Article ID: 2025-0003-OA
A 0.04% mitomycin C (MMC) ophthalmic solution is not commercially available and is prepared in hospitals as required. The physical properties and stability of the MMC ophthalmic solution have not been clarified because of a lack of data. This study aimed to assess the stability of the MMC ophthalmic solution under various storage conditions. The MMC ophthalmic solution was prepared by dissolving 2 mg of MMC in 5 mL of saline solution. Each batch of ophthalmic solution was stored under three conditions [cold/shaded light, room temperature/shaded light, and room temperature/scattered light (approximately 4000 lx)], and MMC concentration was determined using high-performance liquid chromatography on days 0, 1, 3, 5, 7, 14, and 21. The expiry date was calculated based on the results of the degradation analysis. pH measurements and bacterial culture tests were conducted for each storage condition. The MMC concentrations on day 21 under cold/shaded light, room temperature/shaded light, and room temperature/scattered light were 340, 287, and 227 µg/mL, respectively. MMC concentrations decreased over time, and the decrease was highest when the samples were stored at room temperature and exposed to light, and lowest under cold conditions and protected from light. The pH of MMC solutions was 5.8 after preparation, which increased to 6.9 with time under all storage conditions. Culture tests did not detect any bacteria under any storage conditions. The MMC ophthalmic solution was most stable under cold/shaded conditions, and our study clarifies its expiry date for clinical use.
In Japan, the drugs used in clinical practice must adhere to the “Order for Enforcement of the Act on Securing Quality, Efficacy and Safety of Products Including Pharmaceuticals and Medical Devices in Japan.” However, providing optimal drug therapy using commercially available drugs alone is not always possible. In such cases, pharmacists rely on in-hospital preparation of drugs for treatment under the direction of physicians. In-hospital preparations are pharmaceuticals prepared using reagents or by changing the dosage form of a marketed drug. Although various new drugs have been developed, in-hospital preparations for treating rare diseases remain indispensable in clinical practice. However, given that in-hospital preparations are based on in-house protocols, there is a lack of data on the stability and expiry dates of such preparations.
Corneal conjunctival tumors develop on the eye surface and cause symptoms such as swelling, conjunctival hyperemia, and vision loss. The incidence of corneal conjunctival tumors is low at approximately 0.03–1.9 per 100,000 individuals.1 The primary treatment modalities are surgical resection of the tumor and cryocoagulation. However, because wide surgical margins are difficult to achieve in the ocular region, the recurrence rate is high, and antineoplastic eye drops are often used in combination with surgery. Ophthalmic solutions are often administered with antimetabolites such as 5-fluorouracil and mitomycin C (MMC). These anticancer ophthalmic solutions are used both as primary therapies and for tumor reduction prior to surgical management. MMC is an alkylating agent that acts mainly during the G1 and S phases of the cell cycle to generate free radicals. Free radicals induce apoptosis by causing DNA strand breaks and impairing DNA synthesis, ultimately leading to cell death.2,3
Although treatment of corneal conjunctival tumors with MMC ophthalmic solution is effective and widely used worldwide, insurance coverage for the use of such eye drops is lacking. Moreover, an MMC ophthalmic solution is an in-hospital preparation from an MMC injectable drug for off-label use.
A 0.04% MMC ophthalmic solution can achieve complete remission in 90% of cases without the need for surgical excision, highlighting its critical role in treating ocular surface squamous neoplasia (OSSN).4,5 However, the duration of drug usage after preparation of MMC solution varies between hospitals. Although MMC ophthalmic solutions are known to have a limited duration of efficacy, sufficient evidence of their stability is lacking. At the Cancer Institute Hospital of the Japanese Foundation for Cancer Research, MMC ophthalmic solution is administered four times a day for seven consecutive days, followed by a 7-day drug-free period, in a cycle that is repeated for three to four times. However, because of the limited data on the stability of MMC ophthalmic solutions, many institutions determine their own storage duration and conditions for their use.
Given that OSSN is a malignant disease, treatment failure can affect visual function and survival prognosis, which necessitates appropriate management strategies. Nonetheless, the widespread use of MMC eye drops without ensuring their stability remains a significant concern. Therefore, this study aimed to provide detailed information on the stability of MMC ophthalmic solutions to aid in setting the expiry date.
The following reagents and equipment were used to prepare the MMC ophthalmic solution: Mitomycin Injection 10 mg (Kyowa Hakko Kirin, Tokyo, Japan), sterile injection solution (20-mL plastic vial; Otsuka Pharmaceutical, Tokyo, Japan), 10-mL Terumo syringe and Terumo 18G injection needle (Terumo, Tokyo, Japan), and Mylux-GV filter unit (0.22 μm, polyvinylidene fluoride, 33 mm; Merck, Tokyo, Japan). Sterile eye-dropper bottles (cap: blue, body: light blue, 5 mL; Sinto Chemical, Tokyo, Japan) were used to store the prepared MMC ophthalmic solution. High-performance liquid chromatography (HPLC)-grade methanol and ammonium acetate (Nacalai Tesque, Kyoto, Japan) were used for HPLC analysis.
Preparation and storage conditions of the MMC ophthalmic solutionThe MMC ophthalmic solution was prepared by adding 5 mL of saline solution to 2 mg of MMC, filtering it using a Mirex GV filter, and dispensing it into eye-drop bottles within a safety cabinet. The MMC ophthalmic solutions were stored for 21 days under three conditions: in shaded light under cold conditions (4 °C), in scattered light (approximately 4000 lx) at room temperature (approximately 25 °C), and in a dark room at room temperature (approximately 25 °C). A digital illuminometer (separate type) (MonotaRO, Hyogo, Japan) was used to check illuminance. A USB temperature data logger (RC-5 temperature recorder; Elitech, Jiangsu, China) was used to monitor the storage temperature.
Study measuresAfter preparation of the MMC ophthalmic solution, the following parameters were analyzed on days 0, 1, 3, 5, 7, 14, and 21: MMC concentration, pH, sterility, and appearance.
MMC concentrationThe concentration of MMC in the ophthalmic solution was measured by HPLC using Shimadzu Nexera-i LC-2040C 3D Plus with a Shimadzu RF-20A detector (Shimadzu, Kyoto, Japan).
pHThe pH of the ophthalmic solution was measured using a pH meter (LAQUAtwin-pH-22B; Horiba, Kyoto, Japan). The instrument was calibrated with phthalate pH standard solution (pH 4.01, 25 °C; FUJIFILM Wako Pure Chemicals, Osaka, Japan) and neutral phosphate pH standard solution (pH 6.86, 25 °C; FUJIFILM Wako Pure Chemicals).
SterilityThe MMC ophthalmic solution was placed on blood agar medium (Kyokuto Pharmaceutical Industries, Tokyo, Japan), BTB agar medium (Eiken Chemical, Tokyo, Japan), Sabouraud dextrose CG agar medium, or Anaero Colombia RS blood agar medium (Nippon Becton Dickinson, Tokyo, Japan), and incubated at 36 °C. The presence or absence of bacterial growth was assessed.
AppearanceAll ophthalmic solutions prepared in the hospital were observed under fluorescent light by a single person to determine whether any crystal had precipitated or whether the color had changed.
HPLC conditionsChromatographic separation was achieved using a Unison UK-C18 column (4.6 × 150 mm; particle size, 3 µm; Imtakt, Kyoto, Japan). The column temperature was 40 °C; flow rate was 1.0 mL/min; injection volume was 1 µL; detection wavelength was 363 nm; and the mobile phase was 10 mM ammonium acetate buffer:methanol (60:40 ratio). The detection time was 4 min. The MMC calibration curve was set to a concentration range of 2.5–400 µg/mL. The calibration curve was prepared using the absolute calibration method, applying a linear equation with a weighting factor of 1/x2.
Accuracy and precisionThe accuracy and precision of the method were validated in compliance with the guidelines of the Food and Drug Administration6 and the European Medicines Agency.7 Intra-day variability was evaluated through five replicates, whereas inter-day variability was assessed over three separate days. The quality controls (QCs) were prepared at three concentration levels: 2.5 µg/mL (lower limit of quantification, LLOQ), 200 µg/mL (medium QC), and 400 µg/mL (high QC).
Statistical analysisStability test data were analyzed using JMP Pro v.17 (SAS Institute Japan, Tokyo, Japan). The expiry date was calculated using “Stability Test” in the “Degradation Analysis” module of JMP. Based on the changes in MMC concentration in the ophthalmic solution stored under each storage condition, a model was selected at a significance level of 0.25, and the expiry date was confirmed using the model. In this study, the concentration cutoff values for setting the expiry date of the MMC ophthalmic solution were 95%, 90%, and 85% of the original MMC concentration.
We constructed the calibration curve independently on three occasions, achieving a correlation coefficient (R2) of 0.9997 ± 0.0007 (n = 3). The intra-day (n = 5) and inter-day (n = 3) accuracy and precision results are presented in Table 1. All values met the acceptance criteria of ±20% at LLOQ and ±15% at medium QC and high QC.
| Intra-day (n=5) | Inter-day (n=3) | |||||||
|---|---|---|---|---|---|---|---|---|
| Level | Target concentration (µg/mL) |
Obtained concentration (µg/mL) |
Precision (%) |
Accuracy (%) |
Obtained concentration (µg/mL) |
Precision (%) |
Accuracy (%) |
|
| LLOQ | 2.5 | 2.41 ± 0.01 | 0.53 | −3.80 | 2.5 ± 0.04 | 1.44 | −2.10 | |
| Medium QC | 200 | 201.1 ± 0.59 | 0.29 | 0.53 | 202.5 ± 1.98 | 0.98 | 1.25 | |
| High QC | 400 | 397.7 ± 1.21 | 0.31 | −0.57 | 400.2 ± 3.19 | 0.79 | 0.07 | |
LLOQ, lower limit of quantification; QC, quality control
HPLC analysis performed immediately after preparing the 0.04% MMC ophthalmic solution revealed a peak elution time of 3.1 min for MMC (Supplementary Fig. 1A). Supplementary Figure 1B-D shows the results of HPLC analysis of MMC ophthalmic solutions stored for 21 days under different conditions. An analogous peak of MMC was observed between 2.3 and 2.8 min, earlier than that of the MMC peak.
Concentration of MMCMMC concentration was calculated from the MMC peak observed in HPLC analysis. Figure 1 shows that MMC concentration decreased over time under all storage conditions in the following order: room temperature/scattered light, followed by room temperature/shaded light and cold/shaded light conditions. Furthermore, on the day following the preparation of the MMC ophthalmic solution, the degree of decrease in MMC concentration was relatively high under room temperature/shaded light and room temperature/scattered light conditions.
Change in concentration of mitomycin C with time after preparation (n =3, mean ± standard deviation).
Triangles, cold/shade; squares, room temperature/shade; circles, room temperature/scattered light.
Figure 2 shows the number of storage days under different conditions. Room temperature/shaded light and room temperature/scattered light conditions were unable to maintain 95% MMC concentration from the start of the experiment, whereas cold/shaded light conditions retained 95% of the original MMC concentration for about 3.2 days. Similarly, the MMC concentration fell below 90% at the start of the experiment when stored under conditions of room temperature/shaded light and room temperature/scattered shaded light, whereas the MMC concentration fell below 90% after 10.8 days when stored under conditions of cold/shaded light. The storage time to maintain 85% MMC concentration was 5.2 days for room temperature/shaded light, 2 days for room temperature/scattered light, and 17.6 days for cold/shaded light (Table 2).
Stability tests of mitomycin C solutions using JMP.
(A) Shaded storage at refrigerated temperature, (B) shaded storage at room temperature, and (C) storage under scattered light at room temperature.
| Conditions | Expiry of mitomycin C ophthalmic solution (days) | ||
|---|---|---|---|
| 95% | 90% | 85% | |
| Room temperature/scattered light | 0 | 0 | 2 |
| Room temperature/shaded light | 0 | 0 | 5.2 |
| Cold/shaded light | 3.2 | 10.8 | 17.6 |
The changes in pH of the MMC ophthalmic solutions during storage are shown in Figure 3. Under all storage conditions, pH remained within the range of 5.8–7. Compared with the pH value immediately after adjustment, pH increased with time under all storage conditions. Under the cold/shaded light conditions, the degree of increase in pH was relatively low, and pH remained relatively stable.
Changes in mitomycin C pH with time after preparation (n =3, mean ± standard deviation).
Triangles, cold/shade; squares, room temperature/shade; circles, room temperature/scattered light.
No bacteria or fungi were detected in the MMC ophthalmic solution during the study period (data not shown).
In this study, we investigated the stability of MMC ophthalmic solution prepared at the Japanese Foundation for Cancer Research (JFCR) based on the “Guidelines for Preparation and Use of In-Hospital Preparations (Version 1.1)” published by the Japanese Society of Hospital Pharmacists (JSHP), using a protocol unique to JFCR. The stability of the MMC ophthalmic solution was tested, and the expiry date was determined. When the MMC ophthalmic solution was stored at room temperature, the MMC concentration decreased below 90% on the day after preparation. Therefore, storage at room temperature is not recommended. When stored under cold/shaded light conditions, the MMC concentration remained above 95% for 3.2 days, above 90% for 10.8 days, and above 85% for 17.6 days.
In this study, the storage conditions were determined based on locations where MMC ophthalmic solution is expected to be stored by either the patients to whom it is prescribed or the healthcare professionals who prepare it. The anticipated storage locations included a refrigerator (cold/shaded light), inside a cabinet (room temperature/shaded light), and on a desk (room temperature/scattered light). Therefore, the study examined these three storage conditions. Conversely, when the ophthalmic solution is used, it may be exposed to scattered light under cold conditions. However, given that exposure under these conditions is not prolonged, we believe it does not correspond to the storage conditions evaluated in this study. This study focused on evaluating the stability of unused ophthalmic solutions under three storage conditions.
HPLC analysis revealed a peak analogous to that of MMC. The area of this peak increased over time under all three storage conditions. However, the rate of increase for storage under conditions of cold/shaded light was lower than the rate of increase at room temperature.
The use of MMC ophthalmic solutions varies from hospital to hospital, and the expiry date is set based on the experience of ophthalmologists. Although the stability of MMC ophthalmic solutions has been previously analyzed, only one study has clarified the solvent used to dissolve MMC.8 In that study, the stability of a 5% dextrose solution and irritation of the ocular mucosa owing to the usage of water for injection have been reported. In the present study, MMC was dissolved in saline solution to avoid such issues. Human tear has buffering properties, with an average pH of 7.6.9 If the pH of the ophthalmic solution is in the range of 5–8.5, the risk of eye irritation and discomfort is very low.10,11,12
The MMC ophthalmic solutions used in this study were prepared with saline to give a pH range of 5.8–7, which is unlikely to cause irritation of the ocular mucosa. Several adverse effects of MMC ophthalmic solutions, including corneal disorders, have been reported. Long-term corneal complications, such as persistent keratoconjunctivitis and corneal problems, occur in 31% of individuals using MMC ophthalmic solutions.13 Corneal disorders are highly frequent when the pH of the ophthalmic solution is less than 6.5.14
MMC is known to exhibit pH-dependent degradation.15 Furthermore, when MMC is dissolved in basic or acidic reagents, the peaks of MMC degradation products appear on the chromatogram.16 In this study, the pH of the ophthalmic solution fluctuated between 5.8 and 7, which could be considered stable compared to the pH immediately after preparation, implying that the solution is unlikely to have undergone significant degradation. In this study, multiple peaks were detected before the MMC peak when the solution was stored under the conditions of room temperature/scattered light. Therefore, in addition to pH, light exposure may cause degradation of MMC.
The analogous peak area for samples stored under scattered light at room temperature was larger, and the peak was narrower than those observed for samples stored under the conditions of cold/shaded light. The peaks that appear before the MMC peak are expected to be decomposition products or related substances. However, no previous reports have revealed the decomposition products of MMC, and their identities were not clarified in this study. Whether these MMC analogs affect drug efficacy was not investigated in this study, which could be considered a limitation.
Several methods have been used to evaluate the stability of pharmaceuticals using predictions based on reaction kinetics, from which the expiry date of a drug is determined. The expiry dates for antiviral drugs and sodium chloride ophthalmic solution have been previously reported.17 In this study, the cutoff values were defined as 85%, 90%, and 95%, and the expiry dates were determined. Although no clear standard has been recommended for defining the cutoff values, various expiry dates have been established.18,19 This study aimed to clarify the time limit for use to guarantee product validity by indicating three different cutoff values.
This study did not conduct a stability assessment of the ophthalmic solution after opening. Unlike commercially available ophthalmic solutions, in-hospital preparations do not utilize airtight or sealed containers used in industrial manufacturing. Therefore, it is assumed that the storage conditions remain unchanged before and after opening. However, if stability after opening were to be compromised, it would likely result from improper handling by patients or deviation from appropriate storage conditions. Consequently, it is imperative to provide thorough patient education regarding adherence to proper storage conditions and appropriate handling post-opening. The objective of this study was to elucidate the fundamental stability of the ophthalmic solution in its unused state. The findings provide critical insights into the shelf life of MMC ophthalmic solutions under optimal storage conditions, offering valuable information for clinical practice.
We thank Editage (www.editage.jp) for English language editing.
The authors have declared that no conflict of interest exists.
