Stability of Bendamustine Solutions: Influence of Sodium Chloride Concentration, Temperature and Container

<jats:title>Abstract</jats:title>                <jats:sec id="j_pthp-2017-0033_s_001_w2aab3b7c35b1b6b1aab1c15b1Aa">                   <jats:title>Background</jats:title>                   <jats:p>Bendamustine is used for the treatment of non-Hodgkin lymphoma, chronic lymphocytic leukaemia and myeloma. The stability of bendamustine is highly dependent on temperature and chloride-ion concentration. Limited stability data are available. The objective of this work was to study the stability of the bendamustine reconstituted solution at 2.5 mg/mL and the diluted solution in normal saline and 1.5 % sodium chloride to evaluate a potential increase in stability.</jats:p>                </jats:sec>                <jats:sec id="j_pthp-2017-0033_s_002_w2aab3b7c35b1b6b1aab1c15b2Aa">                   <jats:title>Methods</jats:title>                   <jats:p>A stability indicating High Performance Liquid Chromatography method with Diode Array Detection was used. A first study was carried out in glass vials and then in polyolefin containers at 0.25 and 0.60 mg/mL. Solutions were stored at room temperature and at 2–8 °C for 7 days.</jats:p>                </jats:sec>                <jats:sec id="j_pthp-2017-0033_s_003_w2aab3b7c35b1b6b1aab1c15b3Aa">                   <jats:title>Results</jats:title>                   <jats:p>Stability was defined as a concentration above 95 % of the initial concentration [10]. The reconstituted solution at 2.5 mg/mL was stable for only 2 hours at room temperature and 8 hours at 2–8 °C. The stability of diluted solutions was in accordance with the manufacturer’s recommendations of 3.5 hours at room temperature and 48 hours at 2–8 °C. The addition of sodium chloride doesn’t increase the stability for preparation in infusion in daily practice.</jats:p>                </jats:sec>                <jats:sec id="j_pthp-2017-0033_s_004_w2aab3b7c35b1b6b1aab1c15b4Aa">                   <jats:title>Conclusions</jats:title>                   <jats:p>The information brought by this study is an 8-hour stability of the reconstituted solution at 2–8 °C.</jats:p>                </jats:sec>

section_content: Bendamustine is an anticancer drug which has an original structure mixing an antimetabolite structure of methylbenzymidazole and a nitrogen mustard structure (Figure 1 ). It is used for the treatment of non-Hodgkin lymphoma, chronic lymphocytic leukaemia and myeloma [1]. The drug is administered as an intravenous infusion diluted in 0. 9 % sodium chloride over 2 hours at doses between 60 and 150 mg/m 2 [1] [2] [3] [4]. 

Bendamustine is available as vials containing either 25 mg or 100 mg of bendamustine hydrochloride powder. The tradename in Europe is Levact ® and several generics are also available. Mannitol is the only excipient in the formulation in all generics. Vials of 25 mg should be reconstituted with 10 mL of water for injection (WFI) and the 100 mg vial with 40 mL to yield a concentration of 2. 5 mg/mL. 

The tradename in North America is Treanda ®. Two presentations are available: as a powder in Europe and a ready-to-dilute solution which contains propyleneglycol and N, N-dimethylacetamide as excipients. However, N, N-dimethylacetamide can react with certain plastics. This drawback has also been seen with the use of Busilvex ® which contains the same excipient and safety alerts have been published by the Food and Drug Administration [5] [6] [7]. 

The manufacturers of the powder in Europe do not give stability data for the reconstituted solution but indicate that "The reconstituted concentrate should be diluted immediately with a 0. 9 % sodium chloride solution". The manufacturers of Treanda ® powder indicate that "The reconstituted solution must be transferred to the infusion bag within 30 minutes of reconstitution". 

About the stability of diluted solutions in 0. 9 % sodium chloride, the European manufacturers indicate 3. 5 hours at room temperature and 48 hours at 2-8 °C [1] [2] [3]. The manufacturer of Treanda ® has more restrictive stability data with 24 hours when stored under refrigerated conditions at 2-8 °C or for 2 hours when stored at room temperature (15-30 °C) and room light after using the ready to dilute solution and 3 hours when using the powder formulation. 

As for melphalan, a compound having a related chemical structure, bendamustine must not be diluted in 5 % dextrose because the stability is dependent on the presence of chloride ions in the solution [8]. 

Krämer et al [9] studied the stability of the solution at 0. 25 mg/mL in 0. 9 % sodium chloride and demonstrated a 9-hour stability at room temperature and a 4-day stability at 2-8 °C. In this study, the stability was defined as not less than 90 % of the initial drug concentration. 

No stability studies have been published about the reconstituted solution. 

The objectives of this work were to study the stability of the reconstituted vials at 2. 5 mg/ml, of the diluted solutions at 0. 25 and 0. 60 mg/mL in 0. 9 % and 1. 5 % sodium chloride to evaluate the influence of the concentration of sodium chloride on the stability. We studied also the influence of the container for the infusion by studying glass and polyolefin containers.
section_name: Introduction

section_name: Materials and methods

section_content: All manipulations were performed under a biological safety cabinet. 

Reconstituted solution: Bendamustine hydrochloride 100 mg vials (Levact ®, batch 90380, Mundipharma, France) were reconstituted with 40 ml of WFI to obtain a 2. 5 mg/mL solution. 

Diluted solutions at 0. 25 and 0. 60 mg/mL: We worked in two steps according to the recommendations of the European Guideline for practical stability studies in oncology [10]. The intrinsic stability profiles were investigated with small volumes of solutions in glass tubes and then in relation with the results, we investigated the stability in infusion bags under selected conditions. 

Stability study in glass tubes: 2 mL of Bendamustine hydrochloride at 2. 5 mg/mL were diluted with 18 ml of sodium chloride 1 % to obtain a 0. 9 % sodium chloride concentration, with 18 ml of 1. 67 % sodium chloride to obtain a 1. 5 % sodium chloride concentration. The test solutions were stored at 2-8 °C with protection from light or at room temperature without protection from light. For each storage condition, 3 solutions were prepared. 

Stability study in polyolefin infusion bags: Freeflex ® 0. 9 % sodium chloride 100 mL (Fresenius, batch 13 ELS) were used. Bendamustine (Levact ® 25 mg batch 94066) were reconstituted with 10 mL of WFI to obtain a 2. 5 mg/mL solution. To take into account the overfilling of the infusion bag (109 mL instead of 100 ml), 19 mL of 0. 9 % sodium chloride were withdrawn from the bag and 10 mL of bendamustine reconstituted solution were injected into the bag to obtain a 0. 25 mg/mL concentration. For the preparation of the concentration at 0. 6 mg/mL, 33 mL were withdrawn and 24 ml of reconstituted solution were injected. For the 0. 25 mg/mL infusions bags, the exact concentration of sodium chloride is 0. 81 g/100 mL and 0. 68 g/mL for the 0. 60 mg/mL infusions bags. Two bags were prepared for each concentration.
section_name: Preparation of test solutions

section_content: Bendamustine hydrochloride concentrations were analysed by a stability indicating reversed-phase high-performance liquid chromatography (RP-HPLC) assay with photodiode array detection adapted from Krämer [9]. 

The HPLC system consisted of an ELITE LaChrom VWR/Hitachi plus autosampler, a VWR photodiode array (PDA) detector L-2455 and a VWR L-2130 HPLCpump. Data were acquired and integrated by using EZChrom Elite (VWR, Agilent). The column used was the ULTISPHERE UP50DB-20QS C18, 120 Å x 4. 6 mm, particle size 5 µ (Interchim, France). The mobile phase consisted of 40 % 0. 02 M potassium sodium sulfate solution [2. 841 g sodium sulfate (Merck, France) dissolved in 1 L of ultrapure water produced by Ultra ionic Purelab (Elga Veolia)] and 60 % methanol [methanol HPLC gradient grade (VWR, France)]. The pH of the sodium sulfate solution was adjusted to pH 3 with 1 mol/L sulphuric acid (VWR, France) by using a pHmeter (Fisher Bioblock Scientific 93,313, France). 

The flow rate was set at 0. 8 mL/minute, with an injection volume of 10 µL. The detection wavelength was set at 331 nm. The temperature of the injector was set at 4 °C and the temperature of the column at 20 °C. Under these conditions, the retention time of bendamustine hydrochloride was about 5. 4 minutes. The calibration curve was constructed from plots of peak area versus concentration. The linearity of the method was evaluated at five concentrations (0. 15, 0. 20, 0. 25, 0. 30 and 0. 35 mg/mL). 

Bendamustine hydrochloride 25 mg (Levact ® ) was reconstituted with 10 mL WFI and then diluted to 25 mL. This solution was used to prepare the standard curve by diluting with 0. 9 % sodium chloride or 3 % sodium chloride. The intraday and interday precisions were evaluated at 0. 15, 0. 25 and 0. 35 mg/mL. 

The stability-indicating capability was evaluated by analysing forced degraded bendamustine hydrochloride solutions. 

Acidic conditions: Solution of 2. 5 mg/mL bendamustine hydrochloride 200 µL were diluted with 500 µL HCl 5N stored at room temperature for 60 minutes, neutralized by 500 µL of NaOH 5N and diluted with 800 µL of WFI to obtain a theoretical concentration of 0. 25 mg/mL. 

Alkali degradation: Solution of 2. 5 mg/mL bendamustine hydrochloride 200 µL were diluted with 500 µL NaOH 0. 01N stored at room temperature for 10 minutes, neutralized by 500 µL of HCl 0. 01N and diluted with 800 µL of WFI to obtain a theoretical concentration of 0. 25 mg/mL. 

Influence of heat: Solutions of 2. 5 mg/mL bendamustine hydrochloride 200 µL were diluted with 1800 µL WFI stored at 60 °C for 10 minutes. 

Influence of UV light: Solutions of 2. 5 mg/mL bendamustine hydrochloride 200 µL were diluted with 1800 µL WFI stored under UV light Vilbert Lourmat ® at 254 nm for 10 minutes. 

Run time was set at 12 minutes. Stability was defined as not less than 95 % of the initial bendamustine concentration [10].
section_name: HPLC assay

section_content: The reconstituted solution at 2. 5 mg/mL was analysed after 1, 2, 3, 4, 5, 6, 7, 8 and 24 hours. 100 µL samples were withdrawn from each test solution and diluted 1:10 with WFI in order to obtain a 0. 25 mg/mL concentration (middle of the standard curve). Immediately after dilution, the samples were analysed by RP-HPLC. 

The solution at 0. 25 mg/mL in 0. 9 % or 1. 5 % sodium chloride was injected directly in the column. The solutions at 0. 6 mg/mL mL in 0. 9 % or 1. 5 % sodium chloride were diluted before analysis (400 µL of the solution with 600 µL of 0. 9 % sodium chloride).
section_name: Sample preparation

section_name: Results

section_content: The calibration curve was linear, the correlation coefficient was 0. 999502. There was no influence of the diluent on the standard curve. The intra-day and inter-day reproducibility were evaluated at 3 concentrations (0. 15, 0. 25 and 0. 35 mg/mL, 6 injections by concentration). 

The intra-day precision expressed as relative standard deviation (RSD) was 1. 50 at 0. 15 mg/mL, 0. 58 at 0. 25 mg/mL and 1. 10 at 0. 35 mg/mL. 

The inter-day precision expressed as relative standard deviation (RSD) was 1. 08 at 0. 15 mg/mL, 0. 48 at 0. 25 mg/mL and 0. 73 at 0. 35 mg/mL. 

Stability indicating capacity was proved by using a technique various stressed conditions. Ten degradation products were detected with retention time at 2. 8, 3. 2, 3. 6, 3. 7, 4. 2, 4. 35, 6. 8, 7, 8. 2 and 8. 6. The retention time of bendamustine was 5. 34. The main degradation product had a retention time at 3. 2 and was probably a hydrolysate compound. Chromatograms obtained after various stressed conditions are presented in Figures 2 3 4. 

The mass balance was evaluated and is presented in Table 1. Peaks present in chromatograms obtains after various stressed conditions were compared with chromatograms of a bendamustine solution at 0. 25 mg/ mL, diluted in 0. 9 % sodium chloride, freshly prepared and stored at room temperature, without light protection.
section_name: Reversed phase HPLC

section_content: Bendamustine reconstituted solutions with WFI at 2. 5 mg/ mL were stable for at least 8 hours at 2-8 °C but only 2 hours at room temperature (Table 2 ). B (for Bendamustine) endamustine diluted with 0. 9 % sodium chloride at 0. 25 mg/mL and 0. 60 mg/mL in glass tubes was stable for at least 24 hours at 2-8 °C but only 4 hours at room temperature (Tables 3 and 4 ). The evolution of the degradation product expressed as the percentage of surface area of total products on the chromatogram are presented in Figure 5 for the bendamustine at 0. 60 mg/mL in 0. 9 % sodium chloride at room temperature and in Figure 6 for the storage at 2-8 °C. 

Bendamustine diluted with 1. 5 % sodium chloride at 0. 25 mg/mL in glass tubes was stable for at least 24 hours at 2-8 °C but only 3 hours at room temperature (Tables 5 and 6 ). The solution at 0. 60 mg/mL was stable for 48 hours at 2-8 °C and 3 hours at room temperature. 

The stability of 0. 25 and 0. 60 mg/mL bendamustine diluted with 0. 9 % sodium chloride in polyolefin bags is presented in Tables 7 and 8.
section_name: Stability of solutions

section_content: The manufacturers in Europe give 3. 5-hour for the stability of diluted solutions in 0. 9 % sodium chloride and 48 hours at 2-8 °C. Based on the T95 % of the initial bendamustine concentration we are in accordance with these recommendations. At 2-8 °C, in polyolefin bags, bendamustine solutions at 0. 25 mg/mL and 0. 60 mg/mL diluted in 0. 9 % sodium chloride are stable 48 hours. The increase of the sodium chloride concentration at 1. 5 % only slightly increased the stability at 0. 60 mg/mL. This was not of sufficient importance to be applied in daily practice. The evolution of the main degradation product shows the influence of temperature with a percentage of 25 % after 24 hours in room temperature instead of 5 % at 2-8 °C. 

Our study demonstrated that the reconstituted solution can be stored for 8 hours between 2 and 8 °C. This data allows the re-use of the reconstituted vial within the day. 

The influence of the plastic container was investigated on only two bags by storage condition because of the cost of the drug at the beginning of the study. The results were the same as in glass vials, there were no interferences with the polyolefin container tested, no adsorption or absorption were detected under the storage condition tested.
section_name: Discussion

section_content: Based on the definition of stability at not less than 95 % of the initial concentration, bendamustine reconstituted solutions at 2. 5 mg/mL were stable for 8 hours at 2-8 °C. 

The increase of the concentration of sodium chloride (1. 5 % versus 0. 9 %) has little influence on the stability of diluted solutions and no application for the practice. 

There was no influence of the polyolefin container on the stability of bendamustine diluted solutions at 0. 25 and 0. 60 mg/mL. No adsorption or absorption of the drug was observed. The stability of diluted solutions were in accordance with the manufacturer's recommendations.
section_name: Conclusion

section_content: Acknowledgements: Assistance with the study: Thank you to Jacques Kuhnlé for reading through it all and making meticulous corrections. Thanks to Nathalie Sobalak for her help and involvement throughout this stability study. Financial support and sponsorship: none

section_content: Conflict of interest statement: 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.