Concentrations of Betaxolol in Ocular Tissues of Patients with Glaucoma and Normal Monkeys after 1 Month of Topical Ocular Administration

Abstract

purpose. To measure the concentration of betaxolol in tissues of humans with glaucoma and normal monkeys after topical administration.

methods. Enucleated eyes (n = 7) of patients with glaucoma (age range, 27–79 years), without apparent anatomic disruption that would be likely to influence betaxolol absorption and intraocular distribution (exceptions: one pseudophakic, one aphakic) or other disease, were analyzed for betaxolol concentrations after self-administration of 0.25% betaxolol twice daily for 28 days or longer. The last instillation was made within 6 hours of surgery. Cynomolgus monkeys (n = 3) received 0.25% betaxolol twice daily unilaterally for 30 days. Betaxolol was measured by HPLC and tandem mass spectrometry (MS/MS) in plasma and ocular tissues.

results. In humans, mean betaxolol concentrations (excluding the aphakic patient) were 71.4 ± 41.8 ng/g in the retina, 31.2 ± 14.8 ng/g in the optic nerve head, and 1290 ± 1170 ng/g in the choroid. Mean concentrations in the iris and ciliary body were 73,200 ± 89,600 and 4,250 ± 3,020 ng/g, respectively. Betaxolol concentration was higher in all ocular tissues than in the plasma (0.59 ± 0.32 ng/mL). In the monkeys the concentrations in the posterior tissues of the treated eyes were higher than in the untreated eyes, with mean differences in the retina and optic nerve head of 121 and 130 ng/g, respectively.

conclusions. Topically applied betaxolol was bioavailable to posterior ocular tissues, including the retina and optic nerve head, of patients with glaucoma and of normal cynomolgus monkeys. The higher betaxolol levels in the treated versus untreated monkey eyes are consistent with betaxolol’s reaching posterior tissues by local absorption and distribution.

Topical delivery of glaucoma drugs is convenient to the patient and can mitigate systemic side effects encountered with much higher oral doses. Many drugs applied topically enter the eye via corneal and noncorneal (conjunctival–scleral) routes and reach their apparent highest concentrations within the anterior ocular tissues (e.g., iris and ciliary body). ¹ Topically instilled betaxolol and timolol have been shown to accumulate significantly in the periocular tissues and the sclera ² of patients with glaucoma after long-term use, suggesting the possibility of drug delivery along the orbital tissues and scleral penetration into the posterior segment. Similar periocular accumulation and penetration were found for topical nipradilol in monkeys ³ and topical iganidipine in rabbits. ⁴ Moreover, studies of rabbits have shown that topically applied betaxolol reaches posterior segment tissues in the eye, possibly through periocular accumulation and penetration as well as systemic distribution pathways. ⁵  

Reports of in laboratory studies of betaxolol’s having vasorelaxant and neuroprotective properties and apparently promoting the preservation of the visual field in a manner unrelated to lowering of intraocular pressure (IOP), ^{6 7 8 9 10 11} have raised interest in whether this drug may reach pharmacologically active concentrations (nanomolar and higher) in the posterior segment tissues of patients on chronic betaxolol therapy for glaucoma or ocular hypertension. In fact, multiple published clinical study results indicate that betaxolol may modify blood flow parameters at the back of the eye and slow the rate of progression of visual field changes in patients. Although the clinical reports have shown conflicting results, the point would be moot if the drug does not reach pharmacologically active concentrations in the potential target tissues, such as the retina, choroid, and optic nerve head. Prompted by these reports and the absence of any reported data in monkeys or humans on the distribution of betaxolol to the back of the eye, we undertook this investigation. The objectives of these experiments were to determine the distribution of betaxolol within anterior and posterior segment eye tissues of normal nonhuman primates and of patients with glaucoma scheduled for enucleation and to assess the contribution of its local and systemic absorption. 

Materials and Methods

Human Study

The study protocol was approved by the Institutional Review Boards of the Semmelweis University and the University of Texas Southwestern Medical Center in accordance with the Declaration of Helsinki. Seven patients, four female and three male (six white, one black), age 27 to 79 years, scheduled for enucleation due to glaucoma-related ocular pain, severe visual impairment or blindness were included in this open-label, multiple dose, nonrandomized study. A written informed consent was obtained from each patient by the principal investigators, or designees, before enrollment. Patients with ocular trauma, intraocular and periocular tumor, prior vitrectomy, intravitreal silicon oil implantation, endophthalmitis, uveitis, or any anatomic abnormality that could influence the ocular tissue distribution of betaxolol, were excluded from the study. By exception, one patient was aphakic after aspiration of congenital cataract in infancy and another pseudophakic with an intact posterior capsule. Patients with hypersensitivity to oral or topical β-adrenergic blocking agents, a history of pulmonary or cardiac disease, or overt cardiac failure, were also excluded. None of the patients used betaxolol systematically. Patients continued their glaucoma treatments, except for any ophthalmic β-blocking agent other than betaxolol. Subjects self-administered one drop of betaxolol 0.25% ophthalmic suspension (Betoptic S; Alcon Laboratories Inc., Fort Worth, TX), twice daily for a minimum of 28 days before the scheduled enucleation surgery. Patients who used any other topical IOP-lowering medication in addition to betaxolol in the study period (Table 1)were instructed to instill the eye drops at least 30 minutes apart from the use of betaxolol. The final betaxolol instillation occurred generally 0.25 to 2 hours before blood sampling (in one patient, 5 hours), and approximately 1.5 to 6 hours before surgery (Table 1) . At the time of surgery and in the study period, the corneal epithelium was intact in each case. 

Immediately after enucleation surgery, all tissues were dissected, whole and undamaged if possible, rinsed thoroughly with sterile saline, blotted dry with sterile gauze, accurately weighed, and stored frozen until analysis. Conjunctiva, muscle, and connective tissue were carefully removed from the sclera and optic nerve. The optic nerve was cut close to the insertion point, preserving the optic nerve head. Approximately 0.1 mL of aqueous humor was aspirated into a 1-mL tuberculin syringe with a 27-gauge needle and transferred to a sample storage tube. Approximately 0.4 to 0.8 mL of vitreous humor was collected, with care taken not to aspirate fragments of ciliary body or retina. When necessary, a microsponge was used to remove excess vitreous humor from the posterior tissues. After collection of the posterior segment tissues, retina, choroid, and optic nerve head, the anterior segment was dissected, to obtain samples of lens (if present), iris, ciliary body, cornea, and sclera. The tissue samples were weighed and stored at −80°C until analysis. 

Monkey Study

The experiment was performed in compliance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. Each right eye of cynomolgus monkeys (Macaca fascicularis; two males, one female; 3.3–5.0 kg) was instilled with a single 30-μL dose of 0.25% betaxolol, twice daily for 30 days. The twice-daily doses were given 12 hours apart on day 1 and days 27 to 29 (near the pharmacokinetic samplings), and 8 hours apart on days 2 to 26. On days 1 and 30, blood samples were drawn at 0.5, 1, 3, 6, and 12 hours after instillation. At 12 hours after application on day 30, the animals were euthanatized with intravenous ketamine and pentobarbital sodium by methods approved by the facility’s Animal Care and Use Committee, and ocular tissues were collected from the right (treated) and left (untreated) eyes. Anterior sclera, vitreous humor, retina, choroid, optic nerve, and optic nerve head were collected from both, while aqueous humor and iris–ciliary body were collected from the treated eye only. Tissue samples were weighed and stored frozen at approximately −80°C. 

Sample Analysis

Human ocular tissue and plasma samples were analyzed for betaxolol by a sensitive high-performance liquid chromatography–tandem mass spectrometry (HPLC/MS/MS) method. Plasma, aqueous humor, or homogenized ocular tissues (in water) were spiked with the cyclobutyl analogue of betaxolol as an internal standard, adjusted to basic pH, and extracted with 60:40 n-hexane-ethyl acetate. The organic layer was evaporated to dryness and reconstituted in 1:1 acetonitrile-water. Chromatographic separation was performed on a reversed-phase HPLC column (2 mm internal diameter, pentylsilica), using a mobile phase consisting of (70:30) 0.005 M formate buffer (pH 6.3)-acetonitrile. The protonated molecular ions for both betaxolol and the internal standard were subjected to electrospray ionization. Quantitation was performed by multiple-reaction monitoring of the m/z 308.3→115.9 and 321.2→115.9 transitions for betaxolol and internal standard, respectively. The working range for the method was 0.05 to 25 ng/extract of tissues. 

Monkey samples were also analyzed by HPLC/MS/MS, but the method differed slightly from the human tissue assay. Tissues were homogenized on ice in HPLC-grade water by sonication to a total volume of 1.0 mL. The homogenates were then spiked with internal standard (metoprolol) and extracted with ethyl acetate. After centrifugation, removal of the organic layer, and evaporation to dryness, residues were reconstituted in the mobile phase and chromatographed on a reversed-phase HPLC column. The chromatograph was interfaced with an MS/MS system by positive-ion electrospray ionization. The multiple-reaction monitoring transitions of m/z 308 to 116 and m/z 268 to 191 were monitored for betaxolol and internal standard, respectively. The analytical working range was typically 0.05 to 10.0 ng per extract. 

Statistics

Descriptive statistics were calculated for the betaxolol concentrations in ocular tissues and plasma. Mean (±SD) levels in anterior segment tissues and plasma included data from all the human subjects: phakic, pseudophakic, and aphakic. Those of posterior segment tissues (vitreous humor, retina, choroid, optic nerve, and optic nerve head) did not include values from the aphakic patient because the lack of a lens could affect drug distribution toward the posterior segment. Statistical comparisons were not performed on the human data. A two-sided paired t-test was used to compare concentration in tissues of the treated and untreated eyes in the monkey experiment. P < 0.05 was considered significant. Elimination half-lives of drug in monkey plasma were determined by log-linear regression of the terminal phase of the plasma concentration versus time curve (WinNonlin; Pharsight Corp., Mountain View, CA). 

Results

Human Study

Patient demographics, diagnoses, previous ophthalmic interventions, concomitant topical medication, IOP, and the duration of topical betaxolol medication are shown in Table 1 . Betaxolol was found in anterior and posterior eye tissues and in the plasma. Table 2shows that the highest concentrations were present in anterior segment tissues, particularly the iris (73,200 ± 89,600 ng/g) and ciliary body (4,250 ± 3,020 ng/g). The rank order of mean concentrations (all eyes) in the anterior segment was iris > ciliary body > cornea > aqueous humor > lens > sclera. Betaxolol levels in the posterior segment tissues were lower, with mean values (excluding the aphakic patient) of 1290 ± 1170 ng/g in the choroid, 71.4 ± 41.8 ng/g in the retina, 31.2 ± 14.8 ng/g in the optic nerve head, 8.34 ± 6.17 ng/g in the optic nerve, and 4.12 ± 2.82 ng/g in the vitreous humor. On a molar basis, the concentrations in retina and optic nerve head were 0.233 ± 0.136 and 0.102 ± 0.048 μM (30 ng/mL is approximately 0.1 μM), respectively. The mean plasma betaxolol concentration was 0.59 ± 0.32 ng/mL (range, 0.16–1.02). 

Monkey Study

Analysis of plasma betaxolol on day 30 showed a maximum concentration (C _max) of 1.42 ± 0.41 ng/mL (mean ± SD, Table 3 ). Comparison of day 1 and day 30 C _max and AUC_0–12h indicate a slight accumulation of betaxolol in the plasma. With an elimination half-life of 4 hours, plasma steady state was considered to have been reached well before 30 days. The plasma concentration versus time data are plotted in Figure 1 . 

Betaxolol was found at higher concentrations in tissues of the treated eye than the untreated eye at the concentration trough of 12 hours (Table 4) . Although the concentration differences between the treated and untreated ocular tissues were all numerically substantial, they were statistically significant (P < 0.05) only in the vitreous humor and the sclera (Table 4) . There was an anterior-to-posterior distribution of betaxolol along a concentration gradient in the treated eye, with highest levels in the iris-ciliary body (78,300 ± 41,400 ng/g), choroid (22,700 ± 17,800 ng/g), and sclera (6390 ± 1570 ng/g), compared with the retina at 148 ± 85 ng/g (0.48 ± 0.28 μM) and optic nerve head at 222 ± 125 ng/g (0.72 ± 0.41 μM). Measurable drug levels were also present in the optic nerve and vitreous humor. 

Estimates of the contribution by local distribution were calculated as the difference in concentration between treated and untreated eyes (Table 4) , with the latter representing drug distributed from the systemic circulation. The data demonstrate that most of the drug in these posterior segment tissues was derived from local absorption that ranged from 59% in optic nerve head to 95% in the vitreous humor. The locally absorbed concentrations in retina (mean, 121 ng/g or 0.39 μM) and optic nerve head (mean, 130 ng/g or 0.42 μM) were among the highest. 

Discussion

These results demonstrate that betaxolol is distributed to posterior segment tissues after a minimum of 4 weeks of twice-daily topical ocular administration to monkey and human eyes. The ocular distribution of betaxolol follows a concentration gradient from the anterior (high) to posterior (low) direction, which is consistent with local distribution of a topically applied drug. 

Because of the nature of the human study, the time between instillation and enucleation varied by patient, but remained within a 1- to 6-hour time frame. Care was taken to exclude patients who had posterior segment diseases that might compromise the blood–retinal barrier or retinal tissue integrity; but, it is recognized that all human eyes in this study were diseased (Table 1)and that this factor could influence the distribution of betaxolol. However, the results of the monkey experiment, comparing tissue levels in the treated versus untreated normal eyes, verify that local absorption occurs in anterior and posterior ocular tissues. In general, betaxolol concentrations were similar between monkey and human tissues, including retina, optic nerve head, optic nerve, vitreous humor, aqueous humor, and iris. Choroidal and scleral concentrations were much higher in the monkey. 

The unilateral regimen used in the monkey experiment allowed the estimation of the individual contributions of systemic and local absorption to ocular tissue levels. The data show that much of the drug in posterior segment tissues was derived from local absorption, in proportions ranging from 59% in the optic nerve head to 95% in the vitreous humor. Whereas treated-eye posterior tissue concentrations were consistently substantially higher than those in the corresponding untreated tissues, the differences in means were not statistically significant, because of the small sample and the variance (Table 4) . For the vitreous humor and sclera, the differences were significant. The presence of betaxolol in posterior tissues of the untreated monkey eye reflects distribution from the circulation. The absence of contralateral untreated eye data in the human subjects does not permit an analysis of local versus systemic contribution to drug absorption. 

One patient was aphakic, with an intact posterior capsule, and another was pseudophakic. The aphakic eye was excluded from calculation of the mean data for posterior tissues (anterior segment tissue calculations included all patients’ data). The limited data do not allow any conclusions to be drawn about whether the absence of the lens affects betaxolol distribution to the back of the eye, although retinal concentrations were not notably different from those in the phakic eyes. 

Apparently the vitreous humor does not serve as a drug depot for the retina and optic nerve head, given its relatively low concentration of betaxolol. The choroid, based on its substantially high level of betaxolol in both monkey and human and its close proximity to the retina and optic nerve head, may be a depot source of drug in these tissues. 

Results reported from experiments in albino and pigmented rabbits similar to the present ones, with topical unilateral ocular application to steady state, also show higher levels in posterior segment tissues of the eye receiving drug treatment. ⁵  

The mean plasma level of 0.59 ng/mL betaxolol reported herein is within the range, 0.4 to 0.7 ng/mL, of that reported by Vuori et al. ¹² for 15 healthy volunteers, measured during a 240-minute period after receiving 200 μg betaxolol in a single dose compared with the 75 μg twice daily that our subjects received. Subsequently, Vainio-Jylhä et al. ¹³ reported mean betaxolol levels in the plasma of 0.4 ng/mL, 12 hours after a topical dose of 100 μg to both eyes of patients with glaucoma. Mean plasma levels of betaxolol varied from 0.7 to 1.6 ng/mL, at various sampling times over the 480-minute period after a second dose, given 12 hours after the first. Thus, the mean plasma level of betaxolol found in the study being reported herein is within the range reported by others. 

Possible binding sites for betaxolol are many and include β-adrenergic receptors, L-type calcium channels, and sodium channels. The affinity and functional potency of betaxolol as a β-adrenergic receptor blocker is in the nanomolar range. The specific binding of betaxolol and its more active isomer, levobetaxolol, to various tissues of the cadaveric human eye, including the choroid and retina, has been reported. ¹⁴ Both isomers of betaxolol bind to L-type calcium channel binding sites. ^{14 15} In addition, betaxolol has been shown to bind to neurotoxin site 2 and to inhibit veratridine-stimulated Na⁺ influx in rat cortical synaptosomes. ¹⁶ The calcium channel and sodium channel activities of betaxolol are thought, in part, to underlie its putative neuroprotective ¹⁷ and vasorelaxant ¹⁸ properties. 

Local absorption of betaxolol to the posterior part of the eye has been considered a real possibility since Sponsel et al. ² reported microgram quantities in the periocular tissues of patients with glaucoma treated long term with betaxolol 0.5% ophthalmic solution. More recently, periocular penetration and distribution of topically applied nipradilol ³ and iganidipine ⁴ were shown in animal studies with whole-head autoradiography. Ahmed and Patton ¹⁹ showed the importance of intraocular drug penetration via the conjunctiva–sclera route of entry for the β-blocker, timolol, in albino rabbits. This observation was later confirmed in pigmented rabbits. ²⁰ Also in pigmented rabbits, a sigmoidal relationship rather than a parabolic one better describes the relationship between lipophilicity and corneal and conjunctival drug permeabilities for a series of β blockers with log partition coefficients (log PCs) varying from −0.62 (sotalol) to +3.44 (betaxolol). ²¹ Betaxolol’s conjunctival and corneal permeability coefficient is 8 and 48 times greater, respectively, than that of the β blocker, sotalol, and 2.8- and 31-fold greater than that of pindolol (log permeability coefficient [PC] = 1.75). A similar sigmoidal relationship was found in rabbit cultured conjunctival epithelial cells where a 100-fold range in the apparent permeability coefficient between sotalol and betaxolol ²² was observed. Thus, the high lipophilicity of betaxolol favors its penetration through the conjunctiva as well as the cornea, providing access to the conjunctival–scleral route. 

The threshold for vasorelaxation of human retinal arterioles in vitro is 10⁻¹² M or <1 pg/g, ^{23 24} which is well below the micromolar concentrations of betaxolol found by us in the choroid and retina. Moreover, betaxolol concentrations in the untreated monkey eyes appeared high enough to be pharmacologically active if the same concentrations are present intraluminally in the retinal and choroidal microcirculation. However, our study design did not allow us to measure the intravascular concentration of betaxolol in these tissues, which makes the direct comparison with the in vitro data impossible. Our findings also suggest that care must be taken to interpret the results of blood flow studies in relatively small species, when the contralateral eye is used as a control, since a pharmacological effect may occur in that eye, even with unilateral application. 

This is the first published report of the distribution of betaxolol among anterior and posterior segment tissues of nonhuman primate and glaucomatous human eyes. The data from these investigations support the conclusions that topically administered betaxolol enters the eye through local absorption, and it may reach concentrations in the pharmacologically active range within many anterior and posterior segment tissues. 

 Supported in part by Hungarian National Health Grant (ETT) 011/2003 (GH) and an unrestricted research grant from Research to Prevent Blindness, Inc. (JTW).

 Submitted for publication July 21, 2005; revised September 19, 2005; accepted November 23, 2005.

 Disclosure: G. Holló, Alcon (C, F, R), Allergan (C, F, R), and Pfizer (C, R); J.T. Whitson, Alcon (C, R), Allergan (C, R), Pfizer (C, R), and Merck Sharp Dohme (C, R); R. Faulkner, Alcon (E); B. McCue, Alcon (E); M. Curtis, None; H. Wieland, Alcon (E); J. Chastain, Alcon (E); M. Sanders, Alcon (E); L. DeSantis, None; J. Przydryga, Alcon (E); D.C. Dahlin, Alcon (E)

 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 Treatment Information

Patient	Age (y)	Sex	Race	Diagnosis of the Study Eye	Previous Interventions on the Study Eye	Concomitant Topical Medication	IOP at the Time of Surgery (mm Hg)	Duration Betaxolol Treatment (mo)	Study Eye	Time Interval, Last Dose to Blood Sample (h:min)	Time Interval, Last Dose to Enucleation (h:min)
1	48	F	African-American	POAG	None	None	5	6	OS	4:45	5:55
2	79	M	White	Secondary glaucoma due to blunt trauma	None	Pilocarpine Latanoprost	58	1	OS	1:50	2:25
3	76	F	White	POAG; pseudophakia (with intact posterior capsule); corneal decompensation	Trabeculectomy 10 years before enucleation (failed); ECCE + PCL implant 3 years before enucleation	None	23	5	OD	2:00	2:42
4	27	M	White	Congenital cataract; aphakia; secondary glaucoma	Aspiration of congenital cataract 27 years before enucleation	Pilocarpine	55	1	OD	0:30	2:00
5	71	F	White	Pseudoexfoliative glaucoma	ALT 7 years before enucleation	Latanoprost	48	7	OD	0:25	2:04
6	70	F	White	POAG; central retinal vein occlusion; secondary (neovascular) glaucoma	None	Pilocarpine Latanoprost	66	6	OD	0:40	1:40
7	75	M	White	CACG; cataract; secondary glaucoma	Trabeculectomy complicated with intraocular hemorrhage	None	39	6	OS	0:15	1:55

 POAG, primary open angle glaucoma; CACG, chronic angle-closure glaucoma; ECCE, extracapsular cataract extraction; PCL, posterior chamber lens; ALT, argon laser trabeculoplasty.

Table 2.

 

Individual Patient Concentrations of Betaxolol in Ocular Tissues and Plasma after Repeated Topical Ocular Administration of Betaxolol 0.25% Suspension

Patient	Lens Status	Plasma	Vitreous Humor	Retina	Optic Nerve Head	Optic Nerve	Choroid	Lens	Sclera	Cornea	Aqueous Humor	Iris	Ciliary Body
1	Phakic	0.16	7.14	NS	26.1	2.80	669	38.7	112	195	143	10100	1900
2	Phakic	1.02	7.88	46.6	35.8	1.07	1500	206	12.5	417	333	48700	9470
3	Pseudophakic*	0.54	4.22	94.4	54.4	5.47	3540	NS	204	661	195	158000	6620
4	Aphakic	0.95	4.80	140	32.8	8.26	468	NS	22.8	1150	154	5670	3130
5	Phakic	0.39	1.68	52.4	38.1	14.2	817	73.6	116	579	75.1	236000	3440
6	Phakic	0.35	2.48	133	18.9	16.1	951	57.8	65.2	430	252	50000	4730
7	Phakic	0.75	1.32	30.5	13.6	10.4	268	93.4	7.49	27.3	29.8	4220	472
Mean, †		0.59	4.12	71.4	31.2	8.34	1290	93.9	77.1	494	169	73200	4250
SD		0.32	2.82	41.8	14.8	6.17	1170	65.8	71.8	361	103	89600	3020

 Concentrations are expressed in nanograms/gram of tissue or nanograms/milliliter of fluid. NS, No sample.

*  With intact posterior lens capsule.

†  Mean (SD) for posterior segment tissues (vitreous humor, retina, choroid, optic nerve, and optic nerve head); includes only phakic eyes.

Table 3.

 

Plasma Pharmacokinetic Parameters Determined after the First Dose and Multiple Topical Administration of Betaxolol to Monkeys

Dose Day	Animal Weight (kg)	Topical Dose (μg)	C max (ng/mL)	C 12h (ng/mL)	AUC0–12h (ng/mL·h)	t 1/2 (h)
1	4.5 ± 1.0	75	1.14 ± 0.14	0.05 ± 0.09	4.51 ± 1.62	2.6 ± 1.2
30	4.3 ± 0.9	75	1.42 ± 0.41	0.19 ± 0.15	6.96 ± 3.65	4.1 ± 0.6

 Data are expressed as the mean ± SD (n = 3).

Figure 1.

 

Mean concentrations of betaxolol in plasma after a single topical ocular dose (•) or after 30 days (○) of twice-daily topical ocular administration of betaxolol in cynomolgus monkeys.

Table 4.

 

Ocular Tissue Concentrations of Betaxolol in the Cynomolgus Monkey and Estimates of Contribution by Local Absorption

Tissue	12-Hour Concentrations of Betaxolol* (ng/g)			P	Estimated Local Contribution	Local Absorption (%)
		Treated Eye	Untreated Eye
Aqueous humor	100 ± 65	ND	—	—	—
Iris-ciliary body	78,300 ± 41,400	ND	—	—	—
Anterior sclera	6,390 ± 1,570	93.1 ± 30.1	0.02	6297	98
Choroid	22,700 ± 17,800	6,050 ± 2,530	0.20	16650	73
Retina	148 ± 85	27.1 ± 4.3	0.13	121	82
Optic nerve head	222 ± 125	91.8 ± 67.8	0.22	130	59
Optic nerve	35.2 ± 26.5	7.35 ± 9.33	0.18	28	80
Vitreous humor	3.83 ± 1.46	0.17 ± 0.09	0.04	3.6	95
Plasma	0.19 ± 0.15	—	—	—	—

 Data are expressed as the mean ± SD (n = 3). ND: Not determined.

*  12 hours after the last dose of the study.