and Enalapril Maleate; angiotensin II receptor blockers, such as Valsartan and Losartan; calcium channel blockers, such as Amlodipine, and Nifedipine; diuretics, such as Furosemide; beta-receptor blockers, such as Metoprolol succinate, Metoprolol tartrate, Carvedilol, and Atenolol; and antilipemic drugs, such as Simvastatin, Atorvastatin, Fenofibrate, Pravastatin, and Rosuvastatin.

(272) In this example, the administration dose referred to the amount of Compound I, although the pharmaceutical composition was administered. Two groups of subjects were administered once a day for 56 consecutive days at the daily doses of 0.5 mg (1 capsule for each administration, 0.5 mg of Compound I per capsule) and 2.5 mg (1 capsule for each administration, 2.5 mg of Compound I per capsule), respectively, with 6 subjects for the 0.5 mg/day dose group and also 6 for the 2.5 mg/day dose group. The capsules used here were the capsules prepared in Example 2, i.e., the capsules of Capsule formulation 3 (the capsule with 0.5 mg of Compound I), and Capsule formulation 4 (the capsule containing 2.5 mg of Compound I).

(273) Plasma samples were collected to measure plasma drug concentration, and the pharmacokinetic parameters were analyzed using the non-compartment model. Serum samples were collected to measure serum potassium levels.

(274) Results

(275) In the CKD patients administered in multiple doses at the daily dose of 0.5 mg, the average steady-state AUC.sub.tau was 652.5±232.2 ng*h/mL, with no elevated serum potassium level observed.

(276) In the CKD patients administered in multiple doses at the daily dose of 2.5 mg, mild increase of the serum potassium level was observed in one subject. The average safe steady-state AUC.sub.tau was 2613±280 ng*h/mL in the patients.

(277) Those CKD patients following multiple-dosing administrations at the dose of 0.5 mg/day or 2.5 mg/day showed significantly decrease in blood pressures and urine albumin-to-creatinine ratio (UACR). At the 8th week, the UACR was decreased by 58.9% and 50.7% in the two groups, respectively. The pharmaceutical composition of the present invention showed evident effect on kidney protection.

(278) The CKD patients had the steady-state AUC.sub.tau of 105.6 ng*h/mL or more when administered for one day at the daily dose of 0.5 mg, and UACR was decreased by 30.5% or more compared to the baseline, indicating the efficacy of the pharmaceutical composition of the present invention.

(279) TABLE-US-00048 TABLE 11 Pharmacodynamics results Pharmacodynamics parameter Dose (mg) AUC.sub.tau (ng*h/mL) 0.1 0.15 0.2 0.25 0.3 0.5 1.0 2.0 2.5 Actual AUC.sub.tau / / / / / 652.5 / / 2613 Predicated AUC.sub.tau 130.5 195.8 261 326.3 391.5 / 1305 2610 3262.5

(280) In the CKD patients taking the pharmaceutical composition of the present invention, AUC.sub.tau of Compound I was in the range of 188 ng*h/mL to 3173 ng*h/mL, suggesting excellent efficacy. Further, no elevated serum potassium level was observed. Thus, the pharmaceutical composition of the present invention met the clinical safety and efficacy requirements.