Stability Study of Meropenem Eye Drops 50 mg/mL in Polypropylene Dropper Bottles

1. Introduction

Meropenem trihydrate is (4R,5S,6S)-3-[(3S,5S)-5-(dimethylcarbamoyl)pyrrolidin-3 -yl]sulfanyl-6-[(1R)-1-hydroxyethyl]-4-methyl-7-oxo-1-azabicyclo [3.2.0]hept-2-ene-2-carboxylic acid trihydrate (Figure 1). It is a white or almost white (or light yellow) crystalline powder, slightly soluble in water and practically insoluble in ethanol and dichloromethane [1].

Meropenem is a carbapenem antibiotic that exerts bactericidal activity through inhibition of bacterial cell wall synthesis in both Gram-negative (G-) and Gram-positive (G+) organisms. Its antimicrobial spectrum encompasses a wide range of aerobic and anaerobic G- bacilli and G+ cocci. Notably, meropenem retains activity against aerobic G- bacilli that produce extended-spectrum β-lactamases (ESBLs) [2,3].

In Spain, Meropenem is marketed in the pharmaceutical form of vials, which contain 500 and 1000 mg of the active ingredient in powder form. For intravenous administration, it must be reconstituted with water for injection (WFI), in the case of administration as an intravenous bolus injection, or with 0.9% aqueous sodium chloride solution (NS) or 5% aqueous dextrose solution (D5W) prior to dilution in NS or D5W for administration by intravenous infusion [2].

Meropenem is indicated for the treatment of the following infections in adults and children over 3 months of age: severe pneumonia, including hospital-acquired pneumonia and ventilator-associated pneumonia, bronchopulmonary infections in cystic fibrosis, complicated urinary tract infections, complicated intra-abdominal infections, intra- and postpartum infections, complicated skin and soft tissue infections and acute bacterial meningitis [2]. Meropenem is not indicated for the treatment of ocular infections such as keratitis and endophthalmitis in its Summary of Product Characteristics; therefore, its use in these indications would be considered an off-label use, according to Spanish legislation [4]. However, the use of meropenem in ocular infections is supported by preclinical studies, animal models, clinical cases, and small case series, in which it has demonstrated efficacy and safety [5,6] for the treatment of severe keratitis caused by multidrug-resistant G- microorganisms [6,7,8,9,10,11], and in endophthalmitis due to resistant pathogens [12,13,14] , as well as a demonstrated feasibility and favorable pharmacokinetics [7], including adequate penetration into ocular tissues [7,14,15] and a low toxicity profile [5,7], when administered both systemically and as ophthalmic eye drops.

When considering the preparation of a meropenem eye drop with a concentration of 50 mg/mL, it is important to take into account should be kept in mind that the physicochemical and microbiological stability of an ophthalmic solution depends on a series of interrelated factors that affect both, the concentration of the active ingredient and the formulation medium, as well as the storage environment. For this reason, the stability of ophthalmic solutions is usually expressed as a specific shelf life (for example, X days at 2–8 °C, either closed or opened), and experimentally defined through stability studies in which concentration, pH, osmolality, organoleptic characteristics, and absence of microbial growth are monitored [16,17,18].

From a stability perspective, meropenem is highly susceptible to degradation both in aqueous solutions and in the solid state due to the significant intrinsic strain of its fused ring system [19]. Its degradation pathways involve multiple chemical interactions in which different structural moieties of the drug participate. The main degradation mechanisms are: β-lactam ring hydrolysis which is the principal degradation pathway of the drug in aqueous solutions and the primary mechanism by which enzymes such as β-lactamases inactivate the antibiotic [20,21] (the hydrolysis of this ring can be observed as a shift to a yellowish coloration [22,23]); Intermolecular aminolysis which is the fundamental cause of concentration-dependent degradation that occurs when the meropenem molecule degrades itself through a nucleophilic attack in which the side chain of one molecule attacks and cleaves the carbonyl of the β-lactam ring of a neighboring second meropenem molecule [20,24], thus also producing the aforementioned color change; Inactivation of meropenem by intramolecular cyclization of the molecule with formation of a β-lactone, which is mediated by certain enzymatic targets (such as class D serine β-lactamases or l,d-transpeptidases) [21]; Thermal degradation and decarboxylation of meropenem in the solid state when subjected to high temperatures, which is due to decarboxylation and aromatization of the pyrrolidine ring (concomitant with hydrolysis of the β-lactam ring), yielding a by-product named 4-methyl-3-(1H-pyrrol-3-ylsulfanil)-5H-pyrrol-2-carboxylic acid [19].

Another issue to consider is the type of container that will hold the preparation. In this study, a PP dropper bottle was chosen. PP is widely regarded as one of the most inert plastics, exhibiting a low tendency to adsorb drug substances. In practice, adsorption to PP is generally negligible; however, it may become significant for drugs at low concentrations or with protein/peptide-like characteristics, as well as for high-value agents such as certain radiopharmaceuticals or insulin. Several studies have demonstrated that the physicochemical stability of most drug formulations is preserved in PP packaging, with no appreciable degradation observed over storage periods ranging from several days to weeks, depending on the specific compound and storage conditions (e.g., refrigeration, protection from light). PP displays low permeability to water vapor and is relatively impermeable to oxygen, which contributes to reduced degradation by oxidation or hydrolysis. Nevertheless, its gas barrier properties are less hermetic than those of glass; therefore, in long-term storage scenarios, the shelf life of particularly sensitive solutions may be compromised. Pharmaceutical-grade PP packaging are specifically designed to minimize particulate contamination and the migration of additives, thereby supporting both product quality and patient safety [25,26,27].

Currently, there are no stability studies on a meropenem 50 mg/mL ophthalmic solution, packaged in PP dropper bottles, under freezing and refrigerated conditions. For this reason, the present study investigates the physicochemical and microbiological stability of such a preparation.

2. Materials and Methods

2.1. Preparation of Meropenem 50 mg/mL Ophthalmic Solution

The manufacturing process was carried out using 1000 mg meropenem vials (Aurovitas Spain, S.A.U., Madrid, Spain) that were reconstituted with 20 mL of WFI (Serra Pamies, S.A., Reus, Spain), in accordance with the specific recommendations for sterile preparations included in the Guide for good preparation practices of medicines in hospital pharmacy services (GPPMHPS) [28]. The contents of each reconstituted vial were transferred to a sterile, pyrogen-free vial (VacuflascTM, Grifols Movaco, S.A., Barcelona, Spain) until a total volume of 420 mL of the ophthalmic solution was obtained. 4 mL of that solution were dosed into 90 sterile white polypropylene dropper bottles of 10 mL of volume with photoprotective treatment (Oiarso S. Coop., Hernani, Spain) (Figure 2).

2.2. Chemical Stability

The eye drops were divided into two groups: frozen stability (FSG) and refrigerated stability after thawing (RSG), in which the eye drop bottles belonging to the FSG were thawed on the day of analysis and moved to the RSG, to be analyzed on the days established according to the analysis protocol. The freezing conditions were -20 ± 2 ºC and the refrigeration conditions 5 ± 3 ºC.

The chemical stability of meropenem 50 mg/mL ophthalmic solution in the FSG was studied over 42 days of storage; days: 0 (D0), 7 (W1), 14 (W2), 21 (W3), 28 (W4), 35 (W5) and 42 (W6), and over 7 days of storage in the RSG after thawing; days: 1 (WnD1) , 2 (WnD2), 3 (WnD3) and 7 (WnD7), where “n” is de number of weeks during which the eye drops remained frozen and, after thawing, were transferred to the refrigerated study (Figure 3).

The preparation was considered stable if the drug concentration remained within 90–110% of the initial concentration throughout the 42-days in the FSG and 7-days in the RSG [29,30,31].

2.3. Chromatographic Method

A Waters Breeze HPLC system (Waters Cromatografía, S.A., Barcelona, Spain) equipped with a XBridge 5 µm C18 reversed-phase column (130 Å pore size, 4.6 × 150 mm; Waters Cromatografía, S.A., Barcelona, Spain) was used for the study. Chromatographic separation was carried out under isocratic conditions using a mobile phase consisting of ultrapure water/acetonitrile/methanol (50/30/20, v/v), adjusting the pH to 7.5 with 10% v/v phosphoric acid. The flow rate was 1 mL/min, with detection at 300 nm, a column temperature of 25 °C, an injection volume of 20 µL, and a total run time of 2 min [32]. HPLC-grade acetonitrile, methanol and phosphoric acid were purchased from Panreac Química S.L.U. (Barcelona, Spain) and ultrapure water was obtained using the Milli-Q® Integral 3/5/10/15 system (Merck KGaA -Darmstadt, Alemania-). The meropenem reference standard was obtained from Merck Life Science, S.L., (Madrid, Spain).

Method validation: The HPLC method was validated for linearity, precision, and accuracy in accordance with the International Council for Harmonization of Technical Requirements for Pharmaceuticals for Human Use (ICH) Q2(R1) guidelines [33]. Linearity between the peak area and the meropenem concentration was assessed across six calibration levels from 0 to 150 µg/mL (0, 15, 30, 60, 90, 120, and 150 µg/mL). A calibration curve was constructed with linear regression analysis to determine the coefficient of determination (R²), slope (a), and y-intercept (b) [31]. Precision was evaluated through intra- and inter-day repeatability studies. The intra-day assessment involved six replicate analyses on the same day at 80%, 100%, and 120% of the target concentration (50 µg/mL), while the inter-day study comprised of three replicates over five different days at the same relative concentrations. Mean, standard deviation, and coefficient of variation were calculated, with acceptance criteria of <1% for intra-day and <2% for inter-day repeatability [31,32]. Accuracy was determined via recovery studies performed in triplicate at meropenem concentrations of 40, 50, and 60 µg/mL. Recovery percentages were calculated and compared to the theoretical value (100%) using Student’s t-test [31,32,34]. The limit of detection (LOD) and limit of quantification (LOQ) for meropenem were calculated using the y-intercept (b) and slope (a) from the calibration curve, applying the equations LOD = 3.3 × b/a and LOQ = 10 × b/a as per ICH recommendations [31,33,35].

This chromatographic method was previously validated in a published stability-indicating study, in which the meropenem samples underwent forced degradation under thermal, photolytic, acidic, alkaline, and oxidative conditions. The investigation confirmed that the degradation products did not interfere with the quantification of intact meropenem, demonstrating the specificity of the method [32].

2.4. Physical Stability

Physical stability assessments comprised of macroscopic visual inspection, pH measurements, and microscopic evaluation for crystallization. On designated evaluation days, 1 mL aliquots from each formulation were withdrawn and subjected to detailed visual examination for anomalies including particulate matter, crystalline precipitates, turbidity, sedimentation and color alterations throughout the storage period. The pH was quantified using a calibrated SevenMulti™ pH meter (Mettler Toledo, Cornellà de Llobregat, Spain). Particle and crystal detection was facilitated by a visual inspection station featuring black/white backgrounds under bright-field illumination, complemented by a SediMAX2™ phase-contrast microscope (77 Elektronika, Budapest, Hungary).

2.5. Microbiological Stability

Microbiological stability was assessed on the evaluation day (day 7 post-refrigerated storage following thawing), after simulating the use conditions of an opened eye drop in the RSG from day 1 to 7. The sample was inoculated into a culture medium for aerobic bacteria and fungi (BD BACTEC™ Peds Plus™/F, Becton Dickinson and Company, San Agustín de Guadalix, Spain), and a dedicated medium for anaerobic bacteria (BD BACTEC™ Lytic/10 Anaerobic/F, Becton Dickinson and Company, San Agustín de Guadalix, Spain).

3. Results

3.1. Validation of the Analytical Method

The HPLC method exhibited excellent linearity across the tested range, with a coefficient of determination of (R2) > 0.9997 and a regression equation of y = 29,661x + 8,019.9 (Supplementary materials Table ST1 and Figure SF1). Intra-day and inter-day precision for three meropenem quality control levels were highly satisfactory: intra-day relative standard deviation (RSD%) ranged from 0.114 to 0.150 (Table 1) and inter-day RSD% from 0.649 to 0.888 (Table 2), meeting the ICH criteria of ≤1% and ≤2% for repeatability and intermediate precision, respectively (Supplementary materials Tables ST2–ST3). Accuracy, determined by recovery rates at three concentration levels, ranged from 99.89% to 100.09%, falling within the accepted 98–102% interval (Supplementary materials Table ST4). The limit of detection (LOD) and limit of quantification (LOQ) were calculated as 0.81 µg·mL−1 and 2.70 µg·mL−1, respectively.

3.2. Stability Study

3.2.1. Freezing Conditions

The chemical stability assessment was performed by determining meropenem concentrations in the PP dropper bottle, on each scheduled sampling day, following the previously described procedure. Mean concentrations were calculated and reported as percentage recovery relative to the initial measurement (D0 = 100%), as shown in Table 3. Representative chromatograms from day 0 (D0) and the first day of the start of weeks 1, 3 and 6 (W1, W3 and W6) for the formulation under freezing conditions are shown in Figure 4. The results show that the concentration remained within the predefined acceptance limits throughout the six-week study period under freezing conditions (Table 3 and Figure 5). Detailed week-by-week concentration data are provided in the Supplementary Materials (Table ST5).

Throughout the study, visual appearance (color and turbidity), pH, visible particulates and crystallization remained unchanged to a statistically non-significant extent (Table 3).

Therefore, the 50 mg/mL meropenem ophthalmic solution in polypropylene bottles, under freezing conditions, was considered physically and chemically stable for up to 42 days.

3.2.2. Refrigerated Conditions

The stability data in Table 4 and the chromatograms in Figure 6 correspond with the results obtained from the eye drops that were stored under freezing conditions for up to 1, 2, 3, 4, 5 and 6 weeks and after thawing, kept under refrigeration for 7 days, simulating real-life use conditions. For illustrative purposes, only W1, W3, and W6 of freezing are presented, followed by the chromatograms corresponding to days 1 (D1), 2 (D2), 3 (D3), and 7 (D7), during which the eye drops were stored under refrigerated conditions. Detailed concentration data (week-by-week and day-by-day of each week) are provided in the Supplementary Materials (Table ST6). During this phase of the study, the concentration observed, decreased progressively from the first day of refrigeration (Table 4 and Figure 7). Between days 1 and 2 of refrigeration, the meropenem concentration reached the acceptance limit (45 mg/mL) relative to the nominal concentration of 50 mg/mL, i.e., it fell below 90% of the nominal value.

No significant changes were monitored in pH, visible particulates, turbidity and crystallization throughout the study, but a progressive change in solution color was observed, evolving from colorless in the freshly prepared solution to a very pale yellow in the thawed samples subsequently stored under refrigeration, with the yellow color intensifying to a deeper shade by the last day (D7) of the refrigeration phase (Figure 8). This color change was reflected by a progressive increase in the chromatographic peak observed at approximately 1 min.

On day 7, aerobic, anaerobic, and fungal cultures of the 50 mg/mL meropenem eye drops stored in refrigerated polypropylene bottles yielded negative results for all analyzed samples.

Thus, the meropenem eye drops 50 mg/ml in PP dropper bottles were regarded as physically and chemically stable for up to 24 hours and microbiologically stable for up to 7 days under refrigeration conditions.

4. Discussion

The HPLC method for meropenem determination was linear, precise, accurate, repeatable, and reproducible, and was validated in accordance with ICH guidelines. The method was demonstrated to be robust and to reliably determine the kinetics run of meropenem in the presence of its degradation products, being consistent with previously published studies [32,36,37,38,39].

Regarding chemical stability, currently, there are no stability studies of a meropenem eye drops at a concentration of 50 mg/mL frozen in a polypropylene dropper bottle for the treatment of keratitis and endophthalmitis caused by multidrug-resistant G- bacteria [40]. Only one published study has evaluated a 50 mg/mL meropenem solution prepared from an injectable formulation in WFI, NS and D5W, stored in glass vials under two conditions: room temperature (21–26 °C and 23–27 °C) and refrigerated storage (4 °C) protected from the light. The study concluded that meropenem 50 mg/mL remained stable in WFI and NS for up to 8 hours at 21–26 °C and up to 7 hours at 23–27 °C in WFI; in D5W the stability was 3 hours at 21–26 °C. Under refrigerated conditions, the stability was 48 hours in WFI and NS, and 24 hours in D5W [41]. This study was classified as level D evidence [42], with the following comments: “Stability indicating capability inadequately assessed, repeatability/reproducibility/standard range: results not provided or results outside specified values, no visual inspection, and no comments on degradation products” [40].

A total of five studies have been published that evaluate the stability of meropenem in polypropylene containers, specifically in polypropylene syringes. Of these, three assess the stability at a concentration near to that of the present study. Two of them evaluated the stability of a 40 mg/mL meropenem solution in NS at room temperature (25 °C); one was not protected from the light and the other did not specify this condition, concluding that the stability was 4 and 8 hours, respectively. The first study, in the order mentioned, was assigned a level of evidence D, with the comment: “Repeatability/reproducibility/standard range: results not provided or results outside specified values” [43]. The second study was classified as level B evidence [44]. The third study evaluated the stability of a 41.7 mg/mL meropenem solution in NS and D5W at room temperature (20–25 °C) without light protection, with a level of evidence C+, concluding that at the aforementioned concentration, the meropenem solution is stable for up to 8 hours in NS and 4 hours in D5W [45].

It is not possible to compare the results of the first phase of this study under freezing conditions, as to date there are no published studies evaluating the stability of a 50 mg/mL meropenem ophthalmic solution under those conditions. With regard to refrigerated storage, although the studies performed were carried out in glass vials, the results of the present study indicate that the stability of the eye drops is lower than that reported in the published studies to date. This may perhaps be influenced by the fact that the refrigerated eye drops were subjected to temperature changes in order to simulate real-life administration conditions, which would involve removing the eye drops from the refrigerator, allowing them to equilibrate to room temperature, administering the prescribed drops, and then storing them again under refrigeration until the next administration. In any case, the result obtained would be aligned with the previously discussed studies, in which a 50 mg/mL meropenem solution in WFI in glass vials was stable for 7 hours when stored at room temperature (23–27 °C) and for up to 48 hours under refrigeration (4 °C). For practical purposes, the present study considered stability under these conditions to be 24 h, although, as shown in Figure 7, the acceptance limit (a decrease in concentration below 90% of the nominal value) would lie between 24 and 48 hours.

With regard to the color change observed in the eye drops during refrigerated storage, this phenomenon is likely to be related to the hydrolytic cleavage of the beta-lactam ring. As previously mentioned in the introduction, this mechanism represents the primary degradation pathway of meropenem in aqueous solution. This ring-opening yields a more polar species with a shorter chromatographic retention time than intact meropenem. Consequently, an increase in peak area is observed at a retention time of 1 minute as refrigeration time progresses, which correlates with both the rising concentration of this degradation product and the intensification of the color.

Although performing X-ray diffraction analysis or differential scanning calorimetry to evaluate the crystalline state of the drug during storage would have strengthened the consistency of our results, these techniques were unavailable at the time of the study.

5. Conclusions

The findings of this study demonstrate that 50 mg/mL meropenem eye drops in polypropylene dropper bottles remain physicochemically stable for up to 42 days under freezing conditions, and for an additional 24 hours after thawing when stored under refrigeration (total stability period: 43 days), both protected from the light. Furthermore, the ophthalmic solution maintained microbiological stability throughout this entire period.