CROSSREFERENCES

[0001] Priority is claimed of European patent application no. 21 181 910.7 that was filed on June 25, 2021

FIELD OF THE INVENTION

[0002] The invention relates to inhibitors of sodium taurocholate co-transporting poly-peptide (NTCP) for use in the treatment of acetaminophen overdose, particularly to ameliorating and preventing the consequences of acetaminophen intoxication.

BACKGROUND ART

[0003] Acetaminophen (APAP) intoxication is the most common cause of acute liver failure in the USA and UK, and is also a major underlying cause in many other countries {Bateman, D.N. in Critical W. et al. N Engl JMed 369, 2525-2534 (2013)). It also represents the second most common reason for liver transplantation worldwide, and occurs more frequently in children than adults (Agrawal, S. et al. in StatP earls (StatPearls PublishingCopyright © 2021, StatP earls Publishing LLC., Treasure Island (FL), 2021)). About half of the intoxications result from unintentional overdoses ( Caparrotta , T.M., Antoine, D.J. et al. Eur J Clin Pharmacol 74, 147-160 (2018); Chiew, A.L., et al. Clin Toxicol (Phila) 58, 1063-1066 (2020)).

[0004] The mechanisms of APAP-induced hepatotoxicity have been intensively studied ( Ni , H.M., et al. J Hepatol 65, 354-362 (2016); Woolbright, B.L. et al. Hepatol 66, 836-848 (2017)). For instance, it is well accepted that APAP is rapidly absorbed from the intestine and transported to the liver, where at therapeutic doses a small fraction is metabolically activated by cytochrome P450 enzymes, mostly Cyp2El and CyplA2, to the reactive N-acetyl-p-benzoquinone imine (NAPQI), which is scavenged by hepatic glutathione (GSH) and excreted through bile ( Ramachandran , A. et al. Semin Liver Dis 39, 221- 234 (2019)). However, an overdose of APAP leads to the depletion of hepatic GSH stores, as well as the formation of NAPQI-protein adducts. Mitochondrial protein adducts trigger oxidative stress, which in turn activates c-jun N-terminal kinase (JNK) in the cytoplasm and induces its translocation to mito chondria, which further enhances oxidative stress that finally causes the opening of the membrane per meability transition pore, membrane potential breakdown and cell death {Ramachandran et al).

[0005] Despite extensive research on the mechanisms of APAP-induced liver injury, only one antidote, N-acetylcysteine (NAC), is approved for clinical application {Akakpo, J.Y., et al. Expert Opin Drug Metab Toxicol 16, 1039-1050 (2020)). NAC helps to restore the depleted GSH stores in hepatocytes, and is maximally effective only when given within 8 hours after APAP ingestion (. Bateman et a ). How ever, patients often seek medical attention only after the initial onset of symptoms, usually ~24 hours after APAP ingestion, which is often too late, and death generally follows 3-4 days after intoxication, or even in some cases after up to 14 days ( Bateman et al). Therefore, there is an urgent need for further therapies, ideally with a longer therapeutic window.

[0006] It is well-known that APAP overdose can lead to increased bile acid (BA) concentrations in blood ( Woolbright , B.L., et al. Toxicol Sci 142, 436-444 (2014)). The initial interpretation that elevated BA concentrations simply signify compromised liver functions has since been challenged ( Woolbright et al. 2014; Trottier, J, et al. PLoS One 6, e22094 (2011); Woolbright, B.L., et al. Toxicol Appl Phar macol 273, 524-531 (2013); Zhang, Y., et al. Liver Int 32, 58-69 (2012)). One hypothesis is that there is a breach of the blood-bile barrier, since BA are present in the biliary tract in millimolar concentrations ( Woolbright et al. 2014). Alternatively, it has been proposed that BA export from hepatocytes may be compromised {Woolbright et al. 2014). Differentiation of these mechanisms is important, since both would require different therapeutic strategies; however, due to technical challenges neither has yet been explored.

[0007] A cholic acid-containing diet given one week prior to APAP administration was reported to reduce liver damage and enhance regeneration; conversely, depletion of BA by cholestyramine feeding aggravated APAP-induced liver injury ( Bhushan , B., et al. Am J Pathol 183, 1518-1526 (2013)).

[0008] US 2007 0049640 Al relates to acetylcysteine compositions in solution, comprising acetylcys teine and which are substantially free of metal chelating agents, such as EDTA. The compositions and methods are useful in the treatment of acetaminophen overdose, acute liver failure, various cancers, methacrylonitrile poisoning, reperfusion injury during cardio bypass surgery, and radiocontrast-induced nephropathy, and can also be used as a mucolytic agent.

[0009] US 2011 0172298 Al relates to the use of erdosteine as antidote in intoxications, especially in heavy metals like lead or mercury, and paracetamol.

[0010] US 2012 0022161 Al relates to a method of treating a patient with a pharmaceutical composi tion, comprising: administering to a patient in need thereof a therapeutically effective amount of a phar maceutically acceptable composition comprising acetylcysteine and a pH adjusting agent, wherein the composition is substantially free of chelating agents, the pharmaceutical composition providing for a reduction in side effects as compared to an acetylcysteine composition containing chelating agents, wherein the composition is administered to treat acetaminophen overdose and the therapeutically effec tive amount is administered intravenously at a dose of about 200 mg/kg over a period of about 3 to about 5 hours followed by a second dose at 100 mg/kg over 16 hours. [0011] US 2013 0039897 A1 relates to a method of reducing the number of neutrophils in a body region comprising administering a dipeptidyl peptidase-IV (DPPIV) composition to the body region in an amount and for a time sufficient to suppress neutrophil movement into the body region or enhancing neutrophil movement out of the body region, wherein the body region comprises the liver, and wherein the disease is acetaminophen overdose.

[0012] US 2015 0080348 A1 relates to pharmaceutical compositions including combinations of pro tective agents selected from isosilybin B, methylsulfonylmethane (MSM), phosphatidylcholine, cysteine (Cys), seleno-cysteine (Se-Cys), ribose-cysteine (RibCys), N-acetylcysteine (NAC), N-acetylcysteine- amide (AD4), methionine (Met) and S-adenosylmethionine (SAM) for reducing and/or preventing drug- induced toxicity, such as acetaminophen-induced toxicity.

[0013] US 2019 0015424 A1 relates to a method of treating and/or protecting against acute liver failure induced by an acetaminophen overdose in an individual, comprising (a) administering to the individual an effective amount of a first active agent which replenishes, or decreases a loss of, functional glutathi one in the individual, wherein the first active agent comprises N-acetylcysteine (NAC), and (b) admin istering an effective amount of a second active agent comprising a manganese complex selected from the group consisting of (i) a calcium manganese mixed metal complex of N,N'-bis-(pyridoxal-5-phos- phate)-ethylenediamine-N,N'-diacetic acid (DPDP) having a molar ratio of calcium to manganese in a range of from 1 to 10, or a pharmaceutically acceptable salt thereof, (ii) a mixture of manganese DPDP (MnDPDP), or a pharmaceutically acceptable salt thereof, and a non-manganese-containing DPDP com pound, or (iii) a mixture of manganese pyridoxyl ethylenediamine (MnPUED), or a pharmaceutically acceptable salt thereof, and a non-manganese-containing pyridoxyl ethylenediamine (PUED) compound, to the individual.

[0014] KR 101 983 298 B1 relates to a pharmaceutical composition for preventing or treating inflam matory disease mediated by inflammation using ezetimibe, which is conventionally known as a choles terol absorption inhibitor, as an active ingredient.

[0015] JP 2011 046677 relates to relates to a pharmaceutical composition containing acetaminophen which can reduce the development of liver function failure by chronic administration, large ingestion, or simultaneous ingestion with alcohol.

[0016] WO 2011/074001 A2 relates to a herbal composition comprises potent liver specific herbs which improves the liver function and is also helpful in cleaning the toxic accumulations and detoxifying poisons in liver. The herbal composition comprises extract of Andrographis paniculata; extract of Cur cuma longa; extract of Glycyrrhiza glabra; and extract of Terminalia chebula. [0017] T.K. Yim et al., Physiother Res, 15(7), 2001, 589-592 relates to the hepatoprotective action of an oleanolic acid-enriched extract of ligustrum lucidum fruits which is mediated through an enhance ment on hepatic glutathione regeneration capacity in mice.

[0018] A. Walubo et al., Hum Exp Toxicol, 23(1), 2004, 49-54 relates to the role of cytochrome-P450 inhibitors in the prevention of hepatotoxicity after paracetamol overdose in rats.

[0019] L. Aleksunes et al., Toxicol Sci., 89(2), 2006, 370-379 relates to coordinated expression of mul tidrug resistance-associated proteins (Mrps) in mouse liver during toxicant-induced injury.

[0020] R. Avizeh et al., J Vet Pharmacol Ther, 33(1), 2010, 95-99 relates to an evaluation of prophy lactic and therapeutic effects of silymarin and N-acetylcysteine in acetaminophen-induced hepatotoxi city in cats.

[0021] Y. Igusa et al., J Gastroent, 47(4), 2011, 433-443 relates to an investigation that loss of autoph- agy promotes murine acetaminophen hepatotoxicity.

[0022] A. A. Fouad et al., Eur J Pharmacol, 693(1), 2012, 64-71 relates to an investigation of the ther apeutic potential of telmisartan in mice exposed to acute hepatotoxicity induced by a single dose of acetaminophen (500 mg/kg, p.o.).

[0023] B.L. Woolbright et al., Toxicol Sci, 142(2), 2014, 436-444 relates to glycodeoxycholic acid levels as prognostic biomarker in acetaminophen-induced acute liver failure patients.

[0024] N.E. Bektur et al., Toxicol Ind Health, 32(4), 2016, 589-600 relates to protective effects of si lymarin against acetaminophen-induced hepatoxicity and nephrotoxicity in mice.

[0025] A. Mathur et al., Food and Chemical Toxicology, 89(6), 2016, 19-31 relates to PHLPP2 down regulation that influences nuclear Nrf2 stability via Akt-1/Gsk3 /Fyn kinase axis in acetaminophen in duced oxidative renal toxicity, wherein protection is accorded by morin.

[0026] O.J. Onaolapo et al., Pharmacother Biomed, 85, 2017, 323-333 relates to a comparison of 1- methionine and silymarin with respect to prophylactic protective capabilities in acetaminophen-induced injuries of the liver, kidney and cerebral cortex.

[0027] Y. Lu et al., Mol Pharm, 93(2), 2018, 63-72 relates to the identification of an oleanane-type triterpene hedragonic acid as famesoid X receptor ligand with liver protective effects and anti-inflam matory activity.

[0028] The known methods of treating acetaminophen overdose, particularly for ameliorating and pre venting the consequences of acetaminophen intoxication are not satisfactory in every respect and there is a demand for alternative or improved methods. [0029] It is an object of the invention to provide medicaments for treating acetaminophen overdose, particularly for ameliorating and preventing the consequences of acetaminophen intoxication, which have advantages compared to the prior art.

[0030] This object has been achieved by the subject-matter of the patent claims.

SUMMARY OF THE INVENTION

[0031] The invention relates to inhibitors of sodium taurocholate co-transporting poly-peptide (NTCP) for use in the treatment of acetaminophen overdose, particularly to ameliorating and preventing the consequences of acetaminophen intoxication.

[0032] The inventors investigated bile acid (BA) transport in mice by intravital two-photon imaging, and quantified endogenous BA concentrations in the serum of mice and patients with acetaminophen (APAP) overdose. Further, the inventors analyzed liver tissue by MS and MALDI-MSI, and assessed the integrity of the blood-bile barrier by immunostaining of tight junction proteins and intravital imaging of fluorescent markers. Furthermore, the inventors identified the intracellular cytotoxic concentrations of BA, and performed interventions to block BA uptake from blood into hepatocytes using the NTCP inhibitor, Myrcludex B and the Oatp knockout mouse model.

[0033] It has been surprisingly found that prior to the onset of cell death, APAP overdose causes a breach of the blood-bile barrier resulting in the leakage of BA from the bile canaliculi into the sinusoidal blood, which is then followed by the uptake of BA into hepatocytes via the basolateral membrane, its secretion into bile canaliculi and repeated cycling.

[0034] Further, it has been surprisingly found that such ‘futile cycling’ of BA leads to increased con centrations of intracellular BA that are high enough to cause hepatocyte death.

[0035] Importantly, however, it has been surprisingly found that the interruption of this cycling by pharmacological NTCP blockage and Oatp knockout strongly reduced APAP -induced hepatotoxicity. Thus, there is strong indication that prevention of paracellular BA cycling represents a therapeutic option after APAP overdose.

[0036] Moreover, it has been surprisingly found that compared to conventional therapy, human patients have more time for therapy with inhibition of NTCP (SLC10A1) according to the invention, namely up to approximately 50 hours after the acetaminophen intoxication. This is important because the conven tional therapy with N-acetylcysteine can only be used with good effectiveness up to about 8 hours after acetaminophen intoxication.

DESCRIPTION OF THE FIGURES [0037] Figure 1 shows ALT activity in plasma of mice after APAP overdose showing significant ele vation at 4 and 8 hours after APAP injection.

[0038] Figure 2 also shows liver enzyme levels after APAP injection.

[0039] Figure 3 shows blebbing of bile canaliculi after APAP overdose. The number of active blebs A and their area B is quantified.

[0040] Figures 4 to 7 show enrichment of bile acids in the pericentral lobular zone after APAP intoxi cation. Figure 4 shows a quantification of the TCA intensity in the MALDI-MSI images (mean and standard error). Figure 5 shows TCA intensity in the periportal and pericentral lobular zone analyzed by MALDI-MSL Figure 6 shows BA concentrations in bile collected from the gallbladder. Figure 7 shows the sum of bile acid concentrations in liver tissues of mice at different time intervals after APAP over dose analyzed by MS.

[0041] Figures 8 to 11 show intracellular bile acid concentrations in relation to cytotoxicity. Figure 8 shows concentration-dependent cytotoxicity of bile acids in the presence of 0, 1 and 4 mM APAP. The inlay gives the ECio and EC ₅₀ values of the fitted curves, the horizontal lines show the EC ₁₀ values and 95% confidence values. The open circles are data of three independent experiments obtained with hepatocytes from different mice, the closed circles represent mean values of the independent experi ments. Figure 9 shows intracellular sum bile acid concentrations of mouse hepatocytes incubated with extrahepatic mouse bile. Figure 10 shows the sum bile acid concentrations in homogenized liver tissue before and 2 hours after i.p. injection of 300 mg/kg APAP. Figure 11 shows the sum concentrations of bile acids in homogenized hepatocytes isolated from mouse livers. Hepatocytes were isolated from the same livers analyzed in Figure 10. The bar plots in D-E show mean values and standard errors and considered significant at p<0.05 (*), p<0.01 (**) and p<0.001 (***), unpaired t test.

[0042] Figures 12 to 17 show the interruption of futile bile acid cycling by Oatp knockout and Myr- cludex B administration protects from APAP-induced hepatotoxicity. Figure 12 shows a quantification of the dead cell area. The bar plots show mean values and standard errors and considered significant at p<0.01 (**) and p<0.001 (***). unpaired t test. E. Unaltered Cyp2El immunostaining in wild-type and Oatp knockout mice. Figure 13 shows liver enzymes. The bar plots again show mean values and standard errors and considered significant at p<0.01 (**) and p<0.001 (***). unpaired t test. Figure 14 shows APAP clearance from blood, APAP glucuronide and sulfate levels, and APAP-GSH, APAP -cysteine and APAP-NAC adduct concentrations between bulevirtide-treated Oatp knockout and WT mice after APAP intoxication. Figure 15 shows similar glutathione levels in wild-type and bulevirtide-treated Oatp knockout mice before and after APAP administration; data are mean values and standard errors and considered significant at p<0.001 (***). Tukey's multiple comparisons test. Figure 16 and Figure 17 show the sum bile acid concentrations in liver tissues (J) and plasma (K) of wild-type and bulevirtide- treated Oatp knockout mice with and without APAP overdose. Data are means and standard errors, and considered significant at p<0.001 (***). Sidak's multiple comparisons test and Tukey's multiple com parisons test, respectively. Data of the individual mice in the bar graphs are illustrated by dots.

[0043] Figures 18 to 20 show clinical results in humans with regard to blood bile acid concentrations and transaminase activities in relation to APAP -induced hepatotoxicity. Figures 18 and 19 show ALT activity and sum of bile acid concentrations in plasma of APAP-intoxicated patients in whom the period between overdose and blood sampling was unknown. Patients were grouped by their combination of ALT and bile acid levels. Figure 20 shows the results of a time-resolved analysis of the sum of blood bile acid concentrations and ALT activity of a 19-year-old woman after APAP overdose. Dashed base lines: reference values in healthy individuals.

[0044] Figure 21 shows the sum of bile acids concentrations and ALT in heart blood after APAP over dose in mice (mean and SE of 4 mice per time point).

DETAILED DESCRIPTION OF THE INVENTION

[0045] In the past years, the inventors established intravital imaging techniques that facilitate the func tional analysis of intact livers at subcellular resolution, including rapid BA transport processes ( Ghallab , A., et al. Bile Microinfarcts in Cholestasis Are Initiated by Rupture of the Apical Hepatocyte Membrane and Cause Shunting of Bile to Sinusoidal Blood. Hepatology 69, 666-683 (2019); Jansen, P.L., et al. The ascending pathophysiology of cholestatic liver disease. Hepatology 65, 722-738 (2017); Koppert, S., et al. Front Immunol 9, 1991 (2018); Vartak, N, et al. Hepatology 73, 1531-1550 (2021)). Using these techniques, the inventors could demonstrate that APAP causes a breach of the blood-bile barrier at the apical hepatocyte membrane, and that paracellular bile acid cycling with subsequent re-uptake is responsible for BA accumulation in hepatocytes and for subsequent cell death.

[0046] In the liver, APAP is metabolically activated to NAPQI, which conjugates to and depletes GSH, binds to proteins, induces oxidative and nitrosative stress, JNK activation and mitochondrial dysfunction ( Jaeschke , H. et al. Arch Toxicol 93, 3491-3502 (2019)). It has been surprisingly found that these mech anisms alone are not sufficient to cause hepatocyte death after the administration of a hepatotoxic APAP dose of 300 mg/kg b.w. to mice. Instead, it could be demonstrated that besides the above-mentioned mechanisms, APAP compromises the blood-bile barrier, thereby causing leakage of bile with high BA concentrations from the canaliculi into the sinusoidal blood, from where BA are taken up by NTCP and OATPs into the hepatocytes and secreted again into the bile canaliculi. This futile cycling leads to in creased BA concentrations in the hepatocytes, which exceed cytotoxic levels and cause cell death. In terruption of this futile cycling by blocking the uptake carriers, NTCP and OATPs strongly reduces intracellular BA concentrations and rescues the hepatocytes from cell death. This finding does not ex clude that APAP is also toxic without the synergistic effect of futile BA cycling, but higher doses may be required. [0047] The therapeutic intervention according to the invention does not interfere with the metabolic activation of APAP nor the leakiness of the blood-bile barrier. However, when BA cycling and accu mulation in hepatocytes is prevented, the liver tissue recovers from APAP-induced stress.

[0048] It has already been reported that APAP compromises tight junctions ( Gamal , W., et al. Sci Rep 7, 37541 (2017); Schafer, C. et al. Cellular physiology and biochemistry : international journal of ex perimental cellular physiology, biochemistry, and pharmacology 32, 431-447 (2013)). However, it has not yet been shown that APAP causes futile BA cycling after compromising the blood-bile barrier. Pre vious studies have addressed the question of whether “systemic increases in BA levels can be a direct cause of liver injury, or whether they are just an indicator of liver dysfunction” ( Woolbright , B.L., et al. 2014). One hypothesis is that elevation of BA concentrations in serum and liver tissue causes hepatocyte death ( Bechmann , L.P. et al. Hepatology 57 , 1394-1406 (2013); Guicciardi, M.E. et al. Comprehensive Physiology 3, 977-1010 (2013); Jang, J.H., et al. Hepatology 56, 209-218 (2012); Rέah, N, et al. Hepa tology 58, 1451-1460 (2013)). However, other studies have challenged this concept {Woolbright, B.L., et al. 2013 and 2014; Trottier, J, et al; Zhang, Y., et al.) hypothesizing that “the source of the BA elevation must be due to either rupture of the biliary tract where BAs are present in millimolar quantities, or direct inhibition of BA export from hepatocytes” {Woolbright, B.L., et al. 2014).

[0049] It has been surprisingly found that the first hypothesis proposing, “rupture of the biliary tract”, is correct but the concept of “inhibition of BA export” does not apply. This insight was made possible by the successful implementation of two-photon based intravital imaging. Using fluorescent high mo lecular weight dextran, APAP-induced leakiness from sinusoidal blood to the lumen of bile canaliculi could be directly observed. In contrast, leakiness from canaliculi to sinusoidal blood was demonstrated by using the intravital non-fluorescent probe, CMFDA that is taken up by hepatocytes, cleaved by in tracellular esterases to release the fluorescent 5-CMF that is secreted into the bile canaliculi and - only after APAP pretreatment - leaked into the sinusoidal blood.

[0050] Moreover, administration of the green-fluorescent BA analogue, CLF demonstrated that the compound is secreted into the bile canaliculi, and - only upon APAP administration - leaks from the canaliculi into the sinusoidal blood and accumulates in pericentral hepatocytes. This accumulation was almost completely prevented by the pharmacological inhibition of NTCP in combination with OATP deletion. Since CLF is an exogenously administered BA analogue, we corroborated the key finding of APAP-induced BA accumulation and its rescue using MALDI-MSI to analyze endogenous BA.

[0051] Intravital two-photon imaging also allows the analysis of BA secretion from hepatocytes into the bile canaliculi {Ghallab, A., et al. (2019)). Interestingly, APAP had no influence on CLF secretion, and consequently the second hypothesis {Woolbright, B.L., et al. 2014) could be disproven. It should be considered that similar to previous studies {Woolbright, B.L., et al. 2014; James, L, et al. PloS one 10, e0131010-e0131010 (2015)), BA concentrations in blood and liver tissue were also analyzed and a transient APAP induced-increase was observed. However, without intravital imaging at subcellular res olution, it would have been impossible to demonstrate this futile cycling of BA as the mechanism re sponsible for BA accumulation in liver tissue.

[0052] The mechanism underlying the present invention are of high clinical relevance, since futile BA cycling after APAP overdose can be interrupted by inhibitors of NTCP and OATPs. Human data show increased serum BA concentrations after accidental or suicidal APAP ingestion, even without a simul taneous increase in liver enzyme activities as seen in some patients, which could correspond to the early period after intoxication in mice when a similar constellation is observed.

[0053] A first aspect of the invention relates to a pharmaceutical dosage form comprising an inhibitor of sodium taurocholate co-transporting poly-peptide (NTCP), a physiologically acceptable salt and/or solvate thereof, for use in the treatment of acetaminophen overdose; preferably wherein the inhibitor of sodium taurocholate co-transporting poly-peptide (NTCP) is bulevirtide (myrcludex B), a physiologi cally acceptable salt and/or solvate thereof.

[0054] In particularly preferred embodiments, the inhibitor of sodium taurocholate co-transporting poly-peptide (NTCP) is bulevirtide (myrcludex B), a physiologically acceptable salt and/or solvate thereof, and the dosage form is administered by subcutaneous injection, preferably once daily. Prefera bly, the dosage form contains bulevirtide acetate equivalent to about 2 mg of bulevirtide (myrcludex B).

[0055] Acetaminophen overdose can be diagnosed by a skilled person. Diagnosis is preferably based on the blood level of paracetamol at specific times after the medication was taken. These values are often plotted on the Rumack-Matthew nomogram to determine level of concern (F. Ferri (2016). Ferri's Clinical Advisor 2017 E-Book: 5 Books in 1. Elsevier Health Sciences). The concentration in serum after a typical dose of paracetamol usually peaks below 30 mg/1, which equals 200 pmol/L (./ Marx et al, (2013). Rosen's Emergency Medicine - Concepts and Clinical Practice. Elsevier Health Sciences). Lev els of 30-300 mg/L (200-2000 pmol/L) are often observed in overdose patients.

[0056] Preferably, the dosage form according to the invention is administered to a patient having an acetaminophen blood concentration of at least 30 mg/1.

[0057] The bile acid blood concentration is usually up to 2.5 pmol/L and may increase to greater than 50 pmol/L following an acetaminophen overdose. Preferably, the dosage form is administered to a pa tient having a total bile acid blood concentration of at least 5 pmol/L, preferably at least 10 pmol/L, more preferably at least 15 pmol/L, still more preferably at least 20 pmol/L, yet more preferably at least 25 pmol/L, even more preferably at least 30 pmol/L, most preferably at least 35 pmol/L, and in partic ular at least 40 pmol/L, based on the total amount of all bile acids.

[0058] The dosage form according to the invention comprises an inhibitor of sodium taurocholate co transporting poly-peptide (NTCP). Suitable inhibitors of sodium taurocholate co-transporting poly- peptide (NTCP) according to the invention are not particularly limited and known to the skilled person. The invention is based inter alia upon the unexpected finding that intracellular concentrations of bile acids can be reduced by blocking bile acid uptake from blood into hepatocytes.

[0059] Preferably, the inhibitor of sodium taurocholate co-transporting poly-peptide (NTCP) is the sole pharmacologically active ingredient that is contained in the pharmaceutical dosage form according to the invention. However, it is contemplated that the pharmaceutical dosage form according to the inven tion comprises a combination of two or more inhibitors of sodium taurocholate co-transporting poly peptide (NTCP).

[0060] NTCP is a bile acid uptake carrier, namely SLC10A1 (Solute Carrier Family 10 Member 1, also known as FHCA2). NTCP belongs to the sodium/bile acid cotransporter family, which are integral mem brane glycoproteins that participate in the enterohepatic circulation of bile acids. Two homologous trans porters are involved in the reabsorption of bile acids; the ileal sodium/bile acid cotransporter with an apical cell localization that absorbs bile acids from the intestinal lumen, bile duct and kidney, and the liver-specific sodium/bile acid cotransporter, represented by NTCP, that is found in the basolateral mem branes of hepatocytes (see e.g. http://www.ncbi.nlm.nih.gov/gene/6554).

[0061] Suitable NTCP inhibitors according to the invention are not particularly limited and known to the skilled person. Various NTCP inhibitors are contained in approved medicaments for other medical indications.

[0062] In a particularly preferred embodiment, the NTCP inhibitor is bulevirtide (myrcludex B), a physiologically acceptable salt and/or solvate thereof (ATC J05AX28, CAS 2012558-47-1, DrugBank DB15248, ChEMBL ChEMBL4297711). Bulevirtide (myrcludex B) is commercially available under the brand name Hepcludex ^® . It is an antiviral medication approved for the treatment of chronic hepatitis D (in the presence of hepatitis B). Bulevirtide (myrcludex B) binds and inactivates the sodium/bile acid cotransporter, blocking both viruses from entering hepatocytes.

[0063] In other preferred embodiments of the invention, the NTCP inhibitor is selected from the group consisting of bendroflumethiazide, ezetimibe, simvastatin, nitrendipine, rosuvastatin, nefazodone, indo- methacin, nifedipine, tioconazole, methylprednisolone, prochlorperazine, rosiglitazone, zafirlukast, TRIAC, Chicago sky blue 6B, sulfasalazine, flufenamic acid, tolfenamic acid, toltrazuril, amlexanox, nelfmavir (e.g. nelfmavir mesylate hydrate), hydroxytacrine (e.g. hydroxytacrine maleate), and physio logically acceptable salts and/or solvates thereof. The capability of the substances to inhibit NTCP is known (see e.g. Z. Dong et al, Mol Pharm. 2013 10(3): 1008-1019; J.M. Donkers et al, Nature Scien tific Reports, 2017, 7:15307, 1-13).

[0064] In further preferred embodiments of the invention, the NTCP inhibitor is selected from betulinic acid, derivatives of betulinic acid, and physiologically acceptable salts and/or solvates thereof; preferably from the group consisting of betulinic acid, 20,29-dihydrobetulonic acid, 3,28-di-O-suc- cinoylbetulin, 28-0(3, 3-dimethylglutaroyl)betulin, 3-0(3, 3-dimethylglutaroyl)betulinic acid, 3,28-di- 0-(3,3-dimethylglutaroyl)betulin, 3-0-acetylbetulin, methyl betulinate, 28-O-succinoylbetulin, betu- lonaldehyde, 28-O-nicotinoylbetulin, 3-0-acetylbetulinic acid, 3,28-di-O-acetylbetulin, 3,28-di-0-(di- hydrocinnamoyl)betulin, 4'-ethyl-T,2',4'-triazoline-3',5'-dione-fused 3,28-di-O-acetylbetulin, betulin, 28-O-cinnamoylbetulin, 3-0-acetyl-28-(tetrahydro-2H-pyran-2-yl)betulin, lupenone, 28-0-(bromoace- tyl)betulin, 3,28-di-0-acetyl-20,30-epoxybetulin, 3,28-di-O-acetyl-18,19-dehydro-20,29-dihydrobetu- lin, lupeol, 28-(tetrahydro-2H-pyran-2-yl)betulin, 3-0-caffeoylbetulin, allobetulin, betulonoyl dime- thyl-L-aspartate, 3-oxoallobetulin, betulinaldehyde oxime, 20,29-dihydrobetulin, 3,28-di-0-acetyl-29- hydroxybetulin, and physiologically acceptable salts and/or solvates thereof. The capability of the sub stances to inhibit NTCP is also known (see e.g. M. Kirstgen et a , Nature Scientific Reports, 2020, 10:21772, 1-16).

[0065] The content of the inhibitor of sodium taurocholate co-transporting poly-peptide (NTCP) within the pharmaceutical dosage form according to the invention is not particularly limited and may vary e.g. within the range of from 0.001 to 95 wt.-%, relative to the total weight of the pharmaceutical dosage form. Preferably, the content of the inhibitor of sodium taurocholate co-transporting poly-peptide (NTCP) within the pharmaceutical dosage form according to the invention is within the range of from 30±25 wt.-%, 50±25 wt.-%, 70±25 wt.-%, 10±9 wt.-%, 15±9 wt.-%, 20±9 wt.-%, 25±9 wt.-%, 30±9 wt.-%, 35±9 wt.-%, 40±9 wt.-%, 45±9 wt.-%, 50±9 wt.-%, 55±9 wt.-%, 60±9 wt.-%, 65±9 wt.-%, 70±9 wt.-%, 75±9 wt.-%, or 80±9 wt.-%, relative to the total weight of the pharmaceutical dosage form.

[0066] The pharmaceutical dosage form according to the invention may be solid, semisolid or liquid. Preferably, the pharmaceutical dosage form according to the invention is liquid.

[0067] Preferably, the pharmaceutical dosage form according to the invention comprises one or more physiologically acceptable pharmaceutical excipients such as carriers, solvents, fdlers, binders and the like. The overall content of all pharmaceutical excipients within the pharmaceutical dosage form ac cording to the invention is not particularly limited and may vary e.g. within the range of from 0.001 to 99.999 wt.-%, relative to the total weight of the pharmaceutical dosage. Preferably, the overall content of all pharmaceutical excipients within the pharmaceutical dosage form according to the invention is within the range of from 30±25 wt.-%, 50±25 wt.-%, 70±25 wt.-%, 20±15 wt.-%, 30±15 wt.-%, 40±15 wt.-%, 50±15 wt.-%, 60±15 wt.-%, 70±15 wt.-%, 80±15 wt.-%, 10±9 wt.-%, 15±9 wt.-%, 20±9 wt.-%, 25±9 wt.-%, 30±9 wt.-%, 35±9 wt.-%, 40±9 wt.-%, 45±9 wt.-%, 50±9 wt.-%, 55±9 wt.-%, 60±9 wt.-%, 65±9 wt.-%, 70±9 wt.-%, 75±9 wt.-%, 80±9 wt.-%, 85±9 wt.-%, or 90±9 wt.-%, relative to the total weight of the pharmaceutical dosage. [0068] The pharmaceutical dosage form according to the invention may be administered by various means, depending on its intended use. Preferably, the dosage form is administered systemically, prefer ably orally or parenterally.

[0069] For example, if the pharmaceutical dosage form according to the invention is to be administered orally, it may be provided in form of tablets, capsules, granules, powders or syrups. Alternatively, the pharmaceutical dosage form according to the invention may be administered parenterally as injections (intravenous, intramuscular or subcutaneous), drop infusion preparations or suppositories. These phar maceutical dosage form may be prepared by conventional means, and, if desired, the pharmaceutical dosage forms may be mixed with any conventional additive, such as an excipient, a binder, a carrier, a disintegrating agent, a buffer, an osmolality adjuster, a surfactant, a lubricant, a solubilizing agent, a suspension aid, an emulsifying agent, a coating agent and any mixtures thereof.

[0070] In pharmaceutical dosage forms of the invention, wetting agents, emulsifiers and lubricants, such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, release agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants may be present.

[0071] The pharmaceutical dosage form may be suitable for oral, nasal, topical (including buccal and sublingual), rectal, vaginal, aerosol and/or parenteral administration. The pharmaceutical dosage forms may conveniently be presented in unit dosage form and may be prepared by any methods well known in the art of pharmacy. The amount of composition that may be combined with a carrier material to produce a single dose varies depending upon the subject being treated, and the particular route of ad ministration.

[0072] Pharmaceutical dosage forms suitable for oral administration may be in the form of capsules, sachets, pills, tablets, lozenges (using a flavored basis, usually sucrose and acacia or tragacanth), pow ders, granules, or as a solution or a suspension in an aqueous or non-aqueous liquid, or as an oil-in-water or water-in-oil liquid emulsion, or as an elixir or syrup, or as pastilles (using an inert base, such as gelatin and glycerin, or sucrose and acacia), each containing a predetermined amount of the inhibitor of sodium taurocholate co-transporting poly-peptide (NTCP), pharmaceutical dosage forms according to the in vention may also be administered as a bolus, electuary, or paste.

[0073] In solid pharmaceutical dosage forms for oral administration (capsules, tablets, pills, dragees, powders, granules and the like), the inhibitor of sodium taurocholate co-transporting poly-peptide (NTCP), is preferably mixed with one or more pharmaceutically acceptable carriers, such as sodium citrate or dicalcium phosphate, and/or any of the following: (1) fillers or extenders, such as starches, lactose, sucrose, glucose, mannitol, and/or silicic acid; (2) binders, such as, for example, carboxymethyl- cellulose, alginates, gelatin, polyvinyl pyrrolidone, sucrose and/or acacia; (3) humectants, such as glyc erol; (4) disintegrating agents, such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate; (5) solution retarding agents, such as paraffin; (6) absorption accelerators, such as quaternary ammonium compounds; (7) wetting agents, such as, e.g., acetyl alcohol and glycerol monostearate; (8) absorbents, such as kaolin and bentonite clay; (9) lubri cants, such a talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sul fate, and mixtures thereof; and (10) coloring agents. In the case of capsules, tablets and pills, the phar maceutical dosage form may also comprise buffering agents. Solid pharmaceutical dosage forms of a similar type may also be employed as fillers in soft and hard-filled gelatin capsules using such excipients as lactose or milk sugars, as well as high molecular weight polyethylene glycols and the like.

[0074] A tablet may be made by compression or molding, optionally with one or more auxiliary ingre dients. Compressed tablets may be prepared using binder (for example, gelatin or hydroxypropyl methyl cellulose), lubricant, inert diluent, preservative, disintegrant (for example, sodium starch glycolate or cross-linked sodium carboxymethyl cellulose), surface-active or dispersing agent. Molded tablets may be made by molding in a suitable machine a mixture of the subject composition moistened with an inert liquid diluent. Tablets, and other solid dosage forms, such as dragees, capsules, pills and granules, may optionally be scored or prepared with coatings and shells, such as enteric coatings and other coatings well known in the pharmaceutical-formulating art.

[0075] Liquid pharmaceutical dosage forms for oral administration include pharmaceutically accepta ble emulsions, microemulsions, solutions, suspensions, syrups and elixirs. In addition, the liquid phar maceutical dosage forms may contain inert diluents commonly used in the art, such as, for example, water or other solvents, solubilizing agents and emulsifiers, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, oils (in particular, cottonseed, groundnut, com, germ, olive, castor and sesame oils), glycerol, tetrahy- drofuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, cyclodextrins and mixtures thereof.

[0076] Suspensions may contain suspending agents such as ethoxylated isostearyl alcohols, polyoxy ethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth, and mixtures thereof.

[0077] Pharmaceutical dosage forms for rectal or vaginal administration may be presented as a suppos itory, which may be prepared by mixing the inhibitor of sodium taurocholate co-transporting poly-pep- tide (NTCP) with one or more suitable non-irritating excipients or carriers comprising, for example, cocoa butter, polyethylene glycol, a suppository wax or a salicylate, and which is solid at room temper ature, but liquid at body temperature and, therefore, will melt in the body cavity and release the active agent pharmaceutical dosage forms which are suitable for vaginal administration also include pessaries, tampons, creams, gels, pastes, foams or spray formulations containing such carriers as are known in the art to be appropriate. [0078] Pharmaceutical dosage forms for parenteral administration preferably comprise the inhibitor of sodium taurocholate co-transporting poly-peptide (NTCP) in combination with one or more pharmaceu- tically-acceptable sterile isotonic aqueous or non-aqueous solutions, dispersions, suspensions or emul sions, or sterile powders which may be reconstituted into sterile injectable solutions or dispersions just prior to use, which may contain antioxidants, buffers, bacteriostats, solutes which render the pharma ceutical dosage form isotonic with the blood of the intended recipient or suspending or thickening agents.

[0079] Examples of suitable aqueous and non-aqueous carriers which may be employed in the pharma ceutical dosage forms according to the invention include water, ethanol, polyols (such as glycerol, pro pylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof, vegetable oils, such as olive oil, and injectable organic esters, such as ethyl oleate and cyclodextrins. Proper fluidity may be maintained, for example, by the use of coating materials, such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.

[0080] The inhibitor of sodium taurocholate co-transporting poly-peptide (NTCP) can be formulated for parenteral administration, as for example, for subcutaneous, intramuscular or intravenous injection, e.g., the inhibitor of sodium taurocholate co-transporting poly-peptide (NTCP) can be provided in a sterile solution or suspension (injectable pharmaceutical dosage form).

[0081] The dosage of any pharmaceutical dosage form according to the invention will vary depending on the symptoms, age and body weight of the patient, the nature and severity of the disorder to be treated or prevented, the route of administration, and the form of the formulation. Any of the pharmaceutical dosage form may be administered in a single dose or in divided doses. Dosages for the pharmaceutical dosage forms according to the invention may be readily determined by techniques known to those of skill in the art. Treatment may be initiated with smaller dosages which are less than the optimum dose of the inhibitor of sodium taurocholate co-transporting poly-peptide (NTCP). Thereafter, the dosage may be increased by small increments until the optimum therapeutic effect is achieved.

[0082] Preferably, the pharmaceutical dosage form according to the invention is adapted for systemic administration, particularly for intravenous administration.

[0083] The pharmaceutical dosage form according to the invention is for use in the treatment of aceta minophen overdose. In this regard, the invention also relates to a method of treating acetaminophen overdose comprising the administration of an effective amount of the pharmaceutical dosage form ac cording to the invention to a subject in need thereof. Further, the invention also relates to the use of the inhibitor of sodium taurocholate co-transporting poly-peptide (NTCP) for the manufacture of a pharma ceutical dosage form according to the invention for the treatment of acetaminophen overdose. [0084] The frequency of administration may depend upon the inhibitor of sodium taurocholate co transporting poly-peptide (NTCP). Preferably, the dosage form is administered once a week, twice a week, once daily, twice daily, or thrice daily.

[0085] In preferred embodiments of the invention, the dosage form is administered not more than 7 days, preferably not more than 4 days, more preferably not more than 3 days, most preferably not more than 2 days after acetaminophen ingestion.

EXAMPLES

[0086] The following examples further illustrate the invention but are not to be construed as limiting its scope. Additional aspects and experimental results have meanwhile also been published in A. Ghallab et ah, Journal of Hepatology 2022, 77, 71-83.

Materials and methods

[0087] Mice and induction of acute liver injury by APAP: Male 8-12 week-old C57BL6/N (Janvier Labs, France) or Oatp-deficient mice (Taconic Biosciences, USA; Cat. No. 10707-M; FVB.129P2- Del(Slcolb2-Slcola5)lAhs) and corresponding wild-type mice (Taconic Biosciences, USA; Cat. No. FVB-M) were used. In order to induce acute liver injury by APAP, a single dose of 300 mg/kg b.w. was intraperitoneally (i.p) injected in warm phosphate-buffered saline (PBS). The mice were starved over night prior to APAP application and were fed ad libitum afterward. For the intervention experiments in Oatp-deficient mice, a dose of 5 mg/kg b.w. Bulevirtide (myrcludex B) was administered intravenously simultaneously with the APAP administration. Blood, as well as liver tissue samples were collected using a standard protocol ( Ghallab , A., etal. J Hepatol 64, 860-871 (2016)).

[0088] Intravital imaging: Functional intravital imaging of mouse livers was performed using an in verted two-photon microscope LSM MP7 (Zeiss, Germany) with an LD C-Apochromat 40 xl 1.1 water immersion objective ( Ghallab , A., etal. Hepatology 69, 666-683 (2019); Ghallab, A., etal. Arch Toxicol (2021); Reif R., etal. Arch Toxicol 91, 1335-1352 (2017)).

[0089] Staining of fixed liver tissue: Two-dimensional (2D) hematoxylin and eosin, immunohisto- chemistry, as well as TUNEL stainings were performed in 5 pm-thick paraformaldehyde (4%)-fixed paraffin-embedded liver tissue sections. The stainings were done on the Discovery Ultra Automated Slide Preparation System (Roche, Germany). TUNEL staining was done using a commercially available kit (Promega, Germany). Three-dimensional (3D) co-staining of CYP2E1 and CD 13 was done in 100 pm-thick liver tissue slices prepared using a cryostat microtome ( Ghallab , A., et al. Influence of Liver Fibrosis on Lobular Zonation. Cells 8 (2019)).

[0090] Image analysis: Quantifications of intravital videos and whole slide scans of the fixed liver tis sue were done using one of the interactive image segmentation toolkits ilastik (Berg, S., et al. Nature Methods 16, 1226-1232 (2019)) (version 1.3.3postl), TiQuant ( Friebel , A., Johann, I, Drasdo, D. & Hoehme, S. ArXiv ahs/2005.07662(2020)) (version 2.0) or QuPath (Bankhead, P., etal. QuPath: Scien tific Reports 7, 16878 (2017)) (version 0.2.3).

[0091] Bile acid assay and MALDI-MSI of taurocholic acid: Bile acid concentrations in plasma, bile, liver tissue and cells were determined by negative electrospray (ESI) liquid chromatography tandem mass spectrometry (LC-MS/MS) in multiple-reaction-monitoring (MRM) mode on an Agilent 6495B triple quadrupole mass spectrometer (Agilent, Germany) coupled to an Agilent Infinity II HPLC system as described previously ( Ghallab , A., etal. 2019; Kuhe, L, etal. Thyroid (2021)). MALDI-MSI analysis of taurocholic acid (TCA) was performed using TIMS TOF Flex ( Ghallab , A., etal. 2019; Sezgin, S., et al. Arch Toxicol 92, 2963-2977 (2018)).

[0092] Glutathione assay: Concentrations of reduced as well as oxidized glutathione (GSH) in liver tissue homogenate were measured by LC-MS/MS ( Sezgin , S., et al). The results were normalized to the wet weight of the liver tissue samples.

[0093] Analysis of APAP, APAP metabolites and APAP adducts: Concentrations of APAP, its glucu- ronide and sulfate metabolites, as well as its glutathione, cysteine, and N-acetylcysteine adducts were measured in plasma separated from hear blood using HPLC-HR-MS, ( Sezgin , S., et al).

[0094] Isolation and cultivation of mouse hepatocytes: Hepatocytes were isolated from 8-10 week-old male C57BL6/N mice and were cultivated in collagen sandwich using a standard protocol (Godoy, P., et al. Archives of Toxicology 87, 1315-1530 (2013)).

[0095] Statistical analysis: Data were analysed using Prism software (GraphPad Prism 7.05 Software, Inc., La Jolla, CA). Statistical significance between experimental groups was analysed using Dunnetfs/ Tukey's/ Sidak's multiple comparisons test, or unpaired t test, as indicated in the figure legends, and concentration-cytotoxicity relationships were described by a four-parameter log-logistic model (4pLL) (Albrecht, W., et al. Arch Toxicol 93, 1609-1637 (2019)).

Results a) Transient cholestasis precedes hepatocvte death after APAP intoxication

[0096] The analysis of blood from patients with APAP overdose on admission to emergency depart ments revealed a heterogeneous pattern, with some patients presenting with both increased ALT and bile acid (BA) levels while others showed increased BA concentrations and almost normal ALT activity. Consequently, no significant correlation between BA concentrations and ALT activity was observed. Since the exact time point of intoxication was not documented for most patients, a time-dependent anal ysis of the sequence of events, i.e. if BA levels increased prior to liver enzymes or vice versa, was not possible. [0097] To study this aspect under controlled conditions, a mouse model was used in which a hepato- toxic dose of APAP was administered and BA concentrations were analyzed in plasma sampled from the portal and hepatic veins, as well as from the right heart chamber. A strong transient increase in BA concentrations was observed in both the hepatic vein and heart blood 2 hours after intoxication, while no significant increase occurred in the portal vein. Liver enzyme levels were only slightly above the control levels 2 hours after APAP injection, but significantly increased at the later timepoints (Figure 1; Figure 2).

[0098] In agreement, the macroscopically visible speckled pattern that indicates zonated cell death was observed at 4 and more clearly 8 hours after intoxication. Furthermore, only few TUNEL positive hepatocytes were present 2 hours after intoxication; thereafter, the number of positive cells and staining intensity increased strongly, where hepatocyte death was exclusively observed in the CYP2E1 positive pericentral area. Thus, the time-dependent analyses clearly show that the initial event was the increase in BA concentrations, followed by hepatocyte death and increased liver enzyme activities in blood. b) Altered bile canalicular morphology after APAP intoxication

[0099] To study if the above-described transient cholestasis is associated with altered bile canalicular morphology, a recently established two-photon microscopy-based intravital imaging toolbox was ap plied that visualizes canalicular secretion by fluorescent BA analogues ( Ghallab , A., et al. Hepatology 69, 666-683 (2019)).

[0100] For this purpose, two functional markers were used: CLF, a green-fluorescent BA analogue; and TMRE, a mitochondrial membrane potential marker. TMRE is more abundantly taken up by peri portal (pp) hepatocytes, thereby outlining the lobular zone. Within 3 minutes after the injection of CLF into control mice, the BA analogue was rapidly taken up from the blood by the hepatocytes and secreted into the bile canaliculi, which appeared homogeneous throughout the liver lobule. Approximately 2 hours after APAP intoxication, CLF was also taken up by hepatocytes and secreted into the bile cana liculi; however, the diameter of the canaliculi were strikingly dilated. Analysis of the intravitally-ac- quired images demonstrated that the dilatation of bile canaliculi occurred only in the pericentral (TMRE intensity bins 0-3) and not in the periportal (TMRE intensity bins 4-9) lobular zone. Moreover, hepato cytes in the pericentral zone enriched CLF.

[0101] In the next step, time-lapse videos were recorded early after APAP administration to character ize the process leading to the alterations of canalicular morphology and the CLF enrichment in hepato cytes. At the beginning of recording (85 minutes after APAP injection), bile canaliculi in the pericentral zone already appeared dilated, with a further increase in diameter over the next 2 hours. In addition, the dilated bile canaliculi formed small CLF -containing protrusions (60 min) that grew larger over time. These canalicular alterations coincided with decreased TMRE-associated red fluorescence indicating a loss in mitochondrial potential, as well as CLF enrichment in hepatocytes. Importantly, these canalicular alterations and CLF enrichment in pericentral hepatocytes occurred prior to cell death as evidenced by propidium iodide exclusion. Segmentation and image analysis of the time-lapse videos showed a time- dependent increase in the number of protrusions (‘active blebs’) and an increase in the total bleb area between 40 and 100 minutes after APAP intoxication (Figure 3). These changes coincided with an over all increase in CLF intensity that was more pronounced in the pericentral than the periportal zone of the liver lobule.

[0102] An advantage of intravital imaging is that morphological and functional changes can directly be observed in a time-resolved manner at subcellular resolution. However, the technique requires the ad ministration of fluorescent markers, such as CLF; thus, controls are required to analyze if key observa tions from intravital imaging can be confirmed under native conditions, i.e. without the administration of fluorescent markers and imaging. Therefore, fixed mouse liver tissues were co-immunostained at various time periods after APAP intoxication using antibodies directed against the bile canalicular marker CD 13, and the pericentral enzyme Cyp2El that is known to metabolically activate APAP.

[0103] In line with the intravital imaging results, marked dilatation of bile canaliculi was also observed in the pericentral Cyp2El positive zone at 1 and 2 hours after APAP injection, which was confirmed by image analysis. Canalicular protrusions into the cytoplasm of hepatocytes were similar to that observed in intravital imaging. Moreover, reconstructions of the canalicular network demonstrated fragmentation and numerous short branches in the pericentral, but not in the periportal region. Thus, the observation of compromised canalicular morphology from intravital imaging was confirmed by immunostaining of fixed liver tissue ex vivo. c) Compromised blood-bile barrier early after APAP intoxication

[0104] The above-described observations led to the hypothesis that the blood-bile barrier may be com promised early on after APAP intoxication. To test this hypothesis, a set of intravital imaging experi ments were performed investigating the tightness of the barrier between the bile canaliculi and sinusoi dal blood by several functional approaches.

[0105] First, BA transport was studied in untreated, as well as in APAP-intoxicated mice using the BA analogue CLF. After a bolus intravenous injection of CLF into control mice, green fluorescence ap peared in the blood of the sinusoids within seconds, was rapidly cleared by uptake into the hepatocytes, and later secreted into the bile canaliculi. Intravital recording performed at minute 85 after APAP ad ministration showed similar CLF uptake by hepatocytes, as well as secretion into the bile canaliculi as in the controls. However, as soon as CLF became enriched in the bile canaliculi its signal also increased in the sinusoidal blood and in hepatocytes of the pericentral compartment of the liver lobule. In addition, CLF clearance from the hepatocytes and canaliculi was delayed compared to controls, particularly in the pericentral region. [0106] The experiments with the CLF bolus injection in APAP-intoxicated mice clearly showed an accumulation of CLF in the pericentral compartment of the liver lobule directly after secretion into the bile canaliculi. A limitation of this approach is the use of an exogenously-administered BA analogue. Therefore, it was studied whether imaging of endogenous BA by MALDI-MSI leads to similar conclu sions. Cyp2El-immunostained frozen liver tissue sections at various time periods after APAP admin istration were superimposed with the MALDI signal for TCA. The result showed a strong, transient TCA accumulation at 2 hours after APAP intoxication, which was more pronounced in the pericentral than the periportal regions (Figure 4 and Figure 5).

[0107] This agreed with the result of MS-based analysis of BA in liver tissue homogenate (Figure 7). Thus, the accumulation of CLF in the pericentral zone 85 minutes after APAP intoxication corresponds to the increased levels of endogenous BA analyzed in liver tissue identified by MALDI-MSI and MS. Collectively, these data suggest that BA leak from the bile canaliculi in the pericentral zone to the sur rounding environment. The analysis of BA concentrations in bile collected from the gallbladder revealed an initial decrease followed by an increase above control levels between hours 2 and 4 after APAP administration (Figure 6); the initial decrease may be due to the transient cholestasis induced by APAP overdose.

[0108] In order to study the mechanism responsible for BA leakage from the canaliculi, immunostain- ing with antibodies directed against the tight junction proteins, ZO-1 and Claudin3 was performed. Both ZOl and Claudin3 immunostaining showed considerably altered right-junction morphology at 1 and 2 hours after APAP intoxication characterized by a strongly increased gap between the two strands and an irregular staining pattern. Importantly, these alterations occurred only in the Cyp2El positive peri central zone of the liver lobule.

[0109] Next, it was studied whether the observed morphological alterations of the blood-bile barrier had any functional consequences. For this purpose, two-photon imaging was performed in mice after tail vein injection of fluorescein-coupled dextran (70 kDa). In healthy livers, dextran appeared in the sinusoids within seconds after i.v. injection, but never passed from the blood into the bile canaliculi within a 30 min imaging period; the canalicular lumen, in contrast to the sinusoids, was free of dextran- associated fluorescence. In contrast, the same analysis 90 min after APAP intoxication demonstrated dextran-associated green fluorescence also present in the canaliculi. More specifically, the time-lapse videos showed that fluorescent dextran first appeared in the sinusoids, after which it continuously in creased in the bile canaliculi. Leakage of dextran from the blood to the bile canaliculi was only observed in the pericentral but not the periportal region. Importantly, dextran-associated fluorescence was ob served in the bile canaliculi of hepatocytes with intact mitochondrial potential as evidenced by the in- travital dye TMRE, and no increase of green fluorescence in the cytoplasm of these cells, overall sug gesting a paracellular passage of dextran directly from the blood to canaliculi. [0110] To study if leakage also occurs in the opposite direction, i.e. from the canaliculi into the sinus oidal blood, CMFDA was used as the intravital dye. The advantage of CMFDA compared to CLF is that it remains non-fluore scent in the blood before its passive uptake into hepatocytes, where it is cleaved by intracellular esterases to produce the green-fluorescent product, 5-CMF. The time-lapse videos of con trol mice showed the cytoplasmic generation of green fluorescence, followed by secretion into canalic uli. Almost no 5-CMF signal was visible in the sinusoidal blood. Interestingly, 80 minutes after APAP intoxication the green fluorescence was present at the interface of pericentral hepatocytes and sinusoids, where the Disse space is located, almost simultaneously or shortly after secretion into the bile canaliculi. Subsequently, green fluorescence became visible in the sinusoids. Quantification of green fluorescence in the sinusoidal blood after CMFDA injection resulted in only very low intensities in the control mice. In contrast, a strong increase in green fluorescence was detected in the sinusoidal blood of APAP-intox- icated mice, particularly in the first 10 minutes after CMFDA injection. This demonstrated that 5-CMF from the bile canaliculi or hepatocytes leaked back into the sinusoidal blood, as seen in the CLF exper iments.

[0111] Collectively, these data suggest that the blood-bile barrier is strongly compromised early after APAP intoxication. This resulted in bile leakage and BA cycling between canaliculi, sinusoids, and hepatocytes in the pericentral compartment of the liver lobule. d) Identification of intracellular cytotoxic concentration ranges of bile acids

[0112] While the previous experiments demonstrated that the total BA concentrations in liver tissue increased after APAP overdose, it remained to be studied if the resulting intracellular BA concentrations are high enough to cause cytotoxicity. Therefore, concentration-dependent incubations of cultivated mouse hepatocytes were performed with bile collected from the extrahepatic bile duct of mice in the presence and absence of APAP in the culture medium. Individual BA concentrations in mouse bile were determined by MS and concentrations in the culture medium of cultivated hepatocytes were adjusted based on the sum concentrations of all BA (Figure 8).

[0113] The EC50 of the sum BA in hepatocytes co-incubated with 0, 1 and 4 mM APAP were 541, 223 and 90 mM, respectively. Bile acid concentrations in homogenized hepatocytes after cultivation for 2 hours with the cytotoxic sum BA concentrations of 0, 10, 100 and 1000 pM were ~7, 170, 260, and 1100 pmol/pg DNA, respectively (Figure 9). Analysis of homogenized liver tissue (Figure 10) and isolated hepatocytes (Figure 11) from the same mice 2 hours after administration of 300 mg/kg APAP showed an increase in BA concentrations.

[0114] To allow a direct comparison of the in vitro- and in vivo-exposed hepatocytes, sum BA concen tration for both were analyzed and normalized to the DNA content. Interestingly, sum BA concentrations in hepatocytes of APAP-intoxicated mice ex vivo (-400 pmol/pg DNA; Figure 11) reached similar lev els as the in vitro cultivated hepatocytes incubated with cytotoxic concentrations of BA (Figure 9). It should be considered that in vivo, mainly the pericentral hepatocytes are damaged by APAP and appear to undergo futile BA cycling; therefore, it is possible that ex vivo analyzed BA concentrations in the pericentral hepatocytes are even higher than the -400 pmol/pg DNA obtained for all isolated hepato cytes, because the pericentral hepatocytes are diluted by less damaged periportal cells. Thus, the extent of the APAP-induced increase in BA concentrations in hepatocytes in vivo is indeed high enough to explain the observed hepatotoxicity. c) Therapeutic intervention by interruption of bile acid cycling

[0115] If the concept of APAP-induced BA cycling is correct, its interruption should reduce BA con centrations in liver tissue and ameliorate APAP-induced hepatotoxicity. To test this hypothesis, Oatp knockout mice were used and additionally the Na ⁺ -taurocholate cotransporting polypeptide ( NTCP , SLC10A1) was inhibited pharmacologically by injecting bulevirtide (myrcludex B) in order to block the carriers responsible for BA uptake into hepatocytes.

[0116] A hepatotoxic dose of APAP (300 mg/kg b.w.) was administered i.p. simultaneously with bulevirtide (myrcludex B) and liver damage was evaluated 24 hours later. This intervention protected the mice from macroscopically visible APAP-induced liver damage, and the extent of pericentral cell death was strongly reduced (Figure 12). In agreement, the increase in blood ALT and AST activities was strongly diminished in comparison to the APAP -treated WT mice (Figure 13).

[0117] Next, it was studied whether alternative explanations, such as decreased metabolic activation or increased detoxification of APAP may explain the observed protective effect. For this purpose, an ex periment was performed in which blood concentrations of APAP and its metabolites and adducts, as well as liver tissue levels of GSH were analyzed time-dependently after APAP administration. Im portantly, no difference in CYP2E1 expression was observed between Oatp knockout and WT mice prior to APAP administration. In agreement, APAP clearance from blood, APAP glucuronide and sul fate levels, and APAP -GSH, APAP -cysteine and APAP-NAC adduct concentrations did not show sys tematic differences between bulevirtide-treated Oatp knockout and WT mice after APAP intoxication (Figure 14). Moreover, no difference was observed in GSH levels in liver tissue prior to APAP injection, and similar depletion was detected afterwards (Figure 15).

[0118] Next, it was investigated whether blocking the BA uptake carriers ameliorated the APAP-in duced alterations in canalicular and right-junction morphology. Although Myrcludex B plus the loss of Oatp ameliorated APAP-induced cell death, they did not prevent the compromised canalicular and right- junction morphology due to APAP overdose, as evidenced by CD 13, ZOl, and Claudin3 immunostain- ing. However, Myrcludex B plus Oatp knockout massively reduced BA concentrations in liver tissue as demonstrated by MALDI-MSI and MS analyses (Figure 16), while BA concentrations strongly in creased in the blood (Figure 17), demonstrating the efficacy of BA uptake inhibition. Altogether, these data suggest that interruption of BA cycling by blocking the sinusoidal uptake carriers strongly amelio rate APAP -induced liver injury. f) Clinical data in humans

[0119] Figures 18 to 20 show clinical results in humans with regard to blood bile acid concentrations and transaminase activities in relation to APAP -induced hepatotoxicity. Figures 18 and 19 show ALT activity and sum of bile acid concentrations in plasma of APAP-intoxicated patients in whom the period between overdose and blood sampling was unknown. Patients were grouped by their combination of ALT and bile acid levels.

[0120] Figure 20 shows the results of a time-resolved analysis of the sum of blood bile acid concentra tions and ALT activity of a 19-year-old woman after APAP overdose. Dashed baselines: reference val ues in healthy individuals.

[0121] Figure 21 shows in comparison the sum of bile acids concentrations and ALT in heart blood after APAP overdose in mice (mean and SE of 4 mice per time point).

[0122] As shown in Figure 20 (human) and Figure 21 (mice), first the concentration of the bile acids increase, only then that of the liver enzymes, e.g. ALT. The situation in mice therefore in principle is the same as in humans, but with a different time factor. Everything happens much earlier in the mouse. However, this means that human patients have more time for therapy with inhibition of NTCP (SLC10A1) according to the invention, namely up to approximately 50 hours after the acetaminophen intoxication. This is important because the conventional therapy with N-acetylcysteine can only be used with good effectiveness up to about 8 hours after acetaminophen intoxication.