JNJ-10280205 and JNJ-10287069: Novel PDE5 inhibitors as clinical candidates for erectile dysfunction.

Phosphodiesterase 5 (PDE5) inhibitors are efficacious in treating patients with erectile dysfunction. New PDE5 inhibitors with different selectivity and pharmacokinetic profiles have been vigorously pursued. Here we report two novel, potent, and selective PDE5 inhibitors, JNJ-10280205 and JNJ-10287069, with Ki values of 0.05 and 0.12 nM, respectively. Both compounds displayed superior selectivity against PDE1-4 and -6 when compared to sildenafil. In the anesthetized dogs, JNJ-10280205 and JNJ-10287069 exhibited similar efficacy as sildenafil in enhancing erectile functions, with no significant effect on cardiovascular parameters. Pharmacokinetic studies showed that JNJ-10287069 had better oral bioavailability than JNJ-10280205 in several animal species. In vitro study suggested that cytochrome P450 (CYP) 3A4 played a major role in the metabolism of both compounds. The compounds inhibited some of the CYP450 enzymes and the human ether-a-go-go (HERG) channel at much higher concentrations than that required to inhibit PDE5, thus, no cross inhibition would be expected at therapeutic doses. Both compounds are suitable clinical candidates.

Induction of a novel conformation in the progesterone receptor by ZK299 involves a defined region of the carboxyl-terminal tail.

Progesterone receptor antagonists are a promising class of therapeutic drugs indicated for the treatment of a variety of reproductive conditions. Understanding their mechanism of action at the molecular level is an important prerequisite for the development of future generations of these drugs. Using limited proteolytic analysis to monitor conformational changes in the progesterone receptor, we can detect three distinct classes of progestin antagonist. The effect of the first, RU486, on the conformation of the carboxyl terminus of the receptor has been previously described. The second, exemplified by RWJ 47626, a nonsteroidal compound with in vitro antiprogestin activity, induces a proteolytic fragment pattern indistinguishable from that induced by the agonist R5020. Finally, ZK299 induces a fragment pattern intermediate between that induced by R5020 and RU486. Site-directed mutagenesis of the carboxyl-terminal tail of the progesterone receptor indicates that the region containing the putative activation function AF-2 is differentially exposed to proteolytic attack depending on the nature of the antagonist bound. The differentially exposed region is most accessible when the antagonist RU486 is bound, less accessible when the antagonist ZK299 is bound, and least accessible when the antagonist RWJ47626 or agonist R5020 is bound. The results suggest that multiple types of antiprogestin can be defined in terms of their effects on the conformation of the carboxyl-terminal activation function of the progesterone receptor.

Nonsteroidal progesterone receptor ligands with unprecedented receptor selectivity.

We have characterized a series of nonsteroidal progesterone receptor ligands, the tetrahydropyridazines. Compounds in this series, exemplified by RWJ 26819, demonstrate high affinity and unprecedented specificity for the progesterone receptor relative to other steroid hormone receptors. Like steroidal progestins, RWJ 26819 induces binding of the receptor to a progesterone response element in vitro, and stimulates gene expression in and proliferation of T47D human breast cancer cells. When administered to rabbits orally or subcutaneously, the compound induces histological changes in the uterine lining comparable to those induced by levonorgestrel. It also inhibits ovulation in monkeys. Though less potent in cells and in animal models than would be predicted from binding affinity alone, their enhanced selectivity suggests that they could be effectively used in a clinical setting. Most of the tetrahydropyridazines synthesized are progestin agonists or mixed agonists and antagonists in vitro; however, one compound with antagonist activity in the rabbit uterine transformation assay has been identified.

Increased expression of stromelysin 3 mRNA in leiomyomas (uterine fibroids) compared with myometrium.

OBJECTIVE: To determine the level of expression of selected matrix metalloproteinases in uterine leiomyoma compared with unaffected myometrium in an effort to explain the abnormal accumulation of extracellular matrix in the leiomyoma. METHODS: The levels of matrix metalloproteinase (MMP) mRNA in leiomyoma and myometrium were measured in samples from 22 patients during either proliferative (n = 6) or secretory phases (n = 16) of the menstrual cycle. Relative amounts of collagenase (MMP-1) and stromelysin (MMP-3) mRNAs were measured by Northern blot analysis, and amounts of stromelysin 3 (MMP-11) and matrilysin (MMP-7) mRNA from each sample were determined by semiquantitative reverse transcriptase-polymerase chain reaction (RT-PCR) and normalized to beta-actin mRNA. RESULTS: The levels of MMP-1 and MMP-3 mRNAs were similar in both leiomyoma and unaffected myometrium. The levels of MMP-11 mRNA were twofold greater in leiomyoma compared with myometrium throughout the menstrual cycle, and the differences in the levels of MMP-11 were significantly different during the secretory phase. The level of MMP-7 mRNA expression was similar in leiomyoma, myometrium, and endometrium. CONCLUSIONS: Among the metalloproteinases examined in this study, only the levels of MMP-11 mRNA were elevated in leiomyoma compared with myometrium. The increased expression of MMP-11 in uterine leiomyoma compared with myometrium is analagous to previously reported elevations of MMP-11 mRNA in dermatofibromas compared with unaffected skin. The increased expression of MMP-11 mRNA in fibroid tumors suggests that this MMP may be involved in the formation of a more fibrous extracellular matrix in leiomyoma relative to unaffected myometrium.

Diverse effects of tyrosine kinase inhibitors on follicle-stimulating hormone-stimulated estradiol and progesterone production from rat granulosa cells in serum-containing medium and serum-free medium containing epidermal growth factor.

Epidermal growth factor (EGF) has been shown to influence FSH-stimulated estradiol (E2) and progesterone (P4) production from granulosa cells. RG 50810, a tyrosine kinase inhibitor (TKI), has previously been shown to inhibit the EGF-receptor tyrosine kinase. RG 50810 has also been shown to inhibit FSH-stimulated increases in mRNA for steroidogenic enzymes, implying a functional role of tyrosine kinases in FSH action in granulosa cells. However, inhibition of FSH-stimulated steroidogenesis by TKIs has not been evaluated in connection with the effects of EGF in granulosa cells. In the present studies, FSH-stimulated E2 production was inhibited similarly by inhibitors of protein kinase A (H-89) and protein kinase C (calphostin C) and by TKIs, and none of the inhibitors were capable of reversing the EGF-induced inhibition of FSH-stimulated E2 production. FSH-stimulated P4 production was enhanced dramatically in serum-containing medium with concentrations of TKI that were near previously reported IC50s. The enhancing effect of TKIs was less evident in serum-free medium. Addition of EGF to serum-free medium enhanced FSH-stimulated P4 production, and the TKIs reversed EGF-enhanced P4 production, but in a manner similar to that of protein kinase A inhibitor H-89. Compared to results in serum-free medium, the potency of RG 50810 and genistein to inhibit the effects of EGF on P4 production was 3- to 8-fold greater relative to H-89. These studies have demonstrated that TKIs RG 50810 and genistein selectively inhibit the effects of EGF on FSH-stimulated P4 production in granulosa cell cultures. In contrast, these studies have demonstrated nonselective inhibition of FSH-stimulated E2 and P4 production by TKIs in serum-free medium, in which it is not clear which enzyme system is affected by the compounds tested.

Identification and functional study of phosphodiesterases in rat urinary bladder.

Abstract Cyclic nucleotides are important secondary messengers involved in modulating the contractility of various smooth muscles. Phosphodiesterases (PDE) play important roles in this process by modulating the levels of cyclic nucleotides and their duration of action. This study was designed to identify and characterize the PDE isoenzymes in rat urinary bladder and to evaluate their roles in regulating bladder smooth muscle tone. The involvement of cAMP and cGMP pathways in this process was also assessed. The studies were carried out with tissues from male and female rats and no significant sex-related difference was found in the results. Utilizing the unique pharmacological properties of different isoenzymes, PDE1, 2, 3, 4, and 5 were identified in rat bladder. Organ bath experiments showed that forskolin was most potent in relaxing pre-contracted rat bladder strips while sodium nitroprusside was moderately effective, suggesting the relaxation was mainly mediated by the cAMP pathway and that the cGMP pathway is moderately involved. For PDE inhibitors, the non-specific inhibitor papaverine was most effective in relaxing pre-contracted bladder strips. Among isoenzyme-selective inhibitors, vinpocetine, EHNA, and sildenafil induced more relaxation than milrinone and rolipram.

Fluorescence polarization assay and SDS-PAGE confirms matrilysin degrades fibronectin and collagen IV whereas gelatinase A degrades collagen IV but not fibronectin.

Matrilysin and gelatinase A are hypothesized to have significant roles in uterine and ovarian function. However, proteolytic activity assays for these enzymes are limited. We describe the development of simple and rapid assays for the proteolysis of fluorescein-labeled full-length substrates, collagen IV (Col-IV) and fibronectin (FN), and demonstrate the selectivity of matrilysin (MMP-7) compared to gelatinase A (MMP-2) for fibronectin. Changes in fluorescence intensity (FIU) and fluorescence polarization (mP) resulting from the protease activity of matrilysin and gelatinase A were measured. These studies show that the fluorescently labeled substrates, Col-IV and FN, are as reliable and amenable to rapid in vitro assay as peptide substrates. In addition, they are easier to use than previously described, non-fluorescent methods. The results demonstrate that assays using full-length, biological matrix proteins are more sensitive indicators of MMP-specific substrate activity than peptide based assays.