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Title: The relevance of microRNAs and circadian rhythms in drug safety
Author: Starkey Lewis, Philip
ISNI:       0000 0004 2745 7086
Awarding Body: University of Liverpool
Current Institution: University of Liverpool
Date of Award: 2012
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Drug-induced liver injury (DILI) is a serious adverse drug reaction (ADR) that is frequently encountered during drug development, representing a major cause of drug attrition. Furthermore, DILI is also a serious concern in the clinic, accounting for approximately half of all acute liver failure cases. Paracetamol overdose (acetaminophen; APAP) accounts for the majority of DILI-associated cases of ALF encountered in patients. The identification and development of novel biomarkers of DILI that are sensitive, specific, and rise early during hepatotoxicity are urgently required in the clinic and in the laboratory. Two liver-specific microRNAs (miRNAs) have recently been described that serve as sensitive and early markers of APAP-induced acute liver injury (APAP-induced ALI) in a mouse model. Together with the superior liver-enrichment of some liver-enriched miRNAs, these potential markers need to be assessed in patients for the clinical promise. Moreover, further work is warranted to test these potential markers in alternative pre-clinical models with other compounds to gain a better understanding regarding sensitivity of release, mechanism of release and circulatory kinetics. Furthermore, in separate work discussed in this thesis, the mammalian biological clock has been found to exert a powerful influence on the physiology of mammalian systems. This regulation hinges on the complex interplay between the clock genes and their products that oscillate over a twenty-four hour period and promote a diurnal variation in numerous output pathways. Emerging evidence suggests that the efficacy and toxicity of many drugs follow a diurnal rhythm and that this may be at least partly attributable to the clock-mediated regulation of drug targets and pathways of drug metabolism. APAP and FS represent two compounds that elicit hepatotoxicity in the mouse through two distinct mechanisms. Both APAP and FS are known to exhibit circadian variation in their toxicology and/or pharmacology. However, little is known about the molecular mechanisms that govern these differences in circadian variation. Two liver-enriched miRNAs (miR-122 and miR-192) were tested alongside serum ALT activity, the gold-standard marker of ALI, for sensitivity and time of release in a mouse model of APAP-induced ALI. At 2 hours after APAP administration, miR-122 (ΔΔCt 75.0, P=0.02) was significantly higher compared to controls (ΔΔCt 4.1) while ALT levels were in the normal range (21 U/L) indicating earlier release of miR-122. In a sensitivity study, miR-122 was not more sensitive than ALT at a 300 mg/kg dose of APAP compared to controls (mean values 300 mg/kg vs 0 mg/kg: ALT = 491 U/L vs 38.1 U/L; miR-122 = ΔΔCt 572.9 vs ΔΔCt 209.4). In patients, serum miR-122 and miR-192 were substantially higher in APAP-induced ALI patients, compared to healthy controls (median ΔΔCt miR-122: 1,265 versus 12.1, P < 0.0001; miR-192: 6.9 versus 0.44 P < 0.0001). A heart-enriched miR-1 showed no difference between APAP-ALI patients and controls, whereas miR-218 (brain-enriched) was slightly higher in the APAP-ALI cohort (ΔΔCt 0.17 versus ΔΔCt 0.07 P = 0.01). In a cohort comprised of patients who presented early (median time of presentation since APAP overdose = 8 hours), miR-122 was significantly raised in patients who develop ALI (> 3 x ULN serum ALT activity) compared to those that did not (median ΔΔCt 3.48 vs ΔΔCt 0.16, P<0.0001). In contrast, presentation ALT levels were not different between patients who developed ALI compared to those that did not (median ALT = 21 U/L vs 19 U/L). Moreover, miR-122 was significantly raised in patients who develop coagulopathy (INR > 1.3) compared to those that did not (ΔΔCt vs 3.48 vs ΔΔCt 0.17, P=0.0004). In contrast, presentation ALT levels were not different between patients who developed ALI iv compared to those that did not (median ALT = 21 U/L vs 19 U/L). In chronotoxicity studies, both APAP exhibited greater toxicity in the evening (mean ALT = 12785, 66% survival) compared to morning (mean ALT = 380, 100% survival) whereas FS showed greater toxicity after morning administration (mean ALT=561, 100% survival) compared to evening administration (mean ALT = 69.2, 100% survival). Circadian variation in APAP-induced ALI was associated with 38% lower (P=0.003) GSH levels and 20% higher (P=0.024) Cyp2e1 levels at 21:00h compared to 09:00. This work confirms that miR-122 is released earlier than ALT in a young mouse model of APAP-induced ALI. Furthermore, it is shown for the first time that circulating liver enriched miRNAs are higher in patients following an APAP overdose. Also, plasma miR-122 is raised at emergency room presentation when serum ALT activity is in the normal range. Further clinical development of blood-based miR-122 is warranted, this work suggests that miR-122 analysis at the point of hospital admission can predict risk of subsequent liver injury in patients. Finally, APAP and FS exhibit circadian variation in their toxicity in a mouse model associated with circadian variation with genes involved in drug metabolism and drug detoxification. Profiling of the hepatic proteome over the circadian phase is now warranted.
Supervisor: Goldring, Christopher; Park, Kevin; Moggs, Jonathan Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available
Keywords: RM Therapeutics. Pharmacology