Trabecular meshwork and lens partitioning of corticosteroids: Implications for elevated intraocular pressure and cataracts

Abstract

Objectives

Incidence of side effects such as elevated intraocular pressure and cataracts is lower with dexamethasone when compared to fluocinolone acetonide or triamcinolone acetonide. We hypothesized that side effects may be explained in part by the differences in drug lipophilicity and partitioning of these drugs into trabecular meshwork and lens.

Methods

n-octanol-phosphate buffer saline (PBS; pH 7.4) partition coefficient (Log D) and bovine/human ocular tissue partition coefficients were determined for triamcinolone (T), prednisolone (P), dexamethasone (D), fluocinolone acetonide (FA), triamcinolone acetonide (TA), and budesonide (B) at 37 °C.

Results

Log D of the corticosteroids ranged from 0.7 to 3. The range of tissue: PBS partition coefficients (Kt:b) following drug incubation at 0.4, 2, and 10 μg/ml were 0.3-1.6, 0.3-2.2, and 0.3-2 for the bovine lens, 0.8-4.1, 0.7-4.4, and 0.6-5.8 for the human lens, and 2.9-9.5, 2.4-9.2, and 1.7-9.9 for the bovine trabecular meshwork. In general, tissue partitioning showed a positive correlation with Log D. Dexamethasone, with lipophilicity less than triamcinolone acetonide and fluocinolone acetonide, exhibited the least amount of partitioning in the trabecular meshwork and lens among these three corticosteroids commonly used for treating back of the eye diseases.

Conclusion

Binding of corticosteroids to the trabecular meshwork and the lens increases as drug lipophilicity increases.

Clinical relevance

Less lipophilic corticosteroids with limited partitioning to the trabecular meshwork and the lens may result in reduced incidence of elevated intraocular pressure and cataracts.

INTRODUCTION

Dexamethasone, fluocinolone, prednisolone, and triamcinolone or their derivatives have been shown to be beneficial in treating several disorders of the eye including uveitis, macular edema, and choroidal neovascularization. However, corticosteroid use is associated with risks including ocular hypertension or elevated intraocular pressure (IOP) (1) and lens opacification (cataracts) (2-4). Several pathological changes have been reported in trabecular meshwork and lens, as a consequence of corticosteroid induced ocular hypertension and cataract, respectively.

Even though it is well established that corticosteroids induce pathological changes in the trabecular meshwork and the lens, it is currently not possible to rank the side effects of various corticosteroids based solely on their glucocorticoid activity(5). This is particularly relevant for corticosteroids administered intravitreally. Currently FDA approved corticosteroid products for the back of the eye include: Ozurdex™ (biodegradable dexamethasone implant; macular edema; 0.7 mg; Allergan), Retisert™ (non-biodegradable fluocinolone acetonide implant; uveitis; 0.59 mg; Bausch & Lomb), and Trivaris™ (Allergan)/Triesence™ (Alcon) (triamcinolone acetonide suspension; uveitis and topical corticosteroid responsive inflammatory conditions; 4 mg). Of these products, the incidence of side effects is the highest for fluocinolone acetonide and the least for dexamethasone. In order to better understand these differences, which might in part depend on the affinity of the drug for trabecular meshwork and lens, the purpose of this study was to determine the lipophilicity of corticosteroids and to further determine their partition coefficients in the trabecular meshwork and the lens.

In this study we used bovine lens, human lens, and bovine trabecular meshwork for partition studies. Due to limited tissue availability, human trabecular meshwork partition coefficients were not determined. Since prior studies have shown that bovine eyes exhibit robust steroid induced ocular hypertensive response, with 100 % occurrence (6, 7), bovine trabecular meshwork was used as a surrogate for human tissue.

MATERIALS AND METHODS

Materials

Triamcinolone, triamcinolone acetonide, fluocinolone acetonide, and budesonide were purchased from Spectrum Chemical and Laboratory Products, a division of Spectrum Chemical Mfg. Corp. (New Brunswick, NJ, USA). Prednisolone, dexamethasone, and n-octanol were purchased from Sigma-Aldrich (St. Louis. MO, USA). High performance liquid chromatography (HPLC) grade acetonitrile and methanol were purchased from Fisher Scientific (Philadelphia, PA, USA). Freshly excised bovine eyes were purchased from G & C Packing Company, Colorado Springs, CO, USA.

Methods

Human cadaver eyes from anonymous donors were obtained from the San Diego Eye bank (San Diego, CA) in accordance with a protocol approved by the Institutional Review Board. All procedures were in compliance with the Declaration of Helsinki for research involving human tissue. The age of the donors ranged from 55-60 years.

Estimation of Log D

n-Octanol–pH 7.4 phosphate buffer saline (PBS) partition coefficients were determined using the shake-flask method. n-Octanol saturated with PBS was used as the organic phase and PBS saturated with n-octanol was used as the aqueous phase. For equilibration of the two phases, solutions were kept at 37 °C for 24 h before use. n-Octanol solutions of each corticosteroid at two different concentrations (0.5 and 2.5 mM) were prepared. An aliquot of 1 mL of these solutions was added to glass vials (Thermo Fisher Scientific Inc., Pittsburgh, PA, USA) containing 2 mL PBS saturated with noctanol. The tubes were shaken for 24 h at 37 °C followed by centrifugation at 10,000 rpm (Fisher Scientific Accuspin Micro17 centrifuge) for 15 min. The concentration of corticosteroids in the aqueous and octanol phases was determined by UV spectrometry. n-Octanol:PBS partition coefficient values were calculated by dividing the concentration of the corticosteroid in the organic phase by that in the aqueous phase. The experiment was run in triplicate for both concentrations.

Tissue Isolation

Enucleated eyes from 3-year old cows were obtained on ice and used within 2 hours after sacrifice. Human eyes maintained at 4 °C were received within 24 hours after death from the eye bank. The experiment was initiated within an hour after receiving the human eyes.

During the entire process of tissue isolation, eyes/tissues were maintained at 4 °C. For isolating trabecular meshwork (TM) from bovine eyes, previously described methods were followed (8). Briefly, the cornea was removed carefully by cutting it away from the limbus region. Beginning at the limbus, approximately 5-10 mm deep circular tissue was isolated. The remaining eye-cup with the intact lens was kept on ice until further use. The circular piece of tissue was cut into four quadrants and each quadrant was processed as follows. With the corneo-scleral surface facing downwards, iris and pectinate ligament layer was removed carefully, exposing the velcro-like structure of TM. Ciliary body posterior to the TM band was separated by an incision, ensuring that it does not contaminate the TM. TM and ciliary body could be clearly distinguished by their colors. TM band appeared grey, while the ciliary body was black in color. Finally, TM band was detached from the sclera. The eye-cup that was kept aside on ice was used for isolating the intact lens. Both tissues were gently rinsed with PBS (pH 7.4). One hundred milligram portions of each tissue were weighed for drug partition studies.

LC/MS/MS Analysis of Corticosteroids

Optimized MS parameters

The MS parameters of sample corticosteroids and internal standard corticosterone were optimized in positive ionization mode by infusing 1 μg/ml solution on PE SCIEX API-3000 LC-MS/MS instrument by syringe infusion mode. The optimized parameters are listed in Table 1.

Table 1.

Optimized mass spectrometry instrument parameters for the corticosteroid analysis. Corticosterone served as the internal standard (IS).

Sr. #	Corticosteroid	MRM	DP	FP	EP	CE	CXP
1	Triamcinolone (T)	395/343	65	200	10	17	15
2	Prednisolone (P)	361/343	65	200	10	17	15
3	Dexamethasone (D)	393/373	60	200	10	13	15
4	Fluocinolone Acetonide (FA)	453/413	60	200	10	18	15
5	Triamcinolone Acetonide (TA)	435/415	30	200	10	15	15
6	Budesonide (B)	431/413	55	200	10	15	15
7	Corticosterone (C; IS)	347/329	40	200	10	23	15

MRM: Multiple Reaction Monitoring; DP: Declustering Potential; FP: Focusing Potential; EP: Entrance Potential; CE: Collision Energy; CXP: Collision Cell Exit Potential

Optimized LC parameters

Sunfire C18 column (2.1 × 50 mm, 5μm; from Waters) was used as the stationary phase; 5 mM ammonium formate in water, adjusted to pH 3.5 with formic acid (A) and acetonitrile: methanol mixture (50:50) (B) was used as the mobile phase at a flow rate of 200 μl/min. The total run time was 6.0 minutes. The gradient elution was set as follows: 80 % A for the first 0.7 min, linear to 15 % A by 2.5 min, 15 % A for the next 1.5 min followed by an increase to 80 % A in the next 0.5 min with a 1.5 min of re-equilibration time before the next injection. A representative LCMS/MS chromatogram is shown in Figure 1.

Figure 1.

Representative LC-MS/MS chromatogram of a standard sample containing a mixture of corticosteroids used in this study.

Corticosteroid Extraction Recovery

For calculating percent extraction recovery, the following formula was used: % Recovery = (Analyte peak area of standard with spiking before extraction×100)/(Analyte peak area of corresponding standard with spiking after extraction procedure). For prespiking or spiking before extraction, 100 mg of tissue mixed with known concentration of analyte mixture (10 μl of 40 μg/ml solution of 6 steroids) and internal standard (10 μl of 50 μg/ml corticosterone) was homogenized in 500 μl of phosphate buffer saline (PBS, pH 7.4). After homogenization, extraction with 2 ml of ethyl acetate was done by vortexing for 15 min using a multitube vortexer (Model VX-2500; VWR International, Marlboro, MA). Organic solvent was separated after centrifugation for 15 min at 3000 rpm. After removing the organic solvent under nitrogen, final reconstitution was achieved with 1 ml acetonitrile. In post-spiking or spiking after extraction, tissues were first homogenized in PBS followed by extraction with organic solvent which was separated after centrifugation. The organic extract was spiked with a known concentration of analyte mixture and internal standard. After mixing, the organic solvent was removed under nitrogen and final reconstitution was done in 1 ml of acetonitrile. Quality control samples were prepared by direct dilution of the analyte and the internal standard in 1 ml of acetonitrile. The matrix effect was calculated using the following formula: % Matrix effect = (Analyte peak area of standard with spiking after extraction procedure×100)/(Analyte peak area of corresponding unextracted standard). After reconstitution, samples were analyzed using LC-MS/MS.

In Vitro Tissue Partitioning Studies

These studies were performed to measure the relative affinity of each corticosteroid towards different ocular tissues and PBS (pH 7.4). All the corticosteroids were used as a cocktail mixture for partitioning studies to minimize any experimental variation. Three concentrations of corticosteroids (0.2, 4, and 10 μg/ml) in PBS were selected for the partitioning study. One hundred milligrams of trabecular meshwork or lens (n = 5) was incubated with 0.5 ml solution of corticosteroids in PBS for 6 h at 37°C. At the end of the incubation period, samples were centrifuged for 15 min at 10,000 rpm. PBS supernatant was removed and tissues were washed with 0.5 ml of fresh PBS. Samples were again centrifuged for 15 min at 10,000 rpm and wash buffer was separated. During the sample processing for drug content estimation in tissue and buffer, corticosterone was added as the internal standard to all tissue samples before extraction, similar to our extraction recovery study. Similarly, internal standard was added to buffer samples prior to analysis. Tissue partitioning was estimated as the tissue:buffer ratio of drug concentration.

Statistical Analyses

All data in this study are expressed as the mean ± SD. Comparison between in vitro tissue partitioning between tissues and six different corticosteroids was performed using one-way ANOVA followed by the Tukey's post-hoc analysis. Statistical significance was set at p < 0.05.

RESULTS

Physicochemical Properties of Corticosteroids

Mean n-octanol-PBS pH 7.4 partition coefficients (Log D) of corticosteroids at 37 °C are summarized in Table 3.

Table 3.

Molecular weight and measured lipophilicity (Log D at pH 7.4) of corticosteroids used in the current study. For Log D measurement, the USP shake flask method has been employed (12, 13) (US EPA 1982). Distribution coefficient was measured at two different concentrations, 0.5 mM (n = 3) and 2.5 mM (n = 3), at 37 °C. Log D data is presented as mean ± SD for n = 6.

Corticosteroid	Molecular Weight	Measured Log D (n=6)
Triamcinolone	394.43	0.712 ± 0.113
Prednisolone	360.44	1.771 ± 0.115
Dexamethasone	392.46	1.955 ± 0.024
Fluocinolone Acetonide	452.48	2.560 ± 0.117
Triamcinolone Acetonide	434.49	2.585 ± 0.036
Budesonide	430.5	2.970 ± 0.098

Estimation of Extraction Recovery

In preparation for our partitioning studies, we initially determined the efficiency of extraction of our corticosteroids from various bovine ocular tissues using ethyl acetate and dichloromethane and determined that ethyl acetate is superior for corticosteroid extraction. Table 2 represents the % extraction recovery of each corticosteroid in the trabecular meshwork and lens using ethyl acetate. All % extraction recovery values fell in the range of ~ 80.7-108.9 %.

Table 2.

Percent extraction recovery (ER) and percent matrix effect (ME) of corticosteroids in bovine trabecular meshwork and lens. Data is expressed as mean ± SD for n = 5.

Corticosteroid Used at 400 ng/ml	Lens		Trabecular meshwork
	% ER	% ME	% ER	% ME
Triamcinolone	80.7± 7.7	104.1±3.9	101± 6.8	91.6±13.5
Prednisolone	85.1± 12.1	103.7±6.1	97.0± 4.5	91.8±4.2
Dexamethasone	89.5± 8.2	93.4±3.1	91.3± 6.0	85.0±8.1
Fluocinolone Acetonide	86.8± 11.4	98.3±2.4	108.9± 9.7	87.3±4.8
Triamcinolone Acetonide	91.7± 1.0	97.7±1.3	99.3± 3.5	109.1±6.2
Budesonide	90.2± 2.8	99.0±2.6	88.6± 5.2	83.8±2.4

Tissue Partition Coefficients

Corticosteroid tissue partition coefficients for TM and lens, are depicted in Figure 2. In general, the tissue partitioning followed the trend: TM > lens (Figure 2). It is noteworthy that for several corticosteroids in each tissue, partition coefficient values were the highest at the lowest concentration used for the partitioning study. For example, triamcinolone showed the highest TM : PBS and lens : PBS, partitioning ratios at 0.4 μg/ml compared to 2 and 10 μg/ml. Trabecular meshwork and lens tissue partition coefficients of corticosteroids at all concentrations showed positive correlation with drug lipophilicity (Figure 3).

Figure 2.

Tissue : PBS (pH 7.4) partition coefficients of various corticosteroids at 0.4, 2 and 10 μg/ml in (A) bovine lens, (B) human lens, and (C) bovine trabecular meshwork. Data is expressed as mean ± SD for n = 5.

Figure 3.

Correlation of tissue partition coefficients with corticosteroid lipophilicity. Log [Tissue : PBS (pH 7.4)] partition coefficients of various corticosteroids at 0.4, 2, 10 μg/ml for (A) bovine lens, (B) human lens, and (C) trabecular meshwork were plotted against lipophilicity (Log D; pH 7.4). Data is expressed as mean ± SD for n = 5.

DISCUSSION

To our knowledge, we are the first to report that corticosteroid lipophilicity and relative partitioning into trabecular meshwork and lens, respectively, may explain elevated intraocular pressure and cataract. We observed that with an increase in lipophilicity, corticosteroids exhibit greater ocular tissue partition coefficients in the trabecular meshwork and lens.

Octanol:buffer (pH 7.4) partition studies at 37 °C indicated that all corticosteroids assessed are lipophilic with Log (distribution coefficient) or Log D values ranging from 0.7 to 3. The relative distribution coefficient (D) of the corticosteroids, between lipophilic octanol and aqueous buffer at physiological pH, ranged from about 2-fold to 1000-fold. For dexamethasone, fluocinolone acetonide, and triamcinolone acetonide, the measured Log D values were 1.95, 2.56, and 2.58, respectively. The distribution coefficients for fluocinolone acetonide and triamcinolone acetonide were 4.1 and 4.3-fold higher compared to dexamethasone, respectively. Typically the literature reports for partition coefficients are determinations at room temperature. Also, on several occasions predicted Log D values as opposed to actual measures are reported. The octanol:buffer partition coefficients measured in this study are expected to be more relevant to interpret physiological differences in drug distributions. Based on these measurements, it is likely that tissue entry of fluocinolone acetonide and triamcinolone acetonide will be greater than dexamethasone. In addition to the above three FDA approved corticosteroids for the back of the eye diseases, we investigated prednisolone, triamcinolone, and budesonide, spanning a broader spectrum of lipophilicity, in order to determine whether corticosteroid lipophilicity correlates with trabecular meshwork and lens partition coefficients. Among the various corticosteroids assessed, triamcinolone was the least lipophilic molecule (Log D: 0.72) and budesonide was the most lipophilic molecule (Log D: 2.97), with the rank order being: triamcinolone < prednisolone ~ dexamethasone < fluocinolone acetonide ~ triamcinolone acetonide < budesonide (Table 3).

Although the mechanism of corticosteroid induced intraocular pressure elevation is much debated, there is general agreement that corticosteroids induce intraocular pressure elevation through their action in the trabecular meshwork. At physiologically relevant pH and temperature, all corticosteroids assessed in this study exhibited preferential accumulation in the trabecular meshwork compared to phosphate buffered saline (partition coefficients > 1, with the range being 1.71 to 9.97). At 0.4 μg/ml buffer concentration, the trabecular meshwork partition coefficients of fluocinolone acetonide and triamcinolone acetonide were 1.85 and 1.56 fold higher, when compared to dexamethasone. Similar trends were also evident when the corticosteroids were incubated at 2 and 10 μg/ml. As the drug concentrations increased from 0.4 to 10 μg/ml, trabecular meshwork partition coefficients for all corticosteroids except budesonide decreased, suggesting that the binding sites in the trabecular meshwork for several corticosteroids could become saturated by an increase in drug concentration. The rank order for trabecular meshwork partition coefficients at 0.4 μg/ml was: triamcinolone < prednisolone < dexamethasone < triamcinolone acetonide < fluocinolone acetonide < budesonide. At 10 μg/ml, there was no difference between the partition coefficients of fluocinolone acetonide and triamcinolone acetonide; the remaining trend mirrored the observations made for 0.4 μg/ml. Thus, the trabecular meshwork preferentially accumulates all corticosteroids compared to the aqueous medium, with the drug accumulation increasing with an increase in drug lipophilicity.

Probably due to its protein-rich and hydrophilic nature, bovine lens accumulation of all corticosteroids was lower (4.9 to 8.4-fold) when compared to the trabecular meshwork. While triamcinolone, prednisolone, and dexamethasone preferentially remained in the aqueous medium (tissue:buffer partition coefficient < 1), triamcinolone acetonide, fluocinolone acetonide, and budesonide remained preferentially in the bovine lens (partition coefficient > 1). The rank order for lens:buffer partition coefficient at 0.4 μg/ml incubation was: triamcinolone < prednisolone < dexamethasone < triamcinolone acetonide < fluocinolone acetonide < budesonide. At 10 μg/ml, the partition coefficient of dexamethasone was less than prednisolone, while the rest of the trend remained the same. A reduction in partition coefficients with an increase in drug concentration was evident for triamcinolone, dexamethasone, and triamcinolone acetonide. At 0.4 μg/ml, the lens:buffer partition coefficients of fluocinolone acetonide and triamcinolone acetonide were 1.53 and 1.44 fold higher compared to dexamethasone. Thus, although the bovine lens has lower affinity for corticosteroids when compared to trabecular meshwork, the tissue partition coefficient increases with an increase in drug lipophilicity. Compounds with Log D equal to 1.95 (dexamethasone) or less exhibited preferential retention in the aqueous buffer when compared to the lens tissue. Similar to bovine lens, human lens partition coefficients also increased with an increase in drug lipophilicity The corticosteroid partition coefficients in human lens were in the order: triamcinolone < prednisolone < dexamethasone < triamcinolone acetonide < fluocinolone acetonide < budesonide. In human lens, all corticosteroids except triamcinolone accumulated preferentially in the tissue, when compared to the aqueous medium. With an increase in drug concentration, there was a decrease in the partition coefficient for all corticosteroids except budesonide.

When compared to the bovine lens, the human lens partition coefficients of corticosteroids were 1.5-3.0-fold higher. This may be explained on the basis of age and species related differences in the lens tissue. It is known that with ageing, the proportion of insoluble proteins inside the lens increases, whereas the proportion of soluble α-crystallin decreases (9, 10). In bovine lens for instance, the percentage of insoluble protein increases from about 5% at birth to 20-30% at 3 years, and 80-90% at 5-6 years (9, 10). Using dynamic light scattering technique, it has been shown that the α-crystallin index (ACI), a measure of soluble α-crystallin in human eyes, decreases significantly (P < 0.001) with ageing in patients in the age range of 7-86 years (11). The value of ACI decreased from 32 % in patients of 7-25 years of age to 15 % in patients of 56-65 years of age, which further reduced to < 5 % in patients of > 75 years of age (11). The human eyes used in our study were obtained from donors in the age group of 55-60 years, whereas the bovine eyes were obtained from 3-year old cows.

To determine whether clinical side effects of corticosteroids correlate with their tissue partitioning data, we collected clinical data (Table 4) from the literature on percent incidences of IOP elevation and cataract after intravitreal treatment with the dexamethasone, fluocinolone acetonide, and triamcinolone acetonide. It is noteworthy that the rank order for ocular side effects parallels the corticosteroid partition coefficients in the tissues. However, since dose, duration of assessment/exposure, and patient disease states were not similar in the clinical reports for the above three drugs, some caution should be exercised in the final interpretations.

Table 4.

Percent occurrence of cataracts and IOP elevation following intravitreal administration of dexamethasone (D), triamcinolone acetonide (TA), and fluocinolone acetonide (FA), based on some clinical studies.

Steroid	Total Dose	Indication (number of patients)	Subjects with IOP Elevation	Duration of Treatment & Effect Assessment	Subjects with cataract	Reference
D	0.7 mg	ME (421)	25% (106/421)	6 months	4% (15/421)	(www.fda.gov; NDA 22315)
FA	0.59/2.1 mg	NIPU (278)	59% (163/278)	7.8 months	13.3% (37/278)	Jaffe et al. (14)
TA	4 mg	CNV/ARMD (75)	41% (31/75)	12 months	5.3% (4/75)	Gillies et al. (15)

ME: Macular edema; NIPU: Non-infectious posterior uveitis; CNV/ARMD: Choroida neovascularization/Age related macular degeneration

In conclusion, corticosteroid partitioning into the trabecular meshwork as well as the lens increases with an increase in lipophilicity. Accumulation in the lens is lower than in the trabecular meshwork for all corticosteroids. All corticosteroids preferentially accumulate in the trabecular meshwork rather than the buffer. All corticosteroids except triamcinolone, prednisolone, and dexamethasone accumulate preferentially in the lens when compared to the buffer in case of bovine tissues. Dexamethasone exhibits lower octanol:water partition coefficient as well as lower lens and trabecular meshwork partition coefficients when compared to fluocinolone acetonide and triamcinolone acetonide. These differences offer an additional explanation for the clinically observed lower incidence of ocular side effects with dexamethasone compared to fluocinolone acetonide and triamcinolone acetonide.