Development and Validation of a New Storage Procedure to Extend the In-Use Stability of Azacitidine in Pharmaceutical Formulations

Abstract

Stability studies performed by the pharmaceutical industry are principally designed to fulfill licensing requirements. Thus, post-dilution or post-reconstitution stability data are frequently limited to 24 h only for bacteriological reasons, regardless of the true physicochemical stability which could, in many cases, be longer. In practice, the pharmacy-based centralized preparation may require preparation in advance for administration, for example, on weekends, holidays, or in general when pharmacies may be closed. We report an innovative strategy for storing resuspended solutions of azacitidine, a well-known chemotherapic agent, for which the manufacturer lists maximum stability of 22 h. By placing the syringe with the azacitidine reconstituted suspension between two refrigerant gel packs and storing it at 4 °C, we found that the concentration of azacitidine remained above 98% of the initial concentration for 48 h, and no change in color nor the physicochemical properties of the suspension were observed throughout the study period. The physicochemical and microbiological properties were evaluated by HPLC-UV and UHPLC-HRMS analysis, FTIR spectroscopy, pH determination, visual and subvisual examination, and sterility assay. The HPLC-UV method used for evaluating the chemical stability of azacitidine was validated according to ICH. Precise control of storage temperature was obtained by a digital data logger. Our study indicates that by changing the storage procedure of azacitidine reconstituted suspension, the usage window of the drug can be significantly extended to a time frame that better copes with its use in the clinical environment.

Keywords: anticancer drugs; azacitidine; drug degradation; in-use stability; limits of use; practical stability.

Figure 1

Chromatogram showing the peak related to water for injection (black line); chromatogram showing the peak related to azacitidine (50 µg/mL) standard solution freshly prepared (blue line); chromatogram of a Vidaza^® (25 mg/mL) preparation subjected to heat stress (violet line). No interfering materials were detected.

Figure 2

Three RGU tautomeric forms.

Figure 3

Path for 5-azacytidine hydrolysis proposed by Notari and Chan with carbinolamine intermediate formation, which is slow at neutral pH.

Figure 4

Comparison of chromatograms between the day the Vidaza^® (25 mg/mL) was reconstituted (a) and the 96 h after the reconstitution (b): we can observe a decrease of the azacitidine peak that favors an increase of the RGU peak.

Figure 5

Typical ESI+ base peak chromatogram of a partially degraded azacitidine sample (a); extracted ion chromatogram (m/z: 263.0986, corresponding to the protonated form of RGU-CHO), showing an additional peak at RT = 4′, attributable to the hydrated form of azacitidine or an RGU-CHO tautomer (b); extracted ion chromatogram (m/z: 235.1037, corresponding to the protonated form of RGU), showing two more peaks attributable to the two RGU tautomers at RT = 2.6′ (c); extracted ion chromatogram (m/z: 245.0881, corresponding to the protonated form of azacitidine) (d).

Figure 6

Trend of the mean percentage of azacitidine lost in the original container over 22 h (red line), in a polypropylene syringe (green line), and in a polypropylene syringe placed between two refrigerant gel packs (blue line) over 96 h. The dotted line represents the maximum 1.86% loss (relative to the initial experimental concentration) identified as the maximum acceptable change of concentration.

Figure 7

Overlap of the IR spectra referring to Vidaza^® (25 mg/mL) at t₀ (blue line), stored in the original container for up to 22 h (red line), in a polypropylene syringe (black line), and in a polypropylene syringe placed between two refrigerant gel packs (green line) for up to 96 h.

Figure 8

Trend of the mean pH of Vidaza^® (25 mg/mL) stored in the original container over 22 h (red line), in a polypropylene syringe (green line), and in a polypropylene syringe placed between two refrigerant gel packs (blue line) over 96 h. The dotted line represents the mean pH in the original container at 22 h.

Figure 9

Morphology of Azacitidine crystals at time zero (a), and after stored Vidaza^® (25 mg/mL) at 2–8 °C in a polypropylene syringe for up to 96 h (b).

Figure 10

Tali^® image of Vidaza^® (25 mg/mL) at time zero (left), and after stored Vidaza^® (25 mg/mL) at 2–8 °C in a polypropylene syringe for up to 96 h (right).

Figure 11

Trend of the temperature of the three Vidaza^® samples (red, green, and blue lines, respectively) stored refrigerated (2–8 °C) between two refrigerant gel packs, obtained placing a temperature data logger into the polypropylene syringes in contact with the drug suspension.

Figure 12

Trend of the temperature inside two refrigerators where the three Vidaza^® lots were placed.

Figure 13

Steps to place syringe containing Vidaza^® suspension between two common refrigerant gel packs: step 1 (left) and step 2 (right).

Figure 14

Percent loss of azacitidine relative to the initial experimental concentration value (red points), and the initial theoretical concentration (blue points) in nine samples at 48 h. The dotted line represents the maximum 1.86% loss (relative to the initial experimental concentration) identified as the maximum acceptable change of concentration.