Treatment with a New Nitric Oxide Donor, a Hybrid Derived from Thalidomide and Hydroxycarbamide 3-(1,3-dioxoisoindolin-2-yl)Benzyl Nitrate, Reverses Priapism in the Sickle Cell Mouse and the Nitric Oxide-Deficient Mouse

Introduction: Sickle cell disease (SCD) men display priapism. Previous studies showed that endothelial nitric oxide synthase (eNOS) gene-deficient mice and combined eNOS and neuronal nitric oxide synthase double gene-deficient mice (dNOS^-/-) display a priapism phenotype associated with phosphodiesterase-5 (PDE5) downregulation. The Berkeley SCD transgenic mice display features of priapism, and, as in SCD human, this appears to be due to decreased nitric oxide (NO) signaling, which leads to lower PDE5 in the penis. Thus, when an erectile stimulus occurs in vivo, cGMP accumulates into the cavernosal smooth muscle cells, rendering the penile vasculature uncontrollably dilated, penile erection persists (i.e. priapism) as cGMP is not degraded as a consequence of PDE5 downregulation. Moreover, oxidative/nitrosative stress have been associated with priapism in SCD mice. In this study, since decreased NO bioavailability leads to lower PDE5 in penis, we aimed to evaluate the effects of new NO donor, a hybrid derived from thalidomide and hydroxycarbamide, 3-(1,3-dioxoisoindolin-2-yl)benzyl nitrate (4C), on functional and molecular alterations of erectile function in SCD and dNOS^-/- mice. We have focused on the dysregulated NO-cGMP-PDE5 pathway and oxidative/nitrosative stress in erectile tissue of SCD and dNOS^-/-mice.

Methods: Wild type (WT, C57BL/6), Berkeley SCD transgenic and dNOS^-/-male mice (3-5 mo old) were treated with compound 4C (100 µmol/kg/day) or its vehicle (20% Cremophor) daily for 3 weeks via intraperitoneal injection. The intracavernous pressure (ICP) was assessed following electrical stimulation of cavernous nerve (CN) in anaesthetized mice. ICP were normalized per mean arterial pressure (MAP). In separate protocols, corpus cavernosum (CC) strips were mounted in isolated organ baths, and the relaxing responses to acetylcholine (ACh; endothelium-dependent response) and sodium nitroprusside (SNP; endothelium-independent response), as well as electrical-field stimulation (EFS; nitrergic relaxation) were obtained in CC strips precontracted with the α1-adrenergic receptor agonist phenylephrine (10 µM).

Results: The CN stimulation (4 V) caused increases of ICP/MAP ratio in WT, SCD and dNOS^-/- mice. However, half detumescence time (sec) and poststimulated ICP/MAP ratio values after 9 min were (P<0.05) higher in SCD compared to the WT group, indicating that SCD mice display priapism. In dNOS^-/- group, maximal ICP/MAP ratio values were higher (P<0.05) compared to WT mice, indicating that dNOS^-/- mice also display priapism. Compound 4C treatment reversed the priapism in SCD and dNOS^-/- mice. The cumulative addition of ACh (0.001-10 µM) produced concentration-dependent CC relaxations in WT and SCD groups, but the ACh potency (pEC₅₀) value was higher (P<0.05) in CC of SCD compared to WT mice, which was reversed by 4C. Likewise, the nitrergic relaxation induced by EFS was also higher (P<0.05) in SCD mice compared to control mice (2 Hz: 12 ± 3 and 37 ± 9 %, respectively; n=5), which was reversed by 4C treatment. Similarly, SNP (0.01- 10 µM) produced concentration-dependent CC relaxations in WT, SCD and dNOS^-/- groups, but, again, the maximal relaxations elicited by this agent were higher (P<0.05) in SCD (93 ± 7%) and dNOS^-/- (105 ± 1%) groups compared to WT mice (72 ± 4%), which were fully restored to WT values with compound 4C treatment (n=5-9). PDE5 protein expressions were reduced (P<0.05) by approximately 48% and 35% in penile tissues from SCD and dNOS^-/- compared to the WT group, respectively. Compound 4C treatment restored (P<0.05) the protein levels of PDE5 in the penises from SCD and dNOS^-/- group. The protein expressions for gp91^phox and 3-NT were significantly higher (P<0.05) in erectile tissues from SCD and dNOS^-/- compared to the WT group, which were reduced (P<0.05) by 4C treatment (n=5-9). In WT mice, PDE5, gp91^phox and 3-NT protein expressions were not affected by 4C, as well as CC relaxations induced by ACh, SNP and EFS.

Conclusion: Compound 4C treatment reversed the priapism, as well as enhanced NO-mediated CC relaxations and normalized PDE5 expressions in the penises from SCD and dNOS^-/- mice. Moreover, increased gp91^phox and 3-nitrotyrosine protein expressions were also decreased by 4C in SCD and dNOS^-/-mice. Compound 4C may constitute an additional strategy to prevent priapism in SCD, since SCD-related priapism therapies are few.

Financial Support: FAPESP