Physicochemical stability of pemetrexed diarginine at 25 mg/mL in partially-used vials and at 3 and 12 mg/mL diluted in dextrose 5% or in sodium chloride 0.9% in polyolefin bags : European Journal of Oncology Pharmacy

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Original Research

Physicochemical stability of pemetrexed diarginine at 25 mg/mL in partially-used vials and at 3 and 12 mg/mL diluted in dextrose 5% or in sodium chloride 0.9% in polyolefin bags

Nisse, Yann-Eric PharmaDa; Sobalak, Nathaliea; Vigneron, Jean PharmDa,∗; Demore, Béatrice PharmD, PhDa,b

Author Information
European Journal of Oncology Pharmacy 5(2):p e00037, April-June 2022. | DOI: 10.1097/OP9.0000000000000037
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Abstract

1 Introduction

Pemetrexed is a multi-targeted anti-cancer antifolate agent that exerts its action by disrupting crucial folate-dependent metabolic processes essential for cell replication. Pemetrexed is used for the treatment of malignant pleural mesothelioma and nonsmall cell lung cancer.

The originator Alimta is available as a disodium salt as a powder for concentrates for solutions for infusion. One hundred and 500 mg vials contain mannitol, hydrochloric acid and sodium hydroxide as excipients. The recommended dose of Alimta is 500 mg/m2 of body surface area administered as an intravenous infusion over 10 minutes on the first day of each 21-day cycle.

The manufacturer indicates that 500 mg vials must be reconstituted with 20 mL of 0.9% sodium chloride (0.9% NaCl) solution for injection, without preservative, resulting in a solution containing 25 mg/mL pemetrexed.

The appropriate volume of reconstituted pemetrexed solution must be further diluted to 100 mL with 0.9% 0.9% NaCl solution for injection, without preservative.

The manufacturer indicates that chemical and physical in-use stability of reconstituted and infusion solutions of pemetrexed disodium were demonstrated for 24 hours at refrigerated temperature.[1]

A previous stability study using this originator Alimta has demonstrated a 31-day stability for the reconstituted solution in polypropylene syringes stored at 23 or 4°C.[2] Zhang et al studied the stability of 2, 10, and 20 mg/mL pemetrexed disodium solutions in polyvinylchloride (PVC) infusion bags of NaCl 0.9% or 5% dextrose (D5W) and demonstrated a 2-day stability at 23°C and a 31-day chemical stability at 4°C. However pemetrexed disodium admixtures developed a large number of microparticles during refrigerated storage exceeding 24 hours.[3] Similar results on the chemical stability were found by Rondelot et al[4] after a 28-day study but the subvisual evaluation was not investigated. Zhang and Trissel[5] studied also the stability of frozen solutions and demonstrated a chemical stability after a 90-day storage but a substantial number of microparticles formed.

Zhang and Trissel[3,5] found that the pemetrexed admixtures in PVC bags developed large numbers of microparticulates during long-term frozen storage. Recently, D’Huart et al focused their study on the formation of microparticles at 2 to 8°C in non-PVC infusion bags and demonstrated that potential microparticles have no importance in daily practice and have no negative impact on the patient if the solution is filtered via a 0.22 μm inline microfilter. The chemical stability was not evaluated in this study.[6]

Another stability study performed by Patel et al using the generic pemetrexed disodium Accord demonstrated a 2-day stability for 2.5, 7.5, and 12.5 mg/mL solutions at 23°C and a 14-day stability at 2 to 8°C in polyolefin containers (Viaflo) or polyethylene containers (Ecoflac). In this study, particle count—using a light-blocking counting system in accordance with the Pharmacopoeia method—did not reveal the presence of microparticles, suggesting that the increase in microparticles observed during the early stages could be due to PVC.[7]

A last stability study focused on a new pemetrexed salt, pemetrexed diarginine (OHRE Pharma). The excipients composition is totally different from pemetrexed disodium salt products, including L-arginine, L-cysteine, propyleneglycol, citric acid and water for injection as excipient. Vidal et al demonstrated that pemetrexed diarginine concentrate solutions of 1000 mg/40 mL and 500 mg/20 mL in vials spiked with a closed system were physicochemically stable for up to 14 days under refrigeration storage. Pemetrexed diarginine diluted at 4, 9, and 12 mg/mL in D5W and NaCl 0.9% was physicochemically stable for up to 4 days when stored under refrigeration and for 1 day at room temperature in polyolefin bags.[8]

A pemetrexed diarginine product was recently marketed by Mylan company. The product is a ready-to-dilute 25 mg/mL solution. Three presentations are available: 140 mg/4 mL, 700 mg/20 mL, and 1400 mg/40 mL.[9]

The manufacturer indicates a 24-hour stability at 2 to 8°C after dilution in D5W. To the best of our knowledge, no extended stability studies to prepare the infusions in advance and no stability study of partially used vials are available in the literature for this new product.

The objectives of this work were to study the stability of: 1°/pemetrexed diarginine in D5W or NaCl 0.9% of polyolefin bags at 3 mg/mL and 12 mg/mL protected from light (PFL) at 2 to 8°C and at 25°C; 2°/pemetrexed diarginine in D5W or NaCl 0.9% polyolefin bags at 3 mg/mL and 12 mg/mL not PFL at room temperature; 3°/pemetrexed diarginine vials at 25 mg/mL partially used perforated with a ChemoClave vented Vial Spike PFL at 2 to 8°C and at 25°C; 4°/pemetrexed diarginine vials at 25 mg/mL partially used perforated with a ChemoClave vented Vial Spike not PFL at room temperature.

2 Material and method

2.1 Preparation of test solutions

All manipulations were performed inside a biological safety cabinet.

The used D5W polyolefin bags (MacoPharma, batch 18G10I) and NaCl 0.9% polyolefin bags (MacoPharma, batch 19E07D) were overfilled compared to the theoretical volume of 100 mL. The overfilling of bags depends on the batches used. It was measured in our laboratory by emptying 3 bags into a graduated cylinder and by calculating the average value. In this stability study, the exact volume of the D5W bags was 108 mL and the volume of the NaCl 0.9% bags was 109 mL. The overfillings (8 mL and 9 mL, respectively) were removed in this stability study.

1°/Stability study in D5W polyolefin bags or in NaCl 0.9% polyolefin bags at 3 mg/mL and 12 mg/mL PFL at 2 to 8°C and at 25°C: Easyflex D5W or Easyflex 0.9% NaCl and pemetrexed diarginine 25 mg/mL (Pemetrexed Mylan, batch 1900138 and batch 1900870) were used. For the preparation of the concentrations 3 mg/mL and 12 mg/mL, 12 mL and 48 mL of D5W or NaCl 0.9% were removed from the polyolefin bags, respectively, and then 12 mL and 48 mL of pemetrexed diarginine 25 mg/mL were injected into the bags, respectively. Two bags were prepared for each concentration, each temperature and each batch of pemetrexed diarginine.

2°/Stability study in D5W polyolefin bags or in NaCl 0.9% polyolefin bags at 3 mg/mL and 12 mg/mL not PFL at room temperature (20–25°C): Easyflex D5W or Easyflex 0.9% NaCl and pemetrexed diarginine 25 mg/mL (Pemetrexed Mylan, batch 1900138) were used. For the preparation of the concentration 3 mg/mL and 12 mg/mL, 12 mL and 48 mL of D5W or NaCl 0.9% were removed from the polyolefin bags, respectively, and then 12 mL and 48 mL of pemetrexed diarginine 25 mg/mL were injected into the bags, respectively. One bag was prepared for each concentration and each temperature.

3°/Stability study of pemetrexed diarginine vials at 25 mg/mL PFL at 2 to 8°C and at 25°C: pemetrexed diarginine 25 mg/mL (Pemetrexed Mylan, batch 1900138 and batch 1900870) were used. Each vial was perforated with a vented vial spike with CLAVE (Icumedical, ref CH-70, batch 4095362). Two vials were used for each temperature and each batch.

4°/Stability study of pemetrexed diarginine vials at 25 mg/mL not PFL at room temperature: pemetrexed diarginine 25 mg/mL (Pemetrexed Mylan, batch 1900138 and batch 1900870) were used. Each vial was perforated with a vented vial spike with CLAVE. One vial was used for each temperature and each batch.

2.2 HPLC assay

Pemetrexed concentrations were analysed by a stability-indicating reversed-phase high-performance liquid chromatography (RP-HPLC) method with photodiode array detection adapted from the analytical method used by Rondelot et al.[4]

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 HPLC-pump. Data were acquired and integrated by using EZChrom Elite (VWR, Agilent). The column used was LiChrospher 100 RP-18, LiChroCART 125–4, length 20 cm and particle size 5 μm (Analytical Chromatography, Merck). The mobile phase was 145 mL acetonitrile (VWR, France) and 1.7 mL glacial acetic acid (VWR, France) added to 1000 mL ultrapure water, and the pH was adjusted to 5.3 with sodium hydroxide 30%.

The flow rate was set at 1 mL/min, with an injection volume of 20 μL. The detection wavelength was set at 285 nm. The temperature of the injector was set at 10°C and the temperature of the column at 30°C. The calibration curve was constructed from plots of peak area versus concentration. The linearity of the method was evaluated with 5 concentrations (0.1, 0.2, 0.3, 0.4, 0.5 mg/mL).

Pemetrexed solutions at 25 mg/mL were diluted in mobile phase to prepare standard curves. The intraday repeatability was evaluated as recommended by International Council for Harmonisation Q2 (R1),[10] using 3 determinations at 0.1 mg/mL, 0.3 mg/mL, and 0.5 mg/mL. For interday precision, 3 injections of pemetrexed at 0.1 mg/mL, 0.3 mg/mL, and 0.5 mg/mL were assayed daily on 3 consecutive days. To demonstrate the specificity of the method, solutions for L-arginine 1 mg/mL (Cooper, batch 17070070/A), L-cysteine 1 mg/mL (Cooper, batch 16060179/B), propylene glycol 1 mg/mL (Cooper, batch 17040023/E), and citric acid 1 mg/mL (Alafa Aesar, batch K27Z061), the excipients of Pemetrexed Mylan, and solutions for D5W (MacoPharma, batch 18G10I), NaCl 0.9% (CDM Lavoisier, batch 19E07D) and 5-hydroxymethylfurfural (5-HMF, the major degradation product of D5W) were realised and analysed by HPLC.

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

Alkali degradation: a solution of 1.2 mg/mL pemetrexed 1 mL was diluted with 1 mL NaOH 1 M (VWR, batch 15K250513), stored at 80°C for 2 hours, neutralised by 1 mL of HCl 1 M, and diluted with 1 mL of mobile phase to obtain a theoretical concentration of 0.3 mg/mL.

Acid degradation: a solution of 1.2 mg/mL pemetrexed 1 mL was diluted with 1 mL HCl 1 M (VWR, batch 18G314010), stored at 80°C for 3 hours, neutralised by 1 mL of NaOH 1 M, and diluted with 1 mL of mobile phase to obtain a theoretical concentration of 0.3 mg/mL.

Oxidative degradation: a solution of 1.2 mg/mL pemetrexed 1 mL was diluted with 1 mL H2O2 30% (Merck; batch K48743810 713), stored at 25°C and diluted with 2 mL of mobile phase to obtain a theoretical concentration of 0.3 mg/mL.

UV degradation: a solution of 0.3 mg/mL pemetrexed was exposed during 40 hours under a sun-like spectrum lamp at 254 nm (Vilbert Lourmat).

Heat degradation: a solution of 0.3 mg/mL pemetrexed was exposed to a temperature of 80°C during 16 hours with a tube heater (J. Toulemonde & Cie).

2.2.1 Sample dilution for analysis by RP-HPLC

The solutions were diluted before analysis to obtain approximatively 0.3 mg/mL concentration (middle of the standard curve).

For the pemetrexed solution at 12 mg/mL: 100 μL were diluted with 4.9 mL of D5W or NaCl 0.9% (240 μg/mL). For the pemetrexed solution at 3 mg/mL: 100 μL were diluted with 0.9 mL of D5W or NaCl 0.9% (300 μg/mL). For the ready-to-dilute pemetrexed solution at 25 mg/mL: 100 μL were diluted with 9.9 mL of mobile phase (250 μg/mL).

The solutions PFL stored at 25°C or at 2 to 8°C were analysed after preparation and after 7 and 14 and 28 days. The solutions not PFL stored at room temperature (20–25°C) were analysed after preparation and after 1, 2, 4, and 7 days.

Total run time was set to 20 minutes. Two samples were taken from each bag each day of the assay.

Chemical stability was defined as not less than 95% of the initial pemetrexed concentration.[11]

2.3 pH measurement

pH measurement was performed using a Bioblock Scientific pH meter. Analysis was carried out for each concentration on each day of the assay. pH values were considered to be acceptable if they did not vary by more than 1 pH unit from the initial measurement.[12] pH was not measured for ready-to-dilute solutions due to the small volume available.

2.4 Determination of physical stability

Physical stability was realized with a visual examination: colour changes and particulate matter every day of the assay. The subvisual aspect was assessed by using a Safas Monaco UV mc2 spectrophotometer. The absorbance light was scanned at 350, 410, and 550 nm.[11]

3 Results

3.1 Reversed phase HPLC

The calibration curve was linear, the correlation coefficient was 1.000. The intraday precision expressed as relative standard deviation (RSD) was between 0.23% and 1.97%. The interday precision expressed as RSD was 1.91% at 0.1 mg/mL, 1.17% at 0.3 mg/mL, and 1.15% at 0.5 mg/mL. The absence of interference with L-arginine, L-cysteine, propylene glycol, citric acid, D5W, NaCl 0.9%, and 5-HMF was validated.

The stability indicating capacity was proved by using various stressed conditions. The retention time of pemetrexed was around 5.6 minutes. The chromatogram obtained without stressed conditions is presented in Figure 1 and the chromatogram after acid stressed conditions is presented in Figure 2 for example.

F1
Figure 1:
Chromatogram of 0.3 mg/mL pemetrexed diarginine solution in D5W without stressed conditions. The peak at 4.687 is 5-HMF. 5-HMF = 5-hydroxymethylfurfural, D5W = 5% dextrose.
F2
Figure 2:
Chromatogram of 0.3 mg/mL pemetrexed diarginine solution after acid stressed conditions (HCl 1 M, 80°C, 3 h).

The mass balance was evaluated and is presented in Table 1. Area for exclusion limit was established at 7548.

Table 1 - Mass balance of pemetrexed diarginine solutions after various stressed degradations.
Peak areas

Peaks Retention time (min) Relative retention Without stressed degradation HCl 1 M, 80°C, 3 h NaOH 1 M, 80°C, 2 h H2O2 30% Heat 80°C, 16 h UV-photolysis 254 nm, 40 h
1 2 0.36 67,940 128,254 221,083 295,001 173,388
2 2.18 0.40 238,596 77,703 379,794
3 2.37 0.43 610,536 37,315,944
4 2.48 0.45 10,259 39,594 50,191 16,245
5 2.96 0.54 25,854 40,151 100,330 72,880
6 3.11 0.57 79,289 22,538 1,705,203 132,269 1,663,462 890,585
7 4.01 0.73 18,177
8 4.26 0.77 13,711 27,525
9 4.7 0.85 42,320 37,304 440,781 104,368 55,288
Pemetrexed 5.5 29,711,929 26,209,900 24,820,726 25,631,137 26,222,749 27,527,958
10 6.5 1.18 43,373 20,824
11 8.6 1.56 70,457 8675
12 9.2 1.67 8022 8586
13 10.14 1.84 9208
14 12 2.18 12,291
15 12.42 2.26 10,009 12,966 14,102
16 12.7 2.31 7956
17 13.31 2.42 10,671
18 15.87 2.89 9367 10,358
19 15.9 2.89 219,527
20 16.1 2.93 2,159,171 8282
21 17.8 3.24 11,220
Total mass balance: 30,101,769 28,714,004 28,403,569 63,368,945 28,509,363 28,780,061
% degradation: 11.8% 16.5% 13.7% 11.7% 7.4%

3.2 Chemical stability of solutions

3.2.1 HPLC assay

The percentages of pemetrexed at 3 mg/mL and 12 mg/mL after storage PFL at 2 to 8°C and at 25°C for various time points are shown in Table 2. The percentages of pemetrexed at 3 mg/mL and 12 mg/mL not PFL at 20 to 25°C for various time points are shown in Table 3. The percentages of ready-to-dilute pemetrexed at 25 mg/mL after storage PFL at 2 to 8°C and at 25°C for various time points are shown in Table 4. The percentages of ready-to-dilute pemetrexed at 25 mg/mL after storage not PFL at 20 to 25°C for various time points are shown in Table 5.

Table 2 - Stability of pemetrexed diluted with D5W or NaCl 0.9% at 25°C and 2–8°C protected from light.
% initial concentration remaining ± RSD

Batch number Theoretical concentration (mg/mL) Initial concentration (mg/mL) 7 d 14 d 28 d
2–8°C in D5W
1900138 3 3.1 99.70 ± 1.20 100.21 ± 1.84 97.22 ± 1.53
3 3.1 98.94 ± 1.30 99.26 ± 0.44 97.78 ± 0.33
1900870 3 3.1 99.00 ± 0.56 98.63 ± 0.34 96.57 ± 0.11
3 3.1 100.48 ± 0.27 99.17 ± 0.38 96.96 ± 0.49
1900138 12 12.2 100.31 ± 0.17 99.23 ± 0.31 97.50 ± 0.90
12 12.0 100.91 ± 0.07 100.21 ± 1.84 98.93 ± 0.14
1900870 12 12.2 99.20 ± 0.16 98.87 ± 0.08 98.12 ± 0.39
12 12.0 102.77 ± 0.13 102.31 ± 0.42 100.14 ± 0.72
25°C in D5W
1900138 3 3.1 97.45 ± 1.91 96.76 ± 1.61 93.47 ± 0.54
3 3.1 99.50 ± 0.62 96.03 ± 0.05 95.18 ± 1.00
1900870 3 3.1 101.11 ± 0.24 97.84 ± 0.73 95.25 ± 0.29
3 3.1 99.86 ± 2.10 100.18 ± 0.56 96.43 ± 0.03
1900138 12 11.9 99.81 ± 0.04 96.55 ± 0.22 93.15 ± 0.91
12 11.9 98.47 ± 0.07 96.33 ± 1.05 94.10 ± 0.67
1900870 12 11.9 101.15 ± 0.70 98.76 ± 0.53 95.73 ± 0.17
12 12.1 99.80 ± 0.23 97.88 ± 0.00 94.01 ± 1.07
2–8°C in NaCl 0.9%
1900138 3 3.0 98.93 ± 1.23 98.93 ± 1.23 98.63 ± 0.62
3 3.0 100.07 ± 0.61 100.07 ± 0.61 98.95 ± 0.68
1900870 3 3.1 100.28 ± 0.24 101.16 ± 1.05 98.45 ± 0.46
3 3.1 99.64 ± 0.67 99.14 ± 1.02 99.56 ± 1.10
1900138 12 11.7 101.30 ± 0.55 100.68 ± 0.24 99.13 ± 0.25
12 11.5 102.09 ± 0.55 99.86 ± 0.16 97.96 ± 1.05
1900870 12 12.2 101.77 ± 0.57 102.17 ± 1.18 100.70 ± 0.01
12 12.0 101.52 ± 0.28 100.79 ± 0.61 101.16 ± 0.63
25°C in NaCl 0.9%
1900138 3 3.1 100.38 ± 0.35 99.45 ± 0.00 96.96 ± 0.97
3 3.0 99.63 ± 0.68 98.41 ± 0.66 96.76 ± 1.03
1900870 3 3.1 96.39 ± 0.23 95.13 ± 0.33 93.89 ± 1.15
3 3.1 99.44 ± 1.01 99.04 ± 0.86 99.44 ± 0.06
1900138 12 11.9 96.29 ± 0.57 94.44 ± 1.41 90.02 ± 0.85
12 11.9 96.97 ± 1.95 97.63 ± 0.72 91.23 ± 0.63
1900870 12 11.9 98.43 ± 0.06 92.89 ± 0.19 94.06 ± 1.03
12 11.9 98.56 ± 0.81 93.25 ± 1.58 93.89 ± 1.15
Drug concentrations in samples taken at time zero were designated as 100% (n = 2).D5W = 5% dextrose, RSD = relative standard deviation.

Table 3 - Stability of pemetrexed diluted with D5W or NaCl 0.9% at 20–25°C not protected from light.
% initial concentration remaining ± RSD

Batch number Theoretical concentration (mg/mL) Initial concentration (mg/mL) 1 d 2 d 4 d 7 d
20–25°C in D5W
1900138 3 3.1 99.06 ± 0.07 99.64 ± 0.04 98.49 ± 0.66 98.79 ± 0.24
12 12.0 99.67 ± 0.60 98.72 ± 0.00 98.17 ± 1.90 98.84 ± 0.35
20–25°C in NaCl 0.9%
1900138 3 2.8 99.93 ± 0.19 97.92 ± 1.51 95.34 ± 1.23 99.77 ± 0.14
12 11.3 99.93 ± 1.16 99.41 ± 0.10 98.17 ± 0.86 98.55 ± 2.43
Drug concentrations in samples taken at time zero were designated as 100% (n = 2).D5W = 5% dextrose, RSD = relative standard deviation.

Table 4 - Stability of ready-to-dilute pemetrexed diarginine vial at 25 mg/mL perforated with a vented vial spike at 25°C and 2–8°C protected from light.
% initial concentration remaining ± RSD

Batch number Theoretical concentration (mg/mL) 7 d 14 d 28 d
2–8°C
1900138 25 102.17 ± 0.44 101.48 ± 0.03 102.36 ± 0.74
25 101.72 ± 1.41 99.90 ± 0.98 99.06 ± 0.05
1900870 25 102.62 ± 0.29 100.12 ± 0.47 101.42 ± 0.36
25 99.69 ± 0.12 99.95 ± 0.47 99.54 ± 0.52
25°C
1900138 25 102.12 ± 1.32 100.01 ± 0.52 97.62 ± 0.02
25 100.58 ± 0.28 100.52 ± 0.44 97.99 ± 0.35
1900870 25 100.10 ± 0.01 100.45 ± 0.29 99.69 ± 097
25 100.19 ± 1.02 99.84 ± 0.91 96.90 ± 0.07
Drug concentrations in samples taken at time zero were designated as 100% (n = 2).RSD = relative standard deviation.

Table 5 - Stability of ready-to-dilute pemetrexed diarginine vial at 25 mg/mL perforated with a vented vial spike at 25 mg/mL at 20–25°C not protected from light.
% initial concentration remaining ± RSD

Batch number Theoretical concentration (mg/mL) 1 d 2 d 4 d 7 d
20–25°C
1900138 25 100.49 ± 0.29 100.42 ± 0.60 101.08 ± 0.62 99.29 ± 0.05
1900870 25 101.59 ± 0.34 100.90 ± 0.18 100.46 ± 0.41 99.31 ± 0.50
Drug concentrations in samples taken at time zero were designated as 100% (n = 2).RSD = relative standard deviation.

3.2.2 Identification of degradation products

Two degradation products were observed during the stability study. The relative retentions were 0.40 and 0.57. They were present after the preparation but their peak areas have grown over time (Table 6). The spectra of these degradation products are shown in Figure 3.

Table 6 - Peak area of the 2 degradation products expressed as percentage of the pemetrexed diarginine peak area at T0.
Percentage of the degradation product peak area compared to the pemetrexed peak area at D0

Relative retention Theorical concentration (mg/mL) Solvent Storage condition D0 D4 D7 D14 D28
0.4 3 D5W 2–8°C PFL 1.69 1.71 1.76 1.95
12 D5W 2–8°C PFL 1.93 1.94 1.98 2.17
3 NaCl 0.9% 2–8°C PFL 0.77 0.90 0.88 0.88
12 NaCl 0.9% 2–8°C PFL 0.79 0.96 0.91 0.92
25 2–8°C PFL 0.50 0.69 0.67 0.72
3 D5W 25°C PFL 1.69 1.71 1.76 1.95
12 D5W 25°C PFL 1.95 2.09 2.13 2.89
3 NaCl 0.9% 25°C PFL 0.82 0.92 0.76 0.92
12 NaCl 0.9% 25°C PFL 0.82 0.99 0.86 1.73
25 25°C PFL 0.52 0.77 0.67 0.98
3 D5W 20–25°C not PFL 1.57 1.72 1.84
12 D5W 20–25°C not PFL 1.81 1.88 2.01
3 NaCl 0.9% 20–25°C not PFL 0.62 0.89 0.87
12 NaCl 0.9% 20–25°C not PFL 0.67 0.84 0.98
25 20–25°C not PFL 0.57 0.77 1.00
0.57 3 D5W 2–8°C PFL 0 0.33 0.52 0.92
12 D5W 2–8°C PFL 0 0.35 0.55 0.99
3 NaCl 0.9% 2–8°C PFL 0 0.54 0.53 0.75
12 NaCl 0.9% 2–8°C PFL 0 0 0.70 0.91
25 2–8°C PFL 0 0.20 0.25 0.63
3 D5W 25°C PFL 0 0.89 1.87 3.34
12 D5W 25°C PFL 0 0.85 2.03 3.96
3 NaCl 0.9% 25°C PFL 0 0.90 1.24 2.49
12 NaCl 0.9% 25°C PFL 0 1.08 1.74 5.30
25 25°C PFL 0.22 0.19 0.58 1.09
3 D5W 20–25°C not PFL 0 0.64 1.22
12 D5W 20–25°C not PFL 0 0.50 0.93
3 NaCl 0.9% 20–25°C not PFL 0 0.59 0.93
12 NaCl 0.9% 20–25°C not PFL 0.25 0.78 1.41
25 20–25°C not PFL 0 0.11 0.21
Pemetrexed diarginine was diluted in D5W or NaCl 0.9% for 3 and 12 mg/mL solutions and was not diluted for 25 mg/mL solutions.D5W = 5% dextrose, PFL = protected from light.

F3
Figure 3:
Spectra of the 2 degradation products identified (A: degradation product at relative retention 0.4; B: degradation product at relative retention 0.57).

3.3 Physical stability of solutions

3.3.1 Visual aspect

Pemetrexed diarginine solutions are colourless after the preparation. During the study, all solutions have gradually turned brown-yellow (Fig. 4). The colour intensity was concentration dependent and temperature dependent. At 25°C, a light yellow colour appeared at day 7 in the highest concentrations and the colouration increased over time to an intense brown-yellow colouration on day 28. At 2 to 8°C, a light yellow colour appeared too but was less intense than at 25°C. No precipitate was observed in the different solutions.

F4
Figure 4:
Color change of the pemetrexed solutions (from left to right: 12 mg/mL solution, 3 mg/mL solution, D5W/NaCl 0.9%). D5W = 5% dextrose.

3.3.2 Subvisual aspect

Concerning turbidity assays, no significant change was observed at 550 nm. The absorbance at 350 nm and 410 nm has significantly increased. At 350 nm, the absorbance was between 0.0189 and 0.3165 on day 0 and between 0.0770 and 0.6967 on day 28. At 410 nm, the absorbance was between 0.0068 and 0.0137 on day 0 and between 0.0500 and 0.3837 on day 28.

3.3.3 pH measurements

All samples had a pH in the range of 8.05 to 8.94 during the study. No significant modification of pH was observed during the whole stability study. For all solutions, the maximum variation observed between the day of assay and day 0 was 0.42 pH unit.

4 Discussion

4.1 Reversed phase HPLC

Pemetrexed solutions were analysed by a stability-indicating RP-HPLC method adapted from Rondelot et al.[4] The stability indicating capacity of this method has been proved with forced degradation of pemetrexed solutions in extreme conditions (acidic, alkaline, oxidative, UV-photolysis, and heat conditions). The degradations obtained were moderate and caused a loss of 7% to 17% of the initial concentration according to the recommendations of Bardin et al.[10] The sums of the peak areas of the various degradation conditions are close to 29 millions and close to the value of the freshly prepared solution. The only discordant value comes from oxidative degradation, which has a significant peak from hydrogen peroxide at a relative retention of 0.43. The withdrawal of this peak leads to a value close to 26 millions consistent with the other degradation conditions.

4.2 Chemical stability

The chemical stability was defined as not less than 95% of the initial pemetrexed concentration. This limit was chosen to permit dose banding. Classically, dose banding authorizes a 5% difference between individual patient dose and standard dose.[13] Five percent degradation limit of the product permits also a maximum variation of 10%.

Pemetrexed solutions PFL

  • - at 3 mg/mL and 12 mg/mL in D5W retained more than 95% of the initial concentration for 14 days at 25°C and for 28 days at 2 to 8°C.
  • - at 3 mg/mL and 12 mg/mL in NaCl 0.9% retained more than 95% of the initial concentration for 28 days at 2 to 8°C.
  • - at 3 mg/mL in NaCl 0.9% retained more than 95% of the initial concentration for 14 days at 25°C.
  • - at 12 mg/mL in NaCl 0.9% retained more than 95% of the initial concentration for 7 days at 25°C.

Pemetrexed solutions not PFL at 3 and 12 mg/mL in D5W or NaCl 0.9% retained more than 95% of the initial concentration for 7 days at room temperature.

Ready-to-dilute pemetrexed diarginine vials at 25 mg/mL perforated with a vented vial spike PFL retained more than 95% of the initial concentration for 28 days at 25°C and at 2 to 8°C.

Ready-to-dilute pemetrexed diarginine vials at 25 mg/mL perforated with a vented vial spike not PFL retained more than 95% of the initial concentration for 7 days at room temperature.

4.3 Degradation products

The 2 degradation peaks observed were already observed after the forced degradation in acidic and alkaline conditions for peak at relative retention 0.4 and in acidic, alkaline, heat, photolytic, and oxidative conditions for peak at relative retention 0.57. The increase of the degradation products is correlated with the increase of the yellow-brown colour. Degradation product were not identified in this study. Two previous studies focused on the degradation products of pemetrexed. These are mainly due to the oxidation of pemetrexed. One of the studies describes pale green to yellow degradation products but these studies are not transposable to ours because of difference in mobile phase.[14,15] In this study, the degradation product B at relative retention 0.57 probably cause the yellow coloration.

4.4 Physical stability of solutions

The summary of product characteristics indicates that the solution can be colourless to slightly brown-yellow.[9] The pemetrexed solution at 12 mg/mL at 25°C PFL is slightly yellow on day 7 but on day 14 the yellow-color has significantly increased that's why we propose to limit the stability of this solution to 7 days. One ready-to-dilute pemetrexed diarginine vial at 25 mg/mL perforated with a ChemoClave vented Vial Spike PFL at 25°C has an intense yellow-brown colour on day 14 so we propose to limit the stability of this solution to 7 days.

Ready-to-dilute pemetrexed diarginine vials at 25 mg/mL perforated with a ChemoClave vented Vial Spike not PFL at 25°C were stable for 7 days with a slight yellow color as acceptance criterion. This preservation condition corresponds to conventional storage inside an isolator in which the vial can remain several days at room temperature. This duration of stability can be used for the preparation of a pemetrexed infusion intended to be administered during the day. For the realization of a preparation in advance, the use of a colorless vial is recommended with a stability defined at 4 days in our study with the criterion of absence of coloring retained.

The subvisual examination showed significant change at 350 and 410 nm. The increase of the absorbance can be explained by the increase of the concentration of the degradation products which absorb at 350 and 410 nm. The absorbance at 550 nm remained unchanged showing no increase of the opalescence of the solutions.[11]

All pH variations were less than 1 pH unit and were considered not significant according to the guideline of Sautou.[12] The summary of product characteristics indicates that the ready-to-dilute solutions can have a pH from 8.3 to 9.[9] Some solutions had a pH between 8.05 and 8.3, also lower than the specification. That can be explained by the solvent used, D5W, which has a theoretical pH between 3.5 and 6.5 but usually around pH 4.[16] There are no specifications for pH values of diluted solutions of pemetrexed diarginine in D5W indicated by the manufacturer. These small variations under pH 8.3 are therefore acceptable.

The chemical stability results in this study are consistent with the previous results obtained by Vidal et al. The differences in the conclusions depend on the choice of the definition of stability. Vidal et al[8] defined physical stability as no change in the colour of the solution, whereas in the study, slight coloration was defined as acceptable in accordance with the summary of product characteristics.[9]

5 Conclusion

Pemetrexed diarginine diluted in D5W or in NaCl 0.9% at 3 and 12 mg/mL PFL was stable for 7 days at 25°C and for 28 days at 2 to 8°C. Pemetrexed diarginine diluted in D5W or in NaCl 0.9% at 3 and 12 mg/mL not PFL was stable for 7 days at 20 to 25°C. These stability data of pemetrexed diarginine diluted in D5W or NaCl 0.9% allow advance preparation and minimize drug wastage.

With a slight yellow color as acceptance criterion, ready-to-dilute pemetrexed diarginine at 25 mg/mL perforated with a ChemoClave vented Vial Spike PFL was stable for 7 days at 25°C and for 28 days at 2 to 8°C. The absence of a color change as an acceptance criterion for the 25 mg/mL ready-to-dilute solution perforated with a ChemoClave vented Vial Spike leads to a stability of 7 days at 2 to 8°C and 4 days at room temperature allowing the use of the vial for a preparation in advance with an optimal stability.

These stability data of ready-to-dilute pemetrexed diarginine perforated with a vented vial spike allows considering feasibility of multiple preparations from a single vial for several days.

Acknowledgments

Thank you to Jacques Kuhnlé for reading through it all and making corrections. Thank you to Nathalie Sobalak for her technical assistance and her help during this study. Thanks to French Society of Pharmacy Oncology (SFPO) for help of publication.

References

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Keywords:

chemotherapy; IV administration; pemetrexed diarginine; stability

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