Azacitidine is a pyrimidine antimetabolite supplied as Vidaza® (Celgene, France). This drug is appropriate for the treatment of various myelodysplastic syndromes [1, 2]. The compound is injected subcutaneously: the administration can be performed at the patient’s home as part of a home care program. Subcutaneous injection and quite easy management of possible adverse side-effects do make Vidaza® an eligible drug for home care program [1–3].
Vidaza® is a lyophilised powder that is reconstituted with sterile water to make a suspension for injection . Yet, azacitidine is unstable in aqueous medium. It is a two-stage degradation. A rapid and reversible hydrolysis occurs, which leads to N-formylribosylguanylurea (RGU-CHO): a balance between the 2 molecules is observed. Then, RGU-CHO is irreversibly hydrolysed into ribosylguanylurea (RGU) (Figure 1) [4–7].
According to the summary of product characteristics (SPC) the suspension can be kept for 45 min at room temperature or for 8 h between 2 and 8 °C. However, when refrigerated water for injection (2–8 °C) is used, the suspension remains stable for another 22 h if stored at 2–8 °C [1, 2]. In fact, azacitidine degradation is a temperature-dependent process: the higher the temperature, the higher the solubility [6, 8–10].
The economic and public health evaluation committee of the French National Authority for Health (HAS) has established that the 100 mg single-use vials do not fit with prescription requirements (75 mg/m2): on average 1.3 vials are needed for an adult, resulting in 2/3 of the second vial being wasted . Celgene therefore committed to the European Medicines Agency to develop a more appropriate packaging . The fact that an optimisation of the packaging has not been carried out so far makes the management of leftovers very difficult and generates considerable financial losses [9, 11].
Published data on azacitidine have reported higher stability than the SPC and a freeze thaw resistance [9, 10, 12, 13]. Vieillard et al shows a 5-day stability at 4 °C . This makes it possible to produce batches that can be used for five-day long treatments. Significant reductions in the costs of production, administration, and operations can therefore be achieved.
The study evaluates the stability of 25 mg/mL azacitidine suspensions kept for 1 month at –20 °C, and then for 5 days at 2–8 °C.
Materials and methods
Three batches of 7 azacitidine syringes were prepared with refrigerated material in a class A isolator by various technicians according to Good Preparation Practices (GPP) and under controlled atmospheric conditions . Every vial of Vidaza® 100 mg was reconstituted with 4 mL of refrigerated sterile water for injection in order to obtain a 25 mg/mL suspension. The content of every vial was transferred into an empty sterile bag in order to improve the homogenisation of the suspension. Seven 3 mL Luer Lock™ (BD Plastipak™, Becton Dickinson, USA) syringes were filled with 2 mL of suspension. Complying with the ICH guidelines, the preparation procedure and syringes were identical to those used in the daily practice of the anticancer preparation unit in the Paris University Hospital Hôtel Dieu (AP-HP) .
When removed out of the isolator, 6 syringes from the batch were immediately placed in a freezer at –20±5 °C (in darkness), whereas the 7th syringe was analysed straight away (Dfab). After 1 month in the freezer (D0), the 6 remaining syringes were defrosted at room temperature for 20 min (resting on the workbench) and then moved into a refrigerator at 5±3 °C (away from light). The temperature levels in both the freezer and the refrigerator were controlled and recorded (Sirius Stockage®, JRI operating software).
The studied physicochemical parameters were: visual aspect of the suspension in the syringes, observation of crystals with the optical microscope (magnification x400, Leica DMLB), turbidity measurements by UV-visible spectroscopy (550 nm, quartz container, Thermo scientific) and determination of concentration. The limit in stability variation was kept at ±5 % as compared with the initial value . Analyses were performed: on the production day (Dfab), after 1 month of freezing (D0) and on a daily basis for 5 days afterwards (D1 to D5).
The azacitidine concentration in the syringes was determined by UV reverse-phase high performance liquid chromatography (UV-visible HPLC) (Dionex). Detection was performed at 200 nm. For every point, after vigorous shaking, two 500-fold dilutions were applied to every syringe in order to reach a target concentration of 50 μg/mL.
The procedure used to assess stability was adapted from Harting et al. . The quantification was conducted with an azacitidine powder (Sigma-Aldrich, France) on a 10–100 µg/mL concentration range (6 concentration levels, 2 dilutions per level). The tests for forced degradation and validation of the procedure (linearity, accuracy, precision: repeatability and intermediate precision, detection limit and quantification limit) were conducted according to the ICH guidelines .
In a Vidaza® vial, mannitol is used as an excipient at a ratio of 1 mg of mannitol for 1 mg of azacitidine . A test under identical conditions was performed with mannitol powder (European Pharmacopoeia quality, Cooper, France): 100 mg of mannitol were diluted in 4 mL of refrigerated sterile water for injection; two 500-fold dilutions were then applied and analysed by UV-visible HPLC.
On the production day, before freezing, all syringes contained a white, milky and uniform suspension. The analysis results were homogeneous for the 3 batches in terms of concentration (24.2 mg/mL, 23.6 mg/mL and 23.9 mg/mL) and turbidity. The chromatogram study showed an azacitidine peak, preceded by 2 other peaks attributed to both its degradation products, RGU-CHO and RGU (Figure 2).
One month after production, D0 azacitidine preparations were removed out of the freezer. The volumes contained in the syringes had increased (+0.1 mL, i. e. +5 %), with a phase separation: one phase very white and milky, the other more translucent. Vigorous shaking resulted into a resuspension . The optical microscopic analysis on D5 showed crystals longer than those observed on D0. The turbidity study did not show any difference during the various stages of the analysis.
Regarding the chromatograms, the RGU-CHO’s area under the curve (AUC) remained relatively stable. On the contrary, a symmetrical evolution of the AUC is observed between the azacitidine and the RGU (Figures 2 and 3). Batches tested with mannitol suspensions revealed no peak.
During the freezing stage, the concentration in azacitidine did not evolve: it remained at an average 99.3 % of the initial concentration. During the refrigeration stage, a concentration loss of 4.5 % on D3, of 6.3 % on D4 and of 8.3 % on D5 occurred (Figure 4).
Although a high temperature can degrade azacitidine, freezing efficiently delays its degradation and refrigeration enables it to be used after freezing. Despite the preliminary freezing, the result obtained in this study is consistent with existing literature. Indeed, regarding the stability between 2 and 8 °C, other studies conducted without any preliminary freezing resulted into stabilities ranging from less than a day to 5 days [9, 10, 12].
The presence of peaks for both degradation products as soon as Dfab can be explained by the immediate hydrolysis of azacitidine since it is reconstituted, despite the precautions taken with the use of refrigerated material via the DPTE® transfer system (Getinge, Sweden). The initial concentration is nearly 5 % below the expected 25 mg/mL; the results of the study are similar to those of Vieillard V. et al and Legeron R. et al but different from those of Walker SE. et al [9, 10, 12].
Regarding the choice of the dosage procedure, the question whether to choose UV wavelength arose. Detection levels at both 240 nm and 200 nm were considered as potential options. The 240-nm level corresponds to the lambda max of both azacitidine and RGU-CHO. However, RGU shows no significant absorption at this wavelength. The other wavelength level is less discriminatory for the first 2 compounds but allows for an appropriate detection for RGU.
The increase in length of the crystals observed on D5 is similar to that described in the study of Vieillard V. et al . Nevertheless, this observation has not been quantified. The possible consequences on safety and efficiency of the drug product have not been assessed in our study.
Conclusion and perspectives
It is possible to produce an anticipated preparation of 25 mg/mL Vidaza® syringes with immediate freezing followed by a 3-day refrigeration between 2 °C and 8 °C. Freezing efficiently delays degradation, and the suspension can be used up to 3 days after defrosting.
Before our study, in the AP-HP HAD (home care program), Vidaza® syringes were delivered and kept between 2–8 °C in the Kalibox® packaging system . Given the azacitidine extended stability when frozen, a distribution network of frozen syringes was established.
In the future, dose-banding could be an option. By determining dose levels, the risks of confusion between different syringes could be eliminated. Moreover, using advance preparations with standard doses would reduce the quantity of leftovers and the corresponding financial losses [9, 11, 18–20].
The authors thank Gerard Gabella for having reviewed the English version.
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About the article
Clara Balouzet is a PharmD candidate. She was resident in the sector of drug manufacturing and control at Paris University hospital Hôtel Dieu. Currently she is resident at the Paris University hospital Pitié Salpêtrière. Her main interests are drug control and risk management.
Cédric Chanat started studying pharmacy at the University of Dijon. Thereafter he was a resident in Paris and graduated as PharmD at the University of Paris Descartes. He worked as a pharmacist assistant in the sector of drug control at Paris University hospital Hôtel Dieu. Currently he is a clinical pharmacist at the Deaconess Saint Simon Cross Hospital Grou being in charge of patients in a home care program. His main interests are correct drug usage and quality processes.
Marion Jobard has been a hospital pharmacist in the sector of drug manufacturing at Paris University hospital Hôtel Dieu since 2013. She is in charge of the quality control unit. She is also involved in quality management in the Pharmacy department. She studied pharmacy at the University of Paris XI (Châtenay-Malabry) where she also completed her training with a Master’s degree in Pharmacology. She is especially interested in drug manufacturing innovations such as robots for automated compounding of intravenous treatments.
Marie-Laure Brandely-Piat studied pharmacy at the University of Paris Descartes. She has been a hospital pharmacist in the Paris University hospital Hôtel-Dieu since 2000. Since 2005 she is responsible for the cytotoxic and ophthalmic preparations unit at the pharmacy. Her special interests concern cancer chemotherapy production, especially automated compounding and eye-drop formulations.
François Chast Pharm D, Ph D in Biopharmacy and Pharmaceutical Sciences, is Head of the Clinical Pharmacy Department of Hôpitaux Universitaires Paris Centre (Hôtel-Dieu, Cochin, Broca – 1, place du Parvis Notre Dame, 75,181 Paris cedex 04) and Associate Professor at the Paris Descartes University in Paris (Clinical Pharmacy and Pharmacokinetics).
He was elected Chairman of the French National Academy of Pharmacy (2010) et Chairman of the Comité d’Education Sanitaire et Sociale de la Pharmacie – Committee for Health and Social Education – (2015). He is also a member of the scientific advisory board of the CNAMTS (French social insurance).
François Chast has developed an extensive activity in hospital compounding, mainly in the field of ophthalmic drugs (immunosuppressive eye-drops as cyclosporine, antibiotic as ceftazidime, antifungal as liposomal amphotericine B, intraocular injectable antibiotics, intravitreous Anti-VEGF). He also developed a laboratory dedicated to microbiota transplant preparations.
Published Online: 2017-03-24
Published in Print: 2017-03-01
Conflict of interest: Authors state no conflict of interest. All authors have read the journal’s Publication ethics and publication malpractice statement available at the journal’s website and hereby confirm that they comply with all its parts applicable to the present scientific work.