Stable pharmaceutical composition of 3-(hydroxymethyl)-5,5-diphenylhydantoin disodium phosphate ester
BACKGROUND OF THE INVENTION 
     5,5-Diphenylhydantoin, phenytoin, is a commonly used drug for the treatment and management of epilepsy and other types of convulsive states. While phenytoin is widely used for the treatment of these conditions, it has an extremely low solubility and, consequently, low bioavailability. Phenytoin is a high melting, weakly acidic drug exhibiting poor solubility in water. These properties lead to erratic absorption after oral dosing with both the free acid and the sodium salt. See the papers by S. A. Varia et al., Journal of Pharmaceutical Sciences 73(8): 1068-190, August 1984. For parenteral use, sodium phenytoin is formulated in an aqueous alkaline medium of pH 12 containing 40% propylene glycol and 10% ethanol. The parenteral dosage form can be painful if the intravenous injection is rapid and the free acid appears to precipitate at intramuscular injection sites. Emergency use of parenteral phenytoin, namely, in cases of controlling seizures in patients with head injuries, may require the administration of the drug intramuscularly. To be clinically acceptable, intramuscular administration of a drug should cause minimal tissue damage at the injection site. Intramuscular administration of sodium phenytoin has been reported to be painful, probably due to the precipitation of phenytoin. It has also been shown to cause hemorrhage, hematoma and necrosis at the injection site in cats and rabbits. 
     U.S. Pat. No. 4,260,769 issued Apr. 7, 1981, and the previously noted articles by S. A. Varia et al. in the Journal of Pharmaceutical Sciences, disclose various prodrugs of phenytoin with more desirable physicochemical properties. In particular, patent 4,260,769 and the noted publications disclose the phenytoin prodrug 3-(hydroxymethyl)-5,5diphenylhydantoin disodium phosphate ester which is shown to have physicochemical properties that are suitable for a prodrug of phenytoin for parenteral use. S. A. Varia and V. J. Stella, at pages 1087-1090 in the Journal of Pharmaceutical Sciences, report that the compound did not exhibit any tissue damage after subcutaneous or intramuscular administration and thus, would be a suitable prodrug candidate for intramuscular delivery of phenytoin. However, this prodrug tends to degrade with the subsequent precipitation of phenytoin. Common methods used to delay the precipitation point involve modifying the formulation to contain agents that might solubilize larger quantities of the degradation product. These agents include alcohol, propylene glycol, L-arginine, sodium desoxycholate, polysorbate-80, and various combinations of these compounds. 
     SUMMARY OF THE INVENTION 
     It has been found that the prodrug, 3-(hydroxymethyl)-5,5-diphenylhydantoin disodium phosphate ester, is stable in an aqueous system when maintained at a pH of about 8.3 to 9.4 to produce diphenylglycinamide as the primary degradant with minimal quantities of phenytoin. A preferred pharmaceutical composition would contain 35 to 130 mg/mL of the prodrug and 0.05 to 0.2 M buffer. 
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
     The stability of the prodrug has always been limited by the occurrence of precipitation in the product. This precipitation has been related to the degradation of prodrug to phenytoin and the subsequent precipitation of phenytoin. 
     A preferred pharmaceutical composition would contain: 
     prodrug: 35 to 180 mg/mL 
     alcohol: USP 0 to 25% 
     propylene glycol: 0 to 25% 
     L-arginine: 0 to 0.2 M 
     sodium desoxycholate: 0 to 0.1 M 
     polysorbate-80: 0 to 1.5% 
     tromethamine: 0.05 to 0.2 M 
     in water for injection with the pH adjusted from 8 to 10 with hydrochloric acid or sodium hydroxide. 
     The pH range found to provide the greatest stability is a pH of about 8.3 to 9.4. In this pH range, the choice of buffers is limited, namely, to buffers effective to maintain a pH of about 8 to 10. In addition to tromethamine, tris-(hydroxymethyl)aminomethane; other buffers which can be used are bicine, N-N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid; tricine, N-tris(hydroxymethyl)methylglycine; sodium bicarbonate; glycylglycine; Hepes, N-2-hydroxyethylpiperazine-N&#39;-2-ethanesulfonic acid; Hepps, N-2-hydroxyethylpiperazine-N&#39;3-propane-sulfonic acid; sodium phosphate; and Taps, 3{[tris(hydroxymethyllmethylamino}propanesulfonic acid. Tromethamine, bicine or tricine are preferred. Suitable wetting agents in addition to polysorbate-80 are: polyoxamer 188 and polyoxyethylene fatty acid esters. A more specific composition would be 75 mg/mL prodrug; 0.1 M tromethamine and a pH of about 8.3 to 9. 
     The following tables illustrate the stability of a composition of the phenytoin prodrug comprising 75.7 mg/mL prodrug, 0.1 M tromethamine, pH adjusted to 9.1 with HCl. 
     
                                           TABLE I                                 
__________________________________________________________________________
MASS BALANCE OF FORMALDEHYDE IN DEGRADED PRODRUG                          
AGE           INITIAL                                                     
                    CURRENT                                               
                           PERCENT AS PRODRUG                             
                                         PERCENT                          
LOT                                                                       
   (MO)                                                                   
       TEMP                                                               
           PH POTENCY                                                     
                    POTENCY                                               
                           DIZ    FORM   RECOVERY                         
__________________________________________________________________________
2131                                                                      
   27  RT  8.9                                                            
              98.22 93.75  0.196  5.553  101.30                           
2140                                                                      
   27  RT  8.9                                                            
              96.42 92.13  0.172  5.042  100.96                           
2183                                                                      
   24  RT  8.8                                                            
              98.06 94.30  0.130  4.450  100.84                           
2183                                                                      
   24  RT  8.8                                                            
              98.06 94.30  0.135  4.314  100.70                           
2211                                                                      
   23  RT  8.3                                                            
              104.52                                                      
                    103.38 0.033  1.509  100.38                           
2224                                                                      
   23  RT  8.9                                                            
              103.75                                                      
                    97.24  0.155  4.349  98.07                            
2293                                                                      
   16  RT  8.5                                                            
              100.91                                                      
                    99.11  0.055  1.747  100.00                           
2295                                                                      
   16  RT  8.3                                                            
              101.49                                                      
                    99.04  0.040  1.303  98.91                            
2327                                                                      
    9  40  8.9                                                            
              100.42                                                      
                    93.74  0.382  7.893  101.59                           
2327                                                                      
    8  40  8.9                                                            
              101.41                                                      
                    94.88  0.342  7.450  101.24                           
2358                                                                      
    7  40  8.9                                                            
              98.70 94.34  0.251  6.653  102.58                           
2360                                                                      
    7  40  8.9                                                            
              99.63 94.61  0.246  5.364  100.59                           
__________________________________________________________________________
 DIZ = 5,5DIPHENYL-4-IMIDAZOLIDINONE                                      
 FORM = FORMALDEHYDE                                                      
 
    
     
                                           TABLE II                                
__________________________________________________________________________
MASS BALANCE OF THE 75 MG/ML PHENYTOIN PRODRUG USING METHOD I             
                          DEGRADATION PRODUCTS (PERCENT                   
AGE          INITIAL     AS PRODRUG)           PERCENT                    
LOT                                                                       
   (MO)                                                                   
       TEMP                                                               
           pH                                                             
             POTENCY                                                      
                   POTENCY                                                
                         PHEN                                             
                             DPG BZP                                      
                                    DIZ                                   
                                       UNKB                               
                                           FORM                           
                                               RECOVERY                   
__________________________________________________________________________
2131                                                                      
   27  RT  8.8                                                            
             98.22 93.75 0.211                                            
                             0.295                                        
                                 0.001                                    
                                    0.196                                 
                                       5.046                              
                                           5.553                          
                                               101.30                     
2140                                                                      
   27  RT  8.8                                                            
             96.42 92.13 0.189                                            
                             0.245                                        
                                 0.004                                    
                                    0.172                                 
                                       4.604                              
                                           5.042                          
                                               100.96                     
2183                                                                      
   24  RT  8.8                                                            
             98.06 94.30 0.131                                            
                             0.053                                        
                                 0.002                                    
                                    0.130                                 
                                       4.264                              
                                           4.450                          
                                               100.84                     
2183                                                                      
   24  RT  8.8                                                            
             98.06 95.57 0.130                                            
                             0.049                                        
                                 0.003                                    
                                    0.135                                 
                                       4.132                              
                                           4.314                          
                                               102.00                     
2211                                                                      
   23  RT  8.3                                                            
             104.52                                                       
                   103.38                                                 
                         1.016                                            
                             0.011                                        
                                 0.0003                                   
                                    0.033                                 
                                       0.482                              
                                           1.509                          
                                               100.38                     
2224                                                                      
   23  RT  8.9                                                            
             103.75                                                       
                   97.24 0.106                                            
                             0.047                                        
                                 0.0015                                   
                                    0.155                                 
                                       4.195                              
                                           4.349                          
                                               98.07                      
2293                                                                      
   16  RT  8.5                                                            
             100.91                                                       
                   99.11 0.053                                            
                             0.000                                        
                                 0.0005                                   
                                    0.055                                 
                                       1.694                              
                                           1.747                          
                                               100.00                     
2295                                                                      
   16  RT  8.3                                                            
             101.49                                                       
                   99.04 0.059                                            
                             0.000                                        
                                 0.0006                                   
                                    0.040                                 
                                       1.243                              
                                           1.303                          
                                               98.91                      
2327                                                                      
   9   40  8.8                                                            
             100.42                                                       
                   93.74 1.485                                            
                             1.147                                        
                                 0.013                                    
                                    0.382                                 
                                       5.248                              
                                           7.893                          
                                               101.59                     
2327                                                                      
   8   40  8.8                                                            
             101.41                                                       
                   94.88 1.071                                            
                             0.746                                        
                                 0.014                                    
                                    0.342                                 
                                       5.619                              
                                           7.450                          
                                               101.24                     
2358                                                                      
   7   40  8.8                                                            
             98.70 94.34 0.769                                            
                             0.404                                        
                                 0.006                                    
                                    0.251                                 
                                       5.474                              
                                           6.653                          
                                               102.58                     
2360                                                                      
   7   40  8.8                                                            
             99.63 94.61 0.800                                            
                             0.419                                        
                                 0.005                                    
                                    0.246                                 
                                       4.140                              
                                           5.364                          
                                               100.59                     
AVERAGE RECOVERY                               100.71                     
__________________________________________________________________________
 PHEN = PHENYTOIN                                                         
 DPG = DIPHENYLGLYCINE                                                    
 BZP = BENZOPHENONE                                                       
 DIZ = 5,5DIPHENYL-4-IMIDAZOLIDINONE                                      
 UNKB* = UNKNOWN B, CALCULATED AS FOLLOWS: UNKB = FORM  PHEN  DPG  BZP    
 FORM = FORMALDEHYDE                                                      
 NOTE: Unknown B (UNKB) has been identified as diphenylglycinamide        
 
    
     
                                           TABLE III                               
__________________________________________________________________________
NEW MASS BALANCE OF 75 MG/ML PRODRUG USING METHOD II                      
                    DEGRADATION                                           
             9653   PRODUCTS                                              
AGE          PERCENT                                                      
                    (PERCENT AS PRODRUG)                                  
                                   PERCENT                                
LOT                                                                       
   (MO)                                                                   
       TEMP                                                               
           pH                                                             
             OF INITIAL                                                   
                    PHEN                                                  
                        DPG DIZ                                           
                               DPGA                                       
                                   RECOVERY                               
__________________________________________________________________________
2131                                                                      
   30  RT  8.8                                                            
             93.06  0.141                                                 
                        0.243                                             
                            0.174                                         
                               5.144                                      
                                    98.76                                 
2140                                                                      
   30  RT  8.8                                                            
             94.79  0.148                                                 
                        0.247                                             
                            0.182                                         
                               5.240                                      
                                   100.61                                 
2183                                                                      
   27  RT  8.8                                                            
             98.40  ND  0.116                                             
                            0.137                                         
                               3.021                                      
                                   101.67                                 
2211                                                                      
   25  RT  8.3                                                            
             98.64  0.097                                                 
                        0.041                                             
                            0.027                                         
                               1.254                                      
                                   100.06                                 
2224                                                                      
   24  RT  8.8                                                            
             94.85  0.104                                                 
                        0.110                                             
                            0.163                                         
                               3.810                                      
                                    99.04                                 
2293                                                                      
   18  RT  8.5                                                            
             98.70  ND  0.080                                             
                            ND 1.631                                      
                                   100.41                                 
2295                                                                      
   18  RT  8.3                                                            
             98.53  ND  0.032                                             
                            0.034                                         
                               1.163                                      
                                    99.76                                 
2327                                                                      
   10  40  8.8                                                            
             92.11  1.491                                                 
                        1.224                                             
                            0.437                                         
                               5.068                                      
                                   100.33                                 
2358                                                                      
    9  40  8.8                                                            
             93.21  1.110                                                 
                        0.757                                             
                            0.329                                         
                               4.153                                      
                                    99.56                                 
2360                                                                      
    9  40  8.8                                                            
             92.34  1.157                                                 
                        0.766                                             
                            0.323                                         
                               4.115                                      
                                    98.70                                 
AVERAGE RECOVERY                   99.89                                  
__________________________________________________________________________
 PHEN = PHENYTOIN                                                         
 DPG = DIPHENYLGLYCINE                                                    
 DIZ = 5,5DIPHENYL-4-IMIDAZOLIDINONE                                      
 DPGA = DIPHENYLGLYCINAMIDE (UNKB in Table II)                            
 ND = NONE DETECTED                                                       
 
    
     Initially, it was proposed that phenytoin prodrug decomposed via a simple two-step pathway resulting in phenytoin. However, it has been determined that phenytoin is not the only decomposition product and could only account for a small portion of the degraded prodrug. The degradation pathway has been determined to proceed from phenytoin prodrug to formaldehyde, 5,5-diphenyl-4-imidazolidinone (DIZ), diphenylglycinamide, diphenylglycine, benzophenone, and phenytoin via the pathways described. 
     Overall Degradation Pathway(s) 
     The overall degradation pathway is shown in Scheme V. Many of the steps involved include consumption of hydroxyl, indicating that the pH of the formulated product should decrease with time. This has been observed in the routine stability program for this product. The addition of a buffer to prevent the pH from dropping significantly from its initial value over the lifetime of the product is required. Small changes in the degradation pathway occur as the pH drops. It is seen that as the pH drops the rate of phenytoin formation increases and the rate of DIZ formation decreases. This indicates that the proposed hydantoin ring opening by the phosphate appears to be the rate limiting step in the primary degradation pathway of phenytoin prodrug. However, formation of phenytoin increases and the solubility of phenytoin decreases as the pH is lowered thus, diminishing the shelf-life of the product due to the saturation of the aqueous solution and eventual precipitation of phenytoin. The chosen pH of the finished product allows the degradation to proceed such that the primary degradant is diphenylglycinamide, while minimal quantities of phenytoin are produced and shelf-life is maximized. 
     Mass Balance Determination 
     Several samples at room temperature and 40° C were analyzed to determine mass balance. Analysis of phenytoin prodrug potency, formaldehyde, 2,2-diphenylglycine, 5,5-diphenylhydantoin, 5,5-diphenyl4-imidazolidinone and benzophenone concentrations were performed using the gradient HPLC method. Analysis of phenytoin prodrug potency, 2,2-diphenylglycine, 2,2-diphenylglycinamide (as 2,2-diphenylglycine). 5,5-diphenylhydantoin, and 5,5-diphenyl-4-imidazolidinone concentrations were determined using the isocratic HPLC method. Molar equivalents were calculated and mass balance results were determined and compared. 
     Method Statistics and Comparison 
     Method I--Gradient Method 
     Several standard curves of each compound were chromatographed for the determination of method linearity. The peak areas and heights were plotted versus respective concentrations to yield calibration curves. All standard curves typically had a correlation coefficient of &gt;0.99. Standard peak heights or areas were shown to be within 4% of the linear regression line thereby passing pre-established system suitability requirements. Replicate injections of a standard solution of each compound were chromatographed. Relative standard deviations were less than 1.7% using peak areas and less than 2.2% using peak heights. These results confirm the method is precise. 
     The same dilutions of standards were used for determination of limit of detection and limit of quantitation. 
     All standards were chromatographed. Peak areas were measured by the data system and linearity was confirmed. Data from standard curve injections before and after a set of sample injections were collected to determine the suitability and effectiveness of the HPLC system. Limits of quantitation were experimentally determined and are shown below. 
     
         ______________________________________                                    
                 Peak Area                                                
                          Peak Height                                     
______________________________________                                    
Diphenylglycine    8.3 μg/mL                                           
                              27.7 μg/mL                               
Diphenylhydantoin  0.5 μg/mL                                           
                              3.0 μg/mL                                
Diphenyl-4-imidazolidinone                                                
                   8.9 μg/mL                                           
                              1.5 μ/mL                                 
Benzophenone       0.05 μg/mL                                          
                              0.05 μg/mL                               
______________________________________                                    
 
    
     Limits of detection were experimentally determined and are shown below. 
     
         ______________________________________                                    
                Peak Area or Height                                       
______________________________________                                    
Diphenylglycine    1.0 μg/mL                                           
Diphenylhydantoin  0.3 μg/mL                                           
Diphenyl-4-imidazolidinone                                                
                   0.5 μg/mL                                           
Benzophenone      0.01 μg/mL                                           
______________________________________                                    
 
    
     A combination standard curve containing 2,2-diphenylglycine, 5,5-diphenylhydantoin and 5,5-diphenyl-4-imidazolidinone was chromatographed several times for the determination of equality with individual standard curves. The peak areas and heights were plotted versus respective concentrations to yield calibration curves. Slopes and y-intercepts were compared to individual standards, confirming their equality. Replicate injections of a combination standard solution were chromatographed. Relative standard deviations were less than 1.7% using peak areas and less than 2.0% using peak heights with the exception of diphenylglycine, which was 3.09% using peak heights. This confirms the method is precise using the combination standard curve. 
     Mass Balance 
     Several samples kept at various lengths of time at room temperature and 40° C. were analyzed to determine mass balance. Initial and current potency, along with current formaldehyde concentration data, were used to determine mass balance. Diphenylglycine, diphenylhydantoin, diphenyl-4-imidazolidinone and benzophenone concentrations were chromatographically determined using Method I. From this data, mass balance was calculated. Initial and current potency values were converted to initial and current moles of phenytoin prodrug. Formaldehyde, diphenylglycine, diphenylhydantoin, diphenyl-4-imidazolidinone and benzophenone concentrations were converted to molar equivalents. Using the value of moles of formaldehyde, diphenylglycine, diphenylhydantoin and benzophenone, the moles of Unknown B (which later was determined to be diphenylglycinamide) were calculated using Equation 1. ##EQU1## The percent recovery of 12 samples analyzed was 98.0 to 102.6% thereby proving mass balance (Table I). 
     Method II - Isocratic Method 
     Linearity 
     Standard curves were prepared and injected to determine the linearity of the isocratic method. In each case linear regression lines for diphenylglycine, 5,5-diphenyl-4-imidazolidinone and phenytoin had correlation coefficients of greater than 0.999. Diphenylglycinamide was identified as a compound of degradation, previously referred to as Unknown B. As no standard for diphenylglycinamide is available, the curve for diphenylglycine was used to determine the concentrations of the samples. 
     Precision of the isocratic method was performed only by analysis of several lots of degraded prodrug and comparing the results to the results of the gradient method (see Tables II and III). The results showed very favorable comparisons for all the degradants. The major difference is seen in the diphenylglycinamide (previously referred to as Unknown B) levels. As it was quantitated using diphenylglycine as a standard rather than by difference (Equation 1), a slight decrease was observed. These results indicate that quantitation by difference is only affected by the variation in the formaldehyde assay. ##STR1##