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EP2544678A2 - Inhibiteurs du metabolisme du glucose pour l'utilisation dans le traitement du syndrome de birt-hogg-dubé - Google Patents

Inhibiteurs du metabolisme du glucose pour l'utilisation dans le traitement du syndrome de birt-hogg-dubé

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Publication number
EP2544678A2
EP2544678A2 EP11709454A EP11709454A EP2544678A2 EP 2544678 A2 EP2544678 A2 EP 2544678A2 EP 11709454 A EP11709454 A EP 11709454A EP 11709454 A EP11709454 A EP 11709454A EP 2544678 A2 EP2544678 A2 EP 2544678A2
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European Patent Office
Prior art keywords
inhibitor
bhd
cells
metabolism
glucose
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German (de)
English (en)
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Andrew Tee
Keith Baar
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Myrovlytis Technology Ventures Ltd
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Myrovlytis Technology Ventures Ltd
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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
    • A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
    • A61K31/19—Carboxylic acids, e.g. valproic acid
    • A61K31/195—Carboxylic acids, e.g. valproic acid having an amino group
    • 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
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7004—Monosaccharides having only carbon, hydrogen and oxygen atoms
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents

Definitions

  • This invention relates to drugs for use in the treatment of diseases. More particularly, this invention relates to compositions, including compositions comprising 2-deoxy-D- glucose and compositions comprising oxamate for use in the treatment of Birt-Hogg- Dube syndrome.
  • Birt-Hogg-Dube (BHD) syndrome is a dominantly inherited familial cancer syndrome associated with susceptibility to renal cell carcinoma (RCC).
  • BHD is also associated with benign skin fibrofolliculomas (hamartomatous tumours of the hair follicle) and multiple lung cysts and spontaneous pneumothorax (Toro et al., J. Med. Genet. 2008; 45: 321 -331 ).
  • RCC renal cell carcinoma
  • RCC is the fourteenth most common cancer in the UK.
  • RCC is a heterogeneous disorder with a number of histopathological subtypes.
  • One subtype is conventional clear cell RCC (ccRCC), which accounts for more than 75% of cases of RCC.
  • Non- clear-cell forms of RCC comprise papillary (or chromophil) RCC, chromophobe tumours, oncocytoma, collecting duct carcinoma and the rare medullary carcinoma.
  • BHD-associated renal tumours are of variable histopathology but are often chromophobe RCC/oncocytoma.
  • BHD syndrome results from inactivating mutations in the folliculin (FLCN or BHD) gene (Nickerson et al., Cancer Cell 2002; 2: 157-164; Schmidt et al., Am J Hum Genet. 2005; 76: 1023-33; Lim et al., Hum Mutat. 2010 Jan 31 (1 ):E1043-51 ) and renal tumours from BHD patients demonstrate somatic BHD loss. Loss of heterozygosity of the BHD gene which encodes folliculin (FLCN also referred to as BHD) is a frequent event in renal cell carcinoma suggesting that folliculin (FLCN) can act as a classical "Knudson" tumour suppressor (Koo 2004).
  • FLCN the BHD gene product
  • FNIP1 Folliculin-interacting protein 1
  • hypoxia-inducible-factor controls many processes including angiogenesis and cellular metabolism, as well as influencing cell proliferation and survival decisions.
  • HIF oc and HIF2oc are transcription factors that interact with HIF- ⁇ (also known as aryl hydrocarbon receptor nuclear translocator (Arnt)) and as a heterodimer enhance expression of over 70 genes containing hypoxia response elements that are involved in angiogenesis, erythropoiesis, glucose metabolism, cell survival and metastasis (Semanza, 2004).
  • HIF- ⁇ proteins are rapidly degraded in an oxygen-dependent manner through hydroxylation of two proline residues within their oxygen-dependent degradation domain catalysed via HIF prolyl hydroxylases (Semenza, 2004).
  • VHL Von Hippel-Lindau
  • TSC tuberous sclerosis complex
  • VHL patients develop clear cell RCC which is caused by loss of function of the tumour suppressor protein VHL (Hemminki, 2002).
  • TSC Tuberous Sclerosis Complex
  • mTOR is a positive regulator of HIF, in an apparently multifaceted manner via the levels of gene expression, translation, protein stability and activity (see review Dunlop and Tee 2009). Glycolysis is a central pathway in biological systems.
  • Glycolysis is a sequence of reactions that converts glucose into pyruvate with concomitant production of ATP.
  • the pyruvate generated by glycolysis feeds into the citric acid cycle (also known as the Krebs or TCA cycle).
  • the citric acid cycle together with the electron-transport chain harvest the energy stored in glucose.
  • Pyruvate enters the mitochondria where the complete oxidation of pyruvate to carbon dioxide and water takes place. Under conditions where the oxygen supply is limited, pyruvate can instead be converted anaerobically to lactate. This however only releases a small amount of the energy stored in glucose.
  • the glycolysis pathway has been completely elucidated and includes ten reactions:
  • Glucose is converted to glucose-6-phosphate, the reaction being catalysed by the enzyme hexokinase.
  • Glucose-6-phosphate is isomerised to fructose-6-phosphate by phosphoglucose isomerise.
  • Fructose-6-phosphate is phosphorylated to fructose1 ,6-bisphosphate by phosphofructokinase.
  • Fructose 1 ,6-bisphosphate is converted to dihydroxyacetone phosphate and glyceraldehyde 3-phosophate by aldolase.
  • Dihydroxyacetone phosphate is isomerised to glyceraldehyde 3-phosphate by triose phosphate isomerise.
  • Glyceraldehyde 3-phosphate is converted to 1 ,3-bisphosphoglycerate by phosphoglycerate kinase.
  • 1 ,3-Bisphosphoglycerate is converted to 3-phosphoglycerate by phosphoglycerate kinase.
  • 3-Phosphoglycerate is converted to 2-phosphoglycerate by phosphoglyceromutase.
  • 2-Phosphoglycerate is converted to phosphoenolpyruvate by enolase.
  • Phosphoenolpyruvate is converted to pyruvate by pyruvate kinase.
  • Glycolysis generates a net gain of two molecules of ATP per molecule of glucose.
  • the net reaction is: Glucose + 2 Pi + 2 ADP + 2 NAD + ⁇ 2 pyruvate + 2 ATP + 2 NADH + 2 H + + 2 H 2 0
  • pyruvate Under aerobic conditions, pyruvate can be converted to acetyl CoA which feeds into the citric acid cycle.
  • NADH and FADH 2 formed by glycolysis and the reactions of the citric acid cycle then undergo electron transfer to molecular oxygen, generating large amounts of ATP by oxidative phosphorylation.
  • lactate regenerates NAD + from NADH. Lactate must be converted back to pyruvate before it can be metabolised.
  • Anaerobic glycolysis is the process by which the normal pathway of glycolysis is routed to produce lactate (rather than feeding pyruvate into the citric acid cycle) and is a result of low oxygen levels preventing mitochondria from carrying out oxidative phosphorylation to produce ATP.
  • Aerobic glycolysis is a term used to denote the production of lactate from glucose in the presence of oxygen. Cells that produce lactate even though they have enough oxygen can be described as using aerobic glycolysis.
  • the reaction steps in glucose metabolism are shown diagrammatically in Figure 9.
  • Renal cell carcinoma (RCC) has traditionally been considered to be largely resistant to radiotherapy and in vitro studies have shown that renal cancer cells are among the least radiosensitive of human cell types.
  • RCC Renal cell carcinoma
  • Surgical resection is therefore currently the preferred treatment for locally confined RCC and can often achieve a cure in the earlier stages of RCC.
  • composition comprising an inhibitor of glucose metabolism for use in the treatment of Birt-Hogg-Dube syndrome.
  • composition comprising an inhibitor of glucose metabolism for use in the inhibition of growth of BHD-null renal cell carcinoma cells.
  • a composition comprising an inhibitor of glucose metabolism for use in the treatment of renal cell carcinoma associated with BHD gene inactivation.
  • composition comprising an inhibitor of glucose metabolism for use in the differential growth inhibition of BHD-null cells over BHD-wild type cells. Understanding the biological differences between normal and BHD-null cells is essential for the design and development of drugs with selective activity against BHD-null cells (i.e. preferential killing of BHD-null cells without significant toxicity to normal cells).
  • Warburg Effect Proliferating cancer cells showing the Warburg Effect consume glucose at a high rate and release L-lactate and not C0 2 .
  • an increase in aerobic glycolysis is seen in a number of types of cancer cells, it is not seen in all cancer cell types. It is not understood what triggers the Warburg effect in cancer cells and it has not been shown that there is any general link established between gene mutation and triggering of the Warburg effect.
  • compositions according to the invention may comprise an optional pharmaceutically acceptable carrier, diluent or excipient, including combinations thereof.
  • compositions may be for human or animal usage in human and veterinary medicine and will typically comprise any one or more of a pharmaceutically acceptable diluent, carrier or excipient.
  • Acceptable carriers or diluents for therapeutic use are well known in the art.
  • the choice of pharmaceutical carrier, excipient or diluent can be selected with regard to the intended route of administration and standard pharmaceutical practice.
  • the pharmaceutical compositions may comprise as, or in addition to the carrier, excipient or diluent, any suitable binder(s), lubricant(s), suspending agent (s), coating agent(s) or solubilising agent(s).
  • the present invention provides compositions comprising inhibitors of all aspects of glucose metabolism.
  • the term "inhibiting" means decreasing, slowing or stopping.
  • compositions comprising inhibitors of enzymes of glucose metabolism.
  • An inhibitor of such an enzyme is a substance which reduces, attenuates, decreases or eliminates the expression and/or activity of such an enzyme.
  • Expression in this context is used to refer to any of the steps of transcription and translation.
  • Activity of the enzyme in this context is used to refer to enzymatic activity of a polypeptide encoded by a gene involved in glucose metabolism.
  • An inhibitor may exert inhibition via any mechanism.
  • a suitable inhibitor may be capable of inhibiting hexokinase, phosphoglucose isomerase, phosphofructokinase, aldolase, triose phosphate isomerase, glyceraldehyde 3-phosphate dehydrogenase, phosphoglycerate kinase, phosphoglycerate mutase, enolase or pyruvate kinase.
  • a suitable inhibitor may be capable of inhibiting more than one enzyme within glucose metabolism, for example two, three, four or more enzymes involved in glucose metabolism.
  • glucose metabolism refers to not only the conversion of glucose to pyruvate, but also the conversion of pyruvate in anaerobic and aerobic glycolysis, and also the uptake of glucose by cells.
  • the invention provides not only compositions comprising inhibitors of the conversion of glucose and sequential metabolites to other intermediates in the glucose metabolism pathways, but also inhibitors of glucose uptake by cells.
  • the invention provides inhibition of glucose metabolism by, for example, down regulation of the glucose transporter GLUT-1 .
  • a composition of the invention can inhibit a tumour cell from becoming larger and/or can prevent the tumour cell from dividing and replicating and increasing the number of tumour cells.
  • the inhibitor of glucose metabolism is a glycolytic inhibitor.
  • glycolytic inhibitors refers to inhibitors that inhibit one or more of the steps involved in the conversion of glucose to pyruvate.
  • the glycolytic inhibitor may be a competitive inhibitor of glucose metabolism.
  • a competitive inhibitor can be bound by an enzyme in place of the substrate.
  • the enzyme cannot bind the inhibitor and the substrate at the same time.
  • a competitive inhibitor may bind in the active site of the enzyme and therefore prevent the binding of the substrate.
  • a competitive inhibitor diminishes the rate of catalysis by reducing the proportion of enzyme molecules bound to a substrate.
  • the glycolytic inhibitor is 2-deoxy-D-glucose (2DG) or a pharmaceutically acceptable salt or solvate thereof.
  • 2-Deoxy-D-glucose is a glucose analogue lacking a hydroxyl group on carbon 2. The 2-hydroxyl group is replaced by hydrogen so that it cannot undergo further glycolysis. 2DG is also taken up by glucose transporters, thereby inhibiting the transport of glucose.
  • the inhibitor of glucose metabolism is an inhibitor of pyruvate metabolism.
  • the inhibitor of pyruvate metabolism is an inhibitor of lactate dehydrogenase.
  • Lactate dehydrogenase catalyses the conversion of pyruvate to lactate with concomitant conversion of NADH to NAD + .
  • the inhibitor of pyruvate metabolism is oxamate or a pharmaceutically acceptable salt or solvate thereof.
  • Oxamate is the ion of amino oxoacetic acid (aminooxoacetate). Aminooxoacetate has formula NH 2 COCOO " .
  • Oxamate inhibits lactate dehydrogenase. Oxamate is available as aminooxoacetic acid sodium salt (NH 2 COCOONa) which is also know as oxalic acid monoamide sodium salt and oxamic acid sodium salt.
  • the invention also provides a method of treating Birt-Hogg-Dube syndrome comprising administering to a subject a composition comprising an inhibitor of glucose metabolism.
  • the "subject" can include domesticated animals, such as cats, dogs etc., livestock (e.g. cattle, horses, pigs, sheep, goats, etc.), laboratory animals (e.g. mouse, rabbit, rat, guinea pig etc.) and birds.
  • the subject is a mammal such as a primate, and more preferably a human.
  • the composition may further comprise a pharmaceutically acceptable carrier.
  • the composition is administered in amount that is effective to treat Birt-Hogg- Dube syndrome in a subject.
  • an "effective amount" of a compound is that amount needed to achieve the desired result or results.
  • a composition comprising a compound of the instant invention may be administered to a subject by any of a number of routes of administration including, for example, orally (for example drenches as in aqueous or non-aqueous solutions or suspension, tablets, boluses, powders, granules, pastes for application to the tongue); sublingually, anally, rectally, or vaginally (for example as a pessary, cream or foam); parenterally (including intramuscularly, intravenously, subcutaneously or intrathecally as for example a sterile solution or suspension); nasally; intraperitoneally; subcutaneously; transdermal ⁇ (for example as a patch applied to the skin); or topically (for example as a cream, ointment or spray applied to the skin).
  • the compound may also be formulated for inhalation.
  • the invention provides a method of inhibiting growth of BHD-null renal cell carcinoma cells comprising administering to a subject a composition comprising an inhibitor of glucose metabolism.
  • the invention also provides a method of treating renal cell carcinoma associated with BHD inactivation comprising administering to a subject a composition comprising an inhibitor of glucose metabolism.
  • the invention also provides a method of differentially inhibiting the growth of BHD- null cells over BHD-wild type cells comprising administering to a subject a composition comprising an inhibitor of glucose metabolism.
  • Figure 1 shows BHD negatively regulates HIF-induced gene expression during hypoxia.
  • the mRNA levels of (a) BNIP3, (b) CCND1 , (c) VEGF-A and (d) G6PD1 were compared within BHD + (UOK257-2) and BHD " (UOK257) cells treated overnight with 50 nM Rapamycin under normoxia (21 % oxygen) and hypoxia (1 % oxygen), where indicated, by qRT-PCR.
  • mRNA levels were standardised against ⁇ -Actin and fold activation was compared to BHD " cells under normoxia.
  • n 3.
  • Figure 2 show increased transcriptional activity of HIFI oc in BHD " cells
  • FIG. 3 shows increased levels of HIF target proteins.
  • Paraffin embedded samples were obtained from a chromophobe renal carcinoma from a patient with BHD.
  • Magnification is x 400 for all panels. Note the different staining patterns for BNIP3 and GLUT1 in chromophobe carcinoma compared to unaffected tissue.
  • For VEGF-A staining is much more intense in the tumor than in normal tissue but the intracellular distribution seems to be more diffuse.
  • erythrocytes are clearly stained, serving as a positive internal control.
  • FIG. 4 shows increased enzyme activity levels of pyruvate kinase and lactate dehydrogenase within BHD " cells, (a) Diagram of glucose and fatty acid metabolism depicting aerobic and anaerobic respiration.
  • FIG. 5 shows inactivation of PDH in BHD " cells.
  • Western blot analysis were carried out on cell lysates prepared from BHD + (UOK257-2) and BHD " (UOK257) cells after 18 h of normoxia (21 % oxygen) or hypoxia (1 % oxygen), with and without 50 nM rapamycin, where indicated.
  • Protein levels of BHD, rpS6, phosphorylated rpS6 at Ser235 and Ser236, Akt, phosphorylated Akt at Thr308, PDH, phosphorylated PDH at Ser293, PDK1 and ⁇ -actin employed as a loading control
  • FIG. 6 shows BHD " cells utilise L-lactate as a metabolic fuel
  • ECAR extracellular acidification rate
  • Figure 7 shows proliferation of the BHD " cells is selectively inhibited with 2- deoxyglucose.
  • BHD + UOK257-2
  • BHD " UOK257
  • the percentage of the original number of cells present in each cell line was then determined after 24, 48 and 72 h.
  • n 3.
  • Figure 8 shows proliferation of the BHD " cells is selectively inhibited with Lactate Dehydrogenase inhibitor oxamate.
  • BHD + UOK257-2
  • BHD " UOK257
  • the percentage of the original number of cells present in each cell line was then determined after 24, 48 and 72 h.
  • Figure 9 is a schematic diagram showing the reaction steps in glucose metabolism. Abbreviations used in the diagram are: HK, hexokinase; PGI, phosphoglucose isomerase; PFK, phosphofructokinase; TPI, triosephosphate isomerase; GAPDH, glyceraldehyde- 3-phosphate dehydrogenase; PGK, phosphoglycerate kinase; PGM, phosphoglycerate mutase; PK, pyruvate kinase; PDH: pyruvate dehydrogenase; LDH:lactate dehydrogenase.
  • HK hexokinase
  • PGI phosphoglucose isomerase
  • PFK phosphofructokinase
  • TPI triosephosphate isomerase
  • GAPDH glyceraldehyde- 3-phosphate dehydrogenase
  • PGK phospho
  • Figure 10 shows HIF activity is enhanced upon knockdown of BHD.
  • Figure 13 shows knockdown of BHD expression increases HIF activity under hypoxia in human kidney ACHN cells.
  • Figure 13a The protein levels of HIF1 a and HIF2a were compared in ACHN cells, stably transfected with either BHD shRNA or scrambled shRNA, after 18 h of normoxia (21 % oxygen) or hypoxia (1 % oxygen) by western blot, ⁇ -actin serves as a loading control and endogenous BHD expression was determined to verify efficient BHD knockdown.
  • G6PD1 HIF-dependent Glucose-6-Phosphate Dehydrogenase 1 (G6PD1 ) gene expression (Gao, 2004).
  • Glucose-6-Phosphate Dehydrogenase 1 (G6PD1 ) is the rate-limiting enzyme within the pentose phosphate pathway (PPP, also referred to as the hexose- monophosphate shunt) that is induced upon oxygen stress.
  • PPP pentose phosphate pathway
  • the PPP maintains the level of co-enzyme nicotinamide adenine dinucleotide phosphate (NADPH), the primary reducing agent within mammalian cells and in so doing helps maintain cellular redox homeostasis (Gao, 2004).
  • NADPH co-enzyme nicotinamide adenine dinucleotide phosphate
  • we observe a higher level of G6PD1 mRNA within the BHD " cells when compared to the BHD + cells ( Figure 1 D).
  • the mRNA levels of G6PD1 under normoxic conditions were not further enhanced when the BHD " cells were cultured in low oxygen suggesting that G6PD1 is maximally transcribed by HIF under normoxic conditions.
  • HIF gene expression is upregulated in a BHD tumour
  • GLUT1 glucose transporters
  • pyruvate kinase Figure 4C
  • LDH lactate dehydrogenase
  • Pyruvate lies at the intersection of two glycolytic pathways: as a substrate for LDH enabling L-lactate production or as a substrate of the PDH reaction to generate the Krebs cycle entry molecule, acetyl CoA (see Figure 4A).
  • PDH is typically inhibited via phosphorylation by PDK1 .
  • pyruvate is preferentially converted to lactic acid by LDH (Wigfield, 2008) and entry of acetyl-CoA from fatty acid oxidation is preferred.
  • rpS6 is routinely employed to determine relative levels of signal transduction through the mTOR/70 kDa ribosomal protein S6 kinase 1 (S6K1 ) signalling pathway. As previously reported, the level of rpS6 phosphorylation is elevated in the BHD " cells ( Figure 5, lane 3), indicating that they have a higher basal level of mTOR signalling (Hartman, 2009) (Proc Natl Acad Sci U S A. 2009 Nov 3;106(44):18722-7. Epub 2009 Oct 22. Hasumi Y et al[A1 ]).
  • HIF activity is enhanced upon knockdown of BHD
  • ACHN is a human renal carcinoma cell line.
  • ACHN adenocarcinoma human
  • the BHD " cells were more sensitive to 2- deoxy-D-glucose (IC50 at 48h: 2.5mM) than the BHD + cells (IC50 at 48h: 10mM) and show significant reduction to cell proliferation after 72h of treatment (Figure 7C). This suggests that the BHD " cells have a higher dependence on glucose as a fuel. This inability to shift fuel oxidation to match availability is know as a loss of "metabolic flexibility" (Galgani, 2008). Oxamate selectively impairs growth of BHD " cells (UOK257)
  • Anti-Folliculin antibodies were kindly provided by Dr Arnim Pause, McGill University, Canada.
  • Anti-PDK1 , Anti-Beta-Actin, Anti-GLUT1 and anti-VEGF-A antibodies were bought from Abeam (Cambridge, United Kingdom).
  • Anti-phospho-ribsosomal protein S6 Thr308, Ser476)
  • anti-ribosomal protein S6, anti-phospho-AKT Thr308, Ser476)
  • total anti-Akt antibodies anti-phospho Acetyl Choline carboxylase and total and phospho AMPKoc antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA).
  • Anti-phospho-Pyruvate Dehydrogenase (Thr306, Ser473) and anti-Pyruvate Dehydrogenase antibodies were purchased from Santa Cruz Biotechnology, inc. (Santa Cruz, CA. USA) and anti-BNIP3 antibodies and all other reagents used if not otherwise stated was obtained from Sigma-Aldrich. Rapamycin was purchased from Calbiochem/Merck (Beeston, Nottingham, UK). The N-terminal Flag-tagged BHD vector was a kind gift from Dr. Laura S. Schmitt (Bethesda, MD USA, described in (Baba, 2006)).
  • UOK257 and UOK257-2 cells that were a kind gift from Dr. Laura S. Schmitt (Bethesda, MD USA) were cultured in Dulbecco's Modified Eagle's Medium supplemented with 10 % (v/v) foetal calf serum, 100 U/ml penicillin and 100 ⁇ g ml streptomycin (Gibco, Paisley, UK).
  • UOK257-2 and UOK257 cells were incubated at either 21 % or 1 % oxygen either with or without 50 nM rapamycin treatment. After 48 h, these cells were harvested.
  • UOK257 is the only RCC cell line that has been derived from a patient with BHD and harbours a germline FLCN frames h if t mutation (c.1285dupC) (predicted, in the absence of nonsense mediated mRNA decay, to lead to premature protein truncation (p.His429ProfsX27) (Yang et al., Cancer Genet Cytogenet. 2008 Jan 15;180(2):100-9[A2]).
  • RNA from each sample (1 ⁇ g) was transcribed into cDNA using Quantitect reverse transcription kit (Qiagen, West Wales, United Kingdom) in a thermal cycler (Applied Biosystems, California, USA).
  • Quantitect reverse transcription kit Qiagen, West Wales, United Kingdom
  • the sequences of the VEGF-A l-ll primers used were Forward 5'-CTGCTGTCTTGGGTGCATTG-3'; Reverse 5'- TTCACAATTTGTTGTGCTGTAG-3' as described in (Roland et al., 2000). All other primer sets were purchased from Qiagen, who have to right to with hold primer sequence information.
  • Quantitative Real Time PCR reactions were conducted in 96 well plates using appropriate primer assays and Sybr Green PCR Master mix (Qiagen, West Wales, United Kingdom).
  • Assays were performed as follows: initial denaturation step (95 °C, 15 min), 40 cycles of denaturation.(94 °C, 15 sec), annealing step (55 °C, 30 sec), extension step (72 °C, 40 sec). The amplification products were quantified during the extension step in the 40th cycle. The results were then determined using the ddCT method, and normalised first to ⁇ -Actin. A dissociation step was performed, which verified that only one PCR product was produced with each primer set and shows their specificity. The correct size of PCR products were also verified via resolution on a 2% polyacrylamide gel.
  • the expected size of the amplified products was approximately 70 for VEGFa, 104 bp for ⁇ -Actin (Catalogue Number QT01680476), 73 bp for BNIP3 (Catalogue Number QT00024178), 96 bp for CCND1 (Catalogue Number QT00495285), 91 bp for G6PD1 (Catalogue number QT00071596), and 104 bp for HIF1 a (Catalogue Number QT00083664).
  • the information given above is in accordance to the minimum information for publication of real time quantitative PCR data as described in (Bustin et al., 2009).
  • lysis buffer (20 mM Tris, 135 mM NaCI, 5 % (v/v) glycerol, 50 mM NaF and 0.1 % (v/v) triton X-100, pH 7.5 supplemented with complete mini protease inhibitor cocktail (Roche Diagnostics Ltd. Burgess Hill, United Kingdom) and 1 mM Dithiothreitol (DTT)) at 4 °C.
  • UOK257 cells were transfected with either pRK7 empty vector or Flag-tagged BHD with the firefly luciferase reporter pGL2-TK-HRE plasmid (a gift from G. Melillo (National Cancer Institute at Frederick, Maryland) using Lipofectamine 2000 transfection reagent (Invitrogen, Paisley, United Kingdom) according to the manufacturer's protocol.
  • the pGL2-TK-HRE plasmid was generated by subcloning three hypoxia response elements (5 -GTGACTACGTGCTGCCTAG-3') from the inducible nitric-oxide synthase promoter into the promoter region of the pGL2-TK vector as previously described (Rapisarda, A., Uranchimeg, B., Scudiero, D. A., Selby, M., Sausville, E. A., Shoemaker, R. H., and Melillo, G. (2002) Cancer Res. 62, 4316-4324).
  • hypoxia response elements 5 -GTGACTACGTGCTGCCTAG-3'
  • Luciferase Reagent 20 mM HEPES (pH 7.7), 5 mM MgS0 4 , 1 mM d-luciferin, and 2mM ATP.
  • the protein amount in each sample was determined using the Bradford assay. Levels of Luciferase present were determined via a luminometer 2 s and 10 s after initial injection.
  • the enzyme assay protocol where carried out as described in Suarez, 1986 where the cells were lysed directly into homogenization buffer (20 mM Hepes (pH 7.4), 2 mM EDTA, 0.1 % (v/v) triton X-100, and 10 mM DTT. Lysates were pulse sonicated and centrifugation at 13,000 rpm for 8 min at 4 °C. Protein levels were quantified by using a Bradford assay. For the Hexokinase, Pyruvate kinase and LDH enzyme assays the samples were assayed in 50 mM imidazole-HCI (pH 7.4) under the following conditions.
  • Hexokinase 5 mM glucose (omitted for control), 1 mM ATP, 5 mM MgCI 2 , 5 mM DTT, 0.5 mM NADP + , and excess glucose-6-phosphate dehydrogenase (GGPDH).
  • Pyruvate kinase 5 mM phospho(enol)pyruvate (omitted for control), 5 mM ADP, 0.15 mM NADH, 10 mM MgC1 2 , 100 mM KCI, 0.02 mM fructose 1 ,6-bisphosphate, 5 mM DTT, excess LDH.
  • LDH assay 4 mM pyruvate (omitted for control), 0.15 mM NADH, 5 mM DTT.
  • Citrate synthase Malate dehydrogenase and HOAD enzyme assays, samples were assayed in 50 mM Tris-CI (pH 8.0).
  • Citrate synthase 0.5 mM oxaloacetate (omitted for control), 0.3 mM acetyl CoA, 0.1 mM 5,5'-dithiobis(2- nitrobenzoic acid) (DTNB).
  • Malate dehydrogenase 50 mM imidazole-HCI (pH 7.4), 10 mM oxaloacetate (omitted for control), 0.15 mM NADH, 5 mM DTT.
  • HOAD 50 mM imidazole-HCI (pH 7.4), 0.1 mM acetoacetyl CoA (omitted for control), 0.15 mM NADH, 1 mM EDTA, 5 mM DTT.
  • Immunohistochemical staining was performed on paraffin embedded samples of a chromophobe renal cell carcinoma (samples obtained during total nephrectomy). After deparaffination with xylene and rehydration, sections were incubated in 3 % (w/v) hydrogen peroxide (H 2 0 2 ) diluted in methanol to inactivate endogenous peroxidases. Antigen retrieval was done by microwave treatment using citrate buffer (pH 6).
  • BHD is considered a tumour suppressor, but it is currently unclear how BHD functions to repress cell growth.
  • Folliculin-interacting protein 1 was discovered to interact with folliculin and this protein complex is phosphorylated in an mTOR and AMP-dependent protein kinase (AMPK)-dependent manner (Baba et al, 2006). This evidence implies that FLCN is a downstream cell signalling component of both mTOR and AMPK.
  • HIF plays a pivotal role for tumour progression in VHL (Kaelin, 2005), HLRCC (Issacs, 2005) and TSC (Liu, 2003).
  • BNIP3's expression pattern has changed in the tumour compared to unaffected tissue.
  • BNIP3 does have a transmembrane domain and has a pro-apoptotic function.
  • BNIP3 translocates to mitochondria (van de Velde et al, 2000).
  • BNIP3 has emerging functions in regulating autophagy and may therefore well have protective functions during hypoxia.
  • the apparent shift in staining intensity from cytoplasm to the cell membrane is a quite interesting observation in this respect and might be consistent with BNIP3 fulfilling a protective rather than apoptotic function in the chromophobe carcinoma.
  • HIF1 and HIF2 are critical players in promoting tumour progression by driving gene expression that leads to cellular changes in energy metabolism, cell survival, angiogenesis, glucose transport, and metastasis (Semenza, 2004).
  • BHD expression is restored in UOK257 cells we see a robust inhibition of HIF-mediated gene expression ( Figure 1 ) and HIF activity ( Figure 2C) implying that BHD negatively regulates HIF.
  • Figure 1 HIF-mediated gene expression
  • Figure 2C HIF activity
  • Oxamate is a structural analogue of pyruvate, and binds to and inhibits LDH. Oxamate competes with enzymes that use pyruvate as a substrate, and therefore competitively inhibits LDH, gluconogenesis and pyruvate entry into the mitochondria.
  • Bustin SA Benes V, Garson JA, Hellemans J, Huggett J, Kubista M, Mueller R, Nolan T, PfaffI MW, Shipley GL, Vandesompele J and Wittwer CT. (2009). Clinical Chemistry, 55, 61 1 -622.
  • Van de Velde C Cizeau J, Dubik D, Alimonti J, Brown T, Israels S, Hakrem R and Greenberg AH. (2000). Molecular Cell Biology, 20. Warburg O. (1956). Science, 123, 309-314.
  • Wigfield SM Wigfield SM, Winter SC, Giatromanolaki A, Taylor J, Koukourakis ML and Harris AL. (2008). British Journal of Cancer, 98, 1975-1984. Woodward ER, Ricketts C, Killick P, Gad S, Morris MR, Kavalier F, Hodgson SV, Giraud S, Paillerets BB-d, Chapman C, Escudier B, Latif F, Richard S and Maher ER. (2008). Clinical Cancer Research, 5925.

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Abstract

La présente invention a pour objet une composition comprenant un inhibiteur du métabolisme du glucose destiné à être utilisé dans le traitement du syndrome de Birt-Hogg-Dubé. L'inhibiteur du métabolisme du glucose peut être un inhibiteur glycolytique. L'inhibiteur du métabolisme du glucose peut être un inhibiteur du métabolisme du pyruvate. Des inhibiteurs du métabolisme du glucose convenables comprennent le 2-désoxy-D-glucose ou son sel ou son solvate pharmaceutiquement acceptable ou un oxamate ou son sel ou son solvate pharmaceutiquement acceptable.
EP11709454A 2010-03-09 2011-03-08 Inhibiteurs du metabolisme du glucose pour l'utilisation dans le traitement du syndrome de birt-hogg-dubé Withdrawn EP2544678A2 (fr)

Applications Claiming Priority (2)

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GB1003894A GB2478556A (en) 2010-03-09 2010-03-09 A composition for use in the treatment of Birt-Hogg-Dubô sydrome
PCT/GB2011/050454 WO2011110842A2 (fr) 2010-03-09 2011-03-08 Inhibiteurs du métabolisme du glucose destinés à être utilisés dans le traitement du syndrome de birt-hogg-dubé

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EP2544678A2 true EP2544678A2 (fr) 2013-01-16

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WO2021007499A1 (fr) * 2019-07-11 2021-01-14 Emory University Polythérapies pour la gestion du cancer
US20230321135A1 (en) * 2020-06-08 2023-10-12 Qatar Foundation For Education, Science And Community Development Targeting of lactate dehydrogenase c, methods of preparing same, and methods of using same in combination with anti-cancer treatments

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US6670330B1 (en) * 2000-05-01 2003-12-30 Theodore J. Lampidis Cancer chemotherapy with 2-deoxy-D-glucose
US6979675B2 (en) * 2003-01-10 2005-12-27 Threshold Pharmaceuticals, Inc. Treatment of cancer with 2-deoxyglucose
WO2004064734A2 (fr) * 2003-01-17 2004-08-05 Threshold Pharmaceuticals, Inc. Polytherapies anticancereuses
LT2481409T (lt) * 2007-03-07 2018-10-25 Abraxis Bioscience, Llc Nanodalelės, apimančios rapamiciną ir albuminą, kaip priešvėžinį agentą
RU2538683C2 (ru) * 2008-10-31 2015-01-10 Новартис Аг КОМБИНАЦИЯ ИНГИБИТОРА ФОСФАТИДИЛИНОЗИТОЛ-3-КИНАЗЫ (Р13К) И ИНГИБИТОРА mTOR

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WO2011110842A3 (fr) 2012-05-10
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GB2478556A (en) 2011-09-14
GB201003894D0 (en) 2010-04-21
GB201218050D0 (en) 2012-11-21

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