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Stability of pemetrexed diarginine concentrates for solution in vials and diluted in 0.9% sodium chloride and dextrose 5% polyolefin infusion bags
  1. Fabrice Vidal,
  2. Camille Cotteret,
  3. Abdel Negbane,
  4. Maria Sebti,
  5. Mélanie Hinterlang,
  6. Salvatore Cisternino,
  7. Joël Schlatter
  1. Hopital universitaire Necker-Enfants malades, Paris, Île-de-France, France
  1. Correspondence to Dr Joël Schlatter, Hopital universitaire Necker-Enfants malades, 75015 Paris, Île-de-France, France; joel.schlatter{at}aphp.fr

Abstract

Objectives To determine the physicochemical stability of pemetrexed diarginine in original vials, and after dilution in two commonly used infusion fluids (0.9% sodium chloride, 5% dextrose) in polyolefin bags, stored under refrigeration (2–8°C) or at ambient temperature (22–25°C) exposed to light.

Methods Stability of pemetrexed diarginine injection concentrate was determined in the original glass vials with closed-system transfer device. Diluted pemetrexed diarginine infusion solutions were aseptically prepared by dilution of pemetrexed diarginine concentrate with either 0.9% sodium chloride or dextrose 5% in polyolefin bags, in amounts yielding pemetrexed diarginine concentrations of 4, 9 and 12 mg/mL. Test solutions were stored under refrigeration (2–8°C) or at ambient temperature (22–25°C) exposed to light. Pemetrexed diarginine concentrations were determined throughout a 14-day storage period using a stability-indicating HPLC assay. In addition, test solutions were visually examined for colour change and precipitation.

Results Pemetrexed diarginine injection concentrate with closed-system transfer device is shown to be physicochemically stable for up to 4 days when stored under refrigeration and for 1 day at room temperature. A browning of the pemetrexed diarginine concentrate solutions appeared 0n day 2 when stored at ambient temperature and on day 5 under refrigeration. Pemetrexed diarginine diluted in dextrose 5% and 0.9% sodium chloride was physicochemically stable for up to 4 days when stored under refrigeration and for 1 day at room temperature. A browning of the diluted solutions appeared on day 2 when stored at room temperature and on day 5 when stored under refrigeration.

Conclusions Pemetrexed diarginine concentrate for solution stored under refrigeration with closed-system transfer device can be retained as a residual to reduce product losses. The analytical stability of pemetrexed diarginine in dextrose 5% and 0.9% sodium chloride under refrigeration enables our centralised unit to prepare this drug in advance.

  • antineoplastic agents
  • pharmaceutical preparations
  • pharmacy administration
  • medical oncology
  • drug compounding
  • pulmonary medicine

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Introduction

Pemetrexed (PXD) is an antifolate antineoplastic agent that exerts its action by disrupting folate-dependent metabolic processes essential for cell replication.1–3 PXD inhibits thymidylate synthase, dihydrofolate reductase, and glycinamide ribonucleotide formyltransferase involved in the novo biosynthesis of thymidine and purine nucleotides.3 4 PXD is transported into cells by the reduced folate carrier and membrane folate binding protein transport systems. In the cell, the drug is converted to polyglutamate forms that are potent inhibitors of thymidylate synthetase and glycinamide ribonucleotide formyltransferase.3 The therapeutic indications of PXD are malignant pleural mesothelioma in combination with cisplatin and non-small cell lung cancer as monotherapy or in combination with cisplatin.5 6 PXD ORHE Pharma, which has been available since 2018 on the French market, is supplied as concentrate solutions for infusion; each vial contains 25 mg/mL (expressed as a base) in 4, 20 or 40 mL.7 The ready-to-use form will facilitate the preparation of the solutions diluted in appropriate solvent. The product is a white to either light yellow or green-yellow for both forms. The pH of the concentrate solution is between 8.3 and 9.0.7 The excipient composition of this generic is not identical to the original form. Unopened vials may be stored at room temperature. The required dose of pemetrexed concentrate solution should be diluted further to 100 mL with either dextrose 5% or 0.9% sodium chloride for injection and administered as an intravenous infusion over 10 min.7 The diluted infusion solution is stable for 24 hours at 2–8°C when protected from light, but the procedures for dilution and administration should be performed in very short periods of time because there are no antimicrobial preservatives present in the formulation.7 PXD from the original form was tested in vials, PVC and polyolefin bags.8–11 The chemical stability of reconstituted PXD 25 mg/mL in glass vials and PXD 5 mg/mL in PVC bags was demonstrated for 28 days at 2–8°C.9 10 At room temperature, PXD was chemically stable for 2 days in dextrose 5% and 0.9% sodium chloride for injection.10 However, substantial numbers of microparticules may form in PXD diluted in the infusion solutions in PVC bags, limiting the refrigerated storage period by the manufacturer for up to 24 hours. Recently, the stability of PXD diarginine solutions in dextrose 5% and 0.9% sodium chloride and PXD diarginine ready-to-use solutions have been studied for 28 days when protected or not protected from light.12 13 PXD diarginine solutions and vials were stable for 3 or 4 days, four at room temperature and 7 days under refrigeration.12 13

The purpose of this study was to determine the stability of PXD diarginine ORHE solution in vials and diluted in 0.9% sodium chloride and dextrose 5% polyolefin bags.

Methods

Chemicals

The following materials were used: PXD (reagent quality, Sigma-Aldrich, Saint-Quentin Fallavier, France, Lot LRAC1932) and PXD impurities mixture CRS (Sigma-Aldrich, identification numbers 008RF3 and 009N20). Pharmaceutical quality PXD diarginine solutions for injection at 1000 mg/40 mL (Lot 1 902 542A) and 500 mg/20 mL (Lot 1 902 538A) were obtained from OHRE Pharma (Tours, France). Potassium dihydrogen phosphate, orthophosphoric acid 85%, acetonitrile HPLC grade and hydrochloric acid were from VWR International (Leuven, Belgium); sodium hydroxide was from Merck (Damstadt, Germany); 3% hydrogen peroxide (H2O2) was from Gilbert (Hérouville, France); 0.9% sodium chloride 100 mL polyolefin bags (Lot 13PAF261) and dextrose 5% 100 mL polyolefin bags (Lot 13NKS141) were obtained from Fresenius Kabi (Freeflex, Sèvres, France). Purified water was from Fresenius Kabi (Versylene).

Sample preparation

Stability studies on the concentrated stock solutions (ie, 1000 mg/40 mL and 500 mg/20 mL) kept in their original PXD OHRE Pharma glass vials were performed in triplicate.

The diluted PXD solutions for injection were prepared in triplicate under aseptic conditions in a laminar air flow hood (Biocyt Class II, Thermofisher, Courtaboeuf, France) by adding PXD OHRE Pharma stock solution to polyolefin 100 mL infusion bags containing either 0.9% sodium chloride or dextrose 5%, in amounts yielding PXD at different concentrations: 4, 9 and 12 mg/mL.

One millilitre (in triplicate) of the PXD solution contained in the original concentrated vials or diluted in the infusion bags was taken for each analysis and diluted in the mobile phase to obtain a theoretical PXD concentration at 250 µg/mL. This solution was then mixed and analysed directly in the HPLC system.

Standard solution preparation

Standard solutions were prepared by dissolving the active substance and impurities in the mobile phase. The final concentration of the PXD standard solution was 2.5 mg/mL. This solution was used as standard for method validation.

Stability study

Three commercially available concentrated PXD stock solutions at 1000 mg/40 mL or 500 mg/20 mL were impacted by a closed system commonly used in practice (Spike SWAN-LOCK needle free adapter, 0.2 µm air eliminating filter; Codan, Bischwiller, France) and were stored at 22–25°C and 2–8°C. Three diluted PXD solutions in 0.9% sodium chloride or dextrose 5% at 4, 9 and 12 mg/mL were stored at 22–25°C and 2–8°C. Chemical determinations were performed in triplicate on day 0, 1, 2, 4, 6, 8 and 14 to define drug stability during the study period. The samples at room temperature were not protected from light. The chemical stability of the preparation was defined by the drug content that contained no less than 90% of the labelled amount of PXD.14 The physical appearance of samples stored under each condition was examined using visual inspection with the naked eye against a white and black screen. Evaluation of subvisual particulates in the studied solutions was not assessed.

Instrumentation and chromatographic conditions

An HPLC method used for the determination of the active pemetrexed drug and impurities mentioned in the European Pharmacopoeia was developed and validated.15 An integral HPLC system Dionex Ultimate 3000 (Thermo-Fisher, Villebon-sur-Yvette, France) with an octadecyl (C18) silica gel HPLC column (Polaris 5 C18, 250×4.6 mm, 5 µm; Agilent, Les Ulis, France) was used for the chromatographic analysis. The mobile phase consisted of the 0.01M phosphate buffer (1.4 g potassium dihydrogen phosphate in 1 L of purified water) and acetonitrile in the ratio 80:20 (v/v); the mobile phase was filtered by a 0.45 µm nylon membrane filter before use. The isocratic flow rate was 1.0 mL/min and the absorbance UV detector was set at a wavelength of 227 nm. Chromatograms of standard solution with UV spectrum and impurities are shown in figure 1.

Figure 1

Typical chromatogram and UV spectrum of 250 µg/mL pemetrexed (PXD) standard solution (2) and typical chromatogram of impurities (1).

Method validation

The method was validated according to the International Council for Harmonisation (ICH) Q2(R1) guidelines.16 In accordance, the precision was determined by analysing six replicate samples and expressed as a relative standard deviation (RSD), which was expected to be lower than 2%. For linearity determination, a calibration curve was created using six points that covered the concentration range of PXD from 125 to 375 µg/mL. The calibration curve was used to confirm the linear relationship between the PXD peak areas and its concentration. The slope, intercept, and regression coefficient (r) were calculated as regression parameters using the least square method. The accuracy for the active compound was determined by analysing three replicates of samples prepared at 80%, 100% and 120% of the target concentration. Accuracy was expressed as percentage of recovery determined by the ratio of the experimental concentration on the theoretical concentration. The acceptance criterion was ±2% deviation from the normal value for the recovery of PXD. The LOD and LOQ for the PXD assay were determined by the calibration curve method by using the following equations:

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The specificity was assessed by subjecting PXD solutions to various forced degradation conditions. Solutions of PXD were mixed with 1M HCl, 1M NaOH, and 3% H2O2 prior to being maintained at 60°C away from light. The UV spectral purity of the PXD peak in chromatograms of the degraded sample was retained to evaluate the final chromatographic system.

Statistical analysis

Data analyses were performed using Prism 6 (Version 6.01, GraphPad Software, San Diego, USA). Descriptive statistics for continuous variables were expressed as mean±SD. ANOVA tests were performed for the linearity test to determine any statistical differences. A p value less than 0.05 was considered statistically significant.

Results

Method validation

The linear relationship between the peak area and the concentration range for PXD was established over the concentration range of 175–375 µg/mL (each solution was injected three times). The equation of the calibration curves was y=0.5762 (±0.0017) x+1.6880 (±0.4650) with r2 greater than 0.999. According to the statistical analysis (ANOVA), the calibration curve was linear (p<0.0001). The relative standard deviation (RSD) values were less than 1.0% for all concentrations tested and confirmed the very good intra-day and inter-day precision of the method (online supplemental table 1). The percentage PXD recoveries was found to be 100.3–100.9%, with RSD range of 1.1–1.3%. The results of recovery studies demonstrated the accuracy of the method. The determined values of LOD and LOQ were 4.8 and 9.9 µg/mL, respectively, calculated using slope and Y-intercept.

Forced degradation

The results of the PXD forced degradation studies are summarised in online supplemental table 2. PXD was found to be more labile and prone to degradation in alkaline conditions. A PXD degradation study using 1M NaOH was associated with a rise in degradation product at relative retention time (RRT) of 24 min (figure 2A). PXD in acidic conditions performed with 1M HCl showed no increase in degradation products (figure 2B). PXD was found to be susceptible to oxidation by 3% H2O2, with detection of two degradation products (figure 2C).

Figure 2

Chromatograms of pemetrexed (PXD 125 µg/mL at T0 (control, blue line), and after 20 hours in stress conditions at 60°C (black line): (A) 1M NaOH; (B) 1M HCl; (C) 3% H2O2.

Physical and chemical stability of PXD solution for injection in vials

PXD concentrates for infusion spiked with a closed system at 1000 mg/40 mL and 500 mg/20 mL and were found to be chemically stable for 14 days when stored at 22–25°C unprotected from light or 2–8°C (table 1). However, a browning appeared on day 2 for both dosages (table 1). Under refrigerated storage conditions, no browning appeared during the study period (table 1). No visual particles or precipitation was observed in any of the samples. The physical instability led to limited stability.

Table 1

Physical and chemical stability of pemetrexed (PXD) concentrate solutions in vials spiked with closed system

Physical and chemical stability of PXD diluted in dextrose 5%

Diluted PXD infusion solutions in dextrose 5% polyolefin bags proved to be chemically stable for 14 days when stored at ambient temperature and under natural light, and under refrigeration (table 2). Under ambient temperature storage conditions, a visual browning of all solutions was observed on day 2 and increased during the study period with apparition of degradation products (figure 3A). Under refrigeration storage conditions, a visual browning of all solutions was observed on day 5 without detection of degradation products (figure 3B). Typical visual colour change is shown in figure 4. No visual particles or precipitation was observed in any of the samples. The physical instability of these solutions led to limited stability.

Figure 3

Typical chromatograms of pemetrexed (PXD) diluted in dextrose 5% on day 14 and UV spectrum: (A) stored at ambient temperature and (B) under refrigeration (12 mg/mL); and typical chromatograms of PXD diluted in 0.9% sodium chloride on day 14 and UV spectrum: (C) stored at ambient temperature and (D) under refrigeration (12 mg/mL).

Figure 4

Typical visual colour change of pemetrexed (PXD) diarginine at 4 mg/mL in polyolefin bags on day 4. No colour change was observed on days 1–3.

Table 2

Chemical stability of pemetrexed (PXD) diluted in dextrose 5% and 0.9% sodium chloride

Physical and chemical stability of PXD diluted in 0.9% sodium chloride

Diluted PXD infusion solutions in 0.9% sodium chloride polyolefin bags proved to be chemically stable for 14 days when stored at ambient temperature and under natural light, and under refrigeration (table 2). Under ambient temperature storage conditions, a visual browning of all solutions was observed on day 2 and increased during the study period with apparition of degradation products (figure 3C). Under refrigeration storage conditions, a visual browning of all solutions was observed on day 5 with apparition of degradation products (figure 3D). Typical visual colour change is shown in figure 4. No visual particles or precipitation was observed in any of the samples. The physical instability of these solutions led to limited stability.

Discussion

To simulate the common practice of centralised cytotoxic preparation units, stability testing of PXD stock solution was carried out in their original vials spiked with a closed system to enable the use of residuals. There was a difference in physical stability according to storage conditions. Storage under refrigeration is highly recommended for the physicochemical and microbial drug stability. Moreover, the physicochemical stability of diluted PXD solutions in dextrose 5% or 0.9% sodium chloride was related to storage conditions. Stored at ambient temperature, diluted PXD solutions were unstable. Diluted PXD solutions were only stable under refrigeration storage for up to 4 days. These data are consistent in part with previously published stability studies on Eli Lilly products whose excipients are different from the ORHE product.8–12 The authors evaluated the stability of PXD disodium (original form) reconstituted with NaCl 0.9% or dextrose 5% injection packaged in polypropylene syringes or in polyvinyl chloride bags.8–10 They reported PXD was chemically stable for 2 days at room temperature and 31 days when refrigerated. However, PXD 2–20 mg/mL in both solvents frozen at −20°C in PVC bags showed the formation of microparticulates relating to the PVC containers.9 In view of the potential precipitation on refrigerated storage, use of the inline filter was recommended to administer PXD solution considering the drug was not adsorbed on the filter material.11 Additionally, the results of the present study are partially consistent with those previously published that noted a colour change on day 4 at room temperature against day 2 in our study, and day 7 under refrigeration against day 4 in our study.12 Degradation products of PXD diarginine identified in the study by Youcef et al13 confirmed the drug was stable for 3 days at 20–25°C and for 7 days at 2–8°C.13 Studies on the degradation chemistry of PXD indicated that PXD degraded mainly by oxidation.17 18 The stability of PXD solutions was significantly influenced by the addition of antioxidants, drug concentration, pH, and dissolved oxygen level in the solution.18 The optimal injectable solution formulation of PXD should include sodium sulfate as an antioxidant ingredient to prevent change of colour, N-acetylcysteine as an antioxidant for prevention of chemical degradation, pH range of 7–8, dissolved oxygen level below 1 ppm, and headspace oxygen level below 1%.18

Conclusion

PXD 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. These data allow PXD residues to be stored in the centralised cytotoxic unit, optimising economic and environmental management. However, the absence of preservatives in the concentrate solutions suggests storage of residues in vials should be limited. PXD prepared as intravenous admixtures of 4, 9 and 12 mg/mL in dextrose 5% injection and 0.9% sodium chloride injection in polyolefin bags is stable for up to 4 days when stored under refrigeration. These results allow drugs to be prepared in advance to anticipate the working flow of the hospital pharmacy.

What this paper adds

What is already known on this subject

  • Stability studies reported pemetrexed disodium was chemically stable for 2 days at room temperature and for 31 days when refrigerated.

  • Stability of pemetrexed solutions was influenced by dissolved oxygen level.

  • Few data exist on the stability of pemetrexed in original vials and in polyolefin bags.

  • No data are published on the stability of pemetrexed diarginine ORHE in original vials and in polyolefin bags.

What this study adds

  • This work gives supplementary data on the physicochemical stability of pemetrexed diarginine concentrate solutions in vials and diluted in dextrose 5% and 0.9% sodium chloride in polyolefin bags.

  • Pemetrexed diarginine concentrate solutions in vials spiked with a closed system were physicochemically stable for up to 14 days when stored under refrigeration

  • Pemetrexed diarginine prepared as intravenous admixtures of 4, 9 and 12 mg/mL in dextrose 5% injection and 0.9% sodium chloride injection in polyolefin bags is stable for up to 4 days when stored under refrigeration.

References

Supplementary materials

  • Supplementary Data

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Footnotes

  • EAHP Statement 3: Production and Compounding.

  • Contributors FV and JS contributed to the planning, conduct, and reporting of the work described in the article. CC, AN, MS, MH contributed to the conduct of the work. SC contributed to the reporting of the work.

  • Funding The authors have not declared a specific grant for this research from any funding agency in the public, commercial or not-for-profit sectors.

  • Competing interests None declared.

  • Patient consent for publication Not required.

  • Provenance and peer review Not commissioned; externally peer reviewed.

  • Data availability statement All data relevant to the study are included in the article or uploaded as supplementary information.

  • Supplemental material This content has been supplied by the author(s). It has not been vetted by BMJ Publishing Group Limited (BMJ) and may not have been peer-reviewed. Any opinions or recommendations discussed are solely those of the author(s) and are not endorsed by BMJ. BMJ disclaims all liability and responsibility arising from any reliance placed on the content. Where the content includes any translated material, BMJ does not warrant the accuracy and reliability of the translations (including but not limited to local regulations, clinical guidelines, terminology, drug names and drug dosages), and is not responsible for any error and/or omissions arising from translation and adaptation or otherwise.