Abstract:
A crystalline form of epirubicin hydrochloride, named herein as “type II” crystalline epirubicin hydrochloride, has excellent thermal stability. Type II crystalline epirubicin hydrochloride has a powder X-ray diffraction pattern having average values of diffraction angle (2θ) and relative intensity P(%) as presented in the following table:  
                                           Diffraction             Angle   Relative Intensity         2Θ   P(%)                                       5.236   9.8         9.212   12.5         13.732   15.5         16.446   4.8         18.234   5         21.114   9.7         22.529   25.5         24.071   29.9         25.879   18.4         27.762   16.5         29.757   10.1         34.392   4.4         38.157   13.1         44.293   5.9         64.699   7.7         77.815   100

Description:
RELATED APPLICATIONS  
       [0001]     This Application claims the benefit of U.S. provisional Application No. 60/484,132 filed on Jul. 2, 2003. U.S. provisional Application No. 60/484,132 is incorporated by reference as if set forth fully herein. 
     
    
     FIELD OF THE INVENTION  
       [0002]     The field of the invention generally relates to crystalline forms of epirubicin hydrochloride, a compound which is useful as an anticancer chemotherapeutic drug. In particular, the field of the invention relates to a particular crystalline form of epirubicin hydrochloride which is distinguished by its improved thermal stability. In addition, the invention relates to methods of manufacturing the aforementioned crystalline form of epirubicin hydrochloride as well as to methods of using the aforementioned crystalline form of epirubicin hydrochloride to treat human and/or animal cancers.  
       BACKGROUND OF THE INVENTION  
       [0003]     Anthracyclines form one of the largest families of naturally occurring bioactive compounds. Several members of this family have shown to be clinically effective anti-neoplastic agents. These include, for example, daunorubicin, doxorubicin, idarubicin, epirubicin, pirarubicin, zorubicin, aclarubicin, and carminomycin. For instance, these compounds have shown to be useful in bone marrow transplants, stem cell transplantation, treatment of breast carcinoma, acute lymphocytic and non-lymphocytic leukemia, chronic lymphocytic leukemia, non-Hodgkin&#39;s lymphoma, and other solid cancerous tumors.  
         [0004]     U.S. Pat. Nos. 4,112,076, 4,345,068, 4,861,870, 5,945,518, and 5,874,550 disclose the preparation of epirubicin hydrochloride and its usage as an anticancer agent, which is represented by the formula:  
                         
 
         [0005]     Currently, there are two major methods of extraction of epirubicin hydrochloride from solutions. The first method involves the treatment of the organic solution of epirubicin base with a solution of hydrogen chloride in methanol. See e.g., U.S. Pat. No. 4,112,076. Alternatively, the second method involves the precipitation of epirubicin hydrochloride from an aqueous or organo-aqueous solution with the aid of acetone. See e.g., U.S. Pat. No. 4,861,870.  
         [0006]     U.S. Pat. No. 6,087,340 discloses an injectable ready-to-use solution containing epirubicin hydrochloride. More specifically, the &#39;340 patent discloses a stable, injectable, sterile, pyrogen-free, anthracycline glycoside solution which consists essentially of a physiologically acceptable salt of an anthracycline glycoside dissolved in a physiologically acceptable solvent therefore, which has not been reconstituted from a lyophilizate, which has a pH of from 2.5 to 3.5 and which is preferably contained in a sealed glass container. While the &#39;340 patent discloses injectable, ready-to-use preparations, the &#39;340 patent does not disclose the stabilization of epirubicin hydrochloride itself as a bulk drug.  
         [0007]     U.S. Pat. No. 6,376,469 discloses a β-Type form of crystalline amrubicin hydrochloride having improved thermal stability.  
         [0008]     It is desirable to have a crystalline form of epirubicin hydrochloride which has improved thermal stability characteristics. Variation of thermal stability for different crystalline forms of epirubicin hydrochloride is described for the first time herein.  
       SUMMARY OF THE INVENTION  
       [0009]     The present invention relates to a novel, strictly defined, crystalline form of epirubicin hydrochloride, named herein as “type II” crystalline epirubicin hydrochloride, that has excellent thermal stability. Variation of thermal stability for different crystalline forms of epirubicin hydrochloride is described herein.  
         [0010]     Type II crystalline epirubicin hydrochloride is characterized by having a powder X-ray diffraction pattern having average values of diffraction angle (2θ) and relative intensity P(%) as presented in the following table:  
                                                                 Diffraction Angle   Relative Intensity           2Θ   P(%)                                        5.236   9.8           9.212   12.5           13.732   15.5           16.446   4.8           18.234   5           21.114   9.7           22.529   25.5           24.071   29.9           25.879   18.4           27.762   16.5           29.757   10.1           34.392   4.4           38.157   13.1           44.293   5.9           64.699   7.7           77.815   100                      
 
         [0011]     Accordingly, several objects of the present invention are as follows:  
         [0012]     (1) Provide a crystalline form (as well as method of making the same) of epirubicin hydrochloride which is distinguished by improved thermal stability characteristics.  
         [0013]     (2) Provide an extraction method in which epirubicin hydrochloride is crystallized from the aqueous portion of an organo-aqueous solution.  
         [0014]     (3) Provide an extraction method in which crystallization is conducted within the range of 2 to 5 pH.  
         [0015]     (4) Provide an extraction method in which crystallization is conducted at temperatures of 20° C. and above.  
         [0016]     (5) Provide an extraction method in which crystallization is conducted with hydrophilic organic solvents such as alcohols, ketone, nitrites, and their mixtures with branched chains of C 1 -C 4 .  
         [0017]     It thus is an object of the invention to provide a crystalline form (i.e., type II) of epirubicin hydrochloride which is distinguished by other crystalline forms of epirubicin hydrochloride by improved thermal stability characteristics. It is a further object of the invention to provide a method of synthesis for the aforementioned type II crystalline form of epirubicin hydrochloride. 
     
    
     BRIEF DESCRIPTION OF THE DRAWINGS  
       [0018]      FIG. 1  illustrates a graph of the temperature vs. heat flow for type II crystalline epirubicin hydrochloride.  
         [0019]      FIG. 2  illustrates an IR-spectrum plot of type II crystalline epirubicin hydrochloride.  
         [0020]      FIG. 3  illustrates a graph of the temperature vs. heat flow for type I crystalline hydrochloride.  
         [0021]      FIG. 4  illustrates an IR-spectrum plot of type I crystalline epirubicin hydrochloride.  
         [0022]      FIG. 5  illustrates the powder x-ray diffraction spectrum of type II crystalline epirubicin hydrochloride.  
         [0023]      FIG. 6  illustrates the powder x-ray diffraction spectrum of type I crystalline epirubicin hydrochloride.  
         [0024]      FIG. 7  illustrates the powder x-ray diffraction spectrum of type I and II crystalline epirubicin hydrochloride. 
     
    
     DETAILED DESCRIPTION OF THE INVENTION  
       [0025]     The present invention is directed to type II crystalline epirubicin hydrochloride which can be produced by crystallizing epirubicin hydrochloride from a suitable solvent such as, for example, water or mixture of water and a hydrophilic organic solvent. Preferably, crystallization of type II epirubicin hydrochloride is performed at a temperature of 20° C. or above.  
         [0026]     Crystallization is conducted by adding a hydrophilic organic solvent, preferably an alcohol with branched carbon chain C 1 -C 3  to a solution of epirubicin hydrochloride in water or solvent-aqueous mixture. Preferably, the concentration by mass of epirubicin hydrochloride in aqueous or solvent-aqueous solution is from 5% to 50%, and more preferably from 10% to 30%. The pH of the solution is preferably maintained between 2 and 5.  
         [0027]     Volume of the solvent preferably exceeds the volume of the epirubicin hydrochloride solution from 2 to 20 times. The crystallization process is conducted at temperatures from 20° C. to 90° C., preferably from 20° C. to 50° C. Time of crystallization varies from between 0.5 to 12 hours, and more preferably between 2 to 5 hours.  
         [0028]     Type II crystalline epirubicin hydrochloride obtained by this method is extracted by standard procedures known to those of ordinary skill in the art (e.g., vacuum-filtration through the glass filter or centrifugal filtration) followed by drying of the crystals. The produced type II crystalline epirubicin hydrochloride can be used for preparation of the final dosage forms of epirubicin hydrochloride. By way of illustration and not limitation, the type II crystalline epirubicin hydrochloride can be lyophilized (e.g. freeze dried) or dissolved in solution for intravenous injection. For intravenous injection applications, the type II crystalline epirubicin hydrochloride can be dissolved in a suitable carrier or carriers known to those skilled in the art.  
         [0029]     The following two examples disclose methods of producing type II crystalline epirubicin hydrochloride.  
       EXAMPLE 1  
       [0030]     (1) A solution of epirubicin hydrochloride (10.0 grams) in water or in ethanol-in-water mixture (pH 3-4) undergoes low-pressure evaporation at a temperature of 40° C. until a gel state of the solution is achieved.  
         [0031]     (2) 1-propanol in the amount of 12-times the volume of the residual solution is then added to the residual solution and stirred for 3 hours.  
         [0032]     (3) Precipitated crystals of epirubicin hydrochloride are then collected by filtration, washed in 10 ml of acetone and dried at room temperature.  
         [0033]     (4) The result is 9.3 grams of type II epirubicin hydrochloride crystals.  
         [0034]     (5) As seen in  FIG. 1 , the melting point of type II crystalline epirubicin hydrochloride is approximately 207° C. with decomposition (hot stage 2° C./min).  FIG. 2  illustrates the IR-spectrum (IR (KBr)) of type II crystalline epirubicin hydrochloride. Peaks/valleys are seen at 3415, 2928, 1720, 1620, 1576, 1510, 1413, 1371, 1284, 1239, 1210, 1162, 1115, 1068, 1019, 991, 930, 908, 880, 814, 768, 719, 693, 595 cm −1 .  
       EXAMPLE 2  
       [0035]     (1) A solution of epirubicin hydrochloride (10.0 grams) in water or in ethanol-in-water mixture (pH 3-4) undergoes low-pressure evaporation at a temperature of 40° C. until a gel state of the solution is achieved.  
         [0036]     (2) Absolute ethanol in the amount of 10-times the volume of the original solution is then added to the residual solution and stirred for 2 hours.  
         [0037]     (3) Precipitated crystals of epirubicin hydrochloride are then collected by filtration, washed in 10 ml of ethanol and 10 ml of acetone and dried at room temperature.  
         [0038]     (4) The result is 7.5 grams of type II epirubicin hydrochloride crystals.  
         [0039]     The following example (Example 3) discloses a method of producing type I epirubicin hydrochloride crystals, namely epirubicin hydrochloride crystals as described in U.S. Pat. No. 4,861,870.  
       EXAMPLE 3  
       [0040]     (1) Step is identical to step 1 in Example 1 above.  
         [0041]     (2) Gel solution of epirubicin hydrochloride is poured into 300 ml of acetone.  
         [0042]     (3) Precipitated crystals of epirubicin hydrochloride are then collected by filtration and washed in 50 ml of acetone.  
         [0043]     (4) The result is 9.7 grams of type I epirubicin hydrochloride crystals.  
         [0044]     As seen in  FIG. 3 , the melting point of type I crystalline epirubicin hydrochloride is approximately 196° C. with decomposition (hot stage 2° C./min).  FIG. 4  illustrates the IR-spectrum (IR (KBr)) of type I crystalline epirubicin hydrochloride. Peaks/valleys are seen at 3430, 2934, 2027, 1724, 1617, 1583, 1508, 1445, 1412, 1284, 1236, 1211, 1162, 1121, 1064, 1018, 992, 931, 909, 876, 814, 792, 767, 738, 721, 693, 588, and 465 cm −1 .  
       EXAMPLE 4  
       [0045]     Optical Microscopy was performed on type I and II crystalline epirubicin hydrochloride as described below:  
         [0046]     Microscope used: Labomed CXRIII optical microscope with polarizing filters. The samples of epirubicin hydrochloride obtained in Example 1 (type II) and Reference Example 3 (type I) both exhibit birefringence and are, therefore, anisotropic crystals.  
       EXAMPLE 5  
       [0047]     In this example, powder X-ray diffraction spectra of crystalline epirubicin hydrochloride of type I and type II were obtained. Powder X-ray diffraction spectra were measured using a Rigaku Cu Anode X-ray Diffractometer (MiniFlex). The conditions for analysis of the samples was as follows:  
         [0048]     Start angle: 3  
         [0049]     Stop angle: 90  
         [0050]     Sampling: 0.02  
         [0051]     Scan speed: 1.00  
         [0052]     X-ray powder diffraction performed with Copper Kα (λ=1.5406 Å incident X-ray)  
         [0053]     Vertical θ: 2θ Bertrano Parafocusing Diffractometer  
         [0054]     Nil scintillating (Pulse height PMT) detector  
         [0055]     Kβ Nickel filter  
         [0056]     The results of the measured powder X-ray diffraction spectra are as follows:  
         [0057]     The X-ray diffraction patterns are dissimilar for the samples obtained in Example 1 (Type II) and Reference Example 3 (Type I). Table 1 shown below illustrates the type II crystalline epirubicin hydrochloride XRD Analysis-Diffraction Angle (2-Θ) versus Relative Intensity (P %). In contrast, Table 2 shown below illustrates the type I crystalline epirubicin hydrochloride XRD Analysis-Diffraction Angle (2-Θ)) versus Relative Intensity (P %).  
                                                                           TABLE 1                           Crystalline Epirubicin hydrochloride type II XRD Analysis-Diffraction       Angle (2-Θ) versus Relative Intensity (P %).            2Θ   d(A)   BG   Peak   P(%)   Area   FWHM                    5.236   16.8641   22   415   9.8   122   0.234       9.212   9.5918   15   531   12.5   207   0.311       13.732   6.4434   40   658   15.5   211   0.256       16.446   5.3855   122   204   4.8   115   0.449       18.234   4.8614   74   214   5   105   0.39       21.114   4.2042   43   411   9.7   233   0.453       22.529   3.9433   323   1084   25.5   405   0.299       24.071   3.6941   102   1272   29.9   422   0.265       25.879   3.44   75   780   18.4   348   0.357       27.762   3.2108   71   701   16.5   319   0.363       29.757   2.9999   109   428   10.1   150   0.279       34.392   2.6055   67   186   4.4   101   0.434       38.157   2.3566   114   558   13.1   196   0.28       44.293   2.0433   78   249   5.9   91   0.292       64.699   1.4395   19   328   7.7   130   0.316       77.815   1.2264   41   4250   100   1817   0.342                  
 
         [0058]    
       
         
               
             
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
             
           
               
                 TABLE 2 
               
             
             
               
                   
               
               
                   
               
               
                 Crystalline Epirubicin hydrochloride type I XRD Analysis-Diffraction 
               
               
                 Angle (2-Θ) versus Relative Intensity (P %). 
               
             
          
           
               
                 2-Θ 
                 d(A) 
                 BG 
                 Peak 
                 P(%) 
                 Area 
                 FWHM 
               
               
                   
               
             
          
           
               
                 38.236 
                 2.3519 
                 8 
                 1750 
                 47.7 
                 585 
                 0.267 
               
               
                 44.453 
                 2.0363 
                 6 
                 802 
                 21.9 
                 302 
                 0.301 
               
               
                 64.825 
                 1.4371 
                 7 
                 373 
                 10.2 
                 141 
                 0.301 
               
               
                 77.955 
                 1.2246 
                 21 
                 3667 
                 100 
                 1520 
                 0.331 
               
               
                 82.139 
                 1.1725 
                 12 
                 277 
                 7.6 
                 120 
                 0.344 
               
               
                   
               
             
          
         
       
     
         [0059]     Type I crystalline epirubicin hydrochloride gives a single strong signal at approximately 38 degrees. In contrast, type II crystalline epirubicin hydrochloride gives multiple strong signals across the entire spectrum.  FIG. 5  illustrates the powder X-ray diffraction spectrum of type II crystalline epirubicin hydrochloride obtained in Example 1.  FIG. 6  illustrates the powder X-ray diffraction spectrum of type I crystalline epirubicin hydrochloride obtained in Example 3 (Reference).  FIG. 7  shows the superimposed X-ray diffraction spectra of type I and type II crystalline epirubicin hydrochloride.  
       EXAMPLE 6  
       [0060]     The following example illustrates the improved thermal stability of type II crystalline epirubicin hydrochloride as compared to type I crystalline epirubicin hydrochloride.  
         [0061]     The type II crystalline epirubicin hydrochloride obtained in Example 1 and type 1 crystalline epirubicin hydrochloride obtained in reference Example 3 were each kept at a temperature 40° C. for six months, thereby mimicking accelerated storage conditions. The thermal stability was investigated and measured by studying the following parameters: (1) assay (HPLC method), (2) doxorubicinone quantity (doxorubicinone, an aglycone of epirubicin, is the major epirubicin degradation product), and (3) total impurities. The results of this investigation is presented in Tables 3 and 4 listed below.  
         [0062]     Anhydrous and solvent-free basis  
                                                                                     TABLE 3                           Stability Data for type II crystalline epirubicin hydrochloride.                Accelerated storage conditions 40° C. ± 2° C.                Batch ESP01   Batch ESP02   Batch ESP03                    Doxo-   Total           Total           Total       Months   Assay*   Rubicinone   impurities   Assay*   Doxorubicinone   impurities   Assay*   Doxorubicinone   impurities               Initial   99.2   0.04   0.39   99.3   Not detected   0.37   99.0   0.06   0.42       3   99.1   0.07   0.44   99.0   0.06   0.44   99.1   0.12   0.48       6   99.1   0.12   0.50   99.0   0.14   0.51   99.0   0.15   0.53                 *Anhydrous and solvent-free basis             
 
         [0063]    
       
         
               
             
               
               
             
               
               
               
               
             
               
               
               
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
               
               
             
           
               
                 TABLE 4 
               
             
             
               
                   
               
               
                   
               
               
                 Stability Data for type I crystalline epirubicin hydrochloride. 
               
             
          
           
               
                   
                 Accelerated storage conditions 40° C. ± 2° C. 
               
             
          
           
               
                   
                 Sample ESP04 
                 Sample ESP05 
                 Sample ESP06 
               
             
          
           
               
                   
                   
                   
                 Total 
                   
                   
                 Total 
                   
                   
                 Total 
               
               
                 Months 
                 Assay* 
                 Doxorubicinone 
                 impurities 
                 Assay* 
                 Doxorubicinone 
                 impurities 
                 Assay* 
                 Doxorubicinone 
                 impurities 
               
               
                   
               
             
          
           
               
                 Initial 
                 98.8 
                 0.17 
                 0.42 
                 99.0 
                 0.21 
                 0.45 
                 99.2 
                 0.07 
                 0.42 
               
               
                 3 
                 94.3 
                 2.1 
                 2.7 
                 94.0 
                 2.4 
                 3.0 
                 95.1 
                 1.8 
                 2.4 
               
               
                 6 
                 89.0 
                 6.0 
                 7.6 
                 90.1 
                 5.8 
                 7.7 
                 90.2 
                 5.4 
                 6.9 
               
               
                   
               
               
                   *Anhydrous and solvent-free basis    
               
             
          
         
       
     
         [0064]     As the results in Tables 3 and 4 confirm, type II crystalline epirubicin hydrochloride exhibits much greater thermal stability than type I crystalline epirubicin hydrochloride. This is particularly advantageous because the type II crystalline epirubicin hydrochloride will retain its efficacy for a longer period of time as compared to type I crystalline epirubicin hydrochloride because there is less degradation and impurities. This also means that the shelf life of type II crystalline epirubicin hydrochloride is longer than the shelf life of type I crystalline epirubicin hydrochloride.  
         [0065]     While embodiments of the present invention have been shown and described, various modifications may be made without departing from the scope of the present invention. The invention, therefore, should not be limited, except to the following claims, and their equivalents.