Spontaneous early-onset glomerulonephritis in a 8-week-old male Crj:CD1 (ICR) mouse.

Glomerular lesions including membranoproliferative glomerulonephritis occur spontaneously in aged mice, but they are rare in young animals. In our laboratory, spontaneous glomerulonephritis was observed in an 8-week-old male Crj:CD1 (ICR) mouse. Macroscopically, the bilateral kidneys were discolored, but no edema or ascites was observed. Glomerular lesions were characterized by a thickening of capillary walls, a double-contoured basement membrane and mesangial expansion due to increased amounts of matrix. Ultrastructurally, mesangial interposition in the capillary wall and subendothelial deposition of basement membrane-like material were observed. No evidence of immune complex deposition or amyloid was found. On the basis of the observed clinical pathology and histopathology, a secondary form of glomerular lesion was excluded. The glomerular lesion was compatible with glomerulonephritis in a young Crj:CD1 (ICR) mouse.

Evaluation of the genotoxicity of tamoxifen in the liver and kidney of F344 gpt delta transgenic rat in 3-week and 13-week repeated dose studies.

Transgenic rat gene mutation assays can be used to assess genotoxicity of chemicals in target organs for carcinogenicity. Mutations in transgenes are genetically neutral and accumulate during a treatment period; thus, the assays are suitable for assessment of the genotoxicity risk of chemicals using a repeated-dose treatment paradigm. However, few such studies have been conducted. To examine the utility of the transgenic rat assays in repeated-dose studies, we treated female F344 gpt delta rats with tamoxifen (TAM) at 20 and 40mg/kg, or toremifene (TOR) at 40mg/kg by gavage daily for 3 weeks. We also fed gpt delta rats with TAM at either 250ppm (15.4-17.6mg/kg) or 500ppm (30.0-32.9mg/kg) for 13 weeks. TAM is carcinogenic in the rat liver and TOR is not carcinogenic. TAM administration significantly increased gpt (point mutations) and Spi(-) (deletions) mutant frequencies (MFs) in the liver, the target organ of carcinogenesis; MFs were higher after treatment for 13 weeks than after treatment for 3 weeks. The MFs in the kidney did not increase in any of the TAM treatment groups. TOR had no effect on MFs (gpt and Spi(-)) in either the liver or the kidney. We conclude that the gpt delta rat assay in the repeated-dose treatment paradigm is sensitive enough to detect gene mutations induced by TAM in the target organ for carcinogenesis. Furthermore, the assay can be integrated into a 13-week dose-finding study for a 2-year cancer bioassay.

Integration of in vivo genotoxicity and short-term carcinogenicity assays using F344 gpt delta transgenic rats: in vivo mutagenicity of 2,4-diaminotoluene and 2,6-diaminotoluene structural isomers.

An important trend in current toxicology is the replacement, reduction, and refinement of the use of experimental animals (the 3R principle). We propose a model in which in vivo genotoxicity and short-term carcinogenicity assays are integrated with F344 gpt delta transgenic rats. Using this model, the genotoxicity of chemicals can be identified in target organs using a shuttle vector lambda EG10 that carries reporter genes for mutations; short-term carcinogenicity is determined by the formation of glutathione S-transferase placenta form (GST-P) foci in the liver. To begin validating this system, we examined the genotoxicity and hepatotoxicity of structural isomers of 2,4-diaminotoluene (2,4-DAT) and 2,6-diaminotoluene (2,6-DAT). Although both compounds are genotoxic in the Ames/Salmonella assay, only 2,4-DAT induces tumors in rat livers. Male F344 gpt delta rats were fed diet containing 2,4-DAT at doses of 125, 250, or 500 ppm for 13 weeks or 2,6-DAT at a dose of 500 ppm for the same period. The mutation frequencies of base substitutions, mainly at G:C base pairs, were significantly increased in the livers of 2,4-DAT-treated rats at all three doses. In contrast, virtually no induction of genotoxicity was identified in the kidneys of 2,4-DAT-treated rats or in the livers of 2,6-DAT-treated rats. GST-P-positive foci were detected in the livers of rats treated with 2,4-DAT at a dose of 500 ppm but not in those treated with 2,6-DAT. Integrated genotoxicity and short-term carcinogenicity assays may be useful for early identifying genotoxic and nongenotoxic carcinogens in a reduced number of experimental animals.

[Convulsive liability of an oral carbapenem antibiotic, tebipenem pivoxil].

Tebipenem pivoxil (TBPM-PI), the first oral carbapenem antibiotic both in Japan and abroad, was examined on its convulsive liability. We used ICR male mice and Sprague-Dawley male rats to examine the pro-convulsive effect and anticonvulsive effect of TBPM-PI and its active metabolite, TBPM. (1) When mice were treated with TBPM-PI (30-1000 mg/kg, p.o.) or TBPM (10-300 mg/kg, i.v.), no convulsion was noted at any dose level. When rats were treated with TBPM (300 mg/kg, i.v.), no convulsant effects were noted in electroencephalography or behavioral observation. In intraventricular injection of TBPM in mice, clonic convulsion was observed in 7/10 animals at 100 microg but no effect at 30 microg. On the other hand, the administration of 10/10 microg imipenem/cilastatin (IPM/CS) resulted in clonic convulsion in all animals and tonic convulsion in 3/10 animals, and 4/10 animals died. The administration of 100 microg meropenem did not cause any effects. (2) When mice were co-administered with pentylenetetrazole (45 mg/kg: maximum dose level at which no convulsion is induced) and TBPM-PI (30-300 mg/kg, p.o.) or TBPM (300 mg/kg, i.v.), convulsion enhancing effect was not noted. On the other hand, the co-administration of pentylenetetrazole with IPM/CS (300/300 mg/kg, i.v.) enhanced a convulsive effect of pentylenetetrazole. (3) When mice were treated with TBPM-PI (30-300 mg/kg, p.o.) or TBPM (100 mg/kg, i.v.), inhibitory effect was not noted on convulsions induced by electrostimulation, pentylenetetrazole or strychinine. In conclusion, there were no pro-convulsive effects or anticonvulsive effect in the oral administration of TBPM-PI or intravenous administration of TBPM. Pro-convulsive effect was observed in the intraventricular injection of TBPM as in the case of other carbapenem antibiotics, but such action was weaker than that in IPM/CS administration. Accordingly, the risk of occurrence of convulsion related to TBPM-PI administration was low compared to IPM/CS administration, and TBPM-PI was considered to be less potential to induce convulsions in clinical use.

Bile acid-induced elevated oxidative stress in the absence of farnesoid X receptor.

The major function of farnesoid X receptor (FXR) is to maintain bile acid and lipid homeostasis. Fxr-null mice, in which the levels of hepatic bile acid and lipid have been elevated, develop spontaneous liver tumors. We evaluated differences in hepatic bile acid and triglyceride concentrations, and in generation of oxidative stress between wild-type mice and Fxr-null mice. The hepatic levels of 8-hydroxy-2'-deoxyguanosine (8OHdG), thiobarbituric acid-reactive substance (TBARS) and hydroperoxides, oxidative stress-related genes, and nuclear factor (erythroid-2 like) factor 2 (Nrf2) protein in Fxr-null mice were significantly higher than those in wild-type mice. An increase in the hepatic bile acid concentration in Fxr-null mice fed a cholic acid (CA) diet resulted in an increase in the hepatic levels of hydroperoxides, TBARS and 8OHdG, whereas a decrease in the hepatic concentration in mice fed a diet containing ME3738 (22beta-methoxyolean-12-ene-3beta,24(4beta)-diol) resulted in a decrease in these oxidative stress marker levels. A good correlation was observed between the hepatic bile acid concentrations and the hepatic oxidative stress marker levels, although there was no significant correlation between the hepatic triglyceride concentrations and oxidative stress. The results show that oxidative stress is spontaneously enhanced in Fxr-null mice, which may be attributable to a continuously high level of hepatic bile acids.

Nonsteroidal progesterone receptor ligands (II); synthesis and SAR of new tetrahydrobenzindolone derivatives.

The human progesterone receptor (PR) binding affinity and the PR agonistic or antagonistic potency of tetrahydronaphthofuranone derivatives were shown previously to be markedly influenced by substituents at the 6- and 7-positions. Here, we synthesized tetrahydrobenzindolones possessing a lactam ring, which enabled us to modify the 6- and 7-positions more freely, since tetrahydrobenzindolones are chemically more stable than tetrahydronaphthofuranones. The tetrahydrobenzindolone derivatives generally showed higher PR binding affinity than the corresponding tetrahydronaphthofuranones. We also succeeded in separating the agonistic and antagonistic activities by choosing suitable substituent groups at the 6- and/or 7-position(s) of the tetrahydrobenzindolone. The effects of representative agonists, 12c (CP8668), and 14a (CP8816), and a representative antagonist, 15f (CP8661), were confirmed in in vivo tests. In this report, we mainly describe the synthesis and structure-activity relationships (SAR) of tetrahydrobenzindolone derivatives, as new nonsteroidal PR ligands.

Nonsteroidal progesterone receptor ligands (I): synthesis and SAR of new tetrahydronaphthofuranone derivatives.

We have synthesized a series of nonsteroidal progesterone receptor (PR) ligands, tetrahydronaphthofuranones, structurally based on the fungal metabolite PF1092C. Structure-activity relationship studies revealed that substituents at the 6- and 7-positions were critical for PR binding affinity and for agonist or antagonist activity. Compounds in this series, exemplified by 19i, exhibited high affinity and high specificity for PR over other steroid hormone receptors and acted as selective PR antagonists. Further modification of PF1092C may generate compounds of potential pharmacological interest.

Endocrinological properties of two novel nonsteroidal progesterone receptor modulators, CP8816 and CP8863.

We have isolated PF1092A, B, and C, novel nonsteroidal progesterone ligands with preferential affinity for the progesterone receptor, from fermentation broth of a fungus [Tabata Y, Miike N, Hatsu M, Kurata Y, Yaguchi T, Someya A, Miyadoh S, Hoshiko S, Tsuruoka T, and Omoto S (1997) J Antibiot 50:304-308; Tabata Y, Hatsu M, Kurata Y, Miyajima K, Tani M, Sasaki T, Kodama Y, Tsuruoka T, and Omoto S (1997) J Antibiot 50:309-313]. The original skeleton of PF1092, tetrahydronaphthofuranone, was modified synthetically to produce a new skeleton, tetrahydrobenzindrone, and in the present study, biological activities of two derivatives, CP8816 [(4aR,5R,6R,7R)-6-(N,N-dimethylaminocarbonyl)oxy-7-methoxy-4a,5,6,7-tetrahydro-1,3,4a,5-tetramethylbenz[f]indol-2(4H)-one] and CP8863 [(4aR,5R,6R,7R)-7-hydroxy-6-(N-methylcarbamoyl)oxy-4a,5,6,7-tetrahydro-1,3,4a,5-tetramethylbenz[f]indol-2(4H)-one], were investigated. Both CP8816 and CP8863 demonstrated selective binding to progesterone receptor and partial agonistic activity in a progesterone-dependent endogenous alkaline phosphatase expression assay. In the Clauberg-McPhail test, progestational activity of CP8816 (0.1 mg/kg s.c. or 10 mg/kg p.o.) was comparable to that of progesterone (0.15 mg/kg s.c.), and oral administration of CP8863 at more than 1.0 mg/kg also exerted similar effects. Anti-estrogenic (antiuterotropic) activity was confirmed on daily oral application of more than 0.1 mg/kg CP8863 for 3 days by inhibition of estrogen-dependent uterine wet weight gain in ovariectomized rats. CP8816 also exerted antiuterotropic activity at doses of 10 mg/kg (s.c.) and 100 mg/kg (p.o.). These results indicate that our nonsteroidal progesterone ligands have affinity for the progesterone receptor with partial progestational activity in vitro and clear progestational effects in vivo. Thus, these progesterone receptor modulator profiles suggest that CP8863 and CP8816 are good candidate compounds for treatment of hormone-dependent gynecological disorders.

CP8668, a novel orally active nonsteroidal progesterone receptor modulator with tetrahydrobenzindolone skeleton.

We investigated progestational activity of a new nonsteroidal compound, CP8668, ((4aR,5R,6R,7R)-7-methoxy-6-(N-propylaminocarbonyl)oxy-4a,5,6,7-tetrahydro-1,3,4a,5-tetramethylbenz[f]indol-2(4H)-one). CP8668 showed selective affinity for human progesterone receptor equal in strength to other steroidal progestins. CP8668 showed no significant affinity for human glucocorticoid receptor or human estrogen receptor and very weak affinity for rat androgen receptor. In endogenous and exogenous progesterone-dependent enzyme expression assays using human mammary carcinoma T47D, CP8668 showed mixed agonist-antagonist activity. However, in a rabbit endometrial transformation test, CP8668 showed good progestational activity following s.c. and p.o. administration. These results suggest that CP8668 is a selective and orally active progesterone receptor modulator, which shows mixed agonist-antagonist activity in in vitro transcription tests and agonist activity in endometrial transformation assays in rabbits, and that it is potentially a promising lead compound for a new type of orally active progesterone receptor modulator.

In vitro and in vivo characterization of novel nonsteroidal progesterone receptor antagonists derived from the fungal metabolite PF1092C.

We studied the pharmacological effects of novel nonsteroidal progesterone receptor antagonists CP8661 and CP8754, which were synthesized from the fungal metabolite PF1092C. CP8661 possess a tetrahydrobenzindolone skeleton and CP8754 possess a tetrahydronaphthofuranone skeleton. In binding assays for steroid receptors, CP8661 and CP8754 inhibited [(3)H]-progesterone binding to human progesterone receptors (hPR), though they are less potent than RU486. CP8661 also showed moderate affinity to rat androgen receptors (rAR), although CP8754 did not. Neither compound showed affinity to human glucocorticoid receptors (hGR) or human estrogen receptors (hER). In exogeneous and endogeneous PR-dependent enzyme expression assays using human mammary carcinoma T47D, CP8661 and CP8754 showed pure antagonistic activity. In a rabbit endometrial transformation test, CP8661 and CP8754 showed anti-progestational activity by s.c. administration in a dose-dependent manner; meanwhile, these compounds showed no progestational activity at the same dose. These results suggested that CP8661 and CP8754 are in vivo effective pure progesterone receptor antagonists and presented the possibility of synthesizing pure progesterone receptor antagonists from both tetrahydronaphthofuranone and tetrahydrobenzindolone skeletons.

Priming effects of novel nonsteroidal progesterone receptor modulators CP8816 and CP8863 on the development of adenomyosis in the mouse uterus.

The possibility of therapeutic application of novel nonsteroidal progesterone receptor modulators CP8816 and CP8863 for preventing the development of uterine adenomyosis was investigated in mice. First priming effects of CP8816 on 17beta-estradiol (E2)-induced cell division in uterine tissues were examined. As a result, pretreatment with CP8816 or progesterone significantly suppressed the elevation of the mitotic activity in the luminal epithelial cells of mice treated with E2 later. Priming with CP8816 had little effect on the stromal cells, but progesterone priming caused an increase of stromal mitotic activity in mice treated with E2 later. To evaluate the inhibitory effect of these compounds on the development of adenomyosis induced experimentally by pituitary grafting, 7-week-old female mice were isografted with a single anterior pituitary in the uterus and divided into four groups. Two groups of mice were given daily subcutaneous injections of 1 mg of CP8816 or the vehicle alone for 6 weeks from the day after the grafting. Remaining two groups of mice were given oral administration of 1 mg of CP8863 or the vehicle only for 5 weeks starting one week after the grafting. The incidence of adenomyosis was significantly lower in the groups of mice treated with CP8816 and CP8863 than in the respective control groups. The mechanism by which CP compounds inhibited the development of adenomyosis might be related to their priming effects, i.e., their inhibitory effect on epithelial cell division and lack of effect on stromal cell division after subsequent exposure to E2.