The potential effect of ambrisentan as monotherapy and combined with tadalafil on diabetic erectile dysfunction in rats

Abstract

Introduction:

This study investigated the role of ambrisentan; the selective endothelin type-A receptor (ETAR) blocker on experimental diabetic erectile dysfunction in rats.

Materials and methods:

Eighty-four adult male Sprague Albino rats were divided randomly into 7 groups. Three control groups received 1 mL saline, 0.2 mg/kg/d ambrisentan and 1.5 mg/kg/d tadalafil, respectively orally for 4 weeks. The remaining four groups were fed high fat diet for 14 days. Diabetes was induced by a single intra-peritoneal injection of 40 mg/kg streptozotocin. After 72 h, diabetes was confirmed by plasma glucose level ⩾250 mg/dL. Diabetic rats were divided randomly into four groups, numbered from 4 to 7. The fourth group was the diabetic-control group, while the fifth and sixth groups received ambrisentan and tadalafil respectively. The seventh group received a combination of both drugs. Treatment continued for 4 weeks then, copulatory, intracavernous pressure measurement, and laboratory tests were conducted.

Results:

In diabetic rats, ambrisentan and tadalafil improved fasting glucose, insulin, insulin resistance, testosterone, nitric oxide, and rho kinase (ROCK) values compared to diabetic group with the maximum improvement achieved in ambrisentan/tadalafil group (p < 0.05). Ambrisentan also enhanced ICP and improved latency to erection and number of mounts with a tolerable SBP. Yet, ambrisentan/tadalafil combined therapy resulted in deterioration in SBP with consecutive worsening in ICP and mating indices.

Conclusion:

Ambrisentan showed significant therapeutic potential to prevent and improve diabetic ED in rats comparable to tadalafil.

Keywords

Ambrisentan diabetic endothelin erectile dysfunction tadalafil

Introduction

Although erectile dysfunction (ED) is not a life-threatening disease, yet it affects social wealth. Diabetes mellitus (DM) was found to result in ED in about 35%–62.9% of adult males. The pathophysiology of diabetic ED is complex, including abnormal neuro-endocrinal signaling and endothelial vascular dysfunction.^1,2 Although phosphodiesterase type 5 inhibitors (PDE5Is) are among the first line therapies of ED, failure of PDE5I was reported in about 51.1% of treated patients.²

Insulin resistance (IR) is one of the features of type 2, non-insulin dependent DM (T2DM). High insulin levels observed in T2DM patients with IR, were found to increase the circulating Endothelin (ET-1) plasma levels through stimulating the transcription of ET-1gene (edn1).^3,4 ET-1 is known as the most potent vasoactive peptide in our bodies and abnormal ET-1 levels were encountered in many vascular disorders such as: pulmonary hypertension, heart failure, systemic hypertension, and some malignancies especially the ovarian cancer.

ET-1 acts upon two types of receptors: ET-type A receptor (ETAR) and ET-type B receptor (ETBR). ETBR are furtherly divided into two subtypes: endothelial ETB1 and ETB2 located on the vascular smooth muscle cells (VSMCs). ET-1 binding to ETAR and ETB2 receptors causes vasoconstriction (VC).⁵ On the other hand, endothelial ETB1 activation results in a nitric oxide (NO)/cGMP dependent vasodilatation (VD). ETBR also contributes in the clearance of ET-1.^6,7

In view of diabetic ED, higher ET-1 levels were observed in patients with ED than in healthy controls.⁷ The upregulation and the long-lasting pressor action of ET-1 on the ETARs in the corpus cavernous (CC) tissue is hypothesized to maintain a flaccid penile state and mediate mitogenic dysfunction that predispose to diabetic ED. This action is at least partially mediated by an rho kinase (ROCK)-dependent Ca²⁺ sensitization mechanism.^8,9 Contrarily, ETBR was found to possess a counter-protective effect and facilitates ET-1 degradation.¹⁰ A previous study by Alkan et al.⁷ demonstrated a beneficial effect of using bosentan (a non-selective endothelin receptor blocker) in treatment of diabetic ED. Alkan and his colleagues reported an abnormal high ET-1 level in DM rats that was decreased after bosentan treatment. They also mentioned that the CC is not only able to respond to ET-1, but also can synthesize it.

In the view of the above-mentioned information, this study aimed to demonstrate the effect of selectively blocking ETAR receptors using ambrisentan on diabetic ED. We hypothesized that ambrisentan can ameliorate the diabetic-dependent ET-1 hazards on the CC through blocking the ETARs. Moreover, by preserving the ETBRs actions, ambrisentan would improve the endothelial NO synthesis and restore the normal erection physiology. Ambrisentan is already approved as a treatment of pulmonary arterial hypertension due to its potent role as a vasodilator and an inhibitor of pulmonary vascular smooth muscle proliferation.^11,12 Accordingly, the role of ambrisentan in diabetic ED was evaluated, either as a monotherapy or in combination with the tadalafil, and compared to tadalafil being a standard therapy for ED. We mainly relied on the copulatory tests to elucidate the real impact of ambrisentan on ED. Yet, for better understanding of the mechanism of action of ambrisentan on diabetic ED, we measured the change in the intracavernous pressure after ambrisentan therapy. We also evaluated the impact of ambrisentan on glucose homeostasis, insulin resistance, pancreatic β cells function, ROCK activity, NO level, and plasma testosterone level.

Material and methods

Drugs and chemicals

Ambrisentan powder (AUG Pharma, Egypt).

Tadalafil powder (EVA Pharma, Egypt).

Streptozotocin powder (Sigma Aldrich, Egypt).

Estradiol benzoate ampoule (MARCH trademark).

Progesterone ampoule (IBSA)

Formalin: Formaldehyde sol. 38%–40% (AlNasr Pharmacy. Chemicals, Egypt).

Kits and instruments

Nitrite/nitrate colorimetric Assay Kits (Sigma-Aldrich, Egypt).

Rho-associated Kinase (ROCK) Activity Assay Kit. (Sigma-Aldrich, Egypt)

Handheld glucometer to measure blood glucose (Gluco Dr auto)

Rat/mouse insulin ELISA kit (Sigma-Aldrich, Egypt).

Testosterone test kit (Sigma-Aldrich, Egypt).

Invasive arterial blood pressure measurement (Powerlab AD Instruments ML866, 430-0820-Australia).

Rat tail sphygmomanometer (Panlab, USA).

Animals used

The study was conducted in accordance with the Institutional Animal Care and Use Committee (IACUC) Faculty of Medicine, Cairo University. Approval number: (CU, III, F, 83, 19)/April 2020, and in accordance with the Guide for the Care and Use of Laboratory Animals as adopted and promulgated by the U.S. National Institutes of Health. Eighty-four adult Sprague Dawley male rats weighting between 300 and 350 g were included in this study. They were housed in standard laboratory conditions under a 12-h light/dark cycle and controlled temperature of 22°C ± 3°C, with free access to standard food and water. Before conducting experiments, animals were acclimatized to laboratory conditions for 7 days.

Induction of diabetes

Diabetes was induced in 48 rats after 14 days of feeding high fat diet (66.5%-commercial feed, 13.5%-lard, and 20%-sugar) to induce insulin resistance,¹³ followed by intra-peritoneal injection of streptozotocin (STZ) at a single dose of 40 mg/kg body weight. After 72 h of STZ injection, fasting blood samples were collected via the tail veins to measure blood glucose levels by a handheld blood glucose monitor. Rats with plasma glucose level of 250 mg or higher were considered to be diabetic (as shown in Supplemental File Table 1).¹⁴ Diabetic rats were then randomized on the four diabetic groups and treatment was started immediately.⁸

Experimental design

As shown in Figure 1, the control rats (n = 36) were subjected to intra-peritoneal injection of 0.5 mL saline and divided randomly into 3groups (n = 12) as follows:

Group 1 (C): Control group, received 1 mL saline by oral gavage.

Group 2 (C-Am): Control-ambrisentan group, treated with ambrisentan suspended in distilled water, prepared freshly for each rat and administrated by oral gavage at a dose of 0.2 mg/kg/d.¹⁵

Group 3 (C-Td): Control-tadalafil group, treated with tadalafil Suspended in distilled water, prepared freshly for each rat and administrated by oral gavage at a dose of 1.5 mg/kg/d.^16,17

Figure 1.

The experimental design of the current study.

As for the diabetic rats, they were randomly divided into four groups (n = 12) as follows:

Group 4 (D): Diabetic group, received STZ as previously described.¹³

Group 5 (D-Am): Diabetic-ambrisentan group, treated with ambrisentan similar to group 2.¹⁵

Group 6 (D-Td): Diabetic-tadalafil group, treated with tadalafil similar to group 3.^16,17

Group 7 (D-Am-Td): Diabetic-ambrisentan-tadalafil group, treated with both ambrisentan and tadalafil for 4 weeks with the same above-mentioned doses.

Treatment lasted for 4 weeks in all groups.⁸

Measurements

Pharmacological studies

Systolic blood pressure measurement

The systolic blood pressure (SBP) of each rat was measured non-invasively using a rat tail sphygmomanometer and was calculated as the mean of three readings on three different days after being trained for 7 days for the measuring procedure before the actual measurement was recorded.¹⁸

Extended Ex. copula erection test

The ex. copula test was performed by restraining the male rat on his back. The penile sheath is then retracted gently to touch the glans smoothly with a cotton pad. Latency for the first penile erection (the time elapsed from touching the glans to erection) and number of penile erections occurring during a 30 min-observation period were recoded.¹⁹

Mating test

According to Mercier et al.²⁰ protocol, this test was carried on between 17:00 and 19:00 pm in a sound attenuated room. Each Male rat was placed individually in an observation glass chamber (30 × 30 × 50 cm) for 5 min for adaptation. The females were subcutaneously injected with 30 μg estradiol benzoate and 500 μg progesterone 24–48 h and 4 h before the test respectively, to induce estrous cycle.²¹ The following were recorded in a period of 30 min: (1) latency to first mount (defined as when a male rat mounts the female from the rear and grasps her flanks with his front feet as shown in the Supplemental Photo S1), (2) number of mounts, (3) number of intromissions, (4) number of ejaculations, and (5) duration of copulation activity. The test was stopped if no intromissions occurred within 15 min. Ejaculations were confirmed by the presence of a semen plug in the female’s vagina.²²

Intra-cavernous penile pressure (ICP) measurement

Under isoflurane 2.5%, male rats were anesthetized and the skin overlying the penis was incised, and the prepuce was degloved to expose the corpora cavernosa. A 26 -gauge needle filled with heparinized saline was carefully inserted into the corpus cavernosum (CC) on one side to measure the ICP. The needle was sealed to a polyethylene 50 tubing of 20 cm length filled with heparinized saline²³ as shown in Supplemental Photo S2, connected to a pressure transducer (Powerlab AD Instruments ML866, 430-0820-Australia).

Biochemical study

At the end of the ICP measurement study and with ensuring 6 h fasting, a blood sample was collected from the lateral vein of the rat tail to test for measuring:

Fasting plasma glucose (FPG) using handheld blood glucose monitor in mmol/L.

Fasting plasma insulin (FPI) using a rat/mouse ELISA kit in mU/L according to the manufacturer instructions.²⁴

Insulin resistance (IR) and pancreatic β (B) cell function were evaluated using the Homeostatic model assessment (HOMA) for insulin resistance (HOMA-IR) and for β cell function (HOMA-%B) as described by Wallace et al.²⁵ using the following equations:

HOMA − IR = (FPI × FPG) / 22.5

HOMA − %B = (20 × FPI)/ (FPG − 3.5)

FPI: is the fasting plasma insulin concentration in mU/L and FPG: is the fasting plasma glucose in mmol/l.

Testosterone level using a rat/mouse ELISA kit in ng/dL according to the manufacturer instructions²⁶

By an esthetic overdose, rats were killed at the end of the experiment. Cavernous tissues were dissected gently to the level of crus penis. The penis was resected from the proximal of the cruses and then the cavernous tissue was frozen at −80°C to be prepared as a tissue homogenate to test the following:

Nitric oxide (NO) in µmol/L using colorimetric laboratory method.²⁷

Rho-Kinase (ROCK) in mU activity according to the manufacturer instructions.²⁸

Statistical analysis

Statistical Package for the Social Sciences (SPSS) version 24 was used for data coding and entering. Data was summarized using mean and standard error of mean (SEM) in quantitative data and using frequency (count) and relative frequency (percentage) for categorical data. Comparisons between the different groups were done using ANOVA with post hoc Tukey test in normally distributed quantitative variables. For non-parametric tests, independent Kruskal Wallis test was used with adjusted Mann Whitney test as the post hoc test. p-Value less than 0.05 was considered as of statistical significance.²⁹

Results

In vivo results

Systolic blood pressure (SBP)

Diabetic group showed non-significant elevation in SBP compared with the control group. Diabetic-ambrisentan didn’t show significant change in SBP in comparison with either the Control or the Diabetic groups. While Diabetic-tadalafil group showed significant reduction in SBP compared with the Diabetic-group, and the Diabetic-ambrisentan-tadalafil group caused significant reduction in SBP compared with the other treated groups (Figure 2(a)).

Figure 2.

(a) Systolic blood pressure (SBP) and (b) intracavernous pressure (ICP) values in the different studied groups.

Ex. copula test

As shown in Table 1, Control-Ambrisentan-group and Control-Tadalafil-group showed non-significant improvement in number of penile reflexes and latency to first erection compared with the Control-group. The Diabetic-group showed significant worsening in both indices compared with the Control-group. Both Diabetic-Ambrisentan and Diabetic-Tadalafil showed significant improvement in latency to first erection. On the other hand, Diabetic-Ambrisentan-Tadalafil group showed significant worsening in both indices compared to the Control-group.

Table 1.

Comparison between the different study groups as regards: Ex. copula test and mating behavior parameters.

		Control	Control-ambrisentan	Control-tadalafil	Diabetic	Diabetic-ambrisentan	Diabetic-tadalafil	Diabetic-ambrisentan-tadalafil
Ex. copula test	Latency to erection	237 ± 66.14	171.17 ± 42.05	173.67 ± 56.11	540.5 ± 142.37*,^#,$	360 ± 59.7^#,$,@	371.33 ± 96.64^#,$,@	651.67 ± 134.82*,^#,$,&,%
Ex. copula test	Number of erections	15.83 ± 3.6	17 ± 3.74	17.5 ± 5.28	6.67 ± 1.75*,^#,$	10.27 ± 1.63	11.33 ± 1.97	6.67 ± 2.16*,^#,$
Mating behavior parameters	Latency to mount (sec)	160 ± 37.42	167.83 ± 65.67	187.33 ± 41.82	350 ± 176.64	251 ± 32.67	296.5 ± 157.92	241.67 ± 113.21
	No of mounts	8.17 ± 1.33	8.17 ± 2.23	6.5 ± 1.64	1.5 ± 1.22*,^#,$	4.33 ± 1.37*,^#,@	2.67 ± 1.51*,^#,$	1.32 ± 0.68*,^#,$,&
	No of Intromissions	6.5 ± 1.22	6.17 ± 1.47	5.33 ± 1.21	0.33 ± 0.52*,^#,$	2.33 ± 1.51*,^#,$	1.33 ± 0.82*,^#,$	0 ± 0*,^#,$,&
	No of ejaculations	5.33 ± 0.52	5.17 ± 1.17	3.67 ± 1.86	0 ± 0*,^#,$	1.33 ± 0.82*,^#,$	0.5 ± 0.55*,^#,$	0 ± 0*,^#,$
	Interval between mounts (seconds)	14.83 ± 3.19	14.17 ± 3.37	21 ± 13.36	127.6 ± 35.08*,^#,$	56.17 ± 24.11*,^#,$	117 ± 60.37*,^#,$	N/A
	The duration of copulatory Activity (min)	10.25 ± 2.12	13.30 ± 4. 05	8.26 ± 1.12^#	N/A	9.13 ± 1.42	7.31 ± 1.05*,^#	N/A

Values are presented as mean ± SD.

HOMA-B: homeostatic model assessment of insulin resistance; HOMA-IR: homeostatic model assessment of insulin resistance.

*,#,$,@,&,%

Statistically significant (p < 0.05) compared to corresponding value in group C, group C-Am, group C-Td, group D, group D-Am, group D-Td respectively.

Mating behavior

Diabetic-group showed significant worsening as regards number of mounts, number of intromissions with failure to accomplish ejaculation and hence the copulatory duration couldn’t be calculated. Diabetic-Ambrisentan rats showed increased sexual motivation evidenced by decreased latency to mount with significant increase in number of mounts, but with non-significant improvement in number of intromissions, number of ejaculations, interval between mounts and copulatory duration compared to Diabetic-group values. Diabetic-Tadalafil group showed overall non-significant improvement in mating parameters in comparison with the Diabetic-group values. Combined ambrisentan and tadalafil-treated diabetic rats showed non-significant improvement in all the mating parameters compared to Diabetic-group as well. Ambrisentan and tadalafil treatment in control groups didn’t add further enhancement for mating behavior compared to the Control-group.

Intracavernous pressure (ICP)

Diabetic-group showed significant reduction in ICP compared with the three control groups. Diabetic-Ambrisentan and Diabetic-Tadalafil groups showed significant elevation in ICP measurement in comparison with the Diabetic-group. However, in diabetic rats, ambrisentan-tadalafil combination didn’t show significant improvement compared to the Diabetic-group (Figure 2(b)).

Biochemical results

Blood samples results

Fasting plasma glucose (FPG)

Control-Ambrisentan and Control-Tadalafil groups didn’t cause alteration in the FPG compared to the Control-group. Diabetic-group showed significant increase in FPG level compared to the Control-group. Treatment of diabetic rats with either ambrisentan, tadalafil, or ambrisentan-tadalafil combination decreased FBG significantly compared to the Diabetic-group, yet showing impaired glucose tolerance levels (Table 2).

Table 2.

Comparison between the different study groups as regards glucose and insulin profiles.

Glucose and insulin profiles	Fasting plasma Glucose (mg/dL) (mmol/L)	Control	Control-ambrisentan	Control-tadalafil	Diabetic	Diabetic-ambrisentan	Diabetic-tadalafil	Diabetic-ambrisentan-tadalafil
		94.83 ± 10.59	93.7 ± 5.81	92.1 ± 5.08	215.77 ± 18.95*,^#,$	151.05 ± 8.16*,^#,$,@	149.07 ± 28.92*,^#,$,@	106.31 ± 26.11^@,&,%
		5.26 ± 0.59	5.2 ± 0.32	5.11 ± 0.28	11.98 ± 1.05*,^#,$	8.38 ± 0.45*,^#,$,@	8.27 ± 1.6*,^#,$,@	5.9 ± 1.45^@,&,%
	Fasting insulin (mIU/mL)	8.12 ± 1.11	6.63 ± 0.6	7.47 ± 0.74	17.78 ± 2.29*,^#,$	12.07 ± 1.5*,^#,$,@	11.48 ± 2.37*,^#,$,@	8.97 ± 0.89^@,&
	HOMA-B%	101.8 ± 40.17	80.51 ± 17.88	95.19 ± 20.75	42.34 ± 6.09*,^$	49.74 ± 7.68*	53.15 ± 20.02	98.24 ± 48.69^@
	HOMA IR	1.91 ± 0.41	1.53 ± 0.17	1.69 ± 0.18	9.51 ± 1.83*,^#,$	4.5 ± 0.61*,^#,$,@	4.2 ± 1.03*,^#,$,@	2.33 ± 0.51^@,&,%

Values are presented as mean ± SD.

HOMA-B: homeostatic model assessment of insulin resistance; HOMA-IR: homeostatic model assessment of insulin resistance.

*,#,$,@,&,%

Statistically significant (p < 0.05) compared to corresponding value in group C, group C-Am, group C-Td, group D, group D-Am, group D-Td respectively.

Fasting plasma insulin (FPI)

Control-Ambrisentan and Control-Tadalafil groups didn’t cause alteration in the FPI compared to the Control-group. Diabetic-group showed significant increase in FPI level compared to the Control-group. Treatment of diabetic rats with either ambrisentan or tadalafil caused significant improvement in FPI level compared to the Diabetic-group with marked improvement in FPI achieved by combining both medications that showed no significant difference than the Control-group (Table 2).

HOMA-IR

Diabetic-group showed significant elevation in the IR values compared to the Control-group. Both Diabetic-Ambrisentan and Diabetic-Tadalafil groups showed significant reduction in IR with marked improvement achieved by combining both medications that showed no significant difference than the Control-group (Table 2).

HOMA-B

Diabetic-group showed significant deterioration in pancreatic β cell function compared to the Control-group. However, Diabetic-Ambrisentan-Tadalafil group showed significant HOMA-B improvement (Table 2).

Testosterone

Diabetic-group showed significant reduction in plasma testosterone level compared to the Control-group. In contrast, treatment of diabetic rats with either ambrisentan, tadalafil, or ambrisentan-tadalafil combination showed significant improvement in plasma testosterone level compared to the Diabetic-group (Figure 3).

Figure 3.

Testosterone values in the different studied groups.

Biochemical results in tissue homogenate

Nitric oxide (NO) level

Ambrisentan and tadalafil caused a slight and a non-significant elevation in NO level compared to Control-group. Diabetic-group caused a significant reduction in NO level that was significantly elevated in Diabetic-Ambrisentan, Diabetic-Tadalafil, and Diabetic-Ambrisentan-Tadalafil groups (Figure 4(a)).

Figure 4.

(a) Nitric oxide and (b) Rho kinase values in the different studied groups.

Rho associated protein kinase (ROCK)

Diabetic-group showed significant elevation in ROCK level that was significantly improved in Diabetic-Ambrisentan, Diabetic-Tadalafil and Diabetic-Ambrisentan-Tadalafil groups (Figure 4(b)).

Discussion

Although the great success in the management of ED using PDE5Is, failure of PDE5Is therapy was reported to range between 30% and 35% in diabetic ED.² Chen et al.³⁰ found that 52% out of 238 men complaining of ED, suffers from IR which was the most prevalent independent factor associated with ED. IR upregulates ET-1, where the latter acts on ETAR receptors causing vasoconstriction and penile flaccidity. High ET-1 can also decrease testosterone production, which would affect the sexual desire and the normal physiology of erection. It can increase the production of pro-inflammatory cytokines that play an important role in diabetic insults as well.^7–9,30 It was noted that prolonged high ET-1 levels result in downregulation of endothelin receptors. Loss of ETBR actions will result in a non-opposed penile vasoconstriction. All together, abnormal ET-1 reported in DM, is speculated to contribute to diabetic-ED.

According to Alkan et al.,⁷ the non-selective ET-1blocker bosentan showed a promising therapeutic potential in the management of diabetic ED through antagonizing the ET-1 medicated VC and decreasing inflammatory cytokines production. However, one of the main contributors of diabetic-ED is the impaired endothelial NO, responsible for penile engorgement and erection. Accordingly, in this study we speculated an additive profit from preserving the vasodilatory actions of ETBR using the selective ETAR blockade, ambrisentan as ETBR is also responsible for almost 80% of ET-1 clearance in the lungs.⁶

In this study, ambrisentan was compared to and combined with tadalafil where the latter role in ED is well established through increasing NO/cGMP levels and augmenting ICP,^31,32 and that was documented also in this study. In addition, tadalafil can decrease ROCK activity possibly through enhancing protein kinase G (PKG) which phosphorylates the ROCK-guanosine triphosphatase unit³³ which explain the significant decrease in ROCK levels in the Diabetic-tadalafil group compared to the Diabetic-group. Concerning tadalafil effects on glucose homeostasis, our results are in concordance with Ho et al.³⁴ who concluded that tadalafil has favorable effects on pancreatic β cells function, possibly through the up-streaming of NO which regulates insulin production and glucose metabolism through cGMP-dependent insulin secretion. Additionally, the vasodilatory properties of NO may increase the delivery of glucose to skeletal muscles and facilitate their metabolism.³⁵ Tadalafil can also improve testosterone production through a cGMP signaling pathway that regulates PKG1 in Leydig cells.^36,37

As regards ambrisentan and concerning the actions of ambrisentan on glucose homeostasis, the improved FPG and HOMA-IR results are in concordance of Lteif et al.³⁸ who found that BQ123; a selective ETAR blockade, increased glucose uptake in hyper-insulinemic euglycemic obese patients due to the enhanced distribution of blood and glucose to skeletal muscles by an insulin-dependent VD. Although Ambrisentan didn’t improve HOMA-B, Eibl et al.³⁹ reported beneficial role of selective ETAR blockade in acute severe pancreatitis by decreasing the capillary permeability possibly by decreasing post-capillary resistance. However, more clinical research is needed to study the role of ambrisentan on pancreatic β cells function.

A realistic evaluation of a medication tested as a potential treatment of ED requires performing a copulatory test to mimic the real physiological situation where there is a harmony between the neuro-endocrinal-vascular and musculoskeletal systems. In this perspective and as regards the ex. Copula test, there was a significant improvement in latency to erection in the Diabetic-Ambrisentan and Diabetic-Tadalafil groups compared to the Diabetic-group. Meanwhile, in the mating test, the Diabetic-Ambrisentan group significantly improved the number of mounts. For a better interpretation of these results, Ferraz et al.⁴⁰ observed that latency to erection/mount is an indicator of sexual motivation being inversely proportional to it. On the other hand, numbers of mounts, intromissions and ejaculations reflect copulatory efficiency. This may point out to the enhanced sexual motivation achieved using ambrisentan due to the significant improvement in NO, ROCK, and testosterone profiles. The significant improvement in number of mounts with ambrisentan can be also explained in the view of Radiloff et al.⁴¹ observation who stated that ambrisentan combination to either ephedrine or methylphenidate could effectively improve exercise duration and decrease high-altitude fatigue by increasing peripheral blood flow and oxygen delivery to hypoxic muscles. However, ambrisentan-tadalafil combination didn’t improve the copulatory efficiency. This may be referred to the marked decrease in SBP and hence the ICP that occurred with this concomitant therapy. As for the ICP measurement, induction of erection using electrical stimulation was intended, but was not performed for some technical troubles. Yet, the baseline ICP provided a good tool for the evaluation of the corpus cavernosa perfusion even in absence of erection. Diabetic-Ambrisentan and Diabetic-Tadalafil groups showed significant improvement compared to the Diabetic-group denoting better penile perfusion. Although this improvement was not achieved with their combination, this finding can be explained in view of Verhaar et al. study⁴² who observed that selective ETAR blocking results in vasodilatation more than that observed with dual ETAR/ETBR blockade, owing to the preserved vasodilatory potential of ETBR. This marked VD decreased the SBP and sequentially the ICP and the copulatory efficiency. Also, bosentan is an CYP3A4 enzyme inducer that can decrease tadalafil exposure by almost 41.5%. Contrarily, ambrisentan isn’t an enzyme inducer, so combining ambrisentan with tadalafil may induce marked hypotension^43,44 affecting the physical ability to perform the mating activity, especially in susceptible patients suffering from volume depletion or other comorbidities which could be the case in our study considering that the diabetic rats didn’t receive any anti-diabetic medication.

Conclusion

A possible potential of ambrisentan on copulation through: improving sexual desire, muscular capacity and increasing ICP can be suggested. This favorable potential can be a direct effect (through improving the penile perfusion and counteracting the ET-1-mediated VC and vascular dysfunction by restoring ROCK/NO balance), or an indirect effect (resulting from its beneficial action on glucose homeostasis, insulin resistance, and testosterone levels). However, long-term clinical trials are recommended to validate these pre-clinical findings on the real scale of diabetic-ED patients

Yet, it would be of interest to test lower doses of ambrisentan (whether alone or with tadalafil) and after ensuring proper glycemic control to elicit the maximum potential of ambrisentan in the management of diabetic ED. Also, it would be of interest to perform a comparative study between bosentan and ambrisentan to elucidate the different mechanisms by which both drugs may improve ED, with special attention to their possible combination with other medications approved for ED management and their interaction with PDE5Is.

Supplemental Material

sj-docx-1-urj-10.1177_03915603231192737 – Supplemental material for The potential effect of ambrisentan as monotherapy and combined with tadalafil on diabetic erectile dysfunction in rats

Supplemental material, sj-docx-1-urj-10.1177_03915603231192737 for The potential effect of ambrisentan as monotherapy and combined with tadalafil on diabetic erectile dysfunction in rats by Omnia Azmy Nabeh, Fatma Ahmed El-Batrawy, Omayma Anwar Khorshid and Ghada Farouk Soliman in Urologia Journal

Footnotes

Acknowledgements

The authors would like to thank professor Dr Laila Rashed, professor of biochemistry Kasr alainy faculty of medicine for the biochemical results in this study.

Authors’ contributions

All authors contributed to the study conception and design. O.A.N was responsible for data collection and analysis. All authors contributed in the scientific writing, read and approved the final manuscript.

Declaration of conflicting interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This research was funded by Kasr Alainy Faculty of medicine, Cairo university, Egypt.

Ethics approval statement

Institutional Animal Care and Use Committee (IACUC) Faculty of Medicine, Cairo University. Approval number: (CU, III, F, 83, 19)/April 2020.

Patient consent statement

Not applicable.

Permission to reproduce material from other sources

Not applicable.

Clinical trial registration

Not applicable.

Data availability statement

All data generated or analysed during this study are included in this published article and the supplementary file.

ORCID iD

Omnia Azmy Nabeh

Supplemental material

Supplemental material for this article is available online.

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