Sildenafil-Induced Reversible Impairment of Rod and Cone Phototransduction in Monkeys

Abstract

Purpose.: To investigate functional alteration of the retina induced by sildenafil in monkeys.

Methods.: Sildenafil was administered intravenously to cynomolgus monkeys at dose levels of 0, 1, 3, and 10 mg/kg, and standard full-field electroretinograms (ERGs) were recorded. The rod and cone a-waves in response to high-intensity flashes were also analyzed by the a-wave fitting model (a-wave analysis). Additionally, the photopic negative responses were recorded.

Results.: Sildenafil at 3 mg/kg or more induced the following alterations in the standard full-field ERGs immediately after dosing: delayed b-wave in the rod response; delayed a-wave in the combined rod-cone response; and attenuated b-waves in the single-flash cone response and in the 30 Hz flicker. Additionally, the following changes were observed in the 10 mg/kg group: attenuated b-wave in the rod response; attenuated a-wave and delayed b-wave in the combined rod-cone response; delayed oscillatory potentials; and attenuated and delayed a-wave in the single-flash cone response. In the a-wave analysis immediately after dosing, sildenafil selectively decreased the sensitivity parameter (S) in the cone a-wave at 3 mg/kg, and in both the rod and cone a-waves at 10 mg/kg. The S value was highly correlated with plasma sildenafil concentration. The above changes fully recovered 24 hours after dosing.

Conclusions.: Sildenafil produced reversible impairment of the rod and cone phototransduction in monkeys. Meanwhile, involvement of the postreceptoral retinal components was suggested. These findings contribute to the clarification of sildenafil-induced visual disturbances. It is suggested that the photoreceptors are predominantly, but not exclusively, affected in the retina of humans with sildenafil-induced visual disturbances.

Introduction

Sildenafil, an inhibitor of phosphodiesterase type-5 (PDE5), is widely used for the treatment of erectile dysfunction and pulmonary arterial hypertension. It is well known that sildenafil can transiently produce dose-dependent and mild alterations in the visual system of patients, such as blue tinge to vision and an increased perception of the brightness of the lights.^1,2 

Alterations in electroretinograms (ERGs) have been reported in sildenafil-treated humans,^3–8 indicating functional involvement of the retina. In addition, sildenafil also has effectiveness as an inhibitor of phosphodiesterase type-6 (PDE6),⁹ which is found in retinal photoreceptors. The enzyme PDE6 is an important component of the phototransduction cascade, which regulates the cation channels in the outer segment of the photoreceptors.¹⁰ Therefore, the site of the sildenafil-induced visual impairment is generally considered to be the photoreceptors. 

In the previous studies of sildenafil in humans, somewhat variable influences on the ERG have been shown as described below. Although attenuation in the amplitude of the rod-driven or the cone-driven ERG has been reported in some studies,^3,4 these findings have not been confirmed in other studies,^5,8,11 which have found delays in the cone-driven ERGs. On the other hand, several studies have reported enhancement in the rod- and cone-driven ERGs.^6,7 One conceivable explanation for these inconsistencies is that the alterations in ERG induced by sildenafil when given at around the clinical doses were mild and therefore partially masked by relatively large inter- and intraindividual differences in human subjects. Differences in the recording condition of ERGs in those studies could also contribute to the inconsistencies. Furthermore, those clinical ERGs in humans seem to be not sufficiently accurate to identify the functional impairment of the photoreceptors. This could be attributable to the limitations of ERGs in clinical practice or studies in humans. For example, there are no waveform components that originate nearly exclusively from the photoreceptors in ERGs as generally assessed in clinical practice (i.e., it has been shown that the a-wave of such ERGs includes not a little contribution from postreceptoral components of the retina^12,13). Also, administration of sildenafil to humans at doses that greatly exceed the recommended maximum clinical dosage could raise ethical concerns, even if the purpose is to clarify the mechanism underlying the visual abnormalities. 

One efficient means to thoroughly examine sildenafil-induced alteration of retinal function would be to assess extended-protocol ERGs, including those specialized to the photoreceptors, not in humans but in animals treated with sildenafil at doses that highly exceed clinical doses. However, there are very few reports that describe sildenafil-provoked changes in the ERG in experimental animals. To our knowledge, no studies have examined ERGs with extended protocols in experimental animals with plasma sildenafil levels considerably higher than those reported in humans with visual disturbances. Although Mochida et al.¹⁴ examined retinal function in dogs treated with sildenafil at a suprapharmacologically active dose, they only assessed light-adapted (i.e., cone-driven) ERG to 30-Hz flickering stimuli. 

Taking these observations together, the functional abnormalities of the retina induced by sufficiently high levels of plasma sildenafil have not been characterized adequately in humans or animals, and therefore the exact mechanisms of sildenafil-induced visual disturbances have not been definitively elucidated. Thus, the purpose of this study was to intensively investigate sildenafil-induced alterations of retinal function in experimental animals. For this purpose, we administered sildenafil at sufficiently high dose levels to monkeys, a species in which the physiology and anatomical structure of the retina is widely known to be similar to that in humans, and assessed retinal function by analysis of extended-protocol ERGs in addition to clinically relevant ERGs. 

Methods

Animals

A total of 16 cynomolgus monkeys (Macaca fascicularis) between 3 and 5 years of age were used in this study. The animals were housed individually in stainless steel cages (W 60 cm × D 68 cm × H 75 cm) in an animal study room where the environmental condition was set as follows: room temperature, 24°C; relative humidity, 60%; illumination, 12-hour lighting (7:00 AM to 7:00 PM) at 300 luces. The animals were fed 100 g/animal per day of pellet food for monkeys (PS; Oriental Yeast Co., Ltd., Tokyo, Japan). Tap water from a feed-water nozzle was supplied ad libitum to the animals. All experimental procedures adhered to the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research, and were approved by the Institutional Animal Care Committee of Daiichi Sankyo Co., Ltd. 

Study Design

Sixteen animals were assigned either to the vehicle-treated group or one of the three sildenafil-treated groups (four animals per group) so that the group means and variances for ERG parameters obtained prior to the start of this experiment were as nearly equal as possible. In these animals, a series of ERGs was recorded before, immediately after, and 24 hours after dosing as described below. 

Drug Administration

Sildenafil citrate salt (Sigma-Aldrich, St. Louis, MO, USA) was dissolved in physiological saline (Otsuka normal saline; Otsuka Pharmaceutical Factory, Inc., Tokushima, Japan) at concentrations of 0, 0.5, 1.5, and 5.0 mg/mL, followed by a filter sterilization. Immediately after preparation, the dose formulation was administered intravenously to the saphenous vein for 10 minutes to animals at dose levels of 0, 1, 3, and 10 mg/kg. The lowest dose (1 mg/kg) was expected to produce a plasma sildenafil level similar to the maximum plasma concentration (C_max) in patients who ingest sildenafil at clinical dosages (50–100 mg/body). 

Clinical Observation

Clinical signs were observed every day up to 7 days after dosing. 

Animal Preparation for ERG Recording

The animals were anesthetized with intramuscular injection of ketamine hydrochloride (Ketalar Intramuscular 500 mg; Daiichi Sankyo Co., Ltd., Tokyo, Japan; 10 mg/kg initial dose, 5–10 mg/kg per hour maintenance dose) and 0.6 mg/kg xylazine hydrochloride (Celactal; Bayer Medical, Ltd., Osaka, Japan). The pupils were dilated with topical 0.5% tropicamide and 0.5% phenylephrine hydrochloride (Mydrin-P ophthalmic solution; Santen Pharmaceutical Co., Ltd., Osaka, Japan); the corneas were anesthetized with topical 0.4% oxybuprocaine hydrochloride (Benoxil ophthalmic solution 0.4%; Santen Pharmaceutical Co., Ltd.) and protected with topical hydroxyethylcellulose (Scopisol solution for eye; Takeda Chemical Industries, Ltd., Osaka, Japan). Prior to ERG recording, pupil diameter of the left eye was measured to calculate retinal illuminance (in trolands; Td) for the a-wave analysis. 

Visual Stimulation

Full-field stimulation was produced with a Ganzfeld stimulator (BigShot Ganzfeld; LKC Technologies, Inc., Gaithersburg, MD, USA) that was equipped with two different types of light source: xenon photostrobe and light-emitting diodes (LEDs). White light flashes at an intensity of 60 phot cd·s/m² or more were generated with the xenon photostrobe. The maximum intensity of the white flash generated with the xenon photostrobe was 826.7 phot cd·s/m². The stimulus intensity was altered by varying the capacitance and applied voltage of a capacitor using software (Ganzfeld control panel; LKC Technologies, Inc.) installed in a personal computer. The white light flashes that were below 60 phot cd·s/m², colored-light flashes, and steady background illumination were generated with the following LEDs: red (λ_max = 627 nm), green (λ_max = 530 nm), and blue (λ_max = 470 nm). The white flashes were produced by combining the output from these three LEDs. The duration of all stimuli was less than 5 ms The maximum intensity of white flash generated with the LEDs in the stimulator was 27.3 phot cd·s/m². The stimulus intensity and the background luminance were altered by varying the LED pulse duration using software (Ganzfeld control panel; LKC Technologies, Inc.) installed in the personal computer. The intensities of flashes generated in this system were measured by a calibrated photometer (IL1700; International Light Technologies, Inc., Peabody, MA, USA) and an optical detector (SED033/Y/R; International Light Technologies, Inc.). 

Recording and Analysis

All ERGs described below were recorded from the corneal surface of the left eye of each animal with a bipolar contact lens electrode (H6515NFC; Mayo Corporation, Aichi, Japan). A ground electrode (TN208-016; Unique Medical Co., Ltd., Tokyo, Japan) was attached to the parietal region of the scalp. Responses were amplified 10,000 times and were filtered with a band pass from 0.5 to 1000 Hz. The amplified signals were stored in the evoked potential test equipment (MEB-9104; Nihon Kohden Corporation, Tokyo, Japan). Three to 10 waveforms for each response were averaged. 

Standard Full-Field ERGs.

Standard full-field ERGs were evaluated according to the guideline¹⁵ of the International Society for Clinical Electrophysiology of Vision (ISCEV). Following 40 minutes or more of dark adaptation, the rod-driven response (the rod response) and the rod- and cone-driven response (the combined rod-cone response [standard flash]) were elicited by white light flashes at an intensity of 0.009 and 2.8 phot cd·s/m², respectively. Another rod- and cone-driven response (the combined rod-cone response [bright flash]) and the oscillatory potentials were simultaneously elicited by white light flashes at an intensity of 17.7 phot cd·s/m². Subsequently, after 10 minutes of light adaptation with a white background light at 29.0 phot cd/m², the single-flash cone response (W/W) and the 30 Hz flicker response were elicited by white light flashes at an intensity of 2.8 phot cd·s/m² under the white background light. 

In the waveform analysis, the amplitude and implicit time of the a-wave were measured from baseline to the a-wave trough and from stimulus onset to the a-wave trough, respectively, for the combined rod-cone response and the single-flash cone response; the amplitude and implicit time of the b-wave were measured from the a-wave trough to the b-wave peak and from stimulus onset to the b-wave peak, respectively, for all the responses. For the oscillatory potentials, the amplitudes of the first and second positive peaks (i.e., OP1 and OP2, respectively) were measured from baseline to each respective peak. The implicit time of the OP1 and OP2 was also measured from stimulus onset to each respective peak. 

A-Wave Analysis.

Immediately before the photopic ERG section of the standard full-field ERGs recording period, responses for the a-wave analysis were recorded in the same manner as that described in our previous report.¹⁶ Briefly, three steps of stimuli were used to elicit the rod- and cone-driven response in the fully dark-adapted state. Thereafter, the cone-driven response in the fully dark-adapted state was elicited by means of a transient rod-saturation procedure, in which the white test stimulus was delivered 500 ms after the conditioning white flash of 4.0 log scot td-s. To elicit the cone-driven response, three steps of test stimuli, which were identical to those used to elicit the rod- and cone-driven response, were used. Rod-isolated responses were obtained by subtracting the cone-driven responses from the rod- and cone-driven responses. To evaluate rod function, the leading edge of the a-wave of the rod-isolated response (i.e., the rod a-wave) was fitted to a curve by the Hood and Birch modification¹⁷ of the Lamb and Pugh model¹⁸:  

 

 

In Equation 1, I is the flash intensity (log scot td-s); t_d is the time delay (ms); t is the time after the flash onset (ms); S is the sensitivity (s⁻²(td-s)⁻¹); and R_max is the maximum response amplitude (μV). The value of t_d was fixed at 3.1 ms, which is the mean value from age-matched healthy monkeys, and S and R_max were varied for the best fit. To evaluate cone function, the leading edge of the a-wave of the cone-driven response (i.e., the cone a-wave) was fitted to a Michaelis-Menten version¹⁹ of Equation 1 combined with an exponential filter:  

 

 

In Equation 2, I is the flash intensity (log phot td-s); t_d is the time delay (ms); t is the time after the flash onset (ms); S is the sensitivity (s⁻³(td-s)⁻¹); and R_max is the maximum response amplitude (μV). The value of t_d was fixed at 1.8 ms, which is the mean value from age-matched healthy monkeys, and S and R_max were varied for the best fit. 

Single-Flash Cone Response (R/B).

Immediately after the photopic ERG section of the standard full-field ERGs recording period, another photopic ERG was elicited by red light flash at an intensity of 2.9 phot cd s/m² under blue background light at 6.9 phot cd/m². For the waveform analysis, amplitude of the b-wave and the photopic negative response (PhNR) were measured from the a-wave trough to the b-wave peak and from baseline to the PhNR trough, respectively. Additionally, the PhNR/b-wave amplitude ratio was calculated as the ratio of the PhNR amplitude to the b-wave amplitude. 

Ophthalmoscopy

Immediately after recording the ERGs at each time point, the fundi of both eyes were observed with a binocular indirect ophthalmoscope (HEINE OMEGA 500; HEINE Optotechnik GmbH & Co. KG, Herrsching, Germany). 

Toxicokinetics

Approximately 0.5 mL of blood was collected from the femoral vein immediately after the recording of the ERGs to measure the plasma sildenafil concentration. Blood was collected just after recording the ERGs on the day of dosing and 24 hours after dosing (0.70–0.85 and 24.57–24.93 hours after starting the infusion of sildenafil, respectively). Seven days after recording the ERGs from the 12 monkeys given sildenafil, sildenafil was administered again to the same animals in the same manner. Blood was collected before dosing, and 10 and 30 minutes, and 2, 4, 7, and 24 hours after the start of dosing in the same manner. The plasma was prepared from the blood samples by centrifugation at 11,200g for 5 minutes at 4°C. The plasma was then stored at −80°C until measurement. The plasma concentration of sildenafil was determined by liquid chromatography mass spectrometry/mass spectrometry (LC/MS/MS) (HPLC; Waters Alliance 2795 Separations Module; Waters Corp., Milford, MA, USA; MS/MS; Quattro Premier XE; Waters Corp.). 

Statistics

For statistical analysis of the ERG parameters, the Dunnett's test was used to assess the difference between the vehicle- and sildenafil-treated groups. The differences were considered to be significant when P was less than 0.05. Furthermore, Pearson's correlation coefficient between the a-wave analysis parameters and plasma sildenafil concentration was estimated and hypothesis testing (null hypothesis: the correlation coefficient is equal to zero) was performed. 

Results

Clinical Observation

No change in clinical signs was observed in any group given sildenafil. 

Electroretinogram

Standard Full-Field ERGs.

Typical waveforms of the standard full-field ERGs in vehicle- and sildenafil-treated monkeys are shown in Figure 1. The amplitude and implicit time of the ERG components are summarized in Table 1. In the group given 1 mg/kg, sildenafil induced no obvious alterations in any ERGs. In the groups given 3 mg/kg or more, the following statistically significant differences compared with the vehicle-treated group were detected immediately after dosing: delayed b-wave in the rod response; delayed a-wave in the combined rod-cone response; and attenuated b-waves in the single-flash cone response and in the 30 Hz flicker. Additionally, the following significant differences in the 10 mg/kg group were also observed: attenuated b-wave in the rod response; attenuated a-wave and delayed b-wave in the combined rod-cone response; delayed OP1 and OP2 in the oscillatory potentials; and attenuated and delayed a-wave in the single-flash cone response. Twenty-four hours after dosing, there were no differences between the vehicle- and sildenafil-treated groups in any ERG. 

Figure 1

 

Typical waveforms of the standard full-field ERGs in vehicle- and sildenafil-treated monkeys. Sildenafil was administered at dose levels of 0 (vehicle), 1, 3, and 10 mg/kg, and the standard full-field ERGs were obtained at baseline, immediately after, and 24 hours after dosing as described in the text. Arrowheads indicate onset of the light flashes. The responses at baseline (gray traces) are superimposed on those obtained after dosing (black traces). Each trace represents an average of 3 to 10 responses.

Table 1

 

Effects of Sildenafil on the Standard Full-Field ERGs

	Dose, mg/kg	Amplitude, μV			Implicit Time, ms
		Baseline	Immediately After	24 Hours After	Baseline	Immediately After	24 Hours After
Rod response
b-wave	0	55.1 ± 24.80	59.6 ± 22.10	77.2 ± 20.90	99.6 ± 6.10	96.6 ± 2.60	95.4 ± 2.50
	1	46.9 ± 17.70	36.4 ± 22.60	49.1 ± 21.20	97.8 ± 4.80	106.0 ± 4.70	95.1 ± 5.70
	3	77.7 ± 10.00	34.5 ± 18.80	83.0 ± 14.40	95.4 ± 9.40	114.6 ± 16.00*	89.6 ± 8.30
	10	51.6 ± 5.40	0.0 ± 0.00**	58.9 ± 17.20	93.1 ± 8.20	NC	92.8 ± 8.40
Combined rod-cone response (standard flash)
a-wave	0	74.8 ± 26.50	86.3 ± 24.70	98.0 ± 29.00	19.2 ± 0.80	19.0 ± 0.70	18.3 ± 0.70
	1	77.3 ± 13.30	77.0 ± 11.00	84.4 ± 14.60	18.9 ± 0.50	20.0 ± 0.50	18.7 ± 0.60
	3	82.0 ± 4.20	70.9 ± 20.60	93.2 ± 9.60	18.4 ± 0.60	21.0 ± 1.10*	18.0 ± 0.70
	10	64.4 ± 7.60	25.6 ± 6.60**	70.8 ± 19.70	19.0 ± 0.60	25.4 ± 1.10**	18.9 ± 0.80
b-wave	0	152.3 ± 54.80	163.4 ± 56.00	183.8 ± 183.80	40.7 ± 0.70	39.6 ± 1.10	39.5 ± 0.90
	1	135.1 ± 42.70	128.3 ± 74.40	140.0 ± 140.00	38.1 ± 3.10	40.6 ± 2.70	37.7 ± 2.30
	3	205.1 ± 6.20	200.8 ± 73.20	223.8 ± 16.00	40.0 ± 2.40	48.8 ± 4.90	39.0 ± 1.70
	10	145.3 ± 13.50	159.2 ± 34.90	147.1 ± 32.50	39.4 ± 0.70	64.1 ± 12.90	38.8 ± 0.60
Combined rod-cone response (bright flash)
a-wave	0	126.8 ± 47.90	140.7 ± 43.60	154.3 ± 50.50	13.7 ± 0.60	13.2 ± 0.70	12.9 ± 0.30
	1	125.4 ± 13.30	132.9 ± 18.70	137.8 ± 24.00	13.0 ± 0.30	14.1 ± 0.50	13.3 ± 0.50
	3	132.2 ± 5.10	134.8 ± 26.30	148.5 ± 9.10	12.6 ± 0.30*	14.5 ± 0.70	12.6 ± 0.20
	10	113.1 ± 12.10	70.9 ± 9.90**	117.0 ± 29.20	13.2 ± 0.50	18.7 ± 3.30**	13.0 ± 0.70
b-wave	0	179.3 ± 63.90	192.1 ± 63.50	212.5 ± 61.80	43.3 ± 4.40	43.6 ± 3.50	43.2 ± 4.90
	1	155.2 ± 41.10	156.5 ± 72.40	156.1 ± 37.10	36.9 ± 10.00	41.2 ± 11.20	38.3 ± 8.50
	3	222.5 ± 8.60	263.3 ± 62.00	242.2 ± 15.50	43.0 ± 3.30	53.0 ± 5.80	43.5 ± 3.00
	10	156.3 ± 9.80	196.9 ± 30.40	161.2 ± 30.50	40.0 ± 2.10	59.3 ± 7.80*	40.6 ± 3.20
Oscillatory potentials
OP1	0	17.7 ± 7.40	20.8 ± 12.70	20.8 ± 9.60	16.4 ± 0.60	16.2 ± 0.70	15.7 ± 0.30
	1	18.6 ± 5.00	16.2 ± 3.30	15.6 ± 4.30	15.8 ± 0.60	16.9 ± 0.50	16.3 ± 0.50
	3	18.9 ± 2.30	17.1 ± 4.00	21.0 ± 2.70	15.6 ± 0.40	17.2 ± 0.70	15.6 ± 0.40
	10	16.4 ± 2.50	8.0 ± 2.50	18.6 ± 5.20	16.1 ± 0.70	19.9 ± 0.80**	16.1 ± 0.90
OP2	0	29.4 ± 7.20	31.7 ± 9.70	35.0 ± 5.80	21.5 ± 0.70	21.2 ± 0.70	20.6 ± 0.50
	1	26.0 ± 2.20	23.1 ± 7.50	27.6 ± 4.60	21.0 ± 0.90	22.4 ± 1.30	21.0 ± 0.90
	3	32.8 ± 4.80	36.3 ± 11.10	38.5 ± 4.10	20.7 ± 0.60	22.5 ± 0.60	20.6 ± 0.80
	10	27.2 ± 5.40	30.2 ± 2.50	29.6 ± 5.70	21.3 ± 0.60	25.9 ± 0.40**	21.2 ± 0.90
Single-flash cone response (W/W)
a-wave	0	17.0 ± 7.50	17.1 ± 5.10	19.3 ± 7.80	14.2 ± 0.80	14.0 ± 0.80	13.8 ± 0.70
	1	15.7 ± 1.90	14.3 ± 4.40	17.7 ± 2.10	13.6 ± 0.40	14.5 ± 0.50	13.8 ± 0.30
	3	17.1 ± 3.80	12.9 ± 1.40	17.5 ± 1.90	13.3 ± 0.30	14.7 ± 0.90	13.3 ± 0.20
	10	14.0 ± 3.70	8.0 ± 1.60**	14.9 ± 3.80	14.3 ± 0.30	17.4 ± 0.60**	14.1 ± 0.30
b-wave	0	64.7 ± 24.30	68.6 ± 20.00	70.7 ± 24.70	27.7 ± 0.60	27.2 ± 0.80	27.0 ± 0.40
	1	71.4 ± 8.00	56.5 ± 23.70	77.9 ± 15.70	26.8 ± 0.80	27.1 ± 0.70	26.6 ± 0.60
	3	71.3 ± 17.50	31.0 ± 8.70*	72.9 ± 10.90	26.2 ± 0.50*	26.5 ± 0.70	26.0 ± 0.80
	10	59.4 ± 7.30	9.8 ± 2.60**	63.0 ± 11.00	26.8 ± 0.70	27.5 ± 3.20	26.5 ± 1.00
30 Hz flicker
b-wave	0	62.6 ± 27.90	69.6 ± 26.80	70.2 ± 29.10	26.5 ± 0.10	26.2 ± 0.40	26.1 ± 0.40
	1	68.3 ± 12.60	54.0 ± 20.20	71.0 ± 11.50	26.5 ± 0.70	26.0 ± 0.50	26.4 ± 0.70
	3	69.9 ± 9.40	30.7 ± 6.90*	72.9 ± 3.30	26.2 ± 0.50	25.5 ± 0.60	26.0 ± 0.30
	10	52.9 ± 9.00	4.9 ± 0.60**	58.3 ± 10.50	26.3 ± 0.20	26.8 ± 1.00	26.2 ± 0.30

 Data are expressed as the mean ± SD of four animals. NC, not calculated because no b-wave was identified in any animals in the group.

*  P < 0.05, ** P < 0.01; significantly different in comparison with the vehicle-treated group by the Dunnett's test.

A-Wave Analysis.

Typical waveforms of the rod a-wave and the cone a-wave in vehicle- and sildenafil-treated monkeys are shown in Figures 2 and 3, respectively. Model parameters of the a-wave analysis are summarized in Figure 4 and Table 2. Relationships between the model parameters and plasma sildenafil concentration are shown in Figure 5. In the rod a-wave, sildenafil at 10 mg/kg reduced the S immediately after dosing. Also in the cone a-wave, 3 and 10 mg/kg of sildenafil reduced the S immediately after dosing. These log S values correlated highly with plasma sildenafil concentration; the Pearson's correlation coefficients in the rod and cone a-waves were −0.704 and −0.930, respectively. On the other hand, sildenafil induced no change in the R_max. At 24 hours after dosing, no difference was detected in any model parameter between vehicle- and sildenafil-treated groups. 

Figure 2

 

Typical waveforms of the rod a-wave in response to various stimulus intensities in vehicle- and sildenafil-treated monkeys. Sildenafil was administered at dose levels of 0 (vehicle), 1, 3, and 10 mg/kg, and the rod a-waves were derived by subtracting the cone responses from the combined rod-cone responses as described in the text. The dotted lines signify the curves fit from Equation 1 in the text. The responses obtained at baseline (gray traces) are superimposed on those obtained after dosing (black traces).

Figure 3

 

Typical waveforms of the cone a-wave in response to various stimulus intensities in vehicle- and sildenafil-treated monkeys. Sildenafil was administered at dose levels of 0 (vehicle), 1, 3, and 10 mg/kg, and the dark-adapted cone responses were elicited as described in the text. The dotted lines signify the curves fit from Equation 2 in the text. The responses at baseline (gray traces) are superimposed on those obtained after dosing (black traces). Each trace represents an average of six responses.

Figure 4

 

The effect of sildenafil on the a-wave in monkeys. Sildenafil was administered at dose levels of 0 (vehicle), 1, 3, and 10 mg/kg, and the model parameters (S, sensitivity; R_max, maximum response amplitude) were determined for the rod and cone a-waves as described in the text. Data are expressed as the mean ± SD of four animals. Ranges indicated by gray areas signify the 95% confidence intervals based on the values from age-matched healthy cynomolgus monkeys (N = 92). Significant decreases in the log S were detected for the rod a-wave (10 mg/kg, **P < 0.01) and for the cone a-wave (3 mg/kg, *P < 0.05; 10 mg/kg, **P < 0.01) by the Dunnett's test.

Figure 5

 

Relationships between the model parameters of the a-wave analysis and plasma sildenafil concentration in monkeys. Sildenafil was administered at dose levels of 0 (vehicle), 1, 3, and 10 mg/kg, and the model parameters (S, sensitivity; R_max, maximum response amplitude) were determined for the rod and cone a-waves as described in the text. The symbols are the data from individual animals obtained immediately after dosing. Log S highly correlated with plasma sildenafil concentration: 95% confidence intervals of Pearson's correlation coefficient for the rod and cone a-waves were −0.890 to −0.321 and −0.976 to −0.806, respectively.

Table 2

 

Effects of Sildenafil on the Model Parameters of the A-Wave Analysis

	Dose, mg/kg	Time After Dosing
		Baseline	Immediately	24 Hours
Rod a-wave
Log S, s−2(td-s)−1	0	1.15 ± 0.090	1.12 ± 0.110	1.20 ± 0.040
	1	1.17 ± 0.140	1.14 ± 0.150	1.12 ± 0.110
	3	1.22 ± 0.080	1.15 ± 0.070	1.21 ± 0.070
	10	1.12 ± 0.060	0.77 ± 0.170**	1.13 ± 0.120
Log Rmax, μV	0	2.07 ± 0.220	2.10 ± 0.190	2.11 ± 0.190
	1	2.06 ± 0.040	2.12 ± 0.070	2.11 ± 0.080
	3	2.11 ± 0.070	2.14 ± 0.060	2.13 ± 0.080
	10	2.04 ± 0.050	2.08 ± 0.030	2.06 ± 0.090
Cone a-wave
Log S, s−3(td-s)−1	0	3.81 ± 0.120	3.77 ± 0.110	3.77 ± 0.140
	1	3.79 ± 0.100	3.56 ± 0.090	3.70 ± 0.110
	3	3.80 ± 0.040	3.12 ± 0.170**	3.83 ± 0.060
	10	3.84 ± 0.080	2.56 ± 0.080**	3.92 ± 0.090
Log Rmax, μV	0	1.72 ± 0.180	1.75 ± 0.140	1.76 ± 0.140
	1	1.74 ± 0.050	1.80 ± 0.040	1.79 ± 0.050
	3	1.75 ± 0.050	1.84 ± 0.100	1.77 ± 0.030
	10	1.68 ± 0.100	1.71 ± 0.120	1.68 ± 0.130

 Data are expressed as the mean ± SD of four animals.

*  P < 0.05, ** P < 0.01; significantly different in comparison with the vehicle-treated group by the Dunnett's test.

Single-Flash Cone Response (R/B).

ERG parameters for this response are summarized in Table 3. Sildenafil induced a general attenuation immediately after dosing. The b-wave amplitude in the 10 mg/kg group was significantly lower in comparison with the vehicle-treated group. No statistically significant difference was detected in the PhNR amplitude or the PhNR/b-wave amplitude ratio between vehicle- and sildenafil-treated groups. At 24 hours after dosing, no difference was detected in any ERG parameter between vehicle- and sildenafil-treated groups. 

Table 3

 

Effects of Sildenafil on the PhNR

Single-Flash Cone Response (R/B)	Dose, mg/kg	Time After Dosing
		Baseline	Immediately	24 Hours
b-wave amplitude, μV	0	61.8 ± 20.36	63.2 ± 13.83	66.2 ± 19.53
	1	62.0 ± 11.31	59.4 ± 25.55	67.6 ± 11.04
	3	59.1 ± 17.99	39.7 ± 11.59	57.9 ± 14.16
	10	49.2 ± 3.83	23.8 ± 4.03*	49.9 ± 2.59
PhNR amplitude, μV	0	18.3 ± 8.50	18.4 ± 7.00	18.5 ± 7.80
	1	18.0 ± 9.00	17.0 ± 11.30	20.3 ± 8.00
	3	17.8 ± 5.00	12.9 ± 3.90	17.7 ± 7.00
	10	11.4 ± 3.10	7.8 ± 1.80	7.7 ± 3.40
PhNR/b-wave amplitude ratio	0	0.29 ± 0.056	0.29 ± 0.051	0.28 ± 0.068
	1	0.28 ± 0.120	0.26 ± 0.113	0.30 ± 0.107
	3	0.33 ± 0.153	0.36 ± 0.181	0.33 ± 0.186
	10	0.23 ± 0.047	0.33 ± 0.057	0.15 ± 0.066

 Data are expressed as the mean ± SD of four animals.

*  P < 0.05; significantly different in comparison with the vehicle-treated group by the Dunnett's test.

Ophthalmoscopy

No changes in the fundus oculi were observed in any animals treated with sildenafil. 

Toxicokinetics

Plasma sildenafil concentrations at the time of ERG recording are shown in Table 4, and plasma concentrations and toxicokinetic parameters of sildenafil after single dosing are shown in Table 5. The plasma concentration of sildenafil at the time of recording the ERGs immediately after dosing in the 1, 3, and 10 mg/kg groups were 378 ± 82.9, 1190 ± 181, and 3610 ± 639 ng/mL, respectively. The maximum concentration (C_max) and the area under the concentration-time curve of plasma sildenafil up to infinity (AUC_0-inf) after single dosing generally increased with the dose ranging from 1 to 10 mg/kg. 

Table 4

 

Plasma Sildenafil Concentrations at the Time of ERG Recording

Dose, mg/kg	Time After Dosing	Plasma Concentration, ng/mL
1	Immediately	378 ± 83
	24 hours	NC *
3	Immediately	1190 ± 181
	24 hours	NC *
10	Immediately	3610 ± 639
	24 hours	NC *

 Data are expressed as the mean ± SD of four animals. NC, not calculated.

*  Individual values were below the lower limit of quantitation (1 ng/mL).

Discussion

Comparison of Plasma Sildenafil Levels in Monkeys and in Humans

In several studies in human subjects, the mean C_max and AUC_0-inf of sildenafil after single oral administration of clinical doses (50 to 100 mg/body) ranged from 238 to 377 ng/mL and from 620 to 1295 ng·h/mL, respectively.^20–22 Therefore, the plasma sildenafil concentration at the time of ERG recording immediately after dosing in monkeys receiving 1 mg/kg in this study (378 ± 82.9 ng/mL) was basically comparable with the C_max of sildenafil in humans treated with a clinical dose of sildenafil. Also, the AUC_0-inf of plasma sildenafil in monkeys given 1 mg/kg (1000 ± 114 ng·h/mL) basically corresponded to that in the sildenafil-treated humans. Meanwhile, both the plasma sildenafil concentration at the time of ERG recording immediately after dosing and the AUC_0-inf of sildenafil in monkeys given 10 mg/kg substantially exceeded those reported in humans treated with clinical dosage of sildenafil. 

Effect of Clinical Plasma Level of Sildenafil on ERGs in Monkeys and in Humans

In the present study, no obvious alterations were found in the ERGs recorded from monkeys treated with sildenafil at 1 mg/kg, at which dose the plasma sildenafil concentration at the time of ERG recording was similar to the plasma C_max in humans treated with sildenafil at clinical dosages. In a randomized and double-blind clinical trial of 20 men who received a single oral dose of placebo or 100 mg (clinical dosage) of sildenafil, assessment of the standard full-field ERGs just showed statistically significant but slight prolongations in the implicit times in several ERG components.¹¹ Furthermore, another randomized and double-masked clinical trial, in which subjects were treated with placebo (n = 82) or 50 mg of sildenafil (n = 77) daily for 6 months, failed to observe any significant changes in the standard full-field ERGs.²³ Therefore, the plasma sildenafil level after administration of clinical doses was insufficient to produce clear-cut alteration in the standard full-field ERGs either in monkeys in this study or in humans. 

Retinal Function

Outer and Middle Retina.

For an in-depth investigation into the functional effects of sildenafil on the photoreceptors, we conducted an a-wave analysis based on the methods by Hood and Birch.^17,19 Sildenafil-induced selective and reversible decreases in the sensitivity parameter S were detected in both the rod and cone a-waves. Additionally, the S values were correlated highly with the plasma sildenafil concentrations. According to Hood and Birch,^17,19 the S provides a measure of the gain and/or efficiency of the phototransduction cascade, which is composed of several activation steps. Therefore, the selective reduction in S that highly correlated with plasma sildenafil concentration indicates that sildenafil affected the activation steps of phototransduction in monkeys in this study. Our interpretation is strongly supported by the observation that sildenafil inhibited PDE6,⁹ which is a central enzyme in the phototransduction cascade. 

The susceptibility of the rods and cones to sildenafil was evaluated by examination of the S in the rod and cone a-waves. In the middle dose group (i.e., sildenafil at 3 mg/kg), the mean S in the cone a-wave immediately after dosing was lower than that in the vehicle-treated group by 0.65 log units (78% reduction). On the other hand, the mean S in the rod a-wave was comparable to that in the vehicle-treated group. These results suggested that the cones were more susceptible to sildenafil than the rods in monkeys. Our data are consistent with the previous in vitro study,⁹ in which purified PDE6 from bovines was utilized, to demonstrate that PDE5 inhibitors including sildenafil showed several-fold preference for inhibiting cone PDE6 compared with the rod isozyme. 

The alterations in several ERGs recorded from sildenafil-treated monkeys in this study also suggest a direct effect of sildenafil on a retinal component other than the photoreceptors, as described below. Sildenafil at 3 mg/kg notably depressed the rod response, which originates from the rod pathway of the retina (i.e., mainly from the rods and the rod-driven bipolar cells). On the other hand, the a-wave analysis at the 3 mg/kg dose level revealed unchanged model parameters from the rod photoreceptors, indicating that the function of the rod photoreceptors was generally preserved at this plasma level of sildenafil. Therefore, it is speculated that sildenafil directly affected the function of the rod-driven bipolar cells in the monkeys. A similar phenomenon was also observed in the ERGs recorded from isolated superfused retinas of both bovines and humans; attenuation of the b-wave, which originates from the postreceptoral components of the retina, was observed at a lower sildenafil concentration than that which attenuated the a-wave, which mostly originates from the photoreceptors.²⁴ Furthermore, the PDE5 enzyme, the pharmacological target of sildenafil, has been shown to be expressed not only on the vasculature but also on the bipolar cells in human retinal tissues.²⁵ These findings support our speculation on the direct effect of sildenafil on the bipolar cell function. 

To produce clear-cut alteration in the ERGs recorded from sildenafil-treated monkeys, we administered sildenafil at doses that highly exceed clinical doses to monkeys. As a result, the following drastic alterations in the standard full-field ERGs were observed in monkeys given 10 mg/kg, at which dose the plasma sildenafil concentration at the time of ERG recording was greatly higher than the plasma C_max in humans treated with sildenafil at clinical dosages: markedly delayed response accompanied by reduction in the dark-adapted ERGs, and markedly reduced response in the light-adapted ERGs. Interestingly, each waveform of the standard full-field ERGs recorded immediately after sildenafil dosing generally appeared as if the ERG had been elicited by weaker light flashes than were actually used. As shown in our previous report,¹⁶ in which intensity-response functions of the ERGs were examined in cynomolgus monkeys, attenuating the intensity of light flashes produces increased implicit time and decreased amplitude in the dark-adapted ERGs, and decreased amplitude in the light-adapted ERGs. According to Hood and Birch,²⁶ selective decrease in the sensitivity parameter S in the a-wave analysis is assumed to be a change that acts as if the flash energy was decreased. Taking these findings and the assumption together, we consider that alterations noted in the standard full-field ERGs in this study are generally attributable to an effect on phototransduction, as indicated by the selectively reduced S in the a-wave analysis. 

Inner Retina.

Although sildenafil-induced visual symptoms are generally mild and reversible, vision-threatening ocular complications such as nonarteritic ischemic optic neuropathy (NAION) have been reported to occur rarely in sildenafil-treated patients.^27–29 However, there is a lack of conclusive evidence to indicate a direct cause-effect relationship between sildenafil use and onset of NAION.³⁰ To address this question in sildenafil-treated monkeys, we assessed the PhNR, a waveform component of the ERG that has been shown to originate mainly from the retinal ganglion cells (RGCs) in monkeys.³¹ As a result, no marked change was detected in the PhNR/b-wave amplitude ratio, an indicator of selective PhNR loss,³² at a substantially higher plasma sildenafil level than that reported in humans. Meanwhile in this study, sildenafil induced a marked delay in dark-adapted oscillatory potentials, which reflect function of the inner retina.³³ However, it is likely that the delayed oscillatory potentials were not suggestive of abnormality in the inner retina but were secondary to the impaired phototransduction in the first-order neurons, indicated by selectively reduced model parameter S in both the rod and cone a-waves. Therefore, our data indicate that the function of the inner retina including the RGCs was generally preserved in sildenafil-treated monkeys. 

Conclusions

Sildenafil produced reversible impairment of the rod and cone phototransduction in monkeys, as indicated by the selectively reduced S in the rod and cone a-waves. Meanwhile, involvement of the postreceptoral retinal components was suggested by the altered rod response and unchanged rod a-wave parameters in the middle dose group. These findings contribute to the clarification of sildenafil-induced visual disturbances. It is suggested that the photoreceptors are predominantly, but not exclusively, affected in the retina of humans with sildenafil-induced visual disturbances. 

Acknowledgments

The authors thank Hidetaka Kudo and Eiichiro Nagasaka of Mayo Corporation for technical assistance. 

Disclosure: J. Kinoshita, None; N. Iwata, None; H. Shimoda, None; T. Kimotsuki, None; M. Yasuda, None