Abstract:
The present invention relates to compositions for lowering the total amount of cholesterol in the blood and methods of using the compositions. The compositions are a mixture of pravastatin and one or more vitamins selected from riboflavins, d-α-tocopherols, ascorbic acids and inositol hexanicotinate.

Description:
This is a Continuation-in-Part Application of U.S. Ser. No. 10/420,442, filed Apr. 22, 2003 now abandoned, which is a Continuation-in-part Application of International Application No. PCT/JP01/09257 filed Oct. 22, 2001 which is incorporated herein by reference in its entirety. 
    
    
     BACKGROUND OF THE INVENTION 
     The present invention relates to compositions for lowering the total amount of cholesterol in the blood, said compositions comprising pravastatin and one or more vitamins selected from the group consisting of riboflavins, d-α-tocopherols, ascorbic acids and inositol hexanicotinate. 
     Pravastatin exhibits activity in lowering the total amount of cholesterol in the blood due to HMG-CoA reductase inhibition in vivo. In addition, it is known that each of riboflavins, d-α-tocopherols, ascorbic acids and inositol hexanicotinate themselves have activity in lowering the total amount of cholesterol in the blood. Furthermore, it is also known that the total amount of cholesterol in the blood can be kept at a low level and the amount of d-α-tocopherols and ascorbic acid in the body is decreased by HMG-CoA reductase inhibitors and this can be supplemented by the combination of an HMG-CoA reductase inhibitor and a d-α-tocopherol or an ascorbic acid (Japanese Patent Application Publication (Kohyo) No. Hei 8-505853). 
     However it has not previously been disclosed that the total amount of cholesterol in the blood is synergistically lowered by a combination of pravastatin and a riboflavin, d-α-tocopherol, ascorbic acid or inositol hexanicotinate. Pravastatin is a safe pharmaceutical agent, but it is administered for a long period. Therefore it has been required that lowering the total amount of cholesterol in the blood could be accomplished with a lower administered amount of pravastatin. 
     BRIEF DESCRIPTION OF THE INVENTION 
     The inventors of this invention have made a great effort to study compositions for lowering the total amount of cholesterol in the blood and found that lowering the total amount of cholesterol in the blood can be accomplished by a combination of pravastatin and a certain vitamin(s), even though a lower amount of pravastatin than that usually used before is administered. 
     The present invention is a composition for lowering the total amount of cholesterol in the blood, said composition comprising pravastatin and one or more vitamins selected from the group consisting of riboflavins, d-α-tocopherols, ascorbic acids and inositol hexanicotinate. Preferably, said composition comprises a combination of pravastatin and one or more vitamins selected from the group consisting of riboflavin tetrabutyrate, d-α-tocopherol butyrate, ascorbic acid and inositol hexanicotinate. 
     DETAILED DESCRIPTION OF THE INVENTION 
     Pravastatin (compound name: (+)-(3R,5R)-3,5-dihydroxy-7-[(1S,2S,6S,8S,8aR)-6-hydroxy-2-methyl-8-[(S)-2-methylbutyryloxy]-1,2,6,7,8,8a-hexahydro-1-naphthyl]heptane) includes the compound of the following formula and a salt (particularly sodium salt) thereof; and is prepared according to the description of the specification of Japanese Patent Application Publication No. Sho 57-2240 and is commercially available. 
                        
 
     Riboflavins refer to riboflavin itself and esters of riboflavin such as riboflavin tetrabutyrate. 
     Tocopherols refer to tocopherol itself (racemic form and optically active form) and esters of tocopherol such as tocopherol butyrate (racemic form and optically active form). 
     Ascorbic acids refer to ascorbic acid itself, salts of ascorbic acid such as the sodium salt of ascorbic acid and esters of ascorbic acid such as the stearate of ascorbic acid. 
     Inositol hexanicotinate refers to the ester of inositol wherein six hydroxyl groups are esterified with nicotinic acid. 
     The total amount of cholesterol in the blood refers to the total amount of cholesterol and esters of cholesterol existing in the blood. 
     “Lowering” of the total amount of cholesterol in the blood means lowering to a clinically significant degree. 
     The solid dosage form of the composition for improving lipid content in the blood of this invention usually contains 0.01 to 5 wt % (preferably 0.05 to 3 wt %) of pravastatin; 0.002 to 40 wt % (preferably 0.01 to 20 wt %) of riboflavins; 0.05 to 50 wt % (preferably 0.5 to 25 wt %) of ascorbic acids; 0.002 to 40 wt % (preferably 0.02 to 20 wt %) of tocopherols and 0.05 to 50 wt % (preferably 0.5 to 25 wt %) of inositol hexanicotinate. 
     The liquid dosage form of the composition for lowering the total amount of cholesterol in the blood of this invention usually contains 0.01 to 10 mg/ml (preferably 0.05 to 5 mg/ml) of pravastatin; 0.05 to 5 mg/ml (preferably 0.1 to 3 mg/ml) of riboflavins; 1 to 10 mg/ml (preferably 3 to 7 mg/ml) of ascorbic acids; 0.5 to 5 mg/ml (preferably 1.5 to 3 mg/ml) of tocopherols; and 1 to 40 mg/ml (preferably 2 to 20 mg/ml) of inositol hexanicotinate. 
     An exemplary dosage form of the composition of this invention for lowering the total amount of cholesterol in the blood includes, for example, a tablet, a fine granule (including a powder), a capsule or a liquid dosage form. Each dosage form can be prepared by using an appropriate additive(s) and an active ingredient(s) according to a conventional procedure described in literature such as the Pharmacopeia of Japan. 
     In the dosage forms described above, various additives usually used can be employed depending on each dosage form. 
     For example, in the case of tablets, lactose, crystalline cellulose or the like can be used as an excipient; magnesium aluminometasilicate or the like can be used as a stabilizing agent; hydroxypropylcellulose or the like can be used as a binding agent; and magnesium stearate or the like can be used as a lubricating agent. 
     In the case of fine granules or capsules, lactose, purified sucrose or the like can be used as an excipient; magnesium aluminometasilicate or the like can be used as a stabilizing agent; corn starch or the like can be used an absorbing agent; and hydroxypropylcellulose, polysorbate or the like can be used as a binding agent. 
     In the case of liquid dosage forms, D-sorbitol solution, honey or the like can be used as a sweetening agent; dl-malic acid or the like can be used as a corrigent; disodium edatate or the like can be used a stabilizing agent; ethanol or the like can be used as a solubility-adjuvant agent; and polyoxyethylene stearate and hydrogenated castor oil 60 can be used as a solubilizing agent. 
     In each dosage form described hereinbefore, if necessary, a disintegrating agent such as crospovidone or the like; an absorbing agent such as calcium silicate or the like; a coloring agent such as iron sesquioxide, caramel or the like; a pH-adjusting agent such as sodium benzoate or the like and a flavoring agent can be added. 
    
    
     EXAMPLES 
     Throughout the Tables the following abbreviations are used with the following meanings.
     ribo.: riboflavin, asco.: ascorbic acid, toco.: tocopherol   inos.: inositol hexanicotinate,   asco.+toco.: ascorbic acid and tocopherol   a.a.: appropriate amount, t.a.: trace amount   

     Example 1 
     Tablet 
     (1) Ingredients 
     
       
         
               
               
               
               
             
               
               
             
               
               
               
               
             
               
               
               
               
               
               
               
             
               
               
               
               
             
           
               
                   
                 TABLE 1 
               
             
             
               
                   
                   
               
               
                   
                 ribo. 
                 asco. 
                 toco. 
               
             
          
           
               
                   
                 in four tablets 
               
             
          
           
               
                   
                 (680 mg) 
                 (1440 mg) 
                 (840 mg) 
               
               
                   
                   
               
             
          
           
               
                 pravastatin sodium 
                 20 
                 mg 
                  20 
                 mg 
                  20 
                 mg 
               
               
                 riboflavin tetrabutyrate 
                 12 
                 mg 
                 — 
                   
                 — 
               
               
                 ascorbic acid 
                 — 
                   
                 500 
                 mg 
                 — 
               
               
                 tocopherol succinate 
                 — 
                   
                 — 
                   
                 200 
                 mg 
               
               
                 crystalline cellulose 
                 120 
                 mg 
                 12 
                 mg 
                 12 
                 mg 
               
               
                 magnesium aluminometasilicate 
                 144 
                 mg 
                 — 
                   
                 — 
               
               
                 fatty acid ester of sucrose 
                 — 
                   
                 140 
                 mg 
                 108 
                 mg 
               
               
                 Hydroxypropylcellulose 
                 96 
                 mg 
                 48 
                 mg 
                 48 
                 mg 
               
               
                 magnesium stearate 
                 24 
                 mg 
                 24 
                 mg 
                 24 
                 mg 
               
               
                 crospovidone 
                 100 
                 mg 
                 48 
                 mg 
                 48 
                 mg 
               
             
          
           
               
                 Lactose 
                 a.a 
                 a.a 
                 a.a 
               
               
                   
               
             
          
         
       
     
                                                                                                         TABLE 2                           inos.   asco. + taco.                in four tablets                    (1400 mg)   (1400 mg)                            pravastatin sodium    20   mg    20   mg           inositol hexanicotinate   500   mg   —           Ascorbic acid   —       500   mg           tocopherol succinate   —       200   mg           crystalline cellulose   12   mg   12   mg           fatty acid ester of sucrose   140   mg   140   mg           hydroxypropylcellulose   96   mg   48   mg           magnesium stearate   24   mg   24   mg           crospovidone   100   mg   48   mg                Lactose   a.a   a.a                        
(2) Method for Preparation
 
     Tablets are prepared in a similar procedure to that described in the general rules for preparation in the “tablet” section of the Pharmacopeia of Japan using the ingredients shown in Tables 1 and 2. 
     Example 2 
     Fine Granules 
     (1) Ingredients 
     
       
         
               
               
               
               
             
               
               
             
               
               
               
               
             
               
               
               
               
               
               
               
             
               
               
               
               
             
           
               
                   
                 TABLE 3 
               
             
             
               
                   
                   
               
               
                   
                 ribo. 
                 asco. 
                 toco. 
               
             
          
           
               
                   
                 in four unit dosages 
               
             
          
           
               
                   
                 (4 g) 
                 (5.2 g) 
                 (4.8 g) 
               
               
                   
                   
               
             
          
           
               
                 pravastatin sodium 
                  20 
                 mg 
                  20 
                 mg 
                 20 
                 mg 
               
               
                 riboflavin tetrabutyrate 
                 12 
                 mg 
                 — 
                   
                 — 
               
               
                 ascorbic acid 
                 — 
                   
                 1.0 
                 g 
                 — 
               
               
                 tocopherol succinate 
                 — 
                   
                 — 
                   
                 200 
                 mg 
               
               
                 purified sucrose 
                 1.4 
                 g 
                 1.6 
                 g 
                 1.4 
                 g 
               
               
                 extract from stevia 
                 — 
                   
                 16 
                 mg 
                 — 
               
               
                 corn starch 
                 1.2 
                 g 
                 1.2 
                 g 
                 1.2 
                 g 
               
               
                 polysorbate-80 
                 80 
                 mg 
                 48 
                 mg 
                 48 
                 mg 
               
               
                 magnesium aluminometasilicate 
                 144 
                 mg 
                 — 
                   
                 128 
                 mg 
               
               
                 magnesium stearate 
                 24 
                 mg 
                 24 
                 mg 
                 24 
                 mg 
               
             
          
           
               
                 Lactose 
                 a.a 
                 a.a 
                     a.a 
               
               
                   
               
             
          
         
       
     
                                                                                                         TABLE 4                           inos.   asco. + toco.                in four unit dosages                    (5 g)           (5 g)                            pravastatin sodium    20   mg    20   mg           inositol hexanicotinate   1000   mg   —           ascorbic acid   —       1000   mg           tocopherol succinate   —       200   mg           purified sucrose   1400   mg   1600   mg           extract from stevia   16   mg   16   mg           corn starch   1200   mg   1200   mg           polysorbate-80   80   mg   48   mg           magnesium aluminometasilicate   144   mg   144   mg           magnesium stearate   24   mg   24   mg                lactose   a.a   a.a                        
(2) Method for Preparation
 
     Fine granules are prepared in a similar procedure to that described in the general rules for preparation of the “granule” section of the Pharmacopeia of Japan using the ingredients shown in Tables 3 and 4. 
     Example 3 
     Capsules 
     (1) Ingredients 
     
       
         
               
               
               
               
             
               
               
               
               
               
               
               
             
               
               
               
               
             
               
               
               
               
               
               
               
             
           
               
                   
                 TABLE 5 
               
               
                   
                   
               
               
                   
                 ribo. 
                 asco. 
                   toco. 
               
               
                   
                 in 4 
                 in 8 
                 in 4 
               
               
                   
                 capsules 
                 capsules 
                 capsules 
               
               
                   
                   
               
             
             
               
                   
               
             
          
           
               
                 pravastatin sodium 
                  20 
                 mg 
                  20 
                 mg 
                  20 
                 mg 
               
               
                 riboflavin tetrabutyrate 
                 12 
                 mg 
                 — 
                   
                 — 
               
               
                 Ascorbic acid 
                 — 
                   
                 500 
                 mg 
                 — 
               
               
                 tocopherol succinate 
                 — 
                   
                 — 
                   
                 200 
                 mg 
               
               
                 corn starch 
                 960 
                 mg 
                 960 
                 mg 
                 840 
                 mg 
               
               
                 polysorbate-80 
                 80 
                 mg 
                 48 
                 mg 
                 48 
                 mg 
               
               
                 magnesium aluminometasilicate 
                 144 
                 mg 
                 — 
                   
                 128 
                 mg 
               
               
                 magnesium stearate 
                 24 
                 mg 
                 24 
                 mg 
                 24 
                 mg 
               
             
          
           
               
                 Lactose 
                 a.a 
                 a.a 
                 a.a 
               
             
          
           
               
                 Subtotal 
                 1520 
                 mg 
                 1940 
                 mg 
                 1580 
                 mg 
               
               
                 Capsule 
                 320 
                 mg 
                 640 
                 mg 
                 320 
                 mg 
               
               
                 Total 
                 1840 
                 mg 
                 2580 
                 mg 
                 1900 
                 mg 
               
               
                   
               
             
          
         
       
     
                                                                                                                     TABLE 6                           inos.   asco. + toco.                in 8 capsules                            pravastatin sodium    20   mg    20   mg           inositol hexanicotinate   500   mg   —           Ascorbic acid   —       500   mg           tocopherol succinate   —       200   mg           corn starch   960   mg   960   mg           polysorbate-80   80   mg   48   mg           magnesium aluminometasilicate   144   mg   144   mg           magnesium stearate   24   mg   24   mg                Lactose   a.a   a.a                Subtotal   2000   mg   2000   mg           Capsule   640   mg   640   mg           Total   2640   mg   2640   mg                        
(2) Method for Preparation
 
     Capsules are prepared in a similar procedure to that described in the general rules for preparation in the “granule” section of the Pharmacopeia of Japan using the ingredients shown in Tables 5 and 6, followed by filling the resulting fine granules into each capsule. 
     Example 4 
     Liquid Dosage Forms 
     (1) Ingredients 
     
       
         
               
               
               
               
             
               
               
             
               
               
               
               
               
               
               
             
               
               
               
               
             
           
               
                   
                 TABLE 7 
               
             
             
               
                   
                   
               
               
                   
                 ribo. 
                 asco. 
                 toco. 
               
             
          
           
               
                   
                 in 100 ml 
               
               
                   
                   
               
             
          
           
               
                 pravastatin sodium 
                  20 
                 mg 
                  20 
                 mg 
                  20 
                 mg 
               
               
                 riboflavin sodium phosphate 
                 20 
                 mg 
                 — 
                   
                 — 
               
               
                 ascorbic acid 
                 — 
                   
                 500 
                 mg 
                 — 
               
               
                 d-α-tocopherol acetate 
                 — 
                   
                 — 
                   
                 50 
                 mg 
               
               
                 D-sorbitol solution (70%) 
                 4 
                 g 
                 6 
                 g 
                 4 
                 g 
               
               
                 Honey 
                 7 
                 g 
                 8 
                 g 
                 7 
                 g 
               
               
                 dl-malic acid 
                 200 
                 mg 
                 — 
                   
                 200 
                 mg 
               
               
                 sodium edatate 
                 20 
                 mg 
                 20 
                 mg 
                 20 
                 mg 
               
               
                 Ethanol 
                 2 
                 ml 
                 2 
                 ml 
                 2 
                 ml 
               
               
                 polyoxyethylene stearate 
                 100 
                 mg 
                 100 
                 mg 
                 100 
                 mg 
               
               
                 hydrogenated castor oil 60 
               
               
                 sodium benzoate 
                 60 
                 mg 
                 60 
                 mg 
                 60 
                 mg 
               
             
          
           
               
                 flavoring agent 
                 t.a. 
                 t.a. 
                     t.a. 
               
               
                 purified water 
                 a.a. 
                 a.a. 
                 a.a 
               
               
                   
               
             
          
         
       
     
                                                                                           TABLE 8                           inos.   asco. + toco.                in 100 ml                            pravastatin sodium    20   mg    20   mg           inositol hexanicotinate   500   mg   —           ascorbic acid   —       500   mg           d-α-tocopherol acetate   —       50   mg           D-sorbitol solution (70%)   4   g   6   g           honey   7   g   8   g           dl-malic acid   200   mg   200   mg           sodium edetate   20   mg   20   mg           ethanol   2   ml   2   ml           polyoxyethylene stearate   100   mg   100   mg           hydrogenated castor oil 60           sodium benzoate   60   mg   60   mg                flavoring agent   t.a.   t.a.           purified water   a.a.   a.a.                        
(2) Method for Preparation
 
     Liquid dosage forms are prepared in a similar procedure to that described in the general rules for preparation in the “liquid dosage form” section of the Pharmacopeia of Japan using the ingredients shown in Tables 5 and 6. 
     Example 5 
     Evaluation of Serum Lipid Level 
     Test Method 
     (1) Test Substance 
     Pravastatin with a purity of 99.4%, manufactured at Sankyo Co. Ltd., was employed in the study. Riboflavin acetate, d-α-tocopherol acetate, ascorbic acid, and inositol hexanicotinate were purchased from Mitsubishi Tokyo Pharmaceutical Co., Eisai, Nippon Roche K.K., and Shiratori Pharmaceutical Co. Ltd., respectively. 
     (2) Test Animal 
     Male beagle dogs were purchased at 5 months old from Covance Research Products Inc., as the test animals, and were used after quarantine and acclimatization periods of approximately 1 month. 
     (3) Dosage Form, Preparation and Storage of the Dosage Form 
     The required amounts of pravastatin or each combination drug as calculated based on the body weight of each animal were placed in a gelatin capsule (½-ounce volume) purchased from TORPAC Co. Capsules filled with pravastatin were stored in a cold room and with combination drugs at room temperature until use. 
     Combination drugs were put in identical geltin capsules. 
     (4) Route of Administration and Administration Period 
     Capsules filled with pravastatin or combination drugs were orally administered once daily between 9:00 and 12:30 to the test animals. All test animals were fasted 2-3 hr prior to administration. The administration period was 11 successive days. 
     (5) Preparation of Test Samples and Assay Methods 
     Approximately 10 ml of blood were collected from the cephalic vein on 14 and 7 days before administration (2 and one week before the drug administration) and 4, 8, and 12 days after administration of the capsules. The animals were fasted for approximate 18 hr prior to blood collection. Collected blood was placed into test tubes and left for 0.5-1 hr at room temperature. The test tubes were then centrifuged at 3,000 rpm for 10 min to isolate serum. Levels of total cholesterol and ALP in the serum were determined by the CEH-COD-POD and Bessey-Lowry methods, respectively. 
     For quantitative analyses, an automatic analyzer, Monarch (Instrumentation Laboratory), was used. 
     Results 
     Levels of serum lipids following single or combined administration of pravastatin, riboflavin acetate, d-α-tocopherol acetate, ascorbic acid, and inositol hexanicotinate relative to their average serum levels 2 and one week before administration (100) were calculated. Each value represents the mean value calculated from 5 animals. 
     (Effects of Co-administered Pravastatin and Riboflavin Acetate) 
     
       
         
               
               
             
               
               
               
               
             
               
               
               
               
             
           
               
                         TABLE 9 
               
             
             
               
                   
               
               
                   
                     Total Cholesterol Level in Serum 
               
               
                 Test substance 
                                                After Administration 
               
             
          
           
               
                 (mg/kg) 
                 4 days 
                 8 days 
                 12 days 
               
               
                   
               
             
          
           
               
                 Pravastatin alone (2) 
                 93.6 
                 90.0 
                 93.0 
               
               
                 Riboflavin acetate alone (6) 
                 103.9 
                 101.6 
                 100.5 
               
               
                 Pravastatin (2) + 
                 91.4 
                 82.6 
                 85.8 
               
               
                 Riboflavin acetate (6) 
               
               
                   
               
             
          
         
       
     
                                           TABLE 10                   Test substance   ALP level (after administration)            (mg/kg)   4 days   8 days   12 days               Pravastatin alone (2)   97.4   96.7   92.2       Riboflavin acetate alone (6)   98.1   98.8   93.9       Pravastatin (2) +   90.8   89.1   89.5       Riboflavin acetate (6)                    
(Effects of Co-administered Pravastatin and d-α-tocopherol Acetate)
 
                                           TABLE 11                       Total Cholesterol Level in Serum       Test substance   After Administration            (mg/kg)   4 days   8 days   12 days               Pravastatin alone (2)   93.6   90.0   93.0       d-α-tocopherol acetate alone (10)   96.3   92.8   95.9       Pravastatin (2) +   92.8   82.7   79.3       d-α-tocopherol acetate (10)                    
(Effects of Co-administered Pravastatin and Ascorbic Acid)
 
                                                                             TABLE 12                               Total Cholesterol Level in Serum           Test substance   After Administration                (mg/kg)   4 days   8 days   12 days                            Pravastatin alone (2)   93.6   90.0   93.0           Ascorbic acid alone (50)   98.7   98.2   103.4           Pravastatin (2) +   89.4   84.1   80.9           Ascorbic acid (50)                        
(Effects of Co-administered Pravastatin and Inositol Hexanicotinate)
 
                                                             TABLE 13                       Total Cholesterol Level in Serum       Test substance   After Administration            (mg/kg)   4 days   8 days   12 days                    Pravastatin alone (2)   93.6   90.0   93.0       Inositol hexanicotinate (400)   99.2   99.8   100.0       Pravastatin (2) +   86.5   83.3   81.6       Inositol hexanicotinate (400)                    
(Effects of Co-administered Pravastatin, d-α-tocopherol Acetate, and Ascorbic Acid)
 
     
       
         
               
               
             
               
               
               
               
             
           
               
                 TABLE 14 
               
             
             
               
                   
               
               
                   
                 Total Cholesterol Level in Serum 
               
               
                 Test substance 
                  After Administration 
               
             
          
           
               
                 (mg/kg) 
                 4 days 
                 8 days 
                 12 days 
               
               
                   
               
               
                 Pravastatin alone (2) 
                 93.6 
                 90.0 
                 93.0 
               
               
                 d-α-tocopherol acetate alone (10) 
                 97.8 
                 96.4 
                 96.1 
               
               
                 Pravastatin (2) + 
                 89.3 
                 87.8 
                 82.4 
               
               
                 d-α-Aocopherol acetate (10) + 
               
               
                 ascorbic acid (50) 
               
               
                   
               
             
          
         
       
     
     The composition of the present invention comprising a combination of pravastatin and ascorbic acid and/or the like exhibits excellent activity for lowering the total amount of cholesterol in the blood and is useful as an agent for lowering the total amount of cholesterol in the blood. 
     Although the dose of compounds used according to the invention may widely vary depending on the extent of diseases and age of patients, (e.g. a human patient), the dose of one administration of pravastatin is normally within the range of from 0.01 mg/kg to 10 mg/kg, preferably from 0.1 mg/kg to 1 mg/kg, administered once or several times a day depending on the extent of diseases. 
     The dose of one administration of riboflavins is normally within the range of from 0.004 mg/kg to 24 mg/kg, preferably from 0.04 mg/kg to 2.4 mg/kg, administered once or several times a day depending on the extent of diseases. 
     The dose of one administration of tocopherols is normally within the range of from 0.02 mg/kg to 60 mg/kg, preferably from 0.2 mg/kg to 6 mg/kg, administered once or several times a day depending on the extent of diseases. 
     The dose of one administration of ascorbic acids is normally within the range of from 0.1 mg/kg to 400 mg/kg, preferably from 1 mg/kg to 40 mg/kg, administered once or several times a day depending on the extent of diseases. 
     The dose of one administration of inositol hexanicotinate is normally within the range of from 0.16 mg/kg to 36 mg/kg, preferably from 1.6 mg/kg to 3.6 mg/kg, administered once or several times a day depending on the extent of diseases.