Identification of the transcriptional regulatory sequences of human calponin promoter and their use in targeting a conditionally replicating herpes vector to malignant human soft tissue and bone tumors.

The calponin (basic or h1) gene, normally expressed in maturated smooth muscle cells, is aberrantly expressed in a variety of human soft tissue and bone tumors. In this study, we show that expression of the calponin gene in human soft tissue and bone tumor cells is regulated at the transcriptional level by the sequence between positions -260 and -219 upstream of the translation initiation site. A novel conditionally replicating herpes simplex virus-1 vector (d12.CALP) in which the calponin promoter drives expression of ICP4, a major trans-activating factor for viral genes was constructed and tested as an experimental treatment for malignant human soft tissue and bone tumors. In cell culture, d12.CALP at low multiplicity of infection (0.001 plaque-forming unit/cell) selectively killed calponin-positive human synovial sarcoma, leiomyosarcoma, and osteosarcoma cells. For in vivo studies, 10 animals harboring SK-LMS-1 human leiomyosarcoma cells were randomly divided and treated twice on days 0 and 9 intraneoplastically with either 1 x 10(7) plaque-forming units of d12.CALP/100 mm(3) of tumor volume or with medium alone. The viral treatment group showed stable and significant inhibition of tumorigenicity with apparent cure in four of five mice by day 35. Replication of viral DNA demonstrated by PCR amplification and expression of the inserted LacZ gene visualized by 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside histochemistry was associated with oncolysis of d12.CALP-treated tumors, while sparing normal vascular smooth muscle cells. In mice harboring two SK-LMS-1 tumors, replication of d12.CALP was detected in a nontreated tumor distant from the site of virus inoculation. These results indicate that replication-competent virus vectors controlled by the calponin transcriptional regulatory sequence may be a new therapeutic strategy for treatment of malignant human soft tissue and bone tumors.

Studies on aromatase inhibitors. II. Synthesis and biological evaluation of 1-amino-1H-1,2,4-triazole derivatives.

1-N,N-Disubstituted amino-1-H-1,2,4-triazole derivatives were prepared and evaluated for aromatase-inhibitory activity (in vitro) and for the inhibitory activity on pregnant mare serum gonadotropin (PMSG)-induced estrogen synthesis (in vivo). 1-N-para-Substituted benzylamino derivatives, having an electron-withdrawing group on the phenyl moiety, exhibited aromatase-inhibitory activity in vitro and in vivo. Among them, 1-[(4-nitrobenzyl)(4-nitrophenyl) amino]-1H,1,2,4-triazole (5b) was the most potent aromatase inhibitor. These 1-N-benzylamino derivatives also showed relatively strong inhibitory activity on aldosterone synthesis, indicating that the selectivity of these derivatives for aromatase inhibition was not sufficient in comparison with that of the 4-amino-4H-1,2,4-triazole derivatives.

Studies on aromatase inhibitors. III. Synthesis and biological evaluation of [(4-bromobenzyl)(4-cyanophenyl)amino]azoles and their azine analogs.

A series of [(4-bromobenzyl)(4-cyanophenyl)amino]azoles and their azine analogs, which have the side chain of the selective aromatase inhibitor YM511, were synthesized and evaluated for aromatase-inhibitory activity (in vitro) and for pregnant mare serum gonadotropin (PMSG)-induced estrogen synthesis inhibitory activity (in vivo). Among these aza-heterocycles, the pyrimidin-5-yl derivative (6a) was the most potent aromatase inhibitor and its in vitro inhibitory activity was comparable to that of YM511. Compound 6a also showed weak inhibitory activity on aldosterone synthesis. These data indicated that the pyrimidin-5-yl moiety is useful as a new azole fragment in place of the 4H-1,2,4-triazol-4-yl moiety of the aromatase inhibitor YM511.

Studies on aromatase inhibitors. IV. Synthesis and biological evaluation of N,N-disubstituted-5-aminopyrimidine derivatives.

In order to study the potency of the 5-aminopyrimidine skeleton as an aromatase inhibitor, we synthesized various N,N-disubstituted-5-aminopyrimidine derivatives and evaluated their aromatase-inhibitory activity (in vitro) and their inhibitory activity on pregnant mare serum gonadotropin (PMSG)-induced estrogen synthesis (in vivo). Compounds with the fluoro-substituted benzyl group showed potent aromatase inhibition. Among them, 5-[(4-cyanophenyl)(3,5-difluorobenzyl)amino]pyrimidine (5w, YM553) was a highly potent compound with an IC50 value of 0.038 nM for aromatase from human placenta. Its inhibitory effect was approximately four times greater than that of YM511. In addition, YM553 was a weak inhibitor of other enzymes involved in steroid hormone synthesis. These results indicate that YM553, as well as YM511 (a 4-amino-4H-1,2,4-triazole derivative), is a promising agent for the treatment of estrogen-dependent diseases.

Studies on aromatase inhibitors. I. Synthesis and biological evaluation of 4-amino-4H-1,2,4-triazole derivatives.

Various 4-N-substituted amino-4H-1,2,4-triazole derivatives were synthesized and evaluated for aromatase-inhibitory activity (in vitro) and for pregnant mare serum gonadotropin (PMSG)-induced estrogen synthesis-inhibitory activity (in vivo). The 4-(4-cyanophenyl) amino derivative and 4-(4-nitrophenyl)amino derivative, each possessing a strong electron-withdrawing group on the phenyl moiety, showed potent aromatase-inhibitory activity. Structure-activity relationship studies indicated that 4-[(4-bromobenzyl)(4-cyanophenyl)amino]-4H-1,2,4-triazole (5k, YM511) is a highly potent aromatase inhibitor with IC50 values of 0.4 and 0.12 nM in in vitro experiments using rat ovary and human placenta, respectively, and an in vivo ED50 of 0.002 mg/kg in rats on oral administration. YM511 was also a weak inhibitor of other steroid hormone synthesis enzymes. These data suggest that YM511 is a highly selective aromatase inhibitor and may be a useful agent for the treatment of estrogen-dependent diseases such as breast cancer.

Novel potassium channel activators: synthesis and structure-activity relationship studies of 3,4-dihydro-2H-1,4-benzoxazine derivatives.

Strong potassium channel-activating effects were found among a series of novel 4-substituted 3,4-dihydro-2H-1,4-benzoxazine derivatives. The key step in preparation was the nucleophilic substitution of 3,4-dihydro-2H-1,4-benzoxazine (3) with activated halogenopyridines, such as halogenopyridine N-oxides (15a--c) and the borane adduct (15d) of 4-bromopyridine. Structure-activity relationship studies identified 2-(3,4-dihydro-2,2-dimethyl-6-nitro-2H-1,4-benzoxazin-4-yl)pyridin e-1-oxide (16a: YM934) as the optimal compound. This compound (16a) showed a more potent oral antihypertensive effect than cromakalim in conscious spontaneously hypertensive rats.

Novel nonsteroidal inhibitor of cytochrome P450(17alpha) (17alpha-hydroxylase/C17-20 lyase), YM116, decreased prostatic weights by reducing serum concentrations of testosterone and adrenal androgens in rats.

BACKGROUND: The purpose of this study was to determine the effects of a nonsteroidal C17-20 lyase inhibitor, 2-(1H-imidazol-4-ylmethyl)-9H-carbazole (YM116), on serum concentrations of androgens and ventral prostatic weight in rats. METHODS: Serum concentrations of testosterone and of dehydroepiandrosterone sulfate and prostatic weights were measured in rats treated with YM116. RESULTS: YM116 inhibited testicular C17-20 lyase competitively (Ki, 0.38 nM), and decreased the serum testosterone concentration in gonadotropin-releasing hormone-treated rats (ED50, 0.7 mg/kg), indicating that YM116 was about 21-24 times more potent than other C17-20 lyase inhibitors such as ketoconazole and liarozole, and was twice as potent as CB7630. YM116 also reduced dehydroepiandrosterone sulfate levels in ACTH-treated castrated rats (ED50, 11 mg/kg). YM116 (40 mg/kg, p.o., for 2 weeks) was almost comparable to bilateral orchiectomy with respect to the time course and magnitude of the reduction in prostatic weight. Each of these two treatments decreased the prostatic weight 3 days following the treatment. Contrarily, leuprolide transiently increased the prostatic weight and then decreased it. YM116 (100 mg/kg) had no effect on the serum cortisol level in guinea pigs, and slightly decreased the serum aldosterone level in rats. CONCLUSIONS: YM116 is a selective C17-20 lyase inhibitor which decreases rat prostatic weight by reducing androgen production in the testes and adrenal glands.