The corticosteroid dexamethasone, which is used to treat numerous health conditions, remains the first-line treatment for patients hospitalized with COVID-19 requiring oxygen. Current British National Formulary prescribing advice warns of a "severe theoretical" or "severe anecdotal" risk of drug-drug interactions between dexamethasone and 138 different medications. In humans, dexamethasone is eliminated via the cytochrome P450 monooxygenase system, particularly CYP3A4. It is also described as a human cytochrome P450-inducing agent. To establish factors that affect concomitant therapy and dexamethasone efficacy in the treatment of COVID-19, we used a unique mouse model humanized for cytochrome P450s and the transcription factors that regulate their expression, the pregnane X receptor, and the constitutive androstane receptor. We found that induction of CYP3A4 with the anticancer drug dabrafenib or the herbal medicine St John's wort profoundly reduced dexamethasone exposure. These data suggest that comedications that induce cytochrome P450 expression can have a marked effect on dexamethasone exposure and, potentially, clinical efficacy. We also observed that rather than increasing CYP3A4 expression, dexamethasone at doses equivalent to or higher than those used in the treatment of COVID-19 reduced CYP3A4 expression and increased exposure to dabrafenib. These data indicate the need for a clinical trial to establish the risk of overexposure to comedications during dexamethasone treatment, including the treatment of COVID-19. SIGNIFICANCE STATEMENT: Current prescribing advice identifies a potential theoretical risk of severe side effects when dexamethasone, one of the most widely used drugs in clinical practice, is coadministered with many other drugs; it is, however, difficult to define the magnitude of this risk for specific drug combinations. We describe the use of cytochrome P450-humanized 8HUM mice to predict drug-drug interactions in patients on polypharmacy, a means of generating data that could better inform clinicians regarding foreseeable drug-drug interactions involving dexamethasone.
Transforming growth factor-β (TGF-β) is a pleiotropic growth factor implicated in the development of atherosclerosis for its role in mediating glycosaminoglycan (GAG) chain hyperelongation on the proteoglycan biglycan, a phenomenon that increases the binding of atherogenic lipoproteins in the vessel wall. Phosphorylation of the transcription factor Smad has emerged as a critical step in the signaling pathways that control the synthesis of biglycan, both the core protein and the GAG chains. We have used flavopiridol, a well-known cyclin-dependent kinase inhibitor, to study the role of linker region phosphorylation in the TGF-β-stimulated synthesis of biglycan. We used radiosulfate incorporation and SDS-PAGE to assess proteoglycan synthesis, real-time polymerase chain reaction to assess gene expression, and chromatin immunoprecipitation to assess the binding of Smads to the promoter region of GAG Synthesizing genes. Flavopiridol blocked TGF-β-stimulated synthesis of mRNA for the GAG synthesizing enzymes, and chondroitin 4-sulfotransferase (C4ST-1), chondroitin sulfate synthase-1 (ChSy-1) and TGF-β-mediated proteoglycans synthesis as well as GAG hyperelongation. Flavopiridol blocked TGF-β-stimulated Smad2 phosphorylation at both the serine triplet and the isolated threonine residue in the linker region. The binding of Smad to the promoter region of the C4ST-1 and ChSy-1 genes was stimulated by TGF-β, and this response was blocked by flavopiridol, demonstrating that linker region phosphorylated Smad can pass to the nucleus and positively regulate transcription. These results demonstrate the validity of the kinases, which phosphorylate the Smad linker region as potential therapeutic target(s) for the development of an agent to prevent atherosclerosis.
Inflammatory injury of the endothelium leads to apoptosis and endothelial dysfunction. The current study explored the effect and mechanisms of paeonol in inflammation-induced apoptosis and endothelial dysfunction induced by lipopolysaccharides (LPSs). The effects of paeonol on LPS-induced inflammatory injury were assessed by Western blotting, flow cytometry and reactive oxygen species (ROS) measurement in human umbilical vein endothelial cells (HUVECs) and C57BL/6J mice. Vascular reactivity of isolated mouse aortae was examined using wire myographs. The exposure of HUVECs to LPS increased the protein presence of Toll-like receptor 4 (TLR4), bone morphogenic protein 4 (BMP4), BMP receptor type 1A, nicotinamide adenine dinucleotide phosphate oxidase subunit 2, mitogen-activated protein kinase (MAPK), inducible nitric oxide synthase (iNOS), and cleaved caspase 3, as well as decreased it in phosphorylated endothelial nitric oxide synthase; these effects were prevented by treatment with paeonol. Similarly, cotreatment with paeonol reversed BMP4-induced apoptosis in HUVECs. Relaxation in response to the endothelium-dependent vasodilator acetylcholine were impaired in mouse aortae after exposure to LPSs; this endothelial dysfunction was reversed by cotreatment with paeonol, noggin (a BMP4 inhibitor), TAK242 (TLR4 antagonist), apocynin (an ROS scavenger), MAPK inhibitors, and AG (an iNOS inhibitor). BMP4 small interfering RNAs (siRNAs) abolished LPS-induced upregulation of BMP4 and cleaved caspase 3 protein, but not in cells treated with TLR4 siRNA and vice versa. The silencing of TLR4 and BMP4 abolished the inhibitory effects of paeonol on LPS-induced activation of cleaved caspase 3. The present results demonstrate that paeonol reduces LPS-induced endothelial dysfunction and apoptosis by inhibiting TLR4 and BMP4 signaling independently.