CHAPTER 1 Pharmaceutical development, analysis & manufacture of EO-9 15 16 Chapter 1.1 Development of a bladder instillation of the indoloquinone anticancer agent EO-9 using tert-butyl alcohol as lyophilization vehicle S.C. van der Schoot, B. Nuijen, F.M. Flesch, A. Gore, D. Mirejovsky ,L. Lenaz, J.H. Beijnen Submitted for publication 17 18 Chapter 1.1 Abstract EO-9 is a bioreductive alkylating indoloquinone and an analogue of the antitumor antibiotic mitomycin C. EO-9 is an inactive prodrug, which is activated by reduction of the quinone moiety to semiquinone or hydroquinone, generating an intermediate with an electrophilic aziridine ring system, which serves as a target for nucleophilic DNA. For the treatment of superficial bladder cancer a pharmaceutical formulation for a bladder instillation with EO-9 was developed. To improve solubility and stability of EO-9, tert-butyl alcohol was chosen as co-solvent for the solution vehicle in the freeze-drying process. Because EO-9 is most stable at alkaline pH sodium bicarbonate was used as alkalizer. Stability and dissolution studies revealed an optimal formulation solution for freeze-drying composed of 4 mg/ml EO-9, 10 mg/ml sodium bicarbonate, and 25 mg/ml mannitol in 40% v/v tert-butyl alcohol in water for injection. Optimization of the freeze drying process was performed by determination of the freeze drying characteristics of tert-butyl alcohol/water systems and differential scanning calorimetry analysis of the formulation solution. Furthermore, the influence of the freeze drying process on crystallinity and morphology of the freeze dried product was determined with X-ray diffraction analysis and scanning electron microscopy, respectively. Subsequently, a reconstitution solution was developed. A stable bladder instillation was obtained after reconstitution of freeze dried product containing 8 mg of EO-9 per vial to 20 ml with a reconstitution solution composed of propylene glycol/water for injection/sodium bicarbonate/sodium edetate 60/40/2/0.02% v/v/w/w, followed by dilution with water for injection to a final volume of 40ml. This pharmaceutical product of EO-9, named EOquinTM, is currently used in Phase II clinical trials. Introduction EO-9, 3-hydroxymethyl-5-aziridinyl-1-methyl-2-(1H-indole-4,7-dione)-prop-ß-en-α- ol, is a bioreductive alkylating indoloquinone and a synthetic analogue of the antitumor antibiotic mitomycin C (MMC) (Figure I). The use of MMC is limited due to its dose-limiting toxicities. To improve the applicability of MMC, analogues such as EO-9 are developed. EO-9 is an inactive prodrug, like MMC, which is activated by reduction of the quinone moiety to semiquinone or hydroquinone, generating an 19 Chapter 1.1 Figure I. Molecular structure of EO-9 (Mw = 288Da) 15 O 14 OH 3 16 N 5 4 9 11 2 6 13 7 8 N 12 1 CH3 OH O 10 intermediate with an electrophilic aziridine ring system. This electrophilic ring system serves as a target for nucleophilic DNA. Reduction occurs under conditions of either low oxygen tension (hypoxic areas) or in presence of high levels of specific reducing enzymes. Preferential cytotoxicity of EO-9 towards hypoxic versus aerobic tumor cells and lack of bone marrow toxicity were seen 1. Because solid tumors contain hypoxic areas, due to a discrepancy between capillary growth and oxygen need of the growing tumor 2, EO-9 is considered a promising treatment option for those tumors. Furthermore, EO9 is a good substrate for several reducing enzymes, e.g. NADPH:cytochrome P450 3 and DT-diaphorase 4. Therefore, EO-9 may be a promising alkylating indoloquinone for the treatment of cancer. A freeze dried product for intravenous administration of EO-9 was developed by Jonkman-de Vries et al 5. They dissolved EO-9 and lactose in water for injection. Due to its low solubility rate, EO-9 drug substance had to be grinded prior to dissolution. Furthermore, the aziridine moiety of EO-9 is easily protonated in acidic and neutral environment, followed by nucleophilic attack of water and formation of EO-5a. Therefore, Jonkman-de Vries et al. added sodium hydroxide to the formulation solution to pH 9-9.5 and freeze dried aliquots of 40 ml in 50 ml vials, resulting in a product composed of 8 mg EO-9 and 200 mg lactose per vial. However, after intravenous administration, no tumor response of EO-9 was seen 6,7. This is likely due to instability of EO-9 in physiological environment of pH 7.4 (i.e. EO-9 is rapidly converted into the inactive EO-5a before it reaches the tumor)8. To administer EO-9 more directly to the tumor, the route of administration was changed to intravesical administration for the treatment of superficial bladder cancer and a formulation for a bladder instillation had to be developed. A complete new formulation was developed with the aim to resolve the disadvantages of the formulation for intravenous administration (i.e. instability of EO-9 after administration, grinding of a hazardous drug substance and the large filling volume per vial). 20 Chapter 1.1 This paper describes the development of a freeze dried intravesical formulation of EO- 9 with use of the organic solvent tert-butyl alcohol in the manufacturing process to improve solubility and stability of EO-9, resulting in much smaller filling volumes and making grinding of EO-9 redundant. Furthermore, the freeze drying program was optimized by determination of the freeze drying characteristics of tert-butyl alcohol/water systems and by differential scanning calorimetry analysis of the formulation solution. Subsequently, the influence of the freeze drying process on crystallinity of the freeze dried product was determined with X-ray diffraction analysis. Furthermore, a reconstitution solution was designed to obtain a stable bladder instillation. Materials and methods Chemicals EO-9 drug substance was supplied by Spectrum Pharmaceuticals, Inc. (Irvine, CA, United States). Mannitol and sodium bicarbonate (Ph.Eur. grade) were purchased from BUFA (Uitgeest, The Netherlands). Tert-butyl alcohol was obtained from Merck (Darmstadt, Germany). Sterile water for injection was purchased from B. Braun (Melsungen, Germany). Phosphate buffer (5mM) was prepared in-house at the Department of Pharmacy & Pharmacology of the Slotervaart Hospital (Amsterdam, The Netherlands). Methanol was purchased from Biosolve B.V. (Amsterdam, The Netherlands). All chemicals obtained were of analytical grade and used without further purification. High Pressure Liquid Chromatography (HPLC) HPLC analysis was performed using an isocratic P1000 pump, AS 3000 autosampler and an UV 1000 UV/VIS detector, all from Thermo Separation Products (Breda, The Netherlands). The mobile phase consisted of 5mM phosphate buffer pH 7/methanol 70/30% v/v. A Zorbax SB-C18 analytical column (750 x 4.6mm ID, particle size 3.5 µm, Agilent Technologies, Palo Alto, California, USA) preceded by a guard column (reversed phase 10 x 3mm, Varian, Palo Alto, California, USA) was used. Detection was performed at 270 nm. An injection volume of 10 µL, flow rate of 0.7 ml/min and a run time of 10 minutes were used. The calibration curve showed a linear relationship with a correlation coefficient > 0.999. The retention time of EO-9 was approximately 8.2 min. The method showed to be stability indicating with EO-5a, the main degradation product of EO-9, at a relative retention time to EO-9 of 0.68. Therefore, 21 Chapter 1.1 the purity (expressed as the peak area of EO-9 as percentage of the total area of all peaks present in the chromatogram) was taken as stability parameter in this study. Gas chromatography (GC) Traces of residual tert-butyl alcohol (TBA) in freeze dried products were determined by gas chromatographic analysis. The GC system was composed of a Model 5890 gas chromatograph equipped with a flame ionisation detector (FID), a split-splitless injector and a Model 6890 series autosampler (Hewlett Packard). Separation was achieved with a crossbond 6% cyanopropylphenyl - 94% dimethylpolysiloxane (30m x 0.53mm ID x 3.0µm film thickness) column. Calibration solutions of 30-500µg/ml TBA in dimethylsulfoxide (DMSO) were used. A sharp signal of TBA appeared at 2 minutes. A large signal of DMSO was seen at 9.3 minutes. The calibration curve was linear with a correlation coefficient of 0.9999. No internal standard was required. Pharmaceutical formulation development Solubility and stability in tert-butyl alcohol (TBA) The maximum solubility and stability of EO-9 in TBA solutions were assessed. Solutions of 0, 10, 20, 30, 40, and 50% v/v TBA in water for injection (WfI) containing 1% w/v sodium bicarbonate were saturated with EO-9. The pH of the solutions increased with increasing TBA content and varied from pH 8.4 to 9.5 for solutions containing 0% to 50% v/v TBA, respectively. The solutions were shaken for 24 hours at room temperature and ambient light. After 6 and 24 hours samples were filtered using Millex HV filters (0.45µm x 4mm, Millipore, Etten-Leur, The Netherlands) and the content and purity of the filtrates were determined with HPLC- UV. Furthermore, the stability of EO-9 in freeze dried products containing a bulking agent and an alkalizing agent was determined. The bulking agents mannitol (crystalline bulking agent) and polyvinylpyrrolidone (an amorphous bulking agent) and the alkalizing agents sodium bicarbonate, sodium hydroxide and meglumine were tested. Sodium bicarbonate has also been used for bladder instillations of MMC. It was shown that addition of sodium bicarbonate to MMC bladder instillations increased time to recurrence of patients with superficial bladder cancer 9. For each solution the bulking agent, alkalizing agent and EO-9 were dissolved in 40% v/v TBA to concentrations of 25mg/ml, 10mg/ml and 4mg/ml, respectively.