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.

Validation of the in vivo CD1 mouse splenocyte micronucleus test.

In order to validate the in vivo micronucleus test in mouse splenocytes using the cytokinesis block method, 14 compounds with various mechanisms of action were tested: three direct alkylating agents (mitomycin C, ethylnitrosourea, beta-propiolactone), seven indirect alkylating agents (cyclophosphamide, benzo[a]pyrene, diethylnitrosamine, dimethylnitrosamine, 4-aminophenol, 4-aminobiphenyl, 1,1-dimethylhydrazine), two intercalating agents (acridine orange, ethidium bromide) and two spindle poisons (vincristine, colchicine). Male mice were dosed once with the compound, and spleen samples were taken 2 or 14 days after treatment. A significant increase in the binucleated micronucleated splenocyte rate was observed with all the alkylating and intercalating agents at at least one sampling time. In contrast, no increase in the binucleated micronucleated splenocyte rate was observed with the spindle poisons. In conclusion, under these experimental conditions, this in vivo test seems appropriate for the detection of clastogenic compounds including compounds that cannot be detected in the bone marrow micronucleus test. The limit of this test, as expected, is the lack of detection of aneugenic compounds.

Detection of micronuclei after exposure to mitomycin C, cyclophosphamide and diethylnitrosamine by the in vivo micronucleus test in mouse splenocytes.

A micronucleus detection test using mouse splenocytes has been adapted from a method previously carried out using human lymphocytes. An ex vivo protocol was chosen: male C57B16 mice were treated with various compounds. Splenocytes were then isolated and placed in culture for 48 h and stimulated with concanavalin A and conditioned medium. The cytokinesis-block method reported by Fenech and Morley was used to detect and score micronuclei in the proliferating lymphocytes (3 micrograms/ml of cytochalasin B for 16 h). Three mutagenic clastogens, mitomycin C (MMC), a direct alkylating agent (0.4, 0.8 and 1.6 mg/kg), cyclophosphamide (CP), an indirect alkylating agent (25, 50 and 100 mg/kg) and diethylnitrosamine (DEN), an indirect alkylating agent with labile metabolites (25, 50 and 100 mg/kg), were tested at four sampling times (2, 4, 8 and 15 days). All three compounds were detected from 48 h after treatment. This method was indeed able to detect clastogenic compounds normally detected by the mouse bone marrow micronucleus test (MMC, CP) as well as a compound with labile metabolites which is not usually detected by this test (DEN). Maximum micronucleus induction was observed after 4 days for MMC, 2 days for CP and 15 days for DEN. This method thus appears to offer a potentially useful toxicological test for assessing in vivo clastogenicity.

Flow cytometric detection of erythropoietic cytotoxicity in mouse bone marrow.

Erythroblast proliferation and maturation in bone marrow are the processes leading to the formation of polychromatic erythrocytes (PE) and normochromatic erythrocytes (NE), respectively. PE contain RNA but no DNA, and can therefore be distinguished both from NE (which lack both RNA and DNA) and from nucleated cells (which contain both DNA and RNA). Cytotoxic agents that induce impairment of the maturation process change the PE:NE ratio. We have developed a simple and rapid method of determining the PE:NE ratio, based on flow cytometric analysis of formaldehyde-fixed, acridine orange (AO)-stained cells. The effects of cyclophosphamide (CP), mitomycin C (MMC), and vincristine (VC) were tested and the PE:NE ratio was evaluated over 7 days of treatment. In this study we monitored the kinetics of these compounds and were able to demonstrate both a time- and a dose-dependent effect. We detected a difference between the effects of the alkylating agents tested and those induced by the spindle inhibitor tested. Flow cytometry of fixed bone marrow samples stained with AO provides more information, better and more rapid statistical analysis, than conventional microscopic methods for counting the PE:NE ratio.