WO2011063469A1

WO2011063469A1 - Tubulin biomarker assay

Info

Publication number: WO2011063469A1
Application number: PCT/AU2010/001595
Authority: WO
Inventors: Gabriel Kremmidiotis; Clayton Ashley Matthews
Original assignee: Bionomics Ltd
Current assignee: Bionomics Ltd
Priority date: 2009-11-27
Filing date: 2010-11-26
Publication date: 2011-06-03
Anticipated expiration: 2012-05-27

Abstract

The invention provides a method for assessing the activity of a tubulin targeting agent. The method involves lysing cells which have been exposed to the tubulin targeting agent under conditions which substantially maintains the polymerisation state of the tubulin present in the cells. The resulting preparation is then centrifuged to separate polymerised and unpolymerised tubulin and an assessment made as to level of polymerised and/or unpolymerised tubulin.

Description

TUBULIN BIOMARKER ASSAY

FILING DATA This application is associated with and claims priority from Australian patent application no. 2009905806 filed on 27 November 2009, the entire contents of which is incorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates generally to a tubulin biomarker assay. In particular, the present invention provides a method for quantifying tubulin polymerisation status changes in a cell following exposure of the cell to a tubulin targeting agent. BACKGROUND OF THE INVENTION

Tubulin is a potential target for treating disease states that are dependent or result from the abnormal formation of new blood vessels including cancer. Tubulin is composed of a heterodimer of two related proteins called a- and β-tubulin, and polymerises to form structures called microtubules which play an active role in mitosis or cell division.

Compounds that interfere with tubulin's ability to polymerise or depolymerise, interfere with microtubule function causing disruption of the cytoskeleton and altering cell shape and ability to undergo cell division. The capacity to correlate drug exposure levels with on-target activity is of central importance in establishing pharmacokinetic/pharmacodynamic relationships in clinical trials. In the case of agents operating through tubulin targeting, the ability to perform both qualitative and quantative analyses of tubulin polymerisation state, would provide a reliable means in evaluating on-target activity in cancer patients. In addition the range of methods available to assess on target activity of tubulin targeting agents is limited. The provision of alternate methods of assessing in vivo activity would be advantageous.

Furthermore, a method for quantitating tubulin polymerisation state would allow the discovery of new tubulin targeting agents and evaluation of their on-target activity. The method would be used to measure tubulin polymer/monomer ratio in cell lines or primary cells cultured in vitro or in vivo in samples derived from animals treated with the agent. SUMMARY OF THE INVENTION

In one aspect of the present invention there is provided a method for assessing the in vivo activity of a tubulin targeting agent, the method comprising:

(i) adrninistering a tubulin targeting agent to an animal;

(ii) obtaining a cell sample from the animal;

(iii) lysing the cells obtained in step (ii) under conditions which substantially maintains the polymerisation state of the tubulin present in the cells to produce a lysed cell preparation;

(iv) centrifuging the lysed cell preparation to obtain a lysed cell precipitate comprising polymerised tubulin and a supernatant comprising unpolymerised tubulin; and

(v) measuring the level of tubulin in the precipitate and in the supernatant.

In another aspect of the present invention there is provided a method for assessing the activity of a tubulin targeting agent, the method comprising:

(i) contacting cells with a tubulin targeting agent;

(ii) lysing the cells from step (i) under conditions which substantially maintains the polymerisation state of the tubulin present in the cells to produce a lysed cell preparation;

(iii) centrifuging the lysed cell preparation to obtain a lysed cell precipitate comprising polymerised tubulin and a supernatant comprising unpolymerised tubulin; and

(iv) measuring the level of tubulin in the precipitate and in the supernatant.

In a further aspect of the present invention there is provided a method for assessing the activity of a tubulin targeting agent, the method comprising:

(i) providing two samples of cells and contacting cells of one sample with a tubulin targeting agent;

(ii) lysing the cells of both samples from step (i) under conditions which

substantially maintains the polymerisation state of the tubulin present in the cells to produce two lysed cell preparations;

(iii) centrifuging the lysed cell preparations to obtain a lysed cell precipitate comprising polymerised tubulin and a supernatant comprising unpolymerised tubulin from each of the cell samples; and

(iv) comparing the level of polymerised or unpolymerised tubulin in each of the cell samples. BRIEF DESCRIPTION OF THE FIGURES

Figure 1 shows tubulin response in A2780 cells following exposure to various concentrations of Compound Example 2 for 1 hour: "P" = pellet fraction following cell lysis, "S" = supernatant fraction following cell lysis, "C" = control (i.e. no cell exposure to Compound Example 2), "D" = DMSO vehicle control. (A) total tubulin and actin content in pellet and supernatant fractions from cells following exposure to 10, 20, 25 and 30 nM Compound Example 2; (B) total tubulin content in pellet fraction of cells following exposure to 2, 5, 10, 20, 25, 30, 40, 50, 100, 200 and 500 nM concentrations of Compound Example 2; (C) total tubulin content in supernatant fraction of cells following exposure to 2, 5, 10, 20, 25, 30, 40, 50, 100, 200 and 500 nM concentrations of Compound Example 2.

Figure 2 shows tubulin response in human peripheral blood mononuclear cells (PBMCs) following exposure to 0.537 //m of Compound Example 2 for 30 mins in vitro: "P" = pellet fraction following PBMC lysis, "S" = supernatant fraction following PBMC lysis, "+" = exposure to Compound Example 2, "-" = control (i.e. no exposure to

Compound Example 2). (A) shows total tubulin and actin content for volunteer A and volunteer B; (B) normalized tubulin in human PBMC pellet fraction for volunteer A and volunteer B; (C) normalized tubulin in human PBMC supernatant fraction for volunteer A and volunteer B.

Figure 3 shows tubulin response in peripheral blood mononuclear cells (PBMCs) of a patient havmg oesophageal cancer following administration of 18.9 mg/m of Compound Example 2: "P" = pellet fraction following PBMC lysis, "S" = supernatant fraction following PBMC lysis, "pre" = pre-bleed PBMCs prior to administration of Compound Example 2. (A) total tubulin and actin content of pre-bleed PBMCs or PBMCs obtained from the patient either 1 or 2 h following administration of Compound Example 2; (B) normalized tubulin from PBMC pellet fraction; (C) normalized tubulin from PBMC supernatant fraction.

Figure 4 shows tubulin response in peripheral blood mononuclear cells (PBMCs) of a patient having renal cancer following administration of 12.6 mg m² of Compound Example 2: "P" = pellet fraction following PBMC lysis, "S" = supernatant fraction following PBMC lysis, "pre" = pre-bleed PBMCs prior to administration of Compound Example 2. (A) total tubulin and actin content of pre-bleed PBMCs or PBMCs obtained from the patient 1, 2, 3-5, 7 or 24 h following administration of Compound Example 2; (B) normalized tubulin from PBMC pellet fraction as a function of time.

Figure 5 shows tubulin response in peripheral blood mononuclear cells (PBMCs) of a patient having rectosigmoid following administration of 12.6 mg/m of Compound Example 2: "P" = pellet fraction following PBMC lysis, "S" = supernatant fraction following PBMC lysis, "pre" = pre-bleed PBMCs prior to administration of Compound Example 2. (A) total tubulin and actin content of pre-bleed PBMCs or PBMCs obtained from the patient 1, 2, 3-5, 7 or 24 h following administration of Compound Example 2; (B) normalized tubulin from PBMC pellet fraction as a function of time.

Figure 6 shows tubulin response in peripheral blood mononuclear cells (PBMCs) of a patient having colorectal cancer following administration of 16 mg/m of Compound Example 2: "P" = pellet fraction following PBMC lysis, "S" = supernatant fraction following PBMC lysis, "pre" = pre-bleed PBMCs prior to administration of Compound Example 2. (A) total tubulin and actin content of pre-bleed PBMCs or PBMCs obtained from the patient 1, 2, 3-5, 7 or 24 h following administration of Compound Example 2; (B) normalized tubulin from PBMC pellet fraction as a function of time.

Figure 7 shows the tubulin response of A2780 cells in vitro following a 4 hour exposure to paclitaxel. Using the methodology described here, polymerized tubulin was shown to be highly concentrated in the cell pellet in response to the tubulin-polymerizing drug, paclitaxel. Pel+ indicates pellet exposed to paclitaxel; Pel- indicates pellet not exposed to paclitaxel; Sup+ indicates supernatant exposed to paclitaxel; Sup- indicates supernatant not exposed to paclitaxel. The response to this tubulin polymerizing drug was exactly opposite to the response detected following exposure to the tubulin depolymerising drugof Example 2.

Figure 8 shows combined results from patients dosed at 12.6mg/m to 18.9mg/m of the compound Example 2, inclusive. These data clarify the natural variability at each time point between patients. At the 2 and 3-5 hour time points most patient PBMC pellets contain less than 20% of their predose tubulin concentrations indicating a strong response to the drug across patient cohorts. Figure 9 shows average tubulin concentration in cell pellets from each dose cohort at the 3-5 hour post-dose time point. No tubulin reduction was observed at or below the 8.4 mg/m² dose level. A possible dose-response was observed at doses >12.6mg/m². DESCRIPTION OF THE PREFERRED EMBODIMENTS

Throughout this specification the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element or integer or group of elements or integers but not the exclusion of any other element or integer or group of elements or integers.

The reference in this specification to any prior publication (or information derived from it), or to any matter which is known, is not, and should not be taken as an acknowledgment or admission or any form of suggestion that that prior publication (or information derived from it) or known matter forms part of the common general knowledge in Australia in the field of endeavour to which this specification relates.

It is to be understood that unless otherwise indicated, the subject invention is not limited to specific manufacturing methods, formulation components, dosage regimens, or the like, as such may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, preferred methods and materials are described. For the purposes of the present invention, the following terms are defined below. The articles "a" and "an" are used herein to refer to one or to more than one (i.e. to at least one) of the grammatical object of the article. By way of example, "a cell" means one cell, more than one cell or even a population of cells.

The term "tubulin targeting agent" is intended to mean any compound which prevents microtubule formation or which acts to destabilize polymerized tubulin, causing it to depolymerize. Compounds that act to either stabilize or destabilize microtubules have an effect on the amount of tubulin in the polymerized state. Since polymerized tubulin can be separated from depolymerized tubulin fraction by high speed centrifugation, the pellet (polymerized) tubulin fraction and the supernatant (depolymerized) fraction can be analyzed to measure shifts in tubulin concentration in each fraction in response to changes in polymerization state.

Accordingly, in one aspect of the present invention there is provided a method for assessing the in vivo activity of a tubulin targeting agent, the method comprising:

(i) administering a tubulin targeting agent to an animal;

(ii) obtaining a cell sample from the animal;

(vi) lysing the cells obtained in step (ii) under conditions which substantially maintains the polymerisation state of the tubulin present in the cells to produce a lysed cell preparation;

(vii) centrifuging the lysed cell preparation to obtain a lysed cell precipitate comprising polymerised tubulin and a supernatant comprising unpolymerised tubulin; and

(viii) measuring the level of tubulin in the precipitate and in the supernatant.

The level of tubulin may be measured using a number of methods known in the art. It is preferred however that the level of tubulin is measured using an anti-β tubulin antibody. Preferably the level of tubulin is measured by Western blot or ELIS A.

In one embodiment of this aspect of the invention the animal has cancer. The animal may be a human. The method of this aspect of the present invention is particularly useful in a clinical trial setting. In these embodiments various cells may be used as surrogates to measure the affect of the tubulin targeting agent. In one embodiment the cell sample may comprises PBMC. This is particularly useful as these cells can be obtained in a relatively noninvasive manner. It will be appreciated however that other cells such as circulating endothelial cells and tumour cells may be used.

(i) contacting cells with a tubulin targeting agent;

(ii) lysing the cells from step (i) under conditions which substantially maintains the polymerisation state of the tubulin present in the cells to produce a lysed cell preparation; (iii) centrifuging the lysed cell preparation to obtain a lysed cell precipitate comprising polymerised tubulin and a supernatant comprising unpolymerised tubulin; and

(iv) measuring the level of tubulin in the precipitate and in the supernatant.

(ii) lysing the cells of both samples from step (i) under conditions which

(iv) comparing the level of polymerised or unpolymerised tubulin in each of the cell samples.

The tubulin targeting agent may be any of a range of such compounds including

a pharmaceutically acceptable salt, solvate or prodrug thereof. The methods according to the present invention can be used to screen compounds having therapy potential, particularly in the treatment of cancer.

Accordingly, in yet another aspect of the present invention there is provided a method for identifying a target compound as having tubulin targeting activity, the method comprising:

(i) contacting cells with a target tubulin targeting agent;

(iv) measuring the level of tubulin in the precipitate and in the supernatant, wherein an increase in the level of tubulin in the supernatant is indicative of tubulin targeting activity. By detecting the amount of β-tubulin, as opposed to a-tubulin, the assay or method of the present invention more accurately quantifies the total amount of tubulin present, whether in the polymerized state or the depolymerized state. In our experience, the use of available anti-alpha tubulin antibodies (Molecular Probes cat# Al 1126) was not as sensitive as tubulin detection using anti-beta tubulin antibodies (Sigma cat# T7816). However, it may not be possible to generalise this finding to include all commercially available anti-alpha tubulin antibodies without further testing.

In an embodiment of the present invention the cancer cell line is selected from the group consisting of A2780, FADU, A549, 786-0, DU145, and peripheral blood mononuclear cells (PBMCs).

It will be appreciated by a person skilled in the art that the lysis buffer will contain components sufficient to cause cell lysis, while maintaining the cell suspension at a desired pH level. For instance, the lysis buffer used in the methods according to the present invention may contain one or more detergents sufficient to disrupt the cell membrane and cause cell lysis. Suitable detergents may include Triton X-100 or Nonidet P40. Further, the skilled person will appreciate that the lysis buffer will contain an organic compound suitable to prevent rapid pH shifts following cell lysis. Examples of suitable organic compounds include piperazine-N,N'-bis(2-ethanesulfonic acid) (PIPES), 4-(2- hydroxyethyl)- 1 -piperazineethanesulfonic acid (HEPES),

acid (MES), 3-(N-morpholino)propanesulfonic acid (MOPS), 3-[4-(2-Hydroxyethyl)-l- piperazinyl]propanesulfonic acid (HEPPS), tris(hydroxymethyl)aminomethane (TRIS) and the like.

The cell lysis buffer used in the methods according to the present invention may also comprise one or more chelating agents including but not limited to ethylene glycol tetraacetic acid (EGTA), ethylenediaminetetraacetic acid (EDTA) etc. The cell lysis buffer may also comprise one or more protease inhibitors, including but not limited to, metalloprotease inhibitors, cysteine protease inhibitors, serine protease inhibitors, threonine protease inhibitors, aspartic protease inhibitors. Examples of protease inhibitors include aprotinin, bestatin, calpain, chymostatin, E-64, leupeptin, pepstatin, and trypsin inhibitors.

The cell lysis buffer is adjusted to a pH in the range of 6.0-8.0, preferably 6.5-7.5, more preferably 6.5-7.0 and most preferably 6.9.

In an embodiment of the present invention, the cell lysis buffer comprises EGTA, MgS0₄, glycerol, DMSO, Triton X-100, PIPES, GTP, one or more protease inhibitors, and is adjusted to a pH of 6.9. Depending on the intended application of the assay or method of the present invention, paclitaxel may be added to the lysis buffer if tubulin yields are low, so as to maintain polymerized tubulin.

The step of separating the lysed cells from the cell lysis buffer involves removing the lysed cells from the buffer. This may be achieved by centrifuging the lysed cell suspension and aspirating the supernatant to leave the lysed cells. Centrifugation is typically carried out at 180,000 xg at 37°C for 1 hour.

The step of detecting the amount of β-l tubulin present in cells involves identifying the presence of β-Ι tubulin and quantifying the amount present. In an embodiment of the present invention, β-Ι tubulin is detected by western blot using an anti- β-Ι tubulin antibody and the bound antibody is used to quantify the amount of β-Ι tubulin present. To increase detection sensitivity, reliability, and increase sample throughput, an ELISA-based detection method may be developed to quantify tubulin in cell lysates.

The method or assay of the present invention can be applied to quantify the efficacy of known tubulin targeting agents such as, for example, nocodazole, paclitaxel (and other taxanes), vinblastine, dolastatin, estramustine, podophyllotoxin, rhizoxin, vinorelbine, trifluralin, vindesine, ixabepilone, SB-715992 and SB-743921 (GlaxoSrnithKline). Other examples of tubulin targeting agents include those which act at the colchicine binding site including colchicine and annulated furans (e.g., benzofurans, furo[2,3-d]pyrimidin-2(lH)- ones, etc), benzothiophene and indole structural scaffolds, such as those disclosed in US 7,456,214, US 7,429,681, US 7,071,190, US 6,849,656, US 5,886,025, US 6,162,930, US 6,350,777, US 5,340,062, WO 06/084338, WO 02/060872, WO 07/087684, and WO 08/070908 (which are incorporated herein in their entirety by reference). However it will be appreciated that another advantage of the present assay is its ability to identify new active tubulin targeting agents which function to destabilize or stabilize tubulin.

Those skilled in the art will appreciate that the invention described herein is susceptible to variations and modifications other than those specifically described. It is to be understood that the invention includes all such variations and modifications which fall within the spirit and scope. The invention also includes all of the steps, features, compositions and compounds referred to or indicated in this specification, individually or collectively, and any and all combinations of any two or more of said steps or features.

Certain embodiments of the invention will now be described with reference to the following examples which are intended for the purpose of illustration only and are not intended to limit the scope of the generality hereinbefore described.

EXAMPLES Preparation of Tubulin Targeting Agent (MTA)

Preparation of a tubulin targeting agents 6-methoxy-2-methyl-3-(3,4,5- trimemoxybenzoyl)benzofuran-7-yl (herein referred to as "Compound Example 1 ") used in Experiments described in Example 4 and disodium 6-methoxy-2-methyl-3-(3,4,5- trimethoxybenzoyl)benzofuran-7-yl phosphate (hereinafter referred to as "Compound

Example 2"), used in the experiments described in Example 4, were prepared according to the following synthetic protocol.

Preparation of 2-Bromo- 7-acetox -3-(3,4,5-trimethoxybenzoyl)-6-methoxybenzqfuran

Step 1: 2-t-ButyldimethyMlyl-3-(t-butyldimethyIs yloxymethylene)-6-metho^-7- isopropoxybenzofuran (Larock coupling)

A suspension of 2-isopropoxy-3-methoxy-5-iodophenol (4.41 mmol), l-(/ert- butyldimemylsilyl)-3-(tert-butyldimethylsilyloxy)propyne (1.5 g, 5.28 mmol), lithium chloride (189 mg, 4.45 mmol) and sodium carbonate (2.34 g, 22.08 mmol) in dry dimethylformamide (5 mL) at 100 °C was deoxygenated 4 times by evacuation and backfilling with nitrogen. Palladium acetate (135 mg, 0.60 mmol) was added and the reaction vessel was degassed twice with nitrogen. The reaction mixture was then stirred at this temperature for 4 hours (tic) and the solvent was removed by distillation under vacuum. The residue was dissolved in ethyl acetate (75 mL), stirred well, filtered and treated with triemylarnine (5 mL). The solution was concentrated onto silica gel (10 g) and purified by flash chromatography (silica gel, eluent = hexane/diethyl emer/lriethylamine; 95:5:1%) to afforded the title compound as a yellow oil (1.45 g, 96 %); 1H NMR (300 MHz, CDC1₃) δ 7.24(d, 1H, J= 8.45 Hz), 6.88(d, 1H, J= 8.47 Hz), 4.80(s, 2H, CH₂), 4.73(m, 1H), 3.88(s, 3H, OMe), 1.36(d, 6H, J= 6.17 Hz), 0.94(s, 9H), 0.92(s, 9H), 0.35(s, 6H), 0.12(s, 6H).

Step 2: 2-t-Butyldimethykilyl-3-formyl-6-methoxy-7-isopropo^benzofuran

To a solution of 2-t-butyldimemylsilyl-3-(t-butyldimemylsilyloxymethylene)-6-methoxy- 7-isopropoxybenzofuran (2.69 mmol) in methanol (100 mL) was added concentrated hydrochloric acid (200 μί) and the reaction was stirred for 30 minutes (monitored by tic), quenched with triemylarnine (2 mL) and the solvent removed by distillation under vacuum. The residue was dissolved in dichloromethane (20 mL), washed with water (10 mL), dried over magnesium sulfate, concentrated under vacuum and co-distilled with toluene (20 mL). The crude product was dissolved in dry dichloromethane (4 mL) and added to a stirred solution of Collin's reagent (chromium trioxide (1.01 g), pyridine (1.65 mL) in dry dichloromethane (30 mL)). The suspension was stirred for 10 minutes, filtered and the residue washed with diethyl ether (20 mL). The filtrate was concentrated onto silica (10 g) and purified by flash chromatography (silica gel, eluent = hexane/diethyl- emer/triemylamine (90:9:1) to afford the title compound as a light yellow oil (503 mg, 48%); 1H NMR (300 MHz, CDC1₃) δ 10.25(s, 1H, CHO), 7.79(d, 1H, J= 8.45 Hz), 6.98(d, 1H, J= 8.46 Hz), 4.65(m, 1H), 3.89(s, 3H, OMe), 1.35(d, 6H, J= 6.17 Hz), 0.97(s, 9H), 0.45(s, 6H).

Step 3: 2-t-ButyUimethykilyl-3-(3,4,5-trimethoxybenzoyl)-6-methoxy-7- isopropoxybenzofuran

To a stirred solution of 3,4,5-trimethoxyiodobenzene (377 mg, 1.27 mmol) in dry tetrahydrofuran (1 mL) at -78 °C under nitrogen was added n-butyllithium (795 LL, 1.59 mmol, 2M solution in cyclohexane) and the reaction mixture was stirred at this

temperature for 40 minutes. After this time a solution of 2-t-butyldimethylsilyl-3-formyl-6- memoxy-7-isoproxybenzofuran (1.07 mmol) in dry tetrahydrofuran (1 mL) was added to the reaction dropwise via syringe pipette. The reaction mixture was stirred at -60 °C for 20 minutes and then allowed to warm to 0°C, stirred for 10 minutes, quenched with saturated ammonium chloride solution (2 mL) and diluted with ethyl acetate (20 mL). The organic layer was washed with water (10 mL), dried over magnesium sulfate and the solvent was removed under vacuum to give a residue that was co-distilled with toluene. The crude product (908 mg) was dissolved in dry tetrahydrofuran (10 mL) and treated with 2,3- dichloro-5,6-dicyano-l,4-benzoquinone (900 mg, 1.59 mmol) was added. The reaction mixture was stirred at room temperature for 16 hours (monitored by tic) and then loaded onto silica (10 g) and purified by flash chromatography (silica gel, eluent = hexane/diethyl emer/triemylarnine, 90:9:1) to afford the title compound as a light yellow oil (498 mg, 69%); 1H NMR (300 MHz, CDC1₃) δ 7.14(s, 2H, benzoyl Hs), 6.81(d, 1H, J= 8.64 Hz), 6.77(d, 1H, J= 8.64 Hz) 4.74(m, 1H), 3.93(s, 3H, OMe), 3.86(s, 3H, OMe), 3.78(s, 6H, 2 x OMe), 1.39(d, 6H, J= 6.14 Hz), 1.01(s, 9H), 0.26(s, 6H).

Step 4: 2-(tert-butyldimethylsttyloxy)- 7-acetoxy-3-(3,4,5-trimethoxybenzoyl)-6- methoxybenzofuran

To a stirred solution of 2-(t-butyldimethylsilyloxy)-7-isopropoxy-3-(3,4,5- trimemoxybenzoyl)-6-memoxy-benzofuran (160 mg, 0.31 mmol) in dry DCM (2 mL) at room temperature under nitrogen was added solid aluminium trichloride (83 mg, 0.62 mmol) and the reaction mixture was stirred for 15 minutes (monitored by tic). The reaction was quenched with a saturated solution of ammonium chloride, extracted with dichloromethane and dried over magnesium sulfate. The solvent was removed by distillation and residue was dried by azeotropic removal of water with toluene. The crude product was dissolved in pyridine (2 mL), acetic anhydride (1 mL) was added and reaction mixture was stirred for 2 hours at room temperature. The solvent was distilled under vacuum and the residue was loaded onto silica gel (1 g) and purified by column chromatography (silica gel, eluent, hexane:diethyl-ether; 80:20) (134 mg, 84%); 1H NMR (300 MHz, CDC1₃) δ 7.14(s, 2H, benzoyl Hs), 6.98(d, 1H, J= 8.72 Hz), 6.85(d, 1H, J= 8.72 Hz), 3.93(s, 3H, OMe), 3.86(s, 3H, OMe), 3.80(s, 6H, 2 x OMe), 2.41(s, 3H), 0.99(s, 9H), 0.25(s, 6H).

Step 5: 2-Bromo- 7-acetoxy-3-(3,4,5-trimethoxybenzoyl)-6-ntethoxybenzofuran

To a stirred solution of 2-t-butyldimemylsilyl-7-acetoxy-3-(3,4,5-trimethoxybenzoyl)-6- memoxybenzofuran (120 mg, 0.44 mmol) in 1,2-dichloroethane (1 mL) at room

temperature under nitrogen was added bromine (12 μΐ, 0.44 mmol) dropwise and the reaction mixture was stirred at this temperature for 10 minutes. After this time the reaction was quenched with saturated sodium thiosulfate solution, extracted with ethyl acetate (20 mL), dried over magnesium sulfate and the solvent removed by distillation under vacuum. The crude product was purified by silica gel column chromatography (eluent =

Hexane:diethyl ether; 8:2 - 7:3) to afford the title compound as a colourless crystalline solid (91 mg, 81%); Ή NMR (300 MHz, CDC1₃) δ 7.40(d, 1H, J= 8.70 Hz), 7.14(s, 2H, benzoyl-Hs), 6.98(d, 1H, J= 8.75 Hz), 3.94(s, 3H, OMe), 3.89(s, 3H, OMe), 3.86(s, 6H, 2 x OMe), 2.43(s, 3H); ¹³C NMR (75 MHz, CDC1₃) δ 187.95(CO), 167.71, 152.75, 149.54, 147.49, 142.59, 131.92, 131.80, 123.91, 121.84, 119.89, 117.72, 109.89, 106.92, 60.69, 56.61, 56.00, 20.09.

Preparation of Compound Example 1 Preparation of 2-Methyl-7-hydroxy-3-(3,4,5-trimethoxybenzoyl)-6-methoxybenzofuran

Preparation A

To a stirred solution of 2-Bromo-7-acetoxy-3-(3,4,5-trimethoxybenzoyl)-6- methoxybenzofuran (20 mg, 0.042 mmol), methyl-boronic acid (40 mg, 0.67 mmol), in 1,4- dioxane (2 mL) at 90 °C was added te/ra&w-triphenylphosphine palladium (11 mg, 0.01 mmol) followed by the addition of a solution of sodium bicarbonate (40 mg, 0.48 mmol) in distilled water (0.5 mL). The reaction mixture turned red after 5 minutes. After 2 hours (tic) the reaction mixture was brought to room temperature and was added saturated ammonium chloride (2 mL) and diluted with dichloromethane (20 mL). The organic layer was separated and washed with water, dried over magnesium sulfate and the solvent was removed by distillation under vacuum. The residue was purified by PTLC (eluent = Dichloromethane/Methanol, 1:1) to give the title compound (actate cleaved during rection) as a fluffy white solid; (3 mg, 19%). Preparation B (Negishi Coupling)

To a stirred solution of zinc-bromide (592 mg, 2.63 mmol) in dry THF(1.5 mL) at 0°C was added the solution of methyl lithium (1.6 M solution in diethyl-ether, 2.6 mL, 4.15 mmol) and the reaction mixture was stirred for 2 hours. Solid 2-bromo-7-acetoxy-3-(3,4,5- trimemoxybenzoyl)-6-memoxy-berizofuran (300 mg, 0.63 mmol) was added and the ether was removed under vacuum and to the rest suspension was added

dicUorobis(triphenylphosphine)palladium catalyst (21 mg) and catalytic amount of copper (I) iodide. The reaction mixture was stirred at room temperature for 36 hours (monitored by tic), quenched with saturated ammonium chloride solution and extracted with dichloromethane (10 mL), dried over magnesium sulfate and solvent distilled under vacuum and the product was purified by silica gel column (eluent = hexane/ethyl acetate; 8:2). The product was crystallized in methanol (106 mg, 46%); 1H NMR (300 MHz, CDC1₃) δ 7.09(s, 2H, benzoyl Hs), 6.93(d, 1H, J= 8.54 Hz), 6.83(d, 1H, J= 8.56 Hz), 5.70(bs, 1H, OH), 3.93(s, 3H, OMe), 3.92(s, 3H, OMe), 3.83(s, 6H, 2 x OMe), 2.54(s, 3H, 2-Me) Preparation of Compound Example 2

Preparation of Disodium 6-methoxy-2-methyl-3-(3,4,5-trimethoxybenzoyl)benzofuran-7- yl phosphate

Step 1: Dibenzyl 6-methoxy-2-methyl-3-(3,4,5-trimethoxybenzoyl)benzofuran-7-yl phosphate:

To a mixture of 0.081 g (0.22 mmol) of (7-hydroxy-6-methoxy-2-memylbenzofuran-3- yl)(3,4,5-trimethoxyphenyl)methanone, 0.086 g (0.261 mmol) of carbon tetrabromide and 0.063 ml (0.283 mmol) of dibenzylphosphite in 2.5 ml of anhydrous acetonitrile 0.046 ml of anhydrous triethylamine was added dropwise at 0°C under nitrogen atmosphere. The resulting mixture was stirred for 2h at room temperature, then diluted to 20 ml with ethyl acetate, washed with water brine, dried over anhydrous magnesium sulfate, filtered off and evaporated to dryness under reduced pressure. The residue was purified by flash column chromatography (dichloromethane/ ethyl acetate, 9:1) to give the title compound as a colorless foam (0.13g, 94%); 1H NMR (CDC1₃) δ 2.42 (s, 3H, Me-2); 3.83 (s, 1H, OMe); 3.93 (s, 3H, OMe); 5.33 (m, 4H, CH₂Ph); 6.89 (d, CH aromatic, J= 8.7 Hz); 7.21 (dd, 1H, CH aromatic, J= 8.72 Hz; J = 1.2 Hz); 7.08 (s, 2H, CH aromatic); 7.29 - 7.43 (m, 10 H, CH aromatic).

Step 2: Disodium 6-methoxy-2-methyl-3-(3,4,5-trimethoxybenzoyl)benzqfuran-7-yl phosphate:

To a stirred solution of 0.122 g (0.193 mmol) of the product from Step 1 in 1 ml of anhydrous acetonitrile 0.075 ml (0.58 mmol) of bromotrimethylsilane was added at -5°C under nitrogen atmosphere. The resulting mixture was stirred for 1 h at 0°C, then evaporated to dryness in vacuo. The residue was diluted to 5 ml with anhydrous methanol and pH of the solution was brought up about 10 by the addition of sodium methoxide. After evaporation of the resulting mixture under reduced pressure the solid residue was washed with anhydrous isopropanol (4 x 1.5 ml) and anhydrous ethanol (3 x 1.5 ml) and dried under vacuum to give 0.062 g (65 % yield) of title compound as an colorless solid; 1H NMR (D₂0) δ 2.37 (s, 3H, Me-2); 3.76 (s, 6H, OMe); 3.79 (s, 3H, OMe); 3.82 (s, 3H, OMe); 4.66 (s, H₂0); 6,93 (d, 1H, CH aromatic, J= 8.6 Hz); 7.04 (d, 1H, CH aromatic, J = 8.6 Hz); 7.10 (s, 2H, CH aromatic).

Example 2: Reagent Preparation lOOmM GTP solution

Dissolve GTP to lOOmM in water and store 30μ1 aliquots at -80°C. Protease inhibitor (PI)

Dissolve protease inhibitor cocktail to 50^χ in water and store 20μ1 aliquots at -20°C. Lysis buffer

PBMC lysis buffer (make up to 5ml with MQH₂0) at pH 6.9

Immediately prior to use add lOmM GTP and 1 *PI to lysis buffer.

10 mM GTP 1 ΟΟμΙ added to 1 ml of lysis buffer

1 x protease inhibitor (PI) 20μ1 added to 1 ml of lysis buffer

Paclitaxel may be included in the lysis buffer if tubulin levels were low, so as to maintain polymerized tubulin if desired. Example 3: Tubulin Extraction and Analysis

Compounds that act to either stabilize or destabilize microtubules have an effect on the amount of tubulin in the polymerized state. The polymerized tubulin can be separated from the depolymerized tubulin fraction by centrifugation at high speed. The pellet tubulin fraction and the supernatant (depolymerized) fraction can be analyzed by Western blot to measure shifts in tubulin concentration in each fraction in response to tubulin targeting induced changes in polymerization state. Tubulin Extraction

Before commencing extraction:

1. thaw GTP and PI and place on ice

2. set ultracentrifuge to 37°C and water bath to 37°C

3. pre- warm ultracentrifuge rotor in water bath to 37°C

4. set Eppendorf tube heater to 37°C and warm the required amount of lysis buffer

5. pre- warm PBS to 37°C in water bath

Extraction ofPBMC Tubulin

1. take patient PBMCs from liquid nitrogen and thaw quickly in 37°C water bath

2. transfer to Eppendorf tubes and centrifuge at 300 RCF for 5 minutes at 37°C if possible, or at room temperature

3. remove supernatant and re-suspend cell pellets in 37°C PBS to wash

4. centrifuge at 300 RCF and remove PBS

5. re-suspend PBMC pellets in 50μ1 of PBMC lysis buffer containing GTP and PI at 37°C

6. transfer lysates to 4ml Beckman ultracentrifuge tubes (polyallomer or

similar)

7. centrifuge samples at 180,000g (~70,000RPM) at 37°C for 1 h

8. carefully aspirate supernatant without disturbing pellet and transfer to

Eppendorf tube

9. re-suspend the pellet in a 50μ1 volume of lysis buffer (-equal to the

supernatant)

10. use the homogenizer to grind the pellet against the polyallomer tube wall in lysis buffer in order to solubilize polymerized tubulin

11. transfer solubilized pellet to an Eppendorf tube and add 15μ1 of 4χ NuPage sample loading buffer containing 30mg/ml DTT to both the pellet and the supernatant fractions

12. boil samples at 100°C for 4 minutes followed by centrifugation at

13,000RPM for a 5 seconds

13. use a fine gauge needle (~29G or 0.33mm) and syringe to shear the DNA by pulling sample in and out of the syringe.

Western Blotting ofPBMC Tubulin

While the supernatant fraction is informative after in vitro cell line experiments, it was not informative for patient PBMC samples. The pellet tubulin fraction was used for patient PBMCs.

1. load equal volumes of polymerized into SDS-PAGE gels and subject to electrophoresis at 130 volts for ~1 hour

2. transfer proteins to nitrocellulose (N/C) membrane at 30 volts for 1 hour

3. block non-specific proteins by rocking N/C membrane in TBS-T + 5% nonfat milk powder at RT for 1 hour

4. rinse N/C membrane in TBS-T for 2 x 5 minutes

5. prepare primary antibodies specific for βΐ-tubulin (concentration 1 :2000) in 5ml of TBS-T

6. encapsulate N/C membrane in sealed plastic bag, add tubulin antibodies and seal

7. incubate at 4°C overnight on a rotating wheel

8. wash N/C membrane for 3 χ 5 minutes in TBS-T at RT in a plastic tray

9. prepare secondary anti-mouse-HRP antibody at 1 :2000 in TBS-T

10. transfer N/C membrane to plastic bag, add prepared secondary antibody and seal

1 1. incubate at room temperature rocking for 1 hour

12. wash N/C membrane 3 x 5 minutes in TBS-T at room temperature

13. combine 0.7ml of each ECL reagent, part A and part B, and pour onto N/C membrane

14. carry in small tray to the camera/detector apparatus along with a clear

plastic sheet to cover the N/C membrane before placing it on the ECL tray in the detector 1 . set the open aperture time to 5 seconds and observe the bands detected on the screen for position and intensity

16. estimate the open aperture time that will give a good intensity without overexposure so that subtle differences in the bands can be observed; set camera to this time and photograph the N/C membrane

17. save the image

18. rinse the N/C membrane in TBS-T for 10 minutes

19. prepare primary antibodies specific for βΐ-actin (concentration 1 :5000) in 5ml of TBS-T

20. Encapsulate N/C membrane in sealed plastic bag, add tubulin antibodies and seal

21. rock at room temperature for 1 hour

22. wash N/C membrane for 3 * 5 minutes in TBS-T at room temperature in a plastic tray

23. prepare secondary anti-mouse-HRP antibody at 1 :2000 in TBS-T

24. transfer N/C membrane to plastic bag, add prepared secondary antibody and seal

25. incubate at room temperature rocking for 1 hour

26. wash N/C membrane 3 ^χ 5 minutes in TBS-T at room temperature

27. combine 0.7ml of each ECL reagent, part A and part B, and pour onto N/C membrane

28. carry in small tray to the camera / detector apparatus along with a clear plastic sheet to cover the N/C membrane before placing it on the ECL tray in the detector

29. set the open aperture time to 5 seconds and observe the bands detected on the screen for position and intensity

30. estimate the open aperture time that will give a good intensity without overexposure; set camera to this time and photograph the N/C membrane

31. save the image.

Analysis of PBMC Tubulin

1. determine quantitative densitometry values of actin and tubulin bands from images

2. divide each tubulin band by its associated actin control to normalize

3. convert the amount of tubulin in the pellet and the supernatant of each

sample to a percentage of the total amount of tubulin present if necessary using the following formula: tubulin band / (pellet tubulin + supernatant tubulin) xlOO

4. generate a graphical representation using Microsoft Excel to compare

tubulin fractions between samples.

Example 5: Results

Figure 1 shows the tubulin response of A2780 cells in vitro when exposed to Compound Example 1. Figure 1 A shows the total amount of tubulin and actin present in the pellet ("P") and supernatant ("S") fractions following exposure of the cells to 10, 20, 25 and 30 nM Compound Example 1. Figures IB and 1C show the amount of tubulin (as a percentage of total tubulin present in cells) present in the pellet and supernatant fractions over various concentrations of Compound Example 1. These data indicate that at concentrations of > 25 nM Compound Example 1, tubulin begins to depolymerize and ends up in the soluble (i.e. supernatant) fraction. At concentrations of < 25 nM Compound Example 1, there is no effect on tubulin polymerization where the total amount of polymerized tubulin in the pellet fraction is similar to the control (C) and vehicle control (DMSO) samples. Figure 2 shows the tubulin response in human peripheral blood mononuclear cells

(PBMCs) in vitro following exposure to Compound Example 1. Figure 2A shows the total amount of tubulin and actin present in the pellet ("P") and supernatant ("S") fractions from two different volunteers following exposure of cells to 0.5 μΜ Compound Example 1 for 30 mins. Figures 2B and 2C show the normalized tubulin in the pellet and supernatant fractions of PBMCs. These data show the shift in tubulin content between the pellet and supernatant fractions evidencing tubulin depolymerisation following exposure to

Compound Example 1.

Figures 3-6 show the tubulin response in PBMCs from patients suffering from oesophageal cancer (Figure 3), renal cancer (Figure 4), rectosigmoid (Figure 5) and colorectal cancer (Figure 6) following administration of Compound Example 2. The dose of Compound Example 2 administered was between 12.6-16 mg/m². The data presented in Figures 3-6 show a reduction in tubulin polymerization following administration of Compound

Example 2. The data presented in Figures 4-6 show at approximately 7 hours post- administration the level of tubulin polymerization returns to normal (i.e. to a level similar to non-treated cells). Further, no haematological toxicity was observed in patients following administration of 12.6 mg/m² Compound Example 2. No dose limiting toxicities have been reported at the higher dose level of 16 mg/m².

In summary, these data show that the methods according to the present invention provide an effective means to determine whether tubulin targeting agents are reaching and affecting target cells.

Example 6:

Introduction

The novel anti-cancer drug of Example 1 causes occlusion of tumor vasculature and suppresses cancer cell proliferation by interfering with tubulin polymerization. The compound of Example 1 is formulated as the disodium phosphate of Example 2 for IV administration. The compound of Example 2 is rapidly converted to the active agent of Example 1 after entering the blood stream. Treatment of mice bearing solid tumors derived from subcutaneous injection of human cancer cell lines results in dramatic disruption of tumor vasculature and significant suppression of tumor growth. The safety, pharmacokinetics and preliminary therapeutic activity of the compound of Example 1 were assessed in a phase I clinical trial in patients with advanced cancer indications. The method of the present invention was used to enable detection of changes in polymerized tubulin in PBMCs in patients following the administration of the compound of Example 2.

Materials and methods PBMCs from patients enrolled in the Phase I clinical trial of the compound of Example 2 were assayed for changes in tubulin polymerization (** indicates a one week delay for cycle 2). Table 1 : the compound of Example 2 Phase I clinical trial patients; tumor types and the compound of Example 2 dose levels.

Patient Tumor type Dose Level Analysis of number (mg/m²) tubulin

polymerization status

2201 Colon 2.1 completed

3302 Ovarian 4.2 completed

1103 Thyroid 8.4 completed

2204 Renal 8.4 completed

3305 Mesothelioma 8.4 completed

2206 Renal 12.6 completed

2207 Colon 12.6 completed

3308 Duodenal 12.6 completed

4412 Rectosigmoid 12.6 completed adenocarcinoma

1115 Leiomyosarcoma 12.6 N/D

1116 Upper GK, unknown primary 12.6 completed

2220 Colon 16.0 completed

1118 Head and Neck 16.0 completed

1122 Adrenocortical carcinoma 16.0 completed

4421 Conon 16.0 N/D

2223 Leiomyosarcoma 16.0 completed

1124 Mesothelioma 16.0 N/D

1109 Esophageal 18.9 completed

2210** Colon 18.9 N/D

4413 Melanoma 18.9 completed

3311 Salivary 18.9 N/D Sampling

Blood samples (approximately 4mls) from patients were obtained from a catheter port in the arm prior to dosing, 1 hour (± 10 minutes), 2 hours (± 10 minutes), 3-5 hours (± 10 minutes), 7 hours (± 10 minutes) and 24 hours (± 10 minutes) post BNC105P

administration, and collected into BD Vacutainer CPT Cell Preparation Tubes (Becton Dickinson, USA).

PBMC Isolation

Blood samples collected into BD Vacutainers were centrifuged to separate PBMCs, platelets, and blood plasma according to the manufactures protocol. PBMCs were isolated and washed in PBS prior to resuspension in 7% DMSO/FCS. Cells were aliquoted into Cryovials and cooled at -l°C/min to -80°C prior to being transferred to liquid nitrogen for long term storage.

Quantitation of PBMC Tubulin

Tubulin extracted from PBMC samples was quantitated as described above. Briefly, samples were thawed in a 37°C water bath and centrifuged to isolate PBMCs from freezing media. PBMC cell pellets were washed in PBS and resuspended in lysis buffer. Cell lysates were centrifuged in a Beckman Optima TLX ultracentrifuge at 180,000xg for 1 hour. Supernatant was aspirated and the insoluble cell pellets were washed in PBS before the addition of 50μί of lysis buffer and homogenization. Cell pellet lysates were subjected to SDS-PAGE and Western blot analysis using anti-tubulin or anti-actin primary antibodies. Protein bands were photographed and analyzed using ImageJ software.

Image and Data analysis Tubulin bands representing polymerized tubulin from PBMCs were quantitated by densitometry, and normalized to the actin loading control for each sample to indicate relative tubulin concentration. Normalized data from each patient sample was expressed as a percentage of pre-dose tubulin concentration and plotted using Microsoft Excel software. Results

This study demonstrates that the compound of Example 1 causes tubulin depolymensation in the PBMC's of patients treated intravenously with the pro-drug, compound of Example 2. The time points identified for tubulin depolymerization and recovery correlate well with PK data. A dose-response effect on tubulin depolymerization was demonstrated across three dosing cohorts. At doses >12.6mg/m² polymerized tubulin in PBMCs was reduced proportionally to the dose of the compound of Example 2 administered. Furthermore, the duration of the depolymerizing effect was also proportional to the dose administered. Patients dosed with 12.6mg/m the compound of Example 2 showed recovery of polymerized tubulin back to pre-dose levels by the 7 hour time point. Patients dosed with

16mg/m 2 and 18.9mg/m 2 showed recovery of polymerized tubulin by the 24 hour time point. Collectively, these results demonstrate on-target activity of the compound of Example 1 at well tolerated dose levels in phase I clinical trial cancer patients.

The combined results are shown in Table 2 and Figure 8. The tubulin concentration at th3- 5hour time point across all patient cohorts is set out in Table 3 while Figure 9 shows the average tubulin concentration in cell pellets from each dose cohort at the 3-5 hour post- dose time point. No tubulin reduction was observed at or below the 8.4 mg/m2 dose level. A possible dose-response was observed at doses >12.6mg/m .

Table 2

Table 3

% Tubulin

compared to Average

Day

Patient Dose predose at 3- for dose

# mg/m² 5hr cohort

2201 2.1 8 100 100

3302 4.2 8 100 100

2204 8.4 1 100 100

3305 8.4 1 100

2206 12.6 8 5 30.5

2207 12.6 1 100

4412 12.6 1 8

1116 12.6 8 9

1118 16 1 10 14.75

1122 16 8 11

2220 16 1 25

2223 16 8 13

1109 18.9 1 3 3 Conclusions

The IV administration of the compound of Example 2 to phase I clinical trial patients resulted in exposure of tissues to the active tubulin polymerization inhibiting agent, compound of Example 1. Given that the agent is administered intravenously it is reasonable to expect that PBMC's will be directly affected by the agent and can therefore be useful as surrogate tissue biomarkers for the analysis of the compound of Example 1- induced tubulin depolymerization. Following the administration of the compound of Example 2 at concentrations >12.6mg/m a reduction in polymerized tubulin was detected in the insoluble (polymerised) tubulin fraction at time points that correlated well with PK data. Although a number of patients remain to be tested to complete this study, the results obtained following the analysis of fourteen phase I patient PBMC samples indicate that the compound of Example 1 administered at concentrations >12.6mg/m² causes tubulin depolymerization in PBMCs. This result shows that the compound of Example 1 causes peripheral cell tubulin depolymerization, and thereby indicates that tumor vasculature is exposed to a drug concentration capable of causing blood vessel occlusion.

Claims

THE CLAIMS DEFINING THE INVENTION ARE AS FOLLOWS:

1. A method for assessing the in vivo activity of a tubulin targeting agent, the method comprising:

(i) administering a tubulin targeting agent to an animal;

(ii) obtaining a cell sample from the animal;

(v) measuring the level of tubulin in the precipitate and in the supernatant.

2. A method for assessing the activity of a tubulin targeting agent, the method comprising:

(i) contacting cells with a tubulin targeting agent;

(iv) measuring the level of tubulin in the precipitate and in the supernatant.

3. A method for assessing the activity of a tubulin targeting agent, the method comprising:

(ii) lysing the cells of both samples from step (i) under conditions which

4. A method according to any one of claims 1 to 3 wherein the level of tubulin is measured using an anti-β tubulin antibody.

5. A method according to any one of claims 1 to 4 wherein the level of tubulin is measured by Western blot or ELIS A.

6. A method according to any one of claims 1 to 5 wherein step (iii) is conducted at a pH of about 6.9.

7. A method according to any one of claims 1 to 6 wherein step (iii) is conducted at a temperature of about 37°C.

8. A method according to any one of claims 1 to 7 wherein step (iii) is conducted in a buffer comprising glycerol, DMSO, Triton X-100, and GTP.

9. A method according to any one of claims 1 to 8 wherein step (iv) is conducted at about 180,000 x g.

10. A method according to any one of claims 1 or 4 to 7 wherein the animal has cancer.

11. A method according to claim 10 wherein the animal is human.

12. A method according to claim 10 or claim 11 wherein the cell sample comprises PBMC.

13. A method according to claim 10 or claim 11 wherein the cell sample comprises circulating endothelial cells.

14. A method according to any one of claims 1 to 11 wherein the cell sample comprises tumour cells.

15. A method according to any one of claims 3 or 4 to 7 wherein the samples of cells are taken from an animal which has cancer.

16. A method according to claim 15 wherein the animal is human.

17. A method according to claim 15 or claim 16 wherein the cell sample comprises PBMC.

18. A method according to any one of claims 1 to 17 wherein the tubulin targeting agent is

or a pharmaceutically acceptable salt, solvate or prodrug thereof.

19. A method for identifying a target compound as having tubulin targeting activity, the method comprising:

(i) contacting cells with a target tubulin targeting agent;

(iv) measuring the level of tubulin in the precipitate and in the supernatant, wherein an increase in the level of tubulin in the supernatant is indicative of tubulin targeting activity.

20. A method according to claim 19 wherein the level of tubulin is measured using an anti-β tubulin antibody.

21. A method according to claim 19 or claim 20 wherein the level of tubulin is measured by Western blot or ELISA.

22. A method according to any one of claims 19 to 21 wherein step (ii) is conducted at a pH of about 6.9.

23. A method according to any one of claims 19 to 22 wherein step (ii) is conducted at a temperature of about 37°C.

24. A method according to any one of claims 19 to 23 wherein step (ii) is conducted in a buffer comprising glycerol, DMSO, Triton X-100, and GTP.

25. A method according to any one of claims 19 to 24 wherein step (iii) is conducted at about 180,000 x g.

26. A method according to any one of claims 19 to 25 wherein the cell is a tumour cell.