The discovery of RPR 200765A, a p38 MAP kinase inhibitor displaying a good oral anti-arthritic efficacy.

RPR132331, a 2-(2-dioxanyl)imidazole, was identified as an inhibitor of tumour necrosis factor (TNF)alpha release from lipopolysaccharide (LPS)-stimulated human monocytes. An intensive programme of work exploring the biology, toxicity and physical chemistry of a novel series of inhibitors, derived from RPR132331, has led to the identification of RPR200765A, a development candidate for the treatment of rheumatoid arthritis (RA). RPR200765A is a potent and selective inhibitor of p38 MAP kinase (IC50 = 50 nM). It inhibits LPS-stimulated TNFalpha release both in vitro, from human monocytes (EC50 = 110 nM), and in vivo in Balb/c mice (ED50 = 6 mg/kg). At oral doses between 10 and 30 mg/kg/day it reduces the incidence and progression in the rat streptococcal cell wall (SCW) arthritis model when administered in either prophylactic or therapeutic dosing regimens. The compound, which is a mesylate salt and exists as a stable monohydrate, shows good oral bioavailabiltiy (F = 50% in the rat) and excellent chemical stability. The data from the SCW disease model suggests that RPR200765A could exhibit a profile of disease modifying activity in rheumatoid arthritis (RA) patients which is not observed with current drug therapies.

Mouse lymphoma thymidine kinase locus gene mutation assay: International Workshop on Genotoxicity Test Procedures Workgroup Report.

The Mouse Lymphoma Assay (MLA) Workgroup addressed and reached consensus on a number of issues. Discussion focused on five areas: (1) acceptable assay versions; (2) cytotoxicity measure; (3) 24-hr treatment; (4) microwell colony counting and sizing; and (5) data acceptability/statistical analysis. Although the International Conference on Harmonisation (ICH) indicated a preference for the microwell over the soft agar method, all of the workgroup members agreed that both versions of the MLA are equally acceptable. The workgroup agreed that it is desirable for both assay versions to use the same measure of cytotoxicity to define the acceptable and required concentration range. Currently, laboratories using the microwell version use the relative survival (RS) determined by cloning immediately after the treatment. Laboratories using the soft agar method do not obtain an RS but use the relative total growth (RTG), a combination of the relative suspension growth (RSG) during the expression period and the relative cloning efficiency determined at the time of mutant selection. The workgroup agreed to investigate the RSG, the RS, and the RTG and to develop further guidance. In the interim, the workgroup reached consensus that the RTG be used as the standard measure of cytotoxicity. The ICH recommended a 24-hr treatment in the absence of S9 when negative results are obtained with short (3-4 hr) treatments. The workgroup agreed to retain this requirement but acknowledged that more data are needed prior to making final recommendations concerning the need for and the specific protocol for the 24-hr treatment. Environ. Mol. Mutagen. 35:185-190, 2000 Published 2000 Wiley-Liss, Inc.

Modulation of P-450 IIC7 and IIIA1,2 mRNA in pre-neoplastic liver. Effect of promotion by phenobarbital.

P-450 IIC7 and IIIA2 mRNAs are constitutively expressed in the hepatic tissue under developmental control. Both forms--as well as IIIA1, 90% homologous to IIIA2 mRNA--display positive modulation by phenobarbital a prototype inducer of the liver monooxygenases and a strong promoter of experimental chemical hepatocarcinogenesis. In the present work the variations in the concentration of these P-450 mRNA were studied in rats submitted to the hepatocarcinogenic protocol of Solt and Farber. We demonstrate that a decrease in the relative concentrations of P-450 IIC7 and IIIA1, 2 mRNA is set up along the tumor promotion stage. Animals--starting the experimental carcinogenic protocol at pubertal age--show a partial inhibition of the physiological expression of P-450 IIIA1,2 mRNA associated to male sex maturation. Administration of phenobarbital results in an acceleration of the pre-neoplastic process which is concomitant with an induction of P-450 IIC7 as well as IIIA1,2 at the earlier promotion stages. P-450 mRNA concentration markedly decreases as the preneoplastic process develops. While an impaired P-450 IIIA1,2 mRNA relative abundance is observed, an inversion of the modulation of P-450 IIC7 as well as of the male phenotype marker alpha-2u-globulin mRNA arises as the tumor promotion stage progresses, both mRNA becoming repressed in response to phenobarbital.

[Modification of the cellular cytoskeleton and hepatic tumor promotion]. / Altérations du cytosquelette cellulaire et promotion tumorale hépatique.

Non transformed epithelial hepatic cells (established cell line and adult rat hepatocytes) treated by liver tumor promoters, phenobarbital and biliverdin, for 24 and 48 h showed a fragmentation and loss of F-actin and a depolymerisation of microtubules. This pattern closely resembles that of transformed cells which were not susceptible to the action of promoters. In liver preneoplastic nodules obtained from rats submitted to an initiation-promotion process, actin almost completely disappeared with the concomitant appearance of a characteristic enzymatic pattern rich in GGT and GST-P. Therefore, cytoskeleton of hepatic cells is a target for tumor promoters and could play a role in promotion mechanism.

Effect of rat developmental stage at initiation on the expression of biochemical markers during liver tumor promotion.

The phenotypic response of rat liver to a carcinogenic protocol involving initiation/selection and promotion with and without phenobarbital (PB) feeding was studied in pubertal and adult male rats. Considering the early presence of preneoplastic nodular areas, it appeared that pubertal rats, initiated at 6-7 weeks, presented a higher susceptibility to the protocol than adult rats initiated at 9-10 weeks. Altered liver phenotype was characterized by: (1) gamma-glutamyl-transpeptidase (GGT) and glutathione S-transferase (GST) activities; (2) the expression of two forms of cytochrome P-450; de novo PB-inducible P-450 II B 1,2 and P-450 II C 7 normally expressed in 45-day-old rats and PB-inducible, and (3) the expression of albumin and alpha-fetoprotein cDNAs. In the absence of PB, the susceptibility of pubertal rat liver to hepatocarcinogenesis was related to a special metabolic phenotype enriched in GGT and GST activities by comparison with the quasi-normal expression of both P-450s. Adult rat liver presented a less altered pattern closer to that of noninitiated rat liver. During PB promotion, the loss of PB inducibility of P-450 II C 7 in pubertal rat liver suggested that the hormonal status of the animals could interact with initiation to modulate specific gene expression. The late phase of PB promotion revealed the loss of highly differentiated functions (P-450s, albumin), whereas enzymatic markers associated with preneoplastic foci showed a persistent high expression.