WO2010089580A1

WO2010089580A1 - Use of a mct1 inhibitor in the treatment of cancers expressing mct1 over mct4

Info

Publication number: WO2010089580A1
Application number: PCT/GB2010/050096
Authority: WO
Inventors: Susan Elizabeth Critchlow; Lorna Tate
Original assignee: Astrazeneca Ab; Astrazeneca Uk Limited
Priority date: 2009-02-06
Filing date: 2010-01-22
Publication date: 2010-08-12

Abstract

The invention relates to a novel method of treatment or prophylaxis of cancer, which comprises administering to a patient in need thereof a therapeutically effective amount of a compound which inhibits monocarboxylate transport.

Description

USE OF A MCT1 INHIBITOR IN THE TREATMENT OF CANCERS EXPRESSING MCT1 OVER MCT4

The present invention relates to a method of treating cancer by administering a compound capable of inhibiting monocarboxylate transport.

Monocarboxylate transporters (MCTs) 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. Direct demonstration of proton- linked lactate and pyruvate transports has been demonstrated for MCTl to MCT4. The nomenclature for the MCT family is taken from Halestrap and Price. Biochemical Journal (1999) 343: 281-299. MCTl and MCT4 have been shown to interact directly with CD147 (also known as basigin and EMMPRIN), a member of the immunoglobulin superfamily with a single transmembrane helix. CD 147 acts an essential chaperone to take MCTl and MCT4 to the plasma membrane where the transporter and CD 147 remain tightly associated (Kirk et al. (2000) EMBO J. 19: 3896-3904). PCT Published Patent Application WO2005/054852 shows that in T-lymphocytes, lactate efflux occurs via MCTl, as small molecule inhibitors of MCTl result in accumulation of intracellular lactate. Data disclosed in this published patent application also links the specific inhibition of human monocarboxylate transporters, in particular MCTl to MCT4, and modulation of lactate transport, with the inhibition of human cellular proliferation for the treatment of cancer

It is well established that tumours display altered metabolism (Kroemer and Pouyssegur (2008) 13: 472-482). Tumours are composed of well oxygenated (aerobic) and poorly oxygenated (hypoxic) regions. Compared to normal cells, tumour cells have an increased dependency on the glycolytic pathway for ATP generation either via aerobic glycolysis (the Warburg effect) or anaerobic glycolysis as a consequence of tumour hypoxia. Highly proliferating tumours and hypoxic tumours appear to be particularly dependent upon glycolysis to meet their energy and biosynthetic requirements. This fundamental difference in cellular metabolism between tumour cells and normal cells, termed the Warburg effect, has been used for diagnostic purposes, but has not yet been exploited for therapeutic benefit.

Widespread clinical use of FDG-PET (Fluorodeoxyglucose Positron Emission Tomography) - PET scanning with the tracer fluorine- 18_(F- 18) fluorodeoxyglucose (FDG), has demonstrated that this glycolytic phenotype is observed in range of solid and haematological tumours. The tracer, F- 18, is a glucose analogue that is taken up by glucose-using cells and phosphorylated by hexokinase_but cannot undergo further glycolysis. This means that FDG is trapped in any cell which takes it up, until it decays, since phosphorylated sugars, due to their ionic charge, cannot exit from the cell. This results in intense radiolabeling of tissues with high glucose uptake, such as the brain, the liver, and most cancers. As a result, FDG-PET can be used for diagnosis, staging, and monitoring treatment of cancers, particularly in Hodgkin's disease, non Hodgkin's lymphoma, and lung cancer. FDG-PET combined with computer tomography has a >90% sensitivity and specificity for the detection of metastases of most epithelial tumours (Mankoff et al. (2007) Clin. Cancer Res. 13:3460-3469).

A by-product of the increased glycolytic rates in tumours is the accumulation of lactate. Intracellular lactate can be transported out of tumour cells via the monocarboxylate transporters (MCTs 1, 2, 3 & 4) (Halestrap, AP and Price, NT. Biochem J. (1999) 343; 291-299). Lactate that is produced by tumour cells can be taken up by stromal and oxygenated tumour cells (via the monocarboxylate transporters MCTl and MCT2) to regenerate pyruvate that can be used to fuel oxidative phosphorylation (OXPHOS) (Koukouris et al., Cancer Res. (2006) 66; 632- 637; Sonveaux et al., J. Clin. Invest. (2008) 118; 3930-3942). It is reported that MCTl and MCT4 are over-expressed in colorectal tumours compared to normal epithelium (Pinheiro et al., Virchows Arch. (2008) 452; 139-146).

The molecular mechanisms that underlie metabolic reprogramming of cancer cells are complex. One of the key factors in driving the glycolytic phenotype of tumours is the activation of hypoxia-inducible factor (HIF), a transcription factor that is activated by hypoxic stress. Furthermore, deregulation of the MYC oncogene occurs in a wide range of cancers and recent studies indicate that HIF and c-Myc cooperate in the control of tumour cell metabolism (Gordan et al., Cancer Cell (2007) 12:108-113; Dang et al., Nature Reviews Cancer (2008) 8: 51-56). MYC is a helix- loop-helix leucine zipper transcription factor that when complexed with MAX binds DNA at the E-boxes and other related sequences and activates transcription. MYC binds to and regulates virtually all glycolytic enzyme genes and is thought to stimulate the glycolytic flux (Kim et al, MoL Cell. Biol. (2004) 24:5923-5936. Furthermore MCTl is regulated by MYC (Coller et al., Proc. Natl. Acad. Sci (2000) 97: 3260- 3265; Kim et al., Oncogene (2006) 25: 130-138). Deregulation of MYC occurs in -30% of human cancers including solid tumours such as colon, lung, prostate and breast. Furthmore MYC translocations/gene amplifications are observed in a range of haematological tumours including lymphomas, ALL, CLL and multiple myeloma.

We have now found that certain solid and haematological cancer cell lines are specifically dependent upon the MCTl monocarboxylate transporter isoform. In particular, we have demonstrated that the anti-proliferative activity of a selective MCTl inhibitor in a wide panel of tumour cell lines is strongly correlated with the MCTl :MCT4 expression profile. Potent anti-proliferative activity of an MCTl inhibitor is observed in glycolytic cell lines that express high levels of MCTl and undetectable levels of MCT4. Furthermore, cell lines that express high levels of MCT4 are insensitive to the selective MCTl inhibitor.

It is known that some patients respond well when treated with an anti-cancer drug whereas some patients have a more limited response. There is an increasing body of evidence that suggests a patient's response to a drug may be related to the patient's genetic profile and/or the characteristics of the disease being treated. It is important to understand the basis of a response to a therapeutic agent since this would allow clinicians to maximise the risk/benefit ratio for each patient, potentially via the development of diagnostic tests to identify patients most likely to benefit from the treatment.

The present invention is based on the discovery of a link between the expression levels of the monocarboxylate transporters, MCTl to MCT4, in certain tumours and susceptibility to treatment of certain tumours with an MCTl inhibitor. This discovery therefore, provides opportunities, methods and tools for selecting patients for treatment with an MCTl inhibitor, while at the same time avoiding treatment of patients less likely to respond therapeutically to the treatment and thus avoiding such patients having to experience or suffer any adverse drug-associated side effects. The inventors have discovered, using cancer cell line models, that tumours that selectively express MCTl over MCT4 have an increased likelihood of responding to treatment with a MCTl inhibitor. Therefore, patients whose tumours express MCT4 at a low level are likely to respond better to an MCTl inhibitor than those patients whose tumours express MCT4 at a higher level. Generally, patients whose tumours have a high MCTl :MCT4 expression ratio (due to the low level of MCT4 expression), are likely to show a better response, so evaluating the relative expression levels of both MCTl and MCT4 provides an additional or alternative means for selecting patients for treatment with a MCTl inhibitor. According to a first aspect of the invention there is provided a method of selecting a patient having cancer in need of treatment with an MCTl inhibitor which comprises testing a tumour sample obtained from the patient for selective expression of MCTl over MCT4..

The expression levels of MCTl and MCT4 in a tumour sample may therefore be used to identify subjects suitable for treatment with a MCTl inhibitor. Patients having tumours that do not selectively express MCTl over MCT4 have a decreased likelihood of responding to a MCTl inhibitor.

The phrase 'selectively express of MCTl over MCT4' means the ratio MCTl :MCT4 is greater than 1 : 1. In another embodiment the ratio of MCTl :MCT4 is greater than 2. In another embodiment the ratio of MCTl :MCT4 is greater than 5. In another embodiment the ratio of MCTl :MCT4 is greater than 10. In yet another embodiment the ratio of MCTl :MCT4 is greater than 100.

The ratio MCTl :MCT4 can be determined by measuring the MCTl and MCT4 protein levels or mRNA levels using the methods described below. According to another aspect of the invention there is provided a method of identifying a patient having cancer most likely to benefit from treatment with an MCTl inhibitor comprising measuring the levels of MCTl in a tumour sample obtained from the patient and identifying whether or not the patient is likely to benefit from treatment with a MCTl inhibitor according to the levels present. Patients which have tumours which selectively express MCTl over MCT4 are more likely to benefit from treatment with a MCTl inhibitor and other patients will benefit from not having to be subjected to treatment and thus unnecessary suffering and risk from adverse events.

According to further aspect of the invention there is provided a method of treating cancer comprising (i) testing a tumour sample obtained from a patient suffering from or likely to suffer from cancer for selective expression of MCTl over MCT4 and (ii) administering to the patient having the tumour which selectively expresses MCTl over MCT4, a MCTl inhibitor.

The MCT inhibitor is preferably a compound of formula (1):

as disclosed in PCT Published Patent Application WO2004065394, wherein: R¹ and R² each independently represent a Ci_₆alkyl, C₃-₆alkenyl, C₃-₅cycloalkylCi_ ₃alkyl or C₃_₆Cycloalkyl; each of which may be optionally substituted by 1 to 3 halogen atoms; R³ is a group CO-G or SO₂-G where G is a 5- or 6-membered ring containing a nitrogen atom and a second heteroatom selected from oxygen and sulphur adjacent to the nitrogen; the ring being substituted by at least one group selected from halogen or Ci_4 alkyl, (which may be optionally substituted by up to five halogen atoms), and optionally substituted by up to a further 4 groups independently selected from halogen, hydroxyl and Ci_4 alkyl, (which may be optionally substituted by up to five halogen atoms);

Q is CR⁴R⁵ where R⁴ is hydrogen, fluorine or Ci_6 alkyl and R⁵ is hydrogen, fluorine or hydroxy; Ar is 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 Ci_4alkyl (optionally substituted by 1, 2 or 3 hydroxy groups), Ci_ ₄alkoxy, halogen, haloalkyl, dihaloalkyl, trihaloalkyl, Ci_₄alkoxyCi_₄alkyl, Ci- 4alkylthio, Ci_4alkoxycarbonyl, C2-4alkanoyl, oxo, thioxo, nitro, cyano, -N(R⁶)R⁷ and - (CH₂)pN(R⁸)R⁹, hydroxy,

Ci_₄alkylsulphonyl, Ci_₄alkylsulphinyl, carbamoyl, Ci_₄alkylcarbamoyl, di-(Ci_₄alkyl)carbamoyl, carboxy, SO₂N(R⁶)R⁷, additionally Ar may be optionally substituted by a 5 or 6 membered aromatic ring containing up to 4 heteroatoms independently selected from nitrogen, oxygen and sulphur, and which is optionally substituted by one or more substituents independently selected from Ci_₄alkyl (optionally substituted by 1,2 or 3 hydroxy groups), Ci_₄alkoxy, halogen, haloalkyl, dihaloalkyl, trihaloalkyl, Ci_₄alkoxyCi_₄alkyl, Ci_₄alkylthio, Ci_₄alkoxycarbonyl, C₂_₄alkanoyl, oxo, thioxo, nitro, cyano, -N(R⁶)R⁷ and -(CH₂)pN(R⁸)R⁹, hydroxy,

Ci_₄alkylsulphonyl, Ci_₄alkylsulphinyl, carbamoyl, C^alkylcarbamoyl, di-(Ci_₄alkyl)carbamoyl, carboxy, SO₂ N(R⁶)R⁷, p is 1, 2, 3 or 4;

R⁶ and R⁷ each independently represent a hydrogen atom, Ci_₄alkanoyl or Ci_₄alkyl, or together with the nitrogen atom to which they are attached form a 5- to 7-membered saturated heterocyclic ring; and R⁸ and R⁹ each independently represent a hydrogen atom, Ci_₄alkanoyl or Ci_₄alkyl, or together with the nitrogen atom to which they are attached form a 5- to 7-membered saturated heterocyclic ring; and pharmaceutically acceptable salts thereof.

In one embodiment the MCT inhibitor is selected from 6-[[3,5-dimethyl-l-(2- pyridinyl)-l/f-pyrazol-4-yl]methyl]-5-[[(4S)-4-hydroxy-4-methyl-2- isoxazolidinyl]carbonyl]-3-methyl- 1 -(I -methylethyl)thieno[2,3-d]pyrimidine-2,4- (l/f,3H)-dione (Compound A); 5-[[(45)-4-hydroxy-4-methyl-2- isoxazolidiny 1] carbonyl] -3 -methyl- 1 -( 1 -methylethyl)-6- [ [5 -methyl-3 - (trifluoromethyl)-lH-pyrazol-4-yl]methyl]-thieno[2,3-(i]pyrimidine-2,4(lH,3H)-dione (Compound B); and (45)-4-methyl-2-[[l,2,3,4-tetrahydro-3-methyl-l-(l-methylethyl)- 6-[[5-methyl-l-(2-pyrimidinyl)-3-(trifluoromethyl)-lH-pyrazol-4-yl]methyl]-2,4- dioxothieno[2,3-<i]pyrimidin-5-yl]carbonyl]- 4-isoxazolidinol (Compound C) and pharmaceutically acceptable salts thereof. According to another aspect of the invention, there is provided a method of selecting a patient having cancer in need of treatment with an MCTl inhibitor which comprises testing a tumour sample obtained from the patient for selective expression of MCTl over MCT4, MCTl over MCT2 and MCTl over MCT3. According to another aspect of the invention there is provided a method of identifying a patient having cancer most likely to benefit from treatment with an MCTl inhibitor comprising measuring the expression levels of MCTl, MCT2, MCT3 and MCT4 in a tumour sample obtained from the patient and identifying whether or not the patient is likely to benefit from treatment with an MCTl inhibitor according to the levels present.

In another embodiment, the expression levels of the monocarboxylate transporters MCTl to MCT4 can be used as a biomarker of susceptibility to effective cancer treatment with an MCTl inhibitor.

Selective expression of MCTl over MCT4, MCT2 and MCT3 means that the ratios MCTl :MCT4, MCTl :MCT2 and MCTl :MCT3 are, in one embodiment, all greater than 1 :1. In another embodiment the ratio of MCTl :MCT2 is greater than 2. In another embodiment the ratio of MCTl :MCT2 is greater than 5. In another embodiment the ratio of MCTl :MCT2 is greater than 10. In yet another embodiment the ratio of MCTl :MCT2 is greater than 100. In another embodiment the ratio of MCTl :MCT3 is greater than 2. In another embodiment the ratio of MCTl :MCT3 is greater than 2. In another embodiment the ratio of MCTl :MCT3 is greater than 5. In another embodiment the ratio of MCTl :MCT3 is greater than 10. In yet another embodiment the ratio of MCTl :MCT3 is greater than 100. Preferably, the sample would have been obtained using a minimally invasive technique to obtain a small sample of the tumour, or suspected tumour, from which to determine the MCT expression levels.

In one embodiment the tumour is selected from haemological tumours, such as acute myeloid leukemia (AML), and lung, colorectal, gastric, breast and prostate cancer. Haemological tumours include, for example, diffuse large B cell lymphoma (DLBCL), Burkitt's lymphoma, B-cell acute lymphoblastic leukemia , chronic myeloid leukemia, B-cell non-hodgkins lymphoma chronic lymphocytic B-leukemia and myeloma.

According to a further aspect of the invention there is provided the use of an MCTl inhibitor in the manufacture of a medicament for the treatment of cancer in a patient wherein the cancer tumour selectively expresses MCTl over MCT4.

According to another aspect of the invention, there is provided the use of an MCTl inhibitor selected from 5-[[(45)-4-Hydroxy-4-methyl-2- isoxazolidiny 1] carbonyl] -3 -methyl- 1 -( 1 -methylethyl)-6- [ [5 -methyl-3 - (trifluoromethyl)-lH-pyrazol-4-yl]methyl]-thieno[2,3-(i]pyrimidine-2,4(lH,3H)-dione; 6-[[3,5-Dimethyl-l-(2-pyridinyl)-l/f-pyrazol-4-yl]methyl]-5-[[(4S)-4-hydroxy-4- methyl-2-isoxazolidinyl] carbonyl] -3 -methyl- 1 -(I -methylethyl)thieno[2,3- J]pyrimidine-2,4-(l/f,3H)-dione; and (45)-4-Methyl-2-[[l,2,3,4-tetrahydro-3-methyl- 1 -( 1 -methy lethyl)-6- [ [5 -methyl- 1 -(2-pyrimidinyl)-3 -(trifluoromethyl)- lH-pyrazol-4- yl]methyl]-2,4-dioxothieno[2,3-(i]pyrimidin-5-yl]carbonyl]- 4-isoxazolidinol and pharmaceutically acceptable salts thereof in the manufacture of a medicament for use in the treatment of cancer in a patient wherein the cancer tumour selectively expresses MCTl over MCT4.

The MCTl inhibitor for use in treating a patient according to the invention will normally be administered via the oral, parenteral, intravenous, intramuscular, subcutaneous or in other injectable ways, buccal, rectal, vaginal, transdermal and/or nasal route and/or via inhalation, in the form of pharmaceutical preparations comprising the active ingredient or a pharmaceutically acceptable addition salt, in a pharmaceutically acceptable dosage form. Depending upon the tumour type and patient to be treated and the route of administration, the compositions may be administered at varying doses.

Suitable daily doses of the MCTl inhibitor, for use in the therapeutic treatment of humans according to the methods of the invention, are about 0.001-10 mg/kg body weight, preferably 0.01-3 mg/kg body weight.

Oral formulations are preferred particularly tablets or capsules which may be formulated by methods known to those skilled in the art to provide doses of the active compound in the range of 0.5mg to 500mg for example 1 mg, 2 mg, 4 mg, 6 mg, 10 mg, 15mg, 20mg, 25mg, 50mg, lOOmg and 300mg.

The MCTl inhibitor may be applied as a sole therapy or may involve, in addition to the compound of the invention, conventional surgery or radiotherapy or chemotherapy. Such chemotherapy may include one or more of the following categories of anti-tumour agents :-

(i) other antiproliferative/antineoplastic drugs and combinations thereof, as used in medical oncology, such as alkylating agents (for example cis-platin, oxaliplatin, carboplatin, cyclophosphamide, nitrogen mustard, melphalan, chlorambucil, busulphan, temozolamide and nitrosoureas); antimetabolites (for example gemcitabine and antifolates such as fluoropyrimidines like 5-fluorouracil and tegafur, raltitrexed, methotrexate, cytosine arabinoside, and hydroxyurea); antitumour antibiotics (for example anthracyclines like adriamycin, bleomycin, doxorubicin, daunomycin, epirubicin, idarubicin, mitomycin-C, dactinomycin and mithramycin); antimitotic agents (for example vinca alkaloids like vincristine, vinblastine, vindesine and vinorelbine and taxoids like taxol and taxotere and polokinase inhibitors); and topoisomerase inhibitors (for example epipodophyllotoxins like etoposide and teniposide, amsacrine, topotecan and camptothecin); CHOP regimen (cyclophosphamide, hydroxydoxorubicin, vincristine and prednisone). (ii) cytostatic agents such as antioestrogens (for example tamoxifen, fulvestrant, toremifene, raloxifene, droloxifene and iodoxyfene), antiandrogens (for example bicalutamide, flutamide, nilutamide and cyproterone acetate), LHRH antagonists or LHRH agonists (for example goserelin, leuprorelin and buserelin), progestogens (for example megestrol acetate), aromatase inhibitors (for example as anastrozole, letrozole, vorazole and exemestane) and inhibitors of 5α-reductase such as finasteride; (iii) anti-invasion agents [for example c-Src kinase family inhibitors like 4-(6-chloro- 2,3 -methylenedioxyanilino)-7- [2-(4-methylpiperazin- 1 -yl)ethoxy] -5 -tetrahydropyran- 4-yloxyquinazoline (AZD0530; International Patent Application WO 01/94341), N-(2- chloro-6-methylphenyl)-2- {6-[4-(2-hydroxyethyl)piperazin- 1 -yl]-2-methylpyrimidin- 4-ylamino}thiazole-5-carboxamide (dasatinib, BMS-354825; J. Med. Chem., 2004, 47, 6658-6661) and bosutinib (SKI-606), and metalloproteinase inhibitors like marimastat, inhibitors of urokinase plasminogen activator receptor function or antibodies to Heparanase];

(iv) inhibitors of growth factor function: for example such inhibitors include growth factor antibodies and growth factor receptor antibodies (for example the anti-erbB2 antibody trastuzumab [Herceptin™], the anti-EGFR antibody panitumumab, the anti-erbBl antibody cetuximab [Erbitux, C225] and any growth factor or growth factor receptor antibodies disclosed by Stern et al. Critical reviews in oncology/haematology, 2005, Vol. 54, pp 11-29); such inhibitors also include tyrosine kinase inhibitors, for example inhibitors of the epidermal growth factor family (for example EGFR family tyrosine kinase inhibitors such as Λ/-(3-chloro-4-fluorophenyl)-7-methoxy-6-(3- morpholinopropoxy)quinazolin-4-amine (gefitinib, ZD 1839), JV-(3-ethynylphenyl)- 6,7-bis(2-methoxyethoxy)quinazolin-4-amine (erlotinib, OSI-774) and 6-acrylamido- Λ/-(3-chloro-4-fluorophenyl)-7-(3-morpholinopropoxy)-quinazolin-4-amine (CI 1033), erbB2 tyrosine kinase inhibitors such as lapatinib); inhibitors of the hepatocyte growth factor family; inhibitors of the insulin growth factor family; inhibitors of the platelet- derived growth factor family such as imatinib and/or nilotinib (AMN 107); inhibitors of serine/threonine kinases (for example Ras/Raf signalling inhibitors such as farnesyl transferase inhibitors, for example sorafenib (BAY 43-9006), tipifarnib (Rl 15777) and lonafarnib (SCH66336)), inhibitors of cell signalling through MEK and/or AKT kinases, c-kit inhibitors, abl kinase inhibitors, PI3 kinase inhibitors, Plt3 kinase inhibitors, CSF-IR kinase inhibitors, IGF receptor (insulin-like growth factor) kinase inhibitors; aurora kinase inhibitors (for example AZDl 152, PH739358, VX-680, MLN8054, R763, MP235, MP529, VX-528 AND AX39459) and cyclin dependent kinase inhibitors such as CDK2 and/or CDK4 inhibitors; (v) antiangiogenic agents such as those which inhibit the effects of vascular endothelial growth factor, [for example the anti-vascular endothelial cell growth factor antibody bevacizumab (Avastin™) and for example, a VEGF receptor tyrosine kinase inhibitor such as vandetanib (ZD6474), vatalanib (PTK787), sunitinib (SUl 1248), axitinib (AG-013736), pazopanib (GW 786034) and 4-(4-fluoro-2-methylindol-5- yloxy)-6-methoxy-7-(3 -pyrrolidin- 1 -ylpropoxy)quinazoline (AZD2171; Example 240 within WO 00/47212), compounds such as those disclosed in International Patent Applications WO97/22596, WO 97/30035, WO 97/32856 and WO 98/13354 and compounds that work by other mechanisms (for example linomide, inhibitors of integrin αvβ3 function and angiostatin)];

(vi) vascular damaging agents such as Combretastatin A4 and compounds disclosed in International Patent Applications WO 99/02166, WO 00/40529, WO 00/41669,

WO 01/92224, WO 02/04434 and WO 02/08213;

(vii) an endothelin receptor antagonist, for example zibotentan (ZD4054) or atrasentan;

(viii) antisense therapies, for example those which are directed to the targets listed above, such as ISIS 2503, an anti-ras antisense;

(ix) gene therapy approaches, including for example approaches to replace aberrant genes such as aberrant p53 or aberrant BRCAl or BRCA2, GDEPT (gene-directed enzyme pro-drug therapy) approaches such as those using cytosine deaminase, thymidine kinase or a bacterial nitroreductase enzyme and approaches to increase patient tolerance to chemotherapy or radiotherapy such as multi-drug resistance gene therapy;

(x) immunotherapy approaches, including for example ex-vivo and in-vivo approaches to increase the immunogenicity of patient tumour cells, such as transfection with cytokines such as interleukin 2, interleukin 4 or granulocyte-macrophage colony stimulating factor, approaches to decrease T-cell anergy, approaches using transfected immune cells such as cytokine -transfected dendritic cells, approaches using cytokine-transfected tumour cell lines and approaches using anti-idiotypic antibodies; and

(xi) targeted immune modulation approaches, including for example monoclonal antibodies such as rituximab (rituxan) that targets the CD20 antigen on the surface of malignant and normal B cells and kills tumour cells through complement-dependent cytotoxicity, antibody-dependent cellular cytotoxicity, and induction of apoptosis.

The invention also provides a kit for use in identifying the likely response that a human will have upon administration of an MCTl inhibitor, the kit comprising reagents for detection of levels of MCTl and MCT4 in a sample. The kit may further comprise instructions for the use of the reagents. The reagents may comprise antibodies or antibody fragments against MCTl or MCT4, or any other binding member capable of binding to MCTl or MCT4. The following methods can be used to determine the expression levels of MCTl, MCT2, MCT3 and MCT4 Protein by Western, FACS, ELISA and IHC or mRNA expression levels of MCTl, MCT2, MCT3 and MCT4.

The sample obtained from the patient may be any tumour tissue or any biological sample that contains material which originated from the tumour, for example a blood sample containing circulating tumour cells or DNA. In one embodiment the blood sample may be whole blood, plasma, serum or pelleted blood. In one embodiment a tumour sample is a tumour tissue sample. The tumour tissue sample may be a fixed or unfixed sample. In another embodiment the biological sample can be obtained using a minimally invasive technique to obtain a small sample of tumour, or suspected tumour, from which to determine the MCT isoform expression profile. In another embodiment the biological sample comprises either a single sample, which may be tested for any MCT isoform expression as described herein, or multiple samples, which may be tested for MCT isoform expression as described herein.

Various methods for determining the expression of mRNA or protein include, but are not limited to, gene expression profiling, polymerase chain reaction (PCR) including quantitative real time PCR (qRT-PCR), microarray analysis, serial analysis of gene expression (SAGE), MassARRAY, Gene Expression Analysis by Massively Parallel Signature Sequencing (MPSS), proteomics, and immunohistochemistry (IHC).

(i) Gene Expression Profiling In general, methods of gene expression profiling can be divided into large groups: methods based on hybridisation analysis of polynucleotides, and methods based on sequencing of polynucleotides. The most commonly used methods known in the art for the quantification of mRNA expression in a sample include northern blotting and in situ hybridisation (Parker & Barnes, Methods in Molecular Biology 106: 247-283 (1999); RNAse protection assays (Hod, Biotechniques 13:852-854 (1992)); and polymerase chain reaction (PCR) (Weis et al, Trends in Genetics 8: 263- 264 (1992)).

Alternatively, antibodies may be employed that can recognise specific duplexes, including DNA duplexes, RNA duplexes, and DNA-RNA hybrid duplexes or DNA-protein duplexes. Representative methods for sequencing-based gene expression analysis include Serial Analysis of Gene Expression (SAGE), and gene expression analysis by massively parallel signature sequencing (MPSS).

Preferably mRNA is also quantified. Normally mRNA analysis is performed using the technique of polymerase chain reaction (PCR), or by microarray analysis. Where PCR is employed, a preferred form of PCR is quantitative real time PCR (qRT- PCR).

Various exemplary methods for determining gene expression will now be described in more detail.

(H) Quantitative Real Time PCR (qRT-PCR). Of the techniques listed above, a sensitive and flexible quantitative method is

PCR, which can be used to compare mRNA levels in different sample populations, in normal and tumour tissues, with or without drug treatment, to characterise patterns of gene expression, to discriminate between closely related mRNAs.

In this method the first step is the isolation of mRNA from a target sample. The starting material is typically total RNA isolated from human tumours or tumour cell lines, and corresponding normal tissues or cell lines, respectively. Thus RNA can be isolated from a variety of primary tumours, including haemato logical, breast, lung, colon, prostate, brain, liver, kidney, pancreas, spleen, thymus, testis, ovary, uterus, etc., tumour, or tumour cell lines, with pooled DNA from healthy donors. If the source of mRNA is a primary tumour, mRNA can be extracted, from frozen or archived paraffin-embedded and fixed (e.g formalin-fixed) tissue samples. General methods for mRNA extraction are well known and are disclosed in standard textbooks of molecular biology, including Ausubel et al, Current Protocols of Molecular Biology, John Wiley and Sons (1997). Methods for RNA extraction from paraffin embedded tissues are disclosed, for example, in Rupp and Locker, Lab Invest 56: A67 (1987), and De Andres et al, BioTechniques 18:42044 (1995). In particular, total RNA isolation can be performed using purification kit, buffer set and protease from commercial manufacturers, such as Qiagen, according to the manufacturer's instructions. For example, total RNA from cells in culture can be isolated using Qiagen RNeasy mini-columns. Other commercially available RNA isolation kits include MASTERPURE® Complete DNA and RNA Purification Kit (EPICENTRE®, Madison, Wis), and Paraffin Block RNA Isolation Kit (Ambion Inc.). Total RNA from tissue samples can be isolated using RNA Stat-60 (Tel-Test). RNA prepared from tumour can be isolated, for example, by caesium chloride density gradient centrifugation. As RNA cannot serve as a template for PCR, the first step in gene expression profiling by PCR is the reverse transcription of the RNA template into cDNA, followed by its exponential amplification in a PCR reaction. The two most commonly used reverse transcriptases are avilo myeloblastosis virus reverse transcriptase (AMV-RT) and Moloney murine leukemia virus reverse transcriptase (MMLV-RT). The reverse transcription step is typically primed using specific primers, random hexamers, or oligo-dT primers, depending on the circumstances and the goal of expression profiling. For example, extracted RNA can be reverse- transcribed using a GENEAMP™ RNA PCR kit (Perkin Elmer, Calif. USA), following the manufacturer's instructions. The derived cDNA can then be used as a template in the subsequent PCR reaction. Although the PCR step can use a variety of thermostable DNA-dependent DNA polymerases, it typically employs the Taq DNA polymerase, which has a 5 '-3' nuclease activity but lacks a 3 '-5' proofreading endonuclease activity. Thus, TAQMAN® PCR typically utilizes the 5 '-nuclease activity of Taq or Tth polymerase to hydro lyse a hybridisation probe bound to its target amplicon, but any enzyme with equivalent 5 ' nuclease activity can be used. Two gene specific oligonucleotide primers are used to generate an amplicon typical of a PCR reaction. A third oligonucleotide, or probe, is designed to detect nucleotide sequence located between the two PCR primers. The probe is non-extendible by Taq DNA polymerase enzyme, and is labelled with a reporter fluorescent dye and a quencher fluorescent dye. Any laser-induced emission from the reporter dye is quenched by the quenching dye when the two dyes are located close together as they are on the probe. During the amplification reaction, the Taq DNA polymerase enzyme cleaves the probe in a template-dependent manner. The resultant probe fragments disassociate in solution, and signal from the released reporter dye is free from the quenching effect of the second fluorophore. One molecule of reporter dye is liberated for each new molecule synthesized, and detection of the unquenched reporter dye provides the basis for quantitative interpretation of the data.TAQMAN® PCR can be performed using commercially available equipment, such as, for example, ABI PRISM 7700® Sequence Detection System ® (Perkin-Elmer-Applied Biosystems, Foster City, Calif, USA) or Lightcycler (Roche Molecular Biochemicals, Mannheim, Germany). These systems consist of a thermocycler, laser, charge-coupled device (CCD), camera and computer. The system amplifies samples in a 96-well format on a thermocycler. During amplification, laser-induced fluorescent signal is collected in real-time through fibre optics cables for all 96 wells, and detected at the CCD. The system includes software for running the instrument and for analysing the data.

5 '-Nuclease assay data are initially expressed as Ct, or the threshold cycle. As discussed above, fluorescence values are recorded during every cycle and represent the amount of product amplified to that point in the amplification reaction. The point when the fluorescent signal is first recorded as statistically significant is the threshold cycle (Ct). To minimise errors and the effect of sample-to-sample variation, PCR is usually performed using an internal standard. The ideal standard is expressed at a constant level among different tissues, and is unaffected by the experimental treatment. RNAs most frequently used to normalise patterns of gene expression are mRNAs for the housekeeping genes glyceraldehyde-3-phosphate-dehydrogenase (GAPDH), 18S, HPRT, and P-actin.

Wherever possible primer and probe sets are designed as described below, but pre-designed and pre-optimised sets are also available from Applied Biosystems ('Assay on Demand' primer and probe sets). To design primer and probe sets target gene mRNA sequences were obtained from the RefSeqN database. Primer and probe sequences were designed to the target gene cDNA using Primer Express software

(Version 1.5a, Applied Biosystems). Factors considered in PCR primer design include primer length, melting temperature (Tm) , and G/C content, specificity, complementary primer sequences, and 3 '-end sequence. In general, optimal PCR primers are generally 17-30 bases in length, and contain about 20-80%, such as, for example, about 50-60% G+C bases. Tm's between 50 and 80⁰C, e.g about 50 to 70⁰C, are typically preferred. For further guidelines for PCR primer and probe designs see e.g Dieffenbach et al, "General Concepts for PCR Primer Design" in PCR Primer, A Laboratory Manual, Cold Spring Harbour Laboratory Press, New York, 1995, pp 133- 155; Innis and Gelfand, "Optimisation of PCRs " in PCR Protocols, A Guide to Methods and Applications, CFC Press, London, 1994, pp 5-11; and Plasterer, T.N.Primerselect: Primer and probe design. Methods MoI. Biol. 70:520-527 (1997), the disclosures of which are incorporated herein by reference.

(Ha) Extraction of RNA from Paraffin Embedded Tissue

The steps of a representative protocol for profiling gene expression using fixed paraffin-embedded tissues at the RNA source, including mRNA isolation, purification, primer extension and amplification are given in various published journal articles (for example: Godfrey et al., J. Molec. Diagnostics 2: 84-91 (2000); Specht et al., Am. J. Pathol 158: 419-29 (2001)). Briefly, a representative process starts with cutting about 10 micrometre thick sections of paraffin-embedded tumour tissue samples. The RNA is then extracted, and protein and DNA are removed. After analysis of the RNA concentration, RNA repair and/or amplification steps may be included, if necessary, and RNA is reverse transcribed using gene specific promoters followed by PCR.

(Hi) Microarrays

Differential gene expression can also be identified, or confirmed using microarray techniques. Thus, the expression profile of tumour-associated genes can be measured in either fresh or paraffin-embedded tumour issue, using microarray technology. In this method, polynucleotide sequences of interest (including cDNAs and oligonucleotides) are plated, or arrayed, on a microchip substrate. The arrayed sequences are then hybridised with specific DNA probes from cells or tissues of interest. Just as in the PCR method, the source of mRNA typically is totally RNA isolated from human tumours or tumour cell lines, and corresponding normal tissues or cell lines. Thus RNA can be isolated from a variety of primary tumours or tumour cell lines. If the source of mRNA is a primary tumour, mRNA can be extracted, for example, from frozen or archived paraffin-embedded and fixed (e.g. formalin-fixed) tissue examples, which are routinely prepared and preserved in everyday clinical practice.

In a specific embodiment of the microarray technique, PCR amplified inserts of cDNA clones are applied to a substrate in a dense array. Preferably at least 10,000 nucleotide sequences are applied to the substrate. The microarrayed genes, immobilised on the microchip at 10,000 elements each, are suitable for hybridisation under stringent conditions. Fluorescently labelled cDNA probes may be generated through incorporation of fluorescent nucleotides by reverse transcription of RNA extracted from tissues of interest. Labelled cDNA probes applied to the chip hybridise with specificity to each spot of DNA on the array. After stringent washing to remove non-specifically bound probes, the chip is scanned by confocal laser microscopy or by another detection method, such as CCD camera. Quantitation of hybridisation of each arrayed element allows for assessment of corresponding mRNA abundance. With dual colour fluorescence, separately labelled cDNA probes generated from two sources of RNA are hybridised pairwise to the array. The relative abundance of the transcripts from the two sources corresponding to each specified gene is thus determined simultaneously. The miniaturised scale of the hybridisation affords a convenient and rapid evaluation of the expression pattern for large numbers of genes. Such methods have been shown to have the sensitivity required to detect rare transcripts, which are expressed at a few copies per cell, and to reproducibly detect at least approximately two-fold differences in the expression levels (Schena et ah, Proc Natl Acad Sci USA 93 (2): 106-149 (1996)). Microarray analysis can be performed by commercially available equipment, following manufacturer's protocols such as by using the Affymetrix GENCHIP™ technology, or Incyte's microarray technology.

The development of microarray methods for large-scale analysis of gene expression makes it possible to search systematically for molecular markers of cancer classification and outcome prediction in a variety of tumour types. (iv) Serial Analysis of Gene Expression (SAGE)

Serial analysis of gene expression (SAGE) is a method that allows the simultaneous and quantitative analysis of a large number of gene transcripts, without the need of providing an individual hybridisation probe for each transcript. First, a short sequence tag (about 10-14 bp) is generated that contains sufficient information to uniquely identify a transcript, provided that the tag is obtained from a unique position within each transcript. Then, many transcripts are linked together to form long serial molecules that can be sequenced, revealing the identity of the multiple tags simultaneously. The expression pattern of any population of transcripts can be quantitatively evaluated by determining the abundance of individual tags, and identifying the gene corresponding to each tag. For more details see, e.g Velculescu et ah, Science 270@ 484-487 (1995); and Velculescu et al, Cell 88: 243-51 (\991).(v)

MassARRAY technologyThe MassARRAY (Sequenom, San Diego, Calif.) technology is an automated, high-throughput method of gene expression analysis using mass spectrometry (MS) for detection. According to this method, following the isolation of RNA, reverse transcription and PCR amplification, the cDNAs are subjected to primer extension. The cDNA-derived primer extension are purified, and dispensed on a chip array that is pre-loaded with the components need for MALTI- TOF MS sample preparation. The various cDNAs present in the reaction are quantitated by analysing the peak areas in the mass spectrum obtained.

(vi) Gene expression Analysis by Massively Parallel Signature Sequencing (MPSS)

The method, described by Brenner et al., Nature Biotechnology 18: 630-634 (2000), is a sequencing approach that combines non-gel based signature sequencing with in vitro cloning of millions of templates on separate 5 microgram diameter microbeads. First, a microbead library of DNA templates is constructed by in vitro cloning. This is followed by the assembly of a planar array of the template-containing microbeads in a flow cell at a high density (typically greater than 3x106 microbeads/cm2). The free ends of the cloned templates on each microbead are analysed simultaneously, using a fluorescence-based signature sequencing method that does not require DNA fragment separation. This method has been shown to simultaneously and accurately provide, in a single operation, hundreds of thousands of gene signature sequences from a yeast cDNA library.

(vii) Immunohistochemistry

Immunohistochemistry methods are also suitable for detecting the expression levels of the prognostic markers of the present invention. Thus, antibodies or antisera, preferably polyclonal antisera, and most preferably monoclonal antibodies specific for each marker are used to detect expression. The antibodies can be detected by direct labelling of the antibodies themselves, for example, with radioactive labels, fluorescent labels, hapten labels such as, biotin, or an enzyme such as horseradish peroxidase or alkaline phosphatase. Alternatively, unlabelled primary antibody is used in conjunction with a labelled secondary antibody, comprising antisera, polyclonal antisera or a monoclonal antibody specific for the primary antibody. Immunohistochemistry protocols and kits are well known in the art and are commercially available.

(a) Antibody generation

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. Following booster immunizations, small samples of serum are collected and tested for reactivity to antigen. Examples of various assays 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 (CIEP), radioimmunoassay, radioimmunoprecipitation, enzyme-linked immuno-sorbent assays (ELISA), dot blot assays and Western blots.

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 hybidoma 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 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 can also be produced using techniques, such as those described by Alting-Mees et al., "Monclonal 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 claim antibodies (see for example, US Patent 4,946,778), Fab fragments may also be developed against the polypeptides of the invention (Huse et al. Science 256: 1275-1281, 1989) using methods known in the art.

Antibodies are defined to be specifically binding if they bind the particular MCT, i.e. MCTl, MCT2, MCT3 or MCT4, with a K_Ά 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. N. Y. 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)). (b) Detection of MCTl and MCT4 by immunohistochemistry

Expression of MCTl, MCT2, MCT3 and MCT4 can be assessed using immunohistochemistry of fixed paraffin-embedded tissues. Briefly a representative process starts with cutting 4 micron sections of paraffin-embedded tumour tissue samples. Slides of tumour sections are dewaxed and taken to water. Antigen retrieval is performed using the RHS-2 microwave processor on a setting of 110⁰C for 2 minutes at full pressure, using 1OmM Citrate target retrieval solution. Slides subjected to antigen retrieval are then cooled, washed in TBS/Tween and placed on a Lab Vision autostainer. Endogenous peroxidase activity is blocked by incubating samples with 3% H₂O₂ for 20 minutes. Protein blocking is achieved by incubating samples with Dako Protein Block X0909 for 20 minutes. Slides are then incubated with 1/50 dilutions of either MCTl or MCT4 polyclonal antibodies in 0.05% TBS/Tween for two hours. Slides are then washed in TBS/Tween and incubated with ChemMate Envision for 30 minutes. Following a second set of TBS/Tween washes, slides are incubated with DAB chromogen (ChemMateTM Envision), counterstained, dehydrated and coverslipped.

Tumour sample immunohistochemistry scoring is performed by a skilled human pathologist and the intensity of staining scored using a 0, +1, +2, +3 (criteria follow) scoring system for the relative staining intensity of the nuclear, cytoplasm and plasma membrane. Score 0, no staining is observed in less than 10% of tumour cells. Score 1+, a faint staining is detected in more than 10% of the tumour cells. Score 2+, a weak to moderate staining is observed in more than 10% of the tumour cells. Score 3+, a moderate to strong staining is observed in more than 10% of the tumour cells.

(viii) Proteomics

The term "proteome" is defined as the totality of the proteins present in a sample (e.g tissue, organism, or cell culture) at a certain point of time. Proteomics includes, among other things, study of the global changes of protein expression in a sample (also referred to as "expression proteomics"). Proteomics typically includes the following steps: (1) separation of individual proteins in a sample by 2-D gel electrophoresis (2-D PAGE); (2) identification of the individual proteins recovered from the gel, e.g. by mass spectrometry or N-terminal sequencing;, and (3) analysis of the data using bioinformatics. Proteomics methods are valuable supplements to other methods of gene expression profiling, and can be used, alone or in combination with other methods.

The MCTl polypeptide sequence is disclosed as SEQ ID No.l.

The MCTl DNA sequence is disclosed as SED ID No. 2.

The MCT 2 polypeptide sequence is disclosed as SEQ ID No. 3.

The MCT 2 DNA sequence is disclosed as SED ID No. 4.

The MCT 3 polypeptide sequence is disclosed as SEQ ID No. 5.

The MCT 3 DNA sequence is disclosed as SED ID No. 6.

The MCT 4 polypeptide sequence is disclosed as SEQ ID No. 7.

The MCT 4 DNA sequence is disclosed as SED ID No. 8.

In the Figures:

Figure 1 shows the activity of Compound A in cell lines that express MCTl and /or

MCT4;

Figure 2 shows a plot of cell count versus dose of compound A in a D0HH2 follicular lymphoma cell line and a plot of cell viability versus dose of compound A in the same cell line;

Figure 3 shows show a plot of tumour volume versus dosage time in a Raji xenograft model.

The invention is further described by the following non- limiting examples. In the examples MTS is 3-(4,5-dimethylthiazol-2-yl)- 5-(3-carboxymethonyphenol)-2-(4-sulfophenyl)-

2H-tetrazolium;

DMSO is dimethyl sulphoxide; and SDS is sodium dodecyl sulphate.

Example 1 - Activity of MCTl inhibitors in panels of human tumour cell lines.

The anti-pro liferative effect of MCTl inhibition was investigated across a broad panel of solid and haematological tumour cell lines. Cells were routinely cultured in their appropriate growth media (according to ATCC cell biology collection guidelines) for between 5-10 passages prior to compound testing. On day 1, between 1000-5000 cells/well were plated into the internal 60 wells of black 96 well plates. lOOμl Hanks Buffered salt solution (HBSS) was added to the external wells to prevent media evaporation and plates incubated overnight at 37⁰C in the presence of 5% CO₂. On day 2, dry weight compound stocks were dissolved to a concentration of 1OmM in 100% DMSO. Compounds were further diluted in 100% DMSO to generate a dose range of between 600 μM to 100 nM. 5 μL of compound dilution was then added to 95 μl of cells to give a final dose range of 30 μM to 5 nM compound in 0.3% DMSO. Plates were then incubated at 37⁰C in the presence of 5% CO₂ for a further 72 hours post-dosing. On day 5, 20 μl of CellTiter 96^® AQ MTS Reagent was added to each well and the plate returned to the incubator for a further 2 hours. MTS is bioreduced by NADPH (Nicotinamide adenine dinucleotide phosphate) or NADH (reduced form of nicotinamide adenine dinucleotide) produced by dehydrogenase enzymes in metabolically active cells into a coloured formazan product that is soluble in tissue culture medium. The amount of coloured formazan product is directly proportional to the number of living cells in culture. 25 μl of 10% SDS was added to each well and the absorbance of the plates read on a Tecan Ultra plate reader using a 490 nM measurement wavelength. Dose response curves were plotted and GI50 values calculated using Origin 7.5 client. The GI50 value is equivalent to the concentration of compound that causes 50% inhibition of growth calculated from the mean maximum signal to the day 0 minimum signal.

Table 1 gives the activity of Compound A across a panel of solid and haematological tumour cell lines. Table 2 gives the activity of Compound A across a panel of haematological tumour cell lines. (Further details can be found below in examples 3 and 4). This broad cell line profiling indicates that MCTl inhibitors do not display wide-spectrum anti-proliferative activity particularly in solid tumour cell lines. However, surprisingly a panel of lymphoma cell lines including diffuse large B cell lymphoma (DLBCL), Burkitt's lymphoma and other haematological cell lines are exquisitely sensitive to the MCTl inhibitors. As shown below the data in Table 2 gives MCT isoform expression profiling which supports the hypothesis that activity is restricted to cell lines that express predominantly MCTl and not MCT4.

Table 1

Table 2

Example 2 - Correlation between MCTl and MCT4 niRNA and protein expression and sensitivity to MCTl inhibition

Studies on the expression of MCTl and MCT4 in tumour cell lines were been performed. Tumour cell lysates were generated using standard procedures and subsequent protein lysates analysed by Western Blotting using the antibodies as described above ( see page 18, line 8 etc). Following incubation with primary polyclonal antibodies, nitrocellulose membranes were incubated with LiCOR IRDye^® 680 nM goat-anti Rabbit IgG secondary antibodies followed by Tris-buffered saline (TBS)-T ween washes. Expression levels of MCTl and MCT4 were determined by scanning resultant blots on an Odyssey^® Infrared Imaging system. Expression levels are expressed as integrated intensity unit following correction for equal protein loading on the Western blot using a vinculin control. mRNA Expression data was also extracted from an internal Gene Expression database to assess the relative mRNA expression of MCTl and MCT4 across a large panel of tumour cell lines. Five affymetrix probe sets were compared for MCTl and three probesets for MCT4. Normalisation of mRNA expression levels was performed using the Affymetrix MAS5 algorithm with a target intensity (TGT) of 100 and mRNA expression levels expressed as linear intensity values.

Western blot protein profiling indicates that a number of lymphoma cell lines express predominantly MCTl (Table 2). There is a strong correlation between predominant expression of MCTl and sensitivity to an MCTl inhibitor. Cell lines that express predominantly MCTl including Raji, Jeko-1, SC-I, OCI-LY-19, A375, DMSl 14 and K562 are extremely sensitive to growth inhibition by a MCTl inhibitor. Cell lines that express high levels of MCT4, such as U937, and MDAMB231 are resistant to compound A (Table 3 and Figure 1). Furthermore mRNA profiling across a broad range of tumour cell lines indicates that MCTl mRNA is preferentially expressed (over MCT4mRNA) in a range of haemato logical cell lines including B- CLL, B-ALL, B-NHL, Burkitt's lymphoma, CML and myeloma cell lines (Table 4).

Table 3

The colour coding for the protein expression levels is as follows :-

Protein Expression

>0-0.25

Table 4

Example 3 - Sensitivity of haematological cell lines to MCTl inhibition

The anti-pro liferative effect of MCTl inhibition was investigated across a broad panel of haematological tumour cell lines. Cells were routinely cultured in their appropriate growth media (according to ATCC cell biology collection guidelines) for between 5-10 passages prior to compound testing. On day 1, between 1000-5000 cells/well were plated into the internal 60 wells of black 96 well plates. lOOμl Hanks Buffered salt solution (HBSS) was added to the external wells to prevent media evaporation and plates incubated overnight at 37⁰C in the presence of 5% CO₂. On day 2, dry weight compound stocks of Compound A were dissolved to a concentration of 1OmM in 100% in DMSO. Compounds were further diluted in 100% DMSO to generate a dose range of between 600 μM to 100 nM. 5 μL of compound dilution was then added to 95 μl of cells to give a final dose range of 30 μM to 5 nM compound in 0.3% DMSO. Plates were then incubated at 37⁰C in the presence of 5% CO₂ for a further 72 hours post-dosing. On day 5, lOμl Alamar Blue solution (Invitrogen) was added to each well and the plate returned to the incubator for a further 4 hours. Alamar Blue is an oxidation-reduction indicator dye, which monitors metabolic activity (high level of reduction) as a readout of cellular proliferation. 25 μl of 0.5% SDS was added to each well and the absorbance of the plates read on the Tecan Ultra using 54OnM measurement wavelength and 62OnM reference wavelength. Dose response curves were plotted and IC50 values calculated using Origin 7.5 client. The IC50 value is equivalent to the concentration of compound A that causes 50% growth inhibition.

The results in Table 2 show that the MCTl inhibitor, compound A, demonstrates anti-pro liferative activity against a wider set of haemato logical cell lines including B-ALL, CLL and CML cell lines. Furthermore, anti-proliferative activity correlates with cell lines that express high levels of MCTl mRNA and low levels of MCT4 mRNA.

Example 4 - Analysis of lymphoma cell fate following treatment with MCTl inhibitor.

Cell viability was analysed using the Guava^® Viacount Assay. Cells were routinely cultured in their appropriate growth media (according to ATCC cell biology collection guidelines) for between 5-10 passages prior to compound testing. On day 1, 10,000 cells were plated in the internal 60 wells of black 96 well plates (Costar). lOOμl Hanks Buffered salt solution (HBSS) was added to the external wells to prevent media evaporation and plates incubated overnight at 37⁰C in the presence of 5% CO₂. On day 2, dry weight compound stocks (compound A) were dissolved to a concentration of 1OmM in 100% DMSO. Compounds were further diluted in 100% DMSO to generate a dose range of between 200 μM to 20 nM. 5 μL of compound dilution was then added to 95 μl of cells to give a final dose range of 10 μM to 1 nM compound in 0.3% DMSO. Plates were then incubated at 37⁰C in the presence of 5% CO₂ for a further 48 or 72 hours post dosing. On day 4 or 5, control wells were counted using a Coulter^® Counter to determine the required dilution factor to give cells between 1 x 10⁴ - 5 x 10⁵/ml and cells were diluted in ViaCount^® reagent (Guava) to give final volume of 200μl/well. Cells were repeatedly pipetted to disrupt clumps of cells before the single cell suspension was transferred to a ViaCount^® plate.

Cell viability analysis was performed using a Guava^® PCA system. Briefly, bead checks were carried out on the Guava system before each experiment to ensure correct flow. Assay plates were analysed using the Viacount algorithm with 1000 events (cells) counted per well. Viacount categorises cells as viable, apoptotic or dead depending on the uptake of 2 DNA intercalating dyes. The first dye is membrane permeant to stain all nucleated cells while the second dye only penetrates and stains cells with compromised membrane integrity. Cells that have partial uptake of the 2ⁿd dye have been shown to also react with Annexin V, indicating they have entered the apoptotic pathway. Treatment of selected sensitive lymphoma cell lines results in a loss of viable cell number consistent with the anti-proliferative activity observed in MTS and Alamar Blue proliferation endpoints (Figure 2). Furthermore, the Guava Viacount analysis supports the hypothesis that increasing concentrations of MCTl inhibitor induces apoptosis in the DOHH-2 lymphoma cell line (Figure 2).

Example 5 - In vivo activity of MCTl inhibitors in xenograft models

The in vivo efficacy of MCTl inhibitors has been tested in human xenograft models. The Raji Burkitt's lymphoma cell line can be grown as a subcutaneous xenograft in female severe combined immunodeficient (SCID) mice and tumour volume calculated from bilateral caliper measurements. For efficacy studies, five million Raji cells were inoculated subcutaneously onto the left flank of the animal in a volume of 0.1ml in phosphate buffer solution (PBS). Animals were assigned into treatment groups 7 days after cell implantation and received either 25mg/kg, 50mg/kg or 100mg/kg of a Compound A by oral gavage twice daily (b.i.d) or 0.5% hydroxy propyl methyl cellulose/0.1% Tween 80 at the same schedule. Dosing was continued for 14 days (until day 21) and tumour volume, body weight and tumour condition were recorded twice weekly for the duration of the study. On day 21 the final dose of compound was administered and the tumours allowed to regrow until the study was completed 32 days after inoculation of the animals. At each dose of compound significant anti-tumour activity was observed (25 mg/kg - 72% growth inhibition, 50 mg/kg - 82% growth inhibition and at 100 mg/kg - 72% growth inhibition; Figure 3).

Claims

1. A method of selecting a patient having cancer in need of treatment with an MCTl inhibitor which comprises testing a tumour sample obtained from the patient for selective expression of MCTl over MCT4.

2 A method according to claim 1 wherein the cancer is selected from haemological tumours and lung, colorectal, breast and prostate cancer.

3. Use of an MCT 1 inhibitor in the manufacture of a medicament for use in the treatment of cancer in a patient wherein the cancer tumour selectively expresses MCTl over MCT4.

4. Use of a MCTl inhibitor according to claim 3 where the MCTl inhibitor is selected from 5-[[(45)-4-Hydroxy-4-methyl-2-isoxazolidinyl]carbonyl]-3-methyl-l-(l- methylethyl)-6-[[5-methyl-3-(trifluoromethyl)-lH-pyrazol-4-yl]methyl]-thieno[2,3- J]pyrimidine-2,4(l/f,3H)-dione; 6-[[3,5-Dimethyl-l-(2-pyridinyl)-l/f-pyrazol-4- yl]methyl]-5-[[(4S)-4-hydroxy-4-methyl-2-isoxazolidinyl]carbonyl]-3-methyl-l-(l- methylethyl)thieno[2,3-<i]pyrimidine-2,4-(l/f,3H)-dione; and (4S)-4-Methyl-2- [[ 1 ,2,3,4-tetrahydro-3-methyl- 1 -(I -methylethyl)-6-[ [5 -methyl- 1 -(2-pyrimidinyl)-3- (trifluoromethyl)-lH-pyrazol-4-yl]methyl]-2,4-dioxothieno[2,3-(i]pyrimidin-5- yljcarbonyl]- 4-isoxazolidinol and pharmaceutically acceptable salts thereof in the manufacture of a medicament for use in the treatment of cancer in a patient wherein the cancer tumour selectively expresses MCTl over MCT4.

5. Use of expression levels of the monocarboxylate transporters, MCTl to MCT4 as a biomarker of susceptibility to effective cancer treatment with an MCTl inhibitor.

6. The use of 6-[[3,5-Dimethyl-l-(2-pyridinyl)-l/f-pyrazol-4-yl]methyl]-5-[[(4S)- 4-hydroxy-4-methyl-2-isoxazolidinyl]carbonyl]-3-methyl- 1 -(I - methylethyl)thieno[2,3-d]pyrimidine-2,4-(lH,3H)-dione or a pharmaceutically acceptable salt thereof in the manufacture of a medicament for use in the treatment of cancer in a patient where the cancer selectively expresses MCTl over MCT4.

7. The use of 5-[[(4S)-4-Hydroxy-4-methyl-2-isoxazolidinyl]carbonyl]-3- methyl- 1 -(I -methylethyl)-6-[[5-methyl-3-(trifluoromethyl)- lH-pyrazol-4-yl]methyl]- thieno[2,3-d]pyrimidine-2,4(lH,3H)-dione or a pharmaceutically acceptable salt thereof in the manufacture of a medicament for use in the treatment of cancer in a patient where the cancer selectively expresses MCTl over MCT4.

8. A method of treating cancer comprising (i) testing a tumour sample obtained from a patient suffering from or likely to suffer from cancer for selective expression of MCTl over MCT4 and (ii) administering to the patient having the tumour sample which selectively expresses MCTl over MCT4, an MCTl inhibitor.

9. A method of identifying a patient having cancer most likely to benefit from treatment with an MCTl inhibitor comprising measuring the expression levels of MCTl and MCT4 in a tumour sample obtained from the patient and identifying whether or not the patient is likely to benefit from treatment with a MCTl inhibitor according to the levels present.

10. A method of identifying a patient having cancer most likely to benefit from treatment with an MCTl inhibitor comprising measuring the expression levels of MCTl, MCT2, MCT3 and MCT4 in a tumour sample obtained from the patient and identifying whether or not the patient is likely to benefit from treatment with an MCTl inhibitor according to the levels present.