Pharmacological characterization of N1-(2-methoxyphenyl)-N4-hexylpiperazine as a multi-target antagonist of α1A/α1D-adrenoceptors and 5-HT1A receptors that blocks prostate contraction and cell growth

Abstract

Benign prostatic hyperplasia (BPH) is a progressive disease related to the imbalance of cell growth and apoptosis, and it plays a key role in the development of lower urinary tract symptoms (LUTS). The main pharmacological treatment is based on α1A-adrenoceptor blockers, but in several cases monotherapy has failed. Recent studies of prostate pathophysiology have noted the role of α1D-adrenoceptors and 5-HT1A receptors in prostate cell proliferation in addition to the usual role of α1A-adrenoceptors in prostate contraction. N-phenylpiperazine is a scaffold structure that may confer drug affinity for these three receptors. Therefore, the present work aimed to investigate the pharmacological characteristics of N1-(2-methoxyphenyl)-N4-hexylpiperazine (LDT66). Using isometric contraction assays with rat prostate and aorta, LDT66 reduced phenylephrine-induced contractions and showed K B values of 3.4 and 2.2 nM for α1A- and α1D-adrenoceptors, respectively. According to the functional binding assays data, LDT66 showed a high affinity (nanomolar range) for the 5-HT1A receptors, behaving as an antagonist. LDT66 also showed a low affinity (micromolar range) for receptors unrelated to BPH such as α1B-adrenoceptors, α2A-adrenoceptors, muscarinic and 5-HT2A receptors, which is a desirable profile in order to prevent putative side effects. Accordingly, LDT66 (100 μg/kg) showed a marginal hypotensive effect. Using the DU-145 prostate cells, control experiments characterized the α1D-adrenoceptor- and 5-HT1A receptor-mediated cell growth by phenylephrine and 5-HT, respectively. LDT66 (50 nM) prevented both effects similarly. In conclusion, LDT66 is a high-affinity multi-target antagonist of relevant receptors for BPH, and it may be a new starting point for multi-target drug development to treat BPH and LUTS.

New Multi-target Antagonists of 1A-, 1D-Adrenoceptors and 5-HT1A Receptors Reduce Human Hyperplastic Prostate Cell Growth and the Increase of Intraurethral Pressure

“…Note that tamsulosin produced only partial inhibition of phenylephrine-induced growth at 5 nM ( Fig. 2A), a concentration ∼50-fold higher than the K i we reported for the a 1D -adrenoceptors (Table 2; Chagas-Silva et al, 2014). Similar effects of LDTs on BPH cells were obtained using the MTT assay (P , 0.001; Supplemental Fig.…”

“…Rat prostate and thoracic aorta were removed, cleaned, and cut into 10-mm strips (prostate) or 3-mm rings (aorta). Isometric contraction assays were performed as described previously (Chagas-Silva et al, 2014 New multi-target Drugs as Potential Leads for BPH Treatment and denuded aorta segments were preloaded (60 minutes) with 10 or 20 mN, respectively, and washed twice. Tissues were contracted with 1 mM phenylephrine (aorta) or 60 mM KCl depolarizing solution (prostate).…”

“…Male Wistar rats were cannulated, as described previously (Chagas-Silva et al, 2014). For these assays, each animal was used only once, and all drugs were diluted in isotonic saline.…”

“…Nonspecific binding was defined in the presence of 1 mM prazosin. Alternatively, saturation assays were performed using prazosin at concentrations ranging from 0.0001 to 0.1 mM (Chagas-Silva et al, 2014).…”

“…Previously, we showed that the N1-(2-methoxyphenyl)-N4-piperazine moiety confers affinity for a 1A -, a 1D -adrenoceptors, and 5-HT 1A receptors (Chagas-Silva et al, 2014). In this work, we investigate the in vitro and in vivo pharmacological characteristics of three N-phenylpiperazine derivatives, LDT3, LDT5, and LDT8 (European patent office, application 13733873.7-1451; USPTO application 14370646).…”