WO2016198698A2 - P38 inhibitors for the treatment and prophylaxis of liver cancer - Google Patents

P38 inhibitors for the treatment and prophylaxis of liver cancer Download PDF

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WO2016198698A2
WO2016198698A2 PCT/EP2016/063532 EP2016063532W WO2016198698A2 WO 2016198698 A2 WO2016198698 A2 WO 2016198698A2 EP 2016063532 W EP2016063532 W EP 2016063532W WO 2016198698 A2 WO2016198698 A2 WO 2016198698A2
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compound
salt
gamma
composition
formula
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WO2016198698A8 (en
WO2016198698A3 (en
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Guadalupe Sabio Buzo
Antonia TOMÁS LOBA
Ana MARTÍNEZ GIL
Carmen Gil Ayuso-Gontan
Bárbara González Terán
Elisa MANIERI
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Cnic Fundación Centro Nacional De Investigaciones Cardiovasculares Carlos Iii
Consejo Superior De Investigaciones Científicas
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    • A61K31/5375—1,4-Oxazines, e.g. morpholine
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    • C07D213/24—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom with substituted hydrocarbon radicals attached to ring carbon atoms
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Definitions

  • the present invention provides p38 protein kinase (MAPK) inhibitors which are useful for the treatment and prophylaxis of liver cancer.
  • MAPK protein kinase
  • MAPK Mitogen-activated protein kinase
  • MAPK extracellular signal-regulated kinases
  • JNK Jun N-terminal kinases
  • p38 kinases ERK
  • ERK extracellular signal-regulated kinases
  • JNK Jun N-terminal kinases
  • p38 kinases ERK
  • the mammalian MAPK p38 subfamily has four members, ⁇ 38 ⁇ , ⁇ 38 ⁇ , ⁇ 38 ⁇ (from hereinafter indistinctively p38 gamma or ⁇ 38 ⁇ ) and ⁇ 38 ⁇ (from hereinafter indistinctively p38 delta or ⁇ 38 ⁇ ), which share similar protein sequences and are activated by dual phosphorylation mediated by the MAPK kinases MKK3 and MKK6.
  • p38MAPK Based on expression patterns, substrate specificities and sensitivity to chemical inhibitors, p38MAPK can be further divided into two subsets.
  • One group includes p38a and ⁇ 38 ⁇ , which are closely related that might have overlapping functions, whereas ⁇ 38 ⁇ appears to be expressed at very low levels, p38a is abundant in most cell types and is the best-described isoform.
  • ⁇ 38 ⁇ and ⁇ 38 ⁇ have restricted expression patterns and probably have specialized functions.
  • the p38MAPK subfamily members are included in the group of canonical-signalling pathways involved in the cell transformation process.
  • Malignant transformation requires deregulation of at least six cell processes, and cancer cells have to acquire the following capabilities: self sufficiency in growth signals, unlimited replication potential, protection against apoptotic cell death, de novo angiogenesis and tissue invasion and metastasis.
  • p38a negatively regulates cell cycle progression at both the Gl/S and the G2/M transitions and is involved in survival and in the apoptosis induced by many types of cell stress.
  • p38a might also directly affect migration, tumour invasion and angiogenesis by inducing expression of some matrix metalloproteinases (MMP) and of the vascular endothelial growth factor A, a potent inducer of tumor survival and angiogenesis.
  • MMP matrix metalloproteinases
  • the object underlying the present invention is to provide compounds, in particular, p38 inhibitors as well as compositions and formulations thereof that are useful for the treatment and/or prophylaxis of liver cancer.
  • Liver cancer or "Hepatic cancer” as used herein refers to a cancer that originates in the liver. Liver tumours are discovered on medical imaging equipment (often by accident) or present themselves symptomatically as an abdominal mass, abdominal pain, yellow skin, nausea or liver dysfunction. The leading cause of liver cancer is cirrhosis due to either hepatitis B, hepatitis C, or alcohol.
  • the present invention thus provides compounds for use in a method for the therapeutic treatment and/or prophylaxis of liver cancer.
  • the compound is capable of a. inhibiting the intracellular expression of the p38 gamma protein in the hepatocytes of a subject relative to that observed in the absence of the compound; and/or b. inhibiting the intracellular expression of the p38 delta protein in the hepatocytes of a subject relative to that observed in the absence of the compound.
  • an inhibitor refers to any compound, natural or synthetic, which can reduce activity of a gene product. Accordingly, an inhibitor may inhibit the activity of a protein that is encoded by a gene either directly or indirectly. Direct inhibition can be obtained, for instance, by binding to a protein and thereby preventing the protein from binding a target (such as a binding partner) or preventing protein activity (such as enzymatic activity) . Indirect inhibition can be obtained, for instance, by binding to a protein's intended target, such as a binding partner, thereby blocking or reducing activity of the protein.
  • an inhibitor according to the present invention is capable of reducing the intracellular expression of the p38 gamma and/or delta protein or its activity in at least 20%, preferably at least 30%, more preferably at least 50%, still more preferably at least 70%, still more preferably 90% in comparison to that observed in the absence of the compound.
  • the term "compound capable of inhibiting p38 gamma or delta intracellular expression” refers to any compound, natural or synthetic, which results in a decreased activity of p38 gamma or p38 delta. The skilled person in the art can assess whether a given compound is a p38 gamma or delta inhibitor without undue burden.
  • inhibition of p38 gamma or delta intracellular expression by a given compound may be determined in vitro or in vi vo .
  • the present invention provides compounds selected from the list consisting of:
  • a p38 delta and/or p38 gamma gene silencer compound such as a siRNA (small interfering RNA of p38 delta and/or p38 gamma protein) or shRNA (small hairpin RNA of p38 delta and/or p38 gamma protein) or RNA-directed DNA cleavage by the cas9-crisp or talent based technology;
  • Doramapimod (BIRB 796; BIRB-796; BIRB796) or a salt or derivative thereof capable of inhibiting p38 gamma and/or delta intracellular expression;
  • Rl is selected from H, 3-OCH3 or 4-OCH3;
  • Y is selected from CH or N;
  • Ar is 3-0-CH3-Phenyl , 1-pyridyl, 2-pyridyl, 3-pyridyl, 4- pyridyl, phenyl or 2F-4Br-Phenyl ; or a salt or derivative thereof capable of inhibiting p38 gamma and/or delta intracellular expression; and f .
  • the present invention also provides methods of treatment and/or prophylaxis using the p38 inhibitors described herein for the treatment and/or prophylaxis of liver cancer.
  • the present invention relates to a pharmaceutical composition
  • a pharmaceutical composition comprising a therapeutically effective amount of any of the p38 inhibitors described herein or a salt thereof as active ingredient.
  • the pharmaceutical composition of the present invention is preferably formulated as an oral dosage form to allow its convenient administration to the patient .
  • a further aspect of the present invention is the compound of the general Formula (I) for use in the treatment of a human or animal body, in particular for use in the treatment and/or prophylaxis of liver cancer.
  • the present invention provides methods of treatment using the p38 gamma and/or delta inhibitors described herein for the treatment of a human or animal body, in particular a method for the treatment of liver cancer.
  • a still further aspect of the present invention refers to a screening method for obtaining compounds capable of inhibiting p38 gamma or delta intracellular expression, characterized by using IL6 reduction as a biomarker of ⁇ 38 ⁇ / ⁇ inhibition.
  • a still further aspect refers to the use of IL6 as a biomarker IL-6 for monitoring or determining the effectiveness of ⁇ 38 ⁇ / ⁇ inhibitor compounds, to evaluate the treatment with ⁇ 38 ⁇ / ⁇ inhibitors, as well as to identify ⁇ 38 ⁇ / ⁇ inhibitors in vitro.
  • Levels of IL6 are readily detectable and quantifiable in biological samples (e.g., serum samples) .
  • the aforesaid can further include comparing the level of IL6 in the biological sample to a control level of IL6, wherein a decrease in the level of IL6 in the subject relative to that of the control level is indicative of a positive response to the therapy in the subject.
  • the biological sample can be one or more of whole blood, plasma or serum.
  • the level of IL6 can be detected immunologically .
  • FIG. 1 p38 ⁇ expression in Hepatocellular Carcinoma Cell lines (HCCs) : proliferation rate upon protein reduction.
  • HCCs Hepatocellular Carcinoma Cell lines
  • a ⁇ 38 ⁇ and ⁇ 38 ⁇ protein expression in 5 different HCCs cell lines (Snu354, Huh7, HepG2, Snu449 and Snu398) compare to primary hepatocyte.
  • b HepG2 were treated with lentiviral particles containing shRNA against ⁇ 38 ⁇ or ⁇ 38 ⁇ . Cells were lysates and western blot to assay ⁇ 38 ⁇ and ⁇ 38 ⁇ protein levels.
  • c Growth HepG2 treated with shRNA against ⁇ 38 ⁇ , ⁇ 38 ⁇ or scramble as a control.
  • FIG. 2 ⁇ 38 ⁇ is essential for liver proliferation and regeneration after partial hepatectomy (PHx) .
  • WT mice were subjected to two-thirds of PHx or sham procedure. Immunoprecipitation of ⁇ 38 ⁇ from the livers isolated 6 and 24 hours after PHx. Immunoblot analysis was performed by probing with antibodies to P-p38 and ⁇ 38 ⁇ .
  • b
  • mice AlbCRE mice: Alb-Cre-/+
  • ⁇ 38 ⁇ - deficient mice Lp38vKO mice: Alb-Cre-/+ P38YLOXP/LOXP
  • ⁇ 38 ⁇ -deficient mice Lp385 KO mice: Alb-Cre-/+ ⁇ 38 ⁇ LoxP/LoxP
  • mice were subjected to two-thirds of PHx or sham procedure. Representative sections of the liver from mice at 2 days or 15days post-surgery were stained with an antibody against Ki 67.
  • Ki67 Inmunohistochemistry in the liver of AlbCRE, ⁇ 38 ⁇ and ⁇ 38 ⁇ mice in sham group compare to 2 days and 15 days after PxH.
  • e. The expression of cyclin Al, El and Dl mRNA was examined by RT-PCR by qPCR at basal and 48 hours after PxH in AlbCRE, Lp38yKO and Lp385KO mice. The data presented are normalized for the amount of GAPDH mRNA in each sample and represent the mean ⁇ SD (n 5) . Statistically significant differences are indicated.
  • Immunoblot analysis from liver lysates of AlbCRE, Lp38yKO and Lp385KO mice 48 hours after PxH or sham as control was performed by probing with antibodies to PCNA and phospho Rb Ser 807/811.
  • Figure 3 In vitro and in vivo phosphorylation of Rb by ⁇ 38 ⁇ and ⁇ 38 ⁇ .
  • a Endogenous Rb co-immunoprecipitates with endogenous ⁇ 38 ⁇ . We immunoprecipitated ⁇ 38 ⁇ or IgG as a control from liver lysates of WT mice treated with DEN and euthanized after 48h.
  • b Structural organization of RB, indicating ⁇ 38 ⁇ and p385phosphorylation sites found by in vitro kinase assay .
  • AAV Adenoviruses Associated
  • mice were iv injected with AAV-Alb-active-p38Y and livers harvested after 4 weeks. Liver lysates were prepared and analyzed by SDS-PAGE and blotted with the indicated antibodies.
  • Figure 4 Lack of ⁇ 38 ⁇ or ⁇ 38 ⁇ protected against DEN-induced liver cancer.
  • Figure 5 ⁇ 38 ⁇ inhibitor, Pirfenidone, protects against DEN- induced liver tumour a) WT mice were injected with DEN t 15 days old and after 6 months were treated with or without Pirfenidone in the drinking water. Scheme of the timeline treatment. b) MRI images of the livers. c) Quantification of total tumour volume, number of tumour and maximum tumour volume in the livers. d) Immunoblot analysis of Rb phosphorylation at Ser 807/811 in liver tissue (L) and tumours (T) isolated from mice was performed .
  • FIG. 6 Biochemical analysis in serum after Pirfenidone treatment. WT mice were treated with Pirfenidone for 10 weeks and blood concentration of different parameters were assay. a. Alanine aminotransferase (ALT) . b. Aspartate aminotransferase (AST) . c. Total bilirubin was measured as readout of hepatic inj ury . a. Alkaline phosphatase was measured in serum to analyse both hepatic and cardiac damage. b. Creatinine Kinase (CK) and Creatinine were measure in blood to check cardiac and renal injury.
  • ALT Alanine aminotransferase
  • AST Aspartate aminotransferase
  • c Total bilirubin was measured as readout of hepatic inj ury .
  • Alkaline phosphatase was measured in serum to analyse both hepatic and cardiac damage.
  • CK Creatinine Kinase
  • Creatinine were measure
  • Figure 7 Comparative effects of pirfenidone, Urea-derived compounds, BIRB analogues and other compounds in vivo.
  • Phenylpirazolep-Tolil-pyrazole-derived compounds SMP 1- 47, SMP 1-53, SMP 1-54, SMP 1-55, SMP 2-07, SMP 2-04 and SMP
  • Phenylhydrazine-derived compounds SMP1-61, SMP 1-66 and SMP 1-67
  • FIG. 8 IL6 as a biomarker of the ⁇ 38 ⁇ and ⁇ downstream effect .
  • a. Wt mice and p38 ⁇ whole-body knock-out ( ⁇ 38 ⁇ KO) mice were treated with one ip injection of acute DEN (lOOmg/Kg of body weight) .
  • 48h after the treatment IL6 levels in serum were analysed by ELISA and compared with non-treated wt and ⁇ 38 ⁇ KO mice.
  • b. WT mice were injected with DEN t 15 days old and after 6 months were treated with or without Pirfenidone in the drinking water.
  • Figure 10 Inhibition of ⁇ 38 ⁇ in HEPG2 cells was assay by Rb phosphorylation.
  • HepG2 were treated with different compounds for 24 hours and inhibition of ⁇ 38 ⁇ was assay checking Phospho Rb by western blot.
  • Figure 11 Inhibition of ⁇ 38 ⁇ activity in HEPG2 cells was assayed. HepG2 were treated with different compounds 30 mins before 0,5 M sorbitol stimuli. Cells were lysated and inhibition of ⁇ 38 ⁇ activity was assayed checking the phosphorylation of Rb by western blot. HepG2 treated with lentiviral particles containing shRNA against ⁇ 38 ⁇ were used as positive control. We can detect inhibition of p38gamma activity in PGCl-16, PGCl-6, PGCl-3.
  • Figure 12 Inhibition of ⁇ 38 ⁇ activity and phosphorylation in HEPG2 cells was assayed. HepG2 were treated with different compounds 30 mins before 0,5 M sorbitol stimuli. Cells were lysated and inhibition of ⁇ 38 ⁇ was assayed checking the phosphorylation of Rb and p38 by western blot. HepG2 treated with lentiviral particles containing shRNA against ⁇ 38 ⁇ were used as positive control. We can detect inhibition of p38gamma activity in PGCl-16, PGCl-6, ACMl-7. P38gamma phosphorylation was inhibited by PGCl-16, PGCl-6.
  • the present invention provides compounds for use in therapy and/or prophylaxis of liver cancer.
  • the present invention provides compounds for use in a method for the therapeutic treatment and/or prophylaxis of liver cancer capable of a. inhibiting the intracellular expression of the p38 gamma protein in the hepatocytes of a subject relative to that observed in the absence of the compound; and/or b. inhibiting the intracellular expression of the p38 delta protein in the hepatocytes of a subject relative to that observed in the absence of the compound.
  • the compounds of the present invention are selected from the list consisting of: a.
  • a p38 delta and/or p38 gamma gene silencer compound such as a siRNA (small interfering RNA of p38 delta and/or p38 gamma protein) or shRNA (small hairpin RNA of p38 delta and/or p38 gamma protein) or RNA-directed DNA cleavage by the cas9-crisp or talent based technology;
  • a peptide comprising SEQ ID NO 1 (YGRKKRRQRRRARVPKETAL) ;
  • Doramapimod (BIRB 796; BIRB-796; BIRB796) or a salt or derivative thereof capable of inhibiting p38 gamma and/or delta intracellular expression;
  • Rl is selected from H, 3-OCH3 or 4-OCH3;
  • Y is selected from CH or N;
  • X is 0 or S
  • n 0 or 1 ;
  • Ar is 3-0-CH3-Phenyl , 1-pyridyl, 2-pyridyl, 3-pyridyl, 4- pyridyl, phenyl or 2F-4Br-Phenyl ; or a salt or derivative thereof having capable of inhibiting p38 gamma and/or delta intracellular expression; f .
  • the compound is a p38 delta and/or p38 gamma gene silencer selected from the list consisting of siRNA (small interfering RNA) and/or shRNA (small hairpin RNA) or RNA-directed DNA cleavage by the cas9-crisp or talent based technology .
  • siRNA small interfering RNA
  • shRNA small hairpin RNA
  • RNA-directed DNA cleavage by the cas9-crisp or talent based technology RNA-directed DNA cleavage by the cas9-crisp or talent based technology
  • gene silencer is understood as any compound that have the ability to prevent the expression of a certain gene.
  • the compound is a peptide comprising SEQ ID NO 1 (YGRKKRRQRRRARVPKETAL) . It is noted that this compound (peptide) is known to inhibit the binding of ⁇ 38 ⁇ to its substrate through its PDZ domain (Sabio, G. et al .
  • the compound is doramapimod (BIRB 796; BIRB-796; BIRB796) or a salt or derivative thereof capable of inhibiting p38 gamma and/or delta intracellular expression, wherein said derivative is represented by the following formula II:
  • R is selected from the list consisting of any of the following :
  • the compound is pirfenidone
  • pirfernidone or a salt or derivative thereof capable of inhibiting p38 gamma and/or delta intracellular expression wherein derivatives are preferably selected from the group consisting of:
  • R F 3 or a salt thereof.
  • the compound is a thiourea derivative, in particular a compound represented by formula I:
  • Rl is selected from H, 3-OCH3 or 4-OCH3;
  • Y is selected from CH or N;
  • X is 0 or S
  • n 0 or 1 ;
  • Ar is 3-0-CH3-Phenyl , 1-pyridyl, 2-pyridyl, 3-pyridyl, 4- pyridyl, phenyl or 2F-4Br-Phenyl ; or a salt or derivative thereof capable of inhibiting p38 gamma and/or delta intracellular expression.
  • the compound is represented by any of the following formulae shown in the table below:
  • the compound is represented by of the following formulae:
  • SMP1-61 SMP 1-66 SMP1-67 or a salt thereof.
  • the compound is represented by any of the following formulae: F
  • the compounds as described herein can be prepared following general well known convergent synthesis without undue burden for the skilled person.
  • the examples of the present invention described the preparation of the compounds describe herein for the first time.
  • the compounds of the invention include pharmaceutically acceptable salts, amides, and prodrugs therof, including but not limited to carboxylate salts, amino acid addition salts, amides, and prodrugs of the compounds of the present invention which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of patients without undue toxicity, irritation, allergic response, and the like, commensurate with a reasonable benefit/risk ratio, and effective for their intended use, as well as the zwitterionic forms, where possible, of the compounds of the invention.
  • salts refers to the relatively non-toxic, inorganic and organic acid addition salts of compounds of the present invention. These salts can be prepared in situ during the final isolation and purification of the compounds or by separately reacting the purified compound in its free base form with a suitable organic or inorganic acid and isolating the salt thus formed.
  • Representative salts include the hydrobromide, hydrochloride, sulfate, bisulfate, nitrate, acetate, oxalate, valerate, oleate, palmitate, stearate, laurate, borate, benzoate, lactate, phosphate, tosylate, citrate, maleate, fumarate, succinate, tartrate, naphthylate mesylate, glucoheptonate, lactobsonate, and laurylsulphonate salts, and the like.
  • alkali and alkaline earth metals such as sodium, lithium, potassium, calcium, magnesium, and the like
  • non-toxic ammonium, quaternary ammonium, and amine cations including, but not limited to ammonium, tetramethylammonium, tetraethylammonium, methylamine, dimethylamine, trimethylamine, triethylamine, ethylamine, and the like, (See, for example, Berge S.M, et al . , "Pharmaceutical Salts," J. Pharm.
  • a further aspect of the present invention includes pharmaceutical compositions comprising a therapeutically effective amount of one or more compounds of the invention disclosed above, associated with a pharmaceutically acceptable carrier.
  • the compounds are ordinarily combined with one or more adjuvants appropriate for the indicated route of administration.
  • the compounds may be admixed with lactose, sucrose, starch powder, cellulose esters of alkanoic acids, stearic acid, talc, magnesium stearate, magnesium oxide, sodium and calcium salts of phosphoric and sulfuric acids, acacia, gelatin, sodium alginate, polyvinylpyrrolidine, and/or polyvinyl alcohol, and tableted or encapsulated for conventional administration.
  • the compounds of this invention may be dissolved in saline, water, polyethylene glycol, propylene glycol, carboxymethyl cellulose colloidal solutions, ethanol, corn oil, peanut oil, cottonseed oil, sesame oil, tragacanth gum, and/or various buffers.
  • Other adjuvants and modes of administration are well known in the pharmaceutical art.
  • the carrier or diluent may include time delay material, such as glyceryl monostearate or glyceryl distearate alone or with a wax, or other materials well known in the art .
  • Examples of pharmaceutically acceptable, non-toxic amides of the compounds of this invention include amides derived from secondary amines.
  • Amides of the compounds of the invention may be prepared according to conventional methods.
  • the term "prodrug” refers to compounds that are rapidly transformed in vivo to yield the parent compound of the above formulae, for example, by hydrolysis in blood. A thorough discussion of prodrugs is provided in T. Higuchi and V. Stella, "Pro-drugs as Novel Delivery Systems," Vol. 14 of the A.C.S. Symposium Series, and in Bioreversible Carriers in Drug Design, ed. Edward B. Roche, American Pharmaceutical Association and Pergamon Press, 1987, both of which are hereby incorporated, by reference .
  • the compounds of the present invention can be administered individually or in combination, usually in the form of a pharmaceutical composition. Such compositions are prepared in a manner well known in the pharmaceutical art and comprise at least one active compound.
  • compositions comprising as one or more compounds of the invention disclosed above, associated with a pharmaceutically acceptable carrier.
  • the compounds are ordinarily combined with one or more adjuvants appropriate for the indicated route of administration.
  • the compounds may be admixed with lactose, sucrose, starch powder, cellulose esters of alkanoic acids, stearic acid, talc, magnesium stearate, magnesium oxide, sodium alginate, polyvinylpyrrolidine, and/or polyvinyl alcohol, and tableted or encapsulated for conventional administration.
  • the compounds of this invention may be dissolved in saline, water, polyethylene glycol, propylene glycol, carboxymethyl cellulose colloidal solutions, ethanol, corn oil, peanut oil, cottonseed oil, sesame oil, tragacanth gum, and/or various buffers.
  • Other adjuvants and modes of administration are well known in the pharmaceutical art.
  • the carrier or diluent may include time delay material, such as glyceryl monostearate or glyceryl distearate alone or with a wax, or other materials well known in the art.
  • mice were housed in a pathogen-free animal facility under a 12 h light/dark cycle at constant temperature and humidity and fed a standard rodent chow and water ad libitum.
  • PCR polymerase chain reaction
  • mice were treated with a single dose of DEN (Sigma- Aldrich) given dissolved in saline at a dose of 25 mg/kg body weight by i.p. injection.
  • DEN Sigma- Aldrich
  • Mice in one randomly pre-assigned group were killed at 1, 2, 4, 6 and 8 months after DEN administration for histological and bio-chemical analyses.
  • Matched mice in a second group were employed to assess mortality (ongoing) .
  • adult mice were treated with DEN through a single i.p. injection at a dose of 100/k body weight and killed at 1, 2 and 4 h after DEN administration.
  • Liver and tumor tissues were fixed with phosphate-buffered formalin and embedded in paraffin, and the sections were stained with hematoxylin and eosin for histopathological examination. Cell proliferation was assessed by immunohistochemical staining for Ki-67 (abl5580; Abeam) according to the manufacturer's instructions.
  • Tumours were also monitored by MRI injecting i.v. gadoxetate disodium (PRIMOVISTR; invention) as a contrast. Images were taken every 2 weeks since 7 months after DEN injection and finished at 11 months. Tumour volume was measure using osiriX software.
  • Total hepatic proteins were extracted from 30 mg frozen liver or tumor tissue using 500 ⁇ lysis buffer (50 mM Tris-HCl pH 7.5, ImM EGTA, ImM EDTA pH 8.0, 50mM NaF, ImM glicerofosfato-sodico, 5mM pirofosfato, 0.27 M sacarosa, 1% Triton X-100, 0. ImM PMSF, 0.1% 2-mercaptoethanol, ImM sodium-ortovanadate, 1 yg/ml leupeptin, 1 yg/ml aprotinin) .
  • 500 lysis buffer 50 mM Tris-HCl pH 7.5, ImM EGTA, ImM EDTA pH 8.0, 50mM NaF, ImM glicerofosfato-sodico, 5mM pirofosfato, 0.27 M sacarosa, 1% Triton X-100, 0. ImM PMSF, 0.1% 2-mercaptoethanol,
  • Proteins (for western blot 30yg/lane and for immunoprecipitation, 2mg per condition) were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto nitrocellulose membranes, 0,2 ⁇ (Bio Rad) . Immunoblots were performed using primary antibodies for p38 (#9212, 1:1000; Cell Signalling
  • ALT serum alanine aminotransferase
  • AST aspartate aminotransferase
  • CK creatin kinase
  • GST-Rb (1 ⁇ ) was incubated for 1 h at 30 °C with activated GST-SAPK3 (2.0 units/ml), 10 mM magnesium acetate and 100 ⁇ [g-32P]ATP in a total volume of 200 ⁇ of 50 mM Tris/HCl (pH 7.5), 0.1 mM EGTA, 0.1 mM sodium orthovanadate and 0.1% 2-mercaptoethanol .
  • the band corresponding to 32P-labelled SAP90 was excised, digested with chymotrypsin and chromatographed on a Vydac 218TP54 C18 column equilibrated with 10 mM ammonium acetate (pH 6.5), and the column was developed with a linear acetonitrile gradient. The flow rate was 0.8 ml/min and fractions of 0.4 ml were collected. The two major peaks of 32P radioactivity were analysed by gas-phase sequencing and electrospray ionization-mass spectrometry to determine the peptide sequences and to identify the sites of phosphorylation.
  • HCC Hepatocellular Carcinoma
  • HepG2 were used for knocking down experiment.
  • shRNA against ⁇ 38 ⁇ V3LHS_636283 and V3LHS_636282
  • ⁇ 38 ⁇ V2LHS_170527 and V2LHS_170523
  • HepG2 shp38y, HepG2 shp385 and HepG2 shControl were seeded in 24 well plate at the concentration of 15x104 cells/well at different FBS concentrations (0,1%, 0,2%, 2%, 20%) . 48 hours after incubation, the number of cell were counted .
  • HepG2 shp38y, HepG2 shp385 and HepG2 shControl were seeded at 2x103 cells/plOO petri plate. After 2 weeks (without changing the media) the colonies were fixed and stained with 0.1% crystal violet.
  • Lentiviruses were produced as described (4) .
  • Transient calcium phosphate co-transfection of HEK-293 cells was done with the pGIZP empty vector or pGIZP.shEF2 vector (Darmacon) together with ⁇ 8.9 and pVSV-G.
  • the supernatants containing the LV particles were collected 48 and 72 hours after removal of the calcium phosphate precipitate and were centrifuged at 700 g at 4°C for 10 minutes and concentrated (xl65) by ultracentrifugation for 2 hours at 121 986 g at 4°C (Ultraclear Tubes, SW28 rotor and Optima L-100 XP Ultracentrifuge ; Beckman) .
  • Viruses were collected by adding cold sterile PBS and were titrated by qPCR. Associated Adenovisuses were produced by Viral Vector CNIC unit. Mice were injected in the tail vein with 1x1013 Adenoviral particles suspended in PBS.
  • HCC Human hepatocellular carcinoma
  • Mitogen-activated protein kinases and, among them, the p38 family proteins are important signalling components that transduce external stimuli into a wide range of cellular responses such as proliferation, survival, senescence differentiation, migration and apoptosis depending on the stimuli or even the cell type, tissue and organ..
  • MAPKs Mitogen-activated protein kinases
  • ⁇ 38 ⁇ / ⁇ were present in HCC derived from human patient, we assessed ⁇ 38 ⁇ / ⁇ expression in 5 liver cancer cell lines and in primary human hepatocytes. ⁇ 38 ⁇ / ⁇ were remarkably increased in expression in HCC in comparison with primary human hepatocytes mainly in HepG2 and Huh7 ( Figure la) .
  • HepG2 cell line was one of the cell lines with higher ⁇ 38 ⁇ / ⁇ expression level.
  • shp386 treated cell presented, as expected, reduced levels of this kinase.
  • the treatment with shp38y resulted in reduction of this kinase together with increased levels of ⁇ 38 ⁇ (Fig lb) , suggesting that the reduction of ⁇ 38 ⁇ level was compensated by an increased expression of its related kinase ⁇ 38 ⁇ .
  • ⁇ 38 ⁇ / ⁇ deficiency causes reduced DEN- induced IL6 expression (fig.2).
  • ⁇ 38 ⁇ / ⁇ inhibition with pirfenidone resulted in IL6 reduction (fig. 5d) .
  • ⁇ 38 ⁇ / ⁇ inhibitors can be used for treating HCC .
  • IL6 can be monitored to determine effectiveness of ⁇ 38 ⁇ / ⁇ inhibitors, monitor to evaluate the treatment with ⁇ 38 ⁇ / ⁇ inhibitors, as well as to identify ⁇ 38 ⁇ / ⁇ inhibitors in vitro. Levels of IL6 are readily detectable and quantifiable in biological samples (e.g., serum samples) .
  • the method can further include comparing the level of IL6 in the biological sample to a control level of IL6, wherein a decrease in the level of IL6 in the subject relative to that of the control level is indicative of a positive response to the therapy in the subject.
  • the biological sample can be one or more of whole blood, plasma or serum.
  • the level of IL6 can be detected immunologically.
  • the level of IL6 can be detected using a monoclonal antibody such as a monoclonal antibody attached to a solid substrate.
  • this disclosure features a method of monitoring ⁇ 38 ⁇ / ⁇ inhibition in a subject being treated with a ⁇ 38 ⁇ / ⁇ inhibitor.
  • the method includes obtaining a biological sample from a subject being treated with a ⁇ 38 ⁇ / ⁇ inhibitor; determining the level of IL6 in the biological sample; and assessing a level of ⁇ 38 ⁇ / ⁇ inhibition based on the level of IL6 in the biological sample.
  • the ⁇ 38 ⁇ / ⁇ inhibitor can be a compound, a peptide, an antisense oligonucleotide, or a siRNA.
  • the control level can be the level of IL6 in the subject before treatment with the ⁇ 38 ⁇ / ⁇ inhibitor or can be the level of IL6 in a control population.
  • nBuLi 144 DL, 1,1 eq
  • 2- morpholinoethan-l-amine 27.3 mg, 1 eq
  • THF 3 mL
  • prop-l-en-2-yl 2 , 2-diphenylhydrazine-l-carboxylate 56.2 mg, 1 eq
  • THF 3 mL

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Abstract

The present invention provides p38 inhibitors which are useful for the treatment and/or prophylaxis of liver cancer. In particular, the present invention provides compounds capable of inhibiting the intracellular expression of the p38 gamma protein in the hepatocytes of a subject relative to that observed in the absence of the compound, for use in the therapeutic treatment of liver cancer.

Description

p38 inhibitors for the treatment and prophylaxis of liver cancer
Field of the invention
The present invention provides p38 protein kinase (MAPK) inhibitors which are useful for the treatment and prophylaxis of liver cancer.
Background art
Mitogen-activated protein kinase (MAPK) signalling has a critical role in cell processes whose deregulation leads to cancer development and progression. MAPK pathways link extracellular signals to the machinery that controls fundamental cell activities such as growth, proliferation, differentiation, migration and apoptosis.
There are several groups of MAPK in mammalian cells, among which extracellular signal-regulated kinases (ERK) , Jun N-terminal kinases (JNK) and p38 kinases are the best characterized. The mammalian MAPK p38 subfamily has four members, ρ38α, ρ38β, ρ38γ (from hereinafter indistinctively p38 gamma or ρ38γ) and ρ38δ (from hereinafter indistinctively p38 delta or ρ38δ), which share similar protein sequences and are activated by dual phosphorylation mediated by the MAPK kinases MKK3 and MKK6. Based on expression patterns, substrate specificities and sensitivity to chemical inhibitors, p38MAPK can be further divided into two subsets. One group includes p38a and ρ38β, which are closely related that might have overlapping functions, whereas ρ38β appears to be expressed at very low levels, p38a is abundant in most cell types and is the best-described isoform. In contrast, ρ38γ and ρ38δ have restricted expression patterns and probably have specialized functions. The p38MAPK subfamily members are included in the group of canonical-signalling pathways involved in the cell transformation process. Malignant transformation requires deregulation of at least six cell processes, and cancer cells have to acquire the following capabilities: self sufficiency in growth signals, unlimited replication potential, protection against apoptotic cell death, de novo angiogenesis and tissue invasion and metastasis. p38a negatively regulates cell cycle progression at both the Gl/S and the G2/M transitions and is involved in survival and in the apoptosis induced by many types of cell stress. p38a might also directly affect migration, tumour invasion and angiogenesis by inducing expression of some matrix metalloproteinases (MMP) and of the vascular endothelial growth factor A, a potent inducer of tumor survival and angiogenesis.
Whereas most studies of the p38MAPK pathways to date focused on p38a function in the transformation process, there are few and contradictory reports on the role of ρ38γ and ρ38δ.
Our results are the first to provide evidence on the relationship between the inhibition of ρ38γ/ρ38δ MAPK and the therapy of hepatic cancer or liver cancer.
Summary of the invention
Accordingly, the object underlying the present invention is to provide compounds, in particular, p38 inhibitors as well as compositions and formulations thereof that are useful for the treatment and/or prophylaxis of liver cancer. The term "Liver cancer" or "Hepatic cancer" as used herein refers to a cancer that originates in the liver. Liver tumours are discovered on medical imaging equipment (often by accident) or present themselves symptomatically as an abdominal mass, abdominal pain, yellow skin, nausea or liver dysfunction. The leading cause of liver cancer is cirrhosis due to either hepatitis B, hepatitis C, or alcohol.
The present invention thus provides compounds for use in a method for the therapeutic treatment and/or prophylaxis of liver cancer. Preferably, the compound is capable of a. inhibiting the intracellular expression of the p38 gamma protein in the hepatocytes of a subject relative to that observed in the absence of the compound; and/or b. inhibiting the intracellular expression of the p38 delta protein in the hepatocytes of a subject relative to that observed in the absence of the compound.
In the context of the present invention, the term "inhibitor" or "compound capable of inhibiting" refers to any compound, natural or synthetic, which can reduce activity of a gene product. Accordingly, an inhibitor may inhibit the activity of a protein that is encoded by a gene either directly or indirectly. Direct inhibition can be obtained, for instance, by binding to a protein and thereby preventing the protein from binding a target (such as a binding partner) or preventing protein activity (such as enzymatic activity) . Indirect inhibition can be obtained, for instance, by binding to a protein's intended target, such as a binding partner, thereby blocking or reducing activity of the protein. Preferably, an inhibitor according to the present invention is capable of reducing the intracellular expression of the p38 gamma and/or delta protein or its activity in at least 20%, preferably at least 30%, more preferably at least 50%, still more preferably at least 70%, still more preferably 90% in comparison to that observed in the absence of the compound.
As used herein, the term "compound capable of inhibiting p38 gamma or delta intracellular expression" refers to any compound, natural or synthetic, which results in a decreased activity of p38 gamma or p38 delta. The skilled person in the art can assess whether a given compound is a p38 gamma or delta inhibitor without undue burden.
Additionally, inhibition of p38 gamma or delta intracellular expression by a given compound may be determined in vitro or in vi vo .
In particular, the present invention provides compounds selected from the list consisting of:
a. A p38 delta and/or p38 gamma gene silencer compound such as a siRNA (small interfering RNA of p38 delta and/or p38 gamma protein) or shRNA (small hairpin RNA of p38 delta and/or p38 gamma protein) or RNA-directed DNA cleavage by the cas9-crisp or talent based technology;
b. A peptide comprising SEQ ID NO 1
(YGRKKRRQRRRARVPKETAL) ;
c. Doramapimod (BIRB 796; BIRB-796; BIRB796) or a salt or derivative thereof capable of inhibiting p38 gamma and/or delta intracellular expression;
d. Pirfenidone or a salt or derivative thereof capable of inhibiting p38 gamma and/or delta intracellular expression;
e. A compound represented by formula I:
Figure imgf000005_0001
wherein
Rl is selected from H, 3-OCH3 or 4-OCH3;
Y is selected from CH or N;
X is 0 or S; n is 0 or 1 ; and
Ar is 3-0-CH3-Phenyl , 1-pyridyl, 2-pyridyl, 3-pyridyl, 4- pyridyl, phenyl or 2F-4Br-Phenyl ; or a salt or derivative thereof capable of inhibiting p38 gamma and/or delta intracellular expression; and f . A com ound represented by formula:
Figure imgf000006_0001
or a salt or derivative thereof capable of inhibiting p38 gamma and/or delta intracellular expression; or any combination thereof. The present invention also provides methods of treatment and/or prophylaxis using the p38 inhibitors described herein for the treatment and/or prophylaxis of liver cancer.
In a further aspect, the present invention relates to a pharmaceutical composition comprising a therapeutically effective amount of any of the p38 inhibitors described herein or a salt thereof as active ingredient. The pharmaceutical composition of the present invention is preferably formulated as an oral dosage form to allow its convenient administration to the patient .
A further aspect of the present invention is the compound of the general Formula (I) for use in the treatment of a human or animal body, in particular for use in the treatment and/or prophylaxis of liver cancer. In other words, the present invention provides methods of treatment using the p38 gamma and/or delta inhibitors described herein for the treatment of a human or animal body, in particular a method for the treatment of liver cancer.
A still further aspect of the present invention refers to a screening method for obtaining compounds capable of inhibiting p38 gamma or delta intracellular expression, characterized by using IL6 reduction as a biomarker of ρ38γ/δ inhibition. A still further aspect, refers to the use of IL6 as a biomarker IL-6 for monitoring or determining the effectiveness of ρ38γ/δ inhibitor compounds, to evaluate the treatment with ρ38γ/δ inhibitors, as well as to identify ρ38γ/δ inhibitors in vitro. Levels of IL6 are readily detectable and quantifiable in biological samples (e.g., serum samples) . The aforesaid can further include comparing the level of IL6 in the biological sample to a control level of IL6, wherein a decrease in the level of IL6 in the subject relative to that of the control level is indicative of a positive response to the therapy in the subject. The biological sample can be one or more of whole blood, plasma or serum. The level of IL6 can be detected immunologically .
Figures
Figure 1: p38 γδ expression in Hepatocellular Carcinoma Cell lines (HCCs) : proliferation rate upon protein reduction. a. ρ38γ and ρ38δ protein expression in 5 different HCCs cell lines (Snu354, Huh7, HepG2, Snu449 and Snu398) compare to primary hepatocyte. b. HepG2 were treated with lentiviral particles containing shRNA against ρ38γ or ρ38δ. Cells were lysates and western blot to assay ρ38γ and ρ38δ protein levels. c. Growth HepG2 treated with shRNA against ρ38γ, ρ38δ or scramble as a control. These cells were plated and cultured for 2 days in medium supplemented with different concentrations of serum. Relative cell numbers were measured by staining with crystal violet. The data are presented as means ± standard deviations (n = 3) and are representative of results from three independent experiments. Statistically significant differences (P < 0.01) between shp38y HepG2 and control HepG2 are indicated with an asterisk. d. Colony forming by HepG2 requires ρ38γ. Cells were grown in DMEM with 10% serum. Colony formation was assessed afterl5 days. Representative images are shown of colonies formed by HepG2 treated with shRNA against ρ38γ, ρ38δ or scramble as a control. The number of colonies (>10 cells) formed by each cell type was counted (right) . Statistically significant differences (P < 0.01) are indicated with an asterisk .
Figure 2: ρ38γ is essential for liver proliferation and regeneration after partial hepatectomy (PHx) . a. WT mice were subjected to two-thirds of PHx or sham procedure. Immunoprecipitation of ρ38γ from the livers isolated 6 and 24 hours after PHx. Immunoblot analysis was performed by probing with antibodies to P-p38 and ρ38γ. b. Control mice (AlbCRE mice: Alb-Cre-/+) , ρ38γ- deficient mice (Lp38vKO mice: Alb-Cre-/+ P38YLOXP/LOXP) and ρ38δ -deficient mice (Lp385 KO mice: Alb-Cre-/+ ρ38δ LoxP/LoxP) were subjected to two-thirds of PHx or sham procedure. Representative sections of the liver from mice at 2 days or 15days post-surgery were stained with an antibody against Ki 67. c. The liver/tibia length ratio was calculated and expressed as the mean percentage ± SD (n = 6 mice) in AlbCRE, ρ38γΚΟ and ρ38δ KO mice. d. Ki67 Inmunohistochemistry in the liver of AlbCRE, ρ38γΚΟ and ρ38δΚΟ mice in sham group compare to 2 days and 15 days after PxH. e. The expression of cyclin Al, El and Dl mRNA was examined by RT-PCR by qPCR at basal and 48 hours after PxH in AlbCRE, Lp38yKO and Lp385KO mice. The data presented are normalized for the amount of GAPDH mRNA in each sample and represent the mean ± SD (n = 5) . Statistically significant differences are indicated. f. Immunoblot analysis from liver lysates of AlbCRE, Lp38yKO and Lp385KO mice 48 hours after PxH or sham as control was performed by probing with antibodies to PCNA and phospho Rb Ser 807/811.
Figure 3: In vitro and in vivo phosphorylation of Rb by ρ38γ and ρ38δ. a. Endogenous Rb co-immunoprecipitates with endogenous ρ38γ. We immunoprecipitated ρ38γ or IgG as a control from liver lysates of WT mice treated with DEN and euthanized after 48h. b. Structural organization of RB, indicating ρ38γ and p385phosphorylation sites found by in vitro kinase assay . c. in vivo phosphorylation of Rb by p38gamma using Adenoviruses Associated (AAV) type 8 and 9 expressing ρ38γ active . d. In vivo Rb phosphorylation by ρ38γ. ρ38γΚΟ (whole body knock-out) mice were iv injected with AAV-Alb-active-p38Y and livers harvested after 4 weeks. Liver lysates were prepared and analyzed by SDS-PAGE and blotted with the indicated antibodies.
Figure 4: Lack of ρ38γ or ρ38δ protected against DEN-induced liver cancer. a) DEN-induced hepatocellular carcinoma in control mice (AlbCRE mice: Alb-Cre+/-) and Lp38y, Lp385 or Lp38y/5 - deficient mice (ρ38γΚΟ mice: Alb-Cre+/- P38YLOXP/LOXP, ρ38δΚΟ mice: Alb-Cre+/- p385LoxP/LoxP or both Lp38y/5KO: Alb-Cre+/- ρ38γ/δ LoxP/LoxP) at 6 months of age. Representative images of their livers. b) The maximum diameter of individual tumour nodules and c) the mean ± SD (8-10) width of the tumour nodules are presented . c) The tumour incidence and e) the number of tumours per mouse were examined (n) . d) Kaplan-Meier analysis of the survival of mice treated with DEN is presented, (g) Phosphorylation of ρ38γ and ρ38δ in liver 4 months after DEN injection. e) Immunoblot analysis of ρ38γ and ρ38δ phosphorylation in liver tissue (L) and tumours (T) isolated from mice was performed i) Western blot showing PCNA and phosphor Rb Ser 807/811 from livers of AlbCRE, Lp38yKO, Lp385KO and Ιρ38γδΚΟ mice 1 month after DEN injection, j) Representative images of immunohistochemistry of Ki67 in the livers of AlbCRE and Lp38y5KO mice. (right) quantification of Ki67 positive cells in the livers of AlbCRE and Lp38y5KO mice.
Figure 5: ρ38γ inhibitor, Pirfenidone, protects against DEN- induced liver tumour a) WT mice were injected with DEN t 15 days old and after 6 months were treated with or without Pirfenidone in the drinking water. Scheme of the timeline treatment. b) MRI images of the livers. c) Quantification of total tumour volume, number of tumour and maximum tumour volume in the livers. d) Immunoblot analysis of Rb phosphorylation at Ser 807/811 in liver tissue (L) and tumours (T) isolated from mice was performed .
Figure 6: Biochemical analysis in serum after Pirfenidone treatment. WT mice were treated with Pirfenidone for 10 weeks and blood concentration of different parameters were assay. a. Alanine aminotransferase (ALT) . b. Aspartate aminotransferase (AST) . c. Total bilirubin was measured as readout of hepatic inj ury . a. Alkaline phosphatase was measured in serum to analyse both hepatic and cardiac damage. b. Creatinine Kinase (CK) and Creatinine were measure in blood to check cardiac and renal injury. Figure 7: Comparative effects of pirfenidone, Urea-derived compounds, BIRB analogues and other compounds in vivo. Hep534 cells were plated in a 96 wells plate and cultured for 24 hours in medium supplemented with 0,2% of serum and ΙΟμΜ of Pirfenidone and 19 different BIRB796 analogues. Relative cell numbers were measured by staining with crystal violet. The data are presented as means ± standard deviations (n = 3-6) . a. Phenylpirazolep-Tolil-pyrazole-derived compounds: SMP 1- 47, SMP 1-53, SMP 1-54, SMP 1-55, SMP 2-07, SMP 2-04 and SMP
1-59 b. Urea-derived compounds: SMP 2-40B, SMP 2-40C, SMP 2-42, SMP 2-44, SMP 2-47, JC-AM-I-13, JC-AM-I-15, MR 2-33D and MR
2-34D c. Phenylhydrazine-derived compounds: SMP1-61, SMP 1-66 and SMP 1-67
Figure 8: IL6 as a biomarker of the ρ38γ and δ downstream effect . a. Wt mice and p38 γδ whole-body knock-out (ρ38γδ KO) mice were treated with one ip injection of acute DEN (lOOmg/Kg of body weight) . 48h after the treatment IL6 levels in serum were analysed by ELISA and compared with non-treated wt and ρ38γδ KO mice. b. WT mice were injected with DEN t 15 days old and after 6 months were treated with or without Pirfenidone in the drinking water. The blood concentration of IL6 was measured by ELISA and is presented as the mean ± SD (n = 10) .
Figure 9: Cell viability after treatment. HepG2 cells were plated in a 48 wells plate and cultured for 24 or 48 hours in medium supplemented with 0,2% of serum and 10μΜ of BIRB796 or 10μΜ of different compounds. Relative cell numbers were measured by staining with crystal violet. The data are presented as means ± standard deviations (n = 3-6) . We detect reduced viability in JC AM-1 13, JC AM-1 15, SMP2 39, SMP 2 40A, SMP2 40C, MR2 33D, MR 234D and BIRB796. Figure 10: Inhibition of ρ38γ in HEPG2 cells was assay by Rb phosphorylation. HepG2 were treated with different compounds for 24 hours and inhibition of ρ38γ was assay checking Phospho Rb by western blot. We can detect inhibition of p38gamma activity in SMP1-66, SMP1-67, JC AM-1 13, JC AM-1 15, SMP2 39, SMP 2 40B, SMP2 40C.
Figure 11: Inhibition of ρ38γ activity in HEPG2 cells was assayed. HepG2 were treated with different compounds 30 mins before 0,5 M sorbitol stimuli. Cells were lysated and inhibition of ρ38γ activity was assayed checking the phosphorylation of Rb by western blot. HepG2 treated with lentiviral particles containing shRNA against ρ38γ were used as positive control. We can detect inhibition of p38gamma activity in PGCl-16, PGCl-6, PGCl-3.
Figure 12: Inhibition of ρ38γ activity and phosphorylation in HEPG2 cells was assayed. HepG2 were treated with different compounds 30 mins before 0,5 M sorbitol stimuli. Cells were lysated and inhibition of ρ38γ was assayed checking the phosphorylation of Rb and p38 by western blot. HepG2 treated with lentiviral particles containing shRNA against ρ38γ were used as positive control. We can detect inhibition of p38gamma activity in PGCl-16, PGCl-6, ACMl-7. P38gamma phosphorylation was inhibited by PGCl-16, PGCl-6.
Figure 13: Cell viability after treatment. HepG2 cells were plated in a 48 wells plate and cultured for 24 or 48 hours in medium supplemented with 0,2% of serum and 10μΜ of different compounds. Relative cell numbers were measured by staining with crystal violet. The data are presented as means ± standard deviations (n = 3-6) .
Figure 14: HuH cells were plated in a 48 wells plate and cultured for 24 or 48 hours in medium supplemented with 0,2% of serum and ΙΟμΜ of BIRB796 or ΙΟμΜ of different compounds. Relative cell numbers were measured by staining with crystal violet. The data are presented as means ± standard deviations (n = 3-6) . We only observed reduced viability with JC AM 15 and BIRB796.
Figure 15: Cell viability after treatment. HepG2 cells were plated in a 48 wells plate and treated 30 min with sorbitol after that cultured for 24 or 48 hours in medium supplemented with 0,2% of serum and ΙΟμΜ of different compounds. Relative cell numbers were measured by staining with crystal violet. The data are presented as means ± standard deviations (n = 3-6) .
Detailed description
The present invention provides compounds for use in therapy and/or prophylaxis of liver cancer. In particular, the present invention provides compounds for use in a method for the therapeutic treatment and/or prophylaxis of liver cancer capable of a. inhibiting the intracellular expression of the p38 gamma protein in the hepatocytes of a subject relative to that observed in the absence of the compound; and/or b. inhibiting the intracellular expression of the p38 delta protein in the hepatocytes of a subject relative to that observed in the absence of the compound.
Preferably, the compounds of the present invention are selected from the list consisting of: a. A p38 delta and/or p38 gamma gene silencer compound such as a siRNA (small interfering RNA of p38 delta and/or p38 gamma protein) or shRNA (small hairpin RNA of p38 delta and/or p38 gamma protein) or RNA-directed DNA cleavage by the cas9-crisp or talent based technology;
b. A peptide comprising SEQ ID NO 1 (YGRKKRRQRRRARVPKETAL) ;
c. Doramapimod (BIRB 796; BIRB-796; BIRB796) or a salt or derivative thereof capable of inhibiting p38 gamma and/or delta intracellular expression;
d. Pirfenidone or a salt or derivative thereof capable of inhibiting p38 gamma and/or delta intracellular expression;
e. A compound represented by formula I:
Figure imgf000015_0001
wherein
Rl is selected from H, 3-OCH3 or 4-OCH3;
Y is selected from CH or N;
X is 0 or S;
n is 0 or 1 ; and
Ar is 3-0-CH3-Phenyl , 1-pyridyl, 2-pyridyl, 3-pyridyl, 4- pyridyl, phenyl or 2F-4Br-Phenyl ; or a salt or derivative thereof having capable of inhibiting p38 gamma and/or delta intracellular expression; f . A compound represented by formula:
Figure imgf000016_0001
or a salt or derivative thereof capable of inhibiting p38 gamma and/or delta intracellular expression; and g. A compound represented by formula:
Figure imgf000016_0002
Figure imgf000017_0001
or any combination thereof.
In one preferred embodiment, the compound is a p38 delta and/or p38 gamma gene silencer selected from the list consisting of siRNA (small interfering RNA) and/or shRNA (small hairpin RNA) or RNA-directed DNA cleavage by the cas9-crisp or talent based technology . In the context of the present invention, "gene silencer" is understood as any compound that have the ability to prevent the expression of a certain gene. In another preferred embodiment, the compound is a peptide comprising SEQ ID NO 1 (YGRKKRRQRRRARVPKETAL) . It is noted that this compound (peptide) is known to inhibit the binding of ρ38γ to its substrate through its PDZ domain (Sabio, G. et al . Stress- and mitogen-induced phosphorylation of the synapse- associated protein SAP90/PSD-95 by activation of SAPK3/p38gamma and ERK1/ERK2. The Biochemical journal 380, 19-30, doi:10.1042/BJ20031628 (2004)).
In another preferred embodiment, the compound is doramapimod (BIRB 796; BIRB-796; BIRB796) or a salt or derivative thereof capable of inhibiting p38 gamma and/or delta intracellular expression, wherein said derivative is represented by the following formula II:
Figure imgf000018_0001
and wherein R is selected from the list consisting of any of the following :
a.
Figure imgf000018_0002
Figure imgf000019_0001
or a salt thereof.
Preferred compounds pertaining to this embodiment of the invention are as follows:
Figure imgf000019_0002
Doramapimod (BIRB 796; BIRB-796; BIRB796)
Figure imgf000019_0003
Comp R Comp R
Figure imgf000020_0001
In another preferred embodiment, the compound is pirfenidone
Figure imgf000020_0002
pirfernidone or a salt or derivative thereof capable of inhibiting p38 gamma and/or delta intracellular expression, wherein derivatives are preferably selected from the group consisting of:
2a: R = H '
2b: R = F 3 or a salt thereof.
In another preferred embodiment, the compound is a thiourea derivative, in particular a compound represented by formula I:
Figure imgf000021_0001
wherein
Rl is selected from H, 3-OCH3 or 4-OCH3;
Y is selected from CH or N;
X is 0 or S;
n is 0 or 1 ; and
Ar is 3-0-CH3-Phenyl , 1-pyridyl, 2-pyridyl, 3-pyridyl, 4- pyridyl, phenyl or 2F-4Br-Phenyl ; or a salt or derivative thereof capable of inhibiting p38 gamma and/or delta intracellular expression. Preferably, the compound is represented by any of the following formulae shown in the table below:
Compound R1 Y X N Ar SMP 3-OMe CH 0 0 3-OMePh
2.40B
SMP 3-OMe CH 0 1 3- 2.40C pyridyl
SMP 2.42 4-OMe CH 0 1 4- pyridyl
SMP 2.44 4-OMe CH 0 1 2- pyridyl
SMP 2.47 4-OMe CH 0 0 1- naphtyl
JC-AM-I- H N S 1 Ph
13
JC-AM-I- H N S 0 2F-4BrPh
15
MR 2.33D H CH S 0 3- pyridyl
MR 2.34D H CH S 0 2- pyridyl
another preferred embodiment, the compound is represented by of the following formulae:
Figure imgf000022_0001
SMP1-61 SMP 1-66 SMP1-67 or a salt thereof.
In yet another preferred embodiment, the compound is represented by any of the following formulae:
Figure imgf000023_0001
F
283.2
ACM-1-07
7
H H
The compounds as described herein can be prepared following general well known convergent synthesis without undue burden for the skilled person. In addition, the examples of the present invention described the preparation of the compounds describe herein for the first time.
The compounds of the invention include pharmaceutically acceptable salts, amides, and prodrugs therof, including but not limited to carboxylate salts, amino acid addition salts, amides, and prodrugs of the compounds of the present invention which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of patients without undue toxicity, irritation, allergic response, and the like, commensurate with a reasonable benefit/risk ratio, and effective for their intended use, as well as the zwitterionic forms, where possible, of the compounds of the invention.
The term "salts" refers to the relatively non-toxic, inorganic and organic acid addition salts of compounds of the present invention. These salts can be prepared in situ during the final isolation and purification of the compounds or by separately reacting the purified compound in its free base form with a suitable organic or inorganic acid and isolating the salt thus formed. Representative salts include the hydrobromide, hydrochloride, sulfate, bisulfate, nitrate, acetate, oxalate, valerate, oleate, palmitate, stearate, laurate, borate, benzoate, lactate, phosphate, tosylate, citrate, maleate, fumarate, succinate, tartrate, naphthylate mesylate, glucoheptonate, lactobsonate, and laurylsulphonate salts, and the like. These may include cations based on the alkali and alkaline earth metals, such as sodium, lithium, potassium, calcium, magnesium, and the like, as well as non-toxic ammonium, quaternary ammonium, and amine cations including, but not limited to ammonium, tetramethylammonium, tetraethylammonium, methylamine, dimethylamine, trimethylamine, triethylamine, ethylamine, and the like, (See, for example, Berge S.M, et al . , "Pharmaceutical Salts," J. Pharm. Sci., 1977;66:1-19 which is incorporated herein by reference.) A further aspect of the present invention includes pharmaceutical compositions comprising a therapeutically effective amount of one or more compounds of the invention disclosed above, associated with a pharmaceutically acceptable carrier. For administration, the compounds are ordinarily combined with one or more adjuvants appropriate for the indicated route of administration. The compounds may be admixed with lactose, sucrose, starch powder, cellulose esters of alkanoic acids, stearic acid, talc, magnesium stearate, magnesium oxide, sodium and calcium salts of phosphoric and sulfuric acids, acacia, gelatin, sodium alginate, polyvinylpyrrolidine, and/or polyvinyl alcohol, and tableted or encapsulated for conventional administration. Alternatively, the compounds of this invention may be dissolved in saline, water, polyethylene glycol, propylene glycol, carboxymethyl cellulose colloidal solutions, ethanol, corn oil, peanut oil, cottonseed oil, sesame oil, tragacanth gum, and/or various buffers. Other adjuvants and modes of administration are well known in the pharmaceutical art. The carrier or diluent may include time delay material, such as glyceryl monostearate or glyceryl distearate alone or with a wax, or other materials well known in the art .
Examples of pharmaceutically acceptable, non-toxic amides of the compounds of this invention include amides derived from secondary amines. Amides of the compounds of the invention may be prepared according to conventional methods. The term "prodrug" refers to compounds that are rapidly transformed in vivo to yield the parent compound of the above formulae, for example, by hydrolysis in blood. A thorough discussion of prodrugs is provided in T. Higuchi and V. Stella, "Pro-drugs as Novel Delivery Systems," Vol. 14 of the A.C.S. Symposium Series, and in Bioreversible Carriers in Drug Design, ed. Edward B. Roche, American Pharmaceutical Association and Pergamon Press, 1987, both of which are hereby incorporated, by reference . The compounds of the present invention can be administered individually or in combination, usually in the form of a pharmaceutical composition. Such compositions are prepared in a manner well known in the pharmaceutical art and comprise at least one active compound.
Accordingly, a further aspect of the present invention includes pharmaceutical compositions comprising as one or more compounds of the invention disclosed above, associated with a pharmaceutically acceptable carrier. For administration, the compounds are ordinarily combined with one or more adjuvants appropriate for the indicated route of administration. The compounds may be admixed with lactose, sucrose, starch powder, cellulose esters of alkanoic acids, stearic acid, talc, magnesium stearate, magnesium oxide, sodium alginate, polyvinylpyrrolidine, and/or polyvinyl alcohol, and tableted or encapsulated for conventional administration. Alternatively, the compounds of this invention may be dissolved in saline, water, polyethylene glycol, propylene glycol, carboxymethyl cellulose colloidal solutions, ethanol, corn oil, peanut oil, cottonseed oil, sesame oil, tragacanth gum, and/or various buffers. Other adjuvants and modes of administration are well known in the pharmaceutical art. The carrier or diluent may include time delay material, such as glyceryl monostearate or glyceryl distearate alone or with a wax, or other materials well known in the art.
The following examples are intended for the sole purpose of illustrating the present invention.
Examples
Example 1 : Materials and Methods
1.1. Animal maintenance and treatments.
Mice were housed in a pathogen-free animal facility under a 12 h light/dark cycle at constant temperature and humidity and fed a standard rodent chow and water ad libitum. For all of our studies, we employed ρ38γΚΟ (B6.129-Mapkl2tml ) , ρ38δΚΟ (B6.129- Mapkl3tml) and AblCRE (B6. Cg-Tg (Alb-cre) 21Mgn/J) male mice in the C57BL/6 genetic background. Genotypes of mice were determined by polymerase chain reaction (PCR) . Experiments involving animals were conducted in accordance with the Guide for the Care and Use of Laboratory Animals and were approved by the CNIC Animal Care and Use Committee.
For long-term studies of liver tumour development and survival, 15-day-old mice were treated with a single dose of DEN (Sigma- Aldrich) given dissolved in saline at a dose of 25 mg/kg body weight by i.p. injection. Mice in one randomly pre-assigned group were killed at 1, 2, 4, 6 and 8 months after DEN administration for histological and bio-chemical analyses. Matched mice in a second group were employed to assess mortality (ongoing) . For short-term studies assessing DEN-induced hepatic injury and compensatory proliferation, adult mice were treated with DEN through a single i.p. injection at a dose of 100/k body weight and killed at 1, 2 and 4 h after DEN administration.
For partial hepatectomies (PxH) adult male mice, were anesthetized using a mixture of isoflurane/oxygen . Seventy percent of the liver was excised, which involves the medial and left lateral lobes (used for histological and bio-chemical analyses. After the procedure, the mice were kept ad libitum. Liver proliferation, regeneration and histological and biochemical analysis were done at 6h, 48h and 15 days after PxH. 1.2. Immunohistochemical analyses
Liver and tumor tissues were fixed with phosphate-buffered formalin and embedded in paraffin, and the sections were stained with hematoxylin and eosin for histopathological examination. Cell proliferation was assessed by immunohistochemical staining for Ki-67 (abl5580; Abeam) according to the manufacturer's instructions.
1.3. Assessment of HCC, Magnetic Resonance Images
Tumours were also monitored by MRI injecting i.v. gadoxetate disodium (PRIMOVISTR; Bayern) as a contrast. Images were taken every 2 weeks since 7 months after DEN injection and finished at 11 months. Tumour volume was measure using osiriX software.
Other experimental groups were killing at different times after DEN injection (15 days, 1, 2, 4, 6 and 8 months) . Livers were harvested, weighed and the numbers of visible tumours on the liver surface were counted macroscopically and measured by a calliper. Tumours were harvested and frozen for biochemical analyses. The largest lobe was fixed in formalin and embedded in paraffin. Sections were stained with hematoxylin and eosin and examined microscopically. 1.4. Biochemical analysis
Total hepatic proteins were extracted from 30 mg frozen liver or tumor tissue using 500 μΐ lysis buffer (50 mM Tris-HCl pH 7.5, ImM EGTA, ImM EDTA pH 8.0, 50mM NaF, ImM glicerofosfato-sodico, 5mM pirofosfato, 0.27 M sacarosa, 1% Triton X-100, 0. ImM PMSF, 0.1% 2-mercaptoethanol, ImM sodium-ortovanadate, 1 yg/ml leupeptin, 1 yg/ml aprotinin) . Proteins (for western blot 30yg/lane and for immunoprecipitation, 2mg per condition) were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto nitrocellulose membranes, 0,2μ (Bio Rad) . Immunoblots were performed using primary antibodies for p38 (#9212, 1:1000; Cell Signalling
Technology), ρ38γ (#2307, 1:5000; Cell Signalling Technology and homemade), ρ38δ (1:1000, homemade), phospho p38 T180/Y182 (#921 , 1:1000; Cell Signaling Technology), Rb (#9313, 1:1000; Cell Signaling Technology), phopho Rb Ser 807/811 (#8516, 1:1000, Cell Signaling Technology), PCNA (abl897, 1:1000, Abeam), Vinculin (V4505, 1:1000, Sigma) and GAPDH (G9245, 1:1000, Sigma) . Membranes were incubated with an appropriate horseradish peroxidase-conj ugated secondary antibody (GE Healthcare) . Each membrane was developed using an enhanced chemiluminescent substrate for the detection of horseradish peroxidase (GE Healthcare) .
1.6. Serum biomarkers detection
Hepatic, cardiac and renal injury was examined by serum alanine aminotransferase (ALT) , aspartate aminotransferase (AST) and creatin kinase (CK) , creatinine, alkaline phosphatase and total bilirubin measurement was done by the Animal Facility Unit at CNIC. 116 was measured by ELISA from serum samples.
1.7. Kinase assay
As described previously (3) GST-Rb (1 μΜ) was incubated for 1 h at 30 °C with activated GST-SAPK3 (2.0 units/ml), 10 mM magnesium acetate and 100 μΜ [g-32P]ATP in a total volume of 200 μΐ of 50 mM Tris/HCl (pH 7.5), 0.1 mM EGTA, 0.1 mM sodium orthovanadate and 0.1% 2-mercaptoethanol . After SDS/PAGE and autoradiography, the band corresponding to 32P-labelled SAP90 was excised, digested with chymotrypsin and chromatographed on a Vydac 218TP54 C18 column equilibrated with 10 mM ammonium acetate (pH 6.5), and the column was developed with a linear acetonitrile gradient. The flow rate was 0.8 ml/min and fractions of 0.4 ml were collected. The two major peaks of 32P radioactivity were analysed by gas-phase sequencing and electrospray ionization-mass spectrometry to determine the peptide sequences and to identify the sites of phosphorylation.
1.8. RNA isolation and quantitative real-time-PCR analyses
Total RNA was isolated from liver and tumour tissue employing RNeasy Mini Kit (Qiagen) with on-column DNase I-digestion, according to the manufacturer's instructions. RNA was quantified using a NanoDrop spectrophotometer. Subsequently, complementary DNA synthesis was carried out employing a High-Capacity complementary DNA Reverse Transcription Kit (Applied Biosystems) . Sequences of primers used for quantitative real- time-polymerase chain reaction (qRT-PCR) analyses are provided in Table I. For normalization of expression levels, 18S or GAPDH messenger RNA was used. qRT-PCR was performed using Fast SYBRR Green (Applied Biosystem) on a 7900HT Fast Real-time PCR system (Applied Biosystem) . A dissociation curve program was employed after each reaction in order to verify purity of the PCR products. 1.9. Cell lines and proliferation assays
Five different Hepatocellular Carcinoma (HCC) cell lines derived from human patient (HepG2, Huh7, Snu354, Snu398, Snu449; ATTC) and 1 wild type human hepatocytes (HepaRG from Lifetechnologies ) were cultured with DMEM media (Gibco) supplemented with 10%FBS, glutamine and penicillin/streptomycin.
HepG2 were used for knocking down experiment. shRNA against ρ38γ (V3LHS_636283 and V3LHS_636282 ) and ρ38δ (V2LHS_170527 and V2LHS_170523) from Darmacon were used to reduce the protein expression .
For proliferation assay, HepG2 shp38y, HepG2 shp385 and HepG2 shControl were seeded in 24 well plate at the concentration of 15x104 cells/well at different FBS concentrations (0,1%, 0,2%, 2%, 20%) . 48 hours after incubation, the number of cell were counted .
For the colony-forming assay, HepG2 shp38y, HepG2 shp385 and HepG2 shControl were seeded at 2x103 cells/plOO petri plate. After 2 weeks (without changing the media) the colonies were fixed and stained with 0.1% crystal violet.
1.10. Lentivirus and Adenovirus production
Lentiviruses were produced as described (4) . Transient calcium phosphate co-transfection of HEK-293 cells was done with the pGIZP empty vector or pGIZP.shEF2 vector (Darmacon) together with ρΔ8.9 and pVSV-G. The supernatants containing the LV particles were collected 48 and 72 hours after removal of the calcium phosphate precipitate and were centrifuged at 700 g at 4°C for 10 minutes and concentrated (xl65) by ultracentrifugation for 2 hours at 121 986 g at 4°C (Ultraclear Tubes, SW28 rotor and Optima L-100 XP Ultracentrifuge ; Beckman) . Viruses were collected by adding cold sterile PBS and were titrated by qPCR. Associated Adenovisuses were produced by Viral Vector CNIC unit. Mice were injected in the tail vein with 1x1013 Adenoviral particles suspended in PBS.
1.11. Statistical analysis
Data are expressed as means ± SDs. Differences were analyzed by Student' st-test and P values, 0.05 were considered significant. Fisher's exact test was used in comparing incidence of HCC. For overall survival, the log-rank test was used for assessing significance in the Kaplan-Meier analysis
Example 2 : Results
2.1. Role of p38g/d in proliferation and contact growth inhibition in HCC cells.
Human hepatocellular carcinoma (HCC) is the fifth most common cancer in the world and overall, the incidence and mortality rates are ranging from 67% to 91%. Currently, surgical resection is the only effective treatment for HCC if the tumor is resectable. Small molecules, biologies and siRNAs as anti-cancer drugs have been explored for the treatment of HCC. Selective targeting to tumor tissue rather than normal liver in HCC patients is still a challenge. For that reason to find specific proteins that are over expressed in HCC is very important in order to achieve a specific treatment and to reduce secondary effects .
Mitogen-activated protein kinases (MAPKs) and, among them, the p38 family proteins are important signalling components that transduce external stimuli into a wide range of cellular responses such as proliferation, survival, senescence differentiation, migration and apoptosis depending on the stimuli or even the cell type, tissue and organ.. To determine whether ρ38γ/δ were present in HCC derived from human patient, we assessed ρ38γ/δ expression in 5 liver cancer cell lines and in primary human hepatocytes. ρ38γ/δ were remarkably increased in expression in HCC in comparison with primary human hepatocytes mainly in HepG2 and Huh7 (Figure la) .
To explore whether ρ38γ/δ positive cells could exhibit stronger tumorigenicity than ρ38γ/δ negative cells, we chose HepG2 cell line as cell model to address this issue, because it was one of the cell lines with higher ρ38γ/δ expression level. We treated HepG2 cell line with shRNA against ρ38γ or ρ38δ or scramble as a control. We found that shp386 treated cell presented, as expected, reduced levels of this kinase. However, the treatment with shp38y resulted in reduction of this kinase together with increased levels of ρ38δ (Fig lb) , suggesting that the reduction of ρ38γ level was compensated by an increased expression of its related kinase ρ38δ. Finally, we tested their proliferative and tumorigenic ability. We found that ρ38γ deficiency increased cell growth in all concentrations of serum assay. Stronger proliferation ability could reflect stronger colony formation capability. Significantly, the shscramble treated cells exhibited stronger colony formation ability than shp38y cells, where the colonies derived from shscramble cells were obviously larger than those from shp38y cells (fig. Id) .
2.2. p38y deficiency in hepatocytes prevent liver regeneration following 2/3 PHx .
Based on the previous results, we wonder whether the absence of ρ38γ would lead a defect on proliferation in vivo. To address this question, we perform 2/3 PHx, a well-known protocol to study proliferation and tissue regeneration. First, we found that 2/3 PHx causes robust ρ38γ activation (fig. 2a) . To assay whether mice lacking ρ38γ in hepatocytes exhibited a defect in proliferation and regeneration response to 2/3 PHx, we compared hepatic regeneration in control mice (Albcre) and in mice lacking ρ38γ in hepatocytes (Lp38y) after 2/3 PHx or a sham surgical procedure. This analysis demonstrated that hepatic regeneration was suppressed in Lp38y mice (Fig. 2c) . These data indicate that ρ38γ may play an important role in hepatocyte proliferation. To confirm this conclusion, we examined hepatocyte proliferation by measuring the Ki67 by immunostaining (Fig. 1A) . This analysis demonstrated increased Ki67 immunostaining at 48 h and 15 days post-PHx in both Albcre and Lp385 mice while this was reduced in Lp38y mice (Fig. 2b)
Biochemical analysis of the liver of Albcre and Lp38y mice at 48h post-2/3 PHx demonstrated that ρ38γ deficiency significantly changed the expression of Cyclin Al, Cyclin Bl and Cyclin Dl mRNA (Fig 2d) . Moreover, western blot analysis indicated that PCNA increased at 48h post-2/3 PHx in both Albcre and Lp385 mice while remained reduced in Lp38y livers. These results could be explained because of, while in Albcre and Lp385 mice PHx treatment induced Rb phosphorylation, this was drastically reduced in the liver of Lp38y mice (fig.2e) .
2.3. p38y phosphorylates Rb .
We next investigated whether ρ38γ interacted with Rb . Immunoblot analysis of immunoprecipitated ρ38γ from liver lysates, detected co-immunoprecipitation with Rb protein (Fig. 3a) . Rb contains several potential MAPK phosphorylation sites (S/T-P) , located in the C-terminal . To study if the previous interaction between ρ38γ and Rb that we found would lead a phosphorylation in S/T-p sites, we perform an in vitro kinase assay, where we demonstrated that ρ38γ and p385-mediated phosphorylation of Rb at four residues (Ser807, Ser811, Thr821 and Thr826) (Fig. 3b) . No phosphorylation in these residues by p38 was detected. Rb phosphorylation on Ser807/811 residues in the liver was confirmed by immunoblot analysis of liver lysates from mice ρ38γ whole body knock-out infected with adeno-associated viruses expressing ρ38γ active mutants under the Albumin promoter (p38yactive AAV8 and 9) (Fig. 3c) . 2.4. The absence of ρ38γ or in hepatocytes protects against DEN- induced liver cancer.
The conclusion that ρ38γ is essential for hepatic regeneration led us to question whether ρ38γ is required for hepatocyte proliferation in the context of HCC. To answer this question we used the Diethylnitrosamine- (DEN) -induced HCC model1, whose gene expression profile corresponds closely to that of human HCC with unfavourable outcome. Therefore we tested whether ρ38γ in hepatocytes is required for HCC development by treating Albcre, Lp38yKO, Lp385KO and Ι,ρ38γ/δΚΟ mice with DEN. We found that DEN- induced HCC was strongly suppressed in Lp38yKO, Lp385KO and LP38Y/5KO mice compared with Albcre mice (Fig. 4a) . Tumor size in Lp38yKO, Lp385KO and Lp38Y/5KO mice was significantly smaller than in Albcre mice (Fig. 4b, c) as well as tumor incidence and number of tumor nodules were reduced in Lp38Y/5KO mice compared with Albcre mice (Fig. 4d,e) . Kaplan-Meier analysis demonstrated that the reduced tumor burden in Lp38yKO, Lp385KO and Lp38v/5KO mice caused significantly increased in survival (lifespan) compared to Albcre mice (Fig. 4f) . This effect of ρ38γ/δ deficiency to suppress HCC was associated with markedly activation of ρ38γ and ρ38δ at 4 months after DEN treatment (fig. 4g top panel) and this activation remained in the tumor area at 8 months after DEN administration (fig. 4g bottom panel) . Moreover, histological analysis of Ki67 in hepatic tissue sections demonstrated increased proliferation after DEN injection in Albcre mice than in L ρ38γ/δΚΟ mice (Fig. 4h) .
2.5. Use of ρ38γ/δ inhibition as a treatment for HCC .
To address whether inhibition of ρ38γ/δ could be used in HCC treatment, we used DEN to induced HCC in C57BL/6 at 15 days old. At 7 months after DEN administration, tumors were identified by magnetic resonance imaging (MRI) using PRIMOVISTR to contrast the tissue. Tumor-bearing mice were divided into pirfenidone treated mice or control (non treated mice) . On the basis of MRI analyses, treatment with pirfenidone had a pronounced inhibitory effect on tumor volume and number (Fig. 5c) . The reduction in HCC progression in the treatment could be accounted for, in part, by the reduction in Rb phosphorylation observed after pirfenidone treatment (Fig. 5e) .
2.6. ρ38γ/δ inhibition with pirfenidone do not have deleterious effect .
We treated WT mice with pirfenidone for 4 months, such treatment had no apparent effect on body weight throughout the course of the experiment. Similarly, treatment with either drug had no adverse effects on ALT, AST, bilirubin, alkaline phosphatase creatinin kinase and creatinin. 2.7. Synthetic compounds improve pirfenidone effects in HCC proliferation.
To further extend the finding of new drugs to specifically target ρ38γ or ρ38δ, we used 20 new compounds based on a pan p38 inhibitor, BIRB 796. These modifications were classified in 3 different categories: Phenylpirazole-derived compounds, urea- derived compounds and Phenylhydrazine-derived compounds.
To evaluate the effects of the new inhibitors we plated Hep534 cells in 0.2% of FBS and treated with 10yM of each compounds. After 24h of treatment cell proliferation were studied by staining with crystal violet and compared with pirfenidone effect. From phenylpirazole-derived compounds we found 5 new inhibitors that reduced cell proliferation (SMP 1- 34, SMP 1-47, SMP 1-53, SMP 1-54 and SMP 2-07) (fig. 7a) . From the second group, urea-derived compounds 6 new inhibitors reduced proliferation (SMP 2-40B, SMP 2-40C, SMP 2-42, SMP 2-44, JC-AM-I-13, JC-AM-I-15 and MR 2-33D) (fig. 7b) . Finally, 3 new compounds were identified in the last group of Phenylhydrazine- derived compounds (SMP 1-61, SMP 1-66 and SMP 1-67) .
The finding that ρ38γ is a good candidate for treating liver tumour and the fact that pirfenidone reduce tumour growth open the possibility to further look for new inhibitors. Here we found 14 BIRB 796-derived inhibitors that improve pirfenidone effects in cell growth that could have a huge impact in the treatment of liver cancer. 2.8. Interleukin 6 as biomarkers of ρ38γ/δ inhibition
As described herein, ρ38γ/δ deficiency causes reduced DEN- induced IL6 expression (fig.2). In addition, ρ38γ/δ inhibition with pirfenidone resulted in IL6 reduction (fig. 5d) . ρ38γ/δ inhibitors can be used for treating HCC . IL6 can be monitored to determine effectiveness of ρ38γ/δ inhibitors, monitor to evaluate the treatment with ρ38γ/δ inhibitors, as well as to identify ρ38γ/δ inhibitors in vitro. Levels of IL6 are readily detectable and quantifiable in biological samples (e.g., serum samples) . The method can further include comparing the level of IL6 in the biological sample to a control level of IL6, wherein a decrease in the level of IL6 in the subject relative to that of the control level is indicative of a positive response to the therapy in the subject. The biological sample can be one or more of whole blood, plasma or serum. The level of IL6 can be detected immunologically. For example, the level of IL6 can be detected using a monoclonal antibody such as a monoclonal antibody attached to a solid substrate. In one aspect, this disclosure features a method of monitoring ρ38γ/δ inhibition in a subject being treated with a ρ38γ/δ inhibitor. The method includes obtaining a biological sample from a subject being treated with a ρ38γ/δ inhibitor; determining the level of IL6 in the biological sample; and assessing a level of ρ38γ/δ inhibition based on the level of IL6 in the biological sample. The ρ38γ/δ inhibitor can be a compound, a peptide, an antisense oligonucleotide, or a siRNA. The control level can be the level of IL6 in the subject before treatment with the ρ38γ/δ inhibitor or can be the level of IL6 in a control population.
Example 3. Compound synthesis
3.1. N- (3- (tert-butyl) -1- (p-tolyl) -lH-pyrazol-5-yl) -N' - (pyridin- 3-ylmethyl) urea (SMP 1.54):
Over a solution of 3- (tert-butyl) -1- (p-tolyl) -lH-pyrazol-5-amine (50 mg, 1 eq) in DIPEA (28.4 mg, 1 eq) and THF (2 mL) , the isoproprenyl chloroformate (26.5 mg, 1 eq) was added slowly. The reaction mixture was stirred at room temperature 12 h. Afterwards, pyridin-3-ylmethanamine (23.8 mg, 1 eq) was added and stirred under reflux during 3 h. After extracting with ethyl acetate, the organic phase was washed subsequently with NaHC03 and saturated NaCl solutions. The organic phase was dried over magnesium sulfate, and chromatographed on silica gel column using dichlorometane : MeOH (95:5) as eluents. Yield: 47 mg, 59%. 1H RMN (300 MHz, CDC13) δ: 8.69 (m, 2H) , 7.98 (m, 1H) , 7.77 (m, 5H) , 5.54 (s, 1H) , 4.66 (m, 2H) , 2.38 (s, 3H) , 1.34 (s, 3H) . 13C RMN (75 MHz, CDC13) δ: 169.4, 155.5, 150.1, 149.3, 148.4, 137.6, 136.8, 135.9, 135.1, 130.2, 127.3, 122.8, 92.4, 45.9, 32.1, 30.1, 22.3 HPLC/MS: purity>99%. ESI-MS m/z = 365 (M+2H) . 3.2. 2- (2- (lH-indol-3-yl) acetyl) -N- (3- (tert-butyl) -1- (p-tolyl) - lH-pyrazol-5-yl) hydrazine-l-carboxamide (SMP 2.04):
To a solution of the carbamate obtained after the reaction of 3- (tert-butyl) -1- (p-tolyl) -lH-pyrazol-5-amine in DIPEA and THF with the isoproprenyl chloroformate, 2 eq of 2- (lH-indol-3- yl ) acetohydrazide was added and heated 1 h 30 min. After extracting with ethyl acetate, the organic phase was washed subsequently with NaHC03 and saturated NaCl solutions. The organic phase was dried over magnesium sulfate, and chromatographed on silica gel column using dichlorometane : MeOH (95:5) as eluents. Yield: 35 mg, 48%. 1H RMN (300 MHz, acetone- d6) δ: 10.12 (s, 1H) , 9.06 (d, J = 1.9 Ηζ,ΙΗ), 7.95 (s, 1H) , 7.82 (s, 1H) , 7.57 (m, 2H) , 7.33 (m, 2H) , 7.04 (dddd, J = 29.3, 8.0, 7.0, 1.2 Hz, 2H) , 6.30 (s, 1H) , 5.56 (s, 1H) , 3.68 (s, 2H) , 2.33 (s, 3H) , 1.29 (s, 9H) . 13C RMN (75 MHz, acetone-d6) δ: 172.55, 162.44, 156.44, 138.29, 138.18, 137.97, 131.12, 129.11, 125.45, 125.39, 122.90, 120.34, 120.15, 112.82, 109.67, 97.95, 33.61, 32.30, 31.36, 30.59, 30.09, 21.64 HPLC/MS: purity>99%. ESI-MS m/z = 446 (M+2H) .
3.3. N- (3- (tert-butyl) -1- (p-tolyl) -lH-pyrazol-5-yl) -N' -
(pyrimidin-2-yl) urea (SMP1.59):
Over a solution of 3- (tert-butyl) -1- (p-tolyl) -lH-pyrazol-5-amine (50 mg, 1 eq) in DIPEA (28.4 mg, 1 eq) and THF (2 mL) , the isoproprenyl chloroformate (26.5 mg, 1 eq) was added slowly. The reaction mixture was stirred at room temperature 12 h. Afterwards, pyrimidin-2-amine (26.5 mg, 1 eq) was added and stirred under reflux during 3 h. After extracting with ethyl acetate, the organic phase was washed subsequently with NaHC03 and saturated NaCl solutions. The organic phase was dried over magnesium sulfate, and chromatographed on silica gel column using hexane : ethyl acetate (4:1) as eluents. Yield: 6 1H RMN (300 MHz, CDC13) δ: 8.75 (m, 2H) , 7.92 (m, 2H) , 7.68 (m, 2H) , 7.17 (m, 2H) , 5.58 (s, 1H) , 2.37 (s, 3H) , 1.34 (s, 9H) . 13C RMN (75 MHz, CDC13) δ: 168.6, 158.9, 153.2, 151.1, 137.6, 136.3, 130.1, 125.8, 116.1, 91.4, 32.1, 29.8, 20.4 HPLC/MS: purity>99%. ESI-MS m/z = 352 (M+2H) .
3.4. 2- (3- (3- (tert-butyl) -1- (p-tolyl) -lH-pyrazol-5-yl) ureido) -3-
( lH-indol-3-yl) -N- (2 - (piperidin-l-yl) ethyl) propanamide ( SMP
2.07) : To a solution of the carbamate obtained after the reaction of 3- (tert-butyl) -1- (p-tolyl) -lH-pyrazol-5-amine in DIPEA and THF with the isoproprenyl chloroformate, a mixture of
1 eq of 2-amino-3- (lH-indol-3-yl) -N- (2- (piperidin-l- yl ) ethyl ) propanamide in DMF/THF was added a heated under reflux
2 h. After extracting with ethyl acetate, the organic phase was washed subsequently with NaHC03 and saturated NaCl solutions.
The organic phase was dried over magnesium sulfate, and chromatographed on silica gel column using dichlorometane : MeOH
(4:1) as eluents. Yield: 8.2 mg, 17%. 1H RMN (300 MHz, acetone- d6) δ: 10.07 (s, 1H) , 7.85 (s, 1H) , 7.61 (m, 1H) , 7.35 (m, 1H) , 7.23 (d, J = 8.1 Hz, 2H) , 7.16 (m, 1H) , 7.05-6.88 (m, 3H) , 4.61
(m, 1H) , 3.33 (m, 4H) , 2.58 (m, 2H) , 2.23 (s, 3H) , 1.28 (s, 3H) . 13C RMN (75 MHz, acetone-d6) δ: 175.29, 131.10, 125.91, 112.76, 96.23, 95.25, 68.03, 58.49, 54.83, 39.1, 31.64, 29.07, 27.2. HPLC/MS: Rf = 3.08, purity>99%. ESI-MS m/z = 573 (M+2H) .
3.5. 1- (4-bromo-2-fluorophenyl) -3- (pyridin-2-yl) thiourea ( JC-AM- 1-15) :
A solution of 4-bromo-2-fluoro-l-isothiocyanatobenzene (0.5 g, 0.002 mol), 2-aminopyridine (0.2 g, 0.002 mol) in EtOH (7 mL) was prepared. The mixture was heated under microwave irration during 2h 30 min at 110°C. After cooling down, the solid was filtered and recristalized from EtOH. Yield: 95 mg, 14%. 1H RMN (300 MHz, CDC13) δ: 13.99 (s, 1H, NH) , 9.06 (s, 1H, NH) , 8.39 (t, J = 8.6 Hz, J = 1.0 Hz, 1H) , 8.25 (m, 1H) , 7.70 (m, 1H) , 7.39-7.28 (m, 2H) , 7.03 (m, 1H) , 6.88 (dt, J = 8.4 Hz, J = 1.0 Hz, 1H) . 13C RMN (75 MHz, CDC13) δ: 178.8, 156.6, 153.3, 152.8, 145.7, 139.2, 127.1, 127.0, 126.9, 126.4, 126.3, 119.1, 118.8, 118.4, 112.2. HPLC/MS: purity >99%, m/z = 326.1 (80%), 328.1 (100%) .
3.6. Prop-l-en-2-yl 2 , 2-diphenylhydrazine-l-carboxylate (SMP 1.61) :
Over a solution of 2 , 2-diphenylhydrazine (100 mg, 1 eq) in DIPEA (175 mg, 3 eq) and THF (4 mL) , the isoproprenyl chloroformate (54.6 mg, 1 eq) was added slowly. The reaction mixture was stirred at room temperature 12 h. After extracting with ethyl acetate, the organic phase was washed subsequently with NaHC03 and saturated NaCl solutions. The organic phase was dried over magnesium sulfate, and chromatographed on silica gel column using hexane : ethyl acetate (8:1) as eluents. Yield: 224 mg, 49%. 1H RMN (300 MHz, acetone-d6) δ: 9.15 (s, 1H) , 7.67 (m, 4H) , 6.98-6.86 (m, 6H) , 4.64 (m, 2H) , 2.54 (s, 3H) . 13C RMN (75 MHz, acetone-d6) δ: 156.94, 152.92, 152.03, 146.98, 129.75, 121.42, 102.79, 19.62. HPLC/MS: purity>99%. ESI-MS m/z = 269 (M+H) .
3.7. N- (2-morpholinoethyl) -2, 2-diphenylhydrazine-l-carboxamide
(SMP 1-66) :
nBuLi (144 DL, 1,1 eq) was added slowly over a solution of 2- morpholinoethan-l-amine (27.3 mg, 1 eq) in THF (3 mL) at -78 oC . Afterwards, prop-l-en-2-yl 2 , 2-diphenylhydrazine-l-carboxylate (56.2 mg, 1 eq) in THF (3 mL) was added to the mixture. After cooling down to the room temperature, the reaction was stirred overnight. A small amount of water was added and extracted with ethyl acetate. The organic phase was washed with saturated NaCl solution and finally dried over magnesium sulfate, and chromatographed on silica gel column using hexane: ethyl acetate (7:3) as eluents. Yield: 29 mg, 42%.1H RMN ppm (300 MHz, acetone-d6) δ: 7.89 (m, 2H) , 7.23-7.14 (m, 4H) , 7.14-7.04 (m, 4H) , 6.32 (s, 2H) , 3.36 (t, 2H) , 3.14 (d, J = 5.9 Hz, 4H) , 2.28- 2.14 (m, 2H) , 1.96-1.88 (m, 4H) . 13C RMN (75 MHz, acetone-d6) δ: 159.19, 148.66, 130.30, 124.31, 121.07, 67.84, 59.05, 54.64, 51.98. HPLC/MS: purity>99%. ESI-MS m/z = 342 (M+2).

Claims

Claims
1. A composition comprising a compound capable of inhibiting the intracellular expression of the p38 gamma protein in the hepatocytes of a subject relative to that observed in the absence of the compound, for use in the treatment of hepatic cancer.
2. The composition for use according to claim 1, wherein the compound is selected from the list consisting of:
a. Pirfenidone or a salt or derivative thereof capable of inhibiting p38 gamma intracellular expression; b. Doramapimod (BIRB 796; BIRB-796; BIRB796) or a salt or derivative thereof capable of inhibiting p38 gamma intracellular expression;
c. A p38 gamma gene silencer compound such as a siRNA (small interfering RNA of p38 gamma protein) and/or shRNA (small hairpin RNA of p38 gamma protein) and/or RNA-directed DNA cleavage by the cas9-crisp or talent based technology;
d. A peptide comprising SEQ ID NO 1 (YGRKKRRQRRRARVPKETAL) ;
e. A compound represented by formula I:
Figure imgf000043_0001
(Formula I) wherein
-OCH3 or 4-OCH3;
Y is selected from CH or N;
X is 0 or S; n is 0 or 1 ; and
Ar is 3-0-CH3-Phenyl , 1-pyridyl, 2-pyridyl, 3-pyridyl, 4- pyridyl, phenyl or 2F-4Br-Phenyl ; or a salt or derivative thereof capable of inhibiting p38 gamma intracellular expression; f . A com ound represented by formula:
Figure imgf000044_0001
or a salt or derivative thereof capable of inhibiting p38 gamma intracellular expression; and g. A compound represented by formula:
Reference Chemical structure
PCGl-10
H H
Figure imgf000045_0001
or a salt thereof; or any combination thereof.
3 . The composition for use according to claim 2, wherein the compound is pirfenidone or a salt or derivative thereof capable of inhibiting p38 gamma intracellular expression, wherein derivatives are selected from the group consisting of:
Figure imgf000046_0001
or a salt thereof.
4. The composition for use according to claim 2, wherein the compound is represented by formula I:
Figure imgf000046_0002
and wherein
Rl is selected from H, 3-OCH3 or 4-OCH3;
Y is selected from CH or N;
X is 0 or S;
n is 0 or 1 ; and
Ar is 3-0-CH3-Phenyl , 1-pyridyl, 2-pyridyl, 3-pyridyl, 4- pyridyl, phenyl or 2F-4Br-Phenyl ; or a salt thereof
5. The composition for use according to claim 4, wherein the compound is represented by formula I:
Figure imgf000047_0001
(Formula I] and wherein:
Figure imgf000047_0002
6. The composition for use according to claim 2, wherein the compound is represented by formula:
Figure imgf000048_0001
or a salt thereof.
7. The composition for use according to claim 2, wherein the compound is represented by formula:
Figure imgf000048_0002
Figure imgf000049_0001
a salt thereof.
The composition for use according to claim 2, wherein the compound is a p38 gamma gene silencer selected from the list consisting of siRNA (small interfering RNA) and/or shRNA (small hairpin RNA) and/or RNA-directed DNA cleavage by the cas9-crisp or talent based technology. 9. The composition for use according to claim 2, wherein the compound is a peptide comprising SEQ ID NO 1
(YGRKKRRQRRRARVPKETAL) .
The composition for use according to claim 2, wherein the compound is Doramapimod (BIRB 796; BIRB-796; BIRB796) or a salt or derivative thereof capable of inhibiting p38 gamma intracellular expression, wherein said derivative is represented by the following formula II:
Figure imgf000050_0001
(Formula II) ; and wherein R is selected from the list consisting of any of the following :
Figure imgf000050_0002
Figure imgf000050_0003
; or
Figure imgf000050_0004
a salt thereof.
11. A pharmaceutical composition comprising a therapeutically effective amount of any of the compounds as defined in claims 1 to 10 or of a salt thereof as active ingredient .
12. The pharmaceutical composition according to Claim 11, whereby said pharmaceutical composition is an oral dosage form.
13. Use in vitro of IL6 as a biomarker IL-6 for monitoring or determining the effectiveness of ρ38γ inhibitor compounds, to evaluate the treatment with ρ38γ inhibitors, or to identify ρ38γ inhibitors in vitro.
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