EP1697744A1 - Screening method - Google Patents

Screening method

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Publication number
EP1697744A1
EP1697744A1 EP03778501A EP03778501A EP1697744A1 EP 1697744 A1 EP1697744 A1 EP 1697744A1 EP 03778501 A EP03778501 A EP 03778501A EP 03778501 A EP03778501 A EP 03778501A EP 1697744 A1 EP1697744 A1 EP 1697744A1
Authority
EP
European Patent Office
Prior art keywords
compound
monocarboxylate
mct
binding
immune
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP03778501A
Other languages
German (de)
French (fr)
Inventor
John Raymond Bantick
Ian David Cook
David Keith Donald
Raymond Hutchinson
Andrew Paul Jackson
Clive Geoffrey Jackson
Clare Margaret Murray
Michael Sullivan
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
AstraZeneca AB
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AstraZeneca AB
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Filing date
Publication date
Application filed by AstraZeneca AB filed Critical AstraZeneca AB
Publication of EP1697744A1 publication Critical patent/EP1697744A1/en
Withdrawn legal-status Critical Current

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Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6872—Intracellular protein regulatory factors and their receptors, e.g. including ion channels
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5011—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing antineoplastic activity

Definitions

  • This invention arises from our discovery that compounds capable of blocking cellular monocarboxylate transport can inhibit lymphocyte proliferation, offering up these compounds as therapeutic agents in autoimmune disorders, inflammatory, proliferative and hyperproliferative diseases, cancer and irnmunologically-mediated diseases including rejection of transplanted organs or tissues. Accordingly, this invention relates, inter alia, to methods of screening compounds for their ability to treat certain cancers and immune- mediated disorders, particularly transplant rejection and rheumatoid arthritis. The invention also relates to methods of treating these diseases by administering a compound or compounds capable of inhibiting monocarboxylate transport and the use of monocarboxylate transport inhibitor compounds in treating such diseases.
  • the immune system has evolved to detect the presence of foreign organisms such as bacteria, viruses and other pathogens, and to mount protective immune responses to eliminate them. Under certain circumstances, the induction of an immune response against foreign organisms or tissues proves more harmful to the host than ignoring them, for example allergies to food and extrinsic antigens such as pollen, and also asthma are believed to reflect inappropriate hypersensitivity responses to otherwise harmless substances. In addition, strong responses against transplanted tissues are usually observed. These are detrimental to the survival of the transplanted organ and must be limited by administration of potent immunosuppressive drugs. Under normal circumstances the immune system does not produce immune responses to self-tissues and self-antigens.
  • immune responses are mounted against self-tissues in such an aggressive manner that they lead to destructive autoimmune diseases, for example rheumatoid arthritis, multiple sclerosis and type I diabetes. In these situations it would be desirable to reset the immune system so that responses to self- antigens are silenced but without affecting protective host defence mechanisms directed against exogenous antigens.
  • Most immune responses are initiated and controlled by helper T lymphocytes, which respond to antigenic peptide fragments presented in association with MHC Class II molecules and cytotoxic T lymphocytes which respond to peptides presented in association with MHC Class I molecules on specialised antigen presenting cells such as dendritic cells. Full T cell activation requires two distinct signals from the antigen presenting cell.
  • Signal 1 is antigen specific and provided by the interaction of the T cell receptor (TCR) with the MHC-peptide complex displayed by the antigen-presenting cell.
  • Signal 2 is antigen independent and involves the interaction of the co-stimulatory T ceU molecule, CD28, with its ligand, B7, on antigen presenting cells. These cell-surface interactions trigger downstream biochemical signalling pathways, which ultimately result in IL2 transcription and T-cell activation.
  • TCR T cell receptor
  • B7 its ligand
  • NFAT Activated T-cells
  • This complex is composed of nuclear components fos and jun and a cytoplasmic component NFAT C which translocates to the nucleus after dephosphorylation by the phosphatase calcineurin.
  • the immunosuppressive macro lides cyclospori ⁇ A (CsA) and FK506 block the transcription of the IL2 gene in T- lymphocytes by preventing the formation of the NFAT complex (Crabtree, Cell 96:611-614, 1999).
  • CsA and FK506 Block the transcription of the IL2 gene in T- lymphocytes by preventing the formation of the NFAT complex (Crabtree, Cell 96:611-614, 1999).
  • CsA and FK506 are potent immunosuppressive drugs used clinically for the prevention of graft rejection, their long-term use and utility for the treatment of auto- immune disease are limited by their side effect profile including nephrotoxicity. These adverse reactions appear to be related to inhibition of calcineurin activity as the enzyme is expressed widely across mammalian tissues and has multiple functions.
  • additional immunosuppressive therapies have now been developed (Dumont, Opin. Ther. Patents 11:377-404, 2001). These include rapamycin, which disrupts the cytokine (e.g.
  • IL2 IL2-driven proliferation of T-cells, by interfering with the function of TOR (Target Of Rapamycin), a kinase involved in the cytokine signalling pathway (Dumont and Su Life Sci. 58:373-395,1996).
  • TOR Target Of Rapamycin
  • rapamycin has been shown to cause significant side effects including thrombocytopenia and hyperlipidemia (Hong and Kahan, Semin. Nephrol. 20 (2): 108-125, 2000).
  • Antimetabolite approaches are also of utility for immunosuppression as T-lymphocytes have been shown to be dependent on de novo synthesis of ribonucleotides (Fairbanks et al., J. Biol. Chem 270(50):29682-29689, 1995).
  • MMF Mycophenolate mofetil
  • IMPDH inosine monophosphate dehydrogenase
  • IMPDH inosine monophosphate dehydrogenase
  • IMPDH inosine monophosphate dehydrogenase
  • Screening programmes investigating NFAT-mediated transcription directly have been used to identify small molecule inhibitors of JJL2 production without the side effect profile of calcineurin inhibitors. Michne et al.
  • WO 98/46606 discloses a family of pyrazolo[3,4 ⁇ pyrimidinedione compounds
  • WO 98/54190 discloses a family of tMeno[2,3-d]pyrimidinedione compounds
  • WO 98/28301(AstraZeneca) discloses a family of 5- substituted pyrrolo[3,4-rf]pyrimidine-2,4-dione compounds
  • WO 99/29695 (AstraZeneca), incorporated herein by reference, discloses certain pyrrolo-, thieno-, furano-and pyrazolo- [3,4- yridazinone compounds; WO 00/12514 (AstraZeneca), WO 01/83489 (AstraZeneca), PCT/GB02/03399 (AstraZeneca), PCT/GB
  • MCT1 is a member of a family of monocarboxylate transporters, which mediate the influx and efflux of monocarboxylates, such as lactate and pyruvate, across cell membranes.
  • the MCT proteins transport monocarboxylates by a facilitative diffusion mechanism, which requires the co-transport of protons (Poole and Halestrap. AmJ.Physiol. 264:C761-C782, 1993; Halestrap and Price. BiochemJ. 343:281-299, 1999).
  • the MCT1 protein has been enriched from rat red blood cells and is a 55kDa protein (Poole et al, Biochem J. 320:817-824, 1996).
  • MCT4 is adapted to the release of lactate from glyco lytically-active cells whereas the high affinity transporter MCT1 transports lactate required for energy production into cells (Manning Fox et al, J.PhysioL 529:285-293, 2000).
  • Zhao et al. (Diabetes 50:361-366, 2001) propose that, in some forms of Type II diabetes, MCT overexpression in the pancreatic islet cells could contribute to aberrant secretion of insulin, and, therefore postulate that inhibitors of islet cell lactate transport, or of MCT1 gene expression, could provide a therapeutic target for this disease.
  • Froberg et al. (Neuroreport 12(4):761-765, 2001) reported increased MCT1 expression in high grade glial neoplasms leading to the unproven speculation of MCT 1 as a therapeutic target for treatment of some glial neoplasms. Whilst both publications speculate on potential use of inhibitors of lactate transport or
  • the compound identified as having therapeutic potential is further tested in a cellular proliferation assay, for example one that tests whether the compound inhibits proliferation of activated T-cells or inhibits proliferation of cancer cells, either in vivo or in vitro.
  • the compound identified as having therapeutic potential is further tested in an in vivo or in vitro model of inflammation, autoimmune disease or transplantation.
  • the term 'inhibiting monocarboxylate transport activity' also covers the amount of cellular monocarboxalate transporter protein. Thus, it includes inhibition of activity and reduction in the amount of tranporter protein.
  • the method is useful in identifying agents(s) that may have potential in treating an immune-mediated disorder or cancer.
  • the disease is an immune-mediated disorder, such as transplant rejection, or a non-glial epithelial cancer.
  • a method for determining whether a compound not known to be capable of specifically binding to a monocarboxylate transporter can specifically bind to a monocarboxylate transporter which comprises contacting a monocarboxylate transporter protein with the compound under conditions suitable for binding, and detecting specific binding of the compound to the transporter. Such a method is particularly applicable for identifying potentially useful therapeutic compounds.
  • the transporter is present within a cell, cell ghost, a cell membrane fraction or a liposome.
  • the transporter is presented within a natural or synthetic membrane.
  • the transporter could be presented within lipid vesicles as described by Lynch and McGiven (Biochem. J. 244:503-508, 1987).
  • an assay for identifying compounds which inhibit monocarboxylate transport in a cell comprising: (a) contacting a cell or cell lysate comprising a monocarboxylate transport polypeptide with a test compound; and (b) detecting one or more of the following characteristics: (i) the ability of the test compound to inhibit the ability of the monocarboxylate transport polypeptide to transport monocarboxylate, (ii) the ability of the test compound to bind to the monocarboxylate transport polypeptide, and (hi) the ability of the test compound to block expression of the monocarboxylate transport polypeptide.
  • a method for identifying whether or not a compound may have potential in treating an immune-mediated disorder or cancer which comprises contacting cells expressing a monocarboxylate transporter, or cell membrane preparations thereof, with a compound not known to be capable of inhibiting monocarboxylate transport, under conditions suitable for binding, and determining monocarboxylate transport activity, wherein the ability of the compound to inhibit monocarboxylate transport identifies that compound as having potential in treating an immune-mediated disorder or cancer.
  • a method for determining whether a compound not known to be capable of blocking monocarboxylate transport can block monocarboxylate transport, which comprises contacting cells expressing a monocarboxylate transporter, or cell membrane preparations thereof, with the compound under conditions suitable for binding, and determining monocarboxylate transport activity.
  • a compound for assessment as a potential therapeutic agent Potential test therapeutic agents would possess IC50 values of at least lO ⁇ M, preferably at least 1 ⁇ M, for inhibition of monocarboxylate transport (IC50 being the concentration of compound resulting in 50% inhibition of the response).
  • the monocarboxylate transporter is expressed from nucleic acid exogenously introduced into a cell. In another embodiment monocarboxylate transport is blocked as a result of the compound specifically binding to the monocarboxylate transporter. In another embodiment monocarboxylate transport is blocked as a result of the compound impeding expression of the monocarboxylate transporter. In a further embodiment the method is capable of determining whether or not the compound is capable of specifically blocking monocarboxylate transport.
  • a compound is identified as an MCT inhibitor if it exhibits an inhibition constant, Ki, of less than or equal to 10/xM.
  • the inhibition constant, Ki is the concentration of competing ligand in a competition binding assay which would occupy 50% of the binding sites if no radio ligand was present.
  • the Ki is calculated from the IC50 using the Cheng-Prusoff equation. IC50 values are determined as the concentration of inhibitor which would displace 50% of radio ligand A or C (described in Example 1 herein) as measured in filter binding assays and / or the scintillation proximity assay(s) described herein. According to a further aspect of the invention there is provided a method for identifying a compound that may have potential in treating an immune-mediated disorder or cancer, comprising deterrnining whether the compound is capable of inhibiting monocarboxylate transport activity of a cell.
  • a cell or cell membrane preparation comprising a monocarboxylate transporter protein, preferably one selected from the group consisting of MCT 1 through MCT4, in the in vitro screening of compounds for their ability to treat an immune-mediated disorder or cancer.
  • a human monocarboxylate transporter preferably one selected from the group consisting of MCT 1 to MCT4 for use in the in vitro screening of compounds for their ability to treat an immune-mediated disorder or cancer.
  • Monocarboxylate transport activity as used herein, refers to the ability of the transporter protein to facilitate transport of monocarboxylate molecules, such as lactate and pyruvate across a cell membrane.
  • Such activity can be determined using various techniques known to the person skilled in the art.
  • the techniques described herein for determining whether or not a compound can bind to or inhibit an MCT can be employed/adapted for determining whether or not a protein or polypeptide has monocarboxylate transport activity.
  • Potential therapeutic agents which may be tested in the screening methods described herein, include simple organic molecules, commonly known as "small molecules", for example those having a molecular weight of less than 2000 Daltons. Other potential therapeutics include peptides and antibodies.
  • the methods of the invention for example may be used to screen chemical compound libraries or peptide Hbraries, including synthetic peptide hbraries and peptide phage hbraries, particularly antibody display (such as scFV or Fab) phage hbraries.
  • suitable compound molecules include antibodies, nucleotide sequences, and any other molecules, including nucleic acid or protein derived molecules, which bind to an MCT.
  • the compound is a small molecule chemical compound.
  • the terms compound and agent are used interchangeably herein.
  • the screening methods of the invention will prove useful in determining whether or not test compounds (chemical or biological) may be suitable for use, inter aha, in the treatment, including prophylactic treatment, of cancers, autoimmune, inflammatory, proliferative and hyperproliferative diseases and other immune-mediated diseases including, rejection of transplanted organs or tissues. Examples of immune-mediated disorders and cancers are:
  • asthma reversible obstructive airways diseases including asthma, such as bronchial, allergic, intrinsic, extrinsic and dust asthma, particularly chronic or inveterate asthma (e.g. late asthma and airways hyper-responsiveness); bronchitis; acute, allergic, atopic rhinitis and chronic rhinitis including rhinitis caseosa, hypertrophic rhinitis, rhinitis purulenta, rhinitis sicca and rhinitis medicamentosa; membranous rhinitis including croupous, fibrinous and pseudomembranous rhinitis and scrofoulous rhinitis; seasonal rhinitis including rhinitis nervosa (hay fever) and vasomotor rhinitis; sarcoidosis, farmer's lung and related diseases, fibroid lung and idiopathic interstitial pneumonia; (2) (bone and joints) rheumatoid artl
  • Xenografts rejection Hyperacute and acute and chronic following for example transplantation of kidney, heart, liver, lung, bone marrow, skin, pancreatic islet cells, cornea and stem cells; and chronic graft versus host disease.
  • lymphoid lineage including acute lymphocytic leukemia, B cell lymphoma and Burketts lymphoma.
  • myeloid lineage including acute and chronic myelogenous leukemias and promyelocytic leukemia.
  • Tumors of mesenchymal origin including f ⁇ brosarcoma and rhabdomyo sarcoma, and other tumors, including melanoma, seminoma, tetratocarcinoma, neuroblastoma and glioma.
  • the compounds are thus indicated for use in the treatment, or prevention, of rejection of transplanted organs, tissues, or cells such as kidney, heart, lung, bone marrow, skin, pancreatic islet cells, cornea and stem cells; and of autoimmune, inflammatory, proliferative and hyperproliferative diseases, including cancer, and of cutaneous manifestations of immune-mediated disorders: for example rheumatoid arthritis, systemic lupus erythematosus, Hashimoto's thyroiditis, multiple sclerosis, myasthenia gravis, type 1 diabetes, uveitis, nephrotic syndrome, psoriasis, atopic dermatitis, contact dermatitis and further eczematous dermatitides, seborrhoeic dermatitis, Lichen planus, Pemphigus, Epidermo lysis bullosa, urticaria, angioedemas, vasculitides, erythemas, cutaneous eosinophili
  • the inventors have found that compounds, which are capable of binding to MCTl and/or MCT2, and which inhibit lactate transport of activated T cells, inhibit the proliferation of activated T-cells and tumour cell lines such as the ery thro leukaemia cell line K562.
  • the first aspect of the invention is therefore a screening method to identify compounds that may be useful inter aha, in treating conditions or diseases involving T-cell activation, such as transplant rejection and rheumatoid arthritis, or cellular proliferation, such as cancer.
  • the compound is tested for its ability to inhibit MCT activity. This, for example, may be via inhibition of the ability to transport monocarboxylates or via blockage of MCT expression. It is well known that MCT proteins from different species have a high degree of sequence similarity.
  • rat MCTl is reported to possess 86% identity with human MCTl (Jackson et al., Biochem Biophys Acta 1238: 193-196, 1995).
  • the MCT is of human origin, particularly from the group consisting of human MCTl, through to 4, it is envisaged that MCTs from other species, such as rat or mouse would also work in the invention.
  • Suitable monocarboxylate transporters for use in the screening assay/method of the invention are MCTl, MCT2 and MCT4. MCTl is the most preferred.
  • the sequence of MCTl is disclosed in the EMBL/GenBank DDBJ databases (Blum H., Bauersachs S., Mewes H.W., Weil B., Wiemann S, Submitted (15-MAR-2000) to the EMBL/GenBank/DDBJ databases) with the EMBL Accession No. AL162079.
  • the sequence of the cDNA clone encoding human MCTl used herein, is disclosed in SEQ ID NO: 41, and is identical to the sequence disclosed by Blum et al. (supra).
  • the MCTl polypeptide sequence is disclosed as SEQ ID NO: 1. With regard to MCT2, there appears to be no single definitive published sequence.
  • MCT2 sequences deposited in EMBL differ in three locations that lead to amino acid changes.
  • the sequence of the cDNA clone encoding human MCT2 used herein, and disclosed in SEQ ID NO: 42, is a combination of the two.
  • the MCT2 polypeptide sequence is disclosed as SEQ ID NO: 2.
  • AF049608 encodes the amino acid Asparagine at position 154, whilst AF058056 and SEQ ID NO: 42 both encode a Serine at this position;
  • AF049608 encodes the amino acid Proline at position 268, whilst AF058056 and SEQ ID NO: 42 both encode a Leucine at this position;
  • AF058056 encodes the amino acid Serine at position 445, whilst AF049608 and SEQ ID NO: 42 both encode a Threonine at this position.
  • SEQ ID NO: 42 there are no differences between SEQ ID NO: 42 and the MCT2 genomic exon and predicted transcript sequences (UCSC SOFTBERRY Database Accession No.
  • MCT3 compared to the predicted MCT3 amino acid sequence disclosed in Yoon et al., (Genomics. 60(3):366-370, 1990), the inventors have found an amino acid substitution of Tryptophan to Arginine at position 235.
  • MCT3 genomic sequence accesion No. AL031587
  • the MCT3 polypeptide sequence is disclosed as SEQ ID NO: 3.
  • the MCT4 cDNA sequence is disclosed herein as SEQ ED NO: 44 and the MCT4 polypeptide sequence is disclosed herein as SEQ ID NO: 4. Accordingly, the screening assay is not restricted to use of the full-length MCTl protein as depicted in Table 1, but extends to functional variants, including mutants, deletions and chimaeric variants that maintain activity in the test assay.
  • substantially homologous we mean a sequence which possesses at least 70%, and in increasing order of preference at least 75%, 80%, 85%, 90%, 95%, 97% and 99% sequence identity thereto.
  • substantially the same biological activity we mean having the ability to effect monocarboxylate transport.
  • the variant proteins will have at least 10%, and in increasing order of preference at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% and 95% of the monocarboxylate transportation activity of the wild-type protein, whose sequence is depicted in the respective sequence identifier, when recombinantly expressed in a suitable heterologous expression system
  • sequence identity between two sequences can be determined by pair-wise computer ahgnment analysis, using programs such as, BestFit, Gap or FrameAlign.
  • the preferred ahgnment tool is BestFit.
  • isolated refers to molecules, either nucleic acid or amino acid sequences, that are removed from their natural environment and purified or separated from at least one other component with which they are naturally associated. Also encompassed by this term are molecules that are artificially synthesised and purified away from their synthesis materials. Thus, a polynucleotide is said to be isolated when it is substantially separated from other contaminant polynucleotides or nucleotides.
  • the natural MCTl polypeptide depicted in SEQ ID NO. 1 and a variant polypeptide may only possess for example 80% sequence identity, they are actually likely to possess a higher degree of similarity, depending on the number of dissimilar codons that are conservative changes.
  • Similarity between two sequences includes direct matches as well as conserved amino acid substitutes, which possess similar structural or chemical properties, e.g. similar charge.
  • Examples of conservative changes (conserved amino acid substitutes) are shown in Table 2. Suitable conservative substitutions of amino acids are known to those of skill in this art and may be made without altering the biological activity of the resulting polypeptide, regardless of the chosen method of synthesis.
  • the phrase "conservative substitution” includes the use of a chemically derivatized residue in place of a non-derivatized residue provided that such polypeptide displays the desired binding activity. D-isomers as well as other known derivatives may also be substituted for the naturally occurring amino acids. See, e.g., U.S. Patent No. 5,652,369, Amino Acid Derivatives, issued July 29, 1997. Substitutions are preferably, although not exclusively, made in accordance with those set forth in TABLE 2 as follows:
  • the MCT coding nucleotide sequences for use in the present invention may also be engineered in order to alter a coding sequence for a variety of reasons, including but not limited to, alterations which modify the cloning, processing and/or expression of the gene product. For example, mutations may be introduced using techniques which are well known in the art, eg, site-directed mutagenesis to insert new restriction sites, to alter glycosylation patterns, to change codon preference, etc. Because the MCT proteins are predicted to have a transmembrane domain structure, they require a membrane scaffold to retain their structural and / or functional integrity.
  • the preferred assay methods involve use of whole cells, or cell membrane preparations thereof, that contain one or more MCTs
  • the MCT proteins or polypeptides can be presented in alternate formats to retain their structural integrity. For example, reconstituted within lipid vesicles (see for example, Lynch and McGiven. Biochem. J. 244:503-508, 1987).
  • Such alternate means of presenting the monocarboxylate transporter protein are also part of the invention.
  • Whole cells expressing MCT or membrane preparations thereof are particularly useful.
  • Suitable whole cells may either be natural cells or cell lines that comprise endogenous MCTs, such as Jurkat, K562, HeLa, Chinese Hamster Ovary (CHO) cells, or transformed/transfected cells, such as INS1, SF9 cells, wherein the MCT protein has been introduced via recombinant techniques well known to the person skilled in the art.
  • endogenous MCTs such as Jurkat, K562, HeLa, Chinese Hamster Ovary (CHO) cells
  • transformed/transfected cells such as INS1, SF9 cells
  • cells or cellular membrane preparations containing an MCT protein are derived from cells, preferably eukaryotic, particularly mammalian, transformed, transfected or transduced with a recombinant expression construct comprising the nucleotide sequence coding for an MCT protein and sequences sufficient to direct the synthesis of the MCT protein in cultures of said transformed, transfected or transduced cells, are used to determine the binding properties of test compounds in vitro.
  • the MCT protein is expressed in eukaryotic cells, especially mammalian, insect and yeast cells. Eukaryotic cells provide post-translational modifications to recombinantly expressed proteins, which include folding and/or phosphorylation and/or glyco sylation.
  • Nucleic acids coding for an MCT for use in the invention can either be isolated or synthesised, and a variety of expression vector/host systems may be used to express MCT coding sequences. These include, but are not limited to microorganisms such as bacteria expressed with plasmids, cosmids or bacteriophage; yeasts transformed with expression vectors; insect cell systems transformed with either the baculovirus expression system or insect expression plasmids; plant cell systems transfected with plant virus expression systems, such as cauliflower mosaic virus; or mammalian cell systems (for example those transfected or transduced with plasmid or viral derived expression vectors e.g. retroviral or adenoviral vectors); selection of the most appropriate system is a matter of choice.
  • Expression vectors usually include an origin of replication, a promoter, a translation initiation site, optionally a signal peptide, a polyadenylation site, and a transcription termination site. These vectors also usually contain one or more antibiotic resistance marker gene(s) for selection. As noted above, suitable expression vectors may be plasmids, cosmids or viruses such as phage or retroviruses. The coding sequence of the polypeptide is placed under the control of an appropriate promoter, control elements and transcription terminator so that the nucleic acid sequence encoding the polypeptide is transcribed into RNA in the host cell transformed or transfected by the expression vector construct.
  • the coding sequence may or may not contain a signal peptide or leader sequence for secretion of the polypeptide out of the host cell.
  • Preferred vectors will usually comprise at least one multiple cloning site to facilitate cloning of the gene.
  • Examples of host cells which may be transformed or transfected with nucleic acid encoding an MCT protein so as to express said MCT protein are: prokaryotic cells, i.e. bacterial cells such as Escherichia coli and Bacillus subt ⁇ lis; lower eukaryotic cells, i.e yeasts such as Saccharomyces cerevisiae, Schizosaccharmoyces pombe, Pichia pastoris, Candida albicans, Aspergillus nidulans or Neurospora crassa; higher eukaryotic cells, i.e.
  • prokaryotic cells i.e. bacterial cells such as Escherichia coli and Bacillus subt ⁇ lis
  • lower eukaryotic cells i.e yeasts such as Saccharomyces cerevisiae, Schizosaccharmoyces pombe, Pichia pastoris, Candida albicans, Aspergillus nidulans or Neurospora cra
  • mammalian cells such as CHO, NIH-3T3, HEK-293, Jurkat; INS-1, insect cells such as Spodoptera frugiperda 9 and 21 cell lines; and, amphibian cells such as Xenopus laevis oocytes.
  • Performance of the invention is neither dependent on nor limited to any particular strain or type of host cell or vector; those suitable for use in the invention will be apparent to, and a matter of choice for, the person skilled in the art.
  • Host cells transformed or transfected with a vector containing an MCT nucleotide sequence may be cultured under conditions suitable for growth with expression and recovery of membrane fractions containing the encoded proteins from the cell culture. Such expressed proteins will preferably, but not necessarily, be presented on the cell surface.
  • Native cells lines used for detecting binding to MCTs or functional activity in MCTs e.g. K562 (human erythroleukaemia cell line), MB231 (breast carcinoma cell line).
  • Membrane preparations for use in the invention can be made using standard techniques well known to the person skilled in the art, including the method disclosed in Example 10. It will be appreciated that there are many screening methods which may be employed to determine the ability of a test compound to block or inhibit monocarboxylate transport. Indeed, monocarboxylate transport activity can be measured directly or indirectly in a number of ways which will be apparent to the person skilled in the art. This invention incorporates each of these different ways.
  • direct binding to an MCT can be determined by standard ligand binding assays.
  • assays can be performed using whole cells or cell membrane preparations containing MCT proteins.
  • Suitable alternative assays might measure monocarboxylate accumulation within the cell, monocarboxylate efflux from the cell, H+ efflux or accumulation, alterations in the glycolytic rate due to monocarboxylate feedback regulation, decreased DNA synthesis and/or cell division, and the like.
  • suitable screening methods which may be used to identify an inhibitor of monocarboxylate (such as lactate) transport include, rapid filtration of equilibrium binding mixtures, radioimmunoassays (RIA) and fluorescence resonance energy transfer assays (FRET).
  • SPA scintillation proximity assay
  • bound labelled ligands will be in close proximity to the fluoromicro spheres, allowing the emitted energy to activate the fluor and produce light. In contrast, the vast majority of unbound labelled ligands will be too far from the fluoromicrospheres to enable the transfer of energy. Bound ligands produce hght but free ligands do not, allowing the extent of hgand binding to be measured without the need to separate bound and free hgand.
  • suitable screening methods is merely intended to be an overview, and is not intended to reflect the full state of the art.
  • Lactate efflux/accumulation by: 1) Enzymatic measurement of lactate levels using lactate as a substrate for lactate oxidase or lactate dehydrogenase using commercially available kits such as the Sigma LO kit (735- 10) or Sigma LD kit (826); or a glucose/lactate analyser (YSI 2700 analyser). 2) Transport of [ 14 C]lactate (or radiolabelled substrates of the monocarboxylate transporter, pyruvate, ⁇ -hydroxybutyrate, glycolate) such as that described by Poole and Halestrap in Am. J. Physiol. 264:C761-C782 (1993). 3) Lactate-induced decrease in intracellular pH using pH sensitive dyes e.g.
  • the screening assay method is a competitive binding assay.
  • a competitive binding assay for compounds that may have potential in treating an immune-mediated disorder or cancer which comprises contacting host cells expressing MCT protein, or a membrane preparation thereof, with both a first test compound and a labelled second compound known to specifically bind to said MCT protein, under conditions suitable for binding of both compounds, and detecting specific binding of the first compound to the MCT protein by measuring a decrease in the binding of the second compound, to the MCT protein in the presence of the first compound indicating that the first compound binds to the MCT protein and may thus have potential in treating an immune-mediated disorder or cancer.
  • the assay can be conducted with only the second compound.
  • the first compound is a small molecule compound.
  • the second compound is a small molecule compound or an antibody.
  • the MCT is a human MCT.
  • the MCT is selected from the group consisting of: MCTl, 2, 3, and 4.
  • the MCT is human MCTl.
  • detectable labels such as radioisotopes, fluorescent labels, chemiluminescent compounds, labelled binding proteins, magnetic labels, spectroscopic markers and linked enzymes that might be used to label up the second compound. Fluorescent labels are often preferred because they are less hazardous than radio labels, they provide a strong signal with low background and various different fluorophors capable of absorbing light at different wavelengths and/or giving off different colour signals exist to enable comparative analysis in the same analysis.
  • fluorescein gives off a green colour
  • rhodamine gives off a red colour
  • both together give off a yellow colour
  • preferred labels are radioisotopes, particularly 14 C, 3 H and 125 I, or non-radioactive labels such as digoxigenin or biotin.
  • the choice of label and the means of detecting such label can be made by the person skilled in the art.
  • a radio hgand binding assay is performed which comprises contacting the test compound with a cellular membrane preparation containing an MCT, preferably MCTl, and a radio-labelled ligand that is known to bind to said MCT, and measuring displacement of said hgand by the test compound.
  • the test compounds will be specific for a particular MCT subtype.
  • Compounds are deemed specific if they bind to one particular MCT subtype and exhibit 10- fold lower potency (Ki), preferably 25-fold lower potency, more preferably 100- fold lower potency to all other subtypes.
  • Potential drug candidates are identified by choosing chemical compounds which bind with high affinity (IC50 of less than 10/zM) to the expressed MCT, by using for example, ligand binding methods well known to those skilled in the art, examples of which are shown in the binding assays described herein.
  • Drug candidates may have broad specificity acting on more than one MCT subtype, alternatively the drug candidates will be specific for a particular MCT subtype.
  • Compounds are deemed specific if they inhibit monocarboxylate transport at least ten fold, preferably at least 25 fold and more preferably at least 100 fold more strongly of one particular MCT subtype than to any other MCT subtype. Alternatively, compounds are deemed specific if they bind at least ten fold, preferably at least 25 fold and more preferably at least 100 fold more strongly to one particular MCT subtype than to any other MCT subtype.
  • Ligands A and C are examples of suitable radio ligands that can be used in the invention. Such radio ligands can be made by standard techniques. These radio ligands are a further aspect of the invention. Thus, according to a further aspect of the invention there is provided a radio labeled compound capable of binding to an MCT.
  • any of the compounds disclosed in any of: WO 98/46606, WO 98/54190, WO 98/28301, WO 99/29695, WO 00/12514, WO 01/83489; PCT/GB02/03399, PCT/GB02/03250 and GB-A-2363377, could be used.
  • the radiolabelled compound is selected from the group consisting of: hgand A, B and C (as described in Example 1 herein).
  • a radiolabelled compound capable of binding to an MCT in a screening assay to identify compounds capable of binding said MCT.
  • the MCT is MCTl
  • the radiolabelled compound is hgand A, B or C, as described herein.
  • a compound, or a pharmaceutically acceptable salt thereof, identified by any of the screening methods of the invention In a preferred embodiment the compound will be capable of specifically inhibiting monocarboxylate transport.
  • a method of producing a pharmaceutical composition which comprises determining whether or not a compound is an MCT inhibitor using any of the screening methods of the invention and furthermore mixing the compound identified therein, or a derivative thereof with a pharmaceutically acceptable carrier.
  • a derivative is a compound which has been designed, synthesised and tested for MCT inhibitor activity based on the parent compound initially identified in the screen.
  • Such a derivative compound is generally identified using conventional structure activity relationship (SAR) studies.
  • SAR structure activity relationship
  • derivative compounds will generally possess shared structural features with the parent compound, but with one or more structural moieties altered.
  • a derivative compound is likely to be one whose structure has been optimised to make the compound more suitable for therapeutic treatments, such as by removal of groups known to be associated with toxic effects; being more bio available; having a longer half-life in vivo etc.
  • a method of producing a pharmaceutical composition which comprises determining whether or not a compound is an MCT inhibitor using any of the screening methods of the invention; preparing derivative compounds of this 'parent' compound; testing these derivative compounds in one of the screening methods of the invention to identify a more active compound; and, mixing said more active compound identified therein with a pharmaceutically acceptable carrier.
  • the components of the screening methods of the invention can be combined in a suitable kit of parts format.
  • kits for use in a method for screening compounds for their potential in treating an immune-mediated disorder or cancer comprising: (i) a cell capable of expressing a monocarboxylate transporter protein or a cell membrane preparation containing a monocarboxylate transport protein; and, (ii) a labelled compound specific for the monocarboxylate transporter protein in step (i);
  • the kit optionally includes instructions for use.
  • the MCT proteins or convenient fragments thereof may be used to raise antibodies. Such antibodies have a number of uses, which will be evident to the molecular biologist or immunologist of ordinary skill.
  • Such uses include, but are not limited to, use as a biotherapeutic, use as the competitive binding hgand in the screening methods of the invention and monitoring protein expression.
  • Enzyme linked immunosorbant assays are weU known in the art and would be particularly suitable for detecting the MCT polypeptide or fragments thereof.
  • the term includes both monoclonal antibodies, which are a substantially homogeneous population, and polyclonal antibodies, which are heterogeneous populations.
  • the term also includes inter alia, humanised and chimeric antibodies, as well as the various types of antibody constructs such as for example F(ab') 2 , Fab and single chain Fv including bacteriophage derived antibodies . In one embodiment, such antibodies are labelled.
  • Suitable adjuvants include, but are not limited to Freund's (complete and incomplete), aluminium hydroxide, BCG and SAC (Bacille Calmette-Guerin and Staphylococcus aureus Cowan). Following booster immunizations, small samples of serum are collected and tested for reactivity to antigen. Examples of various assays useful for such determination include those described in: Antibodies: A Laboratory Manual, Harlow and Lane (eds.), Cold Spring Harbor Laboratory Press, 1988; as well as procedures such as countercurrent immuno-electrophoresis (CDEP), radioimmunoassay, radio immunoprecipitation, enzyme-linked immuno-sorbent assays (ELISA), dot blot assays, and sandwich assays, see U.S.
  • Monoclonal antibodies may be readily prepared using well-known procedures, see for example, the procedures described in U.S. Patent Nos. RE 32,011; 4,902,614; 4,543,439 and 4,411,993; Monoclonal Antibodies, Hybridomas: A New Dimension in Biological Analyses, Plenum Press, Kennett, McKearn, and Bechtol (eds.), (1980).
  • hybridoma cells may be prepared by fusing spleen cells from an immunised animal, e.g. a mouse, with a tumour cell.
  • Appropriately secreting hybridoma cells may thereafter be selected (Koehler & Milstein. Nature. 256:495- 497, 1975; Cole et al. "Monoclonal antibodies and Cancer Therapy", Alan R Liss Inc, New York N. Y. pp 77-96, 1985).
  • Such antibodies may be of any immunoglobulin class including IgG, IgM, IgE, IgA, IgD and any subclass thereof.
  • Rodent antibodies may be humanised using recombinant DNA technology according to techniques known in the art.
  • the monoclonal antibodies of the invention can be produced using alternative techniques, such as those described by Alting-Mees et al., "Monoclonal Antibody Expression Libraries: A Rapid Alternative to Hybridomas", Strategies in Molecular Biology (1990) 3:1-9, which is incorporated herein by reference.
  • binding partners can be constructed using recombinant DNA techniques to incorporate the variable regions of a gene that encodes a specific binding antibody. Such a technique is described in Larrick et al., Biotechnology, (1989) 7: 394.
  • chimeric antibodies, single chain antibodies see for example, US Patent Ser. No. 4,946,778), Fab fragments may also be developed against the polypeptides of the invention (Huse et al. Science.
  • Antibodies are defined to be specifically binding if they bind the particular MCT with a Ka of greater than or equal to about 10 7 M "1 . Affinity of binding can be determined using conventional techniques, for example those described by Scatchard et al., Ann. NY. Acad Sci., (1949) 51:660. Once isolated and purified, the antibodies may be used to detect the presence of antigen in a sample using established assay protocols, see for example "A Practical Guide to ELISA" by D. M. Kemeny, Pergamon Press, Oxford, England.
  • the compounds identified in these prior art patents fall within the scope of one or other of Formulae I to DC:
  • R 1 is C ⁇ -6 alkyl, C 3-6 alkenyl or C 3-6 cyclo alkyl
  • R 2 is or C 3-6 alkenyl
  • R 3 is 1- or 2-indanyl, 1- or 2-(l,2,3,4-tetrahydronaphthalenyl), 9-fluorenyl, acenaphthyl or CHR 4 (CH 2 ) n Ar where n is 0 or 1, R 4 is hydrogen or C ⁇ - 6 alkyl and Ar is quinolinyl, naphthalenyl, benzodioxolinyl optionally susbstituted by one or more halogen atoms, or phenyl optionally substituted by one or more substituent groups selected from halogen,
  • W is H, CH 2 OH, CO 2 H, C0 2 Ci -6 alkyl, CH 2 NR 5 R 6 , CONR 5 R 6 , where R 5 and R 6 are independently hydrogen or or together with the nitrogen atom to which they are attached form a 3- to 8-membered heterocyclic ring optionally further containing an oxygen atom or a group NR 7 where R 7 is hydrogen or Ci-ealkyl, or W is pyridyl or phenyl, each of which may be optionally substituted by one or more substituent groups selected from halogen, hydroxyl, C h alk ! and C ⁇ -6 alkoxy; X is a bond or C ⁇ alkylene;
  • X is not a bond when W is H, CH 2 OH, CO 2 H, CO 2 C ⁇ -6 alkyl, CH 2 NR 5 R 6 or CONR 5 R 6 and Y is sulfur.
  • Ar 1 is naphthyl, quinolyl, isoquinolyl, indolyl, benzofuranyl or benzothienyl, each of which can be optionally substituted by one or more substituents selected from C1- 4 alkyl, C 1 - alkoxy, halogen or trifluoromethyl, or Ar 1 is phenyl optionally substituted by one or more substituents selected from Cw alkyl, alkoxy, halogen, trifluoromethyl, amino, nitro, cyano, trifluoromethoxy, phenoxy, -CH 2 N(R 0 ) 2 , -NHS0 2 CF 3 , -NHC(O)R 6a , CO 2 R 7 or -C(O)NR 8 R 8a ; R 4 represents H or C 1- alkyl; R 5 represents H or OH; each R 6 independently represents H or CM alkyl;
  • R 6a represents H, C 1-6 alkyl, aryl or arC 1- alkyl, wherein the aryl group or aryl moiety in the aralkyl group is phenyl or pyridyl, each of which may be optionally substituted by one or more substituents selected fromC ⁇ alkyl, C M alkoxy, C M alkylcarbonylamino, halogen or trifluoromethyl;
  • R 7 represents H or C ⁇ -4 alkyl;
  • R 8 and R 8a each independently represent H, C alkyl, phenyl or pyridyl;
  • Ar 2 is acenaphthenyl, indanyl, iminodihydrobenzofuranyl or fluorenyl, each of which can be optionally substituted by one or more substituents selected from OH, C1- 4 alkyl,
  • R 1 and R 2 are independently H, C 1-6 alkyl, C 3-6 alkenyl, CH 2 C 3- 5 cycloalkyl or C 3-6 cycloalkyl;
  • R 3 represents H, X-R 9 or X-Ar 3 ;
  • X represents S(O) n , C(O)NR 10 , C(O)O, NH(CO)NR 10 , NH(CO)O or SO 2 NR 10 ;
  • n is 0, 1 or 2;
  • R 9 represents a methyl group optionally substituted by one or more substituents selected from CN, C0 2 H, C 1 - 5 alkoxycarbonyl, 5-tetrazolyl, SO 2 NH 2 or C(0)NR u R 12 , or R 9 represents C 2 - 6 alkyl or C 3 - 6 alkenyl, each of which may be optionally substituted by one or more substituents selected from OH, CN, C0 2 H, C 1 -5 alkoxy, C 1 .5 alkoxycarbonyl, 5-tetrazolyl, azide, phthalimido, SO 2 NH 2 , C(O)NR ⁇ R 12 , NR 13 R 14 , NHC(O)R 15 or NHSO 2 R 16 where R 11
  • R 15 represents C M alkyl, CM alkoxy, di(C M alkyl) amino, or alkoxyalkylene containing up to 6 carbon atoms, and R 16 represents C M alkyl or trifluoromethyl; or, additionally, in the case where X represents C(O)NR 10 , NH(CO)NR 10 or SO2NR 10 , R 9 and R 10 together with the nitrogen atom to which they are attached may form a 4- to 7-membered heterocyclic ring which may be optionally substituted by one or more OH groups; R 10 represents H, Ci_6 alkyl or is linked to R 9 as defined above; and Ar 3 is phenyl, pyridyl or pyridine N-oxide, each of which may be optionally substituted by one or more substituents selected from OH, NO 2 , NH 2 , NHSO 2 CF 3 , C M alkoxy, bis-C M alkanesulphonylamino, or CMalkoxycarbonylamino;
  • Q represents an aryl group Ar 1 wherein Ar 1 represents naphthyl, phenyl, quinolyl, isoquinolyl, indolyl, benzofuranyl or benzothienyl; in the case where
  • W represents a bond
  • Q represents an aryl group Ar 2 wherein Ar 2 represents acenaphthenyl, fluorenyl or indanyl; wherein the ring systems which Ar 1 and Ar 2 represent may all be optionally substituted by one or more substituents selected from C MI alkyl, - alkoxy, halogen, or trifluoromethyl
  • R 10 represents X — (A) p — Y;
  • A represents C S alkylene; p is 0 or 1;
  • Y represents CN, OR 11 , CO 2 R 12 , CONR 13 R 14 , NR 15 R 16 , NHSO 2 R 17 , NHCOR 18 or an optionally substituted aryl or heteroaryl group, provided that when X represents S(0) ft and Y is other than an optionally substituted ary
  • R represents a group -C(O)Ar 1 or -C(R 4 )(R 5 )Ar 1
  • Ar 1 represents a heterocyclic group comprising a total of from 5 to 10 atoms which include from 1 to 3 hetero atoms independently selected from nitrogen, oxygen and sulfur, which group Ar 1 may be optionally substituted by one or more substituents independently selected fro oxo, hydroxyl, CM alkyl, C alkoxy, halogen, trifluoromethyl, amino, nitro, cyano, trifluoromethoxy, phenoxy, -CH 2 N(R 6 ) 2 , -NHS0 2 CF 3 , CMalkylsulfonylammo, -NHC(O)R 6a , C0 2 R 7 or -C(O)NR 8 R 8a , with the proviso that Ar 1 does not represent an optionally substituted benzofuranyl, benzothienyl, indo
  • R 6a represents a hydrogen atom or a C ⁇ -6 alkyl, aryl or arC 1-4 alkyl group, wherein the aryl group or aryl moiety in the aralkyl group is phenyl or pyridinyl, each of which may be optionally substituted by one or more substituents independently selected from C 1-4 alkyl, C M alkoxy, C 1-4 alkylcarbonylamino, halogen or trifluoromethyl;
  • R 7 represents a hydrogen atom or a C M alkyl group;
  • R 8 and R 8a each independently represent a hydrogen atom or a C M alkyl, phenyl or pyridinyl group
  • R 1 and R 2 each independently represent a hydrogen atom or a C 1-6 alkyl, C 3-6 alkenyl, CH2C 3-5 cycloalkyl or C 3-6 cycloalkyl group;
  • R 3 represents a hydrogen atom or a group X-R 9 or X-Ar 2 ;
  • X represents an oxygen atom, S(0) n , C(0)NR 10 , C(O)O, NH(CO)NR 10 , NH(CO)0 or SO 2 NR 10 , with the proviso that when X represents an oxygen atom and R represents a group - C(R 4 )(R 5 )Ar 1 , then R 4 and R 5 both represent a hydrogen atom; n is 0, 1 or 2; R 9 represents a methyl group optionally substituted by one or more substituents independently selected from cyano, carboxyl, C 1-5 alkoxycarbonyl, 5-tetrazolyl or C(O)NR n R 12 , or R 9 represents a C2-6 alkyl or C 3 _ 6 alkenyl group, each of which may be optionally substituted by one or more substituents independently selected from hydroxyl, cyano, carboxyl, C ⁇ -5 alkoxy, C 1 - 5 alkoxycarbonyl, 5-tetrazolyl, azido
  • R 10 represents a hydrogen atom or a C ⁇ - 6 alkyl group or is linked to R 9 as defined above; and Ar 2 is phenyl, pyridinyl, thienyl, pyridone or pyridine N-oxide, each of which may be optionally substituted by one or more substituents independently selected from halogen, hydroxyl, nitro, amino, NHSO 2 CF 3 , C M alkyl, CM alkoxy, bis-C M alkanesulfonylamino, C 1-4 alkylcarbonylamino or C 1-4 alkoxycarbonylamino; or a pharmaceutically-acceptable salt or solvate thereof.
  • WO 01/83489 is phenyl, pyridinyl, thienyl, pyridone or pyridine N-oxide, each of which may be optionally substituted by one or more substituents independently selected from halogen, hydroxyl, nitro, amino, NHSO 2 CF 3 , C
  • R is -C(O)Ar 1 , -C(R 4 )(R 5 )Ar 1 or Ar 3 ;
  • Ar 1 represents a 5- to 10-membered aromatic ring system wherein up to 3 ring atoms may be heteroatoms independently selected from nitrogen, oxygen and sulphur, the ring system being optionally substituted by one or more substituents independently selected from CM alkyl, Ci-
  • R 1 and R 2 each independently represent a hydrogen atom, C ⁇ -6 alkyl, C 3-6 alkenyl, CH 2 C 3- 5 cycloalkyl or C 3-6 cycloalkyl;
  • R 3 represents a group X- Ar 2 ;
  • X represents a group S(0) n , C(O) or CH(OH); n is 0, 1 or 2;
  • Ar 2 represents a 5- or 6-membered aromatic ring wherein up to 4 ring atoms may be heteroatoms independently selected from nitrogen, oxygen and sulphur, the ring being optionally substituted by one or more substituents independently selected from C M alkyl, Ci.
  • R 4 represents a hydrogen atom or CM alkyl
  • R 5 represents a hydrogen atom or hydroxyl group
  • R 6 and R 7 each independently represent a hydrogen atom or C M alkyl, or together with the nitrogen atom to which they are attached form a 5- to 7-membered saturated heterocyclic ring
  • R 8 and R 9 each independently represent a hydrogen atom or C alkyl, or together with the nitrogen atom to which they are attached form a 5- to 7-membered saturated heterocyclic ring
  • Ar 3 represents acenaphthenyl, indanyl or fluorenyl, each of which may be optionally substituted by one or more substituents independently selected from C 1 - 4 alkyl, C 1-4 alkoxy, halogen or trifluoromethyl; with the proviso that when X represents S(O) n , then Ar 2 does not represent pyridyl or thienyl; or a pharmaceutically acceptable salt or solvate thereof.
  • R is -C(O)Ar 1 , -C(R 4 )(R 5 )Ar 1 or Ar 3 ;
  • Ar 1 represents a 5- to 10-membered aromatic ring system wherein up to 3 ring atoms may be heteroatoms independently selected from nitrogen, oxygen and sulphur, the ring system being optionally substituted by one or more substituents independently selected from C alkyl, Ci. 4 alkoxy, halogen, trifluoromethyl, oxo, nitro, cyano, NR 6 R 7 and -CH 2 NR 8 R 9 ;
  • R 1 and R 2 each independently represent a hydrogen atom, C ⁇ -6 alkyl, C 3-6 alkenyl, CH 2 C 3-5 cycloalkyl or C 3-6 cycloalkyl;
  • R 3 represents a group X-R 10 or Ar 2 ;
  • X represents a bond or a group NR 11 ;
  • Ar 2 represents a 5- or 6-membered aromatic ring wherein up to 4 ring atoms may be heteroatoms independently selected from nitrogen, oxygen and sulphur, the ring being optionally substituted by one or more substituents independently selected from C M alkyl, Ci.
  • R 4 represents a hydrogen atom or CM alkyl
  • R 5 represents a hydrogen atom or hydroxyl group
  • R ⁇ and R 7 each independently represent a hydrogen atom or C M alkyl, or together with the nitrogen atom to which they are attached form a 5- to 7-membered saturated heterocyclic ring;
  • R 8 and R 9 each independently represent a hydrogen atom or C M alkyl, or together with the nitrogen atom to which they are attached form a 5- to 7-membered saturated heterocyclic ring;
  • R x0 represents C ⁇ -6 alkyl, C 2 - 6 alkenyl or C 2- 6 alkynyl, each o f which may be optionally subsituted by one or more substituents independently selected from carboxyl, hydroxyl,
  • R 10 represents a C 3-6 cycloalkylcarbonyl, -C(0)CH 2 CN, halophenylcarbonyl or trifluoromethylcarbonyl group;
  • R 11 represents a hydrogen atom or a C ⁇ - 6 alkyl group;
  • R 12 represents piperazinyl optionally substituted by a C ⁇ -6 alkyl group, or
  • R 12 represents a group -NR 17 R 18 ;
  • R 13 and R 14 each independently represent a hydrogen atom, or a C M alkyl, C M hydroxyalkyl or -C(O)-R 19 group, or R 13 and R 14 , together with the nitrogen atom to which they are attached, form a 5- to
  • R 15 and R 16 each independently represent a 5- or 6-membered aromatic ring wherein up to 4 ring atoms may be heteroatoms independently selected from nitrogen, oxygen and sulphur, the ring being optionally substituted by one or more substituents independently selected from halogen atoms, cyano and CM alkyl;
  • R 17 and R 18 each independently represent a hydrogen atom, or a C M alkyl group optionally substituted by one or more substituents independently selected from halogen atoms and hydroxyl;
  • R 19 represents a C ⁇ -6 alkyl or C 3-6 cycloalkyl group, each of which may be optionally substituted by a hydroxyl group;
  • Ar 3 represents acenaphthenyl, indanyl or fluorenyl, each of which may be optionally substituted by one or more substituents independently selected from C 1 - 4 alkyl, CM alkoxy, halogen and trifluoromethyl; or a pharmaceutically acceptable salt or solvate thereof.
  • R 1 and R 2 each independently represent a Ci-ealkyl, C 3- 6alkenyl, C 3 -5cycloalkyl(C ⁇ -3 )methyl or C3-6cycloalkyl; each of which may be optionally substituted by
  • R 3 represents a group -CON(R 10 )YR U or - SO 2 N(R 10 )YR n ;
  • Y is O, S or NR 12 (wherein R 12 is hydrogen or C ⁇ -6 alkyl); and R 10 and R 11 are independently optionally substituted by halo, hydroxy, amino, C ⁇ -6 alkylamino or di-(C ⁇ - 6 alkyl)amino] ;
  • Q is -CO- or -C(R 4 ) (R 5 )- (wherein R 4 represents a hydrogen atom or CMalkyl and
  • R 5 represents a hydrogen atom or hydroxyl group
  • Ar represents a 5- to 10-membered aromatic ring system wherein up to 4 ring atoms may be heteroatoms independently selected from nitrogen, oxygen and sulphur, the ring system being optionally substituted by one or more substituents independently selected from C alkyl, Ci.
  • R 6 and R 7 each independently represent a hydrogen atom, C M alkanoyl or C M al yl, or together with the nitrogen atom to which they are attached form a 5- to 7-membered saturated heterocyclic ring
  • R 8 and R 9 each independently represent a hydrogen atom, C M alkanoyl or C M alkyl, or together with the nitrogen atom to which they are attached form a 5- to 7-membered saturated heterocyclic ring; or a pharmaceutically acceptable salt or prodrug thereof.
  • R 1 and R 2 each independently represent a C ⁇ - 6 alkyl, C 3-6 alkenyl, C 3-5 cycloalkyl(C ⁇ -3 )methyl or C 3-6 cyclo alkyl; each of which may be optionally substituted by
  • R 3 is isoxazolidin-2-ylcarbonyl or tetrahydroisoxazin-2-ylcarbonyl wherein each ring is optionally substituted by one hydroxy group;
  • Q is -CO- or -C(R 4 ) (R 5 )- (wherein R 4 represents a hydrogen atom or and R 5 represents a hydrogen atom or hydroxyl group) ;
  • Ar represents a 5- to 10-membered aromatic ring system wherein up to 4 ring atoms may be heteroatoms independently selected from nitrogen, oxygen and sulphur, the ring system being optionally substituted by one or more substituents independently selected from C M alkyl
  • halogen haloalkyl, dihaloalkyl, trihaloalkyl, C M al oxyC M alkyl, C M alkylthio,
  • R 6 and R 7 each independently represent a hydrogen atom, C M alkanoyl or C M al yl, or together with the nitrogen atom to which they are attached form a 5- to 7-membered saturated heterocyclic ring; R 8 and R 9 each independently represent a hydrogen atom, CMalkanoyl or C M alkyl, or together with the nitrogen atom to which they are attached form a 5- to 7-membered saturated heterocyclic ring; or a pharmaceutical
  • a method of treating an immune-mediated disorder or cancer comprising administration to a human subject in need of treatment of a compound that inhibits cellular monocarboxylate transporter activity, other than a compound disclosed in any of: International Publication Nos.
  • a method of treating an immune-mediated disorder or cancer comprising administration to a human subject in need of treatment of a compound that inhibits cellular monocarboxylate transporter activity, other than a compound within formulae I to IX.
  • a method of treating an immune-mediated disorder or cancer comprising administration, to a human subject in need of treatment, of a compound that inhibits a monocarboxylate transporter other than MCTl and MCT2.
  • a method of treating an immune-mediated disorder or cancer comprising administration, to a human subject in need of treatment, of a compound that inhibits a monocarboxylate transporter selected from the group consisting of: MCT3 and MCT4.
  • a method of treating an immune-mediated disorder or cancer comprising administering an effective amount of a pharmaceutical composition comprising an MCT inhibitor identifiable or identified by a screening assay method of the invention to a subject in need thereof.
  • the MCT inhibitor is a selective inhibitor.
  • a method of treating an immune-mediated disorder or cancer comprising administering an effective amount of a pharmaceutical composition comprising an MCT inhibitor identifiable or identified by (i) contacting a monocarboxylate transporter protein with a test compound under conditions suitable for binding; and (ii) detecting specific binding of the compound to the transporter protein; to a subject in need thereof.
  • a method of treating an immune-mediated disorder or cancer comprising (i) contacting a monocarboxylate transporter protein with a test compound under conditions suitable for binding; (ii) detecting specific binding of the compound to the transporter protein; (hi) preparing a pharmaceutical composition comprising the compound; and, (iv) administering an effective amount of the pharmaceutical composition to a subject in need thereof.
  • a compound that inhibits a monocarboxylate transporter other than a compound of formulae I to IX in the treatment of an immune-mediated disorder or cancer.
  • a compound that inhibits a monocarboxylate transporter other than a quinazolinedione compound or a compound of formulae I to IX in the treatment of an immune-mediated disorder or cancer.
  • an MCT inhibitor compound that inhibits a monocarboxylate transporter, other than MCTl and MCT2 in the treatment of an immune-mediated disorder or cancer.
  • a compound that inhibits a monocarboxylate transporter other than a compound of formulae I to IX in the manufacture of a medicament for the treatment of an immune-mediated disorder or cancer.
  • a compound that inhibits a monocarboxylate transporter other than a quinazolinedione compound or a compound of formulae I to IX in the manufacture of a medicament for the treatment of an immune-mediated disorder or cancer.
  • an MCT inhibitor compound that blocks a monocarboxylate transporter other than MCTl and MCT2 in the manufacture of a medicament for the treatment of an immune-mediated disorder or cancer.
  • an MCT inhibitor compound that blocks a monocarboxylate transporter selected from the group consisting of: MCT3 and MCT4, in the manufacture of a medicament for the treatment of an immune-mediated disorder or cancer.
  • a method of inhibiting T-cell and B-cell proliferation in a human comprising administration to the human in need of such treatment of a compound capable of specifically inhibiting monocarboxylate transport within the T-cell or B-ceU.
  • a method of treating an immune-mediated disorder or cancer comprising (i) in vitro testing a compound for the ability to inhibit lactate transport in a cell, and (ii) administering, to a human patient in need of treatment, an effective amount of a compound which has been identified from step (i) as a compound capable of blocking lactate transport.
  • a method of treating an immune-mediated disorder or cancer comprising (i) testing a compound for its ability to inhibit monocarboxylate transport in a cell, and (ii) administering to a human patient suffering from or likely to suffer from an immune-mediated disorder or cancer, of an effective amount of a compound which has been identified from step (i) as a compound capable of inhibiting monocarboxylate transport.
  • a method of treating a patient suffering from an immune-mediated disorder or cancer comprising administering to a human patient suffering from or likely to suffer from such a disease or condition of an effective amount of a compound which has been shown (or is known) to be capable of blocking cellular monocarboxylate transport.
  • the medical treatment methods of the invention are particularly suitable for treating rheumatoid arthritis and for use before, during and after transplantation surgery, to prevent host rejection of the transplanted tissue.
  • - 37 Compounds capable of effecting monocarboxylate build up in a cell or preventing monocarboxylate efflux from the cell are particularly suitable for use in the disease treatment methods of the invention.
  • compositions of the invention may be in a form suitable for oral use (for example as tablets, lozenges, hard or soft capsules, aqueous or oily suspensions, emulsions, dispersible powders or granules, syrups or elixirs), for topical use (for example as creams, ointments, gels, or aqueous or oily solutions or suspensions), for administration by inhalation (for example as a finely divided powder or a liquid aerosol), for administration by insufflation (for example as a finely divided powder) or for parenteral administration (for example as a sterile aqueous or oily solution for intravenous, subcutaneous, intramuscular or intraperitoneal dosing or as a suppository for rectal dosing).
  • oral use for example as tablets, lozenges, hard or soft capsules, aqueous or oily suspensions, emulsions, dispersible powders or granules, syrups or elixi
  • compositions of the invention may be obtained by conventional procedures using conventional pharmaceutical excipients, well known in the art.
  • compositions intended for oral use may contain, for example, one or more colouring, sweetening, flavouring and/or preservative agents.
  • the pharmaceutical composition of this invention may also contain, or be co-administered (simultaneously or sequentially) with, one or more pharmacological agents of value in treating one or more disease conditions referred to herein.
  • compositions of this invention will normally be administered to a warm-blooded animal at a unit dose within the range 5-5000 mg per square meter body area of the animal, i.e. approximately 0.1-100 mg/kg, and this normally provides a therapeutically-effective dose.
  • a unit dose form such as a tablet or capsule will usually contain, for example 1-250 mg of active ingredient.
  • Preferably a daily dose in the range of 1-50 mg/kg is employed. In general lower doses will be administered when a parenteral route is employed.
  • a dose in the range for example, 0.5 mg to 30 mg per kg body weight will generally be used.
  • a dose in the range for example, 0.5 mg to 25 mg per kg body weight will be used.
  • Oral administration is however preferred, particularly in tablet form. _, - 38 -
  • unit dosage forms will contain about 1 mg to 500 mg of a compound of this invention.
  • the size of the dose for therapeutic or prophylactic purposes will naturally vary according to the nature and severity of the conditions, the age and sex of the animal or patient and the route of administration, according to well known principles of medicine. Accordingly, the optimum dosage may be determined by the practitioner who is treating a particular patient.
  • a therapeutically effective dose or amount refers to that amount of the agent sufficient to prevent development of or to alleviate the existing symptoms associated with the disorder. Determination of the effective amounts is well within the capabilities of those skilled in the art, especially in hght of the detailed disclosure herein. For example, the therapeutically effective amount can be estimated initially from cell culture assays.
  • a dose can be formulated in animal models to achieve a circulating concentration range that includes the IC50 (concentration at which 50% of the maximal effect is demonstrated) as determined in in vitro cellular assays. Such information can be used to more accurately determine the therapeutically effective dose in humans.
  • the amount of active ingredient that is combined with one or more excipients to produce a single dosage form will necessarily vary depending upon the host treated and the particular route of administration.
  • a formulation intended for oral administration to humans will generally contain, for example, from 0.5 mg to 0.5 g of active agent compounded with an appropriate and convenient amount of excipients which may vary from about 5 to about 98 percent by weight of the total composition.
  • a method for producing an anti-proliferative effect in a warm-blooded animal, such as man, in need of such treatment which comprises administering to said animal an effective amount of a compound capable of inhibiting monocarboxylate transport within a ceU, or a pharmaceutically-acceptable composition thereof, as defined hereinbefore.
  • the compound is a selective inhibitor in another embodiment it has broad spectrum activity.
  • a variety of gene therapy approaches may be used in accordance with the invention to modulate expression of an MCT gene in vivo.
  • One therapeutic means of inhibiting or dampening the expression levels of a particular gene is to use antisense therapy.
  • Antisense therapy utilises antisense nucleic acid molecules that are synthetic segments of DNA or RNA ("oligonucleotides”), designed to mirror specific mRNA sequences and block protein production by inhibiting translation of the native gene transcript. Once formed, the mRNA binds to a ribosome, the cell's protein production "factory" which effectively reads the RNA sequence and manufactures the specific protein molecule dictated by the gene. If an antisense molecule is delivered to the cell (for example as native oligonucleotide or via a suitable antisense expression vector), it binds to the messenger RNA because its sequence is designed to be a complement of the target sequence of bases.
  • oligonucleotides synthetic segments of DNA or RNA
  • the mRNA may no longer dictate the manufacture of the encoded protein by the ribosome and/or is rapidly broken down by the cell's enzymes (e.g. RNaseH), thereby freeing the antisense ohgonucleotide to seek and disable another identical messenger strand of mRNA.
  • Oligonucleotides which are complementary to and hybridisable with any portion of novel gene mRNA disclosed herein, are contemplated for therapeutic use.
  • Expression vectors containing random ohgonucleotide sequences derived from previously known polynucleotides are transformed into cells.
  • the cells are then assayed for a phenotype resulting from the desired activity of the ohgonucleotide.
  • the sequence of the ohgonucleotide having the desired activity can be identified. Identification may be accomplished by recovering the vector or by polymerase chain reaction (PCR) amplification and sequencing the region containing the inserted nucleic acid material.
  • Antisense molecules can be synthesised for antisense therapy.
  • antisense molecules may be DNA, stable derivatives of DNA such as phosphorothioates or methylphosphonates, RNA, stable derivatives of RNA such as 2'-O-alkylRNA, or other ohgonucleotide mimetics.
  • the sequence be at least 17 nucleotides in length in order to achieve sufficiently strong annealing to the target mRNA sequence to prevent translation.
  • Antisense ohgonucleotides targetting MCTl in Caco-2 cells have been described by Hadjiagapiou et al., (Am J. Physio 1. Gastrointest. Liver Physiol. 279:G775-G780, 2000).
  • Antisense molecules may be introduced into cells by microinjection, hposome encapsulation or by expression from vectors harboring the antisense sequence.
  • ribozyme molecules may be designed to cleave and destroy the MCT mRNAs in vivo.
  • Ribozymes are RNA molecules that possess highly specific endoribonuclease activity.
  • Hammerhead ribozymes comprise a hybridising region which is complementary in nucleotide sequence to at least part of the target RNA, and a catalytic region which is adapted to recognise and cleave the target RNA.
  • the hybridising region preferably contains at least 9 nucleotides. The design, construction and use of such ribozymes is well known in the art and is more fully described in Haselhoff and Gerlach, (Nature. 334:585-591, 1988).
  • ohgonucleotides designed to hybridise to the 5'- region of the MCT gene so as to form triple helix structures may be used to block or reduce transcription of the MCT gene.
  • RNA interference (RNAi) ohgonucleotides or short (20-25bp) RNAi MCT sequences cloned into plasmid vectors are designed to introduce double stranded RNA into mammalian cells to inhibit and/or result in the degradation of MCT messenger RNA.
  • MCT RNAi molecules may begin adenine/ adenine (A A) and may comprise of 20 or 21 or 22 or 23, or 24 or 25 base pair double stranded RNA molecules with the preferred length being 21 base pairs and be specific to individual MCT sequences with 2 nucleotide 3' overhangs or hairpin forming 45-50mer RNA molecules.
  • a A adenine/ adenine
  • the design, construction and use of such ribozymes is well known in the art and is more fully described in Elbashir et al, (Nature. 411(6836):428-429, 2001).
  • the antisense, ribozyme, triple helix or RNAi nucleotides are designed to specifically inhibit translation and/or transcription of only one MCT, with minimal effects on the other MCT genes.
  • a method for treating a patient suffering from an immune-mediated disorder or cancer comprising administering to said patient an effective amount of an anti-sense molecule, a ribozyme molecule, triple helix forming molecule or RNAi molecule capable of binding to the mRNA of an MCT, as hereinbefore described, including any nucleic acid or protein derived inhibitor of transcription or translation of MCTs.
  • an antisense nucleic acid molecule a ribozyme molecule, a triple helix forming molecule, RNAi molecule or an antibody directed against an MCT
  • the term “therapy” also includes “prophylaxis” unless there are specific indications to the contrary.
  • the terms “therapeutic” and “therapeutically” should be construed accordingly. Prophylaxis is expected to be particularly relevant to the treatment of persons who have suffered a previous episode of, or are otherwise considered to be at increased risk of, the disease or condition in question.
  • Persons at risk of developing a particular disease or condition generally include those having a family history of the disease or condition, or those who have been identified by genetic testing or screening to be particularly susceptible to developing the disease or condition.
  • the invention is further described, but in no way limited, by the following examples: Example 1 - Design and construction of ligands 1 A Ligand A i) 2.6-Dihvdro-6-rr2-iodo ⁇ henyl methvn-2-methyl-4-(2-methylpropyl -lH- ⁇ yrrolor3.4- dlpyridazin- 1-one
  • step (i) (l.lg) and S-[3-[[(l,l-dimethylethyl)dimethylsilyl]propyloxy]- l-(4- methylphenyl)dioxidosulfanyl-propanethiol 4-methyl-benzenesulfonothioate (1.7g) were combined in THF(40ml) under nitrogen at -78°C, and a solution of lithium diisopropylamide (0.6M, 8.3ml) was added dropwise. The reaction was quenched with saturated aqueous sodium bicarbonate solution after 2hr at -78°C and then the mixture was extracted into ethyl acetate. The organic solution was washed with brine, dried and evaporated. The residue was purified by chromatography to afford the sub- title compound (400 mg).
  • step (ii) 34mg) in dry toluene (1 ml) was degassed by purging with nitrogen, and then hexamethyl ditin (100 micro litre) and tetrakistriphenylphosphine palladium (0) (lOmg) were added. The mixture was heated and stirred under nitrogen in a sealed flask at 95°C for 4 hr. The reaction was cooled, diluted with ethyl acetate and filtered tlirough a pad of silica, and then purified by chromatography to afford the sub- title compound (20mg).
  • step (hi) The product of step (hi) (6mg) was treated with a IM solution of TBAF in THF (0.5ml). After 1.5hr the reaction was diluted with saturated aqueous sodium bicarbonate solution, and then extracted into ethyl acetate. The organic solution was washed with brine, dried and evaporated. Chromatography of the residue gave the sub-title compound (3mg).
  • the radiochemical yield was typically between 50 - 60%. vi) 2.6-Dihvdro-7-r(3-hydroxypropyl thiol-6-r(2-iodo ⁇ henyl methyll-2-methyl-4-(2- methylpropyO - lff-pyrrolo ⁇ 3.4-.fl pyridazin- 1 -one
  • step (i) The product of step (i) (4.24g) was suspended in 75% formic acid (80ml) and Raney Nickel (50% dispersion in 8ml water) was added. The mixture was heated at 90°C under nitrogen for 15 min. After cooling the suspension was filtered (kieselguhr) and evaporated. The residue was dissolved in water (100ml) and extracted into ethyl acetate, each ahquot of extraction was 5 washed with sodium bicarbonate solution. The combined organic extracts were dried and evaporated to yield a white foam, which was dissolved in chloroform (20ml) and heated to 50°C. A solution of bromine (0.4ml) in chloroform (5ml) was added and after stirring for 10 min at 50°C was concentrated in vacuo.
  • step (iv) 350 mg
  • S-[3-[[(l,l-dimethylethyl)dimethylsilyl]propyloxy]-l- (4-methylphenyl)dioxidosulfanyl-propanethiol 4-methyl-benzenesulfonothioate 527mg
  • dry THF 10ml
  • LDA 1.95mmol
  • dry THF 5ml
  • the reaction was quenched by addition of sodium bicarbonate solution (30ml), and extracted into ether.
  • step (v) The product of step (v) (2.28 mg, 5.1 ⁇ mol), 10% Pd/carbon (2.35 mg) and ethanol (0.5 ml) were placed in a 1 ml round-bottomed flask which was attached to a tritium manifold. The contents of the flask were frozen in hquid nitrogen and the flask then evacuated before tritium gas (241 GBq, 2.6 ml, 0.113 mmol) was introduced. The flask was allowed to warm to room temperature and the contents left to stir for 22 hours. The flask was removed from the apparatus and the catalyst removed by filtration. The filtrate was diluted with ethanol (5 ml) and the solvent removed under reduced pressure. This was repeated with a further portion of ethanol (5 ml).
  • the equivalent cuts from the two subsequent injections.
  • the volumes of the three fractions were measured in each case and made up to 10 ml by the addition of 50% w/v aqueous sodium thiosulphate (100 ⁇ l) and ethanol.
  • the radioactive concentration, molar specific activity and radiochemical purity of the three fractions was determined and the details included in the table below.
  • Photolabelling reactions were set up by diluting washed rat red-blood cell membranes 10-fold in assay buffer (50mM HEPES (pH 7.5); 0. ImM EDTA; 150mM NaCI) and by incubating in the presence or absence of l ⁇ M unlabelled competing ligand C The 1 5 I-photoligand solution was added to give a final ligand concentration of 1 nM. The photolabelling reaction was performed by irradiating the sample with a hand held 254 nm UV source for 1 minute at room temperature.
  • the labelled ghost membranes were then collected by centrifugation at 100,000g for 10 minutes at 4°C Finally, the samples were each washed in 1 ml of distilled water and the final membrane pellets again coUected at 100,000g for 10 minutes at 4°C The samples were stored at -20°C until use.
  • the photoaffinity-labelled proteins were analysed by one- and two-dimensional gel electrophoresis. The labelled proteins were excised from the gel and subjected to in gel protein digestion prior to analyses by mass spectrometry. Three peptides matching rat monocarboxylate transporter 1 were identified.
  • Example 3 Cloning of MCT genes 3.1 Human MCTl Ohgonucleotide primers containing unique restriction sites, to allow subsequent cloning, and sequences derived from the optimal Kozak consensus sequence (MCT 1-5'; 5'- GGA-TCC-ACC-ATG-CCA-CCA-GCA-GTT-GGA-GG-3'; SEQ ID No: 6; and MCTl-3'; 5'-GTC-GAC-TCA-GAC-TGG-ACT-TTC-CTC-CTC-CTT-G-3'; SEQ ID No: 7) were used in a PCR to amphfy the MCTl ORF (SEQ ID NO: 41) from a cDNA library.
  • the PCR fragment was subcloned into the vector pCR3.1 uni (Invitrogen). Bacterial colonies containing the MCTl ORF in the vector were identified in a PCR colony screen. A number of MCTl positive colonies were grown, the plasmid DNA isolated and subjected to sequence analysis to ensure that no amino acid encoding mutations had been incorporated into the MCTl ORF.
  • the MCTl ORF (BamHI/Sall) was then sub-cloned into the mammalian expression vector pcDNA3 (Invitrogen) and digested with BamHI XhoI to generate the plasmid pcDNA3-hMCTl.
  • the MCTl ORF was then further subcloned into the insect expression vector pIZv5HIS (Invitrogen) and the S. cerevisiae expression plasmid, pACES14.
  • Expression plasmids pcDNA3-hMCTl, pIZ-hMCTl, and pACES14-hMCTl were purified and used to transform relevant host cells.
  • Rat MCTl The open reading frame encoding rat MCTl was amplified from a rat brain cDNA library (Origene) using the ohgonucleotide pair RM1-5' (5'-TGCATGATCA- ATGCCACCTGCGATTGGCGGGCCAG-3'; SEQ ID No. 8) and RM1-3' (5'- TGCAGCTAGCTCAG-ACTGGGCTCTCCTCCT-3'; SEQ ID No. 9) in a PCR.
  • the resulting amplified DNA was digested with Bcll/Nhel and was hgated with pACES14 pre- digested with BamHI/Nhel.
  • Plasmid pACES14-rMCTl was purified and used to transform relevant host ceUs.
  • the amino acid sequence of rat MCTl is depicted in SEQ ID NO: 5.
  • Human MCT2 The open reading frame encoding human MCT2 (SEQ ID NO: 42) was amplified from a full-length cDNA clone using the ohgonucleotide pair MCT2-5' (5'-AGC-TGG-ATC-CAC- CAT-GCC-ACC-AAT-GCC-AAG-3'; SEQ ID No. 10) andMCT2-3' (5-GAC-TCT-CGA- GTT-AAA-TGT-TAG-TTT-CTC-TTT-CTG-A-3'; SEQ ID No. 11) in a PCR. The PCR fragment was subcloned into the vector pCR3.1 uni (Invitrogen).
  • the human MCT3 ORF (SEQ ID NO: 43) spans 4 exons in the human genome (Yoon et al., Genomics 60 (3), 366-370, 1999). Each of the four exons were amplified by PCR from human genomic DNA using the oligos MCT3-5'#3(5'-ATC-AGG-ATC-CAG-GCA-GCG- ATG-GGC-G-3'; SEQ ID No. 12)/MCT3-11# (5'-GAC-ACG-GGG-CCC-GTG-CCG-TAG- AGC-AT-3'; SEQ ID No.
  • MCT3-10# (5'-CGG-CAC-GGG-CCC-CGT-GTC- CAG-CAT-3'; SEQ LD No.14)/MCT3-13# (5'-AGG-CCC-AGG-CCT-GTG-AGC-ACC- CCA-GC-3'; SEQ ID No. 15) for exon II; M3-G1(5'-GTT-CCC-GGA-TCT-GCT-GGG-TT- 3'; SEQ ID No. 16)/M3-G2 (5'-TGG-AGC-TTC-CCT-GGG-TCT-AA-3'; SEQ ID No.
  • MCT3-14# (5'-CCC-TCT-GCC-GGC-CGC-CTG-GTG- GAT-GCG-TTG-AAG-3'; SEQ ID No. 18)/MCT3-3'#3 (5'-GTC-AAC-TAG-TCA-GAC- ACC-CAG-GGG-ATC-AAC-TGG-AG-3'; SEQ ID No. 19) for exon IV to ⁇ 150bp downstream of the termination codon.
  • Exon III was then isolated the M3-G1/M3-G2 PCR product using the ohgos MCT3-12# (5'-TGC-TCA-CAG-GCC-TGG-GCC-TGG-CCC-TCA- 5 A-3' ; SEQ ID No. 20)/MCT3-15# (5'-ACC-AGG-CGG-CCG-GCA-GAG-GGC-GGT-CC-3' ; SEQ ID No. 21).
  • a PCR product (I+II) was generated from the exon I and exon II PCR products using the ohgos MCT3-5'#3/MCT3-13# and subcloned into pCRBluntll-TOPO to give pTOPOMCT3(I+II).
  • a PCR product (IH+IV) was generated from the exon III and exon IV PCR products using the ohgos MCT3-12#/MCT3-3'#3 and subcloned into pCRII-TOPO
  • Human MCT4 A human dendritic cell cDNA clone (AC-DNA-1819) contains an incomplete copy of the MCT4 open reading frame (SEQ ID NO: 44). This was made full length by inserting ⁇ 190bp GBO212 (5'-TAG-GAA-GAA-GCC-CAA-AGA-GCC-ACA-G-3'; SEQ ID No.
  • Human MCT1/MCT2 chimera The human MCTl and MCT2 cDNAs share a common recognition site for the restriction enzyme Hind III. This restriction site hes in the region that encodes the extracellular domain that separates the predicted transmembrane domains 5 and 6.
  • the common Hind III site was used to create a human MCT1/MCT2 chimeric molecule, in the S. cervisiae expression plasmid pACES 14, consisting of the amino terminus of MCT2 (TMs 1- 5) and the carboxy terminus of MCTl (TMs 6-12) SEQ ID 40. Plasmid DNA, ⁇ ACES14- hMCT2/MCTl, was purified and used to transform relevant host cells.
  • Example 4 Expression of MCT proteins in host cells
  • the human breast cell line MDA-MB-231 had been previously identified as expressing low levels of MCTl (Garcia et al., Cell. 76:865-873, 1994).
  • the MDA-MB-231 cell line was grown.
  • Filter binding assays carried out with the MDA- MB-231 cell line showed that the cells exhibited low level of binding to 3 H-ligand C (-6000 binding sites per cell).
  • the cells were grown and transfected withpcDNA3-hMCTl.
  • INS-1 pancreatic ⁇ -cell line
  • lactate transport activity Sekine et al, J. Biol. Chem 269:4895-4902, 1994
  • MCT proteins Ishihara et al, J. Clinical Invest. 104:1621- 1629,1999; Zhao et al, Diabetes. 50:361-366, 2001.
  • INS-1 cells were grown and transfected with pcDNA3-hMCTl .
  • Yeast cells were made competent for DNA transformation and transformed with plasmid DNA using the Yeast Transformation Kit (SIGMA) according to the manufacturer's instructions. Expression of human MCTs was confirmed by Westem analysis using the corresponding anti- human MCT C-terminal peptide antibody.
  • the DNA sequence encoding the Strep Tag, AWRHPQFGG (SEQ ID No. 30) (Schmidt and Skera, 1993, Protein Engineering 6:109-122) was cloned and expressed at the C-teiminus of hMCTl, hMCT2 and hMCT3, producing the polypeptides depicted in SEQ ID Nos: 37 to 39 respectively.
  • MCT1 A three way ligation containing ⁇ ACES14 (BamHI/Nhel), the 5' end of hMCTl (BamHI/BspEl digested pACES14-MCTl) and annealed ohgonucleotides Mlstr ⁇ -1 (5'- CCG-GAC-CAG-AAA-GAC-ACA-GAA-GGA-GGG-CCC-AAG-GAG-GAG-GAA-AGT- CCA-GTC-GCT-TGG-AGA-CAT-CCA-CAA-TTT-GGT-GGT-TAA-T-3'; SEQ ID No.
  • Mlstrp-2 (5'-CTA-GAT-TAA-CCA-CCA-AAT-TGT-GGA-TGT-CTC-CAA-GCG- ACT-GGA-CTT-TCC-TCC-TTG-GGC-CCT-CCT-TCT-GTG-TCT-TTC-TGG-T-3'; SEQ ID No. 32) encoding the C-terminus of MCTl fused to the DNA encoding the strep-tag was carried out to generate the plasmid pACES14-MCTl- strep tag.
  • MCT2 A three way ligation containing pACES14 (BamHI/Nhel), the 5' end of hMCT2 (BamHI Dralll digested ⁇ IZ-MCT2) and annealed ohgonucleotides FM2strep2 (5'-GTG- TAA-CCT-CAG-AAA-GAG-AAA-CTA-ACA-TTG-CTT-GGA-GAC-ATC-CAC-AAT- TTG-GTG-GTT-AAT-3'; SEQ ID No.
  • RM2strep2 (5'-CTA-GAT-TAA-CCA-CCA- AAT-TGT-GGA-TGT-CTC-CAA-GCA-ATG-TTA-GTT-TCT-CTT-TCT-GAG-GTT- ACACTCT-3'; SEQ ID No. 34) encoding the C-terminus of MCT2 fused to the DNA encoding the strep-tag, was carried out to generate the plasmid pACES 14-MCT2-strep tag. Expression of the tagged material was monitored by Western blotting using anti-hMCT2 Abs and a streptavidin-horseradish peroxidase conjugate (IB A GmbH).
  • MCT3 The 3 ' end of hMCT3 (from Topo-MCT3) was amplified in a PCR reaction with the primer pair Mct3E-2 (5'-GCCATCCTGCTGGTGAACTA-3'; SEQ ID No. 35) and M3-3st (5'-TAG-CTA-GTC-TAG ⁇ ATT-AAC-CAC-CAA-ATT-GTG-GAT-GTC-TCC-AAG-CTA- CAG-ACT-CGG-CAG-CCA-GCC-TCG-GCC-TCG-CC-3'; SEQ ID No. 36).
  • the PCR fragment contains the 3' end of hMCT3 fused to the DNA encoding the strep tag.
  • the PCR product was digested with Notl and Xbal and placed in a three-way ligation with pACES14 BamHI/Nhel and the 5' end of hMCT3 (released fromTopo-MCT3 digested with BamHI/Notl), to generate ⁇ ACES14-hMCT3 strep tag.
  • Expression of the tagged hMCT3 was monitored by Western blotting using anti-hMCT3 Abs and a streptavidin-horseradish peroxidase conjugate (IB A GmbH).
  • Example 6 Yeast expressed MCTl filter binding assay Assay to measure the potency of selected compounds using a filter binding assay Competition assays can be used to measure the affinity of unlabelled compound for MCTl. Tritiated hgand C is included at a constant concentration and the compound to be tested is titrated. lO ⁇ l of 3 H-hgand C that had been diluted with assay buffer (50mM HEPES, O.
  • lmM EDTA, 0.15 M NaCI, pH 7.5, 0.5% BSA was dispensed into wells of a polypropylene plate such that when the assay was made to 200 ⁇ l the final concentration would be 2.5nM.
  • lO ⁇ l of compound, in assay buffer was added to each well to give a final concentration typically covering the range 0.1 to lOOOnM.
  • 180 ⁇ l of yeast membranes expressing MCTl in assay buffer containing typically 0.5 to l ⁇ g total protein was added to start the reaction. Non-specific binding was measured in the presence of 1 ⁇ M unlabelled Ligand 1.
  • the competition assay was incubated at 2 hours at room temperature with shaking. Experimental data points were usuaUy carried out in triplicate.
  • the membranes were harvested onto GF-B filter plates and washed with assay buffer without BSA, dried, scintillant added and counts detected using a tritium program on a Packard Top Count plate reader. The results were analysed by subtraction of the non-specific binding from each of the experimental points and then fitting a sigmoidal curve through a semi- log plot of the data in Microcal Origin. Calculated IC 5 o's are shown in Table 5.
  • Ligand 1 (disclosed in WO 98/054190) 5-[(3-hydroxypropyl)thio]-3-methyl-l-(2- methylpropyl)-6-(l-napthalenylmethyl)t eno[2,3-d]pyrimidine-2,4(lH,3H)-dione
  • Ligand 1 appears in patent WO 98/054190 and has CAS number (Chem Abs Registry No) 5 216685-07-3.
  • Ligand 2 (disclosed in WO 99/029695) 2,6-dihydro-7-[(3-hydroxypropyl)thio]-2-methyl-4- (2-methylpropyl)-6-(l-napthanlenylmethyl)-lH-pyrrolo[3,4-d]pyridazin-l-one Ligand 2 appears in patent WO 99/29695 and has CAS number 227321-12-2.
  • Ligand 3 (disclosed in WO 98/054190) 6-(4-quinolinylmethyl)- -3-methyl-l-(2- 0 methylpropyl)-thieno[2,3-d]pyrimidin-2,4(lH,3H)-dione
  • Ligand 4 (disclosed in WO 00/12514): 6-([benzotl ⁇ iazol-2-yl]methyl)-3-methyl-l-(2- methylpropyl)thieno [2,3-d]pyrimidine-2,4( lH,3H)-dione
  • Example 7 - MCT filter binding assay For yeast cells transformed with expression plasmids containing the human MCT 1-4 5 ORFs the ability of membrane preparations to bind radiohgand was determined using a single ligand concentration/ multi protein concentration binding assay.
  • Example 8 Yeast expressed MCTl based SPA assay Scintillation Proximity Assay Method
  • Streptavidin coated SPA beads were resuspended at 2.5mg/ml in assay buffer (50mM HEPES pH 7.5, 0. ImM EDTA, 150mM NaCI) and diluted to give a final concentration of 187 ⁇ g/ml in assay buffer without BSA.
  • Yeast membranes expressing MCTl with a streptavidin- binding sequence tag were then added to give a final concentration of 37 ⁇ g/ml protein and gently rolled for > 30 minutes at room temperature. The beads/membranes were then washed by centrifugation at ⁇ 650g for 10 minutes and resuspended in assay buffer + 0.05% BSA.
  • This SPA assay is suitable for high throughput screening of large compound libraries.
  • Lactate uptake was measured in each blood sample as follows: uptake was initiated by the addition of 50 ⁇ l of blood to 2 ⁇ l of 14 C-lactate (12.5 ⁇ Ci/ml; Amersham). The samples were incubated at room temperature for 30s and then the reaction was halted by transfer of 20 ⁇ l of each sample onto 1 ml of ice-cold dibutyl- pthalate (Sigma). The red blood cells were separated from the plasma by centrifugation of the samples for 30s at 15000g in a bench-top microfuge. The supernatant was aspirated to waste taking care not to disturb the cell peUet.
  • MCTl inhibitors such as Ligand 1, caused a dose-dependent decrease in the amount of [ 14 C]-lactate uptake by rat red blood ceUs. Values from representative experiments were: Table 8.
  • Example 10 Scintillation proximity assay (SPA) using Jurkat T-cell membranes
  • Human Jurkat T ceUs were grown at 37°C in RPMI 1640 medium supplemented with 5 % foetal calf serum, 2mM glutamine. Cells were harvested by centrifugation at 1500g for 10 minutes. The cell pellets were washed twice with phosphate buffered saline (PBS), and centrifuged as above.
  • PBS phosphate buffered saline
  • the final cell pellet was resuspended in lysis buffer (50mM HEPES (pH 7.8), 50mM KC1, 10% glycerol, 0.1 mM EDTA, ImM DTT) and lysed by nitrogen cavitation. Unlysed ceUs and nuclei were removed by centrifugation at 1500g for 10 minutes. The supernatant was then centrifuged at 100,000g for 30 minutes at 4°C, the pellet resuspended and homogenised in assay buffer (50mM HEPES (pH 7.5), ImM EDTA, 150mM NaCI). Aliquots of membrane preparation were stored at -80° C until use. Frozen Jurkat cell membranes were thawed on ice and then homogenised.
  • the radiohgand was diluted in assay buffer containing 0.5%(w/v) BSA and the final concentration in the assay was approximately 0.1 nM. Assays were set up in 96-well flat-bottomed white opaque plates (Costar. Cat No: 5 3912). lO ⁇ l of test compound and lO ⁇ l of radiohgand were incubated with 180 ⁇ l of SPA beads and membranes (0.04mg beads). Non-specific binding was determined in the presence of l ⁇ M Ligand 1 and total binding was determined in the presence of vehicle alone. The plates were incubated for 3 hours at room temperature before quantitation of radioactivity proximal to the SPA beads by scintillation counting. 0 Compounds including Ligand 1 caused a dose-dependent reduction in the specific binding of 125 I-hgand A to Jurkat T-ceU membranes. Values from a representative experiment were: Table 9.
  • 100ml blood was coUected by venopuncture of normal human volunteers into 3 tubes each containing 3ml of 3.2% tri-sodium citrate solution. Blood was centrifuged at 850g for 10 minutes and the plasma was removed. The cells were dUuted to 50ml with RPMI 1640 medium, and each 30ml of diluted blood was layered over 20ml Lymphoprep (Nycomed). The blood/Lymphoprep layers were centrifuged at 850g for 20 minutes at 18°C (with no brake). CeUs at the interface were removed and were washed in RPMI 1640 by centrifugation at 850g for 10 minutes.
  • ceU peUets were then combined and were washed with 2x50ml RPMI1640 at 680g for 7 minutes.
  • CeUs were resuspended to a concentration of 1 x 10 ceUs/ml in RPMI 1640 medium supplemented with 10% human AB serum (Quest Biomedical), L-glutamine (2mM) and antibiotics (50 ⁇ g penicUlin and streptomycin) (Complete medium).
  • Test compounds were dissolved in DMSO to give lOmM stock solutions and were then diluted in complete medium to 20x the final assay concentration. lO ⁇ l of compound in solution was then added to the 96-weU flat bottom assay plate (final volume of 200 ⁇ l).
  • MCTl inhibitors including Ligand 1, caused inhibition of T-ceU proliferation with maximal inhibition of approximately 60%.
  • IA50 values were obtained from dose response curves using the 4-parameter logistic fit of a data analysis program IA 50 values are defined as the concentration of compound giving 50% of the maximum possible inhibition and were obtained from dose response curves using the 4-parameter logistic fit of a data analysis program
  • IA 50 values are defined as the concentration of compound giving 50% of the maximum possible inhibition and were obtained from dose response curves using the 4-parameter logistic fit of a data analysis program
  • PBMCs were prepared as described above by separation over Lymphoprep and T- lymphocytes were then enriched by purification on a nylon wool column. Briefly, 0.6g nylon wool was inserted into a 10ml syringe and this was autoclaved. The column was equilibrated with RPMI 1640 containing 20% human serum (HS) for 30 min at 37°C.
  • HS human serum
  • the PBMC were resuspended in 1ml of pre-warmed RPMI (20% HS) and were loaded onto the nylon wool column.
  • the column was incubated for 45min at 37 °C foUowed by elution of the T-ceUs by adding 10ml RPMI (20% HS) (prewarmed to 37°C) dropwise to the column.
  • the T-ceUs obtained from the elution of the column were centrifuged (850g for 5min) and resuspended in RPMI containing L- glutamine and 5% human serum and stimulated with 0.5ng/ml PMA and 500ng/ml Ionomycin for 48h.
  • T-ceUs were maintained in this growth medium at an initial cell density of 1x10 s ceU per ml under standard ceU culture conditions (37°C, 5% CO 2 ). After 48h, the ceUs were harvested and prepared for the lactate and DNA synthesis assays by washing twice in RPMI 1640 and resuspending in fresh growth medium at lxlO 6 ceUs per ml. Assay of lactate levels by lactate oxidase enzyme activity a) Intracellular lactate levels: Test compounds were dissolved in DMSO to give lOmM stock solutions and were then diluted in complete medium to lOx the final assay concentration. lOO ⁇ l of each concentration were added to triphcate weUs of a 24-weU plate.
  • T-ceUs were then added (1ml per well) and were cultured for 4h under standard ceU culture conditions. The cells were then transferred from each well to microfuge tubes and were centrifuged at 360g for 5min at 4°C. The supernatant was discarded and the ceU pellet was resuspended in 1ml of ice-cold PBS, pH5.0. The ceUs were washed twice in PBS by centrifugation (360g, 5min, 4°C) foUowed by resuspension of the ceU pellet in lOO ⁇ l deionized water.
  • Lactate Reagent Sigma, catalogue No. 735-10
  • L-lactate was prepared as a lOOmM stock solution in PBS, pH 7.5 and was dUuted in distilled water to give a standard curve in the range of 12.5- 200 ⁇ M.
  • the assay was carried out in 96-weU plates at room temperature. 10 ⁇ l of standard or sample were added to each weU foUowed by 200 ⁇ l of the Lactate Reagent.
  • the plate was incubated for 15 min and absorbance at 540nm was then determined using a SPECTRAmaxPlus spectrophotometer (Molecular Devices). The data were coUected and analysed using SOFTmax PRO software (Molecular Devices). Assay of DNA synthesis in activated T-lymphocytes DNA synthesis was assessed by measuring the incorporation of [ 3 H]-thymidine. The assay was carried out in a 96-weU plate. 20 ⁇ l of test compounds at lOx the final concentration were added to the plate foUowed by 200 ⁇ l of T-ceUs per weU (2xl0 5 cells).
  • ceUs were cultured for 4h under standard ceU culture conditions and cultures were pulsed with 3 H- fhymidine (0.5 ⁇ Ci/weU; Amersham) for the final hour of the incubation.
  • CeUs were harvested on to glass fibre filter mats using a 96-weU harvester (Tomtec inc., Orange, USA) and incorporated radioactivity was determined using a 1450 Microbeta counter (Perkin Elmer Life
  • LPS lipopolysaccharide
  • 8MG 8- mercaptoguanosine
  • IgM goat anti mouse IgM at 40 ⁇ g/ml.
  • Ligand 2 a compound with MCT inhibitory activity was added to the cultures in a concentration range of 10 "10 M to 10 "5 M. The cultures were incubated for 48 hours at 37°C and pulsed with tritiated thymidine for the final 6 hours. The ceUs were harvested as described previously and thymidine uptake used as a measure of DNA synthesis.
  • Example 13 In vivo activity of compounds Graft versus Host Response Compounds have been tested in the rat Graft versus Host Response (GVHR), which represents the immune elements associated with transplant rejection. The model was first described by Ford et al 1970.
  • the assay consisted of injecting a lOOul volume 5x10 - 1x10 spleen ceUs from dark agouti (DA) rats into the right hind footpads of D A/Lewis FI hybrid rats. A similar number of D A/Lewis spleen cells were injected into the right hind footpads to act as controls.
  • the DA grafted ceUs were recognised by the recipient DA/Lewis rats as having "self antigenic components whereas the DA graft ceUs recognised the "Le" elements of the FI hybrids as being foreign and subsequently responded by a prohferative response.
  • the increase in prohferation was measured by an increase of weight of the right lymph node compared to the control left lymph node.
  • mice were immunised with 0.5mg/kg ovalbumin (OVA) and 200mg/kg aluminium hydroxide gel in saline intraperitoneaUy and left for 29 days before being re- challenged intraperotoneaUy with 0.5 mg/kg ovalbumin in buffered saline vehicle and left for a further 10 days.
  • OVA ovalbumin
  • Compound was dosed daily from re-challenge to termination prior to serum coUection.
  • Control groups were dosed with compound vehicle only.
  • serum samples were taken from the mice and analysed for total and specific levels of IgE. For total IgE, microtitre plates were coated with 5ug/ml monoclonal rat anti-mouse
  • the plate was washed for a further four times before adding 50ul streptavidin alkaline phosphatase conjugate appropriately dUuted in 1% BSA in PBS at room temperature for 50 minutes.
  • the plate was washed a further four times before enzyme substrate (paranitrophenyl phoshate in IM diethanolamine buffer pH 9.8) at lmg/ml was added.
  • enzyme substrate paranitrophenyl phoshate in IM diethanolamine buffer pH 9.8
  • the plate was read at 405nm Levels of total IgE in serum samples were extrapolated from curve obtained with IgE standards. For specific IgE, the methodology was similar except for foUowing.
  • the microtitre plates were coated with 50ug/ml rat anti-mouse IgE.
  • mice were immunised with ovalbumin in poly I:C adjuvant (polyinosinic:polycytidylic acid adjuvant) and left for 14 days before being re-chaUenged with ovalbumin in buffered saline vehicle and left for a further 7 days. Serum samples were taken from the mice and analysed for total and specific IgG2a. Compound was dosed dahy from re-challenge to termination prior to serum coUection. For total IgG2A, microtitre plates were coated with 5ug/ml of goat anti-mouse IgG2a in PBS and incubated overnight at 4°C.
  • poly I:C adjuvant polyinosinic:polycytidylic acid adjuvant
  • the plates were then washed four times with PBS containing 0.05% Tween 20 and then blocked with 1% BSA at room temperature for 2 hours. This was foUowed by two further plate washings.
  • the serum samples and IgG2a standards were added to the weUs in duplicate and incubated at 4°C overnight.
  • the plates were then washed a further four times before adding 50ul alkaline phosphatase conjugated goat anti-mouse IgG appropriately diluted in 0.1% BSA in PBS and left at room temperature for an hour.
  • the plate was washed a further four times before enzyme substrate (p-nitrophenyl phosphate in IM diethanolamine buffer pH 9.8) at lmg/ml was added.
  • the absorbance of the weUs at 405nm was read in a spectrophotometer.
  • Levels of total IgG2a in serum samples were extrapolated from the curve obtained with IgG2a standards.
  • IgG2a the methodology was similar except for foUowing.
  • the microtitre plates were coated with 50ug/ml OVA.
  • the serum samples or normal control sera were added to the washed plates.
  • Alkaline phosphatase conjugated goat anti-mouse IgG2a was used as enzyme marker.
  • Example 14 Expression and prohferation studies in tumours and tumour ceU lines
  • Western blotting (using the antibodies described in Example 4) has shown expression of MCTl protein in the human erythroleukaemia cell line K562 and the human colorectal tumour ceU line LoVo. MCT4 protein expression was observed in the human colorectal Colo205 ceU line.
  • Expression data was also extracted from the GeneExpress database to assess disease association of MCTl and MCT4.
  • Three Affymetrix probesets were compared for MCTl and a single probeset for MCT4. Both MCTl and 4 showed disease association.
  • MCTl a fold change increase was seen across aU probesets in cervix squamous cell carcinoma and skin malignant melanoma, which was supported by significant increases in cell ratio.
  • a small fold change decrease is seen in colon and stomach adenocarcinoma.
  • Corresponding significant fold changes are seen across most stages of colorectal and lung tumours.
  • a slight trend for increasing expression with stage is seen in NSCLC. This may therefore offer up MCTl and MCT4 as biomarkers of disease.
  • Example 15 -Lactate uptake studies in cells expressing MCT isoforms by measurement of changes in intracellular pH 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein (BCECF) is a pH- sensitive dye that has been used by Wang et al. (Am J. Physiol. 267 (Heart Circ. Physiol. 36): H1759-69, 1994) to measure changes in intraceUular pH of single cells. As lactate entry into ceUs is proton-linked, the addition of exogenous lactate to ceUs expressing functionally active MCT isoforms results in a significant decrease in intracellular pH.
  • Lactate uptake by this method can be measured in cells endogenously expressing MCT isoforms (e.g. K562 and MDA- MB231 ceUs) or in cells transfected with cloned MCT isoforms (e.g. MCTl, 3 and 4 expressed in SF9 or INS-1 ceUs as foUows.
  • MCT isoforms
  • cloned MCT isoforms e.g. MCTl, 3 and 4 expressed in SF9 or INS-1 ceUs as foUows.
  • the construction of plasmids comprising of human MCTl, 2, 3, and 4 inserted into the mammalian expression vector pCDNA3 is described in example 3.
  • INS1 ceUs were transfected with pcDNA3-hMCTl using the
  • the ceU suspensions were centrifuged (465 x g for 5min) and then washed twice in Tyrodes buffer (140mM NaCI, 4mM KC1, 0.2mM CaCl 2 , ImM MgCl 2 , lOmM HEPES, lOmM glucose, pH 7.4).
  • the BCECF- loaded cells were then resuspended at a concentration of 1.0 x 10 7 ceUs/ml in Tyrodes buffer.
  • the 96 weU plate was then centrifuged at 275g for 5 mins to ensure that the ceUs formed a monolayer on the bottom of the plate.
  • the 96 weU plate was then placed in the FLEXstation and fluorescence was measured using the foUowing wavelengths:
  • the experiment was set up so that a reading at time zero was made, the lactate added to the plate and then readings were made every 3 seconds for 3mins.
  • L(+)-lactate Sigma was prepared at 3 times the final concentration by dilution of a IM lactate solution in Tyrodes buffer. 50 ⁇ l dUuted lactate solution was then added to each weU. The ratio of fluorescence at 490nm/440nm was calculated and used to prepare a pH cahbration curve for weUs containing the pH cahbration buffer. The pH cahbration curve was then used to determine the pH of test weUs from the 490/440nm fluorescence ratio.
  • exogenous lactate (0.3-300mM) caused a significant reduction in the intracellular pH of INS-1 ceUs expressing human MCTl, 3 or 4.
  • No change in intraceUular pH in response to lactate addition was observed in untransfected INS-1 ceUs or in INS-1 ceUs expressing the pCDNA3.1 vector.

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Abstract

This invention arises from the discovery that compounds capable of blocking cellular monocarboxylate transport can inhibit lymphocyte proliferation, offering up these compounds as therapeutic agents in various immune-mediated disorders and cancers. Accordingly, this invention relates, inter alia, to methods of screening compounds for their ability to treat certain cancers and immune-mediated disorders, particularly transplant rejection and rheumatoid arthritis. The invention also relates to methods of treating these diseases by administering a compound or compounds capable of inhibiting monocarboxylate transport and the use of monocarboxylate transport inhibitor compounds in treating such diseases.

Description

SCREENING METHOD
This invention arises from our discovery that compounds capable of blocking cellular monocarboxylate transport can inhibit lymphocyte proliferation, offering up these compounds as therapeutic agents in autoimmune disorders, inflammatory, proliferative and hyperproliferative diseases, cancer and irnmunologically-mediated diseases including rejection of transplanted organs or tissues. Accordingly, this invention relates, inter alia, to methods of screening compounds for their ability to treat certain cancers and immune- mediated disorders, particularly transplant rejection and rheumatoid arthritis. The invention also relates to methods of treating these diseases by administering a compound or compounds capable of inhibiting monocarboxylate transport and the use of monocarboxylate transport inhibitor compounds in treating such diseases. The immune system has evolved to detect the presence of foreign organisms such as bacteria, viruses and other pathogens, and to mount protective immune responses to eliminate them. Under certain circumstances, the induction of an immune response against foreign organisms or tissues proves more harmful to the host than ignoring them, for example allergies to food and extrinsic antigens such as pollen, and also asthma are believed to reflect inappropriate hypersensitivity responses to otherwise harmless substances. In addition, strong responses against transplanted tissues are usually observed. These are detrimental to the survival of the transplanted organ and must be limited by administration of potent immunosuppressive drugs. Under normal circumstances the immune system does not produce immune responses to self-tissues and self-antigens. However, under some conditions immune responses are mounted against self-tissues in such an aggressive manner that they lead to destructive autoimmune diseases, for example rheumatoid arthritis, multiple sclerosis and type I diabetes. In these situations it would be desirable to reset the immune system so that responses to self- antigens are silenced but without affecting protective host defence mechanisms directed against exogenous antigens. Most immune responses are initiated and controlled by helper T lymphocytes, which respond to antigenic peptide fragments presented in association with MHC Class II molecules and cytotoxic T lymphocytes which respond to peptides presented in association with MHC Class I molecules on specialised antigen presenting cells such as dendritic cells. Full T cell activation requires two distinct signals from the antigen presenting cell. Signal 1 is antigen specific and provided by the interaction of the T cell receptor (TCR) with the MHC-peptide complex displayed by the antigen-presenting cell. Signal 2 is antigen independent and involves the interaction of the co-stimulatory T ceU molecule, CD28, with its ligand, B7, on antigen presenting cells. These cell-surface interactions trigger downstream biochemical signalling pathways, which ultimately result in IL2 transcription and T-cell activation. As a means of treating these immune-mediated disorders, research programs to date have concentrated on identifying compounds capable of blocking IL2 transcription. The promoter region of the IL2 gene includes a binding site for the Nuclear Factor of
Activated T-cells (NFAT) transcription factor complex. This complex is composed of nuclear components fos and jun and a cytoplasmic component NFATC which translocates to the nucleus after dephosphorylation by the phosphatase calcineurin. The immunosuppressive macro lides cyclosporiα A (CsA) and FK506 block the transcription of the IL2 gene in T- lymphocytes by preventing the formation of the NFAT complex (Crabtree, Cell 96:611-614, 1999). Complexes formed by the binding of CsA and FK506 to their respective immunophilins, cyclophilin and FKBP12, inhibit calcineurin activity and thus block NFATC translocation. Although CsA and FK506 are potent immunosuppressive drugs used clinically for the prevention of graft rejection, their long-term use and utility for the treatment of auto- immune disease are limited by their side effect profile including nephrotoxicity. These adverse reactions appear to be related to inhibition of calcineurin activity as the enzyme is expressed widely across mammalian tissues and has multiple functions. A number of additional immunosuppressive therapies have now been developed (Dumont, Opin. Ther. Patents 11:377-404, 2001). These include rapamycin, which disrupts the cytokine (e.g. IL2)-driven proliferation of T-cells, by interfering with the function of TOR (Target Of Rapamycin), a kinase involved in the cytokine signalling pathway (Dumont and Su Life Sci. 58:373-395,1996). However rapamycin has been shown to cause significant side effects including thrombocytopenia and hyperlipidemia (Hong and Kahan, Semin. Nephrol. 20 (2): 108-125, 2000). Antimetabolite approaches are also of utility for immunosuppression as T-lymphocytes have been shown to be dependent on de novo synthesis of ribonucleotides (Fairbanks et al., J. Biol. Chem 270(50):29682-29689, 1995). Mycophenolate mofetil (MMF), which inhibits inosine monophosphate dehydrogenase (IMPDH), the regulatory enzyme of guani e nucleotide biosynthesis (Allison and Eugui, Immunopharmacology 47:85- 118, 2000) is effective in reducing T-cell proliferation and has been used for the treatment of graft rejection. However, MMF is rapidly glucoronidated in vivo and is associated with gastrointestinal toxicity (Dumont, Curr. Opin. Invest. Drugs. 2 (3):357-363, 2001). Screening programmes investigating NFAT-mediated transcription directly have been used to identify small molecule inhibitors of JJL2 production without the side effect profile of calcineurin inhibitors. Michne et al. discovered a class of quinazolinedione compounds, which were identified by inhibition of NFAT-mediated gene transciption in a Jurkat human leukemic T-cell line (Michne et al, J. Med. Chem 38:2557-2569,1995). An example of the quinazolinedione class of compounds, WIN 61058, which inhibited NFAT-mediated transcription with a potency of 2μM, was shown to block IL2 production in Jurkat T-cells and to inhibit a human Mixed Lymphocyte Reaction (MLR) (Baine et al, J. Immunol 154:3667- 3677, 1995). A chemical programme based on WIN 61058 resulted in the identification of pyrrolopyrimidinedione inhibitors of NFAT-mediated transcription with potencies as high as 2nM (Michne et al, supra). We have confirmed that examples of these pyrrolopyrimidinediones inhibit NFAT-mediated gene transcription in Jurkat T-cells but that early IL2 production in response to mitogenic stimulation of peripheral blood mononuclear cells (PBMC) is not significantly inhibited. The applicant's own chemical programme has exemplified pyrimidinedione inhibitors, which potently inhibit the human MLR in the absence of inhibition of IL2 transcription. Contrary to that proposed by Michne et al. (supra), the inventors have, for the first time, identified the mechanism of action of these pyrimidinedione compounds as being blockade of monocarboxylate transport through the monocarboxylate transporter MCT1. This pioneering invention explains a totally new mechanism of action not forshadowed in any way by the art in this highly competitive field, opening up new method of treatments by inhibiting this mechanism of action and also new targets for identifying immunosuppressive agents. WO 98/46606 (AstraZeneca), incorporated herein by reference, discloses a family of pyrazolo[3,4~^pyrimidinedione compounds; WO 98/54190 (AstraZeneca), incorporated herein by reference, discloses a family of tMeno[2,3-d]pyrimidinedione compounds; WO 98/28301(AstraZeneca), incorporated herein by reference, discloses a family of 5- substituted pyrrolo[3,4-rf]pyrimidine-2,4-dione compounds; WO 99/29695 (AstraZeneca), incorporated herein by reference, discloses certain pyrrolo-, thieno-, furano-and pyrazolo- [3,4- yridazinone compounds; WO 00/12514 (AstraZeneca), WO 01/83489 (AstraZeneca), PCT/GB02/03399 (AstraZeneca), PCT/GB02/03250(AstraZeneca) and GB-A-2363377 ( AstraZeneca), each incorporated herein by reference, which each disclose certain thieno[2,3- djpyrimidinedione compounds. Each of these compounds, which exhibit pharmacological activity, in particular immunosuppressive activity, is disclosed for the first time herein to affect this via inhibition of monocarboxylate transport. MCT1 is a member of a family of monocarboxylate transporters, which mediate the influx and efflux of monocarboxylates, such as lactate and pyruvate, across cell membranes. The MCT proteins transport monocarboxylates by a facilitative diffusion mechanism, which requires the co-transport of protons (Poole and Halestrap. AmJ.Physiol. 264:C761-C782, 1993; Halestrap and Price. BiochemJ. 343:281-299, 1999). Non specific small molecule inhibitors of the transporter have been identified, such as 4,4'-di-isothiocyanatostilbene-2,2'- disulphonate (DIDS), α-cyano-4-hydroxycinnamate (CHC) and phloretin although these are non-selective inhibitors with potencies in the μM range (IC50 values at rat MCT1: DIDS = 50μM; CHC = 27μM; phloretin = lμM)( Poole and Halestrap, supra). None of these compounds were proposed as therapeutic agents. The MCT1 protein has been enriched from rat red blood cells and is a 55kDa protein (Poole et al, Biochem J. 320:817-824, 1996).
Hydrophobicity analysis and studies on the membrane topology of rat MCT1 have suggested a structure with 12 transmembrane segments and intracellular N- and C- terminal regions (Poole et al, supra). The nomenclature for the MCT family is taken from: Price et al., Biochemical Journal 329 (2):321-328,1998; and, Halestrap and Price. Biochemical Journal. 343:281-299,1999. The role of lactate transport via MCTs in proliferating cells, particularly T- lymphocytes, has not been investigated previously. Roth et al. showed that addition of exogenous lactate to T-cells resulted in inhibition of DNA synthesis and proliferation (Roth et al, Cell. Immunol. 136:95-104, 1991) but that E 2 production was augmented (Droge et al, Cell. Immunol. 108:405-416, 1987). Studies on the metabohsm of activated T-lymphocytes (thymocytes) have demonstrated that the cells derive 86% of their energy supply by aerobic glyco lysis i.e. glycolytic breakdown of glucose to lactate (Brand and Hermfisse. FASEB J. 11:388-39, 1997; Guppy et al, Eur.J.Biochem 212:95-99, 1993). The monocarboxylate transporter MCT4 has been characterised as a transporter with low affinity for lactate and pyruvate (Dimmer et al, BiochemJ. 350:219-227, 2000). Therefore, it has been suggested that MCT4 is adapted to the release of lactate from glyco lytically-active cells whereas the high affinity transporter MCT1 transports lactate required for energy production into cells (Manning Fox et al, J.PhysioL 529:285-293, 2000). The Examples herein, show for the first time that in T-lymphocytes, lactate efflux actually occurs via MCT1 as small molecule inhibitors of MCT 1 result in accumulation of intracellular lactate. Zhao et al., (Diabetes 50:361-366, 2001) propose that, in some forms of Type II diabetes, MCT overexpression in the pancreatic islet cells could contribute to aberrant secretion of insulin, and, therefore postulate that inhibitors of islet cell lactate transport, or of MCT1 gene expression, could provide a therapeutic target for this disease. Froberg et al., (Neuroreport 12(4):761-765, 2001) reported increased MCT1 expression in high grade glial neoplasms leading to the unproven speculation of MCT 1 as a therapeutic target for treatment of some glial neoplasms. Whilst both publications speculate on potential use of inhibitors of lactate transport or
MCT gene expression for the treatment of some forms of type II diabetes and high-grade glial neoplasms and carcinomas, neither paper establishes a credible, substantial and specific link between expression or function of MCTs to the functional effects of MCT inhibition e.g. antiproliferative effects. As far as the inventors are aware the data provided in this patent specification for the first time conclusively links the specific inhibition of human monocarboxylate transporters, in particular MCT1 to -4, and modulation of lactate transport, with the functional inhibition of human cellular proliferation for the treatment of immune-mediated disorder or cancers. The discovery that inhibition of monocarboxylate transport, via blockage of MCT1, can be used as a new mechanism of therapeutic action, also realises medical treatment via blockage of any protein that effects monocarboxylate transport, and opens up the way for identifying new therapeutic agents capable of blocking any monocarboxylate transporter, such as MCT1 through MCT4. The monocarboxalate transporters MCT1 to MCT4, are known to transport monocarboxylate (Halestrap and Price. Biochemical Journal. 343:281-299,1999). Detailed Description According to a first aspect of the invention there is provided a method for identifying compound(s) that may have potential in therapeutic treatments, comprising determining whether the compound is capable of inhibiting monocarboxylate transport activity of a cell. If the compound inhibits cellular monocarboxylate transport activity it has potential therapeutic value. In one embodiment the compound identified as having therapeutic potential is further tested in a cellular proliferation assay, for example one that tests whether the compound inhibits proliferation of activated T-cells or inhibits proliferation of cancer cells, either in vivo or in vitro. In another embodiment, the compound identified as having therapeutic potential is further tested in an in vivo or in vitro model of inflammation, autoimmune disease or transplantation. For the avoidance of doubt, the term 'inhibiting monocarboxylate transport activity' also covers the amount of cellular monocarboxalate transporter protein. Thus, it includes inhibition of activity and reduction in the amount of tranporter protein. In a preferred embodiment, the method is useful in identifying agents(s) that may have potential in treating an immune-mediated disorder or cancer. In a particular embodiment the disease is an immune-mediated disorder, such as transplant rejection, or a non-glial epithelial cancer. According to a further aspect of the invention there is provided a method for determining whether a compound not known to be capable of specifically binding to a monocarboxylate transporter can specifically bind to a monocarboxylate transporter, which comprises contacting a monocarboxylate transporter protein with the compound under conditions suitable for binding, and detecting specific binding of the compound to the transporter. Such a method is particularly applicable for identifying potentially useful therapeutic compounds. In one embodiment the transporter is present within a cell, cell ghost, a cell membrane fraction or a liposome. In another embodiment the transporter is presented within a natural or synthetic membrane. For example, the transporter could be presented within lipid vesicles as described by Lynch and McGiven (Biochem. J. 244:503-508, 1987). According to a further aspect of the invention there is provided an assay for identifying compounds which inhibit monocarboxylate transport in a cell, comprising: (a) contacting a cell or cell lysate comprising a monocarboxylate transport polypeptide with a test compound; and (b) detecting one or more of the following characteristics: (i) the ability of the test compound to inhibit the ability of the monocarboxylate transport polypeptide to transport monocarboxylate, (ii) the ability of the test compound to bind to the monocarboxylate transport polypeptide, and (hi) the ability of the test compound to block expression of the monocarboxylate transport polypeptide. According to a further aspect of the invention there is provided a method for identifying whether or not a compound may have potential in treating an immune-mediated disorder or cancer, which comprises contacting cells expressing a monocarboxylate transporter, or cell membrane preparations thereof, with a compound not known to be capable of inhibiting monocarboxylate transport, under conditions suitable for binding, and determining monocarboxylate transport activity, wherein the ability of the compound to inhibit monocarboxylate transport identifies that compound as having potential in treating an immune-mediated disorder or cancer. According to a further aspect of the invention there is provided a method for determining whether a compound not known to be capable of blocking monocarboxylate transport can block monocarboxylate transport, which comprises contacting cells expressing a monocarboxylate transporter, or cell membrane preparations thereof, with the compound under conditions suitable for binding, and determining monocarboxylate transport activity. In a particular embodiment such method is employed to determine the suitability of a compound for assessment as a potential therapeutic agent. Potential test therapeutic agents would possess IC50 values of at least lOμM, preferably at least 1 μM, for inhibition of monocarboxylate transport (IC50 being the concentration of compound resulting in 50% inhibition of the response). In one embodiment the monocarboxylate transporter is expressed from nucleic acid exogenously introduced into a cell. In another embodiment monocarboxylate transport is blocked as a result of the compound specifically binding to the monocarboxylate transporter. In another embodiment monocarboxylate transport is blocked as a result of the compound impeding expression of the monocarboxylate transporter. In a further embodiment the method is capable of determining whether or not the compound is capable of specifically blocking monocarboxylate transport. A compound is identified as an MCT inhibitor if it exhibits an inhibition constant, Ki, of less than or equal to 10/xM. The inhibition constant, Ki, is the concentration of competing ligand in a competition binding assay which would occupy 50% of the binding sites if no radio ligand was present. The Ki is calculated from the IC50 using the Cheng-Prusoff equation. IC50 values are determined as the concentration of inhibitor which would displace 50% of radio ligand A or C (described in Example 1 herein) as measured in filter binding assays and / or the scintillation proximity assay(s) described herein. According to a further aspect of the invention there is provided a method for identifying a compound that may have potential in treating an immune-mediated disorder or cancer, comprising deterrnining whether the compound is capable of inhibiting monocarboxylate transport activity of a cell. Thus, in a further aspect of the invention there is provided use of a cell or cell membrane preparation comprising a monocarboxylate transporter protein, preferably one selected from the group consisting of MCT 1 through MCT4, in the in vitro screening of compounds for their ability to treat an immune-mediated disorder or cancer. Thus, in a further aspect of the invention there is provided a human monocarboxylate transporter (preferably one selected from the group consisting of MCT 1 to MCT4) for use in the in vitro screening of compounds for their ability to treat an immune-mediated disorder or cancer. Monocarboxylate transport activity as used herein, refers to the ability of the transporter protein to facilitate transport of monocarboxylate molecules, such as lactate and pyruvate across a cell membrane. Such activity can be determined using various techniques known to the person skilled in the art. The techniques described herein for determining whether or not a compound can bind to or inhibit an MCT can be employed/adapted for determining whether or not a protein or polypeptide has monocarboxylate transport activity. Potential therapeutic agents which may be tested in the screening methods described herein, include simple organic molecules, commonly known as "small molecules", for example those having a molecular weight of less than 2000 Daltons. Other potential therapeutics include peptides and antibodies. The methods of the invention, for example may be used to screen chemical compound libraries or peptide Hbraries, including synthetic peptide hbraries and peptide phage hbraries, particularly antibody display (such as scFV or Fab) phage hbraries. Other suitable compound molecules include antibodies, nucleotide sequences, and any other molecules, including nucleic acid or protein derived molecules, which bind to an MCT. Preferably the compound is a small molecule chemical compound. The terms compound and agent are used interchangeably herein. The screening methods of the invention will prove useful in determining whether or not test compounds (chemical or biological) may be suitable for use, inter aha, in the treatment, including prophylactic treatment, of cancers, autoimmune, inflammatory, proliferative and hyperproliferative diseases and other immune-mediated diseases including, rejection of transplanted organs or tissues. Examples of immune-mediated disorders and cancers are:
(1) (the respiratory tract) reversible obstructive airways diseases including asthma, such as bronchial, allergic, intrinsic, extrinsic and dust asthma, particularly chronic or inveterate asthma (e.g. late asthma and airways hyper-responsiveness); bronchitis; acute, allergic, atopic rhinitis and chronic rhinitis including rhinitis caseosa, hypertrophic rhinitis, rhinitis purulenta, rhinitis sicca and rhinitis medicamentosa; membranous rhinitis including croupous, fibrinous and pseudomembranous rhinitis and scrofoulous rhinitis; seasonal rhinitis including rhinitis nervosa (hay fever) and vasomotor rhinitis; sarcoidosis, farmer's lung and related diseases, fibroid lung and idiopathic interstitial pneumonia; (2) (bone and joints) rheumatoid artliritis, osteoarthritis, seronegative spondyloarthropathies (including ahkylosing spondylitis, psoriatic arthritis and Reiter's disease), Behcet's syndrome, Sjogren's syndrome and systemic sclerosis; (3) (skin) psoriasis, atopic dermatitis, contact dermatitis and other eczmatous dermitides, seborrhoetic dermitis, Lichen planus, Pemphigus, bullous Pemphigus, Epidermo lysis bullosa, urticaria, angiodermas, vasculitides, erythemas, cutaneous eosinophilias, uveitis, Alopecia areata and vernal conjunctivitis;
(4) (gastrointestinal tract) Coeliac disease, proctitis, eosinophilic gastro-enteritis, mastocytosis, Crohn's disease, ulcerative colitis, food-related allergies which have effects remote from the gut, e.g., migraine, rhinitis and eczema;
(5) (other tissues and systemic disease) multiple sclerosis, atherosclerosis, systemic lupus erythematosus, Hashimoto's thyroiditis, myasthenia gravis, type I diabetes, nephrotic syndrome, eosinophilia fascitis, hyper IgE syndrome, lepromatous leprosy, Sezary syndrome and idiopathic thrombocytopenia purpura;
(6) (allograft rejection) acute and chronic following for example transplantation of kidney, heart, liver, lung, bone marrow, skin, pancreatic islet cells, cornea and stem cells; and chronic graft versus host disease.
(7) (Xenografts rejection) Hyperacute and acute and chronic following for example transplantation of kidney, heart, liver, lung, bone marrow, skin, pancreatic islet cells, cornea and stem cells; and chronic graft versus host disease.
(8) (cancer) carcinoma, including that of the bladder, breast, colon, kidney, liver, lung, ovary, pancreas, stomach, cervix, thyroid and skin. Hematopoietic tumors of lymphoid lineage, including acute lymphocytic leukemia, B cell lymphoma and Burketts lymphoma. Hematopoietic tumors of myeloid lineage, including acute and chronic myelogenous leukemias and promyelocytic leukemia. Tumors of mesenchymal origin, including fϊbrosarcoma and rhabdomyo sarcoma, and other tumors, including melanoma, seminoma, tetratocarcinoma, neuroblastoma and glioma. The compounds are thus indicated for use in the treatment, or prevention, of rejection of transplanted organs, tissues, or cells such as kidney, heart, lung, bone marrow, skin, pancreatic islet cells, cornea and stem cells; and of autoimmune, inflammatory, proliferative and hyperproliferative diseases, including cancer, and of cutaneous manifestations of immune-mediated disorders: for example rheumatoid arthritis, systemic lupus erythematosus, Hashimoto's thyroiditis, multiple sclerosis, myasthenia gravis, type 1 diabetes, uveitis, nephrotic syndrome, psoriasis, atopic dermatitis, contact dermatitis and further eczematous dermatitides, seborrhoeic dermatitis, Lichen planus, Pemphigus, Epidermo lysis bullosa, urticaria, angioedemas, vasculitides, erythemas, cutaneous eosinophilias, Alopecia areata, eosinophilic fasciitis and atherosclerosis. The inventors have found that compounds, which are capable of binding to MCTl and/or MCT2, and which inhibit lactate transport of activated T cells, inhibit the proliferation of activated T-cells and tumour cell lines such as the ery thro leukaemia cell line K562. The first aspect of the invention is therefore a screening method to identify compounds that may be useful inter aha, in treating conditions or diseases involving T-cell activation, such as transplant rejection and rheumatoid arthritis, or cellular proliferation, such as cancer. In one embodiment the compound is tested for its ability to inhibit MCT activity. This, for example, may be via inhibition of the ability to transport monocarboxylates or via blockage of MCT expression. It is well known that MCT proteins from different species have a high degree of sequence similarity. For example, rat MCTl is reported to possess 86% identity with human MCTl (Jackson et al., Biochem Biophys Acta 1238: 193-196, 1995). Thus, whilst in a preferred embodiment the MCT is of human origin, particularly from the group consisting of human MCTl, through to 4, it is envisaged that MCTs from other species, such as rat or mouse would also work in the invention. Indeed, the inventors have found that the screening method works equally well using rat MCTl protein. Suitable monocarboxylate transporters for use in the screening assay/method of the invention are MCTl, MCT2 and MCT4. MCTl is the most preferred. The sequence of MCTl is disclosed in the EMBL/GenBank DDBJ databases (Blum H., Bauersachs S., Mewes H.W., Weil B., Wiemann S, Submitted (15-MAR-2000) to the EMBL/GenBank/DDBJ databases) with the EMBL Accession No. AL162079. The sequence of the cDNA clone encoding human MCTl used herein, is disclosed in SEQ ID NO: 41, and is identical to the sequence disclosed by Blum et al. (supra). The MCTl polypeptide sequence is disclosed as SEQ ID NO: 1. With regard to MCT2, there appears to be no single definitive published sequence. Two MCT2 sequences deposited in EMBL (Accession Numbers AF049608 and AF058056) differ in three locations that lead to amino acid changes. The sequence of the cDNA clone encoding human MCT2 used herein, and disclosed in SEQ ID NO: 42, is a combination of the two. The MCT2 polypeptide sequence is disclosed as SEQ ID NO: 2. The specific amino acid differences are as follows: (i) AF049608 encodes the amino acid Asparagine at position 154, whilst AF058056 and SEQ ID NO: 42 both encode a Serine at this position; (ii) AF049608 encodes the amino acid Proline at position 268, whilst AF058056 and SEQ ID NO: 42 both encode a Leucine at this position; (hi) and finally, AF058056 encodes the amino acid Serine at position 445, whilst AF049608 and SEQ ID NO: 42 both encode a Threonine at this position. However, there are no differences between SEQ ID NO: 42 and the MCT2 genomic exon and predicted transcript sequences (UCSC SOFTBERRY Database Accession No. C12001042), which confirms that the MCT2 cDNA clone depicted in SEQ ID NO:42 is native. With regard to MCT3, compared to the predicted MCT3 amino acid sequence disclosed in Yoon et al., (Genomics. 60(3):366-370, 1990), the inventors have found an amino acid substitution of Tryptophan to Arginine at position 235. However the MCT3 genomic sequence (Accession No. AL031587) encodes an Arginine at position 235, with no further amino acid substitutions which confirms that the MCT3 cDNA clone depicted in SEQ ID NO:43 is native. The MCT3 polypeptide sequence is disclosed as SEQ ID NO: 3. The MCT4 cDNA sequence is disclosed herein as SEQ ED NO: 44 and the MCT4 polypeptide sequence is disclosed herein as SEQ ID NO: 4. Accordingly, the screening assay is not restricted to use of the full-length MCTl protein as depicted in Table 1, but extends to functional variants, including mutants, deletions and chimaeric variants that maintain activity in the test assay.
Table 1
Each of these publications is incorporated herein by reference. The invention is not restricted to the use of full-length native human MCTs. The use of functional variants also forms part of this invention. In particular, included within the scope of the present invention are alleles of the MCT proteins for use in the present invention. As used herein, an "allele" or "allelic sequence" is an alternative form of the molecule described herein. Alleles result from nucleic acid mutations and mRNA splice- variants, which produce polypeptides whose structure or function may or may not be altered. Any given gene may have none, one or many allelic forms. Common mutational changes, which give rise to alleles, are generally ascribed to natural deletions, additions or substitutions of amino acids. Each of these types of changes may occur alone, or in combination with the others, one or more times in a given sequence. Also useful in the invention are artificially produced polypeptides that share sequence homology with, and consequently have substantially the same ligand binding ability or monocarboxylate transport activity as, the proteins coded for by the nucleotide sequences depicted in SEQ ID Nos: 41 to 44 or depicted in SEQ ID Nos 1 to 4. Within this category are truncated or mutated versions of the disclosed MCT proteins and chimeric proteins including some MCT sequence and some heterologous sequence. By the term "substantially homologous" we mean a sequence which possesses at least 70%, and in increasing order of preference at least 75%, 80%, 85%, 90%, 95%, 97% and 99% sequence identity thereto. By the term "substantially the same biological activity" we mean having the ability to effect monocarboxylate transport. In a preferred embodiment the variant proteins will have at least 10%, and in increasing order of preference at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% and 95% of the monocarboxylate transportation activity of the wild-type protein, whose sequence is depicted in the respective sequence identifier, when recombinantly expressed in a suitable heterologous expression system The sequence identity between two sequences can be determined by pair-wise computer ahgnment analysis, using programs such as, BestFit, Gap or FrameAlign. The preferred ahgnment tool is BestFit. In practise, when searching for similar/identical sequences to the query search, from within a sequence database, it is generally necessary to perform an initial identification of similar sequences using suitable software such as Blast, Blast2, NCBI Blast2, WashU Blast2, FastA, Fasta3, PILEUP and CLUSTALW, and a scoring matrix such as Blosum 62. Such software packages endeavour to closely approximate the "gold-standard" alignment algorithm of Smith- aterman. Thus, the preferred software/search engine programme for use in assessing similarity, i.e how two primary polypeptide sequences line up is Smith- Waterman. Identity refers to direct matches, similarity allows for conservative substitutions. As used herein, the term "isolated" refers to molecules, either nucleic acid or amino acid sequences, that are removed from their natural environment and purified or separated from at least one other component with which they are naturally associated. Also encompassed by this term are molecules that are artificially synthesised and purified away from their synthesis materials. Thus, a polynucleotide is said to be isolated when it is substantially separated from other contaminant polynucleotides or nucleotides. Although the natural MCTl polypeptide depicted in SEQ ID NO. 1 and a variant polypeptide may only possess for example 80% sequence identity, they are actually likely to possess a higher degree of similarity, depending on the number of dissimilar codons that are conservative changes. Similarity between two sequences includes direct matches as well as conserved amino acid substitutes, which possess similar structural or chemical properties, e.g. similar charge. Examples of conservative changes (conserved amino acid substitutes) are shown in Table 2. Suitable conservative substitutions of amino acids are known to those of skill in this art and may be made without altering the biological activity of the resulting polypeptide, regardless of the chosen method of synthesis. The phrase "conservative substitution" includes the use of a chemically derivatized residue in place of a non-derivatized residue provided that such polypeptide displays the desired binding activity. D-isomers as well as other known derivatives may also be substituted for the naturally occurring amino acids. See, e.g., U.S. Patent No. 5,652,369, Amino Acid Derivatives, issued July 29, 1997. Substitutions are preferably, although not exclusively, made in accordance with those set forth in TABLE 2 as follows:
Table 2
The MCT coding nucleotide sequences for use in the present invention may also be engineered in order to alter a coding sequence for a variety of reasons, including but not limited to, alterations which modify the cloning, processing and/or expression of the gene product. For example, mutations may be introduced using techniques which are well known in the art, eg, site-directed mutagenesis to insert new restriction sites, to alter glycosylation patterns, to change codon preference, etc. Because the MCT proteins are predicted to have a transmembrane domain structure, they require a membrane scaffold to retain their structural and / or functional integrity. Whilst the preferred assay methods involve use of whole cells, or cell membrane preparations thereof, that contain one or more MCTs, it will be appreciated, that the MCT proteins or polypeptides can be presented in alternate formats to retain their structural integrity. For example, reconstituted within lipid vesicles (see for example, Lynch and McGiven. Biochem. J. 244:503-508, 1987). Such alternate means of presenting the monocarboxylate transporter protein are also part of the invention. Whole cells expressing MCT or membrane preparations thereof are particularly useful. Suitable whole cells may either be natural cells or cell lines that comprise endogenous MCTs, such as Jurkat, K562, HeLa, Chinese Hamster Ovary (CHO) cells, or transformed/transfected cells, such as INS1, SF9 cells, wherein the MCT protein has been introduced via recombinant techniques well known to the person skilled in the art. In one embodiment, cells or cellular membrane preparations containing an MCT protein, are derived from cells, preferably eukaryotic, particularly mammalian, transformed, transfected or transduced with a recombinant expression construct comprising the nucleotide sequence coding for an MCT protein and sequences sufficient to direct the synthesis of the MCT protein in cultures of said transformed, transfected or transduced cells, are used to determine the binding properties of test compounds in vitro. In one particular embodiment, the MCT protein is expressed in eukaryotic cells, especially mammalian, insect and yeast cells. Eukaryotic cells provide post-translational modifications to recombinantly expressed proteins, which include folding and/or phosphorylation and/or glyco sylation. Nucleic acids coding for an MCT for use in the invention can either be isolated or synthesised, and a variety of expression vector/host systems may be used to express MCT coding sequences. These include, but are not limited to microorganisms such as bacteria expressed with plasmids, cosmids or bacteriophage; yeasts transformed with expression vectors; insect cell systems transformed with either the baculovirus expression system or insect expression plasmids; plant cell systems transfected with plant virus expression systems, such as cauliflower mosaic virus; or mammalian cell systems (for example those transfected or transduced with plasmid or viral derived expression vectors e.g. retroviral or adenoviral vectors); selection of the most appropriate system is a matter of choice. Expression vectors usually include an origin of replication, a promoter, a translation initiation site, optionally a signal peptide, a polyadenylation site, and a transcription termination site. These vectors also usually contain one or more antibiotic resistance marker gene(s) for selection. As noted above, suitable expression vectors may be plasmids, cosmids or viruses such as phage or retroviruses. The coding sequence of the polypeptide is placed under the control of an appropriate promoter, control elements and transcription terminator so that the nucleic acid sequence encoding the polypeptide is transcribed into RNA in the host cell transformed or transfected by the expression vector construct. The coding sequence may or may not contain a signal peptide or leader sequence for secretion of the polypeptide out of the host cell. Preferred vectors will usually comprise at least one multiple cloning site to facilitate cloning of the gene. Methods for the expression of MCT proteins in host cells from cloned genes is well known to those skilled in the art of molecular biology and general techniques are described in such publications as ("Molecular Cloning - A Laboratory Manual" Second Edition, Sambrook, Fritsch and Maniatis (Cold Spring Harbor Laboratory, 1989) or "Current Protocols in Molecular Biology Volumesl-3, Edited by FM Asubel, R Brent, RE Kingston pub John Wiley 1998). Examples of host cells which may be transformed or transfected with nucleic acid encoding an MCT protein so as to express said MCT protein are: prokaryotic cells, i.e. bacterial cells such as Escherichia coli and Bacillus subtϊlis; lower eukaryotic cells, i.e yeasts such as Saccharomyces cerevisiae, Schizosaccharmoyces pombe, Pichia pastoris, Candida albicans, Aspergillus nidulans or Neurospora crassa; higher eukaryotic cells, i.e. mammalian cells such as CHO, NIH-3T3, HEK-293, Jurkat; INS-1, insect cells such as Spodoptera frugiperda 9 and 21 cell lines; and, amphibian cells such as Xenopus laevis oocytes. Performance of the invention is neither dependent on nor limited to any particular strain or type of host cell or vector; those suitable for use in the invention will be apparent to, and a matter of choice for, the person skilled in the art. Host cells transformed or transfected with a vector containing an MCT nucleotide sequence may be cultured under conditions suitable for growth with expression and recovery of membrane fractions containing the encoded proteins from the cell culture. Such expressed proteins will preferably, but not necessarily, be presented on the cell surface. Native cells lines used for detecting binding to MCTs or functional activity in MCTs e.g. K562 (human erythroleukaemia cell line), MB231 (breast carcinoma cell line). Primary cells used for detecting binding to MCTs or functional activity in MCTs. Membrane preparations for use in the invention can be made using standard techniques well known to the person skilled in the art, including the method disclosed in Example 10. It will be appreciated that there are many screening methods which may be employed to determine the ability of a test compound to block or inhibit monocarboxylate transport. Indeed, monocarboxylate transport activity can be measured directly or indirectly in a number of ways which will be apparent to the person skilled in the art. This invention incorporates each of these different ways. For example, direct binding to an MCT, such as MCTl protein, can be determined by standard ligand binding assays. Such assays can be performed using whole cells or cell membrane preparations containing MCT proteins. Suitable alternative assays might measure monocarboxylate accumulation within the cell, monocarboxylate efflux from the cell, H+ efflux or accumulation, alterations in the glycolytic rate due to monocarboxylate feedback regulation, decreased DNA synthesis and/or cell division, and the like. Examples of suitable screening methods, which may be used to identify an inhibitor of monocarboxylate (such as lactate) transport include, rapid filtration of equilibrium binding mixtures, radioimmunoassays (RIA) and fluorescence resonance energy transfer assays (FRET). A particularly useful method for identifying a compound capable of inhibiting monocarboxylate transport is a scintillation proximity assay (SPA). SPA involves the use of fluoromicro spheres coated with acceptor molecules, such as receptors, to which, a hgand will bind selectively in a reversible manner (N Bosworfh & P Towers, Nature, 341:167-168, 1989). The technique requires the use of a hgand labelled with an isotope that emits low energy radiation, which is dissipated easily into an aqueous medium
At any point during an assay, bound labelled ligands will be in close proximity to the fluoromicro spheres, allowing the emitted energy to activate the fluor and produce light. In contrast, the vast majority of unbound labelled ligands will be too far from the fluoromicrospheres to enable the transfer of energy. Bound ligands produce hght but free ligands do not, allowing the extent of hgand binding to be measured without the need to separate bound and free hgand. The following disclosure of suitable screening methods is merely intended to be an overview, and is not intended to reflect the full state of the art. Measurement of lactate efflux/accumulation by: 1) Enzymatic measurement of lactate levels using lactate as a substrate for lactate oxidase or lactate dehydrogenase using commercially available kits such as the Sigma LO kit (735- 10) or Sigma LD kit (826); or a glucose/lactate analyser (YSI 2700 analyser). 2) Transport of [14C]lactate (or radiolabelled substrates of the monocarboxylate transporter, pyruvate, β-hydroxybutyrate, glycolate) such as that described by Poole and Halestrap in Am. J. Physiol. 264:C761-C782 (1993). 3) Lactate-induced decrease in intracellular pH using pH sensitive dyes e.g. 2 ',7'- bis(carboxyethyl)-5(6)-carboxyfluorescein (BCECF) such as that described by Carpenter and Halestrap in Biochem. J. 304:751-760 (1999). 4) Lactate-induced decrease in intracellular pH using pH-sensitive electrodes such as that described by Broer et al., in Biochem. J. 333:167-174 (1998). 5) Measurement of proton efflux/accumulation using a microphysiometer such as, for example, that described by McConnell et al., Science (Washington D C). 257(5078): 1906- 1912 (1992). Each of these publications is incorporated herein by reference. In one embodiment, the screening assay method is a competitive binding assay. Thus, according to a further aspect of the invention there is provided a competitive binding assay for compounds that may have potential in treating an immune-mediated disorder or cancer, which comprises contacting host cells expressing MCT protein, or a membrane preparation thereof, with both a first test compound and a labelled second compound known to specifically bind to said MCT protein, under conditions suitable for binding of both compounds, and detecting specific binding of the first compound to the MCT protein by measuring a decrease in the binding of the second compound, to the MCT protein in the presence of the first compound indicating that the first compound binds to the MCT protein and may thus have potential in treating an immune-mediated disorder or cancer. As a comparative control, the assay can be conducted with only the second compound. In one embodiment, the first compound is a small molecule compound. In a further preferred embodiment, the second compound is a small molecule compound or an antibody. In a further embodiment the MCT is a human MCT. In a further embodiment the MCT is selected from the group consisting of: MCTl, 2, 3, and 4. Preferably, the MCT is human MCTl. There are many conventional detectable labels, such as radioisotopes, fluorescent labels, chemiluminescent compounds, labelled binding proteins, magnetic labels, spectroscopic markers and linked enzymes that might be used to label up the second compound. Fluorescent labels are often preferred because they are less hazardous than radio labels, they provide a strong signal with low background and various different fluorophors capable of absorbing light at different wavelengths and/or giving off different colour signals exist to enable comparative analysis in the same analysis. For example, fluorescein gives off a green colour, rhodamine gives off a red colour and both together give off a yellow colour. For use in the present invention, preferred labels are radioisotopes, particularly 14C, 3H and 125I, or non-radioactive labels such as digoxigenin or biotin. The choice of label and the means of detecting such label (such as via autoradiography or fluorescence microscopy), can be made by the person skilled in the art. In one embodiment of the invention a radio hgand binding assay is performed which comprises contacting the test compound with a cellular membrane preparation containing an MCT, preferably MCTl, and a radio-labelled ligand that is known to bind to said MCT, and measuring displacement of said hgand by the test compound. In one embodiment, the test compounds will be specific for a particular MCT subtype.
Compounds are deemed specific if they bind to one particular MCT subtype and exhibit 10- fold lower potency (Ki), preferably 25-fold lower potency, more preferably 100- fold lower potency to all other subtypes. Potential drug candidates are identified by choosing chemical compounds which bind with high affinity (IC50 of less than 10/zM) to the expressed MCT, by using for example, ligand binding methods well known to those skilled in the art, examples of which are shown in the binding assays described herein. Drug candidates may have broad specificity acting on more than one MCT subtype, alternatively the drug candidates will be specific for a particular MCT subtype. Compounds are deemed specific if they inhibit monocarboxylate transport at least ten fold, preferably at least 25 fold and more preferably at least 100 fold more strongly of one particular MCT subtype than to any other MCT subtype. Alternatively, compounds are deemed specific if they bind at least ten fold, preferably at least 25 fold and more preferably at least 100 fold more strongly to one particular MCT subtype than to any other MCT subtype. Ligands A and C (described in Example 1) are examples of suitable radio ligands that can be used in the invention. Such radio ligands can be made by standard techniques. These radio ligands are a further aspect of the invention. Thus, according to a further aspect of the invention there is provided a radio labeled compound capable of binding to an MCT. In terms of a radioligand for binding to human MCTl, any of the compounds disclosed in any of: WO 98/46606, WO 98/54190, WO 98/28301, WO 99/29695, WO 00/12514, WO 01/83489; PCT/GB02/03399, PCT/GB02/03250 and GB-A-2363377, could be used. In one embodiment the radiolabelled compound is selected from the group consisting of: hgand A, B and C (as described in Example 1 herein). In another aspect of the invention there is provided the use of a radiolabelled compound capable of binding to an MCT in a screening assay to identify compounds capable of binding said MCT. In one embodiment the MCT is MCTl, in another, the radiolabelled compound is hgand A, B or C, as described herein. According to a further aspect of the invention there is provided a compound, or a pharmaceutically acceptable salt thereof, identified by any of the screening methods of the invention. In a preferred embodiment the compound will be capable of specifically inhibiting monocarboxylate transport. According to a further aspect of the invention there is provided a method of producing a pharmaceutical composition, which comprises determining whether or not a compound is an MCT inhibitor using any of the screening methods of the invention and furthermore mixing the compound identified therein, or a derivative thereof with a pharmaceutically acceptable carrier. In the context of this aspect of the invention, a derivative is a compound which has been designed, synthesised and tested for MCT inhibitor activity based on the parent compound initially identified in the screen. Such a derivative compound is generally identified using conventional structure activity relationship (SAR) studies. Furthermore, such derivative compounds will generally possess shared structural features with the parent compound, but with one or more structural moieties altered. A derivative compound is likely to be one whose structure has been optimised to make the compound more suitable for therapeutic treatments, such as by removal of groups known to be associated with toxic effects; being more bio available; having a longer half-life in vivo etc. According to a further aspect of the invention there is provided a method of producing a pharmaceutical composition which comprises determining whether or not a compound is an MCT inhibitor using any of the screening methods of the invention; preparing derivative compounds of this 'parent' compound; testing these derivative compounds in one of the screening methods of the invention to identify a more active compound; and, mixing said more active compound identified therein with a pharmaceutically acceptable carrier. The components of the screening methods of the invention can be combined in a suitable kit of parts format. Thus, according to a further aspect of the invention there is provided a kit for use in a method for screening compounds for their potential in treating an immune-mediated disorder or cancer, comprising: (i) a cell capable of expressing a monocarboxylate transporter protein or a cell membrane preparation containing a monocarboxylate transport protein; and, (ii) a labelled compound specific for the monocarboxylate transporter protein in step (i); The kit optionally includes instructions for use. The MCT proteins or convenient fragments thereof may be used to raise antibodies. Such antibodies have a number of uses, which will be evident to the molecular biologist or immunologist of ordinary skill. Such uses include, but are not limited to, use as a biotherapeutic, use as the competitive binding hgand in the screening methods of the invention and monitoring protein expression. Enzyme linked immunosorbant assays (ELISAs) are weU known in the art and would be particularly suitable for detecting the MCT polypeptide or fragments thereof. The term antibody includes both monoclonal antibodies, which are a substantially homogeneous population, and polyclonal antibodies, which are heterogeneous populations. The term also includes inter alia, humanised and chimeric antibodies, as well as the various types of antibody constructs such as for example F(ab')2, Fab and single chain Fv including bacteriophage derived antibodies . In one embodiment, such antibodies are labelled. Methods of making and detecting labelled antibodies are well known (Campbell; Monoclonal Antibody Technology, in: Laboratory Techniques in Biochemistry and Molecular Biology, Volume 13. Eds: Burdon R et al. Elsevier, Amsterdam (1984)). Polyclonal antibodies can be readily generated from a variety of sources, for example, horses, cows, goats, sheep, dogs, chickens, rabbits, mice or rats, using procedures that are well known in the art. In general, antigen is administered to the host animal typically through parenteral injection. Depending on the host species, various adjuvants may be used to enhance the immunological response against the injected polypeptide. Suitable adjuvants include, but are not limited to Freund's (complete and incomplete), aluminium hydroxide, BCG and SAC (Bacille Calmette-Guerin and Staphylococcus aureus Cowan). Following booster immunizations, small samples of serum are collected and tested for reactivity to antigen. Examples of various assays useful for such determination include those described in: Antibodies: A Laboratory Manual, Harlow and Lane (eds.), Cold Spring Harbor Laboratory Press, 1988; as well as procedures such as countercurrent immuno-electrophoresis (CDEP), radioimmunoassay, radio immunoprecipitation, enzyme-linked immuno-sorbent assays (ELISA), dot blot assays, and sandwich assays, see U.S. Patent Nos. 4,376,110 and 4,486,530. Monoclonal antibodies may be readily prepared using well-known procedures, see for example, the procedures described in U.S. Patent Nos. RE 32,011; 4,902,614; 4,543,439 and 4,411,993; Monoclonal Antibodies, Hybridomas: A New Dimension in Biological Analyses, Plenum Press, Kennett, McKearn, and Bechtol (eds.), (1980). By way of example, for the production of human monoclonal antibodies, hybridoma cells may be prepared by fusing spleen cells from an immunised animal, e.g. a mouse, with a tumour cell. Appropriately secreting hybridoma cells may thereafter be selected (Koehler & Milstein. Nature. 256:495- 497, 1975; Cole et al. "Monoclonal antibodies and Cancer Therapy", Alan R Liss Inc, New York N. Y. pp 77-96, 1985). Such antibodies may be of any immunoglobulin class including IgG, IgM, IgE, IgA, IgD and any subclass thereof. Rodent antibodies may be humanised using recombinant DNA technology according to techniques known in the art. The monoclonal antibodies of the invention can be produced using alternative techniques, such as those described by Alting-Mees et al., "Monoclonal Antibody Expression Libraries: A Rapid Alternative to Hybridomas", Strategies in Molecular Biology (1990) 3:1-9, which is incorporated herein by reference. Similarly, binding partners can be constructed using recombinant DNA techniques to incorporate the variable regions of a gene that encodes a specific binding antibody. Such a technique is described in Larrick et al., Biotechnology, (1989) 7: 394. Alternatively, chimeric antibodies, single chain antibodies (see for example, US Patent Ser. No. 4,946,778), Fab fragments may also be developed against the polypeptides of the invention (Huse et al. Science. 256: 1275-1281, 1989), using skills known in the art. Antibodies are defined to be specifically binding if they bind the particular MCT with a Ka of greater than or equal to about 107 M"1. Affinity of binding can be determined using conventional techniques, for example those described by Scatchard et al., Ann. NY. Acad Sci., (1949) 51:660. Once isolated and purified, the antibodies may be used to detect the presence of antigen in a sample using established assay protocols, see for example "A Practical Guide to ELISA" by D. M. Kemeny, Pergamon Press, Oxford, England. Methods of making and detecting labelled antibodies are well known (Campbell; Monoclonal Antibody Technology, in: Laboratory Techniques in Biochemistry and Molecular Biology, Volume 13. Eds: Burdon R et al. Elsevier, Amsterdam (1984)). Method of treatment The inventors' radioligand studies indicate that the actual mechanism of action by which the compounds in International Publication Nos. WO 98/46606, WO 98/54190, WO 98/28301, WO 99/29695, WO 00/12514, and WO 01/83489; International Application numbers PCT/GB02/03399 and PCT/GB02/03250 and GB-A-2363377, each incorporated herein by reference, operate, is via binding to and inhibiting MCTl and to a lesser extent MCT2, leading inter alia, to a build up of lactate in the cell. The compounds identified in these prior art patents fall within the scope of one or other of Formulae I to DC:
WO 98/46606
(Formula I) in which:
R1 is Cι-6alkyl, C3-6alkenyl or C3-6cyclo alkyl;
R2 is or C3-6alkenyl;
R3 is 1- or 2-indanyl, 1- or 2-(l,2,3,4-tetrahydronaphthalenyl), 9-fluorenyl, acenaphthyl or CHR4(CH2)nAr where n is 0 or 1, R4 is hydrogen or Cι-6alkyl and Ar is quinolinyl, naphthalenyl, benzodioxolinyl optionally susbstituted by one or more halogen atoms, or phenyl optionally substituted by one or more substituent groups selected from halogen,
C1-6alkyl, and phenylsulfonylmethyl;
W is H, CH2OH, CO2H, C02Ci-6alkyl, CH2NR5R6, CONR5R6, where R5 and R6 are independently hydrogen or or together with the nitrogen atom to which they are attached form a 3- to 8-membered heterocyclic ring optionally further containing an oxygen atom or a group NR7 where R7 is hydrogen or Ci-ealkyl, or W is pyridyl or phenyl, each of which may be optionally substituted by one or more substituent groups selected from halogen, hydroxyl, Chalk ! and Cι-6alkoxy; X is a bond or C^alkylene;
Y is S(0)p, C≡C, CH=CH, CH2CH2 or CH2CH=CH; and p is 0, 1 or 2; or a pharmaceutically acceptable salt thereof, provided that:
• X is not a bond when W is H, CH2OH, CO2H, CO2Cι-6alkyl, CH2NR5R6 or CONR5R6 and Y is sulfur.
WO 98/54190
(Formula II) wherein: R is -Q^Ar1, -C(R4)(R5)Ar1, or Ar2;
Ar1 is naphthyl, quinolyl, isoquinolyl, indolyl, benzofuranyl or benzothienyl, each of which can be optionally substituted by one or more substituents selected from C1-4 alkyl, C1- alkoxy, halogen or trifluoromethyl, or Ar1 is phenyl optionally substituted by one or more substituents selected from Cw alkyl, alkoxy, halogen, trifluoromethyl, amino, nitro, cyano, trifluoromethoxy, phenoxy, -CH2N(R0)2, -NHS02CF3, -NHC(O)R6a, CO2R7 or -C(O)NR8R8a; R4 represents H or C1- alkyl; R5 represents H or OH; each R6 independently represents H or CM alkyl;
R6a represents H, C1-6 alkyl, aryl or arC1- alkyl, wherein the aryl group or aryl moiety in the aralkyl group is phenyl or pyridyl, each of which may be optionally substituted by one or more substituents selected fromCμ alkyl, CM alkoxy, CM alkylcarbonylamino, halogen or trifluoromethyl; R7 represents H or Cι-4 alkyl;
R8 and R8a each independently represent H, C alkyl, phenyl or pyridyl;
Ar2 is acenaphthenyl, indanyl, iminodihydrobenzofuranyl or fluorenyl, each of which can be optionally substituted by one or more substituents selected from OH, C1-4 alkyl,
C1-4 alkoxy, halogen, or trifluoromethyl; R1 and R2 are independently H, C1-6 alkyl, C3-6 alkenyl, CH2C3-5 cycloalkyl or C3-6 cycloalkyl;
R3 represents H, X-R9 or X-Ar3;
X represents S(O)n, C(O)NR10, C(O)O, NH(CO)NR10, NH(CO)O or SO2NR10; n is 0, 1 or 2; R9 represents a methyl group optionally substituted by one or more substituents selected from CN, C02H, C1-5 alkoxycarbonyl, 5-tetrazolyl, SO2NH2 or C(0)NRuR12, or R9 represents C2-6 alkyl or C3-6 alkenyl, each of which may be optionally substituted by one or more substituents selected from OH, CN, C02H, C1-5 alkoxy, C1.5 alkoxycarbonyl, 5-tetrazolyl, azide, phthalimido, SO2NH2, C(O)NRπR12, NR13R14, NHC(O)R15 or NHSO2R16 where R11, R12, R13 and R14 each independently represent H or C1- alkyl,
R15 represents CM alkyl, CM alkoxy, di(CMalkyl) amino, or alkoxyalkylene containing up to 6 carbon atoms, and R16 represents CM alkyl or trifluoromethyl; or, additionally, in the case where X represents C(O)NR10, NH(CO)NR10 or SO2NR10, R9 and R10 together with the nitrogen atom to which they are attached may form a 4- to 7-membered heterocyclic ring which may be optionally substituted by one or more OH groups; R10 represents H, Ci_6 alkyl or is linked to R9 as defined above; and Ar 3 is phenyl, pyridyl or pyridine N-oxide, each of which may be optionally substituted by one or more substituents selected from OH, NO2, NH2, NHSO2CF3, CM alkoxy, bis-CMalkanesulphonylamino, or CMalkoxycarbonylamino; or a pharmaceutically-acceptable salt or solvate thereof. WO 99/29695
(Formula III) wherein B represents a group CH or a nitrogen (N), sulfur (S) or oxygen (O) atom; D represents a carbon (C) or nitrogen (N) atom; E represents a group CR3 or a nitrogen (N) atom; when D is a carbon atom, then B is a sulfur or oxygen atom and E is a group CR , and when D is a nitrogen atom, then either B is a group CH and E is a group CR3 or a nitrogen atom, or B is a nitrogen atom and E is a group CR3; R1 represents a group NR'R" where R' represent a hydrogen atom or a Cι-C6 alkyl group, R" represents a Cι-C6 alkyl group, or R' and R" together with the nitrogen atom to which they are attached form a 3- to 7-membered saturated heterocyclic ring, or R1 represents a Cι-C6 alkyl, Cχ-C6 alkoxy, CrC3-alkyloxyCr C3-alkyl, C3-C6-cycloalkyloxyCι-C3-alkyl, C3-C6 alkenyl, phenyl, C3-C cycloalkyl, C3- C5 cycloalkylmethyl or C3-C cycloalkenyl group, each of which may be optionally substituted by one or more halogen atoms; R2 represents a methyl group, or a C2-C6 alkyl group optionally substituted by a Cι-C6 alkoxy group other than in the 1-position; R3 represents a hydrogen atom or a group X-R5 or X-Ar1; X represents a group -O-, S(O)Η, S02N(R6) or C(=0)N(R6); n is 0, 1 or 2; R5 represents an optionally substituted alkyl or alkenyl group, or, additionally, in the case where X represents S02N(R6) or C(=O)N(R6), R5 and R6 together with the nitrogen atom to which they are attached may form an optionally substituted 3- to 7-membered heterocyclic ring; Ar1 represents an optionally substituted phenyl or pyridyl group; R6 represents a hydrogen atom, CrC6 alkyl or is linked to R5 as defined above; R4 represents a group CHR7Ar2 or Ar3 or, additionally, in the case where D represents a carbon atom, a group C(O) Ar2 or CR7(OH) Ar2; Ar2 represents an aryl or heteroaryl group which may be optionally substituted; Ar3 represents an acenaphthenyl, indanyl or fluorenyl group, each of which may be optionally substituted; and R represents a hydrogen atom or a Ci-C4 alkyl group; or a pharmaceutically-acceptable salt or solvate thereof. (Formula IV) wherei — W represents — CH2 — or a bond; Q represents Ar1 or Ar2; in the case where W represents —
CH2 — , Q represents an aryl group Ar1 wherein Ar1 represents naphthyl, phenyl, quinolyl, isoquinolyl, indolyl, benzofuranyl or benzothienyl; in the case where
W represents a bond, Q represents an aryl group Ar2 wherein Ar2 represents acenaphthenyl, fluorenyl or indanyl; wherein the ring systems which Ar1 and Ar2 represent may all be optionally substituted by one or more substituents selected from CMI alkyl, - alkoxy, halogen, or trifluoromethyl; R10 represents X — (A)p — Y; X represents S(O)n, C≡C, (CH2)2, CH=CH or CH2CH=CH; n represents 0, 1 or 2; A represents C S alkylene; p is 0 or 1; Y represents CN, OR11, CO2R12, CONR13R14, NR15R16, NHSO2R17, NHCOR18 or an optionally substituted aryl or heteroaryl group, provided that when X represents S(0)ft and Y is other than an optionally substituted aryl or heteroaryl group, then p is 1 and also provided that when X represents S(O)n, p is 1 and Y represents OH, then n is not 0; R13 and R14 independently represent H, CMS alkyl or phenyl, which latter group may be substituted by one or more substituents selected from CMI alkyl, Cι_ alkoxy, halogen, or CO2R21; and R1, R2, R11, R12, R15, R16, R17, R18 and R21 independently represent H or Ci-s alkyl; or a pharmaceutically acceptable derivative thereof. WO 00/12514
(Formula V) wherein: R represents a group -C(O)Ar1 or -C(R4)(R5)Ar1; Ar1 represents a heterocyclic group comprising a total of from 5 to 10 atoms which include from 1 to 3 hetero atoms independently selected from nitrogen, oxygen and sulfur, which group Ar1 may be optionally substituted by one or more substituents independently selected fro oxo, hydroxyl, CM alkyl, C alkoxy, halogen, trifluoromethyl, amino, nitro, cyano, trifluoromethoxy, phenoxy, -CH2N(R6)2, -NHS02CF3, CMalkylsulfonylammo, -NHC(O)R6a, C02R7 or -C(O)NR8R8a, with the proviso that Ar1 does not represent an optionally substituted benzofuranyl, benzothienyl, indolyl, quinolyl or isoquinolyl group; R4 represents a hydrogen atom or a CM alkyl group; R5 represents a hydrogen atom or a hydroxyl group; each R6 independently represents a hydrogen atom or a CM alkyl group;
R6a represents a hydrogen atom or a Cι-6 alkyl, aryl or arC1-4alkyl group, wherein the aryl group or aryl moiety in the aralkyl group is phenyl or pyridinyl, each of which may be optionally substituted by one or more substituents independently selected from C1-4 alkyl, CM alkoxy, C1-4 alkylcarbonylamino, halogen or trifluoromethyl; R7 represents a hydrogen atom or a CM alkyl group;
R8 and R8a each independently represent a hydrogen atom or a CM alkyl, phenyl or pyridinyl group;
R1 and R2 each independently represent a hydrogen atom or a C1-6 alkyl, C3-6 alkenyl, CH2C3-5 cycloalkyl or C3-6 cycloalkyl group; R3 represents a hydrogen atom or a group X-R9 or X-Ar2;
X represents an oxygen atom, S(0)n, C(0)NR10, C(O)O, NH(CO)NR10, NH(CO)0 or SO2NR10, with the proviso that when X represents an oxygen atom and R represents a group - C(R4)(R5)Ar1, then R4 and R5 both represent a hydrogen atom; n is 0, 1 or 2; R9 represents a methyl group optionally substituted by one or more substituents independently selected from cyano, carboxyl, C1-5 alkoxycarbonyl, 5-tetrazolyl or C(O)NRnR12, or R9 represents a C2-6 alkyl or C3_6 alkenyl group, each of which may be optionally substituted by one or more substituents independently selected from hydroxyl, cyano, carboxyl, Cι-5 alkoxy, C1-5 alkoxycarbonyl, 5-tetrazolyl, azido, phthalimido, SO2NH2, C(O)NRuR12, NR13R14, NHC(O)R15 or NHSO2R16 where R11, R12, R13 and R14 each independently represent a hydrogen atom or a C1- alkyl group, R15 represents a CM alkyl, C alkoxy, amino or (di)Cι_ alkylamino group or an alkoxyalkylene group containing up to 6 carbon atoms, and R16 represents a CM alkyl or trifluoromethyl group; or, additionally, in the case where X represents C(0)NR10, NH(CO)NR10 or SO2NR10, R9 and R10 together with the nitrogen atom to which they are attached may form a 4- to 7-membered saturated heterocychc ring which may be optionally substituted by one or more hydroxyl groups;
R10 represents a hydrogen atom or a Cι-6 alkyl group or is linked to R9 as defined above; and Ar 2 is phenyl, pyridinyl, thienyl, pyridone or pyridine N-oxide, each of which may be optionally substituted by one or more substituents independently selected from halogen, hydroxyl, nitro, amino, NHSO2CF3, CM alkyl, CM alkoxy, bis-CMalkanesulfonylamino, C1-4alkylcarbonylamino or C1-4alkoxycarbonylamino; or a pharmaceutically-acceptable salt or solvate thereof. WO 01/83489
(Formula VI) wherein:
R is -C(O)Ar1, -C(R4)(R5)Ar1 or Ar3; Ar1 represents a 5- to 10-membered aromatic ring system wherein up to 3 ring atoms may be heteroatoms independently selected from nitrogen, oxygen and sulphur, the ring system being optionally substituted by one or more substituents independently selected from CM alkyl, Ci-
4 alkoxy, halogen, trifluoromethyl, oxo, nitro, cyano, NR6R7 and -CH2NR8R9;
R1 and R2 each independently represent a hydrogen atom, Cι-6 alkyl, C3-6 alkenyl, CH2C3-5 cycloalkyl or C3-6 cycloalkyl;
R3 represents a group X- Ar2;
X represents a group S(0)n, C(O) or CH(OH); n is 0, 1 or 2;
Ar2 represents a 5- or 6-membered aromatic ring wherein up to 4 ring atoms may be heteroatoms independently selected from nitrogen, oxygen and sulphur, the ring being optionally substituted by one or more substituents independently selected from CM alkyl, Ci.
4 alkoxy, C alkylthio, halogen, trifluoromethyl, oxo, hydroxyl, amino, nitro, cyano and benzyl;
R4 represents a hydrogen atom or CM alkyl; R5 represents a hydrogen atom or hydroxyl group;
R6 and R7 each independently represent a hydrogen atom or CM alkyl, or together with the nitrogen atom to which they are attached form a 5- to 7-membered saturated heterocyclic ring; R8 and R9 each independently represent a hydrogen atom or C alkyl, or together with the nitrogen atom to which they are attached form a 5- to 7-membered saturated heterocyclic ring; and
Ar3 represents acenaphthenyl, indanyl or fluorenyl, each of which may be optionally substituted by one or more substituents independently selected from C1-4 alkyl, C1-4 alkoxy, halogen or trifluoromethyl; with the proviso that when X represents S(O)n, then Ar2 does not represent pyridyl or thienyl; or a pharmaceutically acceptable salt or solvate thereof. GB-A-2363377
(Formula VII) wherein:
R is -C(O)Ar1, -C(R4)(R5)Ar1 or Ar3;
Ar1 represents a 5- to 10-membered aromatic ring system wherein up to 3 ring atoms may be heteroatoms independently selected from nitrogen, oxygen and sulphur, the ring system being optionally substituted by one or more substituents independently selected from C alkyl, Ci. 4 alkoxy, halogen, trifluoromethyl, oxo, nitro, cyano, NR6R7 and -CH2NR8R9;
R1 and R2 each independently represent a hydrogen atom, Cι-6 alkyl, C3-6 alkenyl, CH2C3-5 cycloalkyl or C3-6 cycloalkyl;
R3 represents a group X-R10 or Ar2;
X represents a bond or a group NR11; Ar2 represents a 5- or 6-membered aromatic ring wherein up to 4 ring atoms may be heteroatoms independently selected from nitrogen, oxygen and sulphur, the ring being optionally substituted by one or more substituents independently selected from CM alkyl, Ci.
4 alkoxy, CM alkylthio, acetyl, halogen, trifluoromethyl, oxo, hydroxyl, amino, nitro, cyano and benzyl; R4 represents a hydrogen atom or CM alkyl;
R5 represents a hydrogen atom or hydroxyl group;
Rδ and R7 each independently represent a hydrogen atom or CM alkyl, or together with the nitrogen atom to which they are attached form a 5- to 7-membered saturated heterocyclic ring;
R8 and R9 each independently represent a hydrogen atom or CM alkyl, or together with the nitrogen atom to which they are attached form a 5- to 7-membered saturated heterocyclic ring;
Rx0 represents Cι-6 alkyl, C2-6 alkenyl or C2-6 alkynyl, each o f which may be optionally subsituted by one or more substituents independently selected from carboxyl, hydroxyl,
-C(0)-R12, C3-6 cycloalkyl, morpholinyl, -NR13R14, -SR15, -OR16, phenyl and halophenyl, or
R10 represents a C3-6 cycloalkylcarbonyl, -C(0)CH2CN, halophenylcarbonyl or trifluoromethylcarbonyl group; R11 represents a hydrogen atom or a Cι-6 alkyl group;
R12 represents piperazinyl optionally substituted by a Cι-6 alkyl group, or
R12 represents a group -NR17R18;
R13 and R14 each independently represent a hydrogen atom, or a CM alkyl, CM hydroxyalkyl or -C(O)-R19 group, or R13 and R14, together with the nitrogen atom to which they are attached, form a 5- to
7-membered saturated heterocyclic ring which may be optionally substituted by one or more substituents independently selected from C1-4 alkyl, hydroxyl and oxo;
R15 and R16 each independently represent a 5- or 6-membered aromatic ring wherein up to 4 ring atoms may be heteroatoms independently selected from nitrogen, oxygen and sulphur, the ring being optionally substituted by one or more substituents independently selected from halogen atoms, cyano and CM alkyl;
R17 and R18 each independently represent a hydrogen atom, or a CM alkyl group optionally substituted by one or more substituents independently selected from halogen atoms and hydroxyl; R19 represents a Cι-6 alkyl or C3-6 cycloalkyl group, each of which may be optionally substituted by a hydroxyl group; and Ar3 represents acenaphthenyl, indanyl or fluorenyl, each of which may be optionally substituted by one or more substituents independently selected from C1-4 alkyl, CM alkoxy, halogen and trifluoromethyl; or a pharmaceutically acceptable salt or solvate thereof. PCT/GB02/03250
(Formula VIII) wherein:
R1 and R2 each independently represent a Ci-ealkyl, C3-6alkenyl, C3-5cycloalkyl(Cι-3)methyl or C3-6cycloalkyl; each of which may be optionally substituted by
1 to 3 halogen atoms;
R3 represents a group -CON(R10)YRU or - SO2N(R10)YRn;
[wherein Y is O, S or NR12 (wherein R12 is hydrogen or Cι-6alkyl); and R10 and R11 are independently optionally substituted by halo, hydroxy, amino, Cι-6alkylamino or di-(Cι-6alkyl)amino] ;
Q is -CO- or -C(R4) (R5)- (wherein R4 represents a hydrogen atom or CMalkyl and
R5 represents a hydrogen atom or hydroxyl group);
Ar represents a 5- to 10-membered aromatic ring system wherein up to 4 ring atoms may be heteroatoms independently selected from nitrogen, oxygen and sulphur, the ring system being optionally substituted by one or more substituents independently selected from C alkyl, Ci.
4alkoxy, halogen, haloalkyl, dihaloalkyl, trihaloalkyl, hydroxyC1-4alkyl,
CMal oxyCMal yl, CMalkylthio, CMalkoxycarbonyl, C -4alkanoyl, oxo, nitro, cyano, -
N(R6)R7 and -(CH2)ρN(R8)R9, hydroxy, CMalkylsulphonyl, CMalkylsulphinyl, carbamoyl, di-(Cι- alkyl)carbamoyl, carboxy; p is 1 to 4
R6 and R7 each independently represent a hydrogen atom, CMalkanoyl or CMal yl, or together with the nitrogen atom to which they are attached form a 5- to 7-membered saturated heterocyclic ring; R8 and R9 each independently represent a hydrogen atom, CMalkanoyl or CM alkyl, or together with the nitrogen atom to which they are attached form a 5- to 7-membered saturated heterocyclic ring; or a pharmaceutically acceptable salt or prodrug thereof. PCT/GB02/03399
(Formula IX) wherein:
R1 and R2 each independently represent a Cι-6alkyl, C3-6alkenyl, C3-5cycloalkyl(Cι-3)methyl or C3-6cyclo alkyl; each of which may be optionally substituted by
1 to 3 halogen atoms;
R3 is isoxazolidin-2-ylcarbonyl or tetrahydroisoxazin-2-ylcarbonyl wherein each ring is optionally substituted by one hydroxy group;
Q is -CO- or -C(R4) (R5)- (wherein R4 represents a hydrogen atom or and R5 represents a hydrogen atom or hydroxyl group) ;
Ar represents a 5- to 10-membered aromatic ring system wherein up to 4 ring atoms may be heteroatoms independently selected from nitrogen, oxygen and sulphur, the ring system being optionally substituted by one or more substituents independently selected from CMalkyl
(optionally substituted by 1,2 or 3 hydroxy groups), halogen, haloalkyl, dihaloalkyl, trihaloalkyl, CMal oxyCMalkyl, CMalkylthio,
C1-4alkoxycarbonyl, C2-4alkanoyl, oxo, thioxo, nitro, cyano, -N(R6)R7 and -(CH2)ρN(R8)R9, hydroxy, CMalkylsulphonyl, CMalkylsulphinyl, carbamoyl, C1- alkylcarbamoyl, di-(C1- alkyl)carbamoyl, carboxy; p is 1 to 4 R6 and R7 each independently represent a hydrogen atom, CMalkanoyl or CMal yl, or together with the nitrogen atom to which they are attached form a 5- to 7-membered saturated heterocyclic ring; R8 and R9 each independently represent a hydrogen atom, CMalkanoyl or CM alkyl, or together with the nitrogen atom to which they are attached form a 5- to 7-membered saturated heterocyclic ring; or a pharmaceutically acceptable salt or prodrug thereof. At the filing dates of these applications the mechanism of action of these compounds was not known and therefore is not taught in these applications/publications. Moreover, the newly identified mechanism of action is distinct from, indeed a radical change, from that acted on by current immunosuppressive pharmaceutical compounds, such as cyclosporin A. According to another aspect of the invention there is provided a method of treating an immune-mediated disorder or cancer, comprising administration to a human subject in need of treatment of a compound that inhibits cellular monocarboxylate transporter activity, other than a compound disclosed in any of: International Publication Nos. WO 98/46606, WO 98/54190, WO 98/28301, WO 99/29695, WO 00/12514, and WO 01/83489; International Application numbers PCT/GB02/03399 and PCT/GB02/03250 and UK Patent application number GB-A-2363377. According to another aspect of the invention there is provided a method of treating an immune-mediated disorder or cancer, comprising administration to a human subject in need of treatment of a compound that inhibits cellular monocarboxylate transporter activity, other than a compound within formulae I to IX. According to another aspect of the invention there is provided a method of treating an immune-mediated disorder or cancer, comprising administration, to a human subject in need of treatment, of a compound that inhibits a monocarboxylate transporter other than MCTl and MCT2. According to another aspect of the invention there is provided a method of treating an immune-mediated disorder or cancer, comprising administration, to a human subject in need of treatment, of a compound that inhibits a monocarboxylate transporter selected from the group consisting of: MCT3 and MCT4. According to another aspect of the invention there is provided a method of treating an immune-mediated disorder or cancer, comprising administering an effective amount of a pharmaceutical composition comprising an MCT inhibitor identifiable or identified by a screening assay method of the invention to a subject in need thereof. In one embodiment the MCT inhibitor is a selective inhibitor. According to another aspect of the invention there is provided a method of treating an immune-mediated disorder or cancer, comprising administering an effective amount of a pharmaceutical composition comprising an MCT inhibitor identifiable or identified by (i) contacting a monocarboxylate transporter protein with a test compound under conditions suitable for binding; and (ii) detecting specific binding of the compound to the transporter protein; to a subject in need thereof. According to another aspect of the invention there is provided a method of treating an immune-mediated disorder or cancer, comprising (i) contacting a monocarboxylate transporter protein with a test compound under conditions suitable for binding; (ii) detecting specific binding of the compound to the transporter protein; (hi) preparing a pharmaceutical composition comprising the compound; and, (iv) administering an effective amount of the pharmaceutical composition to a subject in need thereof. According to another aspect of the invention there is provided the use of a compound that inhibits a monocarboxylate transporter other than a compound of formulae I to IX, in the treatment of an immune-mediated disorder or cancer. According to another aspect of the invention there is provided the use of a compound that inhibits a monocarboxylate transporter other than a quinazolinedione compound or a compound of formulae I to IX, in the treatment of an immune-mediated disorder or cancer. According to another aspect of the invention there is provided the use of an MCT inhibitor compound that inhibits a monocarboxylate transporter, other than MCTl and MCT2, in the treatment of an immune-mediated disorder or cancer. According to another aspect of the invention there is provided the use of an MCT inhibitor compound that inhibits a monocarboxylate transporter selected from the group consisting of: MCT3 and MCT4, in the treatment of an immune-mediated disorder or cancer. According to another aspect of the invention there is provided the use of a compound that inhibits a monocarboxylate transporter other than a compound of formulae I to IX, in the manufacture of a medicament for the treatment of an immune-mediated disorder or cancer. According to another aspect of the invention there is provided the use of a compound that inhibits a monocarboxylate transporter other than a quinazolinedione compound or a compound of formulae I to IX, in the manufacture of a medicament for the treatment of an immune-mediated disorder or cancer. According to another aspect of the invention there is provided the use of an MCT inhibitor compound that blocks a monocarboxylate transporter other than MCTl and MCT2, in the manufacture of a medicament for the treatment of an immune-mediated disorder or cancer. According to another aspect of the invention there is provided the use of an MCT inhibitor compound that blocks a monocarboxylate transporter selected from the group consisting of: MCT3 and MCT4, in the manufacture of a medicament for the treatment of an immune-mediated disorder or cancer. According to a further aspect of the invention there is provided a method of inhibiting T-cell and B-cell proliferation in a human, comprising administration to the human in need of such treatment of a compound capable of specifically inhibiting monocarboxylate transport within the T-cell or B-ceU. According to another aspect of the invention there is provided a method of treating an immune-mediated disorder or cancer, comprising (i) in vitro testing a compound for the ability to inhibit lactate transport in a cell, and (ii) administering, to a human patient in need of treatment, an effective amount of a compound which has been identified from step (i) as a compound capable of blocking lactate transport. According to another aspect of the invention there is provided a method of treating an immune-mediated disorder or cancer, comprising (i) testing a compound for its ability to inhibit monocarboxylate transport in a cell, and (ii) administering to a human patient suffering from or likely to suffer from an immune-mediated disorder or cancer, of an effective amount of a compound which has been identified from step (i) as a compound capable of inhibiting monocarboxylate transport. According to another aspect of the invention there is provided a method of treating a patient suffering from an immune-mediated disorder or cancer, comprising administering to a human patient suffering from or likely to suffer from such a disease or condition of an effective amount of a compound which has been shown (or is known) to be capable of blocking cellular monocarboxylate transport. The medical treatment methods of the invention are particularly suitable for treating rheumatoid arthritis and for use before, during and after transplantation surgery, to prevent host rejection of the transplanted tissue. - 37 - Compounds capable of effecting monocarboxylate build up in a cell or preventing monocarboxylate efflux from the cell are particularly suitable for use in the disease treatment methods of the invention. The compositions of the invention may be in a form suitable for oral use (for example as tablets, lozenges, hard or soft capsules, aqueous or oily suspensions, emulsions, dispersible powders or granules, syrups or elixirs), for topical use (for example as creams, ointments, gels, or aqueous or oily solutions or suspensions), for administration by inhalation (for example as a finely divided powder or a liquid aerosol), for administration by insufflation (for example as a finely divided powder) or for parenteral administration (for example as a sterile aqueous or oily solution for intravenous, subcutaneous, intramuscular or intraperitoneal dosing or as a suppository for rectal dosing). It may be desirable to administer the therapeutic agent directly to or at the site of interest, e.g. injection into arthritic joint. This may, for example, obviate any non-specific cellular side effects that the agent may cause. The compositions of the invention may be obtained by conventional procedures using conventional pharmaceutical excipients, well known in the art. Thus, compositions intended for oral use may contain, for example, one or more colouring, sweetening, flavouring and/or preservative agents. In addition to the compounds of the present invention the pharmaceutical composition of this invention may also contain, or be co-administered (simultaneously or sequentially) with, one or more pharmacological agents of value in treating one or more disease conditions referred to herein. Agents such as FK506, Cyclosporin A, steroids, azathioprine, mycophenolate mofetil, leflunomide, methotrexate and antibodies against TNF and its receptor. The pharmaceutical compositions of this invention will normally be administered to a warm-blooded animal at a unit dose within the range 5-5000 mg per square meter body area of the animal, i.e. approximately 0.1-100 mg/kg, and this normally provides a therapeutically-effective dose. A unit dose form such as a tablet or capsule will usually contain, for example 1-250 mg of active ingredient. Preferably a daily dose in the range of 1-50 mg/kg is employed. In general lower doses will be administered when a parenteral route is employed. Thus, for example, for intravenous administration, a dose in the range, for example, 0.5 mg to 30 mg per kg body weight will generally be used. Similarly, for administration by inhalation, a dose in the range, for example, 0.5 mg to 25 mg per kg body weight will be used. Oral administration is however preferred, particularly in tablet form. _, - 38 -
Typically, unit dosage forms will contain about 1 mg to 500 mg of a compound of this invention. However, the size of the dose for therapeutic or prophylactic purposes will naturally vary according to the nature and severity of the conditions, the age and sex of the animal or patient and the route of administration, according to well known principles of medicine. Accordingly, the optimum dosage may be determined by the practitioner who is treating a particular patient. A therapeutically effective dose or amount refers to that amount of the agent sufficient to prevent development of or to alleviate the existing symptoms associated with the disorder. Determination of the effective amounts is well within the capabilities of those skilled in the art, especially in hght of the detailed disclosure herein. For example, the therapeutically effective amount can be estimated initially from cell culture assays. For example, a dose can be formulated in animal models to achieve a circulating concentration range that includes the IC50 (concentration at which 50% of the maximal effect is demonstrated) as determined in in vitro cellular assays. Such information can be used to more accurately determine the therapeutically effective dose in humans. The amount of active ingredient that is combined with one or more excipients to produce a single dosage form will necessarily vary depending upon the host treated and the particular route of administration. For example, a formulation intended for oral administration to humans will generally contain, for example, from 0.5 mg to 0.5 g of active agent compounded with an appropriate and convenient amount of excipients which may vary from about 5 to about 98 percent by weight of the total composition. Techniques for formulation and administration of agents for use in accordance with the present invention may be found in the latest edition of "Remington's Pharmaceutical Sciences", Mack publishing Co., Easton, Pa. Thus according to this aspect of the invention there is provided the use of a compound capable of inhibiting cellular monocarboxylate transport, or a pharmaceutically-acceptable composition thereof, in the manufacture of a medicament for use in the production of an antiproliferative effect in a warm-blooded animal, such as man. An anti-proliferative effect is defined herein as the ability to prevent cell number expansion. In one embodiment the compound is a selective inhibitor in another embodiment it has broad spectrum activity. According to a further feature of this aspect of the invention there is provided a method for producing an anti-proliferative effect in a wann-blooded animal, such as man, in need of such treatment which comprises administering to said animal an effective amount of a compound capable of inhibiting monocarboxylate transport within a ceU, or a pharmaceutically-acceptable composition thereof, as defined hereinbefore. Again, in one embodiment the compound is a selective inhibitor in another embodiment it has broad spectrum activity. A variety of gene therapy approaches may be used in accordance with the invention to modulate expression of an MCT gene in vivo. One therapeutic means of inhibiting or dampening the expression levels of a particular gene (for example one of the MCT proteins) is to use antisense therapy. Antisense therapy utilises antisense nucleic acid molecules that are synthetic segments of DNA or RNA ("oligonucleotides"), designed to mirror specific mRNA sequences and block protein production by inhibiting translation of the native gene transcript. Once formed, the mRNA binds to a ribosome, the cell's protein production "factory" which effectively reads the RNA sequence and manufactures the specific protein molecule dictated by the gene. If an antisense molecule is delivered to the cell (for example as native oligonucleotide or via a suitable antisense expression vector), it binds to the messenger RNA because its sequence is designed to be a complement of the target sequence of bases. Once the two strands bind, the mRNA may no longer dictate the manufacture of the encoded protein by the ribosome and/or is rapidly broken down by the cell's enzymes (e.g. RNaseH), thereby freeing the antisense ohgonucleotide to seek and disable another identical messenger strand of mRNA. Oligonucleotides, which are complementary to and hybridisable with any portion of novel gene mRNA disclosed herein, are contemplated for therapeutic use. U.S. Patent No. 5,639,595, "Identification of Novel Drugs and Reagents", issued Jun. 17, 1997, wherein methods of identifying ohgonucleotide sequences that display in vivo activity are thoroughly described, is herein incorporated by reference. Expression vectors containing random ohgonucleotide sequences derived from previously known polynucleotides are transformed into cells. The cells are then assayed for a phenotype resulting from the desired activity of the ohgonucleotide. Once cells with the desired phenotype have been identified, the sequence of the ohgonucleotide having the desired activity can be identified. Identification may be accomplished by recovering the vector or by polymerase chain reaction (PCR) amplification and sequencing the region containing the inserted nucleic acid material. Antisense molecules can be synthesised for antisense therapy. These antisense molecules may be DNA, stable derivatives of DNA such as phosphorothioates or methylphosphonates, RNA, stable derivatives of RNA such as 2'-O-alkylRNA, or other ohgonucleotide mimetics. U.S. Patent No. 5,652,355, "Hybrid Ohgonucleotide Phosphorothioates", issued July 29, 1997, and U.S. Patent No. 5,652,356, "Inverted Chimeric and Hybrid Oligonucleotides", issued July 29, 1997, which describe the synthesis and effect of physio logically-stable antisense molecules, are incorporated by reference. It is preferred that the sequence be at least 17 nucleotides in length in order to achieve sufficiently strong annealing to the target mRNA sequence to prevent translation. Antisense ohgonucleotides targetting MCTl (in Caco-2 cells) have been described by Hadjiagapiou et al., (Am J. Physio 1. Gastrointest. Liver Physiol. 279:G775-G780, 2000). Antisense molecules may be introduced into cells by microinjection, hposome encapsulation or by expression from vectors harboring the antisense sequence. Alternatively, ribozyme molecules may be designed to cleave and destroy the MCT mRNAs in vivo. Ribozymes are RNA molecules that possess highly specific endoribonuclease activity. Hammerhead ribozymes comprise a hybridising region which is complementary in nucleotide sequence to at least part of the target RNA, and a catalytic region which is adapted to recognise and cleave the target RNA. The hybridising region preferably contains at least 9 nucleotides. The design, construction and use of such ribozymes is well known in the art and is more fully described in Haselhoff and Gerlach, (Nature. 334:585-591, 1988). In another alternative ohgonucleotides designed to hybridise to the 5'- region of the MCT gene so as to form triple helix structures may be used to block or reduce transcription of the MCT gene. In another alternative RNA interference (RNAi) ohgonucleotides or short (20-25bp) RNAi MCT sequences cloned into plasmid vectors are designed to introduce double stranded RNA into mammalian cells to inhibit and/or result in the degradation of MCT messenger RNA. MCT RNAi molecules may begin adenine/ adenine (A A) and may comprise of 20 or 21 or 22 or 23, or 24 or 25 base pair double stranded RNA molecules with the preferred length being 21 base pairs and be specific to individual MCT sequences with 2 nucleotide 3' overhangs or hairpin forming 45-50mer RNA molecules. The design, construction and use of such ribozymes is well known in the art and is more fully described in Elbashir et al, (Nature. 411(6836):428-429, 2001). In one embodiment, the antisense, ribozyme, triple helix or RNAi nucleotides are designed to specifically inhibit translation and/or transcription of only one MCT, with minimal effects on the other MCT genes. Thus, according to another aspect of the invention there is provided a method for treating a patient suffering from an immune-mediated disorder or cancer, comprising administering to said patient an effective amount of an anti-sense molecule, a ribozyme molecule, triple helix forming molecule or RNAi molecule capable of binding to the mRNA of an MCT, as hereinbefore described, including any nucleic acid or protein derived inhibitor of transcription or translation of MCTs. There is also provided the use of an antisense nucleic acid molecule, a ribozyme molecule, a triple helix forming molecule, RNAi molecule or an antibody directed against an MCT, in the treatment of, or manufacture of a medicament for treating, a cell proliferative disorder. In the context of the present specification, the term "therapy" also includes "prophylaxis" unless there are specific indications to the contrary. The terms "therapeutic" and "therapeutically" should be construed accordingly. Prophylaxis is expected to be particularly relevant to the treatment of persons who have suffered a previous episode of, or are otherwise considered to be at increased risk of, the disease or condition in question. Persons at risk of developing a particular disease or condition generally include those having a family history of the disease or condition, or those who have been identified by genetic testing or screening to be particularly susceptible to developing the disease or condition. The invention is further described, but in no way limited, by the following examples: Example 1 - Design and construction of ligands 1 A Ligand A i) 2.6-Dihvdro-6-rr2-iodoρhenyl methvn-2-methyl-4-(2-methylpropyl -lH-ρyrrolor3.4- dlpyridazin- 1-one
2,6-Dihydro-2-methyl-4-(2-methylpropyl)- lH-pyrrolo [3 ,4-d]pyridazin- 1 -one (205mg) , 1 - chloromethyl-2-iodo-benzene (300mg) [see WO99/29695], and caesium carbonate (360mg) were mixed in dry DMF (1.5ml). After stirring at room temperature for 2.5hr under nitrogen the reaction was evaporated to dryness, and the residue was partitioned between ethyl acetate and dilute HCl. The organic solution was washed with brine, dried and evaporated. The residue was purified by chromatography to give a sohd, which was recrystallised from cyclohexane/ethyl acetate to afford the sub-title compound 270mg.
MS (APCI +ve) (M+H)+ 422 NMR 1H δ(CDCi3) 0.96(6H, d), 2.14(1H, m), 2.56(2H, d), 3.72(3H, s), 5.32(2H, s), 6.86(1H, d), 7.05(1H, d), 7.06(1H, t), 7.33(1H, t), 7.47(1H, d), 7.90(1H, d).
ii) 7-[r3-[rri.l-Ddimethylethv dnnethylsilylloxylproρyllthio1-2.6-dihvdro-6-[r2- iodophenyls)methyll-2-methyl-4-('2-methylpropyl - lH-pyrrolo[3.4-d1pyridazin-l-one
The product of step (i) (l.lg) and S-[3-[[(l,l-dimethylethyl)dimethylsilyl]propyloxy]- l-(4- methylphenyl)dioxidosulfanyl-propanethiol 4-methyl-benzenesulfonothioate (1.7g) were combined in THF(40ml) under nitrogen at -78°C, and a solution of lithium diisopropylamide (0.6M, 8.3ml) was added dropwise. The reaction was quenched with saturated aqueous sodium bicarbonate solution after 2hr at -78°C and then the mixture was extracted into ethyl acetate. The organic solution was washed with brine, dried and evaporated. The residue was purified by chromatography to afford the sub- title compound (400 mg).
NMR 1H 5(CDCI3) 0.0(6H, s), 0.84(9H, s), 0.95(6H, d), 1.70(2H, m), 2.08(1H, m), 2.52(2H, d), 3.09(2H, t), 3.60(2H, t), 3.74(3H, s), 5.47(2H, s), 6.40(1H, d), 6.99(1H, t), 7.07(1H, s), 7.25(1H, t), 7.90(1H, d). iii) 7-ri3-rr .l-Ddimethylet1ιyl, dimethylsilylloxylproρyl1thio1-2.6-dihvdro-2-methyl-4-r2- methylpropyl')-6-[r2-(trimethy1stannyl phenyllmethyll- lH-pyrrolor3.4-dlpyridazin-l-one
The product of step (ii) (34mg) in dry toluene (1 ml) was degassed by purging with nitrogen, and then hexamethyl ditin (100 micro litre) and tetrakistriphenylphosphine palladium (0) (lOmg) were added. The mixture was heated and stirred under nitrogen in a sealed flask at 95°C for 4 hr. The reaction was cooled, diluted with ethyl acetate and filtered tlirough a pad of silica, and then purified by chromatography to afford the sub- title compound (20mg). NMR 1H δ(CDci3) 0.0(6H, s), 0.4(9H, s + Sn satellites at 0.32 and 0.49), 0.86(9H, s), 0.96(6H, d), 1.70(2H, m), 2.08(1H, ), 2.52(2H, d), 3.06(2H, t), 3.58(2H, t), 3.75(3H, s), 5.50(2H, s), 6.54(1H, d), 6.98(1H, t), 7.25-7.29(2H, m), 7.54(1H, d).
iv) 2.6-Ddihydro-7-r(3-hydroxypropyl thiol-2-methyl-4-(2-methylpropy -6-[[2- ftrimethylstannyDphenyllmethyll - lH-pyrrolo [3.4-dlpyridazin- 1 -one
The product of step (hi) (6mg) was treated with a IM solution of TBAF in THF (0.5ml). After 1.5hr the reaction was diluted with saturated aqueous sodium bicarbonate solution, and then extracted into ethyl acetate. The organic solution was washed with brine, dried and evaporated. Chromatography of the residue gave the sub-title compound (3mg).
NMR 1H δ(CDci3) 0.39(9H, s + Sn satellites at 0.33 and 0.46), 0.94(6H, d), 1.73(2H, m), 2.08(1H, m), 2.52(2H, d), 3.00(2H, t), 3.74(3H, s), 3.80(3H, m), 5.50(2H, s), 6.54(1H, d), 6.50(1H, d), 7.01(1H, s), 7.21-7.29(2H, m), 7.54(1H, d).
v) 2.6-Dihvdro-7-[(3-hvdroxypropyl)thio1-6-r('2-r rl25JτIliodophenyl)methyll-2-methyl-4-(2- methylpropyl -lg-pynOlo[3.4-<flρyridazin-l-one
To a solution of sodium [ IJiodide (Amersham Pharmacia Biotech; «2000 Ci mmol" , 1 mCi;
0.5 nmol, 10 μl) was added a solution of the product of step (iv) in methanol (10 μl, 3.65 nmol; 200 μg ml"1, 365 nmol ml"1) followed by cMoramine-T in water (4 μl, 8.8 nmol; 50 μg ml"1, 2192 nmol ml"1). The vial was sealed, shaken vigorously and left to stand at room temperature for 30 min. The product was purified by preparative HPLC. The radiochemical purity was typically >98%. The radioactive concentration was determined by liquid scintillation counting and was normally found to be in the range of 5 to 8 MBq ml"1. The radiochemical yield was typically between 50 - 60%. vi) 2.6-Dihvdro-7-r(3-hydroxypropyl thiol-6-r(2-iodoρhenyl methyll-2-methyl-4-(2- methylpropyO - lff-pyrrolo \3.4-.fl pyridazin- 1 -one
(cold hgand A)
The title compound was prepared by the method of step (iv) using the product of step (ii).
MS (APCI +ve) (M+H)+512 NMR 1H δ (cocas) 0.95(6H, d), 1.77 (2H, quintet), 2.10 (IH, septet), 2.54 (2H, d), 3.08 (2H, t), 3.74 (3H, s), 3.86-3.94 (3H, m), 5.50 (2H, s), 6.37-6.41 (IH, m), 6.99-7.06 (IH, m), 7.10 (IH, d), 7.22-7.28 (IH, m), 7.91 (IH, d).
IB Ligand B i) l-Azido-4-chloromethyl-2-iodo-benzene
4-Azido-3-iodo-benzenemethanol (J. Labelled Compd. Radiopharm., 1996, 38:227-37) (350mg) in dry dichloromethane (20ml) was treated with triethylamine (185 micro htre) and methanesulphonyl chloride (100 micro htre) at room temperature for 20 hr. Volatiles were removed in vacuo and the residue was purified by chromatography to give the sub-title compound 210mg. NMR Η δ(CDα3) 4.51(2H, s), 7.11(1H, d), 7.42(1H, dd), 7.82(1H, d).
ii) 6-[(4-Azido-3-iodophenyl methyn-2.6-dihvdro-2-methyl-4-r2-methylpropylVlH- pyrrolor3.4-dlpyridazin-l-one (unlabelled ligand B
2,6-Dihydro-2-methyl-4-(2-methylpropyl)- lH-ρyrrolo[3,4-d]pyridazin- 1-one (W099/29695) (25mg), the product of step (i) (40mg) and caesium carbonate (40mg) were mixed in dry DMF (5ml). After stirring at room temperature for 3 days under nitrogen the reaction was poured into water and extracted into ethyl acetate. The organic solution was washed with brine, dried and evaporated. The residue was purified by chromatography to give the sub-title compound 50mg.
MS (APCI +ve) (M+H)+463 MP 138-9°C NMR 1H δ (cDci3) 0.97 (6H, d), 2.06-2.20 (IH, m), 2.55 (2H, d), 3.72 (3H, s), 5.20 (2H, s), 6.98 (IH, d), 7.11 (IH, d), 7.17 (IH, dd), 7.47 (IH, d), 7.62 (IH, d).
iii) 6-r(4-Azido-3-(trimethylstannyl)phenyl methyll-2,6-dihydro-2-methyl-4-('2- methylpropyl - lH-pyrrolo [3 ,4-dlpyridazin- 1-one
6-[(4-Azido-3-iodophenyl)methyl]-2,6-dihydro-2-methyl-4-(2-methylpropyl)-lH-pyrrolo[3,4- djpyridazin- 1-one (lOmg), hexamethyl ditin (100 μl) and tetrakis triphenylphosphine Pd (0) (2mg) were combined in dry toluene (5 ml) and heated at 100°C under nitrogen for 4 hours. After cooling aU volatiles were removed in vacuo and the residue was purified by preparative thin layer chiOmatography (Si02/ethyl acetate) to afford the sub-title compound (lOmg).
NMR 1H δ(CDci3) 0.3(9H, s + Sn satellites at 0.21 and 0.4, 2xd), 0.96(6H, d), 2.13(1H, m), 2.55(2H, d), 3.72(3H, s), 5.22(2H, s), 7.01(1H, d), 7.10-7.22(3H, m), 7.46(1H, d).
iv) 6-ir4-Azido-3-[125Iliodophenyl')methyll-2.6-dihvdro-2-methyl-4-(2-methylpropyl -lH- pyrrolo \3.4-dlpyridazin- 1-one
To a solution of sodium [125I]iodide (Amersham Pharmacia Biotech, EVIS30; «2000 Ci mmol"1, 1 mCi; 0.5 nmol, 10 μl) was added a solution of the product of step (hi) (10 μl, 3.0 nmol; 150 μg ml"1, 300 mnol ml"1) followed by chloramine-T in water (2 μl, 3.6 nmol; 500 μg ml"1, 1800 nmol ml"1). The vial was sealed, shaken and left to stand at room temperature for 10 minutes. An aliquot of sodium metabisulphite (2 μl, 16 nmol, 1500 μg ml"1, 8000 nmol ml"1) was added to the reaction followed by methanol (25 μl) and the vial shaken. The iodo-azide product was purified using preparative HPLC. The radiochemical purity was typically >99%. The radioactive concentration was determined by liquid scintillation counting and was normally found to be in the range of 2 to 3 MBq ml"1. The radiochemical yield was typically between 20 - 30%. IC Ligand C i) L2.3.4-Tefrahydro-l-(2-hydroxypropyl -3.6-dim^ethyl-2,4-dioxo-5-pyricnidine carbonitrile
Ethyl N-(2-cyano-3-ethoxy-l-oxo-2-butenyl)-carbamate (Chem. Pharm. Bull, 1972, 20, 1380- 8) (5g) was dissolved in ethanol (50ml) at reflux under nitrogen, and DL-l-amino-2-propanol (1.88ml) added. After 5hr at reflux the reaction was cooled and evaporated to dryness. The resulting gum was suspended in water (50ml), treated with sodium hydroxide (1.46g) and stirred lhr. Dimethyl sulphate (3.45ml) was added and stirring was continued for 1 hr. The precipitate was collected, and the aqueous solution was concentrated, then extracted into dichloromethane. The organic solution was dried and evaporated and the residue was combined with the precipitate (above) to afford the subtitle compound (4.35g). MS (El) (M+H)+ 223 BP 159
ii) l-r2-Hhydroxypropy -3-methyl-6-ri-naphthalenylmethyD-lH-pyrrolo[3.4-dlpyrimidine- 2.4f3H.6H)-dione
The product of step (i) (4.24g) was suspended in 75% formic acid (80ml) and Raney Nickel (50% dispersion in 8ml water) was added. The mixture was heated at 90°C under nitrogen for 15 min. After cooling the suspension was filtered (kieselguhr) and evaporated. The residue was dissolved in water (100ml) and extracted into ethyl acetate, each ahquot of extraction was 5 washed with sodium bicarbonate solution. The combined organic extracts were dried and evaporated to yield a white foam, which was dissolved in chloroform (20ml) and heated to 50°C. A solution of bromine (0.4ml) in chloroform (5ml) was added and after stirring for 10 min at 50°C was concentrated in vacuo. The residue was dissolved in ethanol (25ml), treated ith triethylamine (2.96ml) and then 1-naphthalenylmethylaιτιine (1.55ml) was added. After 10 20hr at room temperature the reaction was poured into 2M HCl (100ml) and extracted with ethyl acetate, dried (MgS04) and then concentrated in vacuo. Purification by chromatography (SiO2/2:l hexane-ethyl acetate) gave the sub-title compound, which was crystallised from hexane/ethyl acetate to afford sub-title compound, llOmg.
15 MS (El) (M+H)+ 363 BP 141 iii) S-Methyl-ό-fl-naphthalenyhnethyD-l-^-oxopropyD- lH-pyrrolor3,4-d1pyrimidine- 2.4GH.6H)-dione
20 A solution of anhydrous DMSO (417 μl) in anhydrous dichloromethane (10ml) was added dropwise to a solution of oxalyl chloride (256 μl) in dichloromethane (20ml) at -78°C under nitrogen. After 15min a solution of the product of step (ii) (970mg) in dichloromethane (20ml) at -78°C was added. After 5min triethylamine (900 μl) was added, the reaction was
25 stirred lOmin then allowed to warm to 0°C Water (100ml) was added and the mixture was extracted with dichloromethane. Drying (MgSO4), evaporation and chromatography gave the sub- title compound (660mg). MS (El) (M+H)+ 361 BP 141
iv} 3-Methyl-l-('2-methyl-2-proρenyl)-6-('l-naphthalenylmethyl)- lH-pyrrolor3.4- dlpyrimidine-2.4 3H.6H)-dione
A stirred suspension of methylene triphenylphosphonium bromide (1.22g) in dry THF (20ml) at -78°C under nitrogen was treated with sodium hexamethyldisilazide (3.1ml of IM solution in THF). Ηie reaction mixture was stirred at room temperature for 1 hr. The resulting solution was added to a solution of the product of step (hi) (560mg) in dry THF (30ml) at 0°C under nitrogen, and stirred at 5°C for 2hr then 20min at room temperature. Ηie mixture was poured into water (50ml) and extracted into ethyl acetate. Drying, evaporation and chromatography gave the sub-title compound (465mg).
MS (EI) (M+H)+339 BP 141
v) 5-r(3-Hydroxypropyl)thiol-3-methyl-l-f2-methyl-2-propenyl)-6-(l- naphthalenylmethyl)-lH-pyrrolo[3.4-dlpyrimidine-2.4r3H.6H)-dione
The product of step (iv) (350 mg) and S-[3-[[(l,l-dimethylethyl)dimethylsilyl]propyloxy]-l- (4-methylphenyl)dioxidosulfanyl-propanethiol 4-methyl-benzenesulfonothioate (527mg) were dissolved in dry THF (10ml) at -78°C under nitrogen. LDA (1.95mmol) in dry THF (5ml) was added, and after lhr the temperature was raised to 0°C The reaction was quenched by addition of sodium bicarbonate solution (30ml), and extracted into ether. Drying and evaporation gave a residue which was dissolved in acetonitrile (10ml) and treated with 40% HF (0.4ml) for 30min. The reaction mixture was poured into sodium bicarbonate solution and extracted into ethyl acetate. Drying, evaporation and chromatography gave the sub-title compound which was recrystallised from hexane-ethyl acetate afford the product (163mg).
NMR 1H nmr δ(CDci3) 1.67(3H, s), 1.8(2h, m), 3.1(2H, t), 3.44(3H, s), 3.83(2H, dd), 4.35(2H, s), 4.72(1H, s), 4.83(1H, s), 5.83(2H, s), 6.37(1H, s), 6.76(1H, d), 7.39(1H, t), 7.58(2H, m), 7.83(1H, d), 7.79(2H, m).
vi) 5-rr3-Hvdroxypropyl)thio1-l-rr2.3.3'- Hlisobutyl)-3-methyl-6-(l- naphthalenylmethyl)--lH-pyrrolor3,4-dlpyrimidine-2.4(3H.6H)-dione 5-[(3- Hydroxypropyl) thio] -3-methyl- 1 - [2 ,3 -di3H-2- (3H-methyl)propyl] - 6- ( 1 -naphthalenylmethyl) - lH-pyιτolo[3,4-^pyrimidine-2,4(3flr,6JΪ)-dione
The product of step (v) (2.28 mg, 5.1 μmol), 10% Pd/carbon (2.35 mg) and ethanol (0.5 ml) were placed in a 1 ml round-bottomed flask which was attached to a tritium manifold. The contents of the flask were frozen in hquid nitrogen and the flask then evacuated before tritium gas (241 GBq, 2.6 ml, 0.113 mmol) was introduced. The flask was allowed to warm to room temperature and the contents left to stir for 22 hours. The flask was removed from the apparatus and the catalyst removed by filtration. The filtrate was diluted with ethanol (5 ml) and the solvent removed under reduced pressure. This was repeated with a further portion of ethanol (5 ml). Purification of the tritiated material was achieved by reversed-phase HPLC using a 5 Waters Novapak 8 200 x 8 mm radial compression module eluting with 45% v/v acetonitrile/0.1% v/v aqueous trifluoro acetic acid at 3 ml min"1 and UV detection at 254 nrn. An aliquot of the stock solution (5ml) was reduced to dryness and re-dissolved in acetonitrile (0.5 ml) and purified in approximately three equal injections. The peak due to hgand C was collected as three separate fractions (front, middle and back) and these were combined with
10 the equivalent cuts from the two subsequent injections. The volumes of the three fractions were measured in each case and made up to 10 ml by the addition of 50% w/v aqueous sodium thiosulphate (100 μl) and ethanol. The radioactive concentration, molar specific activity and radiochemical purity of the three fractions was determined and the details included in the table below.
15 Table 3.
Both the front and middle fractions provided material of a suitable radiochemical purity and specific activity for use in the hgand binding assay. Unlabelled ligand C is disclosed in Michne et al., (J. Med. Chem (1995) 38:2557- 20 2569). Example 2 - Photo affinity labelling of the target protein The unlabelled compounds and radio ligands of Example 1 were used to identify the precise target(s) involved in the binding interaction using photoaffinity labelling, gel electrophoresis, peptide sequencing and mass spectrometry. The target protein was identified 25 as being MCTl. Photoaffinity labelling was performed using 125I-labelled hgand B. Photolabelling reactions were set up by diluting washed rat red-blood cell membranes 10-fold in assay buffer (50mM HEPES (pH 7.5); 0. ImM EDTA; 150mM NaCI) and by incubating in the presence or absence of lμM unlabelled competing ligand C The 1 5I-photoligand solution was added to give a final ligand concentration of 1 nM. The photolabelling reaction was performed by irradiating the sample with a hand held 254 nm UV source for 1 minute at room temperature. The labelled ghost membranes were then collected by centrifugation at 100,000g for 10 minutes at 4°C Finally, the samples were each washed in 1 ml of distilled water and the final membrane pellets again coUected at 100,000g for 10 minutes at 4°C The samples were stored at -20°C until use. The photoaffinity-labelled proteins were analysed by one- and two-dimensional gel electrophoresis. The labelled proteins were excised from the gel and subjected to in gel protein digestion prior to analyses by mass spectrometry. Three peptides matching rat monocarboxylate transporter 1 were identified. Example 3 - Cloning of MCT genes 3.1 Human MCTl Ohgonucleotide primers containing unique restriction sites, to allow subsequent cloning, and sequences derived from the optimal Kozak consensus sequence (MCT 1-5'; 5'- GGA-TCC-ACC-ATG-CCA-CCA-GCA-GTT-GGA-GG-3'; SEQ ID No: 6; and MCTl-3'; 5'-GTC-GAC-TCA-GAC-TGG-ACT-TTC-CTC-CTC-CTT-G-3'; SEQ ID No: 7) were used in a PCR to amphfy the MCTl ORF (SEQ ID NO: 41) from a cDNA library. The PCR fragment was subcloned into the vector pCR3.1 uni (Invitrogen). Bacterial colonies containing the MCTl ORF in the vector were identified in a PCR colony screen. A number of MCTl positive colonies were grown, the plasmid DNA isolated and subjected to sequence analysis to ensure that no amino acid encoding mutations had been incorporated into the MCTl ORF. The MCTl ORF (BamHI/Sall) was then sub-cloned into the mammalian expression vector pcDNA3 (Invitrogen) and digested with BamHI XhoI to generate the plasmid pcDNA3-hMCTl. The MCTl ORF was then further subcloned into the insect expression vector pIZv5HIS (Invitrogen) and the S. cerevisiae expression plasmid, pACES14. Expression plasmids pcDNA3-hMCTl, pIZ-hMCTl, and pACES14-hMCTl were purified and used to transform relevant host cells.
3.2 Rat MCTl The open reading frame encoding rat MCTl was amplified from a rat brain cDNA library (Origene) using the ohgonucleotide pair RM1-5' (5'-TGCATGATCA- ATGCCACCTGCGATTGGCGGGCCAG-3'; SEQ ID No. 8) and RM1-3' (5'- TGCAGCTAGCTCAG-ACTGGGCTCTCCTCCT-3'; SEQ ID No. 9) in a PCR. The resulting amplified DNA was digested with Bcll/Nhel and was hgated with pACES14 pre- digested with BamHI/Nhel. The resulting insert DNA was sequenced to ensure that no mutations had been incorporated. Plasmid pACES14-rMCTl was purified and used to transform relevant host ceUs. The amino acid sequence of rat MCTl is depicted in SEQ ID NO: 5.
3.3 Human MCT2 The open reading frame encoding human MCT2 (SEQ ID NO: 42) was amplified from a full-length cDNA clone using the ohgonucleotide pair MCT2-5' (5'-AGC-TGG-ATC-CAC- CAT-GCC-ACC-AAT-GCC-AAG-3'; SEQ ID No. 10) andMCT2-3' (5-GAC-TCT-CGA- GTT-AAA-TGT-TAG-TTT-CTC-TTT-CTG-A-3'; SEQ ID No. 11) in a PCR. The PCR fragment was subcloned into the vector pCR3.1 uni (Invitrogen). Bacterial colonies containing the MCT2 ORF in the vector were identified in a PCR colony screen. A number of MCT2 positive colonies were grown, the plasmid DNA isolated and subjected to sequence analysis to ensure that no amino acid encoding mutations had been incorporated into the MCT2 ORF. The full length open reading frame for human MCT2 was then subcloned into the S. cerevisiae expression plasmid, pACES14, the insect expression vector pIZv5HIS (Invitrogen), and the mammalian expression vector pcDNA3 (Invitrogen). Plasmids pACES14-hMCT2, ρIZ-hMCT2, and pcDNA3-MCT2 were purified and used to transform relevant host cells. The amino acid sequence of human MCT2 is depicted in SEQ ID NO: 2.
3.4 Human MCT3 The human MCT3 ORF (SEQ ID NO: 43) spans 4 exons in the human genome (Yoon et al., Genomics 60 (3), 366-370, 1999). Each of the four exons were amplified by PCR from human genomic DNA using the oligos MCT3-5'#3(5'-ATC-AGG-ATC-CAG-GCA-GCG- ATG-GGC-G-3'; SEQ ID No. 12)/MCT3-11# (5'-GAC-ACG-GGG-CCC-GTG-CCG-TAG- AGC-AT-3'; SEQ ID No. 13) for exon I; MCT3-10#(5'-CGG-CAC-GGG-CCC-CGT-GTC- CAG-CAT-3'; SEQ LD No.14)/MCT3-13# (5'-AGG-CCC-AGG-CCT-GTG-AGC-ACC- CCA-GC-3'; SEQ ID No. 15) for exon II; M3-G1(5'-GTT-CCC-GGA-TCT-GCT-GGG-TT- 3'; SEQ ID No. 16)/M3-G2 (5'-TGG-AGC-TTC-CCT-GGG-TCT-AA-3'; SEQ ID No. 17) for exon III flanked by intron DNA; MCT3-14# (5'-CCC-TCT-GCC-GGC-CGC-CTG-GTG- GAT-GCG-TTG-AAG-3'; SEQ ID No. 18)/MCT3-3'#3 (5'-GTC-AAC-TAG-TCA-GAC- ACC-CAG-GGG-ATC-AAC-TGG-AG-3'; SEQ ID No. 19) for exon IV to ~150bp downstream of the termination codon. Exon III was then isolated the M3-G1/M3-G2 PCR product using the ohgos MCT3-12# (5'-TGC-TCA-CAG-GCC-TGG-GCC-TGG-CCC-TCA- 5 A-3' ; SEQ ID No. 20)/MCT3-15# (5'-ACC-AGG-CGG-CCG-GCA-GAG-GGC-GGT-CC-3' ; SEQ ID No. 21). A PCR product (I+II) was generated from the exon I and exon II PCR products using the ohgos MCT3-5'#3/MCT3-13# and subcloned into pCRBluntll-TOPO to give pTOPOMCT3(I+II). A PCR product (IH+IV) was generated from the exon III and exon IV PCR products using the ohgos MCT3-12#/MCT3-3'#3 and subcloned into pCRII-TOPO
10 to give pTOPOMCT3(III+IV). The MCT3 fragments fromHindlll/StuI digested pTOPOMCT3(I+II) and from Hindlll/Stul digested pTOPOMCT3(πi+IV) were ligated to give the full length pTOPOMCT3 (in the pCRBlunuT vector). A number of MCT3 positive colonies were grown, the plasmid DNA isolated and subjected to sequence analysis to ensure that no amino acid encoding mutations had been incorporated into the MCTl ORF Following
15 sequence analysis the full length open reading frame of human MCT3 was subcloned into the S. cerevisiae expression plasmid, pACES14, the insect expression vector pIZv5HIS (Invitrogen), and the mammalian expression vector pcDNA3 (Invitrogen). Plasmids pACES14-hMCT3, pIZ-hMCT3 and pcDNA3-hMCT3 were purified and used to transform relevant host cells. The amino acid sequence of human MCT3 is depicted in SEQ ID NO: 3.
20 3.5 Human MCT4 A human dendritic cell cDNA clone (AC-DNA-1819) contains an incomplete copy of the MCT4 open reading frame (SEQ ID NO: 44). This was made full length by inserting ~190bp GBO212 (5'-TAG-GAA-GAA-GCC-CAA-AGA-GCC-ACA-G-3'; SEQ ID No.
25 22)/GB0213 (5'-GAC-TTC-TAG-AGC-CCA-GCC-ACT-CAG-ACA-CTT-GTT-TC-3' ; SEQ LD No. 23) MCT4 3' PCR fragment (amplified from a human naive T cell cDNA stock) on a Notl - Xbal fragment to make pGBAC41. The MCT4 ORF was subsequently amplified using ohgonucleotides GBO214 (5'-GAT-CGG-ATC-CAT-GGG-AGG-GGC-CGT-GGT-3'; SEQ ED No. 24)/GBO215 (5'-GTC-AGA-TAT-CGC-CAC-TCA-GAC-ACT-TG-3'; SEQ ID
30 No. 25) which add BamHI and EcoRV restriction site recognition sequences to the 5'&3' ends respectively) from the full length copy and subcloned into the S. cerevisiae expression plasmid pACES14. The insert was sequenced to ensure that no mutations that would result in incorporation of altered amino acids had been incorporated during amplification and cloning. The human MCT4 ORF was then inserted into the insect expression vector pIZv5HIS (Invitrogen) and the mammalian expression vector pcDNA3. Plasmid DNAs, pACES14- hMCT4, pIZ-hMCT4, and pcDNA3-hMCT4 were purified and used to transform relevant host cells. Ηie amino acid sequence of human MCT4 is depicted in SEQ ID NO: 4.
3.6 Human MCT1/MCT2 chimera The human MCTl and MCT2 cDNAs share a common recognition site for the restriction enzyme Hind III. This restriction site hes in the region that encodes the extracellular domain that separates the predicted transmembrane domains 5 and 6. The common Hind III site was used to create a human MCT1/MCT2 chimeric molecule, in the S. cervisiae expression plasmid pACES 14, consisting of the amino terminus of MCT2 (TMs 1- 5) and the carboxy terminus of MCTl (TMs 6-12) SEQ ID 40. Plasmid DNA, ρACES14- hMCT2/MCTl, was purified and used to transform relevant host cells. A series of further chimeric molecules were constructed by replacing parts of MCTl with MCT2. Each of these bound hgand in the latter binding studies to greater or lesser extent. Example 4 - Expression of MCT proteins in host cells The human breast cell line MDA-MB-231 had been previously identified as expressing low levels of MCTl (Garcia et al., Cell. 76:865-873, 1994). The MDA-MB-231 cell line was grown. Filter binding assays (see Examples 6 and 7) carried out with the MDA- MB-231 cell line showed that the cells exhibited low level of binding to 3H-ligand C (-6000 binding sites per cell). The cells were grown and transfected withpcDNA3-hMCTl. An increase in filter binding of 3H-hgand C was consistently measured for transiently transfected cells when compared with control cells transfected with empty vector. The rat pancreatic β-cell line INS-1 has also been shown to exhibit low levels of lactate transport activity (Sekine et al, J. Biol. Chem 269:4895-4902, 1994) and has been shown to express low levels of MCT proteins (Ishihara et al, J. Clinical Invest. 104:1621- 1629,1999; Zhao et al, Diabetes. 50:361-366, 2001). INS-1 cells were grown and transfected with pcDNA3-hMCTl . An increase in filter binding of 3H-hgand C was consistently measured for transfected cells when compared with control cells transfected with empty vector. Filter binding assays carried out with the insect cell line Sf9 showed that the cells exhibited low level of binding to 3H-ligand C. The Sf9 cell line was grown and transfected with pIZ-hMCTl. An increase in filter binding of H- hgand C was consistently measured for transfected cells when compared with control cells transfected with empty vector. The yeast strain used for expression of MCTs was Saccharomyces cerevisiae Hansen BY4742 (Research Genetics) Mat alpha his3Dl leu2D0 lys2D0 ura3D0 Δjen:Kanr. Yeast cells were made competent for DNA transformation and transformed with plasmid DNA using the Yeast Transformation Kit (SIGMA) according to the manufacturer's instructions. Expression of human MCTs was confirmed by Westem analysis using the corresponding anti- human MCT C-terminal peptide antibody. Antibody generation: Anti-human MCT1-4 C-terminal peptide antibodies were made by Cambridge Research Biochemicals. Peptide sequences used to immunise rabbits are as follows: Table 4.
Three single amino acid variants of human MCTl (Lys204Glu, Cys400Gly, Glu490Asp) were expressed in yeast cells, and membrane preparations containing these three variants were tested in filter binding assays. No alteration to compound binding was detected when compared to human MCTl expressed in yeast. Single nucleotide changes that would result in the desired amino acid changes were incorporated into pACES-hMCTl using the QuickChange Site-Directed Mutagenesis Kit (Stratagene). Incorporation of a mutated nucleotide residue that would result in an altered amino acid was confirmed by sequence analysis. Example 5 - Clonriψ of a Strep tag to the C-terminus of MCT proteins. The DNA sequence encoding the Strep Tag, AWRHPQFGG (SEQ ID No. 30) (Schmidt and Skera, 1993, Protein Engineering 6:109-122) was cloned and expressed at the C-teiminus of hMCTl, hMCT2 and hMCT3, producing the polypeptides depicted in SEQ ID Nos: 37 to 39 respectively. MCT1 A three way ligation containing ρACES14 (BamHI/Nhel), the 5' end of hMCTl (BamHI/BspEl digested pACES14-MCTl) and annealed ohgonucleotides Mlstrρ-1 (5'- CCG-GAC-CAG-AAA-GAC-ACA-GAA-GGA-GGG-CCC-AAG-GAG-GAG-GAA-AGT- CCA-GTC-GCT-TGG-AGA-CAT-CCA-CAA-TTT-GGT-GGT-TAA-T-3'; SEQ ID No. 31) and Mlstrp-2 (5'-CTA-GAT-TAA-CCA-CCA-AAT-TGT-GGA-TGT-CTC-CAA-GCG- ACT-GGA-CTT-TCC-TCC-TCC-TTG-GGC-CCT-CCT-TCT-GTG-TCT-TTC-TGG-T-3'; SEQ ID No. 32) encoding the C-terminus of MCTl fused to the DNA encoding the strep-tag was carried out to generate the plasmid pACES14-MCTl- strep tag. Expression of the tagged material was confirmed by Western blotting using anti-hMCTl Abs and a streptavidin- horseradish peroxidase conjugate (IB A GmbH). Ability of a yeast membrane preparation containing the strep tagged MCTl to bind various radioligands (3H-ligand C, 125I-hgand A) was tested and confirmed in the filter binding assay. Yeast membrane preparations containing the strep tagged MCTl were then used to define conditions for an 384-well SPA assay using radiohgand (125I-ligand A) and streptavidin-coated PVT SPA beads (Amersham Pharmacia Biotech), as described below.
MCT2 A three way ligation containing pACES14 (BamHI/Nhel), the 5' end of hMCT2 (BamHI Dralll digested ρIZ-MCT2) and annealed ohgonucleotides FM2strep2 (5'-GTG- TAA-CCT-CAG-AAA-GAG-AAA-CTA-ACA-TTG-CTT-GGA-GAC-ATC-CAC-AAT- TTG-GTG-GTT-AAT-3'; SEQ ID No. 33) and RM2strep2 (5'-CTA-GAT-TAA-CCA-CCA- AAT-TGT-GGA-TGT-CTC-CAA-GCA-ATG-TTA-GTT-TCT-CTT-TCT-GAG-GTT- ACACTCT-3'; SEQ ID No. 34) encoding the C-terminus of MCT2 fused to the DNA encoding the strep-tag, was carried out to generate the plasmid pACES 14-MCT2-strep tag. Expression of the tagged material was monitored by Western blotting using anti-hMCT2 Abs and a streptavidin-horseradish peroxidase conjugate (IB A GmbH).
MCT3 The 3 ' end of hMCT3 (from Topo-MCT3) was amplified in a PCR reaction with the primer pair Mct3E-2 (5'-GCCATCCTGCTGGTGAACTA-3'; SEQ ID No. 35) and M3-3st (5'-TAG-CTA-GTC-TAG~ATT-AAC-CAC-CAA-ATT-GTG-GAT-GTC-TCC-AAG-CTA- CAG-ACT-CGG-CAG-CCA-GCC-TCG-GCC-TCG-CC-3'; SEQ ID No. 36). The PCR fragment contains the 3' end of hMCT3 fused to the DNA encoding the strep tag. The PCR product was digested with Notl and Xbal and placed in a three-way ligation with pACES14 BamHI/Nhel and the 5' end of hMCT3 (released fromTopo-MCT3 digested with BamHI/Notl), to generate ρACES14-hMCT3 strep tag. Expression of the tagged hMCT3 was monitored by Western blotting using anti-hMCT3 Abs and a streptavidin-horseradish peroxidase conjugate (IB A GmbH). Ability of a yeast membrane preparation containing the strep tagged MCT3 to bind various radio hgands (3H-hgand C, 125I-hgand A) was tested in the filter binding assay. Example 6 - Yeast expressed MCTl filter binding assay Assay to measure the potency of selected compounds using a filter binding assay Competition assays can be used to measure the affinity of unlabelled compound for MCTl. Tritiated hgand C is included at a constant concentration and the compound to be tested is titrated. lOμl of 3H-hgand C that had been diluted with assay buffer (50mM HEPES, O. lmM EDTA, 0.15 M NaCI, pH 7.5, 0.5% BSA) was dispensed into wells of a polypropylene plate such that when the assay was made to 200μl the final concentration would be 2.5nM. lOμl of compound, in assay buffer, was added to each well to give a final concentration typically covering the range 0.1 to lOOOnM. 180μl of yeast membranes expressing MCTl in assay buffer containing typically 0.5 to lμg total protein was added to start the reaction. Non-specific binding was measured in the presence of 1 μM unlabelled Ligand 1. The competition assay was incubated at 2 hours at room temperature with shaking. Experimental data points were usuaUy carried out in triplicate. The membranes were harvested onto GF-B filter plates and washed with assay buffer without BSA, dried, scintillant added and counts detected using a tritium program on a Packard Top Count plate reader. The results were analysed by subtraction of the non-specific binding from each of the experimental points and then fitting a sigmoidal curve through a semi- log plot of the data in Microcal Origin. Calculated IC5o's are shown in Table 5.
Table 5. Compound IC50s calculated from the yeast membrane MCTl filter binding assay.
Key: Ligand 1: (disclosed in WO 98/054190) 5-[(3-hydroxypropyl)thio]-3-methyl-l-(2- methylpropyl)-6-(l-napthalenylmethyl)t eno[2,3-d]pyrimidine-2,4(lH,3H)-dione Ligand 1 appears in patent WO 98/054190 and has CAS number (Chem Abs Registry No) 5 216685-07-3. Ligand 2: (disclosed in WO 99/029695) 2,6-dihydro-7-[(3-hydroxypropyl)thio]-2-methyl-4- (2-methylpropyl)-6-(l-napthanlenylmethyl)-lH-pyrrolo[3,4-d]pyridazin-l-one Ligand 2 appears in patent WO 99/29695 and has CAS number 227321-12-2. Ligand 3: (disclosed in WO 98/054190) 6-(4-quinolinylmethyl)- -3-methyl-l-(2- 0 methylpropyl)-thieno[2,3-d]pyrimidin-2,4(lH,3H)-dione Ligand 4: (disclosed in WO 00/12514): 6-([benzotlιiazol-2-yl]methyl)-3-methyl-l-(2- methylpropyl)thieno [2,3-d]pyrimidine-2,4( lH,3H)-dione Example 7 - MCT filter binding assay For yeast cells transformed with expression plasmids containing the human MCT 1-4 5 ORFs the ability of membrane preparations to bind radiohgand was determined using a single ligand concentration/ multi protein concentration binding assay. This generated an estimate of the amount of active binding protein present in each membrane preparation. The hgand concentration used will give an estimate of Bma that is approximately 90% of the true value. lOμl of hgand A, B or C that had been diluted 50 fold with assay buffer (50mM HEPES,0 0. ImM EDTA, 0.15 M NaCI, pH 7.5, 0.5% BSA) was dispensed into wells of a polypropylene microtitre plate. This gives a final radiolabelled ligand concentration of ~2.5nM. Non-specfic binding was measured in the presence of 1 μM Ligand 1. The membranes were diluted to give a known amount/180μl assay buffer. This is typically in the 2000nl to 0.2nl range. For the membranes with high binding site number a more dilute5 membrane preparation should be added. 180μl of each membrane solution was then added to each well and incubated at 2 hours at room temperature with shaking. The membranes were harvested onto GF-B filter plates and washed with assay buffer without BSA, dried, scintillant added and counts detected using the relevant program for each hgand on a Packard Top Count plate reader. Results from a typical experiment are shown in Table 6.0 Table 6.
The filter binding assays of Examples 6 and 7 are suitable for high throughput screening of large compound hbraries. Example 8 - Yeast expressed MCTl based SPA assay Scintillation Proximity Assay Method
Final conditions: 15 μg beads/ well 3 μg yeast membranes /well 0.1nM 125I hgand A 50mM HEPES pH 7.5, 0. ImM EDTA, 150mM NaCI + 0.05% BSA
Streptavidin coated SPA beads were resuspended at 2.5mg/ml in assay buffer (50mM HEPES pH 7.5, 0. ImM EDTA, 150mM NaCI) and diluted to give a final concentration of 187 μg/ml in assay buffer without BSA. Yeast membranes expressing MCTl with a streptavidin- binding sequence tag were then added to give a final concentration of 37 μg/ml protein and gently rolled for > 30 minutes at room temperature. The beads/membranes were then washed by centrifugation at ~650g for 10 minutes and resuspended in assay buffer + 0.05% BSA. The washing was repeated once before resuspended in the appropriate volume of assay buffer + 0.05% BSA. The iodinated hgand A was diluted from the stock into assay buffer + 0.5% BSA to give a concentration of InM. Non-specific binding was measured in the presence of Ligand 1 5 at lμM. Compound ICsoS were measured in triplicate over a 100,000-fold dilution range with individual dilutions at half log units. lOμl of diluted hgand was added to each well, lOμl of cold competitor hgand 1 was added to the non-specific control wells and lOμl of buffer added to the total counts control wells. Finally, 80μl of the membrane/bead suspension was added to each well. The plates were sealed, incubated at room temperature for 3 hours, centrifuged at 10 650g for 5 minutes before counting in a Packard Top Count plate reader using a protocol appropriate for I . The raw counts were analysed by averaging rephcates, subtraction of non-specific binding and the subsequent calculation of % inhibition of binding of the iodinated hgand. ICso's were calculated using Origin data fitting software and are shown in Table 7.
15 Table 7. Compound IC50s calculated from the yeast membrane MCTl-strep tagged SPA assay.
This SPA assay is suitable for high throughput screening of large compound libraries.
20 Example 9 - Uptake of labelled lactate in rat red blood cells Ligand 1 was dissolved at a concentration of lOmM in DMSO and diluted in assay buffer (50mM HEPES (pH 7.5), ImM EDTA, 150mM NaCI) supplemented with 0.5% (w/v) BSA. lOOx stock solutions were made for each concentration and 2 μl of these stocks diluted into 200 μl of rat blood to give the final compound concentration. The blood samples were
25 incubated at room temperature for 2 hours. Lactate uptake was measured in each blood sample as follows: uptake was initiated by the addition of 50 μl of blood to 2 μl of 14C-lactate (12.5 μCi/ml; Amersham). The samples were incubated at room temperature for 30s and then the reaction was halted by transfer of 20 μl of each sample onto 1 ml of ice-cold dibutyl- pthalate (Sigma). The red blood cells were separated from the plasma by centrifugation of the samples for 30s at 15000g in a bench-top microfuge. The supernatant was aspirated to waste taking care not to disturb the cell peUet. The cells were resuspended in 100 μl of 5% NP40 (v/v) in PBS and were transferred to a scintillation vial. 4 ml of scintillant was added and radioactivity was determined by scintillation counting. MCTl inhibitors, such as Ligand 1, caused a dose-dependent decrease in the amount of [14C]-lactate uptake by rat red blood ceUs. Values from representative experiments were: Table 8.
This method was also used to determine the effect of MCTl inhibitors, including Ligand 1, on lactate uptake in human red blood cells and similar results were obtained. Example 10 - Scintillation proximity assay (SPA) using Jurkat T-cell membranes Human Jurkat T ceUs were grown at 37°C in RPMI 1640 medium supplemented with 5 % foetal calf serum, 2mM glutamine. Cells were harvested by centrifugation at 1500g for 10 minutes. The cell pellets were washed twice with phosphate buffered saline (PBS), and centrifuged as above. The final cell pellet was resuspended in lysis buffer (50mM HEPES (pH 7.8), 50mM KC1, 10% glycerol, 0.1 mM EDTA, ImM DTT) and lysed by nitrogen cavitation. Unlysed ceUs and nuclei were removed by centrifugation at 1500g for 10 minutes. The supernatant was then centrifuged at 100,000g for 30 minutes at 4°C, the pellet resuspended and homogenised in assay buffer (50mM HEPES (pH 7.5), ImM EDTA, 150mM NaCI). Aliquots of membrane preparation were stored at -80° C until use. Frozen Jurkat cell membranes were thawed on ice and then homogenised. Wheatgerm agglutinin-linked Scintillation Proximity Assay beads (Amersham) were rehydrated in assay buffer to a concentration of 100 mg/ml. 2ml of Jurkat membranes were added per 0.5 ml (50 mg) of SPA beads and incubated overnight with constant agitation to allow the beads to coat with Jurkat membranes. The coated beads were then collected by centrifugation at 1500g for 5 minutes and were washed twice in large volumes of assay buffer before being resuspended to a final concentration of 10 mg/ml in assay buffer. 125I-hgand A was prepared as described above at a maximum specific activity of 2000 Ci mmol"1. The radiohgand was diluted in assay buffer containing 0.5%(w/v) BSA and the final concentration in the assay was approximately 0.1 nM. Assays were set up in 96-well flat-bottomed white opaque plates (Costar. Cat No: 5 3912). lOμl of test compound and lOμl of radiohgand were incubated with 180μl of SPA beads and membranes (0.04mg beads). Non-specific binding was determined in the presence of lμM Ligand 1 and total binding was determined in the presence of vehicle alone. The plates were incubated for 3 hours at room temperature before quantitation of radioactivity proximal to the SPA beads by scintillation counting. 0 Compounds including Ligand 1 caused a dose-dependent reduction in the specific binding of 125I-hgand A to Jurkat T-ceU membranes. Values from a representative experiment were: Table 9.
5 The mean Ki for Ligand 1 competition of 125I-ligand A binding to Jurkat T-cell membranes was 0.074nM (n=100). Example 11 - Effect of MCTl inhibitors on T-lymphocytes Our studies have shown that the rate of lactate production by T-lymphocytes increases approximately 15-fold by 48h after mitogenic stimulation (PMA/ionomycin). Western0 blotting analyses of stimulated PBMCs using Abs that recognise MCTl, MCT2 and MCT4 showed that these three MCTs are expressed 48 hours after stimulation. Compounds which bind to MCTl with potencies in the region of 0.05-300nM have been shown to cause a significant accumulation of intracellular lactate in the T-lymphocytes and a reduction in the amount of lactate in the extracellular medium The potency of compound effects on lactate5 accumulation shows a significant correlation with inhibition of T-lymphocyte proliferation as measured by the rate of incorporation of [ Hjthymidine into DNA. Data from compound activity in an SPA binding assay, 3-day lymphocyte prohferation assay and experiments on T- lymphocytes after 48h of mitogenic stimulation (lactate levels and DNA synthesis) are shown. Assay of T-lvmphocyte prohferation In vitro, the ceU signalling pathways triggered by T-ceU activation through the T ceU receptor and CD28 can be mimicked using a phorbol ester, phorbol 12-myristate 13-acetate (PMA), which activates protein kinase C, and ionomycin, which induces calcium release from internal ceUular stores. PMA and ionomycin- stimulated prohferation of peripheral blood mononuclear cells (PBMC), of which the predominant ceU type is T-lymphocytes, provides a suitable assay for measuring the ability of small molecules to block T-ceU proliferation. 100ml blood was coUected by venopuncture of normal human volunteers into 3 tubes each containing 3ml of 3.2% tri-sodium citrate solution. Blood was centrifuged at 850g for 10 minutes and the plasma was removed. The cells were dUuted to 50ml with RPMI 1640 medium, and each 30ml of diluted blood was layered over 20ml Lymphoprep (Nycomed). The blood/Lymphoprep layers were centrifuged at 850g for 20 minutes at 18°C (with no brake). CeUs at the interface were removed and were washed in RPMI 1640 by centrifugation at 850g for 10 minutes. The ceU peUets were then combined and were washed with 2x50ml RPMI1640 at 680g for 7 minutes. CeUs were resuspended to a concentration of 1 x 10 ceUs/ml in RPMI 1640 medium supplemented with 10% human AB serum (Quest Biomedical), L-glutamine (2mM) and antibiotics (50μg penicUlin and streptomycin) (Complete medium). Test compounds were dissolved in DMSO to give lOmM stock solutions and were then diluted in complete medium to 20x the final assay concentration. lOμl of compound in solution was then added to the 96-weU flat bottom assay plate (final volume of 200μl). Compounds were tested at a range of concentrations from 1 x 10"11 M to 1 x 10"6 M. PMA and ionomycin were obtained from Sigma and made up to lmg/ml in DMSO. Both were dUuted to 4x the final assay concentration in complete medium. 50μl of each reagent was dispensed per weU to give a final assay concentration of 0.5ng/ml PMA and 500ng/ml ionomycin. Control weUs received lOOμl cells with PMA only (negative control 1), with ionomycin only (negative control 2) and lOOμl cells with 50μl PMA and 50μl ionomycin with no test compound (positive control). lOOμl complete medium was added to the negative controls. The plates were incubated at 37°C for 72h and the cultures were pulsed with 3H-thymidine (0.5μCi/well; Amersham) for the final 6 hours. CeUs were harvested on to glass fibre filter mats using a 96-weU harvester (Tomtec inc., Orange, USA) and incorporated radioactivity was determined using a 1450 Microbeta counter (Perkin Elmer Life Sciences, Cambridge, England). MCTl inhibitors, including Ligand 1, caused inhibition of T-ceU proliferation with maximal inhibition of approximately 60%. IA50 values were obtained from dose response curves using the 4-parameter logistic fit of a data analysis program IA50 values are defined as the concentration of compound giving 50% of the maximum possible inhibition and were obtained from dose response curves using the 4-parameter logistic fit of a data analysis program Experiments on activated T-lymphocytes PBMCs were prepared as described above by separation over Lymphoprep and T- lymphocytes were then enriched by purification on a nylon wool column. Briefly, 0.6g nylon wool was inserted into a 10ml syringe and this was autoclaved. The column was equilibrated with RPMI 1640 containing 20% human serum (HS) for 30 min at 37°C. The PBMC were resuspended in 1ml of pre-warmed RPMI (20% HS) and were loaded onto the nylon wool column. The column was incubated for 45min at 37 °C foUowed by elution of the T-ceUs by adding 10ml RPMI (20% HS) (prewarmed to 37°C) dropwise to the column. The T-ceUs obtained from the elution of the column were centrifuged (850g for 5min) and resuspended in RPMI containing L- glutamine and 5% human serum and stimulated with 0.5ng/ml PMA and 500ng/ml Ionomycin for 48h. T-ceUs were maintained in this growth medium at an initial cell density of 1x10s ceU per ml under standard ceU culture conditions (37°C, 5% CO2). After 48h, the ceUs were harvested and prepared for the lactate and DNA synthesis assays by washing twice in RPMI 1640 and resuspending in fresh growth medium at lxlO6 ceUs per ml. Assay of lactate levels by lactate oxidase enzyme activity a) Intracellular lactate levels: Test compounds were dissolved in DMSO to give lOmM stock solutions and were then diluted in complete medium to lOx the final assay concentration. lOOμl of each concentration were added to triphcate weUs of a 24-weU plate. T-ceUs were then added (1ml per well) and were cultured for 4h under standard ceU culture conditions. The cells were then transferred from each well to microfuge tubes and were centrifuged at 360g for 5min at 4°C. The supernatant was discarded and the ceU pellet was resuspended in 1ml of ice-cold PBS, pH5.0. The ceUs were washed twice in PBS by centrifugation (360g, 5min, 4°C) foUowed by resuspension of the ceU pellet in lOOμl deionized water. The ceUs were incubated for 15min at 4°C to allow cell lysis to occur and the ceU debris was removed by centrifugation at 15000g for 10 min at 4°C. lOμl of each supernatant sample were used for the lactate determination. b) ExtraceUular lactate levels: Test compounds were dissolved in DMSO to give lOmM stock solutions and were then diluted in complete medium to lOx the final assay concentration. 20μl of each concentration were added to triphcate weUs of a 96-weU plate. T- ceUs were then added (200μl per weU, 2x10 cells) and were cultured for 4h under standard ceU culture conditions. The cell supernatants were coUected from each well after the ceUs were peUeted by centrifugation of the plate at 850g for 5min at 4°C. lOμl of each supernatant sample were used for the lactate determination. Lactate Reagent (Sigma, catalogue No. 735-10) was reconstituted according to the manufacturer's instructions. L-lactate (Sigma) was prepared as a lOOmM stock solution in PBS, pH 7.5 and was dUuted in distilled water to give a standard curve in the range of 12.5- 200μM. The assay was carried out in 96-weU plates at room temperature. 10 μl of standard or sample were added to each weU foUowed by 200μl of the Lactate Reagent. The plate was incubated for 15 min and absorbance at 540nm was then determined using a SPECTRAmaxPlus spectrophotometer (Molecular Devices). The data were coUected and analysed using SOFTmax PRO software (Molecular Devices). Assay of DNA synthesis in activated T-lymphocytes DNA synthesis was assessed by measuring the incorporation of [3H]-thymidine. The assay was carried out in a 96-weU plate. 20μl of test compounds at lOx the final concentration were added to the plate foUowed by 200μl of T-ceUs per weU (2xl05 cells). The ceUs were cultured for 4h under standard ceU culture conditions and cultures were pulsed with 3H- fhymidine (0.5μCi/weU; Amersham) for the final hour of the incubation. CeUs were harvested on to glass fibre filter mats using a 96-weU harvester (Tomtec inc., Orange, USA) and incorporated radioactivity was determined using a 1450 Microbeta counter (Perkin Elmer Life
Sciences, Cambridge, England).
Results Compounds caused a dose-dependent decrease in the rate of [3H] thymidine incorporation and extraceUular lactate concentration in 2 day activated T-lymphocytes. The maximum level of inhibition caused by Ligand 1 was 48.6 + 2.1 % in the assay of [3H] thymidine incorporation and 30 ± 1.9 % inhibition in the assay of extraceUular lactate. These values are representative of the maximum inhibition levels observed for the compounds shown below. Ligand 1 caused a dose-dependent increase in the intraceUular lactate concentration from below the limit of detection (6.25 nmoles per 106 ceUs) to concentrations in the range of 26 - 37 nmoles per 106 ceUs. Mean + s.e.mean of pIA50 values (mean ± range for n=2) Table 10.
methoxy-N,3-din ethyl-l-(2-methylpropyl)-2,4-dioxo-tMeno[2,3-d]pyrimidme-5-carboxamide Ligand 5 appears in International patent Application Number PCT/GB02/03250 Example 12 - Inhibition of B-lymphocyte proliferation Splenic B-lymphocytes were obtained from Balb/c mice by disruption of the spleen through a nylon sieve. The resultant ceUular suspension was washed three times by centrifugation. The spleen cells were then plated out in flat-bottomed 96 weU microtitre plates at a concentration of 2-4x105 ceUs per weU. B-lymphocyte stimulators were added with or without compound. Stimulators employed were lipopolysaccharide (LPS) at 50μg/ml, 8- mercaptoguanosine (8MG) at lOOμg/ml, or goat anti mouse IgM at 40μg/ml. Ligand 2, a compound with MCT inhibitory activity was added to the cultures in a concentration range of 10"10M to 10"5M. The cultures were incubated for 48 hours at 37°C and pulsed with tritiated thymidine for the final 6 hours. The ceUs were harvested as described previously and thymidine uptake used as a measure of DNA synthesis. Ligand 2 partially inhibited the prohferative response of the B-lymphocytes to LPS (IA50 = 5X10"9 M) , and 8MG (IA50 = 10"8M ). Inhibition of the proliferative response to goat anti-mouse IgM was weak (IA50 = > 10"7M). Example 13 - In vivo activity of compounds Graft versus Host Response Compounds have been tested in the rat Graft versus Host Response (GVHR), which represents the immune elements associated with transplant rejection. The model was first described by Ford et al 1970. The assay consisted of injecting a lOOul volume 5x10 - 1x10 spleen ceUs from dark agouti (DA) rats into the right hind footpads of D A/Lewis FI hybrid rats. A similar number of D A/Lewis spleen cells were injected into the right hind footpads to act as controls. The DA grafted ceUs were recognised by the recipient DA/Lewis rats as having "self antigenic components whereas the DA graft ceUs recognised the "Le" elements of the FI hybrids as being foreign and subsequently responded by a prohferative response. The increase in prohferation was measured by an increase of weight of the right lymph node compared to the control left lymph node. When the rats were dosed with compounds active in the MCT binding screen and the in vitro proliferation assays they were found to effectively inhibit the development of the GVHR. Compounds were dosed either once or twice daily from the day of ceUular challenge in the footpads untU termination 7 days later. The most active compounds tested gave an ED50 of 1-3 mg/kg dosed by the subcutaneous route. Inhibition of murine antibody production Balb/c mice were immunised with 0.5mg/kg ovalbumin (OVA) and 200mg/kg aluminium hydroxide gel in saline intraperitoneaUy and left for 29 days before being re- challenged intraperotoneaUy with 0.5 mg/kg ovalbumin in buffered saline vehicle and left for a further 10 days. Compound was dosed daily from re-challenge to termination prior to serum coUection. Control groups were dosed with compound vehicle only. On completion of dosing serum samples were taken from the mice and analysed for total and specific levels of IgE. For total IgE, microtitre plates were coated with 5ug/ml monoclonal rat anti-mouse
IgE in phosphate buffered saline (PBS) and incubated overnight at 4°C. The plates were then washed four times with PBS containing 0.05% Tween 20 and then blocked with 1% BSA in PBS at room temperature for 2 hours. This was foUowed by two further plate washings. The serum samples and IgE standards were added to the weUs in duplicate and incubated overnight. The plates were then washed a further four times before adding 50ul biotinylated monoclonal rat anti-mouse IgE appropriately diluted in 0.1% BSA in PBS for 2 hours. The plate was washed for a further four times before adding 50ul streptavidin alkaline phosphatase conjugate appropriately dUuted in 1% BSA in PBS at room temperature for 50 minutes. The plate was washed a further four times before enzyme substrate (paranitrophenyl phoshate in IM diethanolamine buffer pH 9.8) at lmg/ml was added. Once the colour reaction developed, the plate was read at 405nm Levels of total IgE in serum samples were extrapolated from curve obtained with IgE standards. For specific IgE, the methodology was similar except for foUowing. The microtitre plates were coated with 50ug/ml rat anti-mouse IgE. The serum samples or normal control sera were added to the washed plates. Biotinylated OVA diluted in 0.1% BSA in PBS was used as enzyme marker. The activity of the MCT inhibitor Ligand 1 dosed at 3 and 30mg/kg by the subcutaneous route for 10 days foUowing antigenic boost was 67% and 84% inhibition respectively for total IgE and 64% and 53% inhibition respectively for OVA specific IgE. To measure effects of compound on IgG2a production, Balb/c mice were immunised with ovalbumin in poly I:C adjuvant (polyinosinic:polycytidylic acid adjuvant) and left for 14 days before being re-chaUenged with ovalbumin in buffered saline vehicle and left for a further 7 days. Serum samples were taken from the mice and analysed for total and specific IgG2a. Compound was dosed dahy from re-challenge to termination prior to serum coUection. For total IgG2A, microtitre plates were coated with 5ug/ml of goat anti-mouse IgG2a in PBS and incubated overnight at 4°C. The plates were then washed four times with PBS containing 0.05% Tween 20 and then blocked with 1% BSA at room temperature for 2 hours. This was foUowed by two further plate washings. The serum samples and IgG2a standards were added to the weUs in duplicate and incubated at 4°C overnight. The plates were then washed a further four times before adding 50ul alkaline phosphatase conjugated goat anti-mouse IgG appropriately diluted in 0.1% BSA in PBS and left at room temperature for an hour. The plate was washed a further four times before enzyme substrate (p-nitrophenyl phosphate in IM diethanolamine buffer pH 9.8) at lmg/ml was added. Once the colour reaction developed, the absorbance of the weUs at 405nm was read in a spectrophotometer. Levels of total IgG2a in serum samples were extrapolated from the curve obtained with IgG2a standards. For specific IgG2a, the methodology was similar except for foUowing. The microtitre plates were coated with 50ug/ml OVA. The serum samples or normal control sera were added to the washed plates. Alkaline phosphatase conjugated goat anti-mouse IgG2a was used as enzyme marker. The activity of the MCT inhibitor Ligand 1 at 3 and 30mg/kg dosed by the subcutaneous route for 7 days foUowing antigenic boost on total and specific IgG2a production gave ED50's of 30mg kg and lOmg/kg respectively. Example 14 - Expression and prohferation studies in tumours and tumour ceU lines Studies on the expression of MCTl and MCT4 in tumour tissues and cell lines have been carried out. Western blotting (using the antibodies described in Example 4) has shown expression of MCTl protein in the human erythroleukaemia cell line K562 and the human colorectal tumour ceU line LoVo. MCT4 protein expression was observed in the human colorectal Colo205 ceU line. Expression data was also extracted from the GeneExpress database to assess disease association of MCTl and MCT4. Three Affymetrix probesets were compared for MCTl and a single probeset for MCT4. Both MCTl and 4 showed disease association. For MCTl a fold change increase was seen across aU probesets in cervix squamous cell carcinoma and skin malignant melanoma, which was supported by significant increases in cell ratio. A small fold change decrease is seen in colon and stomach adenocarcinoma. Corresponding significant fold changes are seen across most stages of colorectal and lung tumours. A slight trend for increasing expression with stage is seen in NSCLC. This may therefore offer up MCTl and MCT4 as biomarkers of disease. For MCT4 strong fold change increases were seen in kidney and lung tumours, cervix squamous cell carcinoma, endometrial tumours, bladder transitional cell and thyroid papUlary carcinomas, and ovary adenocarcinoma, most showing corresponding increases in call ratio. Small fold change increases are seen in stomach and colon adenocarcinoma with corresponding increases in call ratio, and colon mucinous, esophagus and rectum adenocarcinomas. Significant increases in call ratio are also seen in breast tumours, skin malignant melanoma, pancreas adenocarcinoma and hver hepatoceUular carcinoma. Corresponding increases in fold change are seen across most stages of colorectal adenocarcinoma and NSCLC, and increases caU ratio across breast stages. A trend is seen for increased expression in NSCLC with stage, but this was not found to be statisticaUy significant. The anti-proliferative effect of MCTl inhibition was investigated in the human K562 tumour ceU line. K562 cells were cultured in RPMI1640 medium (Gibco) supplemented with 2mM L-glutamine and 5% foetal calf serum (FCS) (complete medium). Ligand 1 was dissolved at a concentration of lOmM in DMSO and was diluted in absolute ethanol to 20x the final concentration required in the assay. lOμl of the dUuted solutions were then added to the weUs of a 96-weU microtitre plate and the ethanol was allowed to evaporate. The K562 ceUs were diluted to IxlO5 ceUs per ml in complete medium and lOOμl was added to each weU of the plate with the addition of a further lOOμl of complete medium to give a total volume of 200μl. The plates were incubated for a total of 48h at 37°C in 5% C02 with the addition of AlamarBlue (Serotec) for the last 24h AlamarBlue is an oxidation-reduction indicator dye, which monitors metabohc activity (high level of reduction) as a readout of ceUular proliferation. Absorbance was measured at 600nm (OD60o; oxidised form) and 570nm (OD570; reduced form) using a Spectromax spectrophotometer (Molecular Devices). The maximum level of proliferation (OD57o- OD6oo) was determined in the absence of Ligand 1. Ligand 1 caused a significant reduction in OD57o - OD6oo with a mean pIA50 of 8.7 ± 0.2 (n=3). Example 15 -Lactate uptake studies in cells expressing MCT isoforms by measurement of changes in intracellular pH 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein (BCECF) is a pH- sensitive dye that has been used by Wang et al. (Am J. Physiol. 267 (Heart Circ. Physiol. 36): H1759-69, 1994) to measure changes in intraceUular pH of single cells. As lactate entry into ceUs is proton-linked, the addition of exogenous lactate to ceUs expressing functionally active MCT isoforms results in a significant decrease in intracellular pH. Lactate uptake by this method can be measured in cells endogenously expressing MCT isoforms (e.g. K562 and MDA- MB231 ceUs) or in cells transfected with cloned MCT isoforms (e.g. MCTl, 3 and 4 expressed in SF9 or INS-1 ceUs as foUows. The construction of plasmids comprising of human MCTl, 2, 3, and 4 inserted into the mammalian expression vector pCDNA3 is described in example 3. INS1 ceUs were transfected with pcDNA3-hMCTl using the
Fugene™ 6 Transfection Reagent (Roche Molecular Biochemicals, Idianapolis, IN, USA) to manufacturers instructions. Stably transfected ceUs were selected with lOOμg/ml Genetecin (Gibco Laboratories, Grand Island, NY), to generate INS1 MCTl mixed populations, which were subsequently dilution cloned to generate INS1 MCTl clones. The same procedure was performed to generate mixed populations and clones for pCDNA3-hMCT2, pCDNA3- hMCT3 and PCDNA3-hMCT4. A 75cm2 flask of Ins 1 ceUs (clones or mixed populations) transfected with either pCDNA3-hMCTl, pCDNA3-hMCT2, pCDNA3-hMCT3, pCDNA3-hMCT4or pCDNA3.1 was washed with complete media (RPMI 1640 containing 10% FCS, 2mM glutamine, ImM sodium pyruvate, 0.00035% β-mercaptoethanol) and the ceUs removed by incubation with 3ml of accutase (Innovative CeU Technologies, La JoUa, CA, USA.) for 5 mins at 37°C. 3ml of complete media was added to each flask and the ceUs removed by gentle agitation. The ceU suspension was removed and centrifuged (465 x g for 5 min). The resultant ceU pellet was resuspended in 5ml of RPMI 1640 media without serum CeUs were counted using a haemocytometer and resuspended in RPMI 1640 media without serum at 1.0 x 106 ceUs/ml. CeUs were loaded with BCECF by addition of lμl of ImM stock solution per 1ml of cells to give a final concentration of lμM. The BCECF was incubated with the ceUs for 30min at room temperature in the dark. FoUowing loading with BCECF, the ceU suspensions were centrifuged (465 x g for 5min) and then washed twice in Tyrodes buffer (140mM NaCI, 4mM KC1, 0.2mM CaCl2, ImM MgCl2, lOmM HEPES, lOmM glucose, pH 7.4). The BCECF- loaded cells were then resuspended at a concentration of 1.0 x 107 ceUs/ml in Tyrodes buffer. FoUowing BCECF labelling, lOμl of the relevant ceU suspension was added to each weU of a 96 weU plate (Biocat Poly-D-Lysine coated clear-bottomed black plates, Becton Dickinson) and lOμl of inhibitor or vehicle control was added per weU at lOx final concentration. 90μl of Tyrodes or pH calibration buffer (140mM KC1, ImM MgCl2, 20mM HEPES, ImM EGTA, O.OlmM Nigericin at pH values from 5.39 to 8.44) was added to each weU. Plates were incubated for 60 min at room temperature in the dark. The 96 weU plate was then centrifuged at 275g for 5 mins to ensure that the ceUs formed a monolayer on the bottom of the plate. The 96 weU plate was then placed in the FLEXstation and fluorescence was measured using the foUowing wavelengths:
The experiment was set up so that a reading at time zero was made, the lactate added to the plate and then readings were made every 3 seconds for 3mins. L(+)-lactate (Sigma) was prepared at 3 times the final concentration by dilution of a IM lactate solution in Tyrodes buffer. 50μl dUuted lactate solution was then added to each weU. The ratio of fluorescence at 490nm/440nm was calculated and used to prepare a pH cahbration curve for weUs containing the pH cahbration buffer. The pH cahbration curve was then used to determine the pH of test weUs from the 490/440nm fluorescence ratio. The addition of exogenous lactate (0.3-300mM) caused a significant reduction in the intracellular pH of INS-1 ceUs expressing human MCTl, 3 or 4. No change in intraceUular pH in response to lactate addition was observed in untransfected INS-1 ceUs or in INS-1 ceUs expressing the pCDNA3.1 vector. lOOnM Ligand 2 completely abohshed the decrease in intracellular pH observed on addition of exogenous lactate to INS-1 ceUs expressing human MCTl (n=4). For INS-1 ceUs expressing human MCT3, lactate uptake was partially inhibited by lOOnM Ligand 2 and complete blocked by addition of lOμM Ligand 2 (n=2). lOμM Ligand 2 resulted in a significant but partial inhibition of lactate uptake in INS- 1 ceUs transfected with human MCT4 (n=3).

Claims

Claims:
1. A method for identifying a compound having therapeutic potential, comprising determining whether the compound inhibits cellular monocarboxylate transport activity.
2. A method for identifying whether a compound may have potential in treating an immune-mediated disorder or cancer, comprising determining whether the compound is capable of inhibiting ceUular monocarboxylate transport activity.
3. A method as claimed in claim 1 or 2, wherein the transporter protein effecting monocarboxylate transport activity is presented/located within a natural or synthetic ceU membrane.
4. A method for identifying whether or not a compound may have potential in treating an immune-mediated disorder or cancer, which comprises contacting cells expressing a monocarboxylate transporter, or ceU membrane preparations thereof, with a compound not known to be capable of inliibiting monocarboxylate transport, under conditions suitable for binding, and determining monocarboxylate transport activity.
5. An assay method for identifying compounds which inhibit monocarboxylate transport in a ceU, comprising: (a) contacting a cell or ceU lysate comprising a monocarboxylate transport polypeptide with a test compound; and, (b) detecting one or more of the foUowing characteristics: (i) the ability of the test compound to inhibit the ability of the monocarboxylate transport polypeptide to transport monocarboxylate, (ii) the ability of the test compound to bind to the monocarboxylate transport polypeptide, and (iii) the ability of the test compound to reduce the level of expression of the monocarboxylate transport polypeptide.
6. A competitive binding assay for identifying compounds that may have potential in treating an immune-mediated disorder or cancer, which comprises contacting host cells expressing MCT protein, or a membrane preparation thereof, with both a first test compound and a labelled second compound known to specifically bind to said MCT protein, under 5 conditions suitable for binding of both compounds, and detecting specific binding of the first compound to the MCT protein by measuring a decrease in the binding of the second compound, a decrease in the binding of the second compound to the MCT protein in the presence of the first compound indicating that the first compound binds to the MCT protein and may thus have potential in treating an immune-mediated disorder or cancer. 10
7. A method as claimed in claim 6, wherein the second compound is radiolabelled.
8. A method as claimed in any of claims 1 to 7, wherein the transporter protein effecting monocarboxylate transport activity is present within a cell selected from the group
15 consisting of: Jurkat, K562, HeLa, Chinese Hamster Ovary (CHO), INS-1 SF-9, K562 and MB231.
9. A method as claimed in any of claims 1 to 8, wherein the transporter protein effecting monocarboxylate transport activity is expressed from nucleic acid exogenously
20 introduced into a cell.
10. A method as claimed in any of the preceding claims, wherein the monocarboxylate transport activity is inhibited as a result of the compound specifically binding to the monocarboxylate transporter protein.
25 11. A method as claimed in any of the preceding claims, wherein the monocarboxylate transport activity is inhibited as a result of the compound impeding expression of the monocarboxylate transporter.
30 12. A method as claimed in any of the preceding claims, wherein inhibition of monocarboxylate transport activity is determined by measuring: ligand binding, monocarboxylate accumulation within the cell, monocarboxylate efflux from the cell, or, H+ efflux or accumulation.
13. A method as claimed in any of the preceding claims, wherein inhibition of monocarboxylate transport activity is measured by a method selected from the group consisting of: rapid filtration of equilibrium binding mixtures, radioimmunoassays (RIA), fluorescence resonance energy transfer assays (FRET), scintillation proximity assay (SPA), measurement of intracellular pH or the use of labelled substrates to measure transport.
14. A method as claimed in any of claims 1 to 13, wherein the monocarboxylate transporter (MCT) protein is selected from the group consisting of: MCTl, MCT2, MCT3 and MCT4.
15. A method as claimed in claim 14, wherein the MCT is of human origin.
16. A compound identified by a method as claimed in any of the preceding claims, provided the compound is not within any of Formulae I-IX.
17. A compound identified according to a method as claimed in any of the preceding claims, wherein the compound is at least ten times more active against one of MCTl, 2, 3or 4, as against any other of the four.
18. A method of producing a pharmaceutical composition which comprises admixing a compound identified according to a method as claimed in any of claims 1 to 15, or a derivative thereof, with a pharmaceutically acceptable carrier.
19. A method of producing a pharmaceutical composition which comprises determining whether a compound is an MCT inhibitor according to a method as claimed in any of claims 1 to 15; preparing derivative compounds of this 'parent' compound; testing these derivative compounds for their ability to inhibit monocarboxylate transport in a cell to identify a more active compound than the parent compound; and, mixing said more active compound identified therein with a pharmaceutically acceptable carrier.
20. Use of a compound that inhibits a monocarboxylate transporter other than a compound of Formulae I to IX, in the treatment of an immune-mediated disorder or cancer.
21. Use of an MCT inhibitor compound that inhibits a monocarboxylate transporter other than MCTl and MCT2, in the treatment of an immune-mediated disorder or cancer.
22. Use of an MCT inhibitor compound that inhibits a monocarboxylate transporter selected from the group consisting of: MCT3 and MCT4, in the treatment of an immune- mediated disorder or cancer.
23. Use of a compound that inhibits a monocarboxylate transporter other than a compound of formulae I to IX, in the manufacture of a medicament for the treatment of an immune- mediated disorder or cancer.
24. Use of an MCT inhibitor compound that blocks a monocarboxylate transporter other than MCTl and MCT2, in the manufacture of a medicament for the treatment of an immune- mediated disorder or cancer.
25. Use of an MCT inhibitor compound that blocks a monocarboxylate transporter selected from the group consisting of: MCT3 and MCT4, in the manufacture of a medicament for the treatment of an immune-mediated disorder or cancer.
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US20050032135A1 (en) * 2003-07-03 2005-02-10 Xenoport, Inc. Monocarboxylate transporters expressed in cancer cells
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WO2010089580A1 (en) 2009-02-06 2010-08-12 Astrazeneca Ab Use of a mct1 inhibitor in the treatment of cancers expressing mct1 over mct4
US10202350B2 (en) * 2015-06-12 2019-02-12 Vettore, LLC MCT4 inhibitors for treating disease
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