Long term physicochemical stability study of novel
ophthalmic formulations combining ceftazidime and
vancomycin with and without cyclodextrins

Pauline Plaidy, Yassine Bouattour, Mouloud Yessaad, Valérie Sautou, Philip
Chennell

► To cite this version:

Pauline Plaidy, Yassine Bouattour, Mouloud Yessaad, Valérie Sautou, Philip Chennell. Long term physicochemical stability study of novel ophthalmic formulations combining ceftazidime and vancomycin with and without cyclodextrins. Pharmaceutical Technology in Hospital Pharmacy, 2023, 8 (1), pp.20230007. 10.1515/pthp-2023-0007. hal-04448891

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Pauline Plaidy, Yassine Bouattour, Mouloud Yessaad, Valérie Sautou and Philip Chennell*

# Long term physicochemical stability study of novel ophthalmic formulations combining ceftazidime and vancomycin with and without cyclodextrins

https://doi.org/10.1515/pthp-2023-0007

Received May 4, 2023; accepted August 10, 2023;

published online September 8, 2023

## Abstract

**Objectives:** Ceftazidime (CZ) and Vancomycin (VM) are used to treat bacterial keratitis; however, their physicochemical incompatibility does not allow their co-administration. This incompatibility can be managed by buffering the mixture at an alkaline pH or by using cage molecules such as cyclodextrins (CD). The objective of this work was to compare the stability during 168 days of frozen storage of two formulations combining VA and CZ at a final concentration of 25 mg/mL: a CD-free formulation, at a pH=8.5 and a formulation with CD.

**Methods:** Beforehand, a stability indicating method (SIM) was developed. Samples were analysed after 1, 3 and 6 months, and after 12, 24 and 72 h after defrosting. Analyses performed were the following: visual inspection, chromaticity, turbidity, osmolality and pH measurements, particles counting, CZ and VM quantification, breakdown product research, and sterility assay.

**Results:** The developed SIM allowed the simultaneous quantification and breakdown products research of both VM and CZ, without interference of the breakdown products. The analyses showed the presence of a visually detectable precipitate and increased turbidity as early as the first day after thawing for CD-free formulation and on the third day for the formulation with CD. CZ concentrations systematically decreased after thawing for both formulations whilst

VM concentrations remained stable. Osmolality and pH remained unchanged, and no microbial growth was detected throughout the study.

**Conclusions:** CD delayed precipitation by 48 h compared to the CD-free formulation but did not permanently eliminate it. Both formulations showed very limited physicochemical stability after thawing.

**Keywords:** cyclodextrins; vancomycin; ceftazidime; ophthalmic solution; physicochemical stability; bacterial keratitis

## Introduction

Bacterial keratitis is a disease caused by the infiltration of bacteria under the cornea, which can lead to an abscess. The incidence of this illness is estimated to be between 0.03 and 79.9 cases per 10,000 in inhabitants/year [1]. It has become more common in industrialized countries due to the increase in the number of contact lens wearers, for whom it is approximately of 2–20 cases per 10,000 wearers [2]. This ocular infection is responsible for a decrease in visual acuity, particularly through opacification of the cornea, and in the worst cases it can lead to blindness [3]. In the literature published between 2010 and 2020, bacterial keratitis remained the most frequent type of infectious keratitis, representing between 85 and 100 % of all cases [4]. The treatment of bacterial keratitis with a risk of complications requires the patients to be hospitalized in a hospital care unit for treatment using antibiotic eye drops. The antibiotics used in this indication are mostly ceftazidime and vancomycin, generally at high concentrations (25–50 mg/mL) [5]. Ceftazidime (CZ) is a third-generation cephalosporin; it acts on Gram-positive and Gram-negative bacilli by inhibiting the synthesis of peptidoglycan, thus also allowing it to be active against *Pseudomonas aeruginosa*. Vancomycin (VM) is a glycopeptide which also acts by inhibiting peptidoglycan synthesis. VM cannot cross the membrane of gram-negative bacteria, hence the importance of using a dual therapy combining these two active substances to cover the entire

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<footnote>*Corresponding author: Philip Chennell, PharmD PhD, CHU Clermont-Ferrand, Pôle Pharmacie, F-63003 Clermont-Ferrand, France; and Université Clermont Auvergne, CHU Clermont Ferrand, Clermont Auvergne INP, CNRS, ICCF, F-63000 Clermont-Ferrand, France, Phone: +(33)4 73 75 10 36, E-mail: pchennell@chu-clermontferrand.fr. https://orcid.org/0000-0001-8086-7517</footnote>

<footnote>Pauline Plaidy and Mouloud Yessaad, CHU Clermont-Ferrand, Pôle Pharmacie, Clermont-Ferrand, France</footnote>

<footnote>Yassine Bouattour and Valérie Sautou, Université Clermont Auvergne, CHU Clermont Ferrand, Clermont Auvergne INP, CNRS, ICCF, Clermont-Ferrand, France</footnote>
spectrum. To ensure maximum therapeutic activity, a loading dose is required for the administration of these antibiotics. To achieve this, each medication needs to be instilled every 5–10 min during the first hour of treatment, then once every hour for the next 48 h. This dual antibiotic therapy requires an interval of 5 min between each therapy in order to limit the risk of precipitation, especially in case of co-administration of CZ and VM which are incompatible when mixed in solution together [6]. These close instillations have a direct impact on the nurses who are constrained to respect this incompatibility by performing twice as many administrations. In addition, this two-step administration can lead to a decrease in compliance with the treatment or an increase in errors, for example by forgetting an administration.

CZ is a small (molar mass: 546.57 g mol⁻¹) cephalosporin antibiotic (of the β-lactam super family), bearing pyridinium-1-ylmethyl and {[(2Z)-2-(2-amino-1,3-thiazol-4-yl)-2-{[(2-carboxypropan-2-yl)oxy]limino}acetamido groups at positions 3 and 7, respectively, of the cephem skeleton that is very sensitive to hydrolysis. Its strongest acidic and alkaline pKa are respectively of 2.42 and 4.02 [7]. VM is a branched tricyclic glycosylated nonribosomal peptide, and is much larger than CZ (molar mass: 1,485.72 g mol⁻¹). Its strongest acidic and alkaline pKa are respectively of 3.38 and 9.9 [8]. Both drugs are water soluble in their commercial intravenous medication form (VM as a chloride salt, CZ in a pentahydrate form). These medications suffer however from two limiting factors that must be taken into account: their chemical instability in an aqueous media (especially CZ) and their incompatibility when co-administered together. Indeed, once solubilized, the chemical degradation of CZ is very rapid and causes the apparition of breakdown products, including pyridine (degradation product of CZ), which is toxic [9]. Also, in the absence of commercial specialties available, these eye drops must be prepared by compounding pharmacies and used extemporaneously or stored at –20 °C, to be latter thawed and used within 24 h if stored at room temperature, or within 7 days if stored between 4 and 8 °C [10, 11]. Concerning the physicochemical incompatibility between CZ and VM, it results in the appearance of a white precipitate, visible to the naked eye. Park et al. [12] demonstrated an increase in the precipitate when applying the low temperatures, small volumes and high concentrations required to treat bacterial keratitis. Bouattour et al. [13] partially explored the nature of the incompatibility between these drugs and hypothesised that for a pH between 4 and 7.3 the two drugs (with opposite net charges) combined to form an insoluble complex. They also showed that it was possible to mitigate the precipitation by buffering the mixture at an alkaline pH, with the complete elimination of the precipitation being observed for a pH higher than 8.4. However, this

equilibrium remains fragile with notable risks of precipitation under the influence of the contact environment (the eye). This environment could modify the equilibrium between the two molecules and lead to intra-ocular precipitation, as demonstrated in the literature during an intravitreal injection [14].

Cage molecules, such as cyclodextrins (CD), are known to be able to encapsulate compounds. CD are cyclic oligosaccharides of the a(1,4) D glucopyranose, forming ring polymers (see Supplementary data 1) [15, 16]. The most used shapes are the a, β et γ consisting of 6, 7 and 8 units of glucose. They possess unique advantages by allowing the formation of inclusion complexes with many organic, inorganic, and lipophilic substances. Their use in the field of nanotechnology, especially for drug delivery, is constantly increasing [14]. In addition to their encapsulation capabilities, CD are also widely used for the formulation of eye drops to increase the aqueous solubility of the molecule, and reduce local irritation [17, 18]. It has also been shown that their basket-shaped cavity can protect some molecules from degradation [19]. Misiuk et al. [20] demonstrated by various analyses (including NMR and molecular docking techniques) that CZ and hydroxypropyl-γ-CD formed a complex with a 1:1 stoichiometry, with the thiazole ring with sulphur and nitrogen atoms with C–O and C–N bands of CZ inserted into the cavity of HP-γ-CD being the most likely complexation pathway. Zarif et al. [21], on the other hand, worked on the equimolar complexation of VM with HP-β-CD in order to evaluate the complexes’ ability to prolong the release of VM from a solid form of the complex. Building on the works of these two previous authors, Bouattour et al. [13] also showed that it was possible to successfully mitigate the precipitation by encapsulating CZ and VM in CD hydroxypropyl (HP) derivatives (HP-γ-CD and HP-β-CD, respectively) using ratios of 5:1 and 3:1 for respectively (HP-β-CD/VM and HP-γ-CD/CZ), but they did not investigate the stability of the mixture of these fragile antibiotics.

The objective of the work presented here was therefore to study and compare the physicochemical stability over 168 days (samples frozen for about 6 months) of these two formulations combining VM and CZ: one using CDs (CD formulation) and the other one without CD (CD-free) at an alkaline pH (pH higher than 8.4).

# Materials and methods

## Formulation, preparation, and storage

The two ophthalmic formulations were prepared by combining two licenced drugs of CZ (Ceftazidime 1 g, batch 211139, expiring September
2023, Viatris® Santé, Lyon, France) and VM (Vancomycine 1 g, batch D0415, expiring August 2024, Viatris® Santé, Lyon, France), resulting in a final concentration of 25 mg/mL for both antibiotics. The difference between these formulations is based on the method used to address the incompatibility issue: either a predefined appropriate pH (CD-free), or of the use of a mixture of cyclodextrins (CD formulation). The first formulation, called CD-free formulation was prepared with a pH target of 8.5. This formulation did not contain cyclodextrins. The CD formulation was prepared according to the work published by Bouattour et al. [13]. HPβCD (CAS 12844635-5, Sigma-Aldrich, St. Louis, MO, USA) and HPyCD (CAS 128446-34-4, Sigma-Aldrich, St. Louis, MO, USA) were used in this formulation at a final concentration of 125 and 216.8 mg/mL respectively. For each formulation, the antibiotics were reconstituted and diluted separately in sterile deionized water (Fresenius®) to an initial concentration of 50 mg/mL for the CD-free formulation and 200 mg/mL for formulation with CD.

The two antibiotic solutions were mixed in equal volume in the CD-free formulation. Then, pH was adjusted to a pH target of 8.5 using 1 N sodium hydroxide solution (CAS: 1310-73-2, Honeywell Germany). The formulation with CD was prepared as described in the work published by Bouattour et al. [13]. In brief, both cyclodextrins (HPβCDs and HPyCDs) were dissolved in a 30 mM phosphate buffer solution (pH 3 and 4, respectively) to obtain a concentration of 333 and 578.14 mg/mL respectively. The detailed composition of both formulations is presented in Table 1. To obtain the complete dissolution of the CD in the phosphate buffer, the mixture was subjected to ultrasound mediated dissolution for 10 min, using an ultrasonic tank (AL 04-04 Model, Advantage-Lab) followed by 1 h of agitation using a magnetic stirrer (Cimarec, Thermo-scientific). A clarifying filtration was realized for each CD mixture using a 0.45 µm filter (32 mm diameter Supor membrane, reference HP4644, Partisiran model, PALL France). The diluted antibiotics were then mixed with the CD solutions: CZ was

<table><caption>**Table 1:** Composition of the antibiotic's ophthalmic formulations.</caption><thead><tr><th rowspan="3">Chemical components</th><th colspan="2">Final formulation</th></tr><tr><th>CD free formulation</th><th>Formulation with CD</th></tr><tr><th colspan="2">Final solution (100 mL) of 25 mg/mL CZ and VA</th></tr></thead><tbody><tr><td>Initial ceftazidime solution, mL<sup>a</sup></td><td>50</td><td>12.5</td></tr><tr><td>Initial vancomycin solution, mL<sup>a</sup></td><td>50</td><td>12.5</td></tr><tr><td>HPyCD solution, mL</td><td>0</td><td>37.5</td></tr><tr><td>HPβCD solution, mL</td><td>0</td><td>37.5</td></tr><tr><td>Sodium hydroxide (NaOH) 32 % m/v</td><td>Quantity necessary to adjust to pH=8.5</td><td>Quantity necessary to adjust to pH=8</td></tr></tbody></table><table><thead><tr><th rowspan="3">Chemical components</th><th colspan="2">Individual antibiotic solutions</th></tr><tr><th colspan="2">Ceftazidime solution (100 mL)</th></tr><tr><th>CD free formulation</th><th>Formulation with CD</th></tr></thead><tbody><tr><td>Ceftazidime pentahydrate, Viatris®, batch 20103 exp. 08/2022</td><td>5/50</td><td>20/200</td></tr><tr><td>Quantity, g/concentration, mg/mL</td><td></td><td></td></tr><tr><td>Deionized water</td><td>QSP 100 mL</td><td>QSP 100 mL</td></tr><tr><td></td><td colspan="2">Vancomycin solution (for 100 mL)</td></tr><tr><td>Vancomycin hydrochloride, Viatris®, batch C0282, exp. 09/2023</td><td>5/50</td><td>20/200</td></tr><tr><td>Quantity, g/concentration, mg/mL</td><td></td><td></td></tr><tr><td>Deionized water</td><td>QSP 100 mL</td><td>QSP 100 mL</td></tr></tbody></table><table><thead><tr><th rowspan="2">Chemical components</th><th colspan="2">Individual cyclodextrin solutions</th></tr><tr><th>HPyCD solution (100 mL)</th><th>HPβCD solution (100 mL)</th></tr></thead><tbody><tr><td>HPyCD (CAS 128446-34-4), g</td><td>57.81</td><td>0</td></tr><tr><td>HPβCD, g (CAS 128446-35-5)</td><td>0</td><td>33.3</td></tr><tr><td>Phosphate buffer, 30 mM, pH=3</td><td>0</td><td>QSP 100 mL</td></tr><tr><td>Phosphate buffer, 30 mM, pH=4</td><td>QSP 100 mL</td><td>0</td></tr></tbody></table><table><thead><tr><th rowspan="2">Chemical components</th><th colspan="2">Buffer solution (quantity needed for 100 mL)</th></tr><tr><th>Phosphate buffer, 30 mM, pH=3</th><th>Phosphate buffer, 30 mM, pH=4</th></tr></thead><tbody><tr><td>Natrium dihydrogenophosphate dihydrate (NaH<sub>2</sub>PO<sub>4</sub>), mg</td><td>410</td><td>460</td></tr><tr><td>Phosphoric acid (H<sub>3</sub>PO<sub>4</sub>) 85 % (85 g/100 mL), (1 volume diluted in 100 volume of deionized water), µL</td><td>4,250</td><td>475</td></tr><tr><td>Deionized water</td><td>QSP 100 mL</td><td>QSP 100 mL</td></tr></tbody></table>

<footnote><sup>a</sup>Concentrations of the antibiotics were respectively 200 mg/mL for the formulation with cyclodextrins and 50 mg/mL for the cyclodextrines-free formulation. CD, cyclodextrins; HP, hydroxypropyl.</footnote>
mixed with the HPyCD solution for 120 min under magnetic stirring and VM was mixed with HPβCD solution for 30 min. Lastly, the two separate solutions were mixed together to reach a final concentration of 25 mg/mL for both antibiotics, and the pH was adjusted to 8. A sterilizing terminal filtration was realized for each formulation using a 0.22 µm pore diameter filter (40 cm² polyether sulfone membrane, Stericup® Sterile Vacuum filtration Systems, Merck millipore, MC2, Clermont-Ferrand, France), and the aseptic distribution was carried out under the laminar airflow of an ISO 4.8 microbiological safety cabinet using a conditioning pump (Repeater® pump, Baxter, Guyan-court, France), into white multi-dose low density polyethylene eye drop vials (Reference VPL28b10N02, laboratoire CAT, Lorris, France). The caps of these bottles (Novelia® nozzle) are designed and validated by the manufacturer as being able to maintain the sterility of the content after opening. The vials were filled with 8 mL of the studied solution and stored in a freezer (temperature of -20 °C) until analysis.

## Organization of the stability study

The physicochemical stability of the formulations was studied for 168 days at -20 °C. The organisation of the study is presented in Supplementary data 2. The first samples were taken at M0 (immediately after packaging in the bottles), other samples were frozen during 1 month (M1), 3 months (M3) or 6 months (M6). At M1, M3 and M6, some of the samples were analysed immediately after thawing (called M1D0, M3D0 and M6D0), and some samples were thawed then kept at refrigerated temperature (between 4 and 8 °C) to be analysed after 12 h (H12) 1 day (H24) and 3 days (H72) after thawing. After being brought back to ambient temperature (20-25 °C), the bottles containing the samples were homogenized for 10 s before the analyses were performed.

## Analyses

The following analyses were performed on the solutions.

**Visual inspection:** For each sample, the multi-dose vials were homogenized before analysis using an orbital shaker (Vortex Genie 2 Agitator, Thermo-Scientific, France). After this step, 1 mL of each multi-dose vial was introduced into a haemolysis tube, without passing through the delivery system, to be visually inspected under daylight and under a polarized white lamp (LV28, Allen and Co., Liverpool, UK). The aspect and colour of the samples was noted, and solutions were observed for any visible particles, haziness, and gas formation. The operator in charge of visual analysis was a pharmacy resident with a six months experience in a quality control laboratory, and had no known visual deficiency.

**Chromaticity and lightness:** As the evaluation of colour changes by visual examination during stability studies has been shown to be less precise than spectrophotometric methods [22], in order to better detect and characterise minute modifications of the colour of the solutions, the chromaticity and lightness of the samples were measured with a UV-visible spectrophotometer (V670, Jasco Corporation®, Lisses, France) using the “Color Diagnosis mode” (Spectra Manager®, version 2.12.00, Jasco Corporation®, Lisses, France). A quartz cell was used for the measurements. Chromaticity and lightness were determined by using the International Commission Internationale de l'Eclairage (CIE) L*a*b* colour space [23].

The lightness (L*), a* (green to red axis) and b* (blue to yellow axis) allowed the calculation of the colour difference (ΔE) according to the following formula: $ΔE = ((ΔL*)^2 + (Δa*)^2 + (Δb*)^2)/0.5$. The values ΔL*, Δa*, Δb* represent the difference between the initial value and the value obtained at the different measured points [24].

Before each series of analysis, a blank sample with deionized water was analysed and checked. Each sample was taken from a pooled mixture (n=1).

**pH:** Each pH measurement was performed using a Seven Multi™ pH-metre within Lab Micro Pro ISM glass electrode (Mettler Toledo, Viroflayn France). Each series of measurements was preceded by the realization of a control point by a standard buffer solution at pH 7 (Mettler-Toledo, Viroflay, France). A calibration curve was performed weekly on the instrument using 4 standard buffer solutions (pH 2.00; 4.01; 7.01 and 11.01, Mettler-Toledo, Viroflay, France). The pH is expressed as the mean ± 95 % confidence interval.

**Osmolality:** Each osmolality measurement of a sample was performed with a volume of 20 µL, using a freezing osmometer Model 2020 Osmometer® (Advanced instruments Inc, Radiometer, SAS, Neuilly Plaisance, France). Each series of measurements was preceded by the realization of a standard calibration point at 290 mOsm/Kg. As the measure instrument failed to freeze undiluted samples containing cyclodextrins, all samples were diluted to half with deionized water before analysis. Osmolality is expressed as the mean ± 95 % confidence interval.

## Vancomycin and ceftazidime quantification and breakdown products research

**Development of a stability indicating method (SIM):** A SIM method was developed specifically for the study. It was conducted following the methodological guidelines issued by the international Conference on Harmonisation for stability studies [25] and recommendations issued by the European Society of Hospital Pharmaceutical Technologies (GER-PAC) and the French Society of Clinical Pharmacy (SFPC) [26]. The SIM was developed so that VM, CZ and their respective breakdown products could be determined using the same chromatographic method. The method was validated on a reverse-phase HPLC Prominence-I LC2030C 3D with diode array detection (Shimadzu France SAS, Marne La Vallée, France), and the associated software used to record and interpret chromatograms was LabSolutions® version 5.82 (Shimadzu France SAS, Marne La Vallée, France). The mobile phase was composed of acetoni-trile (Chromasolv® for HPLC; Honeywell®, Roissy CDG, France) and an acetate buffer solution (with ammonium acetate for HPLC, MC2, Clermont-Ferrand, France) with a concentration of 100 mM, diluted with sterile deionized (Versylene®; Fresenius Kabi France, Louviers, France) adjusted to pH 5.8. The ratio used of mobile phase was 92 % acetate buffer (pH equal to 5.8) and 8 % acetonitrile (v/v). The stationary phase used was a reverse phase silica column (Nucleodur C18, 250 × 4.6 mm, 5 µm, Macherey-Nagel).

To verify the correct identification of VM and CZ, the standard references of the European Pharmacopoeia were qualified and quantified by the developed chromatographic method (Vancomycin hydrochloride: no. CAS 1404-93-9, reference V0045000 and ceftazidime: n°CAS 78439-06-2, reference C0690500, Sigma Aldrich, Saint Quentin Fallavier, France). The retention times and spectra of the molecules (Pharmacopoeia references) were compared with those of the commercial medications used for this study. Vancomycin for system suitability and
<table><caption>**Table 2:** Degradation conditions applied to molecules of interest.</caption><thead><tr><th rowspan="2">Condition</th><th colspan="2">Vancomycin</th><th colspan="2">Ceftazidime</th></tr><tr><th>Degradation condition</th><th>Contact time</th><th>Degradation condition</th><th>Contact time</th></tr></thead><tbody><tr><td>Acid degradation, HCl</td><td>1 N</td><td>1 h</td><td>0.3 N</td><td>1 h</td></tr><tr><td>Alkaline degradation (NaOH)</td><td>0.5 N</td><td>1 h</td><td>0.002 N</td><td>1 h</td></tr><tr><td>Photolytic degradation</td><td>UVA</td><td>6 h</td><td>UVA</td><td>6 h</td></tr><tr><td>Oxidative degradation</td><td>10 %</td><td>1 h</td><td>3 %</td><td>1 h</td></tr><tr><td>H<sub>2</sub>O<sub>2</sub></td><td></td><td></td><td></td><td></td></tr><tr><td>Heat degradation</td><td>40 °C</td><td>24 h</td><td>80 °C</td><td>1 h</td></tr></tbody></table>

ceftazidime for peak identification (respectively reference Y0002080 and Y0001111) were used to study compound impurities. Pyridine (no. CAS: 110-86-1, pyridine anhydrous, 99.8% reference 270970, Sigma Aldrich, Saint Quentin Fallavier, France) was also used as reference to compare with retention time and spectra of CZ breakdown compound peaks.

Five types of forced degradation were applied to the two molecules of interest. These applied degradations were presented in Table 2.

According to the data available in the European Pharmacopoeia, the detection and quantification of the molecules of interest was performed on HPLC at a wavelength of 256 nm for ceftazidime and 220 nm for Vancomycin [27, 28]. Pyridine was detected and analysed at a wavelength of 256 nm.

**Method validation:** The linearity was verified by the realization of three calibration curves prepared during three successive days for vancomycin, ceftazidime (European Pharmacopoeia reference standard respectively V0045000 and C0690500, from Sigma Aldrich). The calibration points were of 2.5, 10, 15, 40 and 80 µg/mL for ceftazidime and vancomycin. All calibration points were prepared by dilution in sterile deionized water. Each calibration was considered acceptable if the R² was greater than or equal to 0.999. Variance homogeneity was checked using Cochran's test. The applicability of linear model was determined by ANOVA tests.

The repeatability was tested on three successive days, with the realization each day of six samples of each of the two antibiotics at a concentration of 20 µg/mL.

The precision of the method was verified by the repeatability itself determined by the calculation of the relative standard deviation (RSD) of intraday analysis and intermediate precision was evaluated using an RSD of intraday analysis [29]. The acceptability of the method was realized by the application of the RSD. It was judged acceptable if lower than 5 %. The accuracy was checked by recording 5 theoretical concentrations to obtain a linear curve and thus a straight-line equation. The overall accuracy profile was constructed according to Hubert et al. [30–32].

The existence of a matrix effect was evaluated by reproducing the previous methodology with the presence of all excipients (including the CDs) present in the formulation and by comparing the calibration curves and intercepts. As an additional investigation, the excipients (CD) at the concentrations applied in the formulation were also analysed

chromatographically to check the absence of any peaks that could interfere with the quantification of VM and CZ. All excipients used in the formulation were present at the time of verification. The UV spectra of VM, CZ and pyridine were compared with and without excipients.

**Turbidity:** The turbidity of the solutions was measured by pooling the volume of 4 units to obtain a volume greater than or equal to 15 mL. A control point (10 FNU) was performed before each series of measurements on the turbidimeter (2100Q Portable Turbidimeter, Hach Lange, Marne La Vallée, France). The calibration of the instrument was performed using 3 measurement points (10, 100 and 800 FNU).

**Particles counting:** The analyses were performed in accordance with the monograph 1,163 Eye Preparations of the European Pharmacopoeia as well as monograph 2.9.53 Particulate contamination: sub-visible particles in non-injectable liquid preparations [33, 34]. The subvisible particles counting was measured using a HIAC Royco 9703 (Hach Lange, Noisy le Grand, France) equipped with a HRLD 400 EC detector. The results were expressed in particles per millilitre. Two particle sizes were measured: particles larger than 10 µm and particles larger than 25 µm. To obtain the necessary volume for a correct analysis (25 mL), 4 units were pooled for each measurement, and 4 measurements of 5 mL were thus carried out on each pool of solution. The first measurement was systematically excluded from the analyses. Prior to the measurement, a blank sample was performed with deionized water in an identical container as the one used for the analysis of the pooled solution.

**Sterility assay:** Sterility testing was performed as described in the European Pharmacopoeia Monograph 2.9.6 [35].

The analysed samples were opened under the laminar air flow of an ISO 4.8 microbiological safety cabinet then filtered under vacuum using a Nalgene® analytical test filter funnel fitted onto a 47 mm diameter cellulose nitrate membrane with a pore size of 0.45 mm (ref 147-0045, Thermo-Scientific, MC2, Clermont-Ferrand, France). Sterility test method was validated (i.e. growth promotion test, validation of the sterility assay and elimination of the inhibitory effect) by Thibert et al. [36]. The membranes were then rinsed with 500 mL of deionized water (Versylene, Fresenius Kabi, Louviers, France), cut in half and incubated in two different liquid culture media in equal volume (thioglycolate medium and soya trypcase). Each culture medium was incubated for 14 days at 30–35 °C for thioglycolate and 20–25 °C for soya trypcase. The volume of 500 mL of deionized water had been previously shown to remove any inhibitory effect of the antibiotics on the bacterial growth [36].

**Data analysis – acceptability criteria:** A variation of VM and CZ concentrations outside the 90–110 % interval of initial concentration (including the limits of a 95 % confidence interval of the measures) was considered as a being a sign of significant concentration variation. For concentrations fluctuating between a 90 and 95 % or 105 and 110 % range of initial concentration, the risk of instability was assessed in regard to the presence or absence of breakdown products and the variation of the physicochemical parameters. Throughout the study, the appearance of any unidentified breakdown products, or the clear increase in the area under curve of a peak already present and attributed to either an impurity or a possible breakdown product was taken into account in the stability assessment. The observed solutions must be limpid, of unchanged colour, and clear of visible signs of haziness or precipitation.
Since there are no standards that define acceptable pH or osmolality variation, pH measures were considered to be acceptable if they did not vary by more than one pH unit from initial value [26]. Osmolality results were interpreted considering clinical tolerance of the preparation and were considered acceptable if they did not vary by more than 10% from initial values. Turbidity was considered to be acceptable for variations of less than 0.5 Formazin Nephelometric Units (FNU). Lightness and chromaticity measures were interpreted with regards to the calculated colour difference ΔE, for which is has been estimated that if ΔE<1 then the colour change is undetectable to the naked eye of even an experienced observer [37]. Regarding of particles counting, the results were judged acceptable if the mean number of particles does not exceed 1,000 per millilitre for particles equal to or greater than 10 µm in size and does not exceed 100 per millilitre for particles to or greater than 25 µm in size, in concordance with monograph 1,163 of the European Pharmacopoeia [33].

# Results

## Validation of the chromatographic method

### Application of method and realization of calibration curves

With the method used, ceftazidime and vancomycin showed respectively an average retention times of 6.2 ± 0.1 min and 18.5 ± 0.5 min (mean retention time ± standard deviation). The chromatograms with the Pharmacopoeia analytical standards of the molecules of interest are presented in

Figure 1. It can be noted that the area of the peaks obtained from the commercial Viatris® vials were significantly higher (by approximatively 15 %) than the areas obtained with the European Pharmacopoeia standard.

The chromatograms of CD (HPβCD and HPγCD) were presented in Figure 2. No peaks were visible at the retention time of the molecules of interest.

The calibration curves obtained were linear for both molecules for concentrations between 2.5 and 80 µg/mL. The mean linear regression equations for the antibiotics were presented in the form $y = ax + b$, y being the area under the curve of the antibiotics and x the antibiotic concentration expressed in µg/mL. The mean linear equation for vancomycin was $y = 14,116.79x - 5,049.66$. The coefficient of determination was 0.999, and no matrix effect was detected with Vancomycin. The relative mean bias coefficients were less than 3 % for the calibration points, except for the 2.5 µg/mL, for which it was of 30 %. The mean repeatability RSD coefficient and mean intermediate precision RSD coefficient were both of less than 5 %. For CZ, the coefficient of determination was also of 0.999. The mean slope of the calibration curves obtained with the Pharmacopoeia reference was significantly different from the one obtained with the prepared formulation (antibiotics originating from commercialized Viatris® vials + CD), but the intercept was not significantly different. The mean linear equation used to obtain the concentrations was the one obtained from

<figure><img src="image_5.png" /><figcaption>Figure 1: Chromatograms of ceftazidime at 256 nm for (A) the European pharmacopoeia standard, (B) the commercial medication (Viatris laboratory) and vancomycin at 220 nm for (C): the European pharmacopoeia standard and (D): the commercial medication (Viatris laboratory).</figcaption></figure>
<figure><img src="image_6.png" /><figcaption>**Figure 2:** Chromatograms of a solution containing hydroxypropyl-β-cyclodextrin and hydroxypropyl-γ-cyclodextrin, at 220 nm (A) and 256 nm (B).</figcaption></figure>

the pharmacopoeia reference (y = 16,710.19x – 2,262.465). The relative mean bias coefficients were of less than 2 % for the calibration points, except for the calibration point of 2.5 µg/mL, for which it was of 3.75 %. The mean repeatability RSD coefficient and mean intermediate precision RSD coefficient were both less than 5 %.

## Forced degradations

The retention times, areas under the curve and degradation ratios (Breakdown products divided by the molecule of interest, expressed as a percentage) of all the compounds obtained by forced degradation are visible in Supplementary data 3. Chromatograms representing the degradation products formed upon application of the different types of degradation are visible in Figure 3. None of the breakdown products possessed a retention time that could interfere with VM or CZ quantification.

Figure 4 represents the degradation products obtained by forced degradation of the molecules of interest (ceftazidime and vancomycin) as a function of relative retention times. The diameter of the disks is expressed as a percentage of the ratio of the area under the curve of the degradation product to the reference area under the curve (before degradation) of the molecule of interest.

For CZ, numerous breakdown products where noticed, for all forced degradation conditions except UVA radiations. Contact with a 0.002 N NaOH solution resulted in a

degradation of more than 50 % of ceftazidime, indicating a very high sensitivity to alkaline degradation. Heat and acidic degradation also yielded several breakdown products, but to a lesser degree.

For VM, each type of degradation revealed breakdown products. The degradation conditions used allowed to have a degradation of vancomycin ranging from 1 % (UV degradation) to about 25 % (alkaline degradation).

## Research and identification of impurities for ceftazidime and vancomycin

Chromatograms of Ceftazidime of peak identification and Vancomycin suitability were studied. For Ceftazidime, impurity G was potentially identified (retention time: 11.26 min and relative retention time compared with ceftazidime: 1.82). The retention times of impurities found with the Vancomycin suitability solution do not interact with the retention time of vancomycin and ceftazidime.

## Identification of a degradation compound: pyridine

The RT of pyridine was estimated at 11.6 ± 0.4 min, with a relative retention time (RRT, relative to the ceftazidime retention time) of 1.9. The chromatograms and 3D spectra of pyridine and the alkaline forced degradation of ceftazidime are shown in Figure 5. The peak with a RT of 11.2 min corresponds to pyridine.
<figure><img src="image_7.png" /><figcaption>**Figure 3:** Chromatographs of ceftazidime (A) and vancomycin (B) at different conditions of forced degradation (1): alkaline degradation, (2): acid degradation, (3): UV degradation, (4): oxidative degradation, (5): heat degradation.</figcaption></figure>

The limit of detectability of pyridine was estimated at 0.05 µg/mL and the limit of quantification (LOQ) at 0.5 µg/mL.

## Visual inspection, chromaticity measurements, particles counting and turbidity

### Visual inspection

For the CD-free formulation, a haziness and a precipitate were systematically observed 24 h after thawing. This phenomenon was more intense for the samples analysed

on the third day after thawing, resulting in a white opalescent solution. For the formulation with CD, the samples analysed immediately after thawing (M1H0, M3H0, and M6H0) and 12 h post-thawing (M1H12, M3H12 and M6H12) remained limpid, with no visible appearance of particulate matter or haziness. Haziness was present on the third day after thawing, but it remained less intense than the one noticed for the CD-free solution. At the sixth month after thawing (M6H0) time-point, a pinkish colouration was observed for the mixture without cyclodextrins, which was not observed in the formulation containing CD. The visual aspect of the solutions at different time-points is shown in Figure 6.
<figure><img src="image_8.png" /></figure>

<figure><img src="image_9.png" /><figcaption>**Figure 4:** Degradation products of antibiotics applied with conditions of degradation. (A) Degradation products of ceftazidime (CZ) and (B) vancomycin (VM) applied with conditions of degradation. (1): UVA (during 6 h), (2): oxidation degradation ($H_2O_2$ 3 % 1 h for CZ; $H_2O_2$ 10 %, 1 h for VM), (3): heat degradation (80 °C, 1 h for CZ, 40 °C, 24 h for VM), (4): alkaline degradation (NaOH 0.002 N 1 h for CZ, NaOH 0.5 N 1 h for VM) and (5) acid degradation (HCl 0.3 N 1 h for CZ; HCl 1 N 1 h for VM).</figcaption></figure>
<figure><img src="image_10.png" /><figcaption>**Figure 5:** Chromatograms of pyridine and alkaline degradation – spectra of interest molecules. (A) Chromatograms of pyridine (Europeans pharmacopeia reference); (B) ceftazidime alkaline degradation; (C) spectra of degradation product of ceftazidime; (D) spectra of pyridine (reference 270970, Sigma Aldrich).</figcaption></figure>

<figure><img src="image_11.png" /><figcaption>**Figure 6:** Visual aspect of samples 24 and 72 h after thawing (refrigerated storage). CD, cyclodextrins. M0, M1, M3, M6: start of study, then 1, 3 and 6 months of frozen storage. H24 and H72: respectively 24 and 72 h after thawing.</figcaption></figure>
## Turbidity

The results of turbidity are presented in Supplementary data 4.

For the CD-free formulation, turbidity results didn't vary by more than 0.5 FNU only up until M1H24. All other further time points were out of specifications. For the formulation with CD, turbidity measures were within specifications up until M3H24 and M6H12.

## Subvisible particles counting

The result of particles counting (mean and standard deviation) are visible in Figure 7. The number of particles larger than 10 µm remained very high from the first day of sampling for the CD-free formulation, and not measurable at M6H24. For the formulation with CD, the number of particles remained quite low on the first day of thawing but became unquantifiable on the third day. Particle numbers were not measurable for all samples after 72 h of refrigeration after thawing (M1H72, M3H72 and M6H72) (beyond instrumental measuring possibilities).

These results were remained coherent with the visual inspections, notably the appearance of a precipitate.

## Chromaticity and lightness

The chromaticity and luminance results are presented in Table 3. For the formulation with CD, the lightness L* was stable until the third day after thawing, after which a clear decrease was observed (respectively of about 30 and 94 % for M1H72 and M3H72). The increase in ΔE was notable (>1) 72 h after defrosting (M1H72 and M3H72). After 6 months of storage, the change was notable 24 h after thawing (M6H24).

For the CD-free formulation, the decrease in L* was observed from the first day after thawing after the first month of storage and was of the order of 40 %. The increase in a* at M6H0 (2.38 vs. 0.52 at M3H0 and 0.26 at M1H0) was correlated with the evolution of the colour observed during the visual inspection. The increase in ΔE was notable at all time-points, but even more so at M6 after thawing and refrigerated storage.

## pH

The initial pH was of 8.46 ± 0.01 for the CD-free formulation and 8.13 ± 0.01 for formulation with CD. A small decrease in pH was observed over time for both formulations, the minimum observed pH was 7.62 ± 0.01 for the formulation with CD and 8.08 ± 0.03 for the CD-free formulation (at

<figure><img src="image_12.png" /><figcaption>**Figure 7:** Particles counting of two formulations of eye drops. CD, cyclodextrins; HQLT, high quantity limit tolerance. M0, M1, M3, M6: start of study, then 1, 3 and 6 months of frozen storage. H24 and H72: respectively 24 and 72 h after thawing.</figcaption></figure>
<table><caption>**Table 3:** Evolution of lightness and chromaticity for two formulations (pooled samples, n=1).</caption><thead><tr><th rowspan="2"></th><th colspan="4">With CD</th><th colspan="4">CD free</th></tr><tr><th>L*</th><th>a*</th><th>b*</th><th>ΔE</th><th>L*</th><th>a*</th><th>b*</th><th>ΔE</th></tr></thead><tbody><tr><td>M0</td><td>98.36</td><td>-0.99</td><td>5.97</td><td></td><td>99.05</td><td>-1.22</td><td>4.44</td><td></td></tr><tr><td>MIH0</td><td>98.63</td><td>-0.99</td><td>5.95</td><td>0.15</td><td>98.58</td><td>-0.26</td><td>4.63</td><td>2.36</td></tr><tr><td>M1H12</td><td>98.31</td><td>-0.98</td><td>5.76</td><td>0.09</td><td>98.48</td><td>-0.28</td><td>4.82</td><td>2.71</td></tr><tr><td>M1H24</td><td>97.69</td><td>-1.02</td><td>6.02</td><td>0.90</td><td>58.83</td><td>4.81</td><td>26.05</td><td>4,242.00</td></tr><tr><td>M1H72</td><td>67.53</td><td>2.62</td><td>2.175</td><td>2,425.06</td><td>0.2</td><td>0.01</td><td>0.12</td><td>19,583.00</td></tr><tr><td>M3H0</td><td>98.46</td><td>-0.96</td><td>5.92</td><td>0.03</td><td>97.13</td><td>0.52</td><td>5.74</td><td>16.81</td></tr><tr><td>M3H12</td><td>98.5</td><td>-0.89</td><td>5.69</td><td>0.22</td><td>97.79</td><td>0.88</td><td>5.06</td><td>12.76</td></tr><tr><td>M3H24</td><td>98.76</td><td>-1.01</td><td>5.94</td><td>0.32</td><td>98.61</td><td>-0.86</td><td>6.22</td><td>6.98</td></tr><tr><td>M3H72</td><td>6.18</td><td>0.86</td><td>3.96</td><td>17,009.23</td><td>0.17</td><td>0.03</td><td>0.12</td><td>19,594.96</td></tr><tr><td>M6H0</td><td>98.5</td><td>-0.8</td><td>5.81</td><td>0.17</td><td>96.67</td><td>2.38</td><td>5.45</td><td>39.29</td></tr><tr><td>M6H12</td><td>98.41</td><td>-0.92</td><td>5.68</td><td>0.18</td><td>66.13</td><td>6.06</td><td>27.77</td><td>3,362.03</td></tr><tr><td>M6H24</td><td>99.58</td><td>-0.97</td><td>5.61</td><td>3.24</td><td>0.85</td><td>0.46</td><td>0.8</td><td>19,318.62</td></tr><tr><td>M6H72</td><td>2.36</td><td>0.4</td><td>1.72</td><td>18,471.99</td><td>0.09</td><td>0.03</td><td>0.09</td><td>19,627.13</td></tr></tbody></table>

CD, cyclodextrins. M0, M1, M3, M6: start of study, then 1, 3 and 6 months of frozen storage. H0, H12, H24 and H72: storage hours after thawing.

M1H72). The complete results for pH are presented in the Supplementary data 4. The variations in pH measurements at the different sampling times of the two formulations are of less than 1 pH unit, making them compliant with the expected specifications.

## Osmolality

The initial mean osmolality was of 282.25 ± 7.04 mOsm/Kg for formulation with CD, and 75.5 ± 0.92 mOsm/Kg for the CD-free solution. All the results are visible in Supplementary data 4. The variation of the osmolality of the samples remained below 10% for both formulations. These results are therefore consistent with the specifications.

## Vancomycin, ceftazidime and breakdown products analysis

At the beginning of this study (M0H0), the concentrations (mean ± 95% confidence interval) of ceftazidime and vancomycin were respectively of 27.50 ± 1.08 mg/mL and 23.25 ± 2.17 mg/mL for the CD-Free formulation, and of 26.47 ± 1.58 mg/mL and 23.23 ± 0.64 mg/mL for the formulation with CD. Whereas VM concentrations remained relatively stable, CZ concentrations decreased rapidly after thawing for both formulations (Figure 8). For example, for the CD formulation, they were only above the inferior specification limit up until time-points M1H72, M3H72, and from M6H24.

<figure><img src="image_13.png" /><figcaption>**Figure 8:** Concentration of ceftazidime and vancomycin at the different sampling points (expressed in % of the initial quantity, mean ± 95% confidence interval). CD, cyclodextrins. M0, M1, M3, M6: start of study, then 1, 3 and 6 months of frozen storage.</figcaption></figure>
The evolution in pyridine peak area is presented in Figure 9. The areas under the curve were lower than the LOQ of pyridine, not allowing the expression of the results in

pyridine concentration. The formation of pyridine remained low but was more important for the formulation with CD than for the CD-free formulation.

<figure><img src="image_14.png" /><figcaption>**Figure 9:** Pyridine area formed (AUC, area under curve). Comparatively: AUC of ceftazidime at M0 CD-free: 369909 and with CD: 356068. CD, cyclodextrins. M1, M3, M6: respectively 1, 3 and 6 months of frozen storage. x-axis: number of days after thawing.</figcaption></figure>
Sterility assay

The sterility results did not reveal any microbial growth at
the various sampling points.

Discussion

In this study, we proved that the two formulations that we
tested of eye drops made of CZ and VM, formulated so that
the antibiotics were compatible in a mixture together, did
not possess extended stability. Overall, our results showed a
very limited physicochemical stability for both formulations
when stored at refrigerated temperature after thawing, with
the appearance of a physical instability (precipitate) from
the first day for the CD-free formulation and at the third day
for the formulation with CD, as well as a decrease in cefta-
zidime concentrations for both formulations.

The first formulation did not contain CD but was
formulated at a target pH of 8.5: this pH was determined in
order to avoid the phenomenon of precipitation when
ceftazidime and vancomycin are mixed [6], and whilst
respecting as much as possible the physiological pH of the
eye [38]. In the second formulation, we tested the protective
power of the cage molecules over a long period of time,
following the work published by Bouattour et al. [13] that
demonstrated an absence of precipitation of this formula-
tion during 48 h for the optimized molar ratios that were
used here (5:1 and 3:1 for respectively HP-β-CD/VM and
HP-γ-CD/CZ). Regarding the feasibility of the preparation,
the major difficulty lay in the dissolution of the CD in the
buffer solution (3.33 g/L for HPβCD and 5.8 g/L for HPyCD),
where the formation of large clusters delayed the dissolu-
tion, and this despite the concentrations applied here being
far from the theoretical maximum solubility concentra-
tions for these CD [39]. As the pH of this formulation was set
at pH 8.0, thus close to the limit of efficiency of the CD on the
incompatibility, a 30 mM phosphate buffer was added, to
stabilize the pH [13]. To take into account the risk of corneal
calcification described by the European medical agency
(EMA) [40], its molarity was set at 30 mM, thus far from the
concentrations of 50–100 mM described as being at risk
[41, 42]. In regard of the quick degradation of ceftazidime
[43], freezing was the method of choice to delay as much as
possible any physicochemical reaction during storage.

To adequately perform the stability study, a SIM was
developed to analyse both antibiotics simultaneously. The
method allowed us to quantify the antibiotics and research
breakdown products. The analysis of the validation data
highlighted a decreased precision when the excipients were

used, very likely linked to the increased experimental diffi-
culty they induced. The comparison of the calibration curves
of reference ceftazidime (Pharmacopoeia standard) with
the curves of matrix ceftazidime solutions (commercial
specialty with sodium carbonate, HPβCD and HPγCD) also
highlighted a significantly different value between the
slopes. All the calibration points performed with the solution
with matrix had values higher by about 10–15 % on average
compared to the calibration points performed with the
pharmacopoeia reference. No difference in calibration
slopes were found for VM. Moreover, the chromatographic
analysis of the cyclodextrins alone did not show any differ-
ence compared to a classical blank sample. This isolated
increase in CZ peak area was also noted when validation
points were performed at 20 µg/mL without cyclodextrins
(using the commercial Viatris vials®, with several different
batches). Two hypotheses can therefore be considered: a
potential matrix effect of the excipient of the commercial
ceftazidime product (sodium carbonate) or an overfilling
of the commercial ceftazidime vials corresponding to the
tolerance limits of industrial production, such as the one
found for Zavicefta® (Ceftazidime/avibactam), as the EMA
report for that product states that “An overfill is used in
order to ensure that the entire contents of the reconstituted
vial can be accurately removed” [44]. For all the experi-
ments, the reconstitution of ceftazidime was carried out
after removal of the crimp from the vial (metal tray and
rubber stopper), which reduced the loss of active ingredient
compared to conventional sampling with a needle and sy-
ringe through the rubber stopper carried out by the nurses
[45]. In addition, a terminal rinse was performed on all vials
to minimize the loss of active ingredients. It is therefore
highly likely that the excess in ceftazidime found during our
study resulted from the overfill of the ceftazidime Viatris®
vials.

During the stability study, the visual examinations
revealed the presence of a cloudiness as early as 24 h after
thawing for the CD-free mixture; the turbidity found on
these samples was within specifications only for points
M1H0 and M1H12. For the solutions containing CDs, the
turbidity was visible to the naked eye only for the H72 post-
thaw points, where the turbidity was also found to be
outside the specifications for these points as well as for all
the samples taken at month 6 (M6). These results are quite
consistent with particle count data where the number of
particles increased with thawing time regardless of the
month of thawing. The particulate counting limits were
reached for all the H72 points whatever the formulation as
well as the M6H24 for the CD-free formulation.

None of the colour difference (ΔE) measured for
CD-free formulation complied with the specifications. For
the formulation with CD, ΔE was above specifications for all H72 points as well as the M6H24. These results are therefore consistent with the visual inspection and the turbidity results found. pH and osmolality were within specifications and within tolerance with the physiological pH of the eye, so these two parameters were not considered as limiting factors in the stability study.

The quantification of the antibiotics did not show a significant loss of VM at any of the sampling points: the values obtained always remain between 90 and 110 % of the target value for both formulations despite the appearance of precipitate. A more pronounced loss of CZ in both formulations was noticed, especially after thawing for all the H72 samples and for the M6H24 time-point, thus suggesting a higher degree of instability for CZ than for VM, which is consistent with previously published data. Indeed, an experimental study by Moreno et al. showed a loss of 85 % of ceftazidime after an exposure to a temperature of 45 °C for 24 h [46] and the work of Nguyen et al. reported a 10 % loss of ceftazidime after 19 h at room temperature [47]. Our work supports the notion that frozen storage inhibits most if not all of the CZ loss, but that refrigerated storage only slows it down. In our study, the decrease in CZ concentrations is more pronounced in the formulation with CD than the CD-free formulation. This could be caused either by CZ precipitation (possibly more the case in the CD-free formulation) or by chemical degradation. NMR modelling showed good inclusion of CZ in CD [13], however the encapsulation does not seem to confer adequate protection against CZ degradation, perhaps because the fragile part of the molecule may be outside the hydrophobic core of the CD. Another hypothesis could be an interaction of the CD with CZ, thus being able to create a destabilization of the molecule leading to its degradation [48]. This destabilization could be caused by an intrinsic catalytic activity of the CDs mediated by their free hydroxyl groups, as previously illustrated for example by Wouessidjewe et al. [49] or Popielec et al. [50], who in their excellent review of the effects of cyclodextrins on the chemical stability of drugs summarised that CDs could catalyse β-lactam degradation under basic conditions, possibly by ionization/deionization of carboxyl group of β-lactam antibiotic. This degradation may also lead to the appearance of degradation products outside the CD, possibly creating an interaction with VM and the formation of a precipitate. Moreover, the risk of intra-ocular precipitation must be considered: the moderate buffering capacity of tears could decrease the pH initially selected, which could lead to an even greater precipitation [38]. A lacrimation phenomenon could also cause a dilution of the eye drops, and the

balance of H⁺ and OH⁻ ions could re-equilibrate the pH and create the precipitation phenomenon [51].

The peak area of pyridine was also investigated, as it is a known CZ breakdown product; this was notably found in a study from Stendal et al. [52] which showed the formation of 0.4 % pyridine in mass after reconstitution and dilution of ceftazidime to 60 mg/mL stored for 7 days at 4 °C. However, in view of the low pyridine levels (pyridine peak areas were not high enough to allow an accurate quantification), we cannot assert a significant difference between the two formulations. The ocular toxicities mentioned report the absence of toxicity for concentrations of 0.08 M pyridine, a value which we are well below in this study [53]. The systemic passage by the ocular route would also remain quite low [54]. The toxicity found in the literature is only applicable to the IV route and with much higher doses.

Overall, in view of these initial results, neither of the formulations that were tested were good candidates for further more advanced studies, such as a pre-clinical study/safety usage investigation, as they have proven to be too unstable physiochemically.

## Conclusions

In this study, which lasted 168 days, we showed that both formulations (with and without CD) showed very limited physicochemical stability after thawing for the storage conditions that we tested (3 months frozen and 24 h after defrosting for the solution with cyclodextrins). The cyclodextrins-free for formulation proved too instable after freezing and defrosting. In view of the complexity of the formulation, and the instabilities of both formulations, the theoretical advantages of combining VM and CZ seem somewhat nullified.

**Acknowledgements:** The authors gratefully acknowledge Aurélie Hanke for her help during the conditioning process and Amandine Buch, Marie Mula and Lauralee Wnuk for their help in performing the microbiological assay.

**Informed consent:** Not applicable.

**Ethical approval:** Not applicable (experimental in-vitro study).

**Author contributions:** All authors have accepted responsibility for the entire content of this manuscript and approved its submission.

**Competing interests:** The authors state no conflict of interest.

**Research funding:** None declared.
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**Supplementary Material:** This article contains supplementary material (https://doi.org/10.1515/pthp-2023-0007).