(12) INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT)
`
`(19) World Intellectual Property Organization
`International Bureau
`
`( 43) International Publication Date
`16 February 2006 (16.02.2006)
`
`PCT
`
`(51) International Patent Classification7
`:
`C07D 311/72, A61K 31/225, A61P 35/00
`
`C07C 69/716,
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`(21) International Application Number:
`PCT/GB2005/003119
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`(22) International Filing Date: 9 August 2005 (09.08.2005)
`
`(25) Filing Language:
`
`(26) Publication Language:
`
`English
`
`English
`
`(30) Priority Data:
`0417715.0
`0421921.8
`
`9 August 2004 (09.08.2004) GB
`1 October 2004 (01.10.2004) GB
`
`(71) Applicant (for all designated States except US): CANCER
`RESEARCH TECHNOLOGY LIMITED [GB/GB];
`Sardinia House, Sardinia Street, London Greater London
`WC2A 3NL (GB).
`
`(72) Inventors; and
`(75) Inventors/Applicants (for US only): GOTTLIEB, Eyal
`[IL/GB]; The Beatson Institute for Cancer Research,
`Switchback Road, Bearsden, Glasgow Strathclyde G61
`lBD (GB). SELAK, Mary, A. [US/GB]; The Beatson
`Institute for Cancer Research, Switchback Road, Bearsden,
`Glasgow Strathclyde G61 lBD (GB). MACKENZIE,
`Elaine, D. [GB/GB]; The Beatson Institute for Cancer
`Research, Switchback Road, Bearsden, Glasgow Strath(cid:173)
`clyde G61 lBD (GB). WATSON, David, G. [GB/GB];
`Department of Pharmaceutical Sciences, University of
`
`1111111111111111 IIIIII IIIII 11111111111111111111 lllll lllll 11111111111111111111111111111111111111
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`(10) International Publication Number
`WO 2006/016143 Al
`Strathclyde, 27 Taylor Street, Glasgow Strathclyde G4
`ONR (GB).
`
`(74) Agents: WYTENBURG, Wilbelmus et al.; Mewburn El(cid:173)
`lis LLP, York House, 23 Kingsway, London Greater Lon(cid:173)
`don WC2B 6HP (GB).
`
`(81) Designated States (unless otherwise indicated, for every
`kind of national protection available): AE, AG, AL, AM,
`AT, AU, AZ, BA, BB, BG, BR, BW, BY, BZ, CA, CH, CN,
`CO, CR, CU, CZ, DE, DK, DM, DZ, EC, EE, EG, ES, FI,
`GB, GD, GE, GH, GM, HR, HU, ID, IL, IN, IS, JP, KE,
`KG, KM, KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV, MA,
`MD, MG, MK, MN, MW, MX, MZ, NA, NG, NI, NO, NZ,
`OM, PG, PH, PL, PT, RO, RU, SC, SD, SE, SG, SK, SL,
`SM, SY, TJ, TM, TN, TR, TT, TZ, UA, UG, US, UZ, VC,
`VN, YU, ZA, ZM, ZW.
`
`(84) Designated States (unless otherwise indicated, for every
`kind of regional protection available): ARIPO (BW, GH,
`GM, KE, LS, MW, MZ, NA, SD, SL, SZ, TZ, UG, ZM,
`ZW), Eurasian (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM),
`European (AT, BE, BG, CH, CY, CZ, DE, DK, EE, ES, Fl,
`FR, GB, GR, HU, IE, IS, IT, LT, LU, LV, MC, NL, PL, PT,
`RO, SE, SI, SK, TR), OAPI (BF, BJ, CF, CG, CI, CM, GA,
`GN, GQ, GW, ML, MR, NE, SN, TD, TG).
`
`Published:
`with international search report
`
`For two-letter codes and other abbreviations, refer to the "Guid(cid:173)
`ance Notes on Codes and Abbreviations" appearing at the begin(cid:173)
`ning of each regular issue of the PCT Gazette.
`
`(54) Title: ALPHA-KETOGLUTARATES AND THEIR USE AS THERAPEUTIC AGENTS
`
`(57) Abstract: The present invention relates generally to the field of pharmaceuticals and medicine. More particularly, the present
`invention relates to certain compounds (e.g., a-ketoglutarate compounds; compounds that activate HIFa hydroxylase; compounds
`that increases the level of a ketoglutarate, etc.) and their use in medicine, for example, in the treatment of cancer (e.g., cancer in
`which the activity of one of the enzymes in the tricarboxylic acid (TCA) cycle is down regulated), in the treatment of angiogenesis
`(e.g., hypoxia-induced angiogenesis). One preferred class of compounds are a-ketoglutarate compounds having a hydrophobic moi(cid:173)
`ety that is, or is part of, an ester group formed from one of the acid groups of a ketogluartic acid; and pharmaceutically acceptable
`salts, solvates, amides, esters, ethers, N oxides, chemically protected forms, and prodrugs thereof.
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`ALPHA-KETOGLUTARATES AND THEIR USE AS THERAPEUTIC AGENTS
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`RELATED APPLICATIONS
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`This application is related to United Kingdom patent application GB 0417715.0 filed
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`09 August 2004 and United Kingdom patent application GB 0421921.8 filed 01 October
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`2004, the contents of each of which are incorporated herein by reference in their entirety.
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`TECHNICAL FIELD
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`The present invention relates generally to the field of pharmaceuticals and medicine.
`
`More particularly, the present invention relates to certain compounds (e.g.,
`
`a-ketoglutarate compounds; compounds that activate HIFa hydroxylase; compounds that
`
`increases the level of a-ketoglutarate, etc.) and their use in medicine, for example, in the
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`treatment of cancer (e.g., cancer in which the activity of one of the enzymes in the
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`tricarboxylic acid (TCA) cycle is down regulated), in the treatment of angiogenesis (e.g.,
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`hypoxia-induced angiogenesis), etc.
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`BACKGROUND
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`A number of patents and publications are cited herein in order to more fully describe and
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`disclose the invention and the state of the art to which the invention pertains. Full
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`citations for these references are provided herein. Each of these references is
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`incorporated herein by reference in its entirety into the present disclosure.
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`Throughout this specification, including any claims which follow, unless the context
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`requires otherwise, the word "comprise," and variations such as "comprises" and
`
`"comprising," will be understood to imply the inclusion of a stated integer or step or group
`
`of integers or steps, but not the exclusion of any other integer or step or group of integers
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`or steps.
`
`It must be noted that, as used in the specification and any appended claims, the singular
`
`forms "a," "an," and "the" include plural referents unless the context clearly dictates
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`otherwise. Thus, for example, reference to "a pharmaceutical excipient" includes
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`35 mixtures of two or more such excipients, and the like.
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`Ranges are often expressed herein as from "about" one particular value, and/or to "about"
`another particular value. When such a range is expressed, another embodiment includes
`from the one particular value and/or to the other particular value. Similarly, when values
`are expressed as approximations, by the use of the antecedent "about," it will be
`understood that the particular value forms another embodiment.
`
`Cancer is a serious disease and a major killer. Although there have been advances in
`the treatment of certain cancers in recent years, there is still a need for improvements in
`the treatment of the disease.
`
`Cancer is characterised by the uncontrolled growth of cells due to cellular changes, which
`are mostly caused by inherited or somatic mutations of genes. The identification of such
`genes and the elucidation of the mechanism by which these genes affect the
`development of cancer is important in devising strategies of combating cancer.
`
`Enzymes of the mitochondrial tricarboxylic acid (TCA) cycle have long been associated
`with cancer. Several mitochondrial proteins are tumour suppressors including succinate
`dehydrogenase (SDH) and fumarate hydratase (FH). Inherited or somatic mutations in
`subunits B, C or D of the SDH genes are associated with the development of
`phaeochromocytoma and paraganglioma (Baysal et al., 2000; Eng et al., 2003). Recently,
`other types of cancer have also been shown to carry or develop mutations in
`mitochondrial genes. For example, it has been shown that significant SDH down(cid:173)
`regulation occurs in gastric and colorectal carcinoma, particularly during transition to the
`more aggressive Dukes' stage C, colorectal cancer, as compared to the confined Dukes'
`stage B tumours (Frederiksen et al., 2003; Habano et al., 2003).
`
`Eng et al. (2003) discuss the link between mutations of gene encoding FH and SDH and
`cancer. The authors hypothesise that impaired mitochondrial function due to dysfunction
`of enzymes of the TCA cycle leads to severe energy deficiency and large amounts of
`oxygen free radicals. These radicals lead in turn to the induction of Hypoxia-inducible
`Factor - 1a (HIF-1a) promoting cell proliferation or preventing apoptosis and thereby
`leading to neoplasia. The authors also suggest that mutant forms of SDH, which do not
`insert in the mitochondrial membrane, might have anti-apoptotic activity. However, the
`authors are unable to explain the mechanism underlying the anti-apoptotic activity.
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`Baysal (2003) suggests that SDH and FH could be involved in the control of cell
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`proliferation under normal physiological conditions in the affected tissue types. However,
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`the author provides no further suggestion regarding the mechanism of control.
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`Furthermore, tumours similar to phaeochromocytoma and paraganglioma are observed in
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`the apparently unrelated von Hippel-Lindau (VHL) syndrome with a common feature of
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`these tumours being elevated levels of HIF-1a (Eng et al., 2003, Pollard et al., 2003).
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`Importantly, SDH or VHL mutations in these tumours are mutually exclusive (Eng et al.,
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`2003).
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`Hypoxia-inducible factor-1 (HIF-1) is a heterodimer composed of an alpha (a) subunit and
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`a beta(~) subunit. (However, the terms "HIF-1" and "HIF-1a" are often used
`
`interchangeably to mean the complete protein, HIF-1). The beta subunit has been
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`identified as the aryl hydrocarbon receptor nuclear translocator (ARNT/HIF-1 ~) and its
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`protein level is unaffected by oxygen. Similar to HIF-1~, HIF-1a is constitutively
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`expressed regardless of the oxygenation state. However, under normoxic conditions this
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`subunit is rapidly targeted for proteasome-mediated degradation via a protein-ubiquitin
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`ligase complex containing the product of the von Hippel Lindau tumour suppressor
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`protein (pVHL). pVHL recognizes the oxygen degradation domain (ODDO) of HIF-1a only
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`under normoxic conditions. Following exposure to a hypoxic environment, this
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`degradation pathway is blocked, allowing HIF-1a accumulation and subsequent
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`movement to the nucleus where it activates hypoxia-responsive genes. In other words,
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`the physiological function of HIF is to promote adaptation of cells to low oxygen by
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`inducing neovascularization and glycolysis (Semenza et al., 2002; Pugh et al., 2003).
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`HIF-1a stability is controlled by HIFa prolyl hydroxylase (PHO) which hydroxylases two
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`specific prolyl residues. More specifically, PHO hydroxylases the prolyl residues in the
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`ODDO which regulate the binding of the pVHL to HI Fa (Ivan et al., 2001; Jaakkola et al.,
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`2001; Yu et al., 2001). Hydroxylation at the 4-position of Pro-402 and Pro-564 of HI Fa
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`(numbers refers to human HIF-1a) enables formation of two hydrogen bonds to pVHL and
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`increases the binding of pVHL to HIFa by several orders of magnitude (Bruick et al.,
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`2001; Epstein et al., 2001 ). This post-translational modification is catalyzed by the HIFa(cid:173)
`
`prolyl hydroxylases (HPH1-3 or PHD1-3) (Bruick et al., 2001; Epstein et al., 2001; Ivan et
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`35
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`al., 2002). PHO activity is dependent on molecular oxygen and is considered to be an
`important oxygen sensing mechanism in animal cells (Safran et al., 2003). In addition to
`oxygen, the PHDs utilize a-ketoglutarate as a co-substrate and require ferrous iron (Fe2+)
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`and ascorbate as cofactors (Kaelin et al., 2002; Schofield et al., 1999). The PHO
`isozymes belong to the Fe2
`+ - and a-ketoglutarate-dependent family of oxygenases that
`split molecular oxygen in order to hydroxylate their substrates and, in parallel, oxidize and
`decarboxylate a-ketoglutarate to succinate (Schofield et al., 1999).
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`5
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`WO 03/028663 discloses methods and compositions for assaying hypoxia-inducible
`factor prolyl hydroxylation to identify compounds that modulate the hydroxylation;
`however, the document fails to disclose any such compounds.
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`Although the events around the carcinogenic pathway involving HIF-1a stabilisation have
`been investigated, there are still numerous questions that remain unanswered.
`
`In particular, a simple and effective way to inhibit HIF-1a stabilisation - and thereby inhibit
`the carcinogenic pathway - is still very much needed.
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`Furthermore, until now, there has been no clear indication or suggestion about how
`mutations in genes coding for enzymes of the TCA cycle might result in elevated levels of
`HIF-1a. Therefore, the range of treatment available for these cancers is limited. The
`primary treatment of pheochromocytomas and paragangliomas is surgical resection after
`appropriate medical hormonal blockade. Unresectable tumours may be treated with
`palliative chemotherapy with compounds such as cyclophosphamide, decarbazine, and
`vincristine, or external beam radiotherapy for bony metastases or 131 1-labeled MIGB.
`However these therapies are either highly invasive or have large undesired side effects.
`Therefore, there remains a great need for treatments which are less invasive and which
`have little or no side effects. Preferably such a treatment would be tailor-made for the
`biochemical mechanism underlying these specific types of cancers.
`
`Moreover, compounds that inhibit hypoxia-induced angiogenesis are still required as
`treatment for diseases that are characterised by this type of angiogenesis, including
`cancer.
`
`The inventors have demonstrated how mutations and dysfunctions of genes and
`enzymes of the TCA cycle are linked to cancer. The inventors have developed strategies
`for treating cancer and have identified classes of compounds that are useful in these
`treatments.
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`For example, the inventors have demonstrated that inhibition of certain enzymes of the
`TCA cycle, such as SOH and FH, leads to the accumulation of succinate in cells. In turn,
`succinate inhibits the enzymatic activity of HIF-a prolyl hydroxylase (PHO) in the cytosol.
`Also, the inventors have demonstrated that a-ketoglutarate and a-ketoglutarate
`derivatives (e.g., esters) significantly enhance PHO activity under low oxygen conditions,
`thereby reducing HIF dramatically.
`
`In other words, the inventors identified a new way of treating hypoxia-induced
`angiogenesis, which has useful pharmaceutical applications, for example, in the
`treatment of diseases that are characterised by hypoxia-induced angiogenesis.
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`SUMMARY OF THE INVENTION
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`One aspect of the invention pertains to certain compounds (e.g., a-ketoglutarate
`compounds; compounds that activate HIFa hydroxylase; compounds that activate PHD;
`compounds that inhibit or prevent HIF stabilization; compounds that increases the level of
`a-ketoglutarate, etc.).
`
`Another aspect of the invention pertains to a composition comprising an active compound
`as described herein and a pharmaceutically acceptable carrier or diluent.
`
`Another aspect of the present invention pertains to a method of activating PHD in a cell,
`in vitro or in vivo, comprising contacting the cell with an effective amount of an active
`compound, as described herein.
`
`Another aspect of the present invention pertains to a method of inhibiting or preventing
`HIF stabilization in a cell, in vitro or in vivo, comprising contacting the cell with an effective
`amount of an active compound, as described herein.
`
`Another aspect of the present invention pertains to a method of activating HI Fa
`hydroxylase (e.g., HIFa prolyl hydroxylase) in a cell, in vitro or in vivo, comprising
`contacting the cell with an effective amount of an active compound, as described herein.
`
`Another aspect of the present invention pertains to a method of (a) regulating
`(e.g., inhibiting) cell proliferation (e.g., proliferation of a cell), (b) inhibiting cell cycle
`progression, (c) promoting apoptosis, or (d) a combination of one or more these, in vitro
`or in vivo, comprising contacting cells (or the cell) with an effective amount of an active
`compound, as described herein.
`
`Another aspect of the present invention pertains to an active compound, as described
`herein, for use in a method of treatment of the human or animal body by therapy.
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`Another aspect of the present invention pertains to use of an active compound, as
`described herein, in the manufacture of a medicament for use in treatment.
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`Another aspect of the present invention is a method of treatment, comprising
`administering to a patient in need of treatment a therapeutically effective amount of an
`active compound, as described herein.
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`In one embodiment, the treatment is treatment of a condition that encounters hypoxic
`conditions as it proceeds.
`
`In one embodiment, the treatment is treatment of a condition that is characterised by
`inappropriate, excessive, and/or undesirable angiogenesis.
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`In one embodiment, the treatment is treatment of a condition characterised by hypoxia(cid:173)
`induced angiogenesis.
`
`In one embodiment, the treatment is treatment of angiogenesis in which the activity of
`HIF-1a is upregulated due to hypoxia.
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`In one embodiment, the treatment is treatment of a condition selected from: cancer,
`psoriasis, atherosclerosis, menorrhagia, endometrosis, arthritis (both inflammatory and
`rheumatoid), macular degeneration, Paget's disase, retinopathy and its vascular
`complications (including proliferative and diabetic retinopathy), benign vascular
`proliferation, fibroses, obesity and inflammation.
`
`In one embodiment, the treatment is treatment of a proliferative condition.
`In one embodiment, the treatment is treatment of cancer.
`In one embodiment, the treatment is treatment of solid tumour cancer.
`
`In one embodiment, the treatment is treatment of cancer selected from:
`. phaeochromocytoma, paraganglioma, leiomyoma, renal cell carcinoma, gastric
`carcinoma, and colorectal carcinoma.
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`In one embodiment, the treatment is treatment of cancer (e.g., tumours) characterised by
`(e.g., that exhibits) SDH dysfunction.
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`In one embodiment, the treatment is treatment of cancer that develops SDH
`down-regulation in a later stage of the disease.
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`In one embodiment, the treatment is treatment of gastric or colorectal cancer, for example,
`Dukes' stage C of colorectal cancer.
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`- 8 -
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`In one embodiment, the treatment is treatment of oral carcinoma tumours.
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`In one embodiment, the treatment is treatment of cancer in which the activity of HIF-1a is
`upregulated due to hypoxia.
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`1 O
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`In one embodiment, the treatment is treatment of cancer in which the activity of one of the
`enzymes of the TCA cycle (e.g., succinate dehydrogenase, fumarate hydratase) is
`down-regulated.
`
`In one embodiment, the patient being treated has inherited or somatic mutations in
`subunits A, B, C or D of the SDH gene or FH or down regulation of the expression of any
`of the SDH genes (subunits A, B, C or D) or of FH or impaired activity of the enzymes
`encoded by said genes.
`
`Another aspect of the present invention is a method of treatment comprising
`co-administering to a patient in need of treatment: (a) a therapeutically effective amount
`of an active compound, as described herein, and (b) a second agent.
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`In one embodiment, the second agent is a compound that is an enhancer of
`aminolaevulinic acid (ALA) synthase.
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`In one embodiment, the second agent is selected from: barbiturates, anticonvulsants,
`non-narcotic analgetics, and non-steriodal anti-inflammatory compounds.
`
`In one embodiment, the second agent is selected from: Allyl isopropyl acetamide,
`Phenobarbital, Deferoxamine, Felbamate, Lamotrigine, Tiagabine, Cyclophosphamide, N-
`30 methylprotoporphyrin, Succinyl-acetone, Carbamazepine, Ethanol, Phenytoin,
`Azapropazone, Chloroquine, Paracetamol, Griseofulvin, Cadmium, Iron, Pyridoxine.
`
`In one embodiment, the second agent is selected from: Ethosuximide, Diazepam,
`Hydantoins, Methsuximide, Paramethadione, Phenobarbitone, Phensuximide, Phenytoin,
`Primidone, Succinimides, Bromides, Aspirin, Dihydroergotamine- Mesylate, Ergotamine
`Tartrate, Chloramphenicol, Dapsone, Erythromycin, Flucloxacillin, Pyrazinamide,
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`Sulphonamides, Ampicillin, Vancomycin, Sulphonylureas Glipizidelnsulin, Alpha
`tocopheryl acetate, Ascorbic Acid, Folic Acid, Fructose, Glucose, Haem Arginate,
`Amidopyrine, Dichloralphenazone, Diclofenac Na, Dipyrone, Oxyphenbutazone,
`Propyphenazone, Aspitin, Codeine P04, Dihydrocodeine, Canthaxanthin, f1 Carotene.
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`In one embodiment, the method further comprises the step of subjecting the patient to
`photodynamic therapy.
`
`Another aspect of the present invention is a method of treatment comprising the steps of:
`(i) simultaneous, separate, or sequential administration of: (a) a first agent, that is an
`active compound, as described herein, ; and (b) a photosensitizer; followed by (ii) light
`irradiation.
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`Another aspect of the present invention pertains to a kit comprising (a) an active
`compound, as described herein, preferably provided as a pharmaceutical composition
`and in a suitable container and/or with suitable packaging; and (b) instructions for use, for
`example, written instructions on how to administer the active compound.
`
`Another aspect of the present invention pertains to compounds obtainable by a method of
`synthesis as described herein, or a method comprising a method of synthesis as
`described herein.
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`Another aspect of the present invention pertains to compounds obtained by a method of
`synthesis as described herein, or a method comprising a method of synthesis as
`described herein.
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`Another aspect of the present invention pertains to novel intermediates, as described
`herein, which are suitable for use in the methods of synthesis described herein.
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`Another aspect of the present invention pertains to the use of such novel intermediates,
`as described herein, in the methods of synthesis described herein.
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`As will be appreciated by one of skill in the art, features and preferred embodiments of
`one aspect of the invention will also pertain to other aspect of the invention.
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`BRIEF DESCRIPTION OF THE DRAWINGS
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`Figure 1 shows:
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`(A) a photograph of an SOS-PAGE gel for cell extracts mixed with in vitro-translated HA-
`5 ODDO (HA-O) in the presence of Fe2+, ascorbate, and a-ketoglutarate, treated with: 0, 0.1,
`0.5, 1.0, and 5.0 mM dimethyl ester succinic acid (OMS) (lanes 1-5) and with
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`deferoxamine (DFO) (lane 6), showing HA-D and HA-D-OH;
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`(B) a photograph of a western blot gel for cell extracts: treated with CoCl2 (left lane),
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`untreated (2nd, 3rd, and 4th lanes), and treated with dimethyl ester succinic acid (OMS),
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`for 48 hours under normoxic conditions, showing HIF-1a and actin.
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`Briefly, succinate inhibits PHO activity in vitro and induces HIF-1a levels in cells. Cell
`extracts were mixed with in vitro-translated HA-ODDO (HA-D) in the presence of Fe2
`ascorbate and a-ketoglutarate. Where indicated, succinate was added to the reaction.
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`+,
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`Deferoxamine (DFO), an iron chelator, was added to inhibit PHO activity. The
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`hydroxylated polypeptide (HA-D-OH) migrates faster on SOS-PAGE. HIF-1a and actin
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`levels were assessed by western blot of extracts from either untreated cells or from cells
`treated with dimethyl ester succinic acid (OMS) or CoCl2 for 48 hours under normoxic
`conditions.
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`Figure 2 shows:
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`(A) a photograph of a RT-PCR gel for extracts of cells (in triplicate) that were transfected
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`with: scrambled siRNA (scRNAi), siRNA directed at SOHO subunit (Di3 or Di4)), showing
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`SOHO and actin;
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`(B) a bar graph showing succinate-DCIP oxidoreductase (SDH) activity (nmol/min/mg) for
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`the cells transfected as in (A);
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`(C) a photograph of a western blot gel for cell extracts of the cells transfected as in (A),
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`showing HIF-1a and Actin; (D) a bar graph showing HIF transcriptional activity (as
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`measured by the dual luciferase reporter assay system (Promega) using pGL2/HRE-
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`Luciferase as a reporter for HIF activity, and recorded as HRE-luciferase intensity) (light
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`units x 1000) following SDH inhibition for the cells transfected as in (A)
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`Briefly, inhibition of SDH activity increases HIF-1a levels and HIF activity. Cells were
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`transfected (in triplicate) with either scrambled siRNA (scRNAi) or siRNA directed at
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`SDHD subunit (Di3 or Di4). Following transfections, mRNA levels of SDHD and actin
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`were quantified by RT-PCR. Succinate-DCIP oxidoreductase activity was analysed in
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`cells transfected as in panel A, to confirm inhibition of SDH activity. HIF-1a levels were
`detected by western blot analysis following transfection with scRNAi, Di3 or Di4. Actin
`was used as loading control. HIF transcriptional activity following SDH inhibition was
`assessed by the dual luciferase reporter assay system (Promega) using pGL2/HRE-
`Luciferase as a reporter for HIF activity.
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`Figure 3 shows:
`(A) GCMS profiles of selected ionized fragments in extracts from cells transfected with
`scRNAi or Di3 (relative abundance(%) vs. retention time (minutes) vs. mass-to-charge
`ratio (amu); and
`(B) a bar graph showing succinic acid levels (pmol/106 cells) as determined using GCMS
`for cells transfected with: scrambled siRNA (scRNAi), siRNA directed at SDHD subunit
`(Di3 or Di4).
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`Briefly, SDH inhibition leads to increased levels of succinate. GCMS profiles of selected
`ionized fragments in extracts from cells transfected with either scRNAi or Di3 were
`recorded. Deuterated (D4)-succinic acid was used as a reference and was identified by
`its major ion component of 119 amu at retention time of 8.05 min. Succinic acid was
`identified by its major ion component of 115 amu at retention time of 8.15 minutes. An
`increase in the succinic acid level is seen in Di3-transfected cells compared to scRNAi(cid:173)
`transfected cells. Cells were transfected with the indicated siRNA construct and analyzed
`by GCMS as in panel A. Succinic acid levels were calculated as picomol per 106 cells for
`each transfection and the results summarized from two independent experiments each
`done in triplicate.
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`Figure 4 shows:
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`(A) photographs showing GFP fluorescence for each of:
`(i) cells transfected with plasmids encoding GFP, without HA-pVHL, with scRNAi;
`(ii) cells transfected with plasmids encoding GFP, with HA-pVHL, with scRNAi;
`(iii) cells transfected with plasmids encoding GFP-ODDD, without HA-pVHL, with scRNAi;
`(iv) cells transfected with plasmids encoding GFP-ODDD, with HA-pVHL, with scRNAi;
`(v) cells transfected with plasmids encoding GFP-ODDD, with HA-pVHL, with Di3;
`(vi) cells transfected with plasmids encoding GFP-ODDD, with HA-pVHL, with Di4.
`(8) photographs of gels for extracts of cells (in triplicate) of (iv), (v), and (vi) in (A);
`(C) a bar graph showing GFP fluorescence for extracts of cells of (i), (iii), and (v) in (A);
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`(D) photographs of far-western blot gels for cells transfected with plasmids encoding
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`GFP-ODDD and scRNA, Di3, or Di4 (but without HA-pVHL), showing GFP-ODDD,
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`GFP-ODDD/HA-pVHL, and HA-pVHL.
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`Briefly, inhibition of SDH decreases PHO activity. Cells were transfected with plasmids
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`encoding either GFP (i and ii) or GFP-ODDD (iii, iv, v, vi), with (ii, iv, v, vi) or without (i, iii)
`HA-pVHL, and with one of the siRNA constructs: scRNAi (i, ii, iii, iv), Di3 (v) or Di4 (vi).
`GFP fluorescence was detected microscopically. GFP-ODDD and HA-pVHL protein
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`levels were measured for cells transfected (in triplicate) as in panel A, iv, v, vi. Cells were
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`transfected with plasmids encoding GFP or GFP-ODDD together with HA-pVHL and with
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`the indicated siRNA. GFP fluorescence was analyzed in cell extracts before and after
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`immunoprecipitation with an anti-HA antibody. The results are presented as the percent
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`of GFP fluorescence bound to HA-pVHL and are the average and standard deviation of
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`three independent transfections. Direct detection of ODDO hydroxylation was performed
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`by far-western blot analysis. Cells were transfected (in triplicate) with plasmids encoding
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`GFP-ODDD and the indicated siRNA (but without HA-pVHL). Protein extracts were
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`blotted onto nitrocellulose membrane and the binding of immunopurified HA-pVHL to the
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`blotted GFP-ODDD protein was detected using an anti-HA antibody. 10 ng of the
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`immune-purified HA-pVHL protein was loaded on lane 10.
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`Figure 5 shows a schematic model that summarises the role of succinate in the
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`mitochondrion-to-cytosol signalling pathway.
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`Briefly, succinate accumulated in the mitochondria due to SDH inhibition is transported to
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`the cytosol. Elevated cytosolic succinate inhibits PHO and thereby HI Fa hydroxylation.
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`Consequently, pVHL binding to HIFa is decreased and elevated HIF activity induces
`
`expression of genes that facilitate angiogenesis, metastasis and glycolysis, leading to
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`more aggressive tumours.
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`Figure 6 shows:
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`(A) a photograph of an SOS-PAGE gel for cell extracts mixed with in vitro-translated ODD
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`and treated with: 0, 0, 0, 1.0, 1.0, and 1.0 mM succinate (Succ) (lanes 1-6) and 0.01, 0.1,
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`1.0, 0.01, 0.1 and 1.0 mM free a-ketoglutaric acid (a-KG), showing ODD and OH-ODD;
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`(B) a bar graph showing intracellular a-ketoglutarate levels in cells treated with octyl-
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`a-ketoglutarate (octyl-aKG), TFMB-a-ketoglutarate (TFMB a-KG), free a-ketoglutaric acid
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`(aKG) or left untreated (control).
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`Briefly, succinate-mediated inhibition of PHD can be overcome by increasing a(cid:173)
`ketoglutarate levels in vitro. A hydroxylation reaction of the ODD domain was carried out
`in vitro with the indicated amounts of succinate and a-ketoglutarate. Hydroxylation of
`5 ODD (OH-ODD) resulted in a faster migrating band on SDS-PAGE. Cells were either left
`untreated or treated for 5 hours with 1 mM of the indicated a-ketoglutarate ester or with
`free a-ketoglutaratic acid. The intracellular a-ketoglutarate level was analyzed using the
`glutamate dehydrogenase reaction.
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`1 O Figure 7 shows:
`(A) a photograph of an SDS-PAGE gel showing levels of GFP-ODD fusion protein, GFP,
`HA-pVHL or actin (loading control) in extracts from control (Co) cells or clones (C2 and
`C3) co-expressing the GFP-ODD fusion protein and HA-tagged pVHL. Cells were either
`left untreated {U) or treated with CoCl2 (CC);
`(8) a photograph of an SDS-PAGE gel showing levels of GFP-ODD fusion protein and
`actin (loading control) in Clone 3 cells either left untreated (lanes 3-4), or treated with
`CoCl2 (lanes 1-2) or dimethyl succinate (DMS) (lanes 5-10) for 48 hours. a-ketoglutarate
`esters (octyl-a-ketoglutarate (0) (lanes 7-8) or TFMB-a-ketoglutarate (0) (lanes 9-10)
`were added for the final 24 hours of the incubation (with DMS).
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`Briefly, the inhibition of PHD activity by succinate in cells is alleviated by the increase of
`intracellular a-ketoglutarate level. (A) Left panels - Clones (C2 and C3) co-expressing the
`GFP-ODD fusion protein and HA-tagged pVHL were analyzed by western blot. Cells
`transiently transfected with a plasmid encoding for GFP alone were used as a reference
`for GFP molecular weight (Co). Actin was used as a loading control. Right Panels -
`Clone 3 (C3) cells were either left untreated or treated with the hypoxia mimetic
`compound CoCl2 and GFP-ODD and HA-pVHL protein levels were analyzed by western
`blot. (B} Clone 3 cells were treated as in Panel Band GFP-ODD levels were detected by
`western blot. CoC'2-treated cells were used as a positive control for PHD inhibition and
`actin level was used as a loading control.
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`Figure 8 shows:
`(A) a photograph of a western blot