Aqueous Humor Dynamics in Monkeys after Topical R-DOI

Abstract

purpose. To determine the effects of R-DOI, a selective 5-HT₂ agonist, on intraocular pressure (IOP) and aqueous humor dynamics in monkeys.

methods. Normotensive cynomolgus monkeys (n = 8) were treated topically once daily with four 5-μL drops of 0.5% R-DOI in one eye, vehicle in the opposite eye. The 6-hour IOP response (Goldmann applanation tonometry) was determined before the drug application and on the third day of treatment. Aqueous humor formation, or flow (AHF, measured by fluorophotometry), was measured from hours 3 to 8 after the third dose. Beginning 3.5 hours after the fourth or fifth dose, AHF was measured by dilution of radio-iodinated monkey albumin perfused through the anterior chamber and flow to blood by accumulation of albumin in the general circulation. Uveoscleral outflow (Fu) was calculated. Flow to blood was determined at spontaneous and elevated pressures, allowing calculation of trabecular outflow facility. Total outflow facility was determined by two-level constant pressure perfusion from 3.5 to 5 hours and from 5.5 to 6.25 hours after R-DOI treatment.

results. Reduction of IOP in treated eyes was compared to the opposite control eyes corrected for the 6-hour IOP baseline before the first dose. After the third dose of R-DOI, IOP was significantly (P < 0.01, n = 7) decreased by 1.4 to 4.7 mm Hg over the 6 hours. AHF (by fluorophotometry) increased by 13% (P < 0.05, n = 8) in treated compared with control eyes corrected for baseline. AHF (isotope dilution) increased by 30% (P < 0.01, n = 8), flow to blood decreased by 28% (n = 5), and Fu increased by 241% (P < 0.05, n = 5). Total and trabecular outflow facility were unchanged.

conclusions. R-DOI caused a small but significant increase in AHF and lowered IOP in normotensive monkeys primarily by increasing Fu.

Serotonin receptors have been identified in ocular tissues of the anterior segment of the eye in several species, including humans. ^{1 2} Functional mRNA for the serotonin 5-HT₂ receptors coupled to phosphoinositide turnover and [Ca²⁺] mobilization have been confirmed in human trabecular meshwork cells and ciliary muscle cells (Sharif NA, et al. IOVS 2003;44:ARVO E-Abstract 2084). mRNA encoding 5-HT_1A, 5HT2_A–C, and 5HT₇ receptors were detected in human ciliary body samples. ³ These findings suggest that 5-HT may play a role in regulating aqueous humor dynamics and intraocular pressure (IOP). 

There have been conflicting reports on the effects of 5-HT receptor subtype ligands on IOP in various species and as a consequence of activity at other classes of receptors. The 5-HT_1A agonists 8-OH-DPAT and the 5-HT_1A agonist/α₁-antagonist flesinoxan had no effect on IOP in anesthetized ⁴ or conscious ⁵ cynomolgus monkeys, in contrast to their IOP-lowering effects in rabbits. ^{6 7 8} The mechanism for the ocular hypotensive responses to the 5-HT₂ receptor antagonists ketanserin ^{9 10} and sarpogrelate (Takenaka H, et al. IOVS 1995;36:ARVO Abstract 36) in patients with glaucoma is most likely mediated through their α₁-adrenergic antagonist activity and not their 5-HT₂ antagonist activity. ⁵  

Of particular interest, one study demonstrated that 5-HT₂ agonists, but not 5-HT₂ antagonists or 5-HT_1A agonists, are involved in locally mediated control of IOP in conscious cynomolgus monkeys. ⁵ Potent and dose-dependent IOP lowering was observed in both hypertensive (lasered) and normotensive eyes. The response in hypertensive eyes followed an unusual time course, with the peak effect at least 6 hours after the drug was applied. It was thus of interest to determine the mechanism by which 5-HT₂ agonists reduce IOP. R-DOI ((R)(–)-1-(4-iodo-2,5-dimethoxyphenyl)-2-aminopropane)) was selected for this study because it is a prototypic selective 5-HT₂ agonist with a known IOP dose–response relationship in the cynomolgus monkey. ^{5 11}  

We report studies conducted to investigate further the IOP lowering mechanism of R-DOI in normotensive monkeys. 

Methods

Animals and Anesthesia

Eight ocular normotensive cynomolgus monkeys (Macaca fascicularis; five females, three males, 2.6–5.0 kg) were used for IOP, isotope dilution and accumulation studies (AHF, flow to blood, and outflow facility measurements). This group (group 1) of eight monkeys had undergone no invasive ocular procedures before the IOP measurements. These animals were subsequently used in the present study for one outflow facility experiment (Fig. 1)and then, after a rest period, for isotope studies. Eight normotensive cynomolgus monkeys (group 2) with virgin eyes (two females, six males, 3.0–7.5 kg) were used for fluorophotometry AHF studies. All monkeys were free of any ocular abnormalities according to slit lamp biomicroscopy at the time the measurements were taken. All experiments were conducted in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research and the University of Wisconsin Institutional Animal Care and Use Committee. 

The monkeys were anesthetized with intramuscular ketamine (10 mg/kg initial, supplement with 5 mg/kg as needed) for drug administration, 6-hour IOP, and fluorophotometry and with intramuscular ketamine (10 mg/kg) followed by intravenous pentobarbital sodium (15 mg/kg initial, supplemented by 5 to 10 mg/kg as needed) for femoral artery cannulation and isotope and outflow facility studies. 

Subcutaneous or intravenous fluids (lactated Ringers with 5% dextrose, 10 mL/kg per hour) were given during all experiments. For isotope studies, a femoral artery was cannulated for subsequent blood sampling. 

Treatments

The R-DOI dose (100 μg) was selected based on the IOP-lowering response in normal and hypertensive eyes in conscious monkeys measured at Alcon Research, Inc. (Fort Worth, TX). ⁵  

R-DOI (0.5%, formulated at Alcon from commercially available material obtained from Sigma-Aldrich, St. Louis, MO) or vehicle (phosphate-buffered saline with 0.5% hydroxypropyl methyl cellulose; pH 7.4) was administered once daily (8–9 AM) to the central cornea of opposite eyes in four 5-μL drops, separated by 30 seconds, while the eyelids were held open with the animal supine. 

Intraocular Pressure and Slit Lamp Examination

IOP was measured with a “minified” Goldmann applanation tonometer ¹² with cream used as a tear film indicator. ¹³ IOP was measured at 0.5, 1, 1.5, 2, 3, 4, 5, and 6 hours on the baseline day and after the first and third doses on days 1 and 3. Slit lamp examination (to determine the presence of biomicroscopic cells or flare) was performed before the first IOP measurement and at hours 3 and 6 on each day of measurement. 

Isotope Studies: Aqueous Humor Flow, Uveoscleral Outflow, Flow to Blood, and Trabecular Outflow Facility

The IOP-lowering response was verified on day 3, and treatments were continued. After treatment on the fourth or fifth day, the femoral artery was cannulated. Approximately 3 hours after the last treatment with R-DOI or vehicle, the eyes were cannulated with three needles. AHF was measured by dilution of radio-iodinated monkey albumin perfused through the anterior chamber and flow to blood by accumulation of labeled albumin in the general circulation. ^{14 15 16 17 18} Uveoscleral outflow (Fu) was calculated from measurements obtained by circulating I-125 (one eye)- and I-131 (opposite eye)-labeled albumin solution through the anterior chambers. The difference in the gamma emission spectrum of the isotopes permits determination of how much fluid from each eye enters the general circulation during any particular time interval. The dilution of label by newly formed aqueous was monitored with a well detector and allowed calculation of AHF. Accumulation of isotope in the blood within a 2-hour period after its initial introduction into the eye was assumed to be entirely by outflow through the trabecular meshwork. ¹⁹ The difference between the rates of AHF and trabecular outflow was a measure of Fu. 

Flow to blood was determined at both spontaneous IOP from 40 to 90 minutes and elevated IOP (15–16 mm Hg) from 90 to 120 minutes, to guard against any suppression of trabecular outflow due to low spontaneous IOP. ^{20 21} A crude calculation of trabecular outflow facility was performed from the flow-to-blood data collected at two different pressures. 

Outflow Facility

Total outflow facility was determined by two-level constant-pressure perfusion with Bárány’s perfusate ²² on two separate occasions. Pressures were alternated between approximately 15 and 25 mm Hg every 4 minutes. For the first study, before R-DOI treatment on the fourth or fifth day, baseline outflow facility was determined for 35 to 45 minutes. Reservoirs were then closed, and R-DOI or vehicle was administered topically to opposite eyes. After 3.25 hours, reservoirs were opened, and outflow facility was measured for the interval of 3.5 to 5 hours after treatment. On the second occasion, outflow facility was measured in the same monkeys for 35 to 45 minutes at the conclusion of the isotope studies, from approximately 5.5 to 6.25 hours after treatment. ²²  

AHF Measured by Fluorophotometry

Fluorophotometry measurements were performed (Fluorotron Master Ocular Scanning Fluorophotometer; OcuMetrics Inc., Mountain View, CA), and the flow rate was calculated by a modification of the method of Jones and Maurice. ²³ Pachymetry, keratometry, and limbus-to-limbus measurements were taken for calculation of anterior chamber volume. ²⁴ Then, topical anesthetic (proparacaine HCl 0.5% [Alcaine]; Alcon Laboratories) was given and, after 5 minutes, 2% fluorescein drops (usually 5 drops of 2 μL) were administered 1 minute apart to the central cornea. The following day, IOP measurement (with cream) ¹³ and biomicroscopy were performed before the first scan. Baseline fluorophotometry scans were measured hourly from 11 AM to 3 PM. After the last scan, biomicroscopy and IOP were checked. 

R-DOI or vehicle treatment of opposite eyes was begun the following day and the 6-hour IOP response was verified after the third dose, as described earlier. 

Treatments were continued on day 4. On day 5, monkeys were treated with R-DOI or vehicle at approximately 8 AM. Before fluorophotometry scans, at approximately 2.75 hours after treatment, IOP was measured (with cream) and biomicroscopy performed. Fluorophotometry was performed as for the baseline measurements. 

Three to 5 weeks after the last R-DOI treatment, post-treatment baseline fluorophotometry measurements were taken. Baseline values were averaged to obtain one mean value. 

Analysis

Data are the mean ± SEM. Ratios are unitless. Significance was determined by the two-tailed paired t-test for differences compared with 0.0 or ratios compared with 1.0. 

Results

IOP and Slit Lamp Examination

Before any treatment, initial IOP was 15.1 ± 0.5 mm Hg in vehicle-treated eyes and 15.0 ± 0.4 mm Hg in R-DOI-treated eyes (n = 7). IOP, compared with time 0 by two-tailed paired t-test, did not change significantly in either eye at any time point during the 6-hour baseline measurement period, except for an increase at 1 hour in eyes to be treated with R-DOI (to-be-R-DOI eyes; P < 0.05). However, regression analysis of baseline IOP showed a significant decrease in IOP over time in monkeys under ketamine anesthesia in the to-be-vehicle– but not the to-be-R-DOI–treated eyes as follows:  

\[\mathrm{IOP\ vehicle}\ {=}\ 15.2\ {-}\ 0.0895\ {\times}\ \mathrm{time}.\ R^{2}\ {=}\ 36.0\%,\ P\ {=}\ 0.052.\]

 

\[\mathrm{IOP\ R-DOI}\ {=}\ 15.3\ {-}\ 0.0908\ {\times}\ \mathrm{time}.\ R^{2}\ {=}\ 29.8\%,\ P\ {=}\ 0.074.\]

We typically see this type of variability with time under ketamine anesthesia. ²⁵ Slit lamp biomicroscopy findings in both eyes were normal throughout the 6 hours. 

Before the first treatment, IOP was 16.0 ± 0.2 mm Hg in vehicle-treated eyes and 14.9 ± 0.4 mm Hg in R-DOI-treated eyes. After the first treatment, IOP did not change significantly in vehicle-treated eyes over the 6-hour measurement period compared with the 6-hour baseline. IOP in R-DOI-treated eyes was significantly decreased by approximately 2 mm Hg from hours 1 to 6, compared with the corresponding 6-hour baseline measurement (P < 0.05). No biomicroscopic cells or flare were observed with R-DOI or vehicle at any time on day 1 (Fig. 2) . 

Before treatment on day 3, IOP was 15.3 ± 0.3 mm Hg in vehicle-treated eyes and 13.8 ± 0.4 mm Hg in R-DOI-treated eyes. IOP after the third dose of vehicle did not differ from the 6-hour baseline at any time point. IOP before and after R-DOI administration was significantly decreased at all time points compared with the 6-hour baseline (P < 0.05 or less). IOP after R-DOI decreased by approximately 5 mm Hg after 5 to 6 hours (Fig. 2) . This response was similar to that produced in conscious monkeys at Alcon. ⁵  

Slit lamp biomicroscopy in R-DOI-treated eyes revealed that before and at 6 hours after the third dose, one eye (not included in the analysis) had 1+ cells and trace flare. This effect may have been unrelated to the R-DOI treatment because the same monkey was used on separate occasions for outflow facility and isotope experiments and had no signs of inflammation before or after the third or fourth dose of R-DOI in those studies. All other eyes were clear. 

AHF, Trabecular Outflow, and Fu

IOP measured before treatment on day 3 was 14.9 ± 0.3 and 14.2 ± 0.2 mm Hg in vehicle- and R-DOI-treated eyes, respectively. After the third treatment, IOP decreased significantly in R-DOI- compared with vehicle-treated eyes (by 1.1 ± 0.4 to 2.2 ± 0.5 mm Hg; P < 0.05 or less) during hours 1.5 to 6. 

During isotope perfusion experiments on treatment days 4 and 5, in monkeys under pentobarbital anesthesia, IOP was no different between the eyes after cannulation and an initial 10-minute stabilization period at approximately 3.5 hours after treatment (vehicle, 14.1 ± 0.7 mm Hg and R-DOI, 14.1 ± 0.6 mm Hg) and at 5 hours after treatment before opening the reservoirs (vehicle, 9.6 ± 0.6 and R-DOI, 8.6 ± 0.5 mm Hg). 

Aqueous Humor Flow.

AHF determined by isotope dilution at the spontaneous IOP at approximately 4 to 5 hours after the fourth or fifth dose of R-DOI was significantly increased (30%) in R-DOI- compared with vehicle-treated eyes (Table 1) . 

At 5 hours after treatment, the reservoirs were opened and adjusted so that the IOP in both eyes was the same (∼15 mm Hg). Total flow from the reservoirs plus AHF was not significantly different between the eyes when measured by isotope dilution. 

Flow to Blood and Fu.

Comparison of R-DOI- with vehicle-treated eyes showed flow to blood from 4 to 5 hours at the spontaneous IOP after the fourth or fifth topical dose to be reduced significantly (43%; Table 1 ). The ratio of flow to blood to AHF also decreased significantly (57%). 

Because flow to blood can be greatly diminished if the IOP during the experiment becomes less than episcleral venous pressure, additional calculations were performed with the data from animals in which the spontaneous IOP remained above 8 mm Hg (n = 5). Again, the ratio of flow to blood to AHF decreased significantly (28%) in R-DOI- compared with vehicle-treated eyes. 

Fu calculated for eight monkeys indicated a significant 212% increase in R-DOI- compared with vehicle-treated eyes and a 141% increase when expressed relative to AHF. When the calculations were performed in five subjects (i.e., animals with IOP >8 mm Hg during the experiment), similar 241% and 138% increases were found, respectively. 

To compensate for potentially low IOPs and loss of the pressure gradient across Schlemm’s canal, IOP was elevated to the same pressure in both eyes (approximately 15 mm Hg) in all monkeys after 90 minutes by opening the eyes to reservoirs filled with plain Bárány’s solution. These data showed that flow to blood compared with total flow decreased significantly (31%) in R-DOI- versus vehicle-treated eyes. Calculated Fu at the elevated IOP was variable, in part due to the hybrid nature of this determination and the numerous assumptions used in making the calculations, with some values being negative and the resultant SEM very high. However, in every case, Fu was greater in R-DOI-treated eyes, and the arithmetic differences were statistically significant. 

Total and Trabecular Outflow Facility

Baseline total outflow facility on the first occasion (outflow facility 1) before the fourth or fifth treatment was 0.58 ± 0.10 μL/min per mm Hg in R-DOI-treated eyes and 0.53 ± 0.10 μL/min per mm Hg in vehicle-treated eyes. At 3.5 to 5 hours after treatment, total outflow facility 1 was not different between the eyes (Table 2)but had increased in both eyes by 53% to 65% compared with baseline, probably due to washout resulting from the prolonged perfusion (not shown). No change in total outflow facility between the eyes was detected when 30-minute intervals were compared. 

Total outflow facility, measured at the conclusion of isotope studies at approximately 5.5 to 6.25 hours after the fourth or fifth treatment (facility 2) was also no different between the eyes. Baseline measurements were not usually performed as part of the isotope studies, due to the length of the entire experiment. Correction for baseline measurements performed during the single prior perfusion experiment in these same monkeys also revealed no differences between the eyes (not shown). 

Because flow to blood during isotope studies was measured at two pressures (spontaneous and 15 to 16 mm Hg), a crude estimate of trabecular outflow facility was obtainable. There was no change in trabecular outflow facility comparing R-DOI- and vehicle-treated eyes using all data or data only from animals in which the IOP never went below 8 mm Hg. 

AHF Measured by Fluorophotometry

Initial IOP before any treatments was 16.3 ± 0.3 mm Hg in vehicle-treated eyes and 16.0 ± 0.4 mm Hg in R-DOI-treated eyes (n = 8). On day 3, before treatment, IOP in R-DOI-treated eyes was significantly less than in vehicle-treated eyes (by 2.2 ± 0.3 mm Hg; P < 0.001) and continued to decrease throughout the 6 hours of measurements (difference at 6 hours, 4.0 ± 0.5 mm Hg; P < 0.001). IOP before the fifth treatment was significantly less in R-DOI compared with vehicle-treated eyes by 1.8 ± 0.6 mm Hg (P < 0.025) and by 3.9 ± 0.4 mm Hg (P < 0.001) at 8 hours. IOP before the post-treatment baseline fluorophotometry had recovered to the initial pretreatment levels. 

There was no difference in baseline AHF between R-DOI- and vehicle-treated eyes before and 3 to 5 weeks after the last treatment (Table 3) . Also, there was no difference between R-DOI- and vehicle-treated eyes after treatment. However, compared with vehicle and corrected for the average baseline, R-DOI significantly increased AHF by 13% ± 5% (P < 0.05) during the interval 3 to 8 hours after drug application on day 5. No significant difference was detectable when shorter intervals were analyzed. 

Slit lamp biomicroscopy confirmed that all eyes were free of cells and flare at all time points examined during confirmation of the IOP response, baseline, and post-treatment AHF measurements. 

Discussion

Agonists at the 5-HT₂ receptors have been identified as effective hypotensive agents in the nonhuman primate experimental glaucoma model and in normotensive monkey eyes. ⁵ In the present study, we found a similar time course and magnitude of IOP lowering in ketamine-anesthetized monkeys. The study drug was applied once daily, in the morning. It is not known whether the efficacy would be greater if it were applied in the evening instead. Our studies suggest the mechanism for the IOP lowering response is primarily due to an increase in Fu. The discovery of functional 5-HT₂ receptors in the human ciliary muscle (Sharif et al., IOVS 2003;44:ARVO E-Abstract 2084) supports the plausibility of an effect on Fu. 

The magnitude of the IOP lowering, increase in Fu, lack of an effect on outflow facility, and small increase in AHF are similar to responses observed after PGF_2α-ie (topical drops in monkeys). ^{26 27 28} Given the similarity of the responses to R-DOI and PGF_2α-ie, cross-reactivity with prostaglandin receptors should be assessed, and studies with prostaglandin antagonists are indicated. 

5-HT_1A agonists have been shown to increase AHF in nonhuman primates. ⁴ However, the selectivity of R-DOI for the 5-HT₂ receptor ⁵ makes it unlikely that the increase in AHF in the present study is due to cross-reactivity at the 5-HT_1A receptor subtype. The magnitude of the AHF increase in the current studies was greater in the isotope studies than in the fluorophotometry studies, probably because of the lower AHF rate in pentobarbital- versus ketamine-anesthetized monkeys. ²⁹ Increases in AHF after epinephrine treatment are similarly magnified in pentobarbital-anesthetized monkeys. ²⁹  

The lack of an effect of R-DOI on outflow facility in the current studies suggests that functional 5-HT₂ receptors, which were detected in human trabecular meshwork cells (Sharif et al. IOVS 2003;44:ARVO E-Abstract 2084), are unlikely to be involved in the predominant mechanism for IOP lowering in vivo in monkeys. 

In conclusion, R-DOI, a selective 5-HT₂ agonist, caused a small but significant increase in AHF and lowered IOP in normotensive monkeys, primarily by increasing Fu. 5-HT₂ receptor stimulation may represent another pathway for development of IOP-lowering drug therapy. However, the possibility of an overlapping effect by prostaglandin-related mechanisms should be explored. 

 Supported by Alcon Laboratories, National Eye Institute Grant EY02698 (PLK); Research to Prevent Blindness, Inc. (RPB); unrestricted departmental and Physician-Scientist awards (PLK); Ophthalmology Physiology Research and Education Fund (PLK); Fight for Sight (MO), and National Eye Institute Training Grant EY07119 (Department of Biostatistics and Medical Informatics, University of Wisconsin).

 Presented at the annual meeting of the Association for Research in Vision and Ophthalmology, Fort Lauderdale, Florida, April 2004.

 Submitted for publication May 24, 2005; revised July 28, 2005; accepted September 22, 2005.

 Disclosure: B.T. Gabelt, None; M. Okka, None; T.R. Dean, Alcon Research Ltd. (E); P.L. Kaufman, Alcon Research Ltd. (F)

 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.

 

Figure 1.

 

Time line for studies. Rx, treatment (R-DOI or vehicle); Fluoro, fluorophotometry; OF, total outflow facility.

Figure 2.

 

IOP after 100 μg topical R-DOI. IOP was measured for 6 hours before any treatments (baseline) and on days 1 and 3 of once-daily treatment with 100 μg R-DOI in one eye and vehicle in the opposite eye. Time 0 on day 1 was before any treatment; time 0 on day 3 was approximately 24 hours after the second treatment, but before the third treatment. Significance determined by the two-tailed paired t-test for differences compared to 0.0: *P < 0.05 or less for baseline versus time 0 and for days 1 and 3 versus baseline day for each corresponding time point. n = 7.

Table 1.

 

Isotope Study Results: Aqueous Humor Flow, Trabecular Outflow, and Uveoscleral Outflow after 100 μg R-DOI

	IOP	n	R-DOI	Vehicle	R-DOI/Veh	R-DOI/Veh
AHF	Spontaneous	8	1.70 ± 0.13	1.31 ± 0.06	1.30 ± 0.08, †	0.39 ± 0.11, †
	IOP > 8 mm Hg	5	1.82 ± 0.10	1.33 ± 0.07	1.38 ± 0.10*	0.49 ± 0.12*
	Reservoir open	8	4.64 ± 0.58	3.86 ± 0.45	1.22 ± 0.11	0.78 ± 0.40
FTB	Spontaneous	8	0.48 ± 0.14	0.84 ± 0.12	0.57 ± 0.12, †	−0.35 ± 11*
	IOP > 8 mm Hg	5	0.72 ± 0.14	0.98 ± 0.06	0.72 ± 0.12	−0.26 ± 0.10
	Reservoir open	8	2.92 ± 0.34	3.60 ± 0.30	0.84 ± 0.10	−0.68 ± 0.38
Fu	Spontaneous	8	1.22 ± 0.16	0.48 ± 0.13	3.12 ± 0.49, ‡	0.74 ± 0.11, §
	IOP > 8 mm Hg	5	1.10 ± 0.18	0.35 ± 0.05	3.41 ± 0.72*	0.75 ± 0.18*
	Reservoir open	8	1.72 ± 0.47	0.26 ± 0.42	9.20 ± 9.18	1.47 ± 0.29, ‡
FTB/AHF	Spontaneous	8	0.28 ± 0.08, §	0.64 ± 0.09, ‡	0.43 ± 0.09, §	−0.37 ± 0.08, ‡
	IOP > 8 mm Hg	5	0.40 ± 0.08, ‡	0.74 ± 0.03, §	0.54 ± 0.11*	−0.34 ± 0.08*
	Reservoir open	8	0.66 ± 0.07, ‡	0.97 ± 0.07	0.69 ± 0.06, §	−0.31 ± 0.07, ‡
Fu/AHF	Spontaneous	8	0.72 ± 0.08, †	0.36 ± 0.09, §	2.41 ± 0.32, ‡	0.37 ± 0.08, ‡
	IOP > 8 mm Hg	5	0.60 ± 0.08, †	0.26 ± 0.03, §	2.38 ± 0.36*	0.34 ± 0.08*
	Reservoir open	8	0.34 ± 0.07, §	0.03 ± 0.07, §	5.72 ± 5.68	0.31 ± 0.07, ‡

 AHF, aqueous humor flow; FTB, flow to blood; Fu, uveoscleral outflow. Measurements taken at 4 to 5 hours (spontaneous and IOP > 8 mm Hg) and 5 to 5.5 hours (reservoir open) after the fourth or fifth dose of R-DOI or vehicle. Reservoir open indicates that the eyes were opened to an external reservoir, allowing inflow of perfusate that maintained the IOP at the same pressure (approximately 15 mm Hg) in both eyes. Data are the mean ± SEM. AHF, FTB, Fu units are μL/min.

 Significance determined by the two-tailed paired t-test for ratios compared to 1.0 or differences compared to 0.0:

*   P < 0.05;

†   P < 0.01;

‡   P < 0.005;

§   P < 0.001.

Table 2.

 

Trabecular and Total Outflow Facility after Topical R-DOI

		n	R-DOI	Vehicle	R-DOI/Veh
OF1 (3.5–5 h)		8	0.89 ± 0.15	0.90 ± 0.26	1.27 ± 0.16
OF2 (5.5–6.25 h)		8	0.87 ± 0.14	0.69 ± 0.14	1.35 ± 0.17
Ctrab (4–5.5 h)	Spontaneous IOP	8	0.40 ± 0.06	0.56 ± 0.08	0.84 ± 0.11
	IOP > 8 mm Hg	5	0.44 ± 0.08	0.62 ± 0.10	0.74 ± 0.16

 Data are the mean ± SEM. OF, total outflow facility; Ctrab, trabecular outflow facility calculated from flow-to-blood measurements at two different IOPs. Units are μL/min per mmHg for facility.

 Significance determined by the two-tailed paired t-test for ratios compared with 1.0; no differences were significant at P < 0.05.

Table 3.

 

Aqueous Humor Formation (Fluorophotometry) after Topical R-DOI

	Aqueous Humor Formation
		R-DOI	Vehicle	R-DOI/Veh
Pre Rx Baseline 1	1.98 ± 0.31	2.02 ± 0.30	0.99 ± 0.04
Post Rx Baseline 2	2.12 ± 0.29	2.06 ± 0.21	1.02 ± 0.04
Average Baseline	2.05 ± 0.28	2.04 ± 0.23	1.00 ± 0.04
Post Rx	2.57 ± 0.38	2.26 ± 0.23	1.14 ± 0.09
Post Rx/Avg BL	1.26 ± 0.05, †	1.12 ± 0.04*	1.13 ± 0.05*

 Data are mean ± SEM. Aqueous humor formation units are μL/min. Rx, treatment with R-DOI or vehicle to opposite eyes; Post Rx Baseline was measured 3 to 5 weeks after the last treatment with R-DOI or vehicle; n = 8.

 Significance determined by the two-tailed paired t-test for ratios compared with 1.0:

*   P < 0.05;

†   P < 0.005.

The authors thank Theodora J. Bunch, Julie A. Kiland, MS, and Beth Hennes for lending expertise in performing outflow facility measurements and for assistance with fluorophotometry measurements and femoral artery cannulations for blood collection; Timothy S. Grant, MS, for statistical consultation; and Marsha McLaughlin, Jesse A. May, and Mark Hellberg (Alcon Research) for assistance with the serotonin discovery program, study coordination, and drug formulations.