Concentration of Latanoprost Ophthalmic Solution after 4 to 6 Weeks’ Use in an Eye Clinic Setting

Abstract

purpose. To determine the concentration of latanoprost in bottles of latanoprost ophthalmic solution 0.005% after 4 or 6 weeks of use in patients in an eye clinic setting.

methods. Patients treated with latanoprost for open-angle glaucoma or ocular hypertension were randomly assigned to refill their prescriptions either at the Doheny Eye Institute clinic or a local pharmacy. Patients who used latanoprost binocularly were asked to return bottles to the clinic after 4 weeks of use and nonrefrigerated storage, and those who used latanoprost monocularly were asked to return used bottles after 6 weeks. Patients were then interviewed to determine bottle storage information and doses missed. Latanoprost concentration in residual solution was analyzed in a masked fashion, by reversed-phase high-performance liquid chromatography (HPLC).

results. In all, 110 patients were enrolled and 89 returned their bottles. Sixty-nine bottles had sufficient residual volume to conduct HPLC analysis. All patients reported that bottles were stored at room temperature (average high from 70–95°F during the daytime). The mean ± SD latanoprost concentration measured in the residual solutions was 48.31 ± 2.31 μg/mL. Ninety-four percent of the bottles had concentrations within 90% to 110% of the labeled amount. No difference in latanoprost concentrations was found between the bottles used for 4 weeks versus those used for 6 weeks.

conclusions. In an eye clinic setting, latanoprost ophthalmic solution 0.005% remains stable after 4 or 6 weeks of patient use from the same bottle when stored at room temperature.

Commercially available since 1996, latanoprost ophthalmic solution 0.005% (50 μg/mL; Xalatan, Pharmacia & Upjohn Company, Kalamazoo, MI) is the most frequently prescribed ocular hypotensive agent in the United States. According to U.S. labeling, the product should be protected from light, and the unopened bottle should be stored under refrigeration at 2 to 8°C (36–46°F). In European countries, ophthalmic preparations may be used only for a maximum of 4 weeks regardless of stability. ¹ Once opened, the 2.5-mL container may be stored at room temperature up to 25°C (77°F) for 6 weeks. ² Because bottles of topical ophthalmic hypotensive solutions do not typically need refrigeration before opening, the question of stability, particularly when suboptimal storage conditions may occur during patient use has arisen. 

Under experimental storage conditions in the laboratory, extreme temperatures (50 or 70°C) or ultraviolet radiation has been shown to decrease the stability of latanoprost greatly. ³ However, the generalizability of such experimental data from storage under extreme conditions to patient care settings is unclear. Therefore, we undertook this clinical study to evaluate the stability of latanoprost solution after 4 or 6 weeks of actual use in a community setting and to determine whether the type of dispensing (i.e., a sample received from a physician’s office or a prescription filled at a pharmacy) may have affected storage and/or stability before the drug was dispensed. Given the widespread use of latanoprost by patients with ocular hypertension (OHT) and glaucoma, the results of this study should provide patients and practitioners with information about the maintenance of latanoprost concentrations with actual use. 

Methods

Study Participants and Materials

The study was conducted in full conformance with the principles of the Declaration of Helsinki. The Los Angeles County University of Southern California Medical Center Institutional Review Board approved the study protocol. Informed consent was obtained from all patients. Consecutive clinic patients treated at the Doheny Eye Institute of the University of Southern California were eligible for inclusion if they were ≥22 years of age and were using latanoprost ophthalmic solution once daily in one or both eyes, either as monotherapy or as part of a combination regimen for the treatment of open-angle glaucoma or OHT. Exclusion criteria were pregnancy, lactation, or inadequate contraception. Patients provided verbal informed consent which included information about the purpose of the study. Consent was documented in the study records before enrollment. 

Once enrolled, patients were classified according to their need for monocular or binocular treatment with latanoprost and then were randomly assigned either to receive their next bottle of latanoprost ophthalmic solution from the clinic using bottles supplied by the manufacturer’s representative or to receive a prescription to obtain the medication from a local pharmacy. Patients continued to store and use the bottles as they had before enrollment. No special instructions for storage or use were given other than being advised to store the bottle at room temperature after opening, as stated in the prescribing information. 

Monocularly treated patients were instructed to return the used bottles to the clinic after 6 weeks, and binocularly treated patients were to return the bottle to the clinic at the end of 4 weeks. At that time, the patient was interviewed to determine where the bottle had been stored and the approximate number of doses that had been missed during use. Information on the length of usage of ocular hypotensive medications before the initiation of the study was not collected. Returned bottles were refrigerated until they were forwarded to an independent laboratory (Cayman Chemicals, Ann Arbor, MI) for analysis (described later). Before shipping, the existing bottle label was removed and replaced with a standard study label with a patient number and date of shipment. Shipments were made by next-day air in a Styrofoam box with ice packs. 

Before samples were shipped to the laboratory, nine control bottles were prepared by the investigator and randomly interspersed with bottles that had been returned to the clinic. These control bottles each contained 300 to 400 μL of various dilutions of latanoprost ophthalmic solution prepared with a needle and syringe from unopened latanoprost ophthalmic solution bottles and preservative-free sterile water and were labeled identically to the used patient bottles. The identity of the control bottles was masked from the laboratory personnel and maintained at the clinic until all assay results were received from the laboratory. 

High-Performance Liquid Chromatography

The concentration of latanoprost in the residual latanoprost ophthalmic solution (200 μL) taken from the used patient bottles was measured by validated, reversed-phase high-performance liquid chromatography (HPLC) at Cayman Chemical, using an HPLC system (1100 Series HPLC; Agilent, Palo Alto, CA) with autosampler and column compartment module set to 35°C. The analysis of latanoprost samples was conducted over four separate HPLC runs based on the time of return of the latanoprost bottles. Instrument validation (i.e., system precision and performance) was conducted before every run of the latanoprost ophthalmic solution assays. Quality control specifications requires that the HPLC standard response precision and retention time precision vary by no more than 2% relative standard deviation (RSD), that the retention time be 6.0 to 9.0 minutes, and the symmetry factor be between 100 and 170. Aliquots of latanoprost diluted to 4 and 6 μg/mL, bracketing the expected concentrations in the samples, were used as standards. The quality control results are found in Table 2 . Latanoprost samples taken from the patients’ bottles were diluted 10-fold, yielding expected concentrations of approximately 5 μg/mL. Duplicate 100-μL aliquots were removed from the bottle and prepared for analysis by quantitative transfer with mobile phase to a final volume of 1.0 mL each. An RSD between duplicate samples of within 4% was necessary to ensure assay accuracy. Degradation products were not quantified. 

Sample Size Calculation and Statistical Analyses

Since the U.S. Food and Drug Administration considers a decrease in drug concentration of 10% from the labeled concentration to be acceptable, we chose 5 μg/mL (10% of the labeled concentration of latanoprost 50 μg/mL) to be the difference between the 4- and 6-week residual concentrations of latanoprost. A sample size of 25 patients in each of the two arms (4 and 6 weeks) would be able to detect a difference of 5 μg/mL with 99% power and α = 0.05. In determining the final sample size, we considered that as much as 50% of the study population would be lost to follow-up or would not have adequate residual sample in the returned bottles. 

The mean and standard error (SE) of latanoprost concentrations in residual ophthalmic solution were calculated. The statistical significance of differences in means by length of use and location of dispensation were evaluated using a Student’s t-test. 

Results

Patients and Storage Conditions

A total of 110 patients from a glaucoma clinic setting were enrolled in the study between October 2001 and June 2002. All patients who were approached agreed to participate. Most were white women with open-angle glaucoma and all patients were experienced in applying eye drops (Table 1) . Among the 89 patients who returned their latanoprost bottles, 44 obtained their medication from the clinic, and 45 obtained medication from the pharmacy. All bottles had been stored at room temperature and had not been refrigerated. The study was completed in southern California, where the daytime temperatures ranged from a high of 70°F to 95°F (mean 81°F), ⁴ with some highs reaching 110°F. The bottles were commonly kept at the patients’ bedside (43 [48%] patients), the bathroom/medicine cabinet (19 [21%] patients), or the kitchen (11 [12%] patients). The remaining patients reported storing bottles in a combination of these locations or in an alternate location. 

Latanoprost Concentrations in Patient Bottles

A total of 89 used bottles were sent to the laboratory for assessment of residual latanoprost, and 69 of the used bottles contained a sufficient volume (200 μL) to conduct the HPLC analysis. Samples were analyzed in four batches during the following time periods: samples 1 to 26 from July 10 to 12; samples 27 to 55 from July 12 to14; samples 56 to 79 from July 14 to 16 and samples 80 to 98 on September 10. Quality control parameters for the four HPLC runs were within predetermined specifications (Table 2) . 

After 4 to 6 weeks of use, the mean (±SE) concentration of latanoprost in the used bottles was 48.31 ± 0.28 μg/mL (Table 3) . Of the 69 analyzed samples obtained from patient-use bottles, 65 (94%) contained a latanoprost concentration of 45 μg/mL or more. Of the few samples that did not, one bottle contained a latanoprost concentration of 44 μg/mL, two bottles contained 43 μg/mL and one bottle had a concentration of 36 μg/mL. All duplicate sample preparations had a 0.7% RSD. 

No difference in mean latanoprost concentrations was found between the bottles dispensed at the clinic (47.72 ± 0.53 μg/mL) compared with those obtained from pharmacies (48.83 ± 0.21 μg/mL, P = 0.06; Table 3 ). In addition, no difference in mean latanoprost concentrations was found between those samples used for 4 weeks (48.26 ± 0.42 μg/mL) compared with those used for 6 weeks (48.39 ± 0.32 μg/mL, P = 0.82). Latanoprost concentrations in the control bottles ranged from 4.89 μg/mL to 32.04 μg/mL (Fig. 1) . 

Discussion

The susceptibility of latanoprost ophthalmic solution to the environment (heat and light) during and after patient use has been a potential concern due to the physical characteristics of the bottle (clear, pliable polyethylene) used in the commercial packaging. To our knowledge, only one previously published study on the stability of latanoprost reported degradation after exposure to extreme temperatures in the laboratory (10% degradation after 198 hours at 50°C or 31.7 hours at 70°C). ³ Similarly, direct exposure to ultraviolet irradiation from a benchtop transilluminator resulted in degradation of 10% of latanoprost within a few hours. ³ Although these findings suggest that latanoprost solution may degrade under experimental extreme temperature and light exposure conditions, the generalizability of these data to the patient care and community use environment is unclear. Further, they do not demonstrate lack of potency or stability of latanoprost under conditions of actual use. ^{5 6}  

The present study was conducted to evaluate the stability of latanoprost solution during and after use in a “real world” community environment when dispensed from two different settings. After handling by the patients and storage at room temperature, the majority (94%) of used latanoprost ophthalmic solution bottles contained latanoprost at a concentration within 10% of the labeled amount. Mean concentrations of latanoprost in the residual solution from the clinic-dispensed bottles and the pharmacy-dispensed bottles did not differ significantly. In all, one bottle contained residual latanoprost ophthalmic solution, from which latanoprost was measured in a concentration substantially lower than any of the other samples. Possible explanations include degradation of latanoprost under usual storage conditions, lower concentration of latanoprost in the unopened bottle, unusual storage conditions by the patient, errors in study procedures at the clinic, or errors during sampling or assay. 

A change in the labeled concentrations of any pharmaceutical product for outpatient use could potentially negatively affect clinical response. For the latanoprost concentrations evaluated in this study, however, this appears not to be the case. Dose-finding studies with latanoprost have revealed that IOP-lowering activity exists at drug concentrations lower than that provided in the commercially available preparation. ^{7 8 9} In one of the original dose-finding trials, latanoprost at concentrations of 35 μg/mL (0.0035%) and 60 μg/mL (0.006%) administered twice daily produced little difference in reducing IOP, showing no clear dose–response relationship. ⁸ More recently, comparison of once-daily administration of either 0.005% or 0.001% latanoprost demonstrated that even when administered at a concentration of 20% of labeled product, latanoprost lowered IOP within 2 mm Hg of that produced by the labeled (0.005%) concentration. Although this study was not designed to demonstrate a relationship between the potency of latanoprost and its IOP-reducing effects, a decrease in latanoprost concentration (to 35 μg/mL) due to loss of stability, would not be likely to affect the IOP-lowering activity of the drug. ⁸ (In our study, the lowest concentration detected was 36 μg/mL.) 

One limitation of the present study is the lack of baseline reference values for the latanoprost concentration in the unopened bottles. Such data would have allowed determination of whether final concentrations had changed significantly after the patient used the bottle. However, baseline analyses would have necessitated dispensing opened, sampled bottles, which is not feasible in a patient care environment. In addition, latanoprost ophthalmic solution bottles dispensed from pharmacies would not have been available for baseline sampling. Therefore, we are unable to conclude whether the changes in latanoprost concentration occurred before or after dispensation. Another limitation of this study was the homogeneity of the patient population, in that they were accrued from a group of patients with glaucoma and ocular hypertension treated at a private clinic. The applicability of these data to other populations remains unknown. However, we believe that our data suggest that latanoprost ophthalmic solution maintains its concentration when stored at room temperatures similar to that experienced in our community. Further, our data should be cautiously evaluated when extrapolating to other communities particularly those that are not similar to our community, because we do not know what the actual room temperatures were, as patients enrolled in our study may or may not have had room air conditioning. Finally, the scope of this study did not include preservative stability, nor did we evaluate the effect of the preservative on IOP lowering or on the conjunctiva in this setting. 

To our knowledge, this is the first published report of latanoprost stability in latanoprost ophthalmic solution bottles that were used and stored under normal conditions. Our findings demonstrate that latanoprost is stable in an eye clinic setting and are reassuring, in that patients living in warm climatic conditions were successful in maintaining proper storage after receiving routine dispensing instructions. Further, it reassures patients and physicians that latanoprost solution is stable under conditions of normal use. 

 Supported by an unrestricted grant from Research to Prevent Blindness, New York, NY, and an unrestricted grant from Pfizer Inc., New York, NY. RV is a Research to Prevent Blindness Sybil B. Harrington Scholar.

 Submitted for publication November 23, 2004; revised April 6, July 25, and August 22; accepted November 14, 2005.

 Disclosure: R. Varma, None; J. Winarko, None; T. Kiat-Winarko, None; B. Winarko, None

 The publication costs of this article were defrayed in part by page charge payment. This article must therefore be marked “advertisement” in accordance with 18 U.S.C. §1734 solely to indicate this fact.

 

Table 1.

 

Patient Demographics and Disease Characteristics by Dispensing Setting

	Clinic Dispensing* (n = 44)	Pharmacy Dispensing (n = 44)
Age, y (mean ± SD)	74.34 ± 11.5	73.93 ± 8.91
Gender
Male	20	17
Female	24	27
Race, †
White	38	39
Asian	5	3
Other	0	1
Disease type*
POAG	28	31
Other Glaucoma	12	5
OHT	0	8
Combination	4	0

 Other Glaucoma group includes secondary open angle glaucoma including pseudoexfoliation and pigmentary glaucoma. OHT, ocular hypertension; POAG, primary open-angle glaucoma.

*  Data unavailable for one patient.

†  Data unavailable for two patients.

Table 2.

 

High-Performance Liquid Chromatography: Results of Quality Control Assessments

System Requirement	Specification	Run 1 (July 10–12)	Run 2 (July 12–14)	Run 3 (July 14–16)	Run 4 (Sept. 10)
Standard response precision (RSD)	≤2.0%	0.25%	0.45%	0.56%	0.96%
Latanoprost retention time (minutes)	6.0 to 9.0	6.7	6.7	6.6	7.3
Retention time precision (RSD)	≤2.0%	0.11%	0.73%	0.10%	0.7%
Symmetry factor	100 to 170	128	123	117	121
Laboratory standard results average, μg/mL	48.0 to 52.0	49.6	49.3	47.9	50.4

 RSD, relative standard deviation.

Table 3.

 

Latanoprost Concentrations in Residual Ophthalmic Solution

	Latanoprost Concentration (μg/mL)
Control (n = 9)	16.44 ± 10.34
Used bottles (n = 69)	48.31 ± 0.28
By location of dispensation*
Clinic (n = 32)	47.72 ± 0.53
Pharmacy (n = 37)	48.83 ± 0.21
By length of use, †
4 Weeks (n = 40)	48.26 ± 0.42
6 Weeks (n = 29)	48.39 ± 0.32

 Data are expressed as the mean ± SE.

*   P = 0.0606; t-test.

†   P = 0.8183; t-test.

Figure 1.

 

Concentration of latanoprost from all used latanoprost ophthalmic solution bottles with analyzable contents and control bottles. Dashed line: mean concentration in used bottles (48.31 ± 0.28 μg/mL).