Medicament for liver regeneration and for treatment of liver failure

Abstract

The present invention relates to the use of a compound which inhibits the activity of MKK4 as a medicament for the treatment of a patient suffering from an impaired liver function, to the use of a compound as a medicament for the treatment of liver failure, including acute/fulminant or chronic liver failure and/or for increasing the regeneration of liver tissue in a patient.

Claims

1. A method of treatment of liver failure and/or for the protection of hepatocytes against apoptosis and/or for the regeneration of hepatocytes, the method comprising a step of administering to a patient in need of treatment a compound, which is an inhibitor of the activity of MKK4, MKK4 being encoded by the mRNA of SEQ ID NO: 1204, wherein the compound is selected from the group consisting of the following compounds: ##STR00200## ##STR00201## ##STR00202## ##STR00203## ##STR00204##

2. The method according to claim 1, wherein the compound is formulated as liposomes or lipid nanoparticles.

3. The method according to claim 1, wherein the compound is a compound having the following structure: ##STR00205##

4. The method according to claim 1, wherein the patient is a human patient.

Description

DETAILED DESCRIPTION OF THE INVENTION

(1) The invention is now described in greater detail by way of examples with reference to the figures, which show in

(2) FIG. 1 schematic representations of nucleic acid constructs for producing inhibitory RNA,

(3) FIG. 2 a schematic representation of a transposase mediated intrahepatic transfer of an expression cassette encoding an inhibitory RNA (micro RNA based shRNA) of the invention,

(4) FIG. 3 the time course of the body weight of mice after stable transposon mediated intrahepatic transfer of an expression cassette for inhibitory RNA and controls, whereas an increase in body weight correlates with an increase in liver repopulation with the construct,

(5) FIG. 4 GFP-imaging of explanted mouse livers in the process of repopulation by hepatocytes stably expressing shRNA specific for inactivating MKK4,

(6) FIG. 5 a Western blot specific for MKK4 of liver samples of mice stably transfected with an expression cassette encoding shRNA specific for inactivating MKK4,

(7) FIG. 6 immunofluorescence analysis of the livers of mice stably transfected with an expression cassette encoding shRNA specific for inactivating MKK4,

(8) FIG. 7 a Western blot for cyclin A and E of nuclear liver extracts of transfected mouse livers in the indicated time course after partial hepatectomy indicating earlier cell cycle entry of hepatocytes stably expressing shRNA specific for MKK4,

(9) FIG. 8 a Ki67 staining of mouse livers expressing shRNA specific for MKK4 or control shRNA at the indicated time points after partial hepatectomy,

(10) FIG. 9 a quantifying graph of Ki67 positive hepatocytes depicted in FIG. 8,

(11) FIG. 10 TUNEL (upper panel) and H&E staining (lower panel) on liver sections after induction of an acute/fulminant liver failure in control shRNA transfected hepatocytes in comparison to hepatocytes expressing an shRNA specific for RNA of MKK4, which are protected,

(12) FIG. 11 a quantifying graph of apoptotic hepatocytes according to TUNEL staining as depicted in FIG. 10,

(13) FIG. 12 a survival curve of mice expressing the shRNA inactivating MKK4 (shMKK4) compared to control mice (shCtr.) after induction of liver failure,

(14) FIG. 13 a quantifying graph of EdU incorporation into cultured murine hepatocytes with inactivated MKK4 activity (FAHIG-shMKK4) and control hepatocytes (FAHIG-shCtr) with an inset showing phase contrast micrographs of these hepatocytes,

(15) FIG. 14 phase contrast micrographs of cultured hepatocytes with inactivated MKK4 (shMKK4) at day 29 (d29) and of control hepatocytes (shCtr) at day 12 (d12), and at day 3 of hepatocytes replated at day 15 (replating),

(16) FIG. 15 a survival curve of FAH ?/? mice after transplantation of hepatocytes kept one week in culture expressing shRNA specifically inactivating MKK4 or a control shRNA,

(17) FIG. 16 photographs, GFP-imaging, ?-FAH immunostaining and H&E staining of liver of mice aged for 1 year following transplantation of hepatocytes stably expressing shRNA specifically inactivating MKK4, and in

(18) FIG. 17 an overview of the inhibitory effect of preferred small inhibitory compounds.

(19) Using mice and murine liver tissue and hepatocytes as examples, especially representing human patients and human liver tissue and human hepatocytes, respectively, it was found that liver regeneration could be increased by inactivating MKK4 activity, both in vivo and in cultured hepatocytes. Mice harbouring livers with reduced MKK4 activity show increased regenerative capacity under conditions of liver failure, which also resulted in an increased survival. Inactivation of MKK4 activity could efficiently be achieved by inhibitory RNA present in hepatocytes, in vivo and in vitro, which inhibitory RNA could be generated by transcription from a stably or transiently transfected nucleic acid construct containing an expression cassette encoding at least one RNA which under physiological conditions hybridizes to the mRNA of MKK4.

(20) Alternatively, the inhibitory RNA could be introduced, e.g. transfected into hepatocytes in vivo and in culture, e.g. in the form of an siRNA, shRNA or microRNA, preferably in a suitable formulation, e.g. formulated as a liposome preparation or a lipid nanoparticle preparation. In the alternative to the use of inhibitory RNA for use as a medicament for the treatment of liver and hepatocytes, SP600125, myricitine, Genistein, PD98059, 3-(Dimethylamino)-N-[3-[(4-hydroxybenzoyl)-amino]-4-methylphenyl]benzamide (ZM 336372), 2-hydroxy-1-methyl-4-oxo-N-pyridin-4-ylquinoline-3-carboxamide (BAS00525963), 2-(1H-indazol-5-yliminomethyl)-6-nitrophenolate (BAS00697444), 5-[2-(3,5-dimethyl-1H-pyrazol-4-yl)ethyl]-7-oxo-N-phenyl-1H-pyrazolo[1,5-a]pyrimidine-3-carboxamide (SYN22174524), 5-[2-(3,5-dimethyl-1H-pyrazol-4-yl)ethyl]-3-(4-fluorophenyl)-1H-pyrazolo[1,5-a]pyrimidin-7-one (SYN22174787), 5-[2-(3,5-dimethyl-1H-pyrazol-4-yl)ethyl]-3-(4-methylphenyl)-1H-pyrazolo[1,5-a]pyrimidin-7-one (SYN22175977), 3-(4-chlorophenyl)-5-[2-(3,5-dimethyl-1H-pyrazol-4-yl)ethyl]-2-(methoxymethyl)-1H-pyrazolo[1,5-a]pyrimidin-7-one (SYN22176267), -[2-(3,5-dimethyl-1H-pyrazol-4-yl)ethyl]-2-(methoxymethyl)-3-(4-methylphenyl)-1H-yrazolo[1,5-a]pyrimidin-7-one (SYN22176367), 5-[2-(3,5-dimethyl-1H-pyrazol-4-yl)ethyl]-2-(3-methoxyphenyl)-3-methyl-pyrazolo[5,1-b]pyrimidin-7-ol (SYN22176842), 5-[2-(3,5-dimethyl-1H-pyrazol-4-yl)ethyl]-2-(2-methoxyphenyl)-3-methyl-pyrazolo[5,1-b]pyrimidin-7-ol (SYN22176990), 3-(4-chlorophenyl)-5-[2-(3,5-dimethyl-1H-pyrazol-4-yl)ethyl]-2-methyl-1H-pyrazolo[1,5-a]pyrimidin-7-one (SYN22177890), 5-amino-3-[(Z)-1-cyano-2-[3-[(4-methoxy-6-piperidin-1-yl-1,3,5-triazin-2-yl)oxy]phenyl]ethenyl]-1-(2-hydroxyethyl)pyrazole-4-carbonitrile (BAS00896568), 2-(1H-indazol-5-yliminomethyl)-6-methoxy-4-nitrophenolate (BAS00697462), 7-oxobenzo[e]perimidine-4-carboxylic acid (BAS00368055), the further compounds contained in Table 1 given herein, and combinations thereof could be used as medicaments, the presence of which inactivated MKK4 activity at least partially, also resulting in an increase of hepatocyte proliferation, protection against induced apoptosis, and restoration of liver function. These compounds having specific inhibitory activity against MKK4 are also collectively referred to as small inhibitory compounds. Accordingly, both the inhibitory RNA having specificity for the RNA encoding MKK4 and the small inhibitory compounds having specificity for MKK4 protein each inhibit MKK4 and are therefore used as medicaments in the treatment of liver failure and/or for the protection of hepatocytes against apoptosis and/or for the regeneration of hepatocytes. The small inhibitory compounds can be formulated in a pharmaceutically acceptable formulation, comprising e.g. buffer substance and carrier substance as well as formulation additives as known to the pharmacist, e.g. for i.v., i.m., intra-liver administration or oral administration.

(21) During functional in vitro testing of hepatocytes containing nucleic acid constructs with stable expression of FAH, GFP and shRNA, hepatocytes were isolated from mouse livers and cultivated. It was found that only hepatocytes which were transfected with an expression cassette encoding an inhibitory RNA targeting, i.e. specifically hybridizing with, the mRNA encoding MKK4 could be cultivated for extended periods, e.g. for over 30 days. In addition, these hepatocytes could be trypsinized and replated according to standard methods.

(22) Transplantation of primary hepatocytes expressing the shRNA against MKK4 after 1 week in culture into FAH knock-out mice showed the capacity of the hepatocytes in which MKK4 was inactivated to repopulate the liver of these mice and allow survival. In contrast, this result could not be obtained by primary hepatocytes expressing the non-specific control shRNA. This result also indicates that primary hepatocytes expressing the shRNA against MKK4 do not undergo major dedifferentiation during the time of culture.

Example 1: Inactivation of MKK4 by Transcription of Inhibitory RNA from an Expression Cassette Integrated into Liver Tissue

(23) The introduction of inhibitory RNA into hepatocytes, i.e. into the liver of a patient, for inactivating MKK4 in vivo by expression of the inhibitory RNA from a nucleic acid construct encoding the inhibitory RNA in an expression cassette is shown on the example of mice (C57BL/6) using an expression cassette encoding the inhibitory RNA for production of the shRNA hybridizing to the mRNA encoding MKK4. The promoter controlling transcription of the inhibitory RNA was constitutive.

(24) In short, homozygous FAH-negative mice (FAH ?/?) were kept with constant administration of NTBC in order to block the 4-hydroxyphenylpyruvate dioxygenase which would otherwise lead to the accumulation of toxic metabolites in the liver. As inhibitory RNA, SEQ ID NO: 1 or, alternatively, SEQ ID NO: 2 was used, both of which hybridize with the mRNA encoding MKK4. Each inhibitory RNA was introduced by contacting the liver cells in vivo with nucleic acid constructs with transposase-specific inverted repeat sections (IR) on both termini, containing an expression cassette for FAH for complementation of the FAH ?/? genotype upon expression, by hydrodynamic tail vein injection in combination with a second nucleic acid construct encoding transposase sleeping beauty 13 (SB 13) under the control of the PGK promoter.

(25) The nucleic acid constructs are shown in FIG. 1. FIG. 2 schematically shows the steps of the genetic manipulation. A first control construct p/T-FAHIG contains the complementing FAH expression cassette and a green fluorescent protein (GFP) expression cassette comprising the GFP encoding sequence under the control of an IRES element, but encodes no inhibitory RNA. A sequence encoding an inhibitory RNA with no target as a control, which in addition to the GFP expression cassette in 3 to the GFP encoding sequence encodes a microRNA was contained in the construct p/T-FAHIG-shCtr. A sequence encoding an inhibitory RNA according to the invention was contained in construct p/T-FAHIG-shMKK4, which in addition to the GFP expression cassette in 3 to the GFP encoding sequence encodes a microRNA (depicted as a loop) comprising an shRNA as an example for an inhibitory RNA. In the example, SEQ ID NO: 1, alternatively SEQ ID NO: 2 was used as a preferred representative of inhibitory RNA sequences. Following introduction of the nucleic acid constructs, mice were kept in the absence of NTBC for selecting animals having complemented hepatocytes. In cotransfected cells, the transient expression of SB 13 leads to the stable integration of the expression cassette in the genome.

(26) Analyses of mice after introduction of the nucleic acid constructs confirmed stable transcription of the inhibitory RNA from the nucleic acid construct. In detail, analysis of body weight of mice of FIG. 3 shows that the animals having received the control construct p/T-FAHIG (5) as well as the animals having received the control construct p/T-FAHIG-shCtr. (1), which expresses a non-specific RNA could not reconstitute liver function effectively but died.

(27) Animals of those groups having received a nucleic acid construct containing an expression cassette for an inhibitory RNA which is specific for SEQ ID NO: 1, namely p/T-FAHIG-shMKK4.A (2, 4) and p/T-FAHIG-shMKK4.B (3) could reconstitute liver function, as shown by the survival and restoration of body weights.

(28) This result is further supported by FIG. 4 showing livers explanted at day 20 after administration of the nucleic acid construct, where livers are in the process of repopulation by hepatocytes which were co-transfected in vivo with a nucleic acid construct containing an expression cassette for FAH and GFP and including an expression cassette for inhibitory RNA specific for mRNA encoding MKK4 (p/T-FAHIG-shMKK4, both left-hand pictures), or including an expression cassette encoding a non-specific inhibitory RNA (p/T-FAHIG-shCtr, both right-hand pictures). The explanted livers of FIG. 4 show a faster increase of GFP fluorescence over time in vivo from animals co-transfected with the nucleic acid construct which includes the expression cassette encoding an inhibitory RNA specific for the mRNA encoding MKK4 compared to animals co-transfected with the nucleic acid construct which includes the expression cassette encoding an inhibitory RNA with no target.

(29) The result from fluorescence is confirmed in this case in fully repopulated mouse livers by the immunospecific staining for MKK4 in the Western blot shown in FIG. 5 and by the immunofluorescence analyses for expression of MKK4 in the tissue samples of explanted mouse livers which are shown in FIG. 6.

(30) In FIG. 5, shMKK4-224 denotes protein extracts from mouse livers repopulated with an expression cassette encoding an inhibitory RNA against MKK4 and shMKK4-3553 denotes protein extracts from mouse livers repopulated with an expression cassette encoding an independent inhibitory RNA against MKK4; tubulin served as a loading control and was detected by a specific antibody (?-tub), MKK4 was detected by an anti-MKK4 antibody (?-MKK4). In FIG. 6, FAHIG-shCtr denotes a nucleic acid construct containing the expression cassette for the complementing FAH and for GFP, including a non-specific inhibitory RNA (shCtr). shMKK4-A and shMKK4-B denote nucleic acid constructs containing expression cassettes for shRNA which specifically hybridize to the mRNA of MKK4.

(31) Both analyses show that only the nucleic acid construct which includes an expression cassette encoding an inhibitory RNA specific for the mRNA encoding MKK4 results in a decrease of MKK4 expression in hepatocytes.

(32) FIG. 7 shows Western blots for cyclin A and E of nuclear extracts from the mouse livers contacted with the nucleic acid construct expressing the shRNA hybridizing to MKK4 mRNA (shMKK4, +) and expressing the non-specific shRNA (shCtr, +), respectively, at 0, at 24 h, at 38 h, and at 48 h after partial hepatectomy, detected with ?-cyclin A antibody (?-Cyc.A) and ?-cyclin B antibody (?-Cyc.B). This analysis shows that inactivation of MKK4, which is e.g. obtained by the expression of an inhibitory RNA hybridizing to the mRNA encoding MKK4, leads to an earlier entry of the cell cycle after partial hepatectomy.

(33) FIG. 8 shows a Ki67 stain of livers of the experimental animals having received the nucleic acid construct expressing the inhibitory RNA specific for MKK4 mRNA (shMKK4) and of animals having received the construct expressing the non-specific shRNA (shCtr), respectively, at 0 h, 38 h, and 48 h following partial hepatectomy. The analyses show that the inactivation of MKK4, which in the example is obtained by presence of the shRNA which is specific for MKK4 mRNA and is expressed from the nucleic acid construct introduced into the hepatocytes results in an increase of hepatocyte proliferation in vivo.

(34) FIG. 9 shows a quantification of the Ki67-positive cells from the analyses of FIG. 8. The increase in hepatocyte proliferation for the hepatocytes containing the shRNA inhibiting expression of MKK4 (p/t-FAHIG-shMKK4) is significant in comparison to the non-specific shRNA control (p/t-FAHIG-shCtr).

(35) FIG. 10 shows TUNEL staining for identification of apoptotic cells in liver tissue from mice transfected by an integrating nucleic acid construct containing an expression cassette for non-specific shRNA (shCtr.), or an expression cassette for shRNA which specifically hybridizes to mRNA of MKK4 (shMKK4.224 or shMKK4.355, each expressing a mouse-specific siRNA hybridizing to the RNA of MKK4). Apoptosis was induced in vivo at 9 h prior to the analysis experimentally by injection of Jo2 antibody, which interacts with CD95 to induce fulminant liver failure. TUNEL staining reveals less apoptotic hepatocytes in the liver tissue expressing the MKK4-specific shRNA (shMKK4.224, shMKK4.355) than in controls (shCtr.). The upper row of pictures shows fluorescence micrographs of TUNEL analyses, the lower row shows bright field micrographs of H&E stained tissue samples.

(36) The quantification of TUNEL analysis following induction of liver failure is shown in FIG. 11, demonstrating a significantly lower number of apoptotic hepatocytes in those liver tissues containing the shRNA (shMKK4.224 and shMKK4.3553) that hybridizes to mRNA of MKK4 when compared to the control with non-specific shRNA (shCtr.).

(37) FIG. 12 shows the survival rate according to Kaplan Meier of mice transfected with the nucleic acid construct expressing the shRNA hybridizing to mRNA of MKK4 (shMKK4.224 and shMKK4.3553) and of control mice (shCtr.) after the experimental induction of liver failure. The result demonstrates that inactivation of MKK4, which inactivation in the example is obtained by expression of an inhibitory RNA (shRNA) from an expression cassette of a nucleic acid construct, effectively protects hepatocytes in vivo against apoptosis.

Example 2: Inhibition of MKK4 In Vivo by Transcription of Inhibitory RNA from an Expression Cassette Encoding shRNA

(38) For transient transfection of hepatocytes, a nucleic acid construct containing or consisting of an expression cassette encoding an inhibitory RNA which specifically hybridizes to the mRNA encoding MKK4, e.g. containing SEQ ID NO: 1 or SEQ ID NO: 2 (which are both specific for the human and the mouse mRNA of MKK4) was transiently introduced into hepatocytes. For transient transfection in vivo, the nucleic acid construct was formulated in liposomes and administered to the experimental animals. The liposome formulation contained the lipids 3-N-[(qmethoxypoly(ethylene glycol)2000)carbamoyl]-1,2-dimyristyloxy-propylamine (PEG-C-DMA), 1,2-dilinoleyloxy-N,N-dimethyl-3-aminopropane (DLinDMA), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) and cholesterol, in a2:40:10:48 molar percent ratio.

(39) The effect of increasing proliferation of hepatocytes, protection against induction of apoptosis could be shown as laid out for the stable expression of the shRNA in Example 1, indicating that the effect was limited to the period in which the shRNA was present in the hepatocytes using the analytical methods as described in Example 1. This shows that MKK4 activity can effectively be inhibited or inactivated by transient expression of inhibitory RNA, e.g. shRNA or microRNA, from an expression cassette of a nucleic acid construct which does not integrate into the hepatocyte.

Example 3: Inhibition of MKK4 In Vivo by Inhibitory RNA Transfected into Hepatocytes

(40) Suitability of inhibitory RNA for use as a medicament could be shown by transfecting the inhibitory RNA which specifically hybridizes to mRNA of MKK4 into liver tissue in vivo. Inhibitory RNA could be shRNA or microRNA, preferably formulated as liposomes or lipid nanoparticles. Generally, reduction or elimination of MKK4 could be obtained in at least a fraction of the liver tissue contacted by the formulation of the inhibitory RNA using the analytical methods as described in Example 1. This shows that inhibitory RNA specific for mRNA of MKK4 can be used as a medicament, especially for the treatment of impaired liver function.

Example 4: Inhibition of MKK4 In Vivo by SP600125, Myricitine, Genistein, or PD98059 in Hepatocytes

(41) In the alternative to inhibition of MKK4 activity in liver tissue by inhibitory RNA, SP600125, myricitine, Genistein, or PD98059 were used for inactivating MKK4 in the liver. Generally, SP600125, myricitine, Genistein, or PD98059 were administered to mice at a dosage efficient for in vivo inactivation of MKK4. Preferably, the dosage was efficient to inactivate at least 80%, more preferably at least 90 or 95% of mean in vivo MKK4 activity.

(42) It could be found that the inactivation of MKK4 in the liver by administration of SP600125, myricitine, Genistein, or PD98059 as a medicament resulted in a significant increase in liver regeneration, an increase in proliferation, and in protection against induced apoptosis using the analytical methods as described in Example 1.

Example 5: Inhibition of MKK4 in Primary Hepatocytes Cultured In Vitro by Stable or Transient Transcription of Inhibitory RNA from an Expression Cassette Encoding shRNA

(43) For in vitro transfection, cultured primary hepatocytes obtained from experimental animals were contacted by the nucleic acid construct as described in Example 1 or 2. Generally, the nucleic acid construct could be formulated as liposomes according to Example 2.

(44) Generally, stable or transient expression of the inhibitory RNA could be obtained in the cultured hepatocytes, and reduction or elimination of MKK4 could be detected using the analytical methods as described in Example 1.

(45) For experimental purposes, in the alternative to in vitro transfection of primary hepatocytes originating from an experimental animal, stably transfected hepatocytes expressing shRNA specific for MKK4 mRNA were isolated from the experimental mice generated according to Example 1. Analysis of cultured hepatocytes was by quantification of the incorporation of EdU by primary hepatocytes by flow cytometry. The result of cultivated transfected hepatocytes after 3 days culture is shown in FIG. 13. The inset phase contrast micrographs and the relation of hepatocytes containing shRNA specific for mRNA of MKK4, generated by expression from the transfected expression cassette, show that cultured hepatocytes with inactivated MKK4 (FAHIG-shMKK4) show a drastically improved EdU incorporation as a marker for proliferation over controls (FAHIG-shCtr) without inhibition of MKK4 activity in culture.

(46) Replating of the cultured hepatocytes in fresh culture medium shows the increased long-term survival of cultured hepatocytes in which MKK4 activity is essentially inhibited, e.g. by presence of inhibitory RNA (shMKK4) that specifically hybridizes to mRNA of MKK4, as shown in the micrographs of FIG. 14. Hepatocytes with inactivated MKK4 (shMKK4) can be cultured effectively at least to day 29 (d29), and can be cultured by trypsinizing and replating to fresh medium at day 15; right-hand micrographs show day 3 of cells replated after 15 days initial culture. In contrast, transfected cells with a non-specific shRNA (shCtr) show a lower long term survival in culture and no growth upon replating after 15 days initial culture.

(47) These results show that the inactivation of MKK4 activity drastically increases long term survival and replating efficiency of cultured hepatocytes.

(48) Generally, the generally known Eagles medium was used for hepatocyte cultures.

Example 6: Cultured Hepatocytes with Inactivated MKK4 Activity for Use as a Medicament for Liver Regeneration

(49) Hepatocytes from a mouse representing a patient having a compatible or identical blood group, preferably hepatocytes that were immunologically compatible with a later recipient, e.g. a patient, preferably autologous hepatocytes, were cultured. MKK4 activity was inhibited as described in the above Examples, preferably by transfection of cultured hepatocytes with a nucleic acid construct containing an expression cassette for an inhibitory RNA hybridizing to the mRNA encoding MKK4, by transfection with an inhibitory RNA, preferably repeatedly, or by contacting with SP600125, myricitine, Genistein, or PD98059.

(50) Cultured mouse hepatocytes which were stably transfected with a nucleic acid construct expressing the complementing FAH and GFP (FAHIG) and an inhibitory RNA specific for the mRNA encoding MKK4 or a non-specific shRNA (Ctrl), respectively, were harvested by trypsinizing. These hepatocytes were suspended in a pharmaceutically acceptable carrier and transplanted into the spleen or liver of FAH ?/? mice, which subsequently were kept without NTBC. The Kaplan Meier analysis of survival after intraspleenic transplantation of the cultured hepatocytes is shown in FIG. 15. In comparison to mice having received hepatocytes containing the non-specific shRNA (shRNA.Ctrl) that die at day 37-38 (vertical line), mice having received hepatocytes containing shRNA Mkk4 specific for the mRNA of MKK4 by expression from the expression cassette encoding the shRNA have a drastically increased survival.

(51) The experimental FAH ?/? mice that had repopulated livers with hepatocytes with an expression cassette for GFP, including the shRNA specific for the mRNA encoding MKK4 (shRNA.MKK4) were kept for 12 months following repopulation. Analyses of explanted livers in bright field photography (Bright), with GFP imaging (GFP) (left-hand pictures of FIG. 16) and anti-FAH immunofluorescence and H&E staining of liver sections (right-hand pictures of FIG. 16) show no tumor development with stable intrahepatic expression of GFP and of the shRNA specifically inactivating MKK4. These data emphasize that MKK4 inhibition can be used to increase regeneration without triggering tumor growth.

Example 7: Cultivated Hepatocytes with Inactivated MKK4 Activity for Use as a Device for Extracorporeal Blood Purification

(52) Cultured hepatocytes obtained as described above, preferably by cultivating primary hepatocytes which were stably transfected with a nucleic acid construct expressing an shRNA specific for the mRNA encoding MKK4 were grown on a carrier substrate, e.g. a polymer carrier. The cultured hepatocytes adhering to the carrier substrate were arranged in a container which was perfused with blood withdrawn from a patient, exemplified by a mouse or rat. Blood exiting the container could immediately be returned into the patient.

(53) In initial experiments, it could be shown that hepatocytes which are genetically manipulated to stably express an shRNA inactivating the mRNA encoding MKK4 are stable when grown on a carrier substrate, and that these cultures hepatocytes could be used as a blood purification device.

Example 8: Inactivation of MKK4 in In Vitro Analyses

(54) The inhibitory effect of compounds against MKK4 was analysed in an in vitro assay using purified MKK4 protein, e.g. obtained from a cell line that was genetically manipulated to over-express MKK4 from an expression cassette containing the nucleotide sequence SEQ ID NO: 1204 as a coding sequence and affinity purification using e.g. an antibody directed against MKK4 protein.

(55) In the assay, purified active MKK4 protein was incubated with its substrate JNK1a1 and .sup.32P-labelled gATP (5 ?Ci, approx. 10 ?M), without additional active compound, with the small inhibitory compound, or with Genistein as a positive control. For the assay, kinase assay .buffer (20 mM HEPES pH 7.5; 10 mM MgCl.sub.2; 1 mg/ml BSA; 1 mM Na.sub.3VO.sub.4; 1 mM DTT) was used. An inhibitory effect of the small inhibitory compound (final concentration 50 ?M) was detected as a reduction of the phosphorylation activity of MKK4 protein on its substrate JNK1a1 by measuring the amount of radioactive (.sup.32P) phosphate in JNK1a1 Phosphorylation of JNK1a1 was measured in the presence of 2 ml scintillation cocktail per sample by using a scintillation counter (Wallac, Liquid Scintillation Counter). In this assay, Genistein gave an inhibition to approx. 80% activity compared to the assay without additional active compound.

(56) TABLE-US-00001 TABLE 1 small inhibitory compounds assayed for inhibitory activity against MKK4: Inhibition, relative activity of MKK4 compared to control (without additional name Structure compound) (%) ZM336372; 3-(Dimethylamino)- N-[3-[(4- hydroxybenzoyl)- amino]-4- methylphenyl] benzamide 54.2 BAS00525963; 2-hydroxy-1- methyl-4-oxo-N- pyridin-4- ylquinoline-3- carboxamide 72.6 BAS00697444; 2-(1H-indazol-5- yliminomethyl)-6- nitrophenolate 79.0 SYN22174524; 5-[2-(3,5-dimethyl- 1H-pyrazol-4- yl)ethyl]-7-oxo-N- phenyl-1H- pyrazolo[1,5- a]pyrimidine-3- carboxamide 47.8 SYN22174787; 5-[2-(3,5-dimethyl- 1H-pyrazol-4- yl)ethyl]-3-(4- fluorophenyl)-1H- pyrazolo[1,5- a]pyrimidin-7-one 41.5 SYN22175977; 5-[2-(3,5-dimethyl- 1H-pyrazol-4- yl)ethyl]-3-(4- methylphenyl)-1H- pyrazolo[1,5- a]pyrimidin-7-one 55.5 SYN22176267; 3-(4-chlorophenyl)- 5-[2-(3,5-dimethyl- 1H-pyrazol-4- yl)ethyl]-2- (methoxymethyl)- 1H-pyrazolo[1,5- a]pyrimidin-7-one 59.1 SYN22176367; 5-[2-(3,5-dimethyl- 1H-pyrazol-4- yl)ethyl]-2- (methoxymethyl)-3- (4-methylphenyl)- 1H-yrazolo[1,5- a]pyrimidin-7-one 49.9 SYN22176842; 5-[2-(3,5-dimethyl- 1H-pyrazol-4- yl)ethyl]-2-(3- methoxyphenyl)-3- methyl- pyrazolo[5,1- b]pyrimidin-7-ol 54.9 SYN22176990; 5-[2-(3,5-dimethyl- 1H-pyrazol-4- yl)ethyl]-2-(2- methoxyphenyl)-3- methyl-. pyrazolo[5,1- b]pyrimidin-7-ol 0 58.2 SYN22177890; 3-(4-chlorophenyl)- 5-[2-(3,5-dimethyl- 1H-pyrazol-4- yl)ethyl]-2-methyl- 1H-pyrazolo[1,5- a]pyrimidin-7-one 56.3 BAS00896568; 5-amino-3-[(Z)-1- cyano-2-[3-[(4- methoxy-6- piperidin-1-yl- 1,3,5-triazin-2- yl)oxy]phenyl] ethenyl]-1-(2- hydroxyethyl) pyrazole-4- carbonitrile 58.4 BAS00697462; 2-(1H-indazol-5- yliminomethyl)-6- methoxy-4- nitrophenolate 71.1 BAS00368055; 7-oxobenzo[e]- perimidine-4- carboxylic acid 66.2 IUPAC Name: 1- phenyl-2-[[4- phenyl-5-[(5- phenyltetrazol-2- yl)methyl]- 1,2,4-triazol-3- yl]sulfanyl]ethanone IUPAC Name: 2- [[5-[(2,4-dimethyl- anilino)methyl]-4- (furan-2-ylmethyl)- 1,2,4-triazol-3- yl]sulfanylmethyl]- 1H-quinazolin-4- one N-(2-furylmethyl)- N-[1- (isopentylcarbamoyl) ethyl]-5- (morpholinomethyl)- furan-2- carboxamide IUPAC Name: 4-N- benzyl-1-N-[2-(3,4- dimethoxyphenyl)- ethyl]-4-N- ethylbenzene-1,4- disulfonamide IUPAC Name: 3-[2- (2,5- dimethoxyphenyl)- 2- oxoethyl]sulfanyl- 6-methyl-2H-1,2,4- triazin-5-one IUPAC Name: 2-[4- (4-methylbenzoyl)- piperidin-1- yl]sulfonylbenzoate 0 IUPAC Name: 2-[4- [(2,4-dioxo-1,3- thiazolidin-5- ylidene)methyl]-2- methoxyphenoxy]- acetic acid Popular Name: N- (6-ethoxy-1,3- benzothiazol-2-yl)- 2-[[2-(p-tolyl)-9H- purin-6- yl]sulfanyl] acetamide 2-[4-(4-methoxy- phenyl)-piperazin- 1-yl]-N-(3- morpholino- sulfonyl-phenyl)- acetamide Popular Name: 5- [[4-[(2,4,6- trioxohexa- hydropyrimidin-5- ylidene)methyl]- phenoxy]methyl]- furan-2-carboxylic IUPAC Name: 3- (benzimidazol-1- yl)-N-[(2R)-1-[3- (3,4- difluorophenyl)-6- oxopyridazin-1- yl]butan-2- yl]propanamide IUPAC Name: 2- methyl-3-(pyridin- 3- ylmethylamino) benzoate IUPAC Name: N- [2-[[4-amino-6- (dimethylamino)- 1,3,5-triazin-2- yl]oxy]ethyl]-2-(4- chloro-2- methylphenoxy) acetamide IUPAC Name: [3- ethoxy-4-(thiophen- 2- ylmethoxy)phenyl] methyl-(2- morpholin-4-ium-4- ylethyl)azanium IUPAC Name: 2-[4- (2- hydroxyethyl) piperazin-1- yl]-N-(2- pyrrolidin-1- ylsulfonylethyl) pyridine-3- carboxamide IUPAC Name: 1- [[2-(furan-2-yl)- pyrrolo[2,3-b] pyridin-3-yl]methyl methylamino]-3-(4- methoxyphenoxy) propan-2-ol 0 IUPAC Name: N- ethyl-3-[2-(4- methoxyphenoxy) ethoxy]-N- (pyrazolo[1,5- a]pyrimidin-3- ylmethyl)aniline IUPAC Name: N- ethyl-3-[2-(4- fluorophenoxy) ethoxy]-N-[(2- methylpyrimidin-5- yl)methyl]aniline IUPAC Name: 2- methoxy-5- morpholin-4- ylbenzoate IUPAC Name: (1R,2S,3R)-3-(2- aminobenzoyl)-3- methyl-2-N-[(5- methyl-1,2,4- oxadiazol-3- yl)methyl]-1-N- (pyridin-4- ylmethyl) cyclopropane-1,2- dicarboxamide IUPAC Name: (2S,3S)-2,3- bis(ethoxycarbonyl) butanedioate IUPAC Name: diethyl 2-[(1,4- diethoxy-1,4- dioxobut-2-en-2- yl)amino]but-2- enedioate IUPAC Name: (2S,3S)-2,3- bis(ethoxycarbonyl) butanedioate IUPAC Name: diethyl 2-[(1,4- diethoxy-1,4- dioxobut-2-en-2- yl)amino]but-2- enedioate IUPAC Name: 1-ethyl-2-hydroxy-N- (4-hydroxyphenyl)- 4-oxoquinoline-3- carboxamide IUPAC Name: 5- (phenylcarbamoyl- oxy)pentyl N- phenylcarbamate 0 Popular Name: N- [(5-acetamido-2- methoxy- phenyl)methyl]-2- morpholino- acetamide IUPAC Name: 4- [4,6-bis(3- carboxypropyl)- 1,3,5-trioxan-2- yl]butanoic acid IUPAC Name: methyl4- [(4-oxo-2- sulfanylidene-1,3- thiazolidin-5- ylidene)methyl] benzoate IUPAC Name: 2- (3-methylanilino)- N-[(3- nitrophenyl) methylideneamino] acetamide IUPAC Name: [(2R)-2-[3- [bis[3- (dimethylazaniumyl) propyl]amino] propanoyloxy]-3- (dimethylamino) propyl]- dimethylazanium IUPAC Name: 2-hydroxy- 4-oxo-N- pyridin-4-yl- 1H-quinoline-3- carboxamide IUPAC Name: 1-ethyl-2- hydroxy-4-oxo- N-pyridin-4- ylquinoline- 3-carboxamide IUPAC Name: 2,3- bis[2-(2- nitrophenoxy) ethoxy]-1,4-dioxane 4-hydroxy-2-oxo- N-(4- pyridinylmethyl)- 1,2-dihydro-3- quinoline carboxamide IUPAC Name: 7- oxobenzo[e] perimidine-4- carboxylic acid 0 IUPAC Name: 1,3- dioxobenzo[de] isoquinoline-6- carboxylic acid IUPAC Name: diethyl(2S)- 2-[[3-[[(2S)-1,5- diethoxy-1,5- dioxopentan-2- yl]amino]-3- oxopropanoyl] amino]pentanedioate IUPAC Name: diethyl2- [[2-acetamido-3- (4- phenylmethoxyphenyl) propanoyl]amino] pentanedioate IUPAC Name: 3- nitro-N-[(E)-[3- [(E)-[(3- nitrophenyl) hydrazinylidene] methyl]phenyl] methylidene amino]aniline IUPAC Name: (4- methoxyphenyl) methylN-[[4-[2-(3,4- dimethoxyphenyl) ethylamino]-4- oxobutan-2- ylidene]amino] carbamate IUPAC Name: 3- amino-1,5- dihydropyrimido [5,4-b]indole-2,4- dione IUPAC Name: 1- [2-(2- fluorophenoxy) ethyl]-3-[6-[2-(2- fluorophenoxy) ethylcarbamoylamino] hexyl]urea IUPAC Name: methyl4- [[[2-[2-[2-[[4- [hydroxy(methoxy) methyl]phenyl] methylidene] hydrazinyl]-2- oxoethoxy]acetyl] hydrazinylidene] methyl]benzoate IUPAC Name: N- [2-(3,4- dimethoxyphenyl) ethyl]-2-quinolin-8- ylsulfanylacetamide IUPAC Name: (2S,3S)- 2,3-bis(4- butoxyphenoxy)- 1,4-dioxane 0 IUPAC Name: 2-hydroxy-1- methyl-4-oxo-N- pyridin-4- ylquinoline- 3-carboxamide IUPAC Name: quinoline- 2,4-dicarboxylic acid IUPAC Name: 2- [(5Z)-5-[(3- hydroxyphenyl)met hylidene]-4-oxo-2- sulfanylidene-1,3- thiazolidin-3- yl]propanoic acid IUPAC Name: diethyl2- acetamido-2-[[5- amino-2-(2-ethoxy- 2- oxoethoxy)phenyl] methyl]propanedioate IUPAC Name: 4- [2-[2-[2-[(4-amino- 1,2,5-oxadiazol-3- yl)oxy]ethoxy] ethoxy]ethoxy]- 1,2,5-oxadiazol- 3-amine IUPAC Name: (1R,2S,3S, 4S)-2-(thiophen-2- ylmethylcarbamoyl) bicyclo[2.2.l]hept- 5-ene-3- carboxylate IUPAC Name: 3- [4-[2-[(4,4- dimethyl-2,6- dioxocyclohexylidene) methylamino]ethyl] piperazin-1- yl]-1- phenylpyrrolidine- 2,5-dione IUPAC Name: methyl4- [N-[2-(N-(4- methoxy-4- oxobutanoyl)anilino) ethyl]anilino]-4- oxobutanoate IUPAC Name: 7- propan-2-ylidene- 2-(pyridin-3- ylmethylcarbamoyl) bicyclo[2.2.1]hept- 5-ene-3- carboxylic acid 1-[4-[2-hydroxy- 3-(2- pyridylmethylamino) propoxy]phenoxyl- 3-(2- pyridylmethylamino) propan-2-ol 0 IUPAC Name: 3- [5-[(4- hydroxyphenyl) methylidene]-4-oxo-2- sulfanylidene-1,3- thiazolidin-3- yl]propanoic acid 2-(1-benzothiazol- 2- ylaminoiminoethyl- azo)benzoic IUPAC Name: 2- [(3-acetylphenyl)- carbamoyl]bicycle- [2.2.1]hept-5-ene- 3-carboxylate IUPAC Name: 2- (1H-indazol-5- yliminomethyl)-6- nitrophenolate IUPAC Name: 2- bromo-6-[(1H- indazol-5- ylamino)- methylidene]-4- nitrocyclohexa-2,4- dien-1-one IUPAC Name: 2- (1H-indazol-5- yliminomethyl)-6- methoxy-4- nitrophenolate N-{2-[2-(1- methyl-4- piperidinylidene)- hydrazino]-2- oxoethyl}-N-(3- nitrophenyl) benzenesulfonamide (non-preferred name) IUPAC Name: 4- [5-(naphthalen-1- ylmethylidene)-4- oxo-2- sulfanylidene-1,3- thiazolidin-3- yl]butanoate IUPAC Name: 5- amino-3-[(Z)-1- cyano-2-[3-[(4- methoxy-6- piperidin-1-yl- 1,3,5-triazin-2- yl)oxy]phenyl] ethenyl]-1-(2- hydroxyethyl) pyrazole-4- carbonitrile IUPAC Name: 8- [2-methoxy-4-[(1- oxo- [1,3]thiazolo[3,2- a]benzimidazol-2- ylidene)methyl] phenoxy]-1,3,7- trimethylpurine- 2,6-dione 0 IUPAC Name: 2- [2-[2-(4- methylphenyl) sulfonylethoxy] ethoxy]ethyl4- methylbenzenesulfonate IUPAC Name: (2S)-3- acetyl-4-hydroxy- 1-(4- hydroxyphenyl)-2- phenyl-2H-pyrrol- 5-one IUPAC Name: bis[2-(3,4- dimethylphenyl)-2- oxoethyl] cyclohexane-1,2- dicarboxylate IUPAC Name: 8- (butoxymethyl)-3- [2-[[5-(2- chlorophenyl)-1H- 1,2,4-triazol-3- yl]sulfanyl]acetyl]- 3-methyl-2,7- dioxaspiro[4.4] nonane-1,6-dione IUPAC Name: 2- (naphthalen-2- ylsulfonylamino) butanoic acid 1-(3,5- dimethoxyphenyl)- N-[(2- nitrophenyl)methyl] methanamine IUPAC Name: 5- [(2- carboxylatophenyl) sulfamoyl]-2-(3- carboxylatopropylamino) benzoate IUPAC Name: 2- (7H-purin-6- ylazaniumyl)acetate IUPAC Name: [2- (4-bromophenyl)- 2-oxoethyl]6-(5- methyl-2-oxo-1,3- dihydroimidazol-4- yl)-6-oxohexanoate 3-(2,3-dihydro-1H- indol-1- ylcarbonyl)-1,2,2- trimethylcyclo- pentanecarboxylic acid 0 IUPAC Name: benzyl-N- [2-[2-[(4-methoxy- 3-nitrophenyl)- methylidene]- hydrazinyl]-2- oxoethyl]-N- methylcarbamate IUPAC Name: methyl4- [[2-[2-[2-[(4- methoxy-4- oxobutyl)amino]-2- oxoethoxy]phenoxy] acetyl]amino] butanoate IUPAC Name: [2- acetyloxy-4-[2-[5- (ethoxymethyl)-4- imino-2- methylpyrimidin-1- yl]acetyl]phenyl] acetate IUPAC Name: (4- chloro-2- methylphenyl) methylN-[2-[[4- (dimethylamino)-6- [methoxy(methyl) amino]-1,3,5- triazin-2- yl]oxy]ethyl] carbamate 1-[4-[2-hydroxy-3- (2-nitrophenoxy)- propyl]piperazin-1- yl]-3-(2- nitrophenoxy)- propan-2-ol IUPAC Name: 2- [(5R)-3-(4- hydroxyphenyl)- 2,4-dioxo-1,3- thiazolidin-5- yl]acetate IUPAC Name: 2- [2-(2,4- dihydroxyphenyl)- 2- oxoethyl]sulfanyl- 4-hydroxy-1H- pyrimidin-6-one IUPAC Name: 4- hydroxy-2-[2-(1H- indol-3-yl)-2- oxoethyl]sulfanyl- 1H-pyrimidin-6- one IUPAC Name: 3- (3-anilino-2- hydroxypropyl)-1- [[3-(3-anilino-2- hydroxypropyl)- 5,5-dimethyl-2,4- dioxoimidazolidin- 1-yl]methyl]-5,5- dimethylimidazolidine- 2,4-dione 1-(2-furylmethyl)- 4-(3-nitrobenzyl)- piperazine 00 IUPAC Name: 2- nitro-6-[(5-pyridin- 4-yl-1,3,4- thiadiazol-2- yl)carbamoyl] benzoic acid 01 IUPAC Name: [2- [[[2-(4- phenylphenoxy) acetyl]hydrazinylidene] methyl]phenyl] acetate 02 2-((4-HYDROXY- NAPHTHYL)- AZO)BENZOIC ACID 03 8-hydroxy-5,6- dihydro-4H-11- oxa-6a- azabenzo[de] anthracene-7,10- dione 04 IUPAC Name: 2- (pyridin-3- ylmethylcarbamoyl) bicyclo[2.2.1]hept- 5-ene-3- carboxylate 05 4-Ethyl-5-(4- hydroxyphenyl)- 4H-[1,2,4]triazol- 3-ylsulfanyl]-acetic acid 06 IUPAC Name: 5- [[4-(benzylamino)- 3- nitrophenyl] sulfonylamino] benzene- 1,3-dicarboxylate 07 IUPAC Name: 4- [4-(4-carboxylato- phenoxy)phenyl]- sulfonylphthalate 08 N-[(2,4- dihydroxyphenyl)- methyleneamino]- 2-[(8-methoxy-2- methyl-4- quinolyl)- sulfanyl]acetamide 09 IUPAC Name: 1- [2-[2-[2-[2-(2- acetyl- phenoxy)ethoxy]- ethoxy]ethoxy]- phenyl]ethanone 0 IUPAC Name: [2- [4-(4-chloro-2- nitro- phenoxy)phenyl]- 2-oxoethyl] 2- benzamidoacetate IUPAC Name: phenacyl 3- [phenacyl- (phenacylamino)- amino]benzoate IUPAC Name: 1- (4-amino-1,2,5- oxadiazol-3-yl)-5- (1H-benzimidazol- 2- ylsulfanylmethyl)- N-[1-(5-nitrofuran- 2- yl)ethylideneamino]- triazole-4- carboxamide IUPAC Name: 2- benzamido-N-[1- (furan-2- ylmethylamino)-1- oxopropan-2- yl]benzamide IUPAC Name: 2- benzamido-N-[1- (3-imidazol-1- ylpropylamino)-1- oxo-3- phenylpropan-2- yl]benzamide IUPAC Name: ethyl2-[[2- [2-(2,3-dioxoindol- 1- yl)acetyl]oxyacetyl]- amino]-4-methyl- 1,3-thiazole-5- carboxylate IUPAC Name: ethyl5-[[5- ethoxycarbonyl-3- (2-methoxy-2- oxoethyl)-4- methyl-1H-pyrrol- 2-yl]methyl]-4-(3- methoxy-3- oxopropyl)-3- methyl-1H-pyrrole- 2-carboxylate IUPAC Name: ethyl2-[[2-(1H- benzimidazol-2- ylsulfanyl)acetyl]- amino]-6-methyl- 5,7-dihydro-4H- thieno[2,3-c]- pyridine-3- carboxylate keto(3-pyridyl- methylcarbamoyl)- BLAHolate keto(4- pyridylmethyl- carbamoyl)BLAH- olate 0 hydroxy-oxo-N-(4- pyridyl)BLAH- carboxamide IUPAC Name: 3- [[(5R)-2,4-dioxo- 1,3-thiazolidin-5- yl]amino]benzoate methyl N-acetyl-5- (1,3-dioxo-1,3- dihydro-2H- isoindol-2-yl)-2- (2H-tetrazol-5- yl)norvalinate IUPAC Name: 3- [(2,3-dioxo-1,4- dihydroquinoxalin- 6- yl)sulfonyl]propanoate IUPAC Name: N- [2-(3,4- dimethoxyphenyl) ethyl]-2-[[5-[(4,6- dimethylpyrimidin- 2-yl)sulfanyl- methyl]-1,3,4- oxadiazol-2-yl]- sulfanyl]acetamide IUPAC Name: (2S)-1- (2,1,3- benzothiadiazol-4- ylsulfonyl)piperidine- 2-carboxylic acid benzyl(veratryl)- BLAH IUPAC Name: 2- [1-(1,3- benzodioxol-5-yl)- 2,5- dioxopyrrolidin-3- yl]sulfanylpyridine- 3-carboxylate IUPAC Name: 2- [4-(5-acetyl-1- hydroxy-4- methylimidazol-2- yl)-2-ethoxy- phenoxy]-N-(3- methylphenyl)- acetamide (3-chlorophenyl)- keto-BLAH- carboxylate 0 1-naphthyl-oxo- BLAHcarboxylic acid IUPAC Name: 2- (3,4- dimethoxyphenyl)- 1-[4-(2- fluorophenyl)- piperazin-1- yl]ethanone IUPAC Name: 4- (4-ethoxyphenyl)- 5-pyridin-4-yl- 1,2,4-triazole-3- thiolate IUPAC Name: 4- (3-methylphenyl)- 5-pyridin-4-yl- 1,2,4-triazole-3- thiolate IUPAC Name: ethyl 2-(2-benzyl- sulfonylbenzimid- azol-1-yl)acetate IUPAC Name: 2- [(3S)-1-(1,3- benzodioxol-5-yl)- 2,5- dioxopyrrolidin-3- yl]sulfanyl- benzoate Popular Name: 1- [3-(3-methoxy- phenoxy)propyl]-4- [(4-methylphenyl)- sulfonyl]piperazine IUPAC Name: 3- (pyridin-3- ylmethyl- amino)benzoic acid 2-(4-hydroxy- phenyl)quinoline- 4-carboxylic acid IUPAC Name: 6- (2-pyridin-4- ylethylcarbamoyl) cyclohex-3-ene-1- carboxylate 0 IUPAC Name: 2- [(3S)-1-(4- hydroxyphenyl)- 2,5- dioxopyrrolidin-3- yl]sulfanylbenzoate IUPAC Name: 4- (4- methoxyphenyl)-5- pyridin-4-yl-1,2,4- triazole-3-thiolate IUPAC Name: ethyl5-[(2R)-3- (3,5- dimethylpyrazol-1- yl)-2-hydroxy- propoxy]-1,2- dimethylindole-3- carboxylate IUPAC Name: 1- naphthalen-2-yl- sulfonylpyrrolidine- 2-carboxylic acid IUPAC Name: (2S)-2- [(2,3-dioxo-1,4- dihydroquinoxalin- 6-yl)sulfonyl- amino]propanoate IUPAC Name: (2R)-3- acetyl-4-hydroxy- 1-[2-(1H-indol-3- yl)ethyl]-2-pyridin- 2-yl-2H-pyrrol-5- one .2.1.0%1,5&]dec- 8-ene-6-carboxylic acid IUPAC Name: (3S)-3-(1H- indol-3-yl)-3- pyridin-4- ylpropanoic acid IUPAC Name: 5- methyl-2-pyridin- 4-yl-1H- [1,2,4]triazolo[1,5- a]pyrimidin-7-one IUPAC Name: (2S)-1-(1- methyl-2- oxobenzo[cd]indol- 6- yl)sulfonylpyrrolidine- 2-carboxylic acid 0 IUPAC Name: [2- [4-(furan-2- carbonyloxy)phenyl]- 2-oxoethyl]1- (furan-2-ylmethyl)- 5-oxopyrrolidine- 3-carboxylate IUPAC Name: 4- [2-[5-(4- methoxyphenyl)- 1H-pyrazol-4- yl]ethenyl]-6- (trifluoromethyl)- 1H-pyrimidin-2- one IUPAC Name: 2-hydroxy-5-[[(E)- (3-methyl-5-oxo-1H- pyrazol-4-ylidene)- methyl]amino]- benzoate IUPAC Name: 3- oxo-2-(pyridin-4- ylmethyl)-1H- isoindole-4- carboxylic acid 8-hydroxy- [1]benzofuro[3,2- b]quinoline-11- carboxylic acid IUPAC Name: 5- [[3-methoxy-4- (thiophen-2- ylmethoxy)phenyl] methylamino]-2- morpholin-4- ylbenzoate oxylic acid (3R)-4-keto-3- morpholin-4-ium- 4-yl-4-(2- phenoxyethoxy) butyrate (3R)-4-keto-3- morpholin-4-ium- 4-yl-4-[[(2S)- tetrahydrofuran-2- yl]methoxy]butyrate 4-oxo-4-[2-[4-(p- tolylsulfonyl)- piperazin-1- yl]ethoxy]butanoic acid 0 IUPAC Name: 4- (3-chloro-4- fluorophenyl)-3- pyridin-4-yl-1H- 1,2,4-triazole-5- thione 3-[3-(4-pyridyl)-5- thioxo-1H-1,2,4- triazol-4- yl]benzoic IUPAC Name: 1-(3-phenyl- adamantane-1- carbonyl)pyrrolidine- 2-carboxylic acid IUPAC Name: 4-(2,3-dihydro-1,4- benzodioxin-6-yl)- 3-(3- hydroxyphenyl)- 1H-1,2,4-triazole- 5-thione (dimethylBLAHyl)- methyl IUPAC Name: N- [1-(3-imidazol-1- ylpropylamino)-3- methyl-1- oxobutan-2-yl]-2- [(4-methoxy- benzoyl)amino]benz- amide IUPAC Name: 1-ethyl-3-methyl- 2-oxoquinoxaline- 6-carboxylate IUPAC Name: 3-(4-oxo-2H- pyrazolo[3,4- d]pyrimidin-1- yl)propanoate 3-(benzotriazol-1- yl)-1-[(3R)-3-[4- (3-methylisoxazol- 5-yl)-2H-pyrazol- 3-yl]-1-piperidyl]- propan-1-one IUPAC Name: N-[2-(5-oxo-4- phenyltetrazol-1- yl)ethyl]-2-(4-oxo- 3H-phthalazin-1- yl)acetamide 0 IUPAC Name: ethyl4-[[4- (7-amino-2- methyl- pyrazolo[1,5- a]pyrimidin-5- yl)piperidin-1- yl]methyl]-3,5- dimethyl-1H- pyrrole-2- carboxylate IUPAC Name: [4- (4-methyl-5- pyrimidin-4-yl-1,3- thiazol-2- yl)piperidin-1-yl]- (1H-1,2,4-triazol- 5-yl)methanone IUPAC Name: 3- methyl-1-phenyl-5- [(2-pyrrolidin-1- ylsulfonylethylamino) methyl]-2H- pyrazolo[3,4- b]pyridin-6-one 1-[2-(4-methoxy- phenyl)pyrimidin- 5-yl]-N-methyl-N- (4,5,6,7-tetrahydro- 1H-indazol-3- ylmethyl)methan- amine IUPAC Name: N- [[2-(4- methoxyphenyl) pyrimidin-5- yl]methyl]-N- methyl-1-pyridin- 2-ylpropan-2- amine IUPAC Name: N- methyl-N-(2- phenoxyethyl)-2- quinazolin-4- yloxyacetamide IUPAC Name: N- [2-[4-(4- fluorophenyl)-4- hydroxypiperidin- 1-yl]ethyl]-2-[(5- methyl-1,3,4- oxadiazol-2- yl)sulfanyl]- acetamide IUPAC Name: 5- (2-ethoxypyridin- 3-yl)-3-[2-(2- propan-2- ylpyrrolo[2,3- b]pyridin-1- yl)ethyl]-1,2,4- oxadiazole IUPAC Name: N- [1-(3,4-dihydro- 2H-1,5- benzodioxepin-7- yl)-2- methylpropyl]-6- (2-hydroxyethyl)- pyrazolo[1,5- a]pyrimidine-3- carboxamide IUPAC Name: N- [2-(1-methyl- benzimidazol-2- yl)ethyl]-2-[2-(3- methyl-1,2,4- oxadiazol-5- yl)phenoxy]- acetamide 0 2-amino-5-[2-[4- [3-(2,3-dimethyl- phenoxy)propyl]- piperazin-1-yl]-2- oxo-ethyl]-6- methyl-3H- pyrimidin-4- IUPAC Name: N- [1-(3,5-dimethyl- pyrazol-1- yl)propan-2-yl]-1- (1,5- dimethylpyrazol-4- yl)sulfonylpiperidine- 4-carboxamide IUPAC Name: 4- [(3,5-dimethyl- pyrazol-1- yl)methyl]-N-[2- (4-fluorophenoxy)- phenyl]-5-methyl- 1,2-oxazole-3- carboxamide IUPAC Name: 2-(3-methyl-2,6- dioxopurin-7-yl)- N-[1-(7-methyl- 1H-indol-3- yl)propan-2- yl]acetamide IUPAC Name: 7-bicyclo[4.1.0]- heptanyl-[4-[3-(2- methoxyphenoxy)- propyl]piperazin-1- yl]methanone IUPAC Name: 5- [2-(3,5-dimethyl- 1H-pyrazol-4- yl)ethyl]-7-oxo-N- phenyl-1H- pyrazolo[1,5- a]pyrimidine-3- carboxamide IUPAC Name: 5-[2-(3,5-dimethyl- 1H-pyrazol-4-yl)- ethyl]-3-(4-fluoro- phenyl)-1H-pyrazolo- [1,5-a]pyrimidin- 7-one IUPAC Name: 5-[2-(3,5-dimethyl- 1H-pyrazol-4- yl)ethyl]-3-(4- methylphenyl)-1H- pyrazolo[1,5-a]- pyrimidin-7-one IUPAC Name: 5-[2-(3,5-dimethyl- 1H-pyrazol-4-yl)- ethyl]-2-(methoxy- methyl)-3-phenyl- 1H-pyrazolo [1,5-a]pyrimidin-7- one IUPAC Name: 3-(4-chlorophenyl)-5- [2-(3,5-dimethyl-1H- pyrazol-4-yl)ethyl]- 2-(methoxymethyl)- 1H-pyrazolo[1,5-a]- pyrimidin-7-one 0 IUPAC Name: 5-[2-(3,5-dimethyl- 1H-pyrazol- 4-yl)ethyl]-2- (methoxymethyl)- 3-(4-methylphenyl)- 1H-pyrazolo [1,5-a]pyrimidin-7- one 2-(3,4-dimethoxy- phenyl)-5-[2-(3,5- dimethyl-1H- pyrazol-4- yl)ethyl]pyrazolo- [5,1-b]pyrimidin-7- ol 5-[2-(3,5-dimethyl- 1H-pyrazol-4- yl)ethyl]-2-(3- methoxyphenyl)-3- methyl- pyrazolo[5,1- b]pyrimidin-7-ol 5-[2-(3,5-dimethyl- 1H-pyrazol-4- yl)ethyl]-2-(2- methoxyphenyl)-3- methyl- pyrazolo[5,1- b]pyrimidin-7-ol 5-[2-(3,5-dimethyl- 1H-pyrazol-4- yl)ethyl]-3-methyl- 2-(2- thienyl)pyrazolo [5,1-b]pyrimidin-7-ol IUPAC Name: 3-(4-chlorophenyl)- 5-[2-(3,5-dimethyl- 1H-pyrazol-4-yl)- ethyl]-2-methyl-1H- pyrazolo[1,5-a]- pyrimidin-7-one IUPAC Name: 5-[2-(3,5-dimethyl- 1H-pyrazol-4-yl)- ethyl]- 2-methyl-3-phenyl- 1H-pyrazolo[1,5-a]- pyrimidin-7-one 5-[2-(3,5-dimethyl- 1H-pyrazol-4- yl)ethyl]-2-(4- fluorophenyl)- pyrazolo[5,1- b]pyrimidin-7-o IUPAC Name: 3-(4-chlorophenyl)- 5-[2-(3,5-dimethyl- 1H-pyrazol-4-yl)- ethyl]-1H-pyrazolo- [1,5-a]pyrimidin- 7-one

(57) Compounds of Table 1 can be found on http:\\zinc.docking.org.

(58) FIG. 17 gives an overview of the inhibitory effects of these small inhibitory compounds on MKK4 protein in relation to the inhibition by Genistein.

(59) In vitro testing according to Example 4 and in vivo testing according to Example 6 could show that these small inhibitory compounds are suitable for use as a medicament for the treatment of liver failure and/or for the protection of hepatocytes against apoptosis and/or for the regeneration of hepatocytes.