TAS3681, an androgen receptor antagonist, prevents drug resistance driven by aberrant androgen receptor signaling in prostate cancer.

Second-generation androgen receptor (AR) signaling inhibitors (ARSIs), such as abiraterone and enzalutamide, prolong the life of patients with castration-resistant prostate cancer (CRPC). However, patients receiving ARSIs ultimately develop resistance through various complex mechanisms, including AR mutations, constitutively active AR-splice variants (AR-Vs), and AR overexpression. Here, we characterized a novel AR pure antagonist, TAS3681, which inhibits AR transcriptional activity and downregulates AR-full length (AR-FL) and AR-Vs. TAS3681 reduced the protein levels of AR-FL and AR-Vs including AR-V7 in enzalutamide-resistant cells (SAS MDV No. 3-14), in vitro and in vivo, showing strong antitumor efficacy in an AR-V7-positive xenograft model. In AR-overexpressing VCaP (prostate cancer) cells, conversely to enzalutamide, TAS3681 effectively suppressed cell proliferation and downregulated AR expression. Importantly, TAS3681 blocked the transcriptional activity of various mutant ARs, including mutations F877L/T878A and H875Y/T878A, which confer resistance to enzalutamide, and V716M and H875Y mutations, which confer resistance to darolutamide. Our results demonstrate that TAS3681 suppresses the reactivation of AR signaling, which causes resistance to ARSIs, via a newly identified mechanism of action. Therefore, TAS3681 could be a new therapeutic option for CRPC treatment.

Analysis of novel enzalutamide-resistant cells: upregulation of testis-specific Y-encoded protein gene promotes the expression of androgen receptor splicing variant 7.

BACKGROUND: Enzalutamide, a second-generation antiandrogen, is an approved medicine for the treatment of metastatic castration-resistant prostate cancer (CRPC); however, the mechanisms behind the resistance are not completely understood. In the present study, we established enzalutamide-resistant cells derived from lymph node carcinoma of the prostate (LNCaP) cells and characterized their androgen receptor (AR) status and changes in the gene expression with an aim to elucidate these mechanisms. METHODS: SAS MDV No. 3-14 enzalutamide-resistant cells were established from LNCaP xenograft castrated male mice under continuous administration of enzalutamide. Then, the AR status and expression of AR target genes were evaluated by western blotting or real-time polymerase chain reaction analysis. The role of AR in the proliferation was also analyzed using the AR siRNA approach. The gene expression profiling in SAS MDV No. 3-14 cells was evaluated by microarray analysis. The role of testis-specific Y-encoded protein (TSPY), one of the upregulated genes, in the expression of AR and AR target genes and cell growth was also verified using siRNA. RESULTS: SAS MDV No. 3-14 cells expressed AR-v7, leading to the increased expression of AR target genes. Gene silencing of AR showed that both AR-FL and AR-v7 function as proliferative drivers in SAS MDV No. 3-14 cells. Microarray analysis revealed that TSPY is upregulated genes in these cells. TSPY siRNA inhibited cell proliferation, decreased the expression of AR-v7 and AR-v7 targeted genes. CONCLUSIONS: This study demonstrated that SAS MDV No. 3-14 cells increase the expression of AR-v7 by upregulating TSPY, leading to acquired resistance to enzalutamide.

Characterization of muscarinic receptors in the human bladder mucosa: direct quantification of subtypes using 4-DAMP mustard.

OBJECTIVE: To characterize pharmacologically relevant muscarinic receptors in the human bladder mucosa and detrusor muscle using radioligand binding assays with [N-methyl-3H]scopolamine methyl chloride ([3H]NMS) and 4-DAMP mustard. METHODS: Muscarinic receptors in homogenates of bladder mucosa, detrusor muscle, and parotid gland were measured using the radioligand [3H]NMS. 4-DAMP mustard was used to inactivate M3 receptors irreversibly. RESULTS: Specific [3H]NMS binding in the homogenates of the mucosa and detrusor muscle was saturable and of high affinity as shown by dissociation constants (Kd) of 260 ±82 and 237 ±49 pM, respectively. Antimuscarinic agents (oxybutynin, propiverine, tolterodine, and darifenacin) and their active metabolites competed with [3H]NMS for the binding sites in the human mucosa in a concentration-dependent manner. These agents exhibited similar affinity in the detrusor muscle. The Bmax. values of [3H]NMS in the detrusor, bladder mucosa, and parotid gland were significantly decreased by pretreatment with 4-DAMP mustard (36%, 41% and 63%, respectively). CONCLUSION: The density and binding affinity profile of the muscarinic receptor population in the human bladder mucosa was shown to be similar to that of the detrusor muscle. The density of the M3 subtype in the mucosa was similar to that in the detrusor muscle but lower than that in the parotid gland.

Muscarinic Receptor Binding of Imidafenacin in the Human Bladder Mucosa and Detrusor and Parotid Gland.

OBJECTIVES: The current study aimed to characterize comparatively the binding of imidafenacin to muscarinic receptors in the human bladder mucosa and detrusor muscle and parotid gland. METHODS: The muscarinic receptor in homogenates of human tissues (bladder mucosa and detrusor muscle and parotid gland) was measured using a radioligand binding assay with [N-methyl-(3) H]scopolamine methyl chloride ([(3) H]NMS). RESULTS: Imidafenacin competed with [(3) H]NMS for binding sites in the bladder mucosa and detrusor muscle and parotid gland, and its affinity was significantly (2.6-8.7 times) higher than that of oxybutynin. Also, the affinity of imidafenacin for muscarinic receptors was approximately two-fold higher in the parotid gland than bladder tissue. The affinity of imidafenacin in the mucosa was similar to that in the detrusor muscle, suggesting that this agent exhibits therapeutic effects by blocking muscarinic receptors in the mucosa as well as detrusor muscle. Scatchard analysis revealed that imidafenacin increased significantly (approximately four-fold) Kd values for [(3) H]NMS binding in the human detrusor muscle and parotid gland, with little effect on Bmax values. This observation indicates that imidafenacin binds to the muscarinic receptors in human tissues in a competitive and reversible manner. CONCLUSION: Imidafenacin binds to muscarinic receptors in the human bladder mucosa and detrusor muscle and parotid gland with high affinity. This agent was considered to exhibit therapeutic effects on the lower urinary tract symptoms due to an overactive bladder by blocking muscarinic receptors in the urothelium as well as detrusor muscle.

Selective binding of bladder muscarinic receptors in relation to the pharmacokinetics of a novel antimuscarinic agent, imidafenacin, to treat overactive bladder.

The binding of orally administered imidafenacin, used to treat overactive bladders, to muscarinic receptors in rat tissue was characterized based on pharmacokinetics. The binding in six tissues including bladder tissue was measured using [N-methyl-(3)H] scopolamine methyl chloride ([(3)H]NMS). Pharmacokinetic parameters were estimated from measurements of the concentration of imidafenacin in serum, the bladder, and the submaxillary gland by liquid chromatography-mass spectrometry/mass spectrometry. The receptor binding affinity of imidafenacin in vitro was significantly lower in the bladder than submaxillary gland or colon. The oral administration of imidafenacin (0.79, 1.57, and 6.26 µmol/kg) was characterized by a more selective and longer-lasting binding to muscarinic receptors in the bladder than other tissues. Imidafenacin showed little binding to brain muscarinic receptors, consistent with its minor effect on the central nervous system. Pharmacokinetic data showed that orally administered imidafenacin was distributed at a higher concentration in the bladder than the serum or submaxillary gland of rats. After the intravesical instillation of imidafenacin, there was significant binding of muscarinic receptors in the bladder. Furthermore, a significant level of imidafenacin was detected in the urine of rats given a 1.57 µmol/kg concentration of this agent. The present study demonstrated that imidafenacin administered orally distributes predominantly to the bladder and exerts more selective and longer-lasting effect on the bladder than other tissues, such as the submaxillary gland, colon, and brain. Furthermore, the imidafenacin excreted in urine may play an important role in pharmacokinetic and pharmacological selectivity.

The N-oxide metabolite contributes to bladder selectivity resulting from oral propiverine: muscarinic receptor binding and pharmacokinetics.

We characterized contribution of N-oxide metabolites [1-methyl-4-piperidyl diphenylpropoxyacetate N-oxide (M-1) and 1-methyl-4-piperidyl benzilate N-oxide (M-2)] to the binding of muscarinic receptors in relation to the pharmacokinetics of propiverine in rats. The in vitro muscarinic receptor binding activity of M-2 was equipotent to that of propiverine, whereas M-1 was much less active. After the oral administration of propiverine (24.8-248 micromol/kg), there was relatively selective and longer-lasting binding of muscarinic receptors in the rat bladder compared with the submaxillary gland as shown by a significant increase in the apparent dissociation constant (K(d)) for specific binding of [N-methyl-(3)H]scopolamine ([(3)H]NMS). In addition, the intravesical instillation of M-2 produced a significant increase in K(d) for specific [(3)H]NMS binding in the rat bladder. Extremely high concentrations of M-1 and M-2 were detected in plasma after the oral administration of propiverine. The concentration of unbound M-2 was much higher than that of M-1 and propiverine in the rat plasma. The sum of maximal plasma unbound propiverine equivalents (C(max)) after the oral administration of propiverine at doses of 24.8, 74.3, and 248 micromol/kg was 66.0, 303, and 509 nM, respectively. The sum of corresponding area under the time-concentration curve from 0 to 12 h was 194, 2123, and 4645 nM . h, respectively. In fact, the unbound concentration of M-2 comprised more than 90% of sum of unbound propiverine equivalents in the plasma. After oral treatment with propiverine, the bladder showed the highest concentration of M-2, indicating specific distribution of this metabolite into the target organ. Thus, M-2 may contribute greatly to the relatively selective and long-lasting occupation of bladder muscarinic receptors after oral administration of propiverine.

Pharmacological effects of Hachi-mi-jio-gan extract (Harncare) on the contractile response and on pharmacologically relevant receptors in the rat bladder.

Hachi-mi-jio-gan extract (Harncare; HE), a galenical produced from the traditional Chinese herbal mixture Ba-Wei-Die-Huang-Wan, has been reported to improve lower urinary tract symptoms (LUTS) in patients. The present study was undertaken to clarify the pharmacological effects of HE on smooth muscle contraction and on pharmacologically relevant (muscarinic, 1,4-DHP and purinergic) receptors in the rat bladder. Additionally, the effects of repeated oral treatment with HE on the hepatic cytochrome P-450 (CYP) and on blood biochemical values in rats were examined. HE (10 mg/ml) inhibited significantly the acetylcholine-induced contraction of isolated rat bladder strips. The pD(2) value in the absence and presence of HE (10 mg/ml) was 5.14+/-0.16 and 3.99+/-0.17, respectively. HE (0.1 to 10 mg/ml) inhibited the specific binding of [N-methyl-(3)H]scopolamine methyl chloride ([(3)H]NMS), (+)-[(3)H]PN 200-110 and alphabeta-methylene ATP [2,8-(3)H]tetrasodium salt ([(3)H]alphabeta-MeATP) in the rat bladder in a concentration-dependent manner. The respective IC(50) values were 6.85+/-0.94, 7.08+/-0.72 and 1.34+/-0.23 mg/ml. Based on IC(50) values, the binding activity of HE for purinergic receptors was shown to be significantly (about 7 times) greater than that for muscarinic and 1,4-DHP receptors. Repeated oral administration of HE (10, 30 and 100 mg/kg/day) for 4 weeks had little or no effect on the level and activity of hepatic CYP or on blood biochemical values in rats. In conclusion, the present study has shown that HE exerts significant binding activity for pharmacologically relevant receptors in the rat bladder and a relaxant effect on the acetylcholine-induced contraction of isolated muscle strips. HE seemed to exhibit little pharmacokinetic interaction with drugs.

Quantitative analysis of the loss of muscarinic receptors in various peripheral tissues in M1-M5 receptor single knockout mice.

BACKGROUND AND PURPOSE: To compare loss in binding to muscarinic receptor (mAChR) subtypes with their known functions, the total density of muscarinic receptors was measured in peripheral tissues from wild type (WT) and mAChR knockout (KO) mice. EXPERIMENTAL APPROACH: Binding parameters of [N-methyl-3H]scopolamine methyl chloride ([3H]NMS) were determined in 10 peripheral tissues of WT and M1-M5 receptor KO mice. Competition between [3H]NMS and darifenacin (selective M3 receptor antagonist) was also measured. KEY RESULTS: There was an extensive loss of [3H]NMS-binding sites (maximal number of binding sites, Bmax) in heart and smooth muscle from M2KO mice, compared with WT mice. Smooth muscle from M3KO mice also showed a moderate loss of Bmax. Bmax fell in pancreas and bladder of M4KO mice and in prostate in M1KO and M3KO mice. There was a large loss of Bmax in exocrine and endocrine glands of M3KO mice with a moderate decrease in M2KO mice. Darifenacin inhibited specific [3H]NMS binding in submandibular gland and bladder of WT, M2KO and M3KO mice. Ki (inhibition constant) values for darifenacin in the submandibular gland were the same in WT and M2KO mice but increased in M3KO mice. However, Ki values in bladder were decreased in M2KO mice and increased in M3KO mice. CONCLUSIONS AND IMPLICATIONS: Single mAChR KO mice exhibit a loss of mAChR in peripheral tissues that generally paralleled the reported loss of function. Quantitative analysis of data, however, also suggested that, in some instances, normal expression of a receptor subtype depended on expression of other subtypes.