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WO2017216576A1 - Composés induisant l'autophagie - Google Patents

Composés induisant l'autophagie Download PDF

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Publication number
WO2017216576A1
WO2017216576A1 PCT/GB2017/051764 GB2017051764W WO2017216576A1 WO 2017216576 A1 WO2017216576 A1 WO 2017216576A1 GB 2017051764 W GB2017051764 W GB 2017051764W WO 2017216576 A1 WO2017216576 A1 WO 2017216576A1
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Prior art keywords
disease
autophagy
compound
bis
skw137
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Inventor
David John Grainger
Nigel Ramsden
David John Fox
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Epsilon-3 Bio Ltd
University of Warwick
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Epsilon-3 Bio Ltd
University of Warwick
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/40—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil
    • A61K31/4025—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil not condensed and containing further heterocyclic rings, e.g. cromakalim
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D295/00—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms
    • C07D295/04—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms with substituted hydrocarbon radicals attached to ring nitrogen atoms
    • C07D295/08—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms with substituted hydrocarbon radicals attached to ring nitrogen atoms substituted by singly bound oxygen or sulfur atoms
    • C07D295/084—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms with substituted hydrocarbon radicals attached to ring nitrogen atoms substituted by singly bound oxygen or sulfur atoms with the ring nitrogen atoms and the oxygen or sulfur atoms attached to the same carbon chain, which is not interrupted by carbocyclic rings
    • C07D295/088—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms with substituted hydrocarbon radicals attached to ring nitrogen atoms substituted by singly bound oxygen or sulfur atoms with the ring nitrogen atoms and the oxygen or sulfur atoms attached to the same carbon chain, which is not interrupted by carbocyclic rings to an acyclic saturated chain
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the invention relates to difluoro heterocyclic compounds and their medical uses.
  • Autophagy is a highly conserved homeostatic mechanism that involves lysosomal degradation of damaged and unwanted cellular components. It is believed to play an important role in inflammatory diseases such as atherosclerosis and plaque progression, and there is a known correlation between enhancing autophagy and protecting against heart, liver and other common age-related diseases. Autophagy may exert its beneficial effect in atherosclerosis and other diseases by degrading damaged intracellular organelles and thereby preventing oxidative injuries and cellular distresses.
  • tamoxifen a potent inducer of autophagy, inhibited atherosclerosis in mice models by suppressing the diet-induced formation of lipid lesions in the aorta by lowering of low-density lipoprotein (LDL) cholesterol.
  • LDL low-density lipoprotein
  • Tamoxifen (prior art) Tamoxifen (2-[4-[(Z)-l,2-diphenylbut-l-enyl]phenoxy]-N,N-dimethylethan-amine) was originally a failed contraceptive that was redeveloped as a breast cancer drug. Tamoxifen has mixed agonist and antagonist activities that are species-, tissue- and cell- specific. In addition to its well-known antitumor properties derived from its anti-estrogenic activity in breast tissue, tamoxifen has also been found to increase the risk of endometrial cancer.
  • tamoxifen Various analogues of tamoxifen have been developed as anti-cancer agents, including tesmilifene (N,N-Diethyl-2-[4-(phenylmethyl)phenoxy]ethanamine) which binds selectively to the high-affinity microsomal anti-oestrogen binding site but unlike tamoxifen has no affinity for oestrogen receptors.
  • tesmilifene N,N-Diethyl-2-[4-(phenylmethyl)phenoxy]ethanamine
  • WO2013/062079 describes l,l'-difluoro-2,2'-diphenylcycloproprane derivatives as modulators of metabotropic glutamate receptor subtype 2 (mGlu2 receptor) and their uses in the treatment of mental disorders.
  • Miyashita et al. 2005; In: “Environmental Fate and Safety Management of Agrochemicals", American Chemical Society, Vol. 899: 159-166) describes a l,l '-difluoro-2,2'-diphenylcycloproprane derivative (compound 34 in Fig. 2) as a potential oestrogen receptor inhibitor.
  • each X is identical and forms part of an ethoxyamine basic side chain which includes a 5- or 6-membered heterocyclic ring;
  • heterocyclic ring X is selected from the group consisting of:
  • the compound of the invention may be in a pharmaceutically acceptable salt form.
  • pharmaceutically acceptable salt refers to a pharmaceutically acceptable organic or inorganic salt of the compound of the invention. This may include addition salts of inorganic acids such as hydrochloride, hydrobromide, hydroiodide, sulphate, phosphate, diphosphate and nitrate or of organic acids such as acetate, maleate, fumarate, tartrate, succinate, citrate, lactate, methanesulphonate, p-toluenesulphonate, palmoate and stearate.
  • inorganic acids such as hydrochloride, hydrobromide, hydroiodide, sulphate, phosphate, diphosphate and nitrate
  • organic acids such as acetate, maleate, fumarate, tartrate, succinate, citrate, lactate, methanesulphonate, p-toluenesulphonate, palmoate and stearate.
  • Exemplary salts also include oxalate, chloride, bromide, iodide, bisulphate, acid phosphate, isonicotinate, salicylate, acid citrate, oleate, tannate, pantothenate, bitartrate, ascorbate, gentisinate, gluconate, glucuronate, saccharate, formate, benzoate, glutamate, ethanesulfonate, and benzenesulfonate salts.
  • oxalate chloride, bromide, iodide, bisulphate, acid phosphate, isonicotinate, salicylate, acid citrate, oleate, tannate, pantothenate, bitartrate, ascorbate, gentisinate, gluconate, glucuronate, saccharate, formate, benzoate, glutamate, ethanesulfonate, and benzenesulfonate salts.
  • the compound of the invention may be l,l'-((((2,2-difluorocyclopropane-l,l- diyl)bis(4,l-phenylene))bis(oxy))bis(ethane-2,l-diyl))dipyrrolidine (“SKW120").
  • SKW120 l,l'-((((2,2-difluorocyclopropane-l,l- diyl)bis(4,l-phenylene))bis(oxy))bis(ethane-2,l-diyl))dipyrrolidine
  • the compound of the invention may be 4,4'-((((2,2-difluorocyclopropane- 1,1 -diyl)bis(4, 1 -phenylene))bis(oxy))bis(ethane-2, 1 -diyl))bis( 1 -methylpiperazine) ("SKW137").
  • SKW137 4,4'-((((2,2-difluorocyclopropane- 1,1 -diyl)bis(4, 1 -phenylene))bis(oxy))bis(ethane-2, 1 -diyl))bis( 1 -methylpiperazine)
  • a pharmaceutical composition comprising a compound of the invention as described herein and a pharmaceutically or therapeutically acceptable excipient or carrier.
  • pharmaceutically or therapeutically acceptable excipient or carrier refers to a solid or liquid filler, diluent or encapsulating substance which does not interfere with the effectiveness or the biological activity of the active ingredients and which is not toxic to the host, which may be either humans or animals, to which it is administered.
  • a variety of pharmaceutically-acceptable carriers such as those well known in the art may be used.
  • Non-limiting examples include sugars, starches, cellulose and its derivatives, malt, gelatin, talc, calcium sulfate, vegetable oils, synthetic oils, polyols, alginic acid, phosphate buffered solutions, emulsifiers, isotonic saline, and pyrogen-free water.
  • administration of the medicament may be via oral, subcutaneous, direct intravenous, slow intravenous infusion, continuous intravenous infusion, intravenous or epidural patient controlled analgesia (PCA and PCEA), intramuscular, intrathecal, epidural, intracistemal, intraperitoneal, transdermal, topical, buccal, sublingual, transmucosal, inhalation, intra- atricular, intranasal, rectal or ocular routes.
  • the medicament may be formulated in discrete dosage units and can be prepared by any of the methods well known in the art of pharmacy.
  • Administration of the medicament may for example be in the form of oral solutions and suspensions, tablets, capsules, lozenges, effervescent tablets, transmucosal films, suppositories, buccal products, oral mucoretentive products, topical creams, ointments, gels, films and patches, transdermal patches, abuse deterrent and abuse resistant formulations, sterile solutions suspensions and depots for parenteral use, and the like, administered as immediate release, sustained release, delayed release, controlled release, extended release and the like.
  • Another aspect of the invention is the use of a compound of the invention as defined herein in the manufacture of a medicament for the treatment of a disease.
  • a further aspect of the invention is a compound of the invention for use as an autophagy inducer.
  • the invention also encompasses a method of treating a disease, comprising the step of administering the compound or the pharmaceutical composition of the invention as defined herein to a patient in need of same.
  • the invention further encompasses the use of a compound of the invention as an autophagy inducer.
  • the use may be in the treatment of a disease. Additionally or alternatively, the use may be in vitro, for example in an in vitro assay.
  • a disease suitable for treatment according to the relevant aspects of the invention is one which is characterised by defective autophagy or which would benefit from modulation of autophagy.
  • Modified or altered autophagy has been shown to be relevant in neurodegenerative disease, as demonstrated by the accumulation of protein aggregates, for example in Alzheimer disease, Parkinson's disease, polyglutamine diseases, muscle diseases, and amyotrophic lateral sclerosis. Modified autophagy have also been implicated in other neurological diseases including epilepsies, neurometabolic and neurodevelopmental disorders such as schizophrenia. Autophagy inhibition plays a key role in the pathogenesis of inherited autophagic vacuolar myopathies (including Danon disease, X- linked myopathy with excessive autophagy, and infantile autophagic vacuolar myopathy), all of which are characterized by lysosomal defects and an accumulation of autophagic vacuoles.
  • autophagic vacuolar myopathies including Danon disease, X- linked myopathy with excessive autophagy, and infantile autophagic vacuolar myopathy
  • Autophagic vacuolar myopathies and cardiomyopathies can also be secondary to treatment with autophagy-inhibiting drugs (chloroquine, hydroxychloroquine and colchicine), which are used experimentally to interrogate autophagic flux and clinically to treat malaria, rheumatological diseases, and gout.
  • Autophagy impairment has also been implicated in the pathogenesis of inclusion body myositis, an age- associated inflammatory myopathy that is currently refractory to any form of treatment, along with other muscular dystrophies such as tibial muscular dystrophy.
  • definitive tissue diagnosis used to require ultrastructural demonstration of accumulated autophagic vacuoles; more recently, it has been shown that IHC for LC3 and/or SQSTM1 can be used instead.
  • modified basal autophagy levels are seen in rheumatoid arthritis and osteoarthritis.
  • Other aspects of the immune response associated with dysfunctional autophagy are seen in neutrophils from patients with familial Mediterranean fever and in monocytes from patients with TNF receptor-associated periodic syndrome, both of which are autoinflammatory disorders.
  • autophagy regulates an important neutrophil function, the generation of neutrophil extracellular traps (NETs).
  • NETs neutrophil extracellular traps
  • a crucial role for therapy-induced autophagy in cancer cells has recently emerged, in modulating the interface of cancer cells and the immune system; primarily, by affecting the nature of danger signalling (i.e., the signalling cascade that facilitates the exposure and/or release of danger signals) associated with immunogenic cell death (ICD).
  • danger signalling i.e., the signalling cascade that facilitates the exposure and/or release of danger signals
  • ICD immunogenic cell death
  • Various observations have highlighted the important, context-dependent role of therapy-induced autophagy, in modulating the cancer cell immune cell interface by regulating the emission of ICD-associated danger signals. Recent studies also have implicated insufficient autophagy in the pathogenesis of nonresolving vital organ failure and muscle weakness during critical illness, leading causes of death in prolonged critically ill patients.
  • a block of autophagy with consequent accumulation of autophagy substrates is detected in liver fibrosis and lysosomal storage diseases.
  • CMA impairment is associated with several disease conditions, including neurodegenerative disorders, lysosomal storage diseases, nephropathies and diabetes.
  • the disease for treatment according to the present invention may be selected from any of the following as well as other diseases mentioned above: a neurodegenerative disorder (for example, Huntington's disease, Alzheimer's disease or Parkinson's disease), systemic lupus erythematosus (“lupus”), epilepsy, cancer, liver diseases including non- alcoholic fatty liver disease (NAFLD), including its extreme form non-alcoholic steatohepatitis (NASH), and al-antitrypsin deficiency (ATD), Niemann-Pick type C (NPC) disease, fibrinogen storage disease (FSB), inclusion body disease (IBD), lysosomal storage disease, muscular dystrophy (for example Duchenne muscular dystrophy or Limb-girdle muscular dystrophy), myopathy (for example myofibrillar myopathy, hereditary myopathy or diabetic cardiomyopathy), or an anti-inflammatory disorder selected from the group consisting of an autoimmune disease (for example multiple sclerosis, rheumatoid arthritis, l
  • Fig. 1 is a graph showing the dose-dependent effect of SKW120 in an in vitro autophagy assay using human monocyte THP-1 cells.
  • the x-axis shows concentration of SKW120, the right axis shows fluorescence intensity (arbitrary units);
  • Fig. 2 is a graph showing results from a control plate with 5 ⁇ tamoxifen in the assay used in Fig. 1.
  • the x-axis shows the treatment used, the right axis shows fluorescence intensity (arbitrary units);
  • Fig. 3 is a graph showing the dose-dependent effect of SKW120 in an in vitro autophagy assay using human hepatocyte HepG2 cells.
  • the x-axis shows concentration of SKW120, the right axis shows fluorescence intensity (arbitrary units);
  • Fig. 4 is a graph showing results from a control plate using tamoxifen in the assay used in Fig. 3.
  • the x-axis shows concentration of TMX used, the right axis shows fluorescence intensity (arbitrary units);
  • Fig. 5 is a graph showing the dose-dependent effect of SKW137 in an in vitro autophagy assay using human monocyte THP-1 cells.
  • the x-axis shows concentration of SKW137, the right axis shows fluorescence intensity (arbitrary units);
  • Fig. 6 is a graph showing results from a control plate with 5 ⁇ tamoxifen in the assay used in Fig. 5.
  • the x-axis shows the treatment used, the right axis shows fluorescence intensity (arbitrary units);
  • Fig. 7 is a graph showing the dose-dependent effect of SKW137 in an in vitro autophagy assay using human hepatocyte HepG2 cells.
  • the x-axis shows concentration of SKW137, the right axis shows fluorescence intensity (arbitrary units);
  • Fig. 8 is a graph showing results from a control plate with 5 ⁇ tamoxifen in the assay used in Fig. 7.
  • the x-axis shows the treatment used, the right axis shows fluorescence intensity (arbitrary units);
  • Fig. 9 is a graph showing the dose-dependent effect of reference compound 4,4'((((2,2- difluorocyclopropane- 1 , 1 -diyl)bis(4, 1 -phenylene))bis(oxy))bis(ethane-2.1 - diyl))bis(piperazine) ("SKW166”) in an in vitro autophagy assay using human monocyte THP-1 cells.
  • the x-axis shows concentration of SKW166, the right axis shows fluorescence intensity (arbitrary units);
  • Fig. 10 is a graph showing results from a control plate with 5 ⁇ tamoxifen in the assay used in Fig. 9.
  • the x-axis shows the treatment used, the right axis shows fluorescence intensity (arbitrary units);
  • Fig. 11 is a Western blot showing LC3-II levels in HepG2 cells treated with SKW120 in the presence and absence of Bafilomycin A (a fusion blocker), shown as "+” and respectively.
  • Fig. 12 is a Western blot showing LC3-II levels in HepG2 cells treated with SKW137 in the presence and absence of Bafilomycin A (a fusion blocker), shown as "+” and respectively.
  • SKW120 was prepared using the following sequential synthesis procedures (a)-(f).
  • SKW137 was prepared using the following sequential synthesis procedures (a)-(b).
  • the cells were washed twice with fresh media (RPMI phenol red free/5% FBS) and 50 ⁇ 1 RPMI phenol red free/5% FBS containing the Cyto-ID green staining dye provided in a commercially available autophagy kit (Abeam, abl39484) (final concentration IX) and Hoescht (1/1000), and were incubated for 45 minutes at 37°C in the dark. Lysosomal/autophagic vacuoles were detected using the Abeam kit which employs a proprietary dye, a cationic amphiphilic tracer which selectively labels autophagic vacuoles in the perinuclear region of the cell. Finally, cells were washed and fixed in 4% PFA for 10 minutes at RT. The cells were analysed using a SynergyHT plate reader (BioTek).
  • TMX Tamoxifen
  • Fig. 1 show that SKW120 stimulates autophagy in THP-1 cells in a dose- dependent matter, with no cellular toxicity shown at the highest concentration used. SKW120 induces an increase of lysosomal/autophagic vacuoles in THP-1 cells, as measured by an increase in median fluorescence staining by flow cytometry techniques, compared to cells treated with vehicle. The calculated EC50 is 1.1 to 2.3 ⁇ . SKW120 stimulates autophagy in THP-1 cells more effectively than TMX (see Fig. 2).
  • Example 5 Effect of SKW120 in an in vitro autophagy assay using human HepG2 cells
  • the in vitro assay as described in Example 4 was repeated using liver hepatocyte HepG2 cells.
  • HepG2 cells were harvested using trypsin/EDTA then diluted to 1 x 10 5 cells/ml in EMEM (Eagles Minimal Essential Medium)/ 10%FBS, and adhered for 24 h.
  • EMEM Eagles Minimal Essential Medium
  • the data in Fig. 3 show that SKW120 also stimulates autophagy in HepG2 cells in a dose-dependent matter, with no cellular toxicity shown at the highest concentration used.
  • the calculated EC50 is 0.3 to 0.8 ⁇ .
  • SKW120 stimulates autophagy in HepG2 cells more effectively than TMX (see Fig. 4).
  • Example 6 Effect of SKW137 in an in vitro autophagy assay using human monocyte THP-1 cells
  • Example 4 The in vitro assay as described in Example 4 was repeated for SKW137.
  • the data in Fig. 5 show that SKW137 stimulates autophagy in THP-1 cells in a dose-dependent matter at the lower concentrations used.
  • SKW137 induces an increase of lysosomal/autophagic vacuoles in THP-1 cells, as measured by an increase in median fluorescence staining by flow cytometry techniques, compared to cells treated with vehicle.
  • the calculated EC50 is 0.9 to 1.9 ⁇ .
  • SKW137 stimulates autophagy in THP-1 cells more effectively than TMX (see Fig. 6).
  • Example 7 Effect of SKW137 in an in vitro autophagy assay using human HepG2 cells
  • Example 5 The in vitro assay as described in Example 5 was repeated for SKW137.
  • the data in Fig. 7 show that SKW137 also stimulates autophagy in HepG2 cells in a dose-dependent matter at the lower concentrations used.
  • the calculated EC50 is 0.4 to 1.3 ⁇ . SKW137 stimulates autophagy in HepG2 cells more effectively than TMX (see Fig. 8).
  • Example 8 Effect of SKW166 in an in vitro autophagy assay using human monocyte THP-1 cells
  • SKW166 is structurally similar to the compounds of general formula (I) of the present invention, except that each of the symmetric ethoxyamine basic side chains comprises a 6-membered heterocyclic ring which is an unmethylated piperazine group (compared to the corresponding 1-methylpiperazine rings in SKW137).
  • the proprietary fluorescent dye used in the screening assay in Examples 4-7 is a cationic amphiphilic tracer which selectively labels autophagic vacuoles in the perinuclear region of the cell.
  • a population of the proprietary autophagy dye-labelled vesicles co-localise with the microtubule- associated protein 1A/1B light chain-3 (LC3, Mw ⁇ 17kDa,), a ubiquitous key autophagy protein. Changes in cellular LC3-II and the number of LC3-II vesicles correlate with autophagosome abundance, but this does not necessarily reflect autophagic flux (i.e. the rate of autophagosome delivery to the lysosome). This is because blockers of fusion between the autophagosome and the lysosome would result in an increase in the number of autophagosomes (but not flux) and would produce the same signal in this assay.
  • Fig. 11 and Fig. 12 show that treatment with 10 ⁇ SKW120 and SKW137, respectively, resulted in an increased level of LC3-II detected over those measured with BAF-A alone. Chloroquine did not induce levels of LC3-II over that of Baf A (as detected by Western blot). Tamoxifen was synergistic with Baf A in increasing LC3-II levels but not as effective as SKW120 or SKW137.
  • Example 10 Inhibition of cytochrome P450 interactions (Drug-Drug interactions of SKW137)
  • Example 11 In vitro and in vivo properties of SKW137 which predict in vivo hepatic clearance
  • the intrinsic clearance (Clint) and half-life of SKW137 was measured in a mixed hepatocyte suspension of cryopreserved mouse, rat or human hepatocytes. Briefly, compound is incubated with hepatocyte suspensions at 37°C over a time course and remaining compound at each time point is assessed by mass spectrometry (UPLC- MS/MS). Clint in mouse hepatocytes was 20.0 ⁇ /min/lO 6 cells, in rat hepatocytes was 63 ⁇ /min/lO 6 cells, and in human hepatocytes was 10 ⁇ /min/lO 6 cells. Half-life in mouse hepatocytes was >70 min, in rat hepatocytes was 24.3 min and in human hepatocytes was 139 min.
  • SKW 137 bound to plasma proteins such as albumin and alpha-1 acid glycoprotein within human, rat or mouse blood was determined by rapid equilibrium dialysis. Compounds were incubated at 5 ⁇ for 4 hours at 37°C. We found that PPB in mouse cells was 85.35%, in rat cells was 90.68% and in human cells was 75.70%. To understand whether SKW137 was highly bound to red blood cells the Blood: Plasma partitioning was assessed using parallel incubation of the compound in fresh blood and matched plasma. Compound ( ⁇ ) was incubated at 37°C for 30 min at pH7.4 before analysis by UPLC-MS/MS to determine bound vs unbound fractions. The Blood:Plasma ratio was 3.06 in mouse and 5.28 in human.
  • SKW137 was administered to C57BI/6 male mice intravenously (lmg/kg) or orally (5mg/kg) by gavage.
  • Whole blood diluted with water was prepared from these dosed animals over a time course up to 96 hours post dose to allow blood concentrations of drug to be estimated by UPLC-MS/MS.
  • Analysis of the compound levels over the time course allows an estimation of pharmacokinetic properties of the drug.
  • the measurements allowed calculation of the following parameters for SKW137:
  • SKW137 has improved functional activity in autophagy compared with tamoxifen, has good bioavailability with low in vivo clearance resulting in a relatively long half-life in blood. SKW137 does not appear to induce CYP P450s.
  • Example 12 SKW137 efficacy in a murine diet-induced non-alcoholic steatohepatitis ("NASH”) model
  • NASH is a condition in which excess fat accumulates in the liver of patients with no history of alcohol abuse. It is regarded as an hepatic manifestation of metabolic syndrome, for which the incidence is increasing worldwide in line with the prevalence of obesity and type 2 diabetes. It is estimated that around 3% of adults worldwide have NASH (and around 20% have NAFLD). In NASH, not only steatosis but also intralobular inflammation and hepatocellular ballooning, often with progressive fibrosis.
  • the dietary induced mouse model of non-alcoholic steatohepatitis recapitulates many of the histopathological features of the human clinical syndrome (e.g. Clapper et al, 2013, Am J Physiol Gastrointest Liver Physiol 305: G483-G495).
  • the clinical syndrome is quite heterogeneous and reflects a spectrum of disease severity from low grade steatosis, through to marked hepatic steatosis and cellular ballooning with varying degrees of inflammation, finally leading to parenchymal fibrosis.
  • Clinically, a poorer prognostic outcome is associated with inflammation and fibrosis.
  • the murine dietary model presents with characteristic histopathology - microvesicular and macrovesicular steatosis, ballooning degeneration of hepatocytes, inflammation and fibrosis - but, distinct from the human disease - shows a greater degree of spontaneous regeneration (such as biliary regeneration and hepatic regenerative micro-nodules) and variability in the inflammatory response to hepatocyte degeneration. It is an attractive model for delineating cellular sites of action of putative therapeutic agents due to the linear nature of the lesion in the relative absence of co-morbidity.
  • Liver sections were provided from 59 animals from a study set of 64 animals (including animals used for studies not reported here). There were no slides from animals 15, 17, 38, 46 and 59. Three slides were provided from each animal - each slide stained with a different staining protocol - haematoxylin and eosin ("M&E”), Masson's trichome (“MT”) and reticulin (“R”). In general, the quality of the slide processing and staining was good with no rejections on quality grounds.
  • M&E haematoxylin and eosin
  • MT Masson's trichome
  • R reticulin
  • Assignment of grade is based upon the most frequent lesion.
  • fibroplastic foci 0 - no significant pathology
  • 1 low grade fibroplastic foci, often peri-vascular or peri- biliary
  • 2 occasional expansion of fibroplastic expansion of parenchymal chords
  • 3 confluent fibroplastic expansion of parenchymal chords
  • 4 immature fibroplastic foci, with associated inflammation and hepatocyte degeneration
  • 5 - fibrosis foci, with marked inflammation and hepatocyte degeneration.
  • mice maintained on a normal diet showed no significant liver pathology. Autophagy foci were present and levels consistent with normal cell homeostasis.
  • mice maintained on a high fat/ fructose diet developed a mature steatohepatitis with a microvesicular or mixed microvesicular/ macrovesicular steatosis, parenchymal fibrosis, hepatocellular ballooning and necrosis and loss of the anatomical integrity of the reticulin network.
  • Steatohepatitis was associated with a trend towards reduced autophagy foci, but elevated Mallory-Denk bodies - both consistent with reduced clearance of cell debris.
  • M&E haematoxylin and eosin
  • MT Masson' s trichome
  • R reticulin

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Abstract

L'invention concerne des composés hétérocycliques difluorés et leurs utilisations médicales. Les composés de l'invention présentent la formule générale (I) : dans laquelle chaque X est identique et fait partie d'une chaîne latérale basique d'éthoxyamine qui comprend un noyau hétérocyclique à 5 ou 6 chaînons, et le noyau hétérocyclique X est choisi dans le groupe constitué par :
PCT/GB2017/051764 2016-06-16 2017-06-16 Composés induisant l'autophagie Ceased WO2017216576A1 (fr)

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GB1610496.0 2016-06-16
GBGB1610496.0A GB201610496D0 (en) 2016-06-16 2016-06-16 Compounds

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WO2017216576A1 true WO2017216576A1 (fr) 2017-12-21

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Citations (1)

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WO2009049242A1 (fr) * 2007-10-12 2009-04-16 President And Fellows Of Harvard College Composés induisant l'autophagie et utilisations de ceux-ci pour traiter des maladies associées à l'autophagie

Patent Citations (1)

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WO2009049242A1 (fr) * 2007-10-12 2009-04-16 President And Fellows Of Harvard College Composés induisant l'autophagie et utilisations de ceux-ci pour traiter des maladies associées à l'autophagie

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