Abstract:
Physically and chemically stable pharmaceutical compositions useful for administering etanidazole by injection are described. These compositions are essentially aqueous solutions having a pH less than or equal to 5.5, and containing etanidazole, a buffer system, and a tonicity-adjusting agent, and they are optionally stabilized by the addition of a stabilizing agent or by autoclaving.

Description:
This application is a continuation of application Ser. No. 07/709,174 filed Jun. 3, 1991, now abandoned. 
    
    
     BACKGROUND OF THE INVENTION 
     The present invention relates to a pharmaceutical composition for administering etanidazole by injection. More particularly, this invention provides solution formulations containing higher concentrations of etanidazole, and having better physical and chemical stability than other known formulations of etanidazole. 
     Formulations of pharmaceutical compositions and processes for preparing them depend upon the properties of the active ingredient, the desired route of administration and the end use to be obtained. Etanidazole is a substituted nitroimidazole that sensitizes tumor cells to radiation therapy. The compound and methods for its synthesis are described in U.S. Pat. No. 3,679,698. Its use as a radiosensitizing agent is described in U.S. Pat. No. 4,371,540. The preferred route of administration of radiosensitizers is by intravenous injection or infusion. The intravenous route of administration affords rapid delivery of the drug to the target tissue, complete bioavailability, and is more predictable and controllable than other routes. Solution formulations for intravenous administration must be essentially free of particulate matter, and they must be sterile. They must be physically and chemically stable, so that efficacy and safety are predictable. Another property generally needed for cancer chemotherapeutic agents, such as etanidazole, is a high concentration of the active ingredient. This is desirable because therapy is often guided toward the maximum tolerated dose. Etanidazole formulations are subject to all of these requirements. 
     Formulations for intravenous administration can be prepared as solutions that are ready to inject or ready to dilute with an infusion solution, or they can be prepared as dry powders that must be dissolved before use. Solution formulations are preferred over dry powders, when feasible, because of ease of use, ease of manufacture, and reduced cost. 
    
    
     BRIEF DESCRIPTION OF THE DRAWING 
     FIG. 1 is a pH Profile for the Hydrolysis of Etanidazole at 80° C. 
    
    
     SUMMARY OF THE INVENTION 
     According to the present invention it has been discovered that pharmaceutical compositions of the drug etanidazole can be prepared that have improved physical and chemical stability, a high concentration of the active drug, and are ready-to-use solutions. These pharmaceutical compositions are useful for intravenous injection or infusion to treat cancer. More particularly, the compositions contain an effective amount of etanidazole, a suitable buffer system selected to give a pH of the final composition of less than 5.5, a tonicity-adjusting agent, and optionally a stabilizing agent, said composition being optionally autoclaved. Advantages of such a composition include, but are not limited to: ease of use, ease of manufacture, reduced cost, increased shelf-life, and a reduced incidence of particulate formation in the product. 
     DETAILED DESCRIPTION OF THE INVENTION 
     Ready-to-use injectable solution formulations of etanidazole with improved chemical and physical stability are preferably composed of an effective amount of etanidazole, a suitable buffer system to yield a final solution pH &lt;5.5, one or more tonicity adjusting agents, and optionally a stabilizing agent selected from the group consisting of imidazole, ethanolamine, diethanolamine, triethylamine, triethanolamine, or ethylenediamine. Said compositions are optionally autoclaved for sterilization. 
     Specifically preferred compositions use a buffer system of citrate, acetate, or phosphate, wherein the pH is 3.5-4.0. The specifically preferred tonicity adjusting agent is sodium chloride. These compositions are preferably terminally sterilized by autoclaving. A typical autoclaving process is to expose the containers of the composition to steam under pressure for at least 15 minutes at a minimum temperature of 121° C. 
     The preferred concentration of etanidazole in the composition is 20-150 mg/ml. Specifically preferred concentrations are 50-100 mg/ml. Preferred concentrations of the stabilizing agents are 0.001% to 5%. Specifically preferred concentrations are 0.05 to 1%. 
     The k values shown in Tables 1-5 and FIG. 1 should be multipled by a factor of 2.303 to provide the actual k value. 
     EXAMPLE 1 
     The chemical stability of etanidazole was evaluated in solutions of varying composition stored at 80° C. Etanidazole was placed into suitable containers and sufficient buffer was added to result in a 1 mg/ml solution. A constant ionic strength of 0.3 was maintained with potassium chloride. All solutions were prepared in triplicate. The solution was divided into 2 ml sealed glass vials and was placed into cardboard storage boxes to protect the compound from light. At appropriate intervals, samples were removed from the stability chamber and cooled to room temperature. An aliquot of the sample was diluted with mobile phase containing internal standard prior to HPLC analysis. For those pH conditions where degradation was rapid, the sample aliquot was immediately quenched to neutral pH and room temperature. 
     Data were analyzed using methods as described by Martin et al. in &#34;Physical Pharmacy&#34;, 3rd ed, pp 352-395 (1983). The degradation of etanidazole followed apparent first-order kinetics. The degradation rate constants (k obs ) were calculated by least squares regression, and are summarized in Table 1. 
     
                       TABLE 1______________________________________Observed First-Order Rate Constants for theSolution Stability of Etanidazole at 80° C.Buffer           k.sub.obs (day.sup.-1)*______________________________________0.3M HCl pH 0.62 0.263      ± 3.84 × 10.sup.-30.1M HCl pH 1.0  9.61 × 10.sup.-2                       ± 3.36 × 10.sup.-30.01M HCl pH 2.2 8.24 × 10.sup.-3                       ± 1.16 × 10.sup.-30.05M Citrate pH 3.0            5.75 × 10.sup.-3                       ± 5.20 × 10.sup.-50.1M Citrate pH 3.0            8.82 × 10.sup.-3                       ± 6.12 × 10.sup.-40.25M Citrate pH 3.0            1.97 × 10.sup.-2                       ± 1.15 × 10.sup.-30.05M Acetate pH 4.0            1.19 × 10.sup.-3                       ± 1.00 × 10.sup.-50.1M Acetate pH 4.0            1.85 × 10.sup.-3                       ± 5.20 × 10.sup.-50.25M Acetate pH 4.0            3.42 × 10.sup.-3                       ± 1.81 × 10.sup.-40.05M Citrate pH 4.5            3.36 × 10.sup.-3                       ± 1.23 ×  10.sup.-40.1M Citrate pH 4.5            5.87 × 10.sup.-3                       ± 4.06 × 10.sup.-40.18M Citrate pH 4.5            7.89 × 10.sup.-3                       ± 6.11 × 10.sup.-40.05M Acetate pH 5.0            3.86 × 10.sup.-3                       ± 2.69 × 10.sup.-50.1M Acetate pH 5.0            5.16 × 10.sup.-3                       ± 1.04 × 10.sup.-40.25M Acetate pH 5.0            8.01 × 10.sup.-3                       ± 1.10 × 10.sup.-40.02M Citrate pH 5.5            8.43 × 10.sup.-3                       ± 3.51 × 10.sup.-50.05M Citrate pH 5.5            9.57 × 10.sup.-3                       ± 7.37 × 10.sup.-50.1M Citrate pH 5.5            1.01 × 10.sup.-2                       ± 2.08 × 10.sup.-40.013M Citrate pH 6.5            1.06 × 10.sup.-2                       ± 1.53 × 10.sup.-40.03M Citrate pH 6.5            1.19 × 10.sup.-2                       ± 2.08 × 10.sup.-40.065M Citrate pH 6.5            1.32 × 10.sup.-2                       ± 00.05M Phosphate pH 6.5            1.35 × 10.sup.-2                       ± 5.77 × 10.sup.-50.1M Phosphate pH 6.5            1.49 × 10.sup.-2                       ± 1.00 × 10.sup.-40.025M Phosphate pH 6.5            1.95 × 10.sup.-2                       ± 8.00 × 10.sup.-50.05M Phosphate pH 7.4            1.68 × 10.sup.-2                       ± 4.07 × 10.sup.-40.1M Phosphate pH 7.4            1.87 × 10.sup.-2                       ± 2.00 × 10.sup.-40.135M Phosphate pH 7.4            1.83 × 10.sup.-2                       ± 3.50 × 10.sup.-40.05M Borate pH 9.2            3.33 × 10.sup.-2                       ± 1.91 × 10.sup.-30.1M Borate pH 9.2            3.82 × 10.sup.-2                       ± 1.08 × 10.sup.-30.25M Borate pH 9.2            5.96 × 10.sup.-2                       ± 5.83 × 10.sup.-40.1M NaOH pH 12.6            13.1       ± 0.3990.3M NaOH pH 13.1            52.9       ± 3.23______________________________________ *Mean ± standard deviation (n = 3) 
    
     In the intermediate pH range where buffers were employed, the observed first-order rate constant can be defined at any given pH with the following equation; 
     
         k.sub.obs +k.sub.H+ [H+]+k.sub.o +k.sub.OH- [OH-]+k.sub.B [B.sub.T ] 
    
     where k H+   and k OH-   are the second-order specific acid and specifiic base catalysis rate constants, respectively k B  is the second-order rate constant for the catalysis due to the buffer, and [B T  ] is the total buffer concentration. Plotting k obs  vs [B T  ] yields a slope of k B  and a y-intercept of k H+  [H+]+k o  +k OH-  [OH-], the observed rate constant extrapolated to zero buffer concentration (k&#39;). The second order rate constants are provided in Table 2. The observed rate constant extrapolated to zero buffer concentration (k&#39;) are used to generate the pH-rate profile (FIG. 1). 
     The buffer systems permit pH control through the equilibrium of their acidic and basic forms. Utilizing these equilibria, the rate constants for the individual buffer species may be calculated. In the acetate buffers, the second-order rate constant for the catalysis due to the acetate buffer is defined as follows; 
     
         .sup.k B=.sup.f CH.sub.3 COOH .sup.k CH.sub.3 COOH+.sup.f CH.sub.3 COO-.sup.k CH.sub.3 COO- 
    
     where  f  CH 3  COOH is the fraction of the acetate buffer in the neutral form,  k  (CH 3  COOH) is the second-order rate constant for the catalysis due to the acetic acid species,  f  CH 3  COO- is the fraction of the acetate buffer in the ionized form and  k  CH 3  COO- is the second-order rate constant for the catalysis due to the acetate anion. Employing the same buffer for various pH conditions permits the determination of the second-order rate constants for the various species (Table 3). 
     The effect of initial etanidazole concentration on the rate of decomposition was examined in 0.05M citrate pH 5.5, at 80° C. Initial concentrations of 1, 25 and 50 mg/ml were employed. The results indicate that the degradation of etanidazole is not concentration dependent in this range (Table 4). 
     
                       TABLE 2______________________________________The Second-Order Rate Constants for the BufferCatalysis and the First-Order Rate Constant Extrapolatedto Zero Buffer Concentration for the Solution Stabilityof Etanidazole at 80° C.Buffer         k&#39; (day.sup.-1)                     k.sub.B (day.sup.-1 M.sup.-1)______________________________________Citrate pH 3.0 2.04 × 10.sup.-3                     7.04 × 10.sup.-2Acetate pH 4.0 6.88 × 10.sup.-4                     1.10 × 10.sup.-2Citrate pH 4.5 1.97 × 10.sup.-3                     3.40 × 10.sup.-2Acetate pH 5.0 2.96 × 10.sup.-3                     2.03 × 10.sup.-2Citrate pH 5.5 8.24 × 10.sup.-3                     1.98 × 10.sup.-2Citrate pH 6.5 1.02 × 10.sup.-2                     4.81 × 10.sup.-2Phosphate pH 6.5          1.19 × 10.sup.-2                     3.02 × 10.sup.-2Phosphate pH 7.4          1.61 × 10.sup.-2                     1.90 × 10.sup.-2Borate pH 9.2  2.58 × 10.sup.-2                     1.34 × 10.sup.-1______________________________________ 
    
     
                       TABLE 3______________________________________The Second-Order Rate Constants for theVarious Buffer Species for the Solution Stability ofEtanidazole at 80°  C.Buffer Species    k.sub.B (day.sup.-1 M.sup.-1)______________________________________Citric Acid       8.89 × 10.sup.-2Dihydrogen citrate.sup.-1             4.58 × 10.sup.-2Hydrogen citrate.sup.-2             7.11 × 10.sup.-3Citrate.sup.-3    8.12 × 10.sup.-2Acetic acid       8.13 × 10.sup.-3Acetic.sup.-1     2.74 × 10.sup.-2Dihydrogen phosphate.sup.-1             3.41 × 10.sup.-2Hydrogen phosphate.sup.-2             1.01 × 10.sup.-2______________________________________ 
    
     
                       TABLE 4______________________________________Effect of Etanidazole Initial Concentrationon the observed First-Order Rate Constants forthe Degradation of Etanidazole in 0.05M Citrate,pH 5.5, at 80°  C.Ionic Concentration (mg/ml)           k.sub.obs (day.sup.-1)______________________________________ 1              9.57 × 10.sup.-3 ± 7.37 × 10.sup.-525              1.27 × 10.sup.-2 ± 5.86 × 10.sup.-450              1.27 × 10.sup.-2 ± 1.53 × 10.sup.-4______________________________________ 
    
     The impact of the ionic strength of the buffer system on the rate of decomposition was examined in 0.1M acetate, pH 5.0, at 80° C. Ionic strengths of 0.15, 0.30 and 0.60 were employed. The results indicate that the increasing the ionic strength results in a negligible effect on the degradation rate of etanidazole (Table 5). 
     In summary, the hydrolysis of etanidazole followed apparent first-order kinetics over the pH range of 0.6 to 12.6, at 80° C. Citrate and acetate were both catalytic at the pH minimum, with citrate being a stronger catalyst than acetate. Concentration and ionic strength had negligible effects on the stability. Analysis of the degradation product indicated that the primary route of degradation is through the hydrolysis of the amide linkage. The overall rate constant was minimum at a pH of approximately 4. 
     FIG. 1. pH-rate profile for the hydrolysis of etanidazole at 80° C. All rate values have been extropolated to zero buffer concentration. 
     
                       TABLE 5______________________________________Effect of Ionic Strength on the ObservedFirst-Order Rate Constant for the Degradation ofEtanidazole in 0.1M Acetate, pH 5.0, at 80° C.Ionic Strength        k.sub.obs (day.sup.-1)______________________________________0.15         5.90 × 10.sup.-3 ± 1.08 × 10.sup.-40.30         5.16 × 10.sup.-3 ± 1.04 × 10.sup.-40.60         5.18 × 10.sup.-3 ± 3.06 × 10.sup.-4______________________________________ 
    
     EXAMPLE 2 
     Solubility studies were carried out by placing excess etanidazole into a suitable container and rotating end-to-end for twenty four hours at 25° C. The suspension was passed through a 0.2μ filter with the first portion discarded to ensure saturation of the filter. An aliquot of the filtrate was diluted and analyzed by HPLC and the remainder of the filtrate was employed for pH determination. 
     Etanidazole was soluble in water at 68.1 mg/ml, pH 6.5. Changes in pH have a negligible effect on the solubility. The solubility was between 59.2 and 71.7 mg/ml over a pH range of 0.72 to 13.2 with no discernible trends in the data. Initially the etanidazole dissolved at concentrations in excess of 150 mg/ml. However, after rotating for 24 hours crystals appeared. 
     The crystals were isolated and characterized. The material appeared needle-shaped. The retention time of the precipitate was in agreement with that of etanidazole. The precipitate contained 5.6% water as determined by Karl Fischer analysis. Thermal analysis of the precipitate revealed two endothermic peaks at 64.1° C. and 142.2° C. Another sample was heated to 100° C. in a vented pan that permitted volatile evolution. The sample pan was cooled to room temperature and reheated to 200° C. The resulting thermogram was comparable to that of the original drug substance, with an endothermic peak at 165.6° C. The additional peak on thermal analysis of the precipitate can be attributed to water of hydration, with the stoichiometry suggesting a monohydrate. 
     Thus, the solubility studies were actually determining the solubility of the more stable monohydrate form of etanizdazole. The solubility increased as function of temperature to 149 mg/ml and 358 mg/ml at 37° C. and 50° C., respectively. 
     EXAMPLE 3 
     Experimental aqueous buffer solution formulations containing 50 mg/ml etanidazole were observed to occasionally develop crystalline particulates when stored at 4° C. Sixteen samples of etanidazole solutions at various pHs, which contained crystals, were heated in a 56° C. water bath for 1 hour to dissolve the crystals. After heating, the samples were divided evenly. Half of the samples were left as non-autoclaved samples and the other half were autoclaved for 15 minutes at 121° C. After 4 days of storage at 4° C., 3 of 8 non-autoclaved samples contained crystals again. Results are summarized as follows. 
     
         ______________________________________              Autoclaved                        Non-AutoclavedBatch #  pH        # Vials   # Vials______________________________________A        4.0       1         1B        5.0       1         1C        5.5       2         3       *(2)D        4.0       l         1       *(1)E        3.5       1F        3.0       2         2______________________________________ *(# vials with subsequent crystal formation) 
    
     Further studies were done in which etanidazole (50 mg/ml) solutions at pH 3.0, 3.5, and 4.0 were prepared and the effect of autoclaving was evaluated. At pH 3.0, 0 of 48 autoclaved samples and 0 of 48 non-autoclaved samples developed crystals when stored at 4° C. over 43 days. At pH 3.5, 0 of 49 autoclaved samples developed crystals, and 3 of 50 non-autoclaved samples developed crystals when stored at 4° C. over 43 days. At pH 4.0, 0 of 50 autoclaved samples developed crystals when stored at 4° C. for 43 days, but 1 of 50 non-autoclaved samples developed crystals. These studies clearly show that autoclaving prevents the formation of crystals when the etanidazole solutions are subsequently stored at 4° C. 
     EXAMPLE 4 
     The effects of additives on the apparent solubility of etanidazole were evaluated. As described in Example 2, without additives etanidazole was initially soluble at concentrations greater than 150 mg/ml, but eventually a precipitate formed which was apparently etanidazole hydrate, and which had a solubility between 59.2 and 71.7 mg/ml. Possible effects of additives are to inhibit the crystallization of etanidazole hydrate and to solubilize etanidazole hydrate. 
     Solutions were prepared containing 50, 100 and 150 mg/ml etanidazole and concentrations in excess of etanidazole solubility, with 1% imidazole, 1% ethanolamine, or 0.1% diethanolamine as additives. Solutions were mixed by rotating end-to-end for 72 hours at room temperature. For sample containing 1% imidazole and excess etanidazole, the excess solid appeared to have been converted to etanidazole hydrate. Solutions containing 1% imidazole and up to 150 mg/ml etanidazole remained clear and free of particulates. These were placed in 4° C. storage. Within 2 hours the 150 mg/ml solution showed crystal formation, but the 50 and 100 mg/ml solutions remained clear for up to 1 month at 4° C. In the case of solutions containing 1% ethanolamine or 0.1% diethanolamine as additives and excess or 150 mg/ml etanidazole, crystalline material had found within 24 hours at room temperature, but the 50 mg/ml and 100 mg/ml solutions remained clear at room temperature for 72 hours. These samples were placed at 4° C. Within 24 hours, solutions containing 100 mg/ml etanidazole and 1% ethanolamine showed crystallization, whereas those containing 50  mg/ml etanidazole remained clear for at least 1 month. Within 24 hours at 4° C., 1 of 3 vials containing solutions of 100 mg/ml etanidazole and 0.1% diethanolamine showed crystallization. After 2 months at 4° C., 1 of the 3 vials remained free of crystals. The 50 mg/ml etanidazole solution with 0.1% diethanolamine remained clear for at least 2 months at 4° C. These results indicate that these additives stabilize etanidazole solutions, inhibiting crystal formation at room temperature or when stored at 4° C. 
     Equilibrium solubility of etanidazole in 0.05M acetate or citrate buffers at pH 4.0 and the effects of additives were determined. Results are summarized as follows. 
     
         ______________________________________                   Solubility at 4° C.Buffer  Additive        mg/ml, mean ± SD)______________________________________Acetate None            83.4 ± 26.3Acetate 0.13% Ethylenediamine                   54.8 ± 30.9Acetate 0.065% Imidazole                   64.8 ± 32.1Citrate None            36.6 ± 6.4Citrate 0.13% Ethylenediamine                   61.5 ± 33.9Citrate 0.065% Imidazole                   95.4 ± 3.7______________________________________ 
    
     Solutions containing acetate buffer, or ethylenediame as an additive, had greatest variability in etanidazole solubility. Imidazole increased etanidazole solubility in citrate buffer. 
     EXAMPLE 5 
     A ready-made solution formulation of etanidazole is made comprising the following: 
     
         ______________________________________              For 1 ml______________________________________Etanidazole          50      mgCitric Acid          2.03    mgSodium Citrate       1.76    mgSodium Chloride      2.12    mgHydrochloric Acid    To adjust pHSodium Hydroxide     To adjust pHWater for Injection  qs ad 1 ml______________________________________Reasonable variations that may be employed:                Range______________________________________Citric Acid          1-10    mg/mlSodium Citrate       1-10    mg/mlSodium Chloride      0-9     mg/ml______________________________________ 
    
     Required volume of the bulk solution is packaged in appropriate vials to obtain 0.5, 1 and 2 g products. Products are autoclaved at 121° C. for 15 minutes.