An Exploratory Study of a New Vancomycin Eye Drops Formulation for Extemporaneous Compounding

Abstract

Purpose:

Compounded eye drop solutions of vancomycin hydrochloride have important clinical applications, such as postoperative antimicrobial prophylaxis and bacterial keratitis. There exists a plethora of data to support the use of various liquid vehicles to compound vancomycin hydrochloride eye drops. However, there are a number of limitations for implementation, especially the frequent shortage or discontinuation of the vehicle products. This study was designed to investigate the use of an OTC eye wash product as the evergreen vehicle and to evaluate the physical and chemical stability of the new formulation.

Methods:

The Advance Eye Relief^® eye wash and vancomycin hydrochloride for injection vials were used to prepare 10 and 50 mg/mL vancomycin eye drop solutions. The solutions were packaged in Steri-Droppers^® bottles and stored in a freezer for 14 days followed by 28 days in refrigeration. The 14-day period of freezing was included to allow time for sterility testing. At pre-determined stability time points, samples were taken for visual inspection, pH and osmolality measurement, and analysis by a stability-indicating high performance liquid chromatography (HPLC) method.

Results:

Freshly prepared vancomycin eye drops were clear, colorless, and free of particulates. The pH readings were 7.03 and 6.28 for the 10 and 50 mg/mL solutions, respectively. The osmolality of both solutions were within the range of 300-330 mOsmol/kg and considered isotonic. Initial drug concentrations of all samples were confirmed by HPLC to be within 100%-103% of the label claims. Throughout the stability study period, there were no significant changes in the appearance, pH, or osmolality of any samples. The HPLC results also confirmed that the drug concentrations in all stability samples were within 98%-101% of the initial time zero values and no significant degradation product peaks were observed.

Conclusion:

A new compounded vancomycin eye drop formulation was developed to mitigate vehicle sourcing issues. This eye drop formulation was easy to prepare, exhibited satisfactory properties for ophthalmic applications, and remained stable chemically and physically when stored for 14 days in freezer followed by 28 days in refrigerator.

Introduction

Vancomycin is a tricyclic glycopeptide antibiotic originally isolated from Amycolatopasis orientalis.^1,2 First approved by Food and Drug Administration in 1958, it has been used effectively to treat serious infections caused by gram-positive bacteria, such as Staphylococcus aureus and Enterococcus faecalis.^1-4 Many studies and guidelines have also been published over the years to support additional off-label uses, including ophthalmic indications.^4-8 The American Academy of Ophthalmology (AAO) recommends topical vancomycin eye drops (10-50 mg/mL) as a mono- or combination therapy to treat bacterial keratitis. ⁹ Vancomycin eye drops are also prescribed frequently as a prophylactic medication to prevent infections after eye injuries or surgeries.^7,10-12 Despite the extensive use of vancomycin for ophthalmic indications, there are no FDA approved eye drop products of vancomycin, ³ most likely due to inherent drug stability issues in solutions and the lack of commercial interest from pharmaceutical companies. Currently all eye drop prescriptions of vancomycin are filled by extemporaneously compounded preparations. ¹³

In United States, only oral and intravenous (IV) injectable products of vancomycin have received FDA approvals. ³ USP monographs have been published for these products, and numerous generic sources are available.^14-16 Due to the inherent stability issues of vancomycin, the IV injectable products are marketed as sterile lyophilized powder for reconstitution or frozen solution in IV bags. The sterile powder product “Vancomycin Hydrochloride for Injection USP” is available at various strengths, ranging from 500 mg to 10 g per vial.^1,3 Based on the description in the product labeling, this product contains no excipient besides possible hydrochloric acid and/or sodium hydroxide for pH adjustment. The simplicity of the formulation and the flexibility of reconstitution to various drug concentrations in different vehicles make the sterile powder product a desired drug source for extemporaneous compounding of vancomycin ophthalmic solutions.

There are numerous studies that investigated the formulation, stability, and antimicrobial activity of compounded vancomycin ophthalmic.^13,17,18 Most of them selected the injectable sterile product as the drug source ingredient for reasons stated above. However, a wide variety of vehicles were employed in these studies, including over-the-counter (OTC) artificial tears, intravenous (IV) fluids, and balanced salt solutions. ¹³ These liquid vehicles present a number of challenges and limitations for implementation in compounding pharmacies. The artificial tears products contain complex formulas, including polymers and surfactants, ¹⁹ which may negatively impact the activity or stability of vancomycin. Due to the small volume sizes available, the artificial tears products are costly and inconvenient to scale up. Moreover, the OTC artificial tears market is highly competitive which leads to frequent new product launches. As such, studies based on artificial tears as compounding vehicles often become obsolete when the old products become discontinued. The IV fluids, such as 0.9% sodium chloride and 5% dextrose, are standard pharmacy items and do not have product turnover issues. While these IV fluids are sterile and isotonic, they do not contain any buffers or preservatives and are not best suited for multi-dose ophthalmic solutions. Balanced salt solutions, used for irrigation during ophthalmic surgeries, are also available commercially. These solutions are formulated as isotonic solutions with multiple electrolytes to mimic the natural compositions of biological fluids. In similar regard to the IV fluids, the balanced salt solutions lack preservatives, making them suboptimal for the multi-dose ophthalmic solutions.^20-22

Aside from the complexity of vehicle selection, chemical stability of vancomycin should be carefully evaluated in compounded ophthalmic solutions. As shown in Figure 1, vancomycin contains various chemically labile or reactive functional groups, such as phenols and acetals.^1,2 Upon reconstitution and dilution in aqueous vehicle, vancomycin is known to undergo rapid oxidation and hydrolysis.^1,13 Unfortunately some published studies monitored drug stability solely based on visual/physical evaluation instead of stability-indicating assays. ¹³

Figure 1.

Chemical structure and molecular weight (MW) of vancomycin hydrochloride. ¹ The MW of vancomycin base is also included for reference. ¹⁴ All strengths and concentrations listed in this article are expressed based on vancomycin base.

Vancomycin hydrochloride MW: 1485.71.

Vancomycin base MW: 1449.25.

With the multifaceted challenges and limitations described above, this study was initiated to develop a new compounded formulation of multi-dose vancomycin ophthalmic solution. The top priorities of this formulation included ease of preparation, low-risk of vehicle discontinuation, and a satisfactory buffer and preservative system for multi-dose eye drops. In the proposed formulation, the sterile vancomycin powder was reconstituted with an OTC eye wash solution to a final drug concentration of 10-50 mg/mL. A stability study was conducted to confirm the desired storage conditions and beyond-use dating (BUD).

Methods

Materials

All materials for compounding vancomycin eye drop solutions were obtained through pharmacy distributors with the original manufacturers, NDCs, and lot numbers listed below. Vancomycin Hydrochloride for Injection USP, 500 mg vials, Mylan (Rockford, IL), NDC#67457-339-00, and Lot#7693284. Advanced Eye Relief^® Eye Wash (referred to as eye wash in later text), 118 mL bottles, Bausch and Lomb (Rochester, NY), Lot#GH18078. Steri-Droppers^® LDPE eye drop bottles, 7-mL, Medi-Dose Group, Catalog#7621.

The pure drug substance powder, Vancomycin Hydrochloride USP, was used to prepare analytical standards. It was purchased from Medisca (Plattsburgh, NY), Lot#167179/C.

The water used for all sample preparation and analysis was produced on-site by a Milli-Q Direct 8 system from Millipore Sigma (Burlington, MA), and it met the purity requirements for Type I ultrapure water. All other solvents, chemicals, and supplies were purchased from Thermo Fisher Scientific (Waltham, Massachusetts).

Visual Inspection

An IV inspection light box (Cat# 17109) from Health Care Logistics (Circleville, OH), with lighted white and black background, was used for visual inspection.

pH Measurement

A Seven Easy model pH meter from Mettler-Toledo was used with a gel-filled pencil-thin pH electrode from Thermo Fisher Scientific. The pH meter was calibrated on each analysis day with standard pH 4 and 7 buffer solutions from Thermo Fisher Scientific.

Osmolality Measurement

A µOsmette Model 5004 osmometer from Precision Systems (Natick, MA) was used to measure the osmolality of the eye drop solutions. The accuracy of the osmometer was verified on each analysis day by 0.9% Sodium Chloride Injection USP.

High Performance Liquid Chromatography Analysis

The high performance liquid chromatography (HPLC) method was adopted and modified from a previous publication by Enrique et al, ⁶ A Model LC-2010AHT system from Shimadzu Scientific Instruments (Marlborough, MA) was equipped with a Luna C18(2), 3 µm, 100 A, 150 mm× 4.6 mm column from Phenomenex (Torrance, CA) as the stationary phase. The detailed mobile phase composition and gradient program are listed in Table 1. Additional instrument parameters were set as follows: column temperature at 40°C, mobile phase flow rate of 0.8 mL/min, sample injection volume of 20 µL, and UV detection at 280 nm. Data collection and processing were performed using the Shimadzu LC Solution software.

Table 1.

HPLC Mobile Phase and Gradient Program.

Time (min)	% Channel A a	% Channel B b
0	88	12
20	96	4
21	88	12
30	Stop

^aChannel A: mixture of water and phosphoric acid (85%) at a ratio of 99.83:0.17 (v/v), adjusted to pH 3.0 using triethylamine.

^bChannel B: methanol.

Five standard solutions were prepared from USP grade of vancomycin hydrochloride powder in purified water. The concentrations of 8, 9, 10, 11, and 12 mg/mL were selected to cover the 80%-120% range of the expected sample concentration of 10 mg/mL from the stability study. As stated in Figure 1 caption, all drug concentrations were calculated and expressed based on vancomycin base, not the hydrochloride salt.

Forced Degradation Study

A forced degradation study of vancomycin was conducted to verify that the HPLC method developed was stability indicating. The conditions used to force degradation were summarized in Table 2, which included combinations of extreme pH, oxidative stress, high temperature, and/or sun light. The samples were prepared by diluting a reconstituted 50 mg/mL vancomycin for injection solution with deionized water in volumetric flasks to a final concentration of 10 mg/mL. Adequate amount of acid (1N HCl), base (1N NaOH), or 30% hydrogen peroxide was added to the respective sample before the final volume was brought to the qs mark with water. Samples were properly sealed and placed in the respective heat/light conditions. Aliquots from samples were taken frequently for HPLC analysis until at least 10% degradation was observed in any samples. ²³

Table 2.

Forced Degradation Study of 10 mg/mL Vancomycin Solution in Water.

Sample #	Stress conditions	% Drug remaining after 72 hours
1	pH 2 and 40°C	59.6
2	pH 10 and 40°C	35.9
3	1% H2O2 and 40°C	49.5
4	1% H2O2 and sun light and room temperature	56.4

Stability Study of Compounded Vancomycin Eye Drop Solutions

Two vancomycin eye drop solutions, 10 and 50 mg/mL, were prepared and packaged in eye drop bottles for the stability study. To ensure uniform starting composition among all bottles, each solution was prepared as a 100-mL batch (~10% overage) prior to bottle filling.

For the 50 mg/mL samples, the Vancomycin Hydrochloride for Injection USP, 500 mg vials were reconstituted with 10 mL of the eye wash solution based on the diluent volume recommendation from the product labeling. ¹ To achieve the most accurate volume measurement of the diluent, the seal/stopper of each vial was removed, and 10 mL eye wash solution was added to the vial using a sterile serological pipet rather than a syringe. Ten vials were reconstitute in this manner to provide a total of 100 mL. The solution contents from all vials were transferred into a clean sterile container and mixed to obtain a homogenous solution. The solution was filled into 30 eye drop bottles, 3 mL/bottle. All bottles were capped immediately, labeled, and placed on stability study. Please note that sterility was not a requirement of this study which focused on the evaluation of physicochemical stability. Nevertheless, aseptic techniques were used during the preparation of the solutions and eye drop bottles to minimize contamination.

For the 10 mg/mL samples, 3 vials of Vancomycin Hydrochloride for Injection USP, 500 mg/vial, were reconstituted with the eye wash solution in the same manner as described above for the 50 mg/mL samples. The solution contents from the 3 vials (~30 mL in total) was transferred into a clean sterile container and mixed thoroughly. A sterile serological pipet was used to accurately transfer 20 mL of the solution into a 100-mL volumetric flask. A sufficient quantity of the eye wash solution was added to the flask until the calibration line mark was reach. The resulting 10 mg/mL solution was filled into 30 eye drop bottles, 3 mL/bottle. All bottles were capped immediately, labeled, and placed on stability study.

Aside from the initial (Day-0) samples, all remaining eye drop bottles were stored in the freezer for 14 days and then transferred to a refrigerator for 28 days. The total duration of the study was 42 days.

At each pre-determined time point, 3 replicate bottles of each drug concentration were pulled for testing. The solutions from the bottles were transferred into glass vials and inspected visually for color, clarity, microbial growth, and particulates. A 1-mL sample was withdrawn for pH measurement, osmolarity, and HPLC analysis. The 10 mg/mL samples were analyzed directly by HPLC. The 50 mg/mL samples were diluted to 10 mg/mL with water prior to the HPLC analysis.

Results

HPLC Method and Forced Degradation Study

With the HPLC parameters described in the Methods section, vancomycin eluted with a retention time between 7.6 and 7.8 minutes. A representative chromatogram of the 10 mg/mL vancomycin standard is shown in Figure 2. On each analysis day, the calibration curve from the 5 standards (8-12 mg/mL) was linear with R² values greater than 0.97. The intra-day and inter-day coefficients of variation were all below 0.1%.

Figure 2.

A representative HPLC chromatogram of 10 mg/mL vancomycin standard solution.

The results of the forced degradation study are summarized in Table 2. Greater than 10% drug degradation was observed in all 4 stressed samples after 72 hours, and the degradation products were separated from the original drug peak by the gradient HPLC method. The results confirmed that the HPLC method was stability-indicating and suitable for the chemical stability evaluation of the compounded vancomycin eye drop solutions.

Stability Study of Compounded Vancomycin Eye Drop Solutions

The freshly prepared vancomycin hydrochloride eye drop solutions, 10 and 50 mg/mL, appeared clear, colorless, and free of particulates. As shown in Table 3, the initial pH readings were 7.03 ± 0.02 and 6.28 ± 0.05 for the 10 and 50 mg/mL solutions, respectively. As shown in Table 4, the initial osmolality readings were 301 ± 10 and 328 ± 2 mOsmol/kg for the 10 and 50 mg/mL solutions, respectively. Over the stability study period of 14 days in freezer and 28 days in refrigerator, no significant changes were detected for the visual appearance, pH or osmolality of any eye drop solution samples.

Table 3.

pH Results of the Vancomycin Eye Drop Stability Study (n = 3).

Sample	pH
	Day-0	Day-14	Day-21	Day-28	Day-42
10 mg/mL	7.03 ± 0.02	6.98 ± 0.00	6.99 ± 0.01	6.96 ± 0.05	7.01 ± 0.04
50 mg/mL	6.28 ± 0.05	6.28 ± 0.05	6.28 ± 0.11	6.20 ± 0.06	6.28 ± 0.06

Note. All samples were first stored in the freezer for 14 days and then moved to the refrigerator for the remaining study period. In other words, the Day-21, Day-28, and Day-42 samples were stored in refrigerator for 7, 14, and 28 days, respectively.

Table 4.

Osmolality Results of the Vancomycin Eye Drop Stability Study (n = 3).

Sample	Osmolality (mOsmol/kg)
	Day-0	Day-14	Day-21	Day-28	Day-42
10 mg/mL	301 ± 10	300 ± 1	299 ± 3	292 ± 1	296 ± 2
50 mg/mL	328 ± 2	328 ± 4	330 ± 3	325 ± 3	330 ± 4

Note. All samples were first stored in the freezer for 14 days and then moved to the refrigerator for the remaining study period. In other words, the Day-21, Day-28, and Day-42 samples were stored in refrigerator for 7, 14, and 28 days, respectively.

The HPLC results are summarized in Table 5. The initial drug concentrations of the freshly prepared eye drop solution samples were 10.07 ± 0.32 and 51.07 ± 1.73 mg/mL, which were equivalent to 100.7% and 102.1% of the label claim values, respectively. Over the stability study period of 14 days in freezer and 28 days in refrigerator, all sample concentrations remained within 98%-101% of the initial drug concentrations. No significant degradation product peaks were observed in the HPLC chromatograms of any stability samples.

Table 5.

HPLC Results of the Vancomycin Eye Drop Stability Study (n = 3).

Sample	Drug concentration (mg/mL)
	Day-0	Day-14	Day-21	Day-28	Day-42
10 mg/mL	10.07 ± 0.32	10.09 ± 0.33	10.01 ± 0.37	9.92 ± 0.51	9.88 ± 0.53
50 mg/mL	51.07 ± 1.73	51.48 ± 1.96	51.10 ± 1.90	51.16 ± 2.73	51.27 ± 2.65

Note. All samples were first stored in the freezer for 14 days and then moved to the refrigerator for the remaining study period. In other words, the Day-21, Day-28, and Day-42 samples were stored in refrigerator for 7, 14, and 28 days, respectively.

Discussion

The main objective of this study is to develop a multi-dose vancomycin eye drop formulation which is easy to prepare by compounding and has a low risk for ingredient sourcing issues. Towards that end, a simple formulation with only 2 source ingredients was identified and characterized. The vancomycin hydrochloride powder for injection was used as the drug source, as it meets the sterile product requirements and can be readily reconstituted and diluted to the desired concentrations of 10-50 mg/mL for ophthalmic indications. The Advanced Eye Relief^® Eye Wash was selected as the vehicle for a number of reasons. This eye wash formulation is a sterile and isotonic solution containing a standard buffer and preservative system (borate and benzalkonium chloride) for multi-dose eye drop solution products. The eye wash formulation composition is also relatively simple, free of unnecessary excipients, and unlikely to change in future. As an essential OTC pharmacy item, the eye wash product is available in large volume bottles at low cost.

Besides the ingredients for eye drop solutions, packaging is an integral component for the final preparation. The Steri-Droppers^® LDPE bottles were selected, because they are commercially available in sterile packaging and simple to use for bottle filling and tip/cap assembling. The 7 mL bottles were selected over the 3 mL ones to provide head space for solution expansion during freezing.

With the above formula and packaging, the initial time-0 data from the stability study demonstrated that the compounded vancomycin eye drops exhibited satisfactory visual appearance, pH, and tonicity for ophthalmic applications. Both 10 and 50 mg/mL samples also met the requirement for strength label claim.

The physicochemical properties of vancomycin was carefully considered for the selection of storage conditions of the compounded eye drop solutions. As shown in Figure 1, vancomycin has a complex chemical structure with multiple functional groups which are prone to oxidation and hydrolysis, such as the phenols and acetals. To reduce the risks of chemical instability and extend the BUD, a combination of 14 days in freezer and 28 days in refrigerator was proposed as the storage condition. The initial 14 days represent the typical turnaround time for sterility testing, during which the compounded preparations are not dispensed to patients.

The stability-indicating HPLC method was used to monitor the chemical stability of vancomycin eye drops over the storage conditions described above. Additionally visual inspection, pH, and osmolality tests were performed to monitor any potential physical instability. The overall results confirmed that the compounded vancomycin eye drop solution samples remained stable chemically and physically throughout the entire study duration of 14 days in freezer and 28 days in refrigerator.

Because this exploratory study was mainly focused on the physical and chemical stability of the compounded vancomycin eye drops, sterility test or antimicrobial effectiveness test was not performed. Future applications of this study for actual patient use should include all relevant microbiological testing as per USP general chapter 797 on sterile compounding. ²⁴

Conclusion

A new vancomycin eye drop formulation of 10-50 mg/mL was developed for extemporaneous compounding and to address the persistent issues of vehicle shortage and discontinuation. The Vancomycin Hydrochloride for Injection product was reconstituted and diluted with an OTC eye wash solution and packaged in commercially available sterile eye dropper bottles. This compounded eye drop preparation was confirmed to exhibit satisfactory properties for ophthalmic applications, and it remained stable chemically and physically when stored for a combination of 14 days in freezer and 28 days in refrigerator.