Clinical And Basic Research On The Phenomenon Of Adaptation To Drug-induced Liver Injury

Background:Drug-induced liver injury is a major problem that threaten clinical drug safety, but there exists the phenomenon of adaptation to drug-induced liver injury.The implication of adaptation to liver injury is that some drugs,which could induce serious idiosyncratic DILI, could cause temporary increases of liver biochemistry, despite continuing the same drugs and doses, increased serum liver biochemistry could return to normal.An understanding of drug adaptation help to reduce unnecessary drug withdrawal. There were some reports on the the phenomenon of adaptation to drug-induced liver injury in the foreign research and some drug trials of the United States FDA,but there is a lack of the similar studies about the adaptation in China. Therefore, it is necessary to normatively collect hospitalized cases with the phenomenon of adaptation to drug-induced liver injury and analyze clinical information,so that to provide more reliable clinical data about the phenomenon of adaptation of drug-induced liver injury in our country.Antituberculosis drugs are the main cause of acute drug-induced liver injury in our country. Our previous study found that rifampicin could cause cholestasis with typical biochemical changes in mice, biochemical indicators of cholestasis including alkaline phosphatase, total bilirubin and bilirubin showed the tendency of decrease while continuing administration of RFP.Further studies are needed to establish and confirm the model of the phenomenon of adaptation to rifampicin induced cholestasis in rats in the way of dynamic observation of continuous blood testing of the same animals, and then to explore molecular mechanism of the phenomenon of adaptation.Aims:1. To investigate clinical data of inpatients with the phenomenon of adaptationto DILI in recent 10 years in Anhui province, to analyze clinical indicators, and to summarize the clinical characteristics.2. To establish animal model of cholestasis caused by long-term administration of rifampicin, in which the phenomenon of adaptation to cholestasis was obseved from the dynamic changes of rats’ serum liver biochemistry levels, and then to explore molecular mechanism of the phenomenon of adaptation preliminarily.Methods:1. We formulated unified form according to standardized data collection, and used standardized methods for registration and follow-up. According to data integrity, the cases with complete data for analysis were filtered among the cases with the phenomenon of adaptation to DILI that were submitted by 7 hospitals in in Anhui province. Causality of drugs and liver injury in these cases was assessed by using RUCAM scoring system.Each case was made diagnosis or exclusion by the expert conference.In accordance with whether discontinue the drugs after emergence of drug-induced liver injury,we divided the patients into withdrawal group and continuing group respectively. The severity of liver injury was graded according to biochemical parameters and symptoms. It was analyzed the types of patients with adaptation, the relationship between liver injury and the time and features of liver biochemistry.2. A total of 36 rats were randomly divided into RFP and control groups. One hundred mg/kg of RFP was administered by gavage to the rats every morning for 7 weeks, while the same amount of 0.9% saline was administered in the control group. 0.5 milliliter of blood was drawn from the orbital venous plexus at day 0, 7,14,21,28,35,42 and 49 for liver biochemistry assays. The liver tissues were autopsied to observe liver pathology and ultrastructural changes, and examine the expression of multidrug resistance protein 2(Mrp2), bile salt export pump(Bsep), multidrug resistance protein 4(Mrp4) and sodium-dependent taurocholate cotransporting polypeptide(Ntcp) by quantitative RT-PCR and Western blotting.Results:1. Clinical research on the phenomenon of adaptation to drug-induced liver injury1). A total of 50 cases were collected from seven hospitals in Anhui province. 7 cases of incomplete cases were eliminated. No case was considered highly probably related by RUCAM, 16 probably related, 27 possibly related,0 possibly unrelated,0 unrelated. Reports in continuing group scored lower on the RUCAM, with a median score of 4, range:3-6,than in withdrawal group(6;[5-7]). 3 cases were ruled out in expert conference. 40 cases were determined the phenomenon of adaptation to drug-induced liver injury. There are 25 male patients and 15 female patients. The average age was 39.22 years(aged 20 to 74 years old,).There are 24 patients in continuing group and 16 patients in withdrawal group. Drugs of hepatic adaptation were antituberculosis drug(39 cases) and antineoplastic drugs(1 case).2). There were 11 cases with ALT level at grade 0, 12 cases with ALT level at grade 1, 13 cases with ALT level at grade 2, 2 cases with ALT level at grade 3, 2 cases with ALT level at grade 4. There were 11 cases with AST level at grade 0, 9 cases with AST level at grade 1, 17 cases with AST level at grade 2, 2 cases with AST level at grade 3, 1 case with AST level at grade 4. TBIL level in 36 cases was normal range. There were 3 cases with TBIL level at grade 1, 1 case with TBIL level at grade 1. There were 80% of the patients with peak of liver biochemistry for mild to moderate rise(under garde 2), 8 cases with liver biochemistry for more than grade 3.3). 36 cases were classified as hepatocellular injury,3 cases classified as cholestatic injury,and 1 case classified as mixed injury.The severity grading of liver injury was level 1 in 38 patients. The severity grading was level 2 in two patienis.4).The overall median incubation period was 10.5 days(2-138 days). The median time when transaminase levels declined to normal was 8 days(5-67days). The median time of re-treatment in withdrawal group is 12.5 days(7-68days). There was no significant difference in incubation period the time to recovery between withdrawal group and continuing group(P> 0.05).2. Basic research on the phenomenon of adaptation to drug-induced liver injury1). The present study showed no statistically significant change in the ALT, AST, ALP, TBIL and DBIL level at day 7,14 and 49 in the control group. The ALT, AST level of the RFP group had no significantly increased compared with the control group.The serum ALP, TBILand DBIL level and liver and serum TBA level started to rise at day 7(P<0.01),reached the peak in week 1 or 2, and then declined gradually over time(P<0.05) in RFP group. In addition, the TBA level in liver and serum peaked on day 7 and then was significantly decreased on day 49(P < 0.05).Mild hepatic steatosis and inflammation were observed in the RFP group by light microscopy. Swelling mitochondria, dilation of endoplasmic reticulum, bile capillary dilatation and cholestasis were detected under electron microscopy in RFP-treated rats.2).The m RNA levels of Mrp2, Bsep and NTCP were unchanged over time, but MRP4 m RNA levels tended to increase at day 7,14 in the RFP group and were significantly increased compared to the control at day 49.(P<0.01). The Mrp4 levels were significantly increased on weeks 1, 2 and 7. Bsep protein were significantly increased on weeks 2 and 7.The expression of Ntcp protein was significantly decreased at day 49.(P<0.01). Ntcp protein level was decreased compared to the control on week 7. Mrp2 protein did not change during cholestasis. It was preliminary found that the phenomenon of adaptation to rifampicin induced cholestasis was related to the adaptive regulation of hepatobiliary transporters.Conclusions:1. Characteristics of the phenomenon of adaptation to drug-induced liver injury are transient, mild to moderate increase of liver biochemistry, while continuing the same drugs, increased serum liver biochemistry could return to normal. The clinical pattern in the most of the cases with the phenomenon of adaptation to DILI was hepatocellular injury. Cholestatic and mixed liver injury was rare.The severity of liver injury in the cases with the phenomenon of adaptation to DILI was under grade 2.2. The model of the phenomenon of adaptation to rifampicin induced cholestasis in rats was confirmed and established on the basis of dynamic observation of continuous liver biochemistry. It was found that the phenomenon of adaptation to rifampicin induced cholestasis was related to the adaptive regulation of hepatobiliary transporters, which was mediated through increasing export of bile acids by elevating of Mrp4 and Bsep expression, and limiting uptake of bile acids through inhibiting of Ntcp expression.