Stability of mitomycin in polypropylene syringes for use in glaucoma surgery

Abstract

Introduction: 

Outside the oncology field, mitomycin is used in glaucoma surgery. Solutions at 0.2 mg/mL are usually used to fully saturate sterile sponges that are applied to the treatment area. In hospital pharmacies, mitomycin 10 and 40 mg are classically used to prepare the solutions with a lot of losses. The objective of this work was to study the long-term stability to consider preparation in advance and reduce losses in a context of drug shortage.

Methods: 

Two different products were used: Ametycine, which contains sodium chloride as excipient, and Mitomycin medac, which contains urea as excipient.

Solutions at 0.1 mg/mL (sometimes used in our hospital) and the more classically used 0.2 mg/mL were prepared and stored in polypropylene syringes. Solutions were kept at −20°C or at 5°C and analyzed on day 28, 63, and 91. Chemical stability was studied by using a previously validated high-performance liquid chromatography stability-indicating method. pH was measured and a visual inspection was performed.

Results: 

For each condition, solutions were not stable after long-term storage with concentrations below 90% of the initial concentration after 28 days. Frozen solutions were less stable than refrigerated solutions. pH values increased by more than 1 pH unit after 3 months. Ametycine shows precipitate after freezing but not solutions prepared with Mitomycin medac.

The color of frozen solutions was changed from light blue to light green at −20°C but became light blue after thawing.

Conclusion: 

Mitomycin 0.1 and 0.2 mg/mL showed an instability after long-term storage at 5°C or −20°C. These results do not allow the preparation in advance of large batches.

1 Introduction

Mitomycin is an antibiotic with an antineoplastic effect that is isolated from Streptomyces caespitosus. This drug is classified among alkylating agents.

It is given intravenously to treat upper gastro-intestinal cancers (e.g.,ss esophageal carcinoma), anal cancers, and breast cancers, as well as by bladder instillation for superficial bladder tumors.

Mitomycin has also been used by intraperitoneal route and in eye surgery where mitomycin 0.02% is applied topically to prevent scarring during glaucoma filtering surgery. Mitomycin has also been shown to reduce fibrosis in strabismus surgery.

For topical application to the surgical site of glaucoma filtration surgery, the concentration classically used is 0.2 mg/mL. The solution is used to fully saturate sterile sponges that are then applied to the treatment area, in a single layer, with the use of a surgical forceps. Sponges are kept on the treatment area for 2 minutes and then removed.^[1,2]

Mitomycin is available under different formulas with different excipients.

Ametycine 40 mg is presented as lyophilized powder that contains sodium chloride as excipient and should be reconstituted with water for injection (WFI) or normal saline (NS) to get a 1 mg/mL solution.^[3]

Mitomycin Accord contains mannitol instead of sodium chloride and must be reconstituted with NS or 20% dextrose. It may not be reconstituted with WFI.^[4]

Mitomycin MEDAC contains urea as excipient and should be reconstituted with NS. The Summaries of Product Characteristics of these products give only short-term stability.^[5]

Mitosol 0.2 mg has the same composition as Mitomycin Accord but is reserved for eye surgery. It should be reconstituted with 1 mL of WFI giving a 0.2 mg/mL solution with 1-hour stability at room temperature.^[6]

Several stability studies of mitomycin solutions have been published after reconstitution with WFI or NS depending on the product used. Concentrations were from 0.4 mg/mL to1 mg/mL.^[7–14]

After storage at the frozen state, Kinat et al give 23-day stability at −20°C after reconstitution with WFI at 0.4 mg/mL and Stolk et al give 30-day stability at −30°C after reconstitution with NS at 0.6 mg/mL.^[11,14]

Briot et al^[15]give 10-hour stability when reconstituted with NS at 0.2 mg/mL and stored in polypropylene syringes at 25°C.

In our hospital, for glaucoma surgery, mitomycin was used at 0.1 mg/mL. Ametycine 10 mg was used in a first time and then Mitomycin MEDAC 40 mg due to drug shortage. After reconstitution and dilution, the remaining solution was destroyed. Prices were, respectively, 42.8 and 80.1 euros. After Mitomycin MEDAC shortage, French authorities have authorized import of Mitosol from the United States with a price at 250.8 euros for a 0.2 mg vial. To the best of our knowledge, there is no long-term stability of mitomycin at low concentrations (0.1 and 0.2 mg/mL).

Due to the very high price of this product, our objective was to study the long-term stability over at least 3 months, of 0.1 or 0.2 mg / mL solutions stored at −20°C prepared from vials of Ametycine or Mitomycin MEDAC.

2 Materials and methods

2.1 Ethics

This study was carried out without any human, so local ethics board approval was not required.

Recommendations of the European Consensus Conference for practical stability studies of anticancer drugs were followed to perform this stability study.^[16]

2.2 Preparation of test solutions

The drug shortage was anticipated, so some vials of Ametycine and Mitomycin MEDAC were kept to perform this stability study.

Ametycine10 mg (Batch 6086612, Kyowa Kirin Pharma) was reconstituted with 10 mL WFI (Batch 8F447, Chaix et du Marais, France) to get a 1 mg/mL solution. This solution was then diluted with WFI to get a 0.1 or 0.2 mg/mL concentration (200 mL). This solution was divided into 3 mL polypropylene syringes (Medicina ref IVL05). Twenty syringes were stored at 5°C and 20 syringes at −20°C. These syringes were analyzed after preparation and on days 28 and 91. Other syringes were stored at both temperatures for pH measurement.

Mitomycin MEDAC40 mg batch F190353A, (MEDAC, France) was reconstituted with 40 mL NS to get a 1 mg/mL solution. This solution was then diluted with NS to get a 0.2 mg/mL concentration. 200 mL were prepared and treated as previously described for Ametycine solutions. These syringes were analyzed after preparation and on day 63.

At each time point, 3 syringes were analyzed 3 times.

All manipulations were performed inside a biological safety cabinet.

2.3 High-performance liquid chromatography assay

Mitomycin solutions were analyzed by a stability-indicating reversed-phase high-performance liquid chromatography (RP-HPLC) method with photodiode array (PDA) detection previously validated to be stability-indicating.^[15] This previously published method was re-evaluated to ensure reproducibility with our equipment and column.

Briefly, the HPLC system consisted of an ELITE LaChrom VWR/Hitachi plus autosampler, a VWRPDA 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 Uptishere C18, length 20 × 4.6 cm and particle size 5 μm (Interchim).

The mobile phase was composed of a 10 mM dihydrogen phosphate potassium pH 6.5 buffer (65%) (1.36 g KH₂PO₄ was diluted in 1000 mL of ultrapure water and adjusted to pH 6.5 with NaOH 1 M) and methanol (35%).

The flow rate was set at 1 mL/min, with an injection volume of 20 μL. The detection wavelength was set at 216 nm. The temperature of the column was set at 30°C. The calibration curve was constructed from plots of peak area versus concentration.

The linearity of the method was evaluated by 3 standard curves performed on 3 different days from 60 to 140 μg/mL (60, 80, 100, 120 and 140 μg/mL).

The repeatability was assessed by measuring 60, 100, and 140 μg/mL solutions 3 times. The intermediate precision was evaluated by measuring 3 times a day for 3 days at these concentrations.

To demonstrate the specificity of the method, a solution for urea (Cooper, France), the only excipient of Mitomycin MEDAC was analyzed by HPLC.

The stability-indicating capability was verified by analysing forced degraded mitomycin solutions.

Alkali degradation: a solution of 400 μg/mL mitomycin 1 mL was diluted with 1 mL NaOH 0.01 M (VWR, batch 190501), stored at 25°C for 2 minutes, neutralized by 1 mL of HCl 0.01 M and diluted with 1 mL WFI to obtain a theoretical concentration of 100 μg/mL.

Acidic degradation: a solution of 400 μg/mL mitomycin 1 mL was diluted with 1 mL HCl 0.01 M (VWR, batch 180214), stored at 25°C for 2 minutes, neutralized by 1 mL of NaOH 0.01 M and diluted with 1 mL WFI to obtain a theoretical concentration of 100 μg/mL.

Oxidative degradation: a solution of 400 μg/mL mitomycin 1 mL was diluted with 1 mL H₂O₂ 3% (Merck; batch K48743810) stored at 25°C and diluted with 2 mL of WFI to obtain a theoretical concentration of 100 μg/mL.

UV degradation: a solution of 100 μg/mL mitomycin was exposed during 4 hours under a sun-like spectrum lamp at 254 nm (Vilbert Lourmat).

Heat degradation: a solution of 100 μg/mL mitomycin was exposed to a temperature of 90°C during 1 hour.

Mitomycin solutions at 100 μg/mL were directly injected into the column (middle of the standard curve). Solutions at 200 μg/mL were diluted (1:1) with NS to get a 100 μg/mL solution before analysis.

Total run time was set at 10 minutes. Three samples were taken from each syringe each day of the assay.

Chemical stability was defined as not less than 90% of the initial mitomycin concentration.

2.4 pH measurement

pH measurement was performed using a Bioblock Scientific pH meter. pH values were considered to be acceptable if they did not vary by more than 1.0 pH unit from the initial measurement

2.5 Determination of physical stability

Physical stability was realized with a visual examination: particulate matter or change of color.

3 Results

3.1 Reversed phase high-performance liquid chromatography

The calibration curve was linear, the correlation coefficient was 1.000. The intraday precision was evaluated at 60, 100, and 140 μg/mL. Expressed as relative standard deviation (RSD) the intraday precision was between 0.32% and 0.89%. The interday precision expressed as RSD was 3.92% at 60 μg/mL, 3.76% at 100 μg/mL, and 4.03% at 140 μg/mL. The absence of interference by urea was validated.

Stability indicating capacity was verified by using various stressed conditions. The retention time of mitomycin was 5.1 minutes. The chromatogram obtained without stress condition is presented in Figure 1, a chromatogram after heat stressed conditions is presented in Figure 2 and a chromatogram after acidic degradation in Figure 3, for example.

Figure 1: Chromatogram of a freshly prepared 100 μg/mL mitomycin solution without stressed conditions.

Figure 2: Chromatogram of 100 μg/mL mitomycin solution after heat stressed conditions (90°C, 1 hour).

Figure 3: Chromatogram of 100 μg/mL mitomycin solution after acidic stressed conditions (HCL 0.01 M – 2 minutes).

Degradation products (DPs) are clearly separated from mitomycin and appear with relative retention at 0.44, 0.49, 0.55, 0.59, 0.68, 0.75, 0.83, and 1.61.

3.2 Chemical stability of solutions

3.2.1 High-performance liquid chromatography assay

Quantitative results are presented in Tables 1 and 2.

Table 1 - Stability of 100 μg/mL mitomycin solutions stored in polypropylene syringes prepared with Ametycine.

Storage temperature	Day 0: Initial concentration ± CV (%)	Day 28: (%/day 0 ± CV)	Day 91: (%/day 0 ± CV)
−20°C	100% ± 2.88%	85.83% ± 4.84%	74.66% ± 16%
5°C	100% ± 2.88%	88.68% ± 0.76%	86.48% ± 2.54%

Results are expressed as the mean value of 3 determinations of 3 syringes (9 values) ± CV.CV = coefficient of variation.

Table 2 - Stability of 200 μg/mL mitomycin solutions stored in polypropylene syringes.

Product used and storage temperature	Day 0: Initial concentration ± CV (%)	Day 63: (%/day 0 ± CV)
Ametycine
−20°C	100% ± 0.41%	71.57% ± 4.15%
5°C	100% ± 0.41%	79.53% ± 0.82%
Mitomycin Medac
−20°C	100% ± 0.17%	67.78% ± 3.93%
5°C	100% ± 0.17%	81.08% ± 1.06%

Results are expressed as the mean value of 3 determinations of 3 syringes (9 values) ± CV.CV = coefficient of variation.

For the solution at 100 μg/mL, on day 28, DP was present on the chromatograms (Fig. 4), these DP increased on day 91 (Fig. 5).

Figure 4: Focus on degradation products observed on day 28 on solution at 0.1 mg/mL.

Figure 5: Focus on degradation products observed on day 91 on solution at 0.1 mg/mL.

The same situation was observed on day 63 for the 200 μg/mL solution.

3.3 pH measurement

pH values are presented in Table 3.

Table 3 - pH values of mitomycin stored at 5°C and −20°C.

Solution	Initial pH	pH at day 28	pH at day 63	pH at day 91
Ametycine 100 g/mL	6.22	7.00 (5°C)	–	6.96 (−20°C)7.3 (5°C)
Ametycine 200 g/mL	7.08	–	7.00 (−20°C)7.08 (5°C)	–
Mitomycin medac 200 μg/mL	6.75	–	7.08 (−20°C)7.17 (5°C)	–

3.4 Physical stability of solutions

Physical stability was investigated by visual inspection.

Solutions at 100 and 200 μg/mL were initially light blue and kept this color after storage at 5°C. At −20°C, solutions became light green and after thawing at room temperature became light blue like syringes kept in the refrigerator.

For the solution of Ametycine at 200 μg/mL, a precipitate was observed on day 63. It was not observed with Mitomycin medac.

4 Discussion

Recurrent drug shortages of mitomycin used for intravenous injection or for intravesical instillation and the high cost of Mitosol, a product imported from the United States and specifically presented for glaucoma surgery, led us to consider a long-term stability study of mitomycin ready-to-use syringes. Usually, lowering the temperature results in a better stability. So, this project wanted to study the stability after freezing, the stability at 5°C being developed to compare with the frozen solutions.

The production of syringes in advance could have been financially very interesting, a 10 mg vial at 42 euros (French price) being only used to produce 50 syringes or a 40 mg vial at 80 euros used to produce 250 syringes. Unfortunately, both solutions were unstable and the frozen solution was less stable than the refrigerated one. In the context of a shortage of mitomycin vials intended for bladder instillations, we are obliged to use 0.2 mg Mitosol imported vials with a cost much higher than the extemporaneous preparations previously prepared from the 10 mg vials.

5 Conclusion

Mitomycin 0.1 and 0.2 mg/mL were not stable after long-term storage at 5°C or −20°C. Concentrations fall below 90% of the initial concentration after one month and DPs appeared on the chromatograms. Frozen solutions were less stable than refrigerated solutions. These results do not allow the preparation in advance of large batches.