Molecular basis for the anti-inflammatory properties of chlomethiazole

Chlomethiazole (CMZ) exerts neuroprotective effects in animal models of stroke, and its clinical efficacy is currently evaluated in acute stroke patients. CMZ also protects the liver from alcohol- induced oxidative damage by inhibiting the cytochrome P450 2E1 (CYP2E1), as well as inflammation associated with alcohol liver disease (ALD) by inhibiting cytokine expression. The aim of the present study was to characterise the molecular mechanisms of action of CMZ, as a basis to understand the protective effects of the drug in both liver and brain.

First, we described a novel post-translational mechanism of inhibition of CYP2E1 by CMZ in Fao rat hepatoma cells. The effect of CMZ on CYP2E1 was insensitive to cycloheximide treatment, indicating that it might be exerted at the level of enzyme degradation. Thus, direct inhibition of CYP2E1 may account for at least some of the protective effects of CMZ in the liver.

To investigate whether the anti-inflammatory properties of CMZ in the liver could be relevant also for its neuroprotective effects, we used primary cultures of rat cortical glial cells stimulated by lipopolysaccharide (LPS). We found that CMZ inhibited LPS-inducible c-fos and c-jun expression, and subsequently AP-1 activation, through inhibition of p38 MAP kinase (p38 MAPK) activity. CMZ inhibited c-fos expression induced by inflammatory stimuli such as LPS or IL-10 but not glutamate or mitogenic stimuli exemplified by TPA.

The induction by LPS of c-fos expression was found to occur via the activation of Elk 1 and CREB/ATF1 transcription factors at the SIZE and CRE elements of the c-fos proximal promoter. Transactivation mediated by either Elk1 or CREB was found to be dependent on p38 MAPK, and was inhibited by CMZ. The mode of inhibition of p38 MAPK activity by CMZ, albeit yet unidentified, was found to be distinct from the competitive inhibition elicited by pyridinyl imidazoles. However, similarly to pyridinyl imidazoles CMZ inhibited p38 MAPK catalysis but not its activation by phosphorylation.

Since p38 MAPK is involved in cytokine biosynthesis, and the pro-inflammatory cytokine IL-1beta is an important mediator of ischaemic brain damage, we evaluated the effect of CMZ on IL-1beta expression. Inhibition of p38 MAPK activity did not affect IL-1beta biosynthesis or secretion from glial cells but inhibited IL-1beta effects such as iNOS expression and NO production.

These results further establish the anti-inflammatory properties of CMZ, and suggest that inhibition of p38 MAPK-mediated IL-1beta effects in glial cells may be a potential mechanism for the neuroprotection elicited by CMZ in vivo. The inhibition of p38 MAPK by CMZ also uncovers a new potential for the compound in treating, apart from stroke, a panel of acute or chronic inflammatory conditions where p38 MAPK would play a role.

List of scientific papers

I. Simi A, Ingelman-Sundberg M (1999). Post-translational inhibition of cytochrome P-450 2E1 expression by chlomethiazole in Fao hepatoma cells. J Pharmacol Exp Ther. 289(2): 847-52.
https://pubmed.ncbi.nlm.nih.gov/10215662

II. Simi A, Ingelman-Sundberg M, Tindberg N (2000). Neuroprotective agent chlomethiazole attenuates c-fos, c-jun, and AP-1 activation through inhibition of p38 MAP kinase. J Cereb Blood Flow Metab. 20(7): 1077-88.
https://pubmed.ncbi.nlm.nih.gov/10908041

III. Simi A, Porsmyr-Palmertz M, Hjerten A, Ingelman-Sundberg M, Tindberg N (2002). The neuroprotective agents chlomethiazole and SB203580 inhibit IL-1beta signalling but not its biosynthesis in rat cortical glial cells. J Neurochem. 83(3): 727-37.
https://pubmed.ncbi.nlm.nih.gov/12390534

IV. Simi A, Edling Y, Ingelman-Sundberg M, Tindberg N (2003). c-fos activation by lipopolysaccharide in glial cells via p38-MAPK-dependent activation of SRE or CRE elements. [Manuscript]

V. Edling Y, Simi A, Ingelman-Sundberg M, Tindberg N (2003). Glutamate activates c-fos transcription in glial cells through a CREB-independent mechanism. [Manuscript]