Introduction With the aim of overcoming the high toxicity of PnTx2-6 (or δ-CNTX-Pn2a), a toxin from the venom of the armed spider (Phoneutria nigriventer), the 19-aminoacid peptide, PnPP-19 (P nigriventer potentiator peptide), was synthesized based on molecular modeling studies of PnTx2-6. PnPP-19 improved the erectile function of normotensive rats and mice, without eliciting side effects, and no signs of toxicity were observed. In addition, PnPP-19 was able to potentiate the effect of sildenafil. Aim To evaluate the efficacy of PnPP-19 in hypertensive and diabetic mouse/rat models in restoring erectile function, after topical administration; verify the biodistribution of PnPP-19 administration (topical and intravenous), permeation, and cyclic guanosine monophosphate (cGMP)/nitric oxide via implication. Methods Corpus cavernosum relaxation was evaluated using cavernous strips from male spontaneous hypertensive rats (SHR) and from streptozotocin (STZ)-diabetic mice contracted with phenylephrine and submitted to electrical field stimulation before and after incubation with PnPP-19 (10−8 mol/L, 10 minutes) or vehicle. This procedure was also used to determine cGMP/nitric oxide levels, at 8 Hz and to check the effect of PnPP-19 with sildenafil citrate. Biodistribution assays were performed using iodine 123–radiolabeled PnPP-19. In vivo erectile function was evaluated using intracavernosal pressure/main arterial pressure ratio in STZ-diabetic rats after PnPP-19 topical administration. Main Outcome Measures PnPP-19 may become a new drug able to fill the gap in the pharmacologic treatment of erectile dysfunction, especially for hypertensive and diabetic individuals Results PnPP-19 potentiated corpus cavernosum relaxation, in both control and SHR rats. SHR-cavernosal tissue treated with PnPP-19 (1–32 Hz) reached the same relaxation levels as control Wistar rats (16 and 32 Hz). PnPP-19 treatment improved cavernosal tissue relaxation in STZ-diabetic mice and rats. PnPP-19 enhanced cGMP levels in STZ-diabetic mice corpus cavernosum strips. After topical or intravenous administration in rats, 123I-PnPP-19 was mainly recruited to the penis. When topically administered (400 μg/rat), PnPP-19 restores erectile function in STZ-diabetic rats, also improving it in healthy rats by increasing the intracavernosal pressure/main arterial pressure ratio. PnPP-19 exhibited an additive effect when co-administered with sildenafil, showing a novel mode of action regardless of phosphodiesterase type 5 inhibition. Clinical Implications PnPP-19 seems to be an indicated drug to be tested to treat ED in diabetic and hypertensive patients. Strength & Limitations PnPP-19, although active by topical application and showing safety to human beings (not shown), has low permeability, about 10% of the applied dose. Conclusion Our results showed that PnPP-19 may emerge as a potent new drug that can be topically administered, becoming a promising alternative for erectile dysfunction treatment.
Recognition of bacterial lipopolysaccharide (LPS) by innate immune system is mediated by the cluster of differentiation 14/ Toll-like receptor 4/myeloid differentiation protein 2 (MD-2) complex. In this study, we investigated the modulatory effect of gedunin, a limonoid from species of the Meliaceae family described as a heat shock protein Hsp90 inhibitor, on LPS-induced response in immortalized murine macrophages. The pretreatment of wild-type (WT) macrophages with gedunin (0.01-100 mM, noncytotoxic concentrations) inhibited LPS (50 ng/ml)-induced calcium influx, tumor necrosis factor-a, and nitric oxide production in a concentration-dependent manner. The selective effect of gedunin on MyD88-adapter-like/myeloid differentiation primary response 88-and TRIF-related adaptor molecule/TIR domain-containing adapter-inducing interferon-b-dependent signaling pathways was further investigated. The pretreatment of WT, TIR domain-containing adapter-inducing interferon-b knockout, and MyD88 adapter-like knockout macrophages with gedunin (10 mM) significantly inhibited LPS (50 ng/ml)-induced tumor necrosis factor-a and interleukin-6 production, at 6 hours and 24 hours, suggesting that gedunin modulates a common event between both signaling pathways. Furthermore, gedunin (10 mM) inhibited LPS-induced prostaglandin E 2 production, cyclooxygenase-2 expression, and nuclear factor kB translocation into the nucleus of WT macrophages, demonstrating a wide-range effect of this chemical compound. In addition to the ability to inhibit LPS-induced proinflammatory mediators, gedunin also triggered anti-inflammatory factors interleukin-10, heme oxygenase-1, and Hsp70 in macrophages stimulated or not with LPS. In silico modeling studies revealed that gedunin efficiently docked into the MD-2 LPS binding site, a phenomenon further confirmed by surface plasmon resonance. Our results reveal that, in addition to Hsp90 modulation, gedunin acts as a competitive inhibitor of LPS, blocking the formation of the Toll-like receptor 4/MD-2/LPS complex.
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