This invention relates to a method of measuring one or more thiopurine metabolites in a patient treated with a thiopurine drug. The invention also relates to a method of predicting clinical response or tolerance of a patient to a thiopurine drug.

BACKGROUND

Since their introduction into clinical practice more than six decades ago, the purine analogues 6-mercaptopurine (6-MP), azathioprine and 6-thioguanine (6-TG) have been used extensively in the treatment of diseases such as acute childhood leukaemia, inflammatory bowel disease (IBD), auto-immune hepatitis and rheumatoid arthritis. These purine analogues are inactive pro-drugs that are cytotoxic after metabolism within nucleated cells; cytotoxicity is believed to be mediated by their intracellular metabolites which have a variety of effects including inhibition of de novo purine synthesis (DNPS), alterations in DNA methylation ¹ , incorporation of thioguanine nucleotides (TGNs) into DNA and disturbances in G-protein signalling ² .

Thiopurines are likely to be metabolised in the same manner as endogenous purines and require activation by hypoxanthine-guanine phosphoribosyl transferase (HGPRT, E.C. 2.4.2.8) followed by a multi-step metabolism to TGNs or to methylated products (MeTGNs) that inhibit DNPS (Figure 1). Within this pathway several key enzymes such as inosine- monophosphate dehydrogenase (IMPDH) ³ , inosine tri-phosphatase ⁴ (ITPase) and methyl- thioadenosine phosphorylase (MTAP) ⁵ modulate the effects of thiopurines. The most extensively-investigated enzyme of thiopurine metabolism is thiopurine methyl-transferase (TPMT) ⁶ ' ⁷ ; this has a trimodal distribution of activity in the population with about one in 300 people lacking any activity at all ⁸ . If treated with normal doses of thiopurines, patients lacking TPMT activity develop high TGN levels which can lead to life-threatening leukopenia ^9"11 . To monitor and optimise thiopurine therapy, many assays have been developed to measure metabolites in different cellular compartments: erythrocytes ^{9, 12"22} , whole blood ^{21 , 23, 24} , isolated lymphocytes ^25"27 , leukocyte DNA ^28"31 and plasma ^32"36 . Despite the range of methods available, the ease of access to erythrocytes coupled with straightforward HPLC separation techniques has meant that the identification of thiopurine metabolites in erythrocytes has become the standard method for therapeutic monitoring. Although this approach is useful to guide therapy and assess patient compliance, there is significant debate about concordance with therapeutic response ^{37, 38} . These current methods rely on chemical modifications which

l alter the phosphorylation and or methylation status of thiopurine metabolites present in the sample. Non-concordance can be due to methodological issues (review ³⁹ ) but also to the fact that erythrocytes cannot synthesise purines and that nucleotide salvage in these cells is limited by a lack of the crucial enzymes, adenylosuccinate synthase and IMPDH. Thus, TGNs or the thioxanthine monophosphate (TXMP) precursor within erythrocytes cannot be derived from erythrocyte thiopurines directly but must be taken up from the surrounding milieu after metabolism via IMPDH in nucleated cells.

Adverse reactions and non-response are common in patients treated with thiopurine drugs and therapeutic monitoring of thiopurine metabolites is important for guiding clinical treatment. Current methods limit monitoring to the presence of thiopurine metabolites in erythrocytes after chemical derivatisation to facilitate detection. The results of such measurements can show poor correlations with clinical response. There is a need for an improved method to detect thiopurine metabolites in a patient treated with a thiopurine drug.

BRIEF SUMMARY OF THE DISCLOSURE

In one aspect, the invention provides a method of measuring one or more thiopurine metabolites in a patient treated with a thiopurine drug, comprising:

(i) lysing a test cell derived from said patient in a lysis buffer so as to obtain a cell lysate; and

(ii) measuring one or more thiopurine metabolites in said cell lysate,

wherein said lysis buffer consists of one or more reagents that do not chemically alter the phosphorylation and/or methylation state of said one or more thiopurine metabolites in said cell lysate.

In a further aspect, the invention provides a method of predicting clinical response or tolerance of a patient to a thiopurine drug, said method comprising:

(i) lysing a test cell derived from said patient in a lysis buffer so as to obtain a cell lysate; (ii) measuring one or more thiopurine metabolites in said cell lysate,

(iii) correlating the relative amount of said one or more thiopurine metabolites in said cell lysate to the clinical response or tolerance of said patient to said thiopurine drug so as to predict clinical response or tolerance; and optionally

(iv) preparing a treatment regimen based upon said prediction,

wherein said lysis buffer consists of reagents that do not chemically alter the phosphorylation and/or methylation state of said one or more thiopurine metabolites in said cell lysate. In one embodiment, said reagents do not chemically alter said one or more thiopurine metabolites in said cell lysate.

In one embodiment, said thiopurine drug is selected from the group consisting of azathioprine, 6-mercaptopurine, and 6-thioguanine.

In one embodiment, said one or more thiopurine metabolites is selected from the group consisting of 6-thioguanine-mono-phosphate (TGMP), 6-thioguanine-di-phosphate (TGDP), 6-thioguanine-tri-phosphate (TGTP), 6-methylthioguanine-mono-phosphate (meTGMP), 6- methylthioguanine-di-phosphate (meTGDP), 6-methylthioguanine-tri-phosphate (meTGTP), 6-methylthioinosine-mono-phosphate (meTIMP), 6-methylthioinosine-di-phosphate

(meTIDP), 6-methylthioinosine-tri-phosphate (meTITP) and 6-thioinosine-mono-phosphate (TIMP). In one embodiment, said one or more thiopurine metabolites is 6-methylmercaptopurine (6- MMP).

In one embodiment, said patient is a human. In one embodiment, said patient has a disease or disorder selected from the group consisting of an immune-mediated gastrointestinal disorder, an autoimmune disease, acute lymphoblastic leukemia and graft versus host disease, cancer, and hyperproliferative disease. In one embodiment, said cancer is metastatic cancer or non-metastatic cancer. In one embodiment, said patient is an organ transplant recipient.

In one embodiment, said immune-mediated gastrointestinal disorder is inflammatory bowel disease (IBD).

In one embodiment, said autoimmune disease is rheumatoid arthritis.

In one embodiment, said one or more thiopurine metabolites is measured using the mass/charge ratio of said one or more thiopurine metabolites or fragments thereof, optionally wherein said measurement is preceded by chromatographic separation. In one embodiment, said one or more thiopurine metabolites is measured using mass spectrometry, optionally wherein said mass spectrometry is selected from the group consisting of LC-MS, LC-MS/MS and LC- ESI -MS/MS. In one embodiment, lysing said test cell comprises cell sonication.

In one embodiment, said lysis buffer comprises or consists of water, optionally wherein said water is deionised water. In one embodiment, said lysis buffer comprises a detergent, optionally wherein the detergent is selected from the group consisting of Triton X-100, Triton X-114, NP-40, Brij-35, Brij-58, Tween 20, Tween 80, Octyl glucoside, Octyl thioglucoside, sodium lauryl sulphate, sodium lauryl sarcosine and CHAPS (3-[(3-Cholamidopropyl) dimethylammonio]-1- propanesulfonate).

In one embodiment, said lysis buffer comprises EDTA. In one embodiment, said lysis buffer comprises a phosphatase inhibitor. In one embodiment, said lysis buffer comprises DTT. In one embodiment, said test cell is a blood cell.

In one embodiment, said blood cell is an erythrocyte, a leukocyte or a peripheral blood mononucleated cell.

In one embodiment, said test cell is derived from a solid tissue.

In one embodiment, said solid tissue is liver.

In a further aspect, the invention provides a method of measuring thiopurine metabolites in a patient treated with a thiopurine drug as hereinbefore described, with reference to the accompanying drawings. In a further aspect, the invention provides a method for predicting clinical response or tolerance of a patient to a thiopurine drug as hereinbefore described, with reference to the accompanying drawings. BRIEF DESCRIPTION OF THE DRAWINGS

Embodiments of the invention are further described hereinafter with reference to the accompanying drawings, in which:

Figure 1 provides a schematic of the metabolism of azathioprine (AZA) to 6-mercaptopurine (6-MP) and other metabolites via the enzymes inosine-monophosphate dehydrogenase (IMPDH), thiopurine methyl-transferase (TPMT), xanthine oxidase (XO), hypoxanthine- guanine phosphoribosyl transferase (HGPRT), inosine tri-phosphatase (ITPase) and guanosine monophosphate synthetase (GMPS). Metabolites measured by current commercial assays based on the Dervieux method are 6-MMP, MeMPR and TGNs, and by the new LC-MS/MS assay of the invention are MeTIMP, MeTIDP, MeTITP, MeTGTP, MeTGDP, MeTGMP, TGMP, TGDP and TGTP. In the commercial assays, some metabolites are indistinguishable from each other after pre-processing. After acid hydrolysis, 6- methylmercaptopurine (6-MMP) undergoes degradation, leading to 4-amino-5- (methylthio)carbonyl imidazole when using the method by Dervieux ⁴⁶ and this is indistinguishable from the methylmercaptopurine ribosides (MeMPR) and methylated TGNs (MeTGNs). In the case of the thioguanine nucleotides removal of the phosphate groups results in mono- di- and tri- nucleotides being indistinguishable ¹⁹ .

Figure 2 provides LC-MS/MS profiles for metabolite standards.

Figure 3 provides example LC-MS/MS profiles for thiopurine metabolites from PBMC (right panel) and erythrocytes (left panel) from a patient on treatment with low-dose azathioprine with allopurinol.

Figure 4 summarises the key elements of the invention (LC-MS/MS profiles of thiopurine metabolites) in relation to the metabolism of thiopurines.

DETAILED DESCRIPTION

The inventors have surprisingly identified a method of processing cells for thiopurine metabolite measurement that does not result in or require chemical derivatisation of the metabolites to be analysed. The method involves minimal sample processing and advantageously provides a method of sufficient sensitivity to detect thiopurine metabolites in less abundant cell types, which may be better representatives of target cell populations. The methods of the invention can be used to develop more accurate therapeutic monitoring techniques and biomarkers of patient response.

More specifically, the inventors have found that the cell processing method of the invention allows the use of mass spectrometry (liquid chromatography-tandem mass spectrometry (LC- MS/MS)) to measure thiopurine metabolites in peripheral mononuclear cells from inflammatory bowel disease (IBD) and autoimmune hepatitis patients without chemical derivatisation of the metabolites to be analysed. The inventors have demonstrated the advantageous sensitivity provided by the processing method of the invention. The inventors compared PBMC metabolite profiles with erythrocyte metabolite profiles derived from the same patient and have surprisingly found that, using the methods of the invention, they were able to detect marked differences in metabolite profiles between erythrocytes and PBMCs. The method of the invention was used to quantify nine clinically-relevant thiopurine metabolites in PBMCs with minimal sample processing. With commercially available standards, an intra- and inter-assay variability below 8.3 and 1 1.6% respectively was achieved using liquid chromatography and detection by tandem mass spectrometry. All nine metabolites were detected in as few as 50,000 cells and 0.195 pmol/mg of protein. This is particularly beneficial in thiopurine drug target cells which are less abundant than erythrocytes.

The methods of the invention allow for precise discrimination of distinct thiopurine metabolites in different cell types and provides a powerful analytical tool that will facilitate a better understanding of thiopurine metabolism in relation to clinical response and the mechanisms of action of these inexpensive yet effective drugs.

The methods of the invention may therefore be used to identify clinical biomarkers that correlate with clinical response or tolerance. The invention provides a method of measuring one or more thiopurine metabolites in a patient treated with a thiopurine drug, comprising:

(i) lysing a test cell derived from said patient in a lysis buffer so as to obtain a cell lysate; and

(ii) measuring one or more thiopurine metabolites in said cell lysate,

wherein said lysis buffer consists of reagents that do not chemically alter the phosphorylation and/or methylation state of said one or more thiopurine metabolites in said cell lysate. The invention also provides a method for predicting clinical response or tolerance of a patient to a thiopurine drug, said method comprising:

(i) lysing a test cell derived from said patient in a lysis buffer so as to obtain a cell lysate;

(ii) measuring one or more thiopurine metabolites in said cell lysate,

(iii) correlating the relative amount of said one or more thiopurine metabolites in said cell lysate to the clinical response or tolerance of said patient to said thiopurine drug so as to predict clinical response or tolerance; and optionally

(iv) preparing a treatment regimen based upon said prediction,

wherein said lysis buffer consists of reagents that do not chemically alter the phosphorylation and/or methylation state of said one or more thiopurine metabolites in said cell lysate.

As used herein, the terms "drug" and "therapeutic agent" are interchangeable. As used herein, a "thiopurine drug" refers to a broad class of drugs that inhibit cell growth or induce the death of cells, including Natural Killer (NK) cells of the immune system. In the context of the invention, the terms "6-thiopurine drug" and "6-thioguanine drug" can be used interchangeably. Thiopurine drugs are purine antimetabolites which are routinely used in the treatment of a number of diseases and disorders, as defined further herein. 6-thioguanine (6- TG), 6-mercaptopurine (6-MP) and azathioprine are examples of thiopurine drugs, but the invention is not limited to these thiopurine drugs only.

Azathioprine (AZA) is a pro-drug of 6-MP. In the body, 6-MP may be converted to either thioguanine nucleotides (TGNs) or 6-methylmercaptopurine (6-MMP) (see Figure 1). TGNs are the principle metabolites leading to immunosuppression, whereas 6-MMP has been linked to hepatotoxicity and other side effects. The conversion of 6-MP to 6-MMP is catalysed by the polymorphic methyltransferase enzyme thiopurine-S- methyltransferase (TPMT). Pre-treatment measurement of TPMT activity is recommended to allow safe dose rationalisation of thiopurines, since low levels of this enzyme are associated with myelotoxicity at standard drug doses.

6-thiopurine drugs also include, for example, 6-TG. 6-TG is a particularly useful thiopurine drug in patients having high TPMT activity. Patients exhibiting high TPMT activity are expected more easily to convert thiopurine drugs such as 6-MP and AZA to 6-MMP (see Figure 1). High levels of 6-MMP are associated with hepatotoxicity. As used herein, the term "6-thioguanine" or "6-TG" includes 6-thioguanine or analogues thereof, including molecules having the same base structure, for example, 6-thioguanine ribonucleoside, 6-thioguanine ribonucleotide mono-, di- and tri-phosphate, 6-thioguanine deoxyribonucleoside and 6- thioguanine deoxyribonucleotide mono, di, and triphosphate. The term "6-TG" also includes derivatives of 6-thioguanine, including chemical modifications of 6-TG, so long as the structure of the 6-TG base is preserved.

As used herein, the term "6-methylmercaptopurine" or "6-MeMP", or "6-MMP" includes 6- methylmercaptopurine or analogues thereof, including analogues having the same base structure, for example, 6-methylmercaptopurine ribonucleoside, 6- methylmercaptopurine ribonucleotide mono-, di-, and tri-phosphate, 6- methylmercaptopurine deoxyribonucleoside, and 6-methylmercaptopurine deoxyribonucleotide mono-, di- and tri-phosphate. The term "6- MMP" also includes derivatives of 6-methylmercaptopurine, including chemical modifications of 6-MMP, so long as the structure of the 6-MMP base is preserved.

As used herein, the term "metabolite" includes a metabolic product derived from a thiopurine drug in a biological system. The terms "metabolite" and "thiopurine metabolite" are used interchangeably herein unless stated otherwise. Exemplary thiopurine metabolites are shown in Figure 1 and include 6-thioguanine-mono-phosphate (TGMP), 6-thioguanine-di- phosphate (TGDP), 6-thioguanine-tri-phosphate (TGTP), 6-methylthioguanine-mono- phosphate (meTGMP), 6-methylthioguanine-di-phosphate (meTGDP), 6-methylthioguanine- tri-phosphate (meTGTP), 6-methylthioinosine-mono-phosphate (meTIMP), 6- methylthioinosine-di-phosphate (meTIDP), 6-methylthioinosine-tri-phosphate (meTITP), and 6-thioinosine-mono-phosphate (TIMP). The methods of the invention may measure and/or distinguish one or more thiopurine metabolite(s). The methods of the invention may therefore measure and/or distinguish 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10 or more distinct thiopurine metabolites within a test cell sample.

As used herein, a "patient" refers to a subject that is in need of treatment or undergoing medical treatment or therapy. Optionally, the patient is being treated with a thiopurine drug. The patient may be any subject, including but not limited to a human, non-human primate, cat, dog, horse, rabbit, rat or mouse. In a preferred embodiment, the patient is a human.

As used herein, the terms "therapy," "therapeutic," "treating," "treat," "treatment," "treatment regimen," or "treatment regime" are used interchangeably and refer broadly to treating a disease, arresting, or reducing the development of the disease or its clinical symptoms, and/or relieving the disease, causing regression of the disease or its clinical symptoms. Therapy encompasses prophylaxis, treatment, remedy, reduction, alleviation, and/or providing relief from a disease, signs, and/or symptoms of a disease. Therapy encompasses an alleviation of signs and/or symptoms in patients with ongoing disease signs and/or symptoms (e.g., inflammation, pain). Therapy also encompasses "prophylaxis". The term "reduced", for purpose of therapy, refers broadly to the clinical significant reduction in signs and/or symptoms. Therapy includes treating relapses or recurrent signs and/or symptoms (e.g., inflammation, pain). Therapy encompasses but is not limited to precluding the appearance of signs and/or symptoms anytime as well as reducing existing signs and/or symptoms and reducing or eliminating existing signs and/or symptoms. Therapy includes treating chronic disease ("maintenance") and acute disease. For example, treatment includes treating or preventing relapses or the recurrence of signs and/or symptoms (e.g., inflammation, pain).

As used herein, the terms "disease", "disorder" or "condition" are interchangeably used to refer to a condition that is not considered to be the norm, normal or healthy. Preferably, the disease is any disease that can be treated, ameliorated or benefit from the administration of thiopurine, or a pharmaceutical equivalent, analog, derivative, and/or salt thereof, to the individual being treated.

A patient may have one or more disease or disorder selected from an immune-mediated gastrointestinal disorder, an autoimmune disease, acute lymphoblastic leukemia and graft versus host disease, cancer, and hyperproliferative disease. Optionally, said cancer is metastatic cancer or non-metastatic cancer. Optionally, said immune-mediated

gastrointestinal disorder is inflammatory bowel disease (IBD), such as ulcerative colitis or Crohn's disease. Optionally, said autoimmune disease is rheumatoid arthritis. Optionally, said autoimmune disease is autoimmune hepatitis.

Optionally, said patient may be an organ transplant recipient, such as an organ or allograft transplant recipient. Optionally, said patient may be a cell therapy recipient, such as an IPSC or stem cell transplant recipient. The patient may be treated with one thiopurine drug only, with a combination of two or more thiopurine drugs, or may be treated with one or more thiopurine drugs in addition to a further non-thiopurine drug. By way of example, the patient may also be treated with allopurinol. The thiopurine drug may be administered at any appropriate dose. Such doses are well known in the art.

As used herein "preparing a treatment regimen" refers to determining a thiopurine drug treatment regimen and / or administering said thiopurine drug treatment regimen to a patient. As used herein, the term "test cell" refers to a sample comprising one or more cells isolated or obtained from the patient. The sample may be a cellular or biological sample isolated or obtained from the patient. The term "biological sample" as used herein refers to any biological fluid, cell or tissue sample from a subject (e.g. patient), which can be assayed for thiopurine metabolites. The test cell is therefore not limited to any particular sample type, provided that it comprises one or more thiopurine metabolites compounds. By way of example, the test cell may be a nucleated cell. The test cell may be a blood cell, such as, but not limited to an erythrocyte, a leukocyte or a peripheral blood mononucleated cell. In other words, the sample being tested by the methods of the invention may comprise a blood cell such as one (or more) of the blood cells mentioned above. The terms "peripheral blood monocyte", "PBMC" and "peripheral blood mononucleated cell" are used interchangeably herein.

The test cell may be a sample comprising one or more cells isolated or obtained from a tissue (e.g. a solid tissue, such as the liver) of the patient.

For example, the test cell sample may include 1 , 100, 1000, 5,000, 10,000, 20,000, 30,0000, 40,000, 50,000, 60,000, 70,000, 80,000, 90,000, 100,000 or more cells.

The test cell may be obtained or isolated from the patient using any method that is well known in the art (such as, for example, known methods for obtaining blood cells from a patient). In one example, the test cell may be a sample comprising one or more cells isolated or obtained from a tissue (e.g. solid tissue) of the patient, wherein the sample is derived from any bodily tissue by homogenisation or lysis of a tissue biopsy or by lysis after mechanical separation of the tissue into a cell separation.

Within the methods of the invention, a test cell derived from the patient is lysed in a lysis buffer so as to obtain a cell lysate.

As used herein "lyse", "lysing" and "lysed" all refer to the process of lysis i.e. the bursting open of a cell by osmosis. Lysis may be partial or complete. Within the context of the invention, lysis is used to ensure that the thiopurine metabolites present within the cell can be measured, therefore any amount of (partial) lysis that achieves this effect is sufficient. A number of different lysis methods are well known in the art, including chemical methods and mechanical methods. Chemical lysis methods may be achieved by using an appropriate lysis buffer. As used herein, the term "lysis buffer" refers to a buffer or solution which is added to the test cell and in which the lysis step is performed. The contents of the lysis buffer (e.g. reagents that make up the lysis buffer) are not limited in any way provided that the lysis buffer does not chemically alter the phosphorylation and/or methylation state of the one or more thiopurine metabolites in the cell lysate that is obtained after the lysis step has been performed. In other words, the lysis buffer of the invention consists of reagents that do not chemically alter the phosphorylation and/or methylation state of the thiopurine metabolites to be measured. By way of example, the lysis buffer may comprise of water and other reagents that do not chemically alter the phosphorylation and/or methylation state of the thiopurine metabolites to be measured. The lysis buffer may also consist only of water (with no other reagents present). Optionally, the water used in any of the lysis buffers discussed herein is deionised water. In one embodiment, the lysis buffer may comprise or consist of an alcohol, for example propanol.

As used herein, a reagent that "does not chemically alter the phosphorylation and/or methylation state of the thiopurine metabolites" is a reagent that does not (directly or indirectly) change the phosphorylation and/or methylation state of the one or more thiopurine metabolites in the cell lysate. In other words, the reagent does not directly or indirectly increase or decrease the level of phosphorylation (and/or methylation) of the one or more thiopurine metabolites in the cell lysate. In one example, the reagent(s) in the lysis buffer do not chemically alter the one or more thiopurine metabolites in the cell lysate any way. In other words, in this example, the reagent(s) in the lysis buffer do not directly or indirectly induce the formation of chemical derivatives of the thiopurine metabolites in the cell lysate. A skilled person could identify if a reagent would not chemically alter the phosphorylation and/or methylation state of thiopurine metabolites using, for example, an LC-MS/MS assay standard.

The lysis buffer may include a reagent that facilitates lysis, such as a detergent. Examples of appropriate detergents are well known in the art, and include, anionic, cationic, non-ionic or zwitterionic detergents. By way of example but without limitation, such detergents include Triton X-100, Triton X-114, NP-40, Brij-35, Brij-58, Tween 20, Tween 80, Octyl glucoside, Octyl thioglucoside, sodium lauryl sulphate, sodium lauryl sarcosine, CHAPS (3-[(3- Cholamidopropyl) dimethylammonio]-1-propanesulfonate). The lysis buffer may (also) include a reagent that directly or indirectly prevents biological alteration (e.g. alteration of the phosphorylation and/or methylation state) of the thiopurine metabolites in the cell lysate. By way of example, the lysis buffer may include one or more reagents that minimise thiopurine metabolite modifications, such as ethylene diamine tetra- acetic acid (EDTA), which acts to chelate metal ions in the cell lysate. By way of another example, the lysis buffer may include reagents that reduce phosphatase activity (e.g.

phosphatase inhibitors such as imidazole, sodium fluoride, sodium molybdate, sodium orthovanadate, and sodium tartrate dihydrate). The lysis buffer may also include reagents that protect thiol groups, such as dithiothreitol (DTT) or Tris (2 carboxyl ethyl) phosphine HCL (TCEP).

The test cell may (additionally) be mechanically lysed, for example using sonication or vortexing. As shown herein, sonication of a test cell in the lysis buffer of the invention advantageously provides a cell lysate wherein one or more thiopurine metabolites can be measured without chemical modification/derivatisation of the thiopurine metabolites in the cell lysate. The measurements obtained from the methods of the invention therefore provide a more accurate reflection of the thiopurine metabolic status of the test cell. As used herein, a "cell lysate" refers to the sample that is obtained by lysis. The cell lysate is typically a fluid sample that comprises the contents of the lysed cells.

The methods of the invention measure one or more thiopurine metabolites in a test cell (sample) derived from the patient. As used herein, "measure", "measuring" and "measurement" all refer to quantifying the amount (e.g. relative amount or concentration) of one or more thiopurine metabolites that is detectable, measurable or quantifiable in a test cell sample. For example, the amount can be a concentration expressed as pg/L, ng/L, pmol/mg of protein, or nmol/mg of protein, or a relative amount such as 1.1 , 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1 , 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1 , 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 10, 15, 20, 25, and/or 30 times more or less than a reference level. The amount of thiopurine metabolite detected can include, for example the cell-associated thiopurine metabolite(s) that are detectable in the cell lysate. The amount of thiopurine metabolite detected can (also) include, for example, the amount of soluble thiopurine metabolite (e.g. non-cell associated metabolites such as cleaved, secreted, released, or shed metabolites) detectable in the cell lysate. The reference level, can for example, be the average or median concentration of the one or more thiopurine metabolites in a plurality of reference samples (e.g. reference samples obtained from a plurality of subjects treated with the same thiopurine drug as the patient being tested and known to have a particular clinical response or tolerance to the thiopuine drug).

Alternatively, the reference level may be comprised of a thiopurine metabolite amount from a reference database, which may be used to generate a pre-determined cut off value, i.e. a diagnostic score that is statistically predictive of a symptom or disease or lack thereof or may be a pre-determined reference level based on a standard population sample.

The amount of thiopurine metabolite detected may be normalised by correcting the absolute amount of metabolite measured in a sample by comparing its concentration to the

concentration of a reference compound that is not a thiopurine metabolite in the same sample. This normalization allows the comparison of the amount of metabolite in one sample to another sample, or between samples from different sources. This normalized value can then optionally be compared to a reference standard or control. For example, when measuring the amount of thiopurine metabolite present in a blood sample the amount of thiopurine metabolite may be expressed as an absolute concentration or, alternatively, it may be normalized against a known blood marker (or normalised for example per mg of total protein in the sample).

The methods of the invention may be used to predict the clinical response or tolerance of a patient to a thiopurine drug. By way of example, it is possible to predict the clinical response or tolerance of a patient to a thiopurine drug by comparing the level(s) of one or more thiopurine metabolite(s) in the cell lysate with a reference level or value, and correlating the relative amount of the one or more thiopurine metabolites in the cell lysate to the clinical response or tolerance of the patient to the thiopurine drug. The reference level or value may be data obtained from a plurality of subjects treated with the same thiopurine drug as the patient being tested, wherein the subjects are known to have a particular clinical response or tolerance to the thiopuine drug.

The one or more thiopurine metabolites may be measured using any appropriate measurement method known in the art.

By way of example, the one or more thiopurine metabolites may be measured using the mass/charge ratio of said one or more thiopurine metabolites or fragments thereof (e.g. using mass spectrometry). Optionally, the measurement may be preceded by chromatographic separation (e.g. by liquid chromatography).

The inventors have surprisingly found that measuring thiopurine metabolites using a combination of mass spectrometry and chromatographic separation is particularly advantageous as it achieves a high level of specificity. This is particularly the case when the sample to be analysed is prepared using the methods (e.g. lysis buffer and lysing techniques) described herein. Optionally, the type of mass spectrometry is selected from the group consisting of MS, MS/MS and ESI-MS/MS. These and other mass spectrometry methods are well known methods in the art.

As used herein, the term "mass spectrometry" or "MS" analysis refers to a technique for the identification and/or quantitation of molecules in a sample. MS includes ionizing the molecules in a sample, forming charged molecules; separating the charged molecules according to their mass-to-charge ratio; and detecting the charged molecules. MS allows for both the qualitative and quantitative detection of molecules in a sample. The molecules may be ionized and detected by any suitable means known to one of skill in the art. The phrase "tandem mass spectrometry" or "MS/MS" is used herein to refer to a technique for the identification and/or quantitation of molecules in a sample, wherein multiple rounds of mass spectrometry occur, either simultaneously using more than one mass analyzer or sequentially using a single mass analyzer. As used herein, a "mass spectrometer" is an apparatus that includes a means for ionizing molecules and detecting charged molecules.

Preferably, the type of mass spectrometry is selected from the group consisting of LC-MS, LC-MS/MS and LC-ESI-MS/MS. These and other mass spectrometry methods are well known methods in the art. As used herein, the phrase "liquid chromatography" or "LC" is used to refer to a process for the separation of one or more molecules or analytes in a sample from other analytes in the sample. LC involves the slowing of one or more analytes of a fluid solution as the fluid uniformly moves through a column of a finely divided substance. The slowing results from the distribution of the components of the mixture between one or more stationery phases and the mobile phase. LC includes, for example, reverse phase liquid chromatography (RPLC) and high pressure liquid chromatography (HPLC). As used herein, the term "separate" or "purify" or the like are not used necessarily to refer to the removal of all materials other than the analyte of interest from a sample matrix. Instead, in some embodiments, the terms are used to refer to a procedure that enriches at a point in time or space the amount of one or more analytes of interest relative to one or more other components present in the sample matrix. In some embodiments, a "separation" or "purification" may be used to remove or decrease the amount of one or more components from a sample that could interfere with the detection of the analyte, for example, by mass spectrometry.

Chromatographic separation of thiopurine metabolites can be achieved using columns well known in the art. By way of example, a Clarity Oligo-WAX column (150 mm x 4.6 mm) or a SecurityGuard Oligo-WAX column (4 x 3 mm) both from Phenomenex (Cheshire, UK) amy be used.

The column may be maintained at any suitable temperature. For example, the column may be maintained in a range of from 25°C to 35°C (or any smaller range there inbetween, wherein the lower end of the temperature range is selected from 25, 26, 27, 28, 29, 30, 31 , 32, 33 or 34; and the upper end of the temperature range is selected from 26, 27, 28, 29, 30, 31 , 32, 33, 34 and 35). Preferably the temperature is maintained in the range of 28°C to 32°C. Optionally, the column is maintained at 30 ^° C.

Analytes may be eluted with any appropriate buffer. Such buffers are well known in the art. By way of example only, the analytes may be eluted in mobile phases of 0.01 M aqueous ammonium acetate pH 8.0 (A) and 0.01 M aqueous ammonium acetate pH 10.01 (B). The mobile phase system used herein to demonstrate the invention consisted of a starting condition at 90% A followed by a 0.5 min gradient from 90% A to 80% A at 1.5 min. A 2 min gradient from 80% A to 0% A was carried out at 3.5 min with those conditions maintained until 13.5 min when the column was returned to 10% A in a 0.5 min gradient. The flow rate was 0.7 mL/min and a post column flow splitter was utilised to divert 70% of mobile phase to waste and improve ionisation.

As used herein, the term "about", in the context of level of steroid hormone, typically means +/- 5% of the stated value, more typically +/- 4% of the stated value, more typically +/- 3% of the stated value, more typically, +/- 2% of the stated value, even more typically +/- 1 % of the stated value, and even more typically +/- 0.5% of the stated value. The recitation of numerical ranges by endpoints herein includes all numbers and fractions subsumed within that range (e.g. 1 to 5 includes 1 , 1.5, 2, 2.75, 3, 3.90, 4, and 5). It is also to be understood that all numbers and fractions thereof are presumed to be modified by the term "about." Further, it is to be understood that "a," "an," and "the" include plural referents unless the content clearly dictates otherwise. The term "about" means plus or minus 0.1 to 50%, 5-50%, or 10-40%, preferably 10-20%, more preferably 10% or 15%, of the number to which reference is being made. Throughout the description and claims of this specification, the words "comprise" and "contain" and variations of them mean "including but not limited to", and they are not intended to (and do not) exclude other moieties, additives, components, integers or steps. Throughout the description and claims of this specification, the singular encompasses the plural unless the context otherwise requires. In particular, where the indefinite article is used, the specification is to be understood as contemplating plurality as well as singularity, unless the context requires otherwise.

Features, integers, characteristics, compounds, chemical moieties or groups described in conjunction with a particular aspect, embodiment or example of the invention are to be understood to be applicable to any other aspect, embodiment or example described herein unless incompatible therewith. All of the features disclosed in this specification (including any accompanying claims, abstract and drawings), and/or all of the steps of any method or process so disclosed, may be combined in any combination, except combinations where at least some of such features and/or steps are mutually exclusive. The invention is not restricted to the details of any foregoing embodiments. The invention extends to any novel one, or any novel combination, of the features disclosed in this specification (including any accompanying claims, abstract and drawings), or to any novel one, or any novel combination, of the steps of any method or process so disclosed. The reader's attention is directed to all papers and documents which are filed concurrently with or previous to this specification in connection with this application and which are open to public inspection with this specification, and the contents of all such papers and documents are incorporated herein by reference. Examples

Materials and Methods

Chemicals The thiopurine standards 6-thioguanine-mono-phosphate (TGMP), 6-thioguanine-di- phosphate (TGDP), 6-thioguanine-tri-phosphate (TGTP), 6-methylthioguanine-mono- phosphate (MeTGMP), 6-methylthioguanine-di-phosphate (MeTGDP), 6-methylthioguanine- tri-phosphate (MeTGTP), 6-methylthioinosine-mono-phosphate (MeTIMP), 6- methylthioinosine-di-phosphate (MeTIDP), 6-methylthioinosine-tri-phosphate (MeTITP) and 6-thioinosine-mono-phosphate (TIMP) were from Jena Bioscience (Jena, Germany). HPLC grade acetic acid was from Fisher Scientific (Loughborough, UK). Lymphoprep™ was from Axis-Shield and Pierce BCA kit was from Thermo Scientific (Cramlington, UK). All other reagents were from Sigma (Gillingham, UK).

Sample collection and processing

Clinical samples were from inflammatory bowel disease (IBD) patients treated with low-dose azathioprine in combination with allopurinol; the study protocol was approved by the ethics committee of NRES Committee South West - Cornwall & Plymouth, Bristol Research Ethics Committee Centre. Peripheral blood monocytes (PBMC) were isolated from whole blood (collected in EDTA tubes) using Lymphoprep™ following the manufacturers protocol. Briefly, whole blood (4 mL) was diluted 1 : 1 with phosphate buffered saline (PBS) and layered onto 8 mL of sterile Lymphoprep and centrifuged at 800 g for 30 min without braking. The mononuclear layer from the interface was removed, and erythrocytes lysed by the addition of 20 volumes of erythrocyte lysis buffer (0.15 M ammonium chloride, 0.01 mM potassium bicarbonate, 0.1 mM EDTA) and mixed for 10 min. Samples were centrifuged to pellet the cells followed by two washes in PBS. The final pellets were stored at -80 °C to await analysis. Pellets were thawed, re-suspended in 200 of sterile deionised water and sonicated at amplitude 5 for 3 s three times on ice and centrifuged at 300 g for 5 min. The supernatant was then transferred into a suitable tube for analysis and an aliquot kept for protein measurement. A small aliquot of erythrocytes was diluted 1/100, sonicated and prepared for analysis as for PBMC above. Metabolites profiles were similar in duplicate pellets stored and extracted at different times, but cell lysates could not be stored frozen without affecting metabolite profiles in subsequent analyses.

Alternative sample processing

In an alternative sample processing method, frozen PBMC pellets, isolated using Lymphoprep™ following the manufacturers protocol as described above, are thawed and re- suspended in 200 100% propanol, containing a deuterated Methyltmercaptopurine control, vortexed and then dried under nitrogen. The dried sample is then re-suspended in 200 water, and vortexed. After leaving to stand at room temperature, the sample is then centrifuged to remove debris. The generated supernatant is then transferred to an HPLC insert for LCMS analysis. Protein concentration

An aliquot of cell lysate was diluted in sterile deionised water to a total volume of 50 μΙ_. Protein content was determined colourimetrically using either a Pierce BCA kit (Thermo Scientific, Cramlington, UK) for PMBCs, or a Bradford assay (BioRad, Hemel Hempsted, UK) for erythrocytes. Protein standards in the range 0.05 - 1.2 mg/mL were prepared in deionised water using bovine serum albumin (Thermo Scientific, Cramlington, UK). 10 μΙ_ of deionised water, standard or diluted cell lysate were added in quadruplicate to a 96 well plate. For the Pierce BCA assay, 190 μΙ_ of Pierce BCA reagent mixture was added to each well according to the manufacturer's instructions and the plate read at 562 nm using an Omega plate reader after incubation for 30 min at 37° C. For the Bradford assay, 200 μΙ_ of Bradford reagent, diluted 1 in 5 with deionised water, was added to each well and read at 595 nm after 30 min at room temperature.

Liquid chromatography- tandem mass spectrometry (LC-MS/MS)

Chromatographic separation of thiopurine metabolites was achieved using a Shimadzu Prominence UFLC (Shimadzu, Kyoto, Japan) equipped with a Clarity Oligo-WAX column (150 mm x 4.6 mm) and SecurityGuard Oligo-WAX column (4 x 3 mm) both from Phenomenex (Cheshire, UK) maintained at 30 ^° C. Analytes were eluted with mobile phases of 0.01 M aqueous ammonium acetate pH 8.0 (A) and 0.01 M aqueous ammonium acetate pH 10.01 (B). The mobile phase system consisted of a starting condition at 90% A followed by a 0.5 min gradient from 90% A to 80% A at 1.5 min. A 2 min gradient from 80% A to 0% A was carried out at 3.5 min with those conditions maintained until 13.5 min when the column was returned to 10% A in a 0.5 min gradient. The flow rate was 0.7 mL/min and a post column flow splitter was utilised to divert 70% of mobile phase to waste and improve ionisation. Standards and samples were injected in a volume of 50 μΙ_. Standards were freshly diluted from a secondary dilution of stock on the day of analysis, and the secondary dilutions were used within six months of preparation from the primary stocks received from the manufacturer; the standards were stable under this protocol. An API4000 triple quadrupole LC-MS/MS (Applied Biosystems, California, USA) was used for analysis with electrospray ionisation (ESI) performed in positive ion mode using nitrogen gas with the following optimum settings: curtain gas, 10; ion source gas 1 , 40; ion source gas 2, 50; ion spray voltage, 5500; collision gas, 6; entrance potential, 10; ionisation temperature, 400Ό. Optimisation of MS/MS parameters for all analytes was performed by selecting precursor ions and determining the most prominent four product ions. The three best of these were then further optimised for fragmentation and voltage parameters until the most abundant and robust product ion could be ascertained. Quantification of analytes was performed in multiple reaction monitoring (MRM) mode where mass transitions and optimised MS/MS parameters are given in Table 1. Analyst ^® software v1.5 (AB SCIEX, Framingham, USA) was used for sample analysis, peak integration and analyte quantification (Figure 2). Statistical analyses were carried out using R 2.15.1 ⁴⁰ .

Table 1

Table 1 : Mass transitions and optimised MS/MS parameters for analyte quantification.

LC-MS/MS validation

Method validation was adapted from the 2001 FDA guidelines ⁴¹ . It consisted of accuracy, precision, lower limit of quantitation (LLOQ) and calibration curve response. The linear dynamic ranges, limits of detection (LOD), limits of quantification (LOQ), and intra/inter-day assay precision were determined for all analytes. Quality control solutions of analytes were prepared in ultrapure water (Milli-Q; Merck Millipore, Billerica, MA) at the concentrations 30, 15 and 3 pmol/mL and used to determine intra- and inter- assay precision through multiple injections. A standard mix of analytes was prepared in water to study dynamic range, via serial dilution (50 pmol/mL to 0.195 pmol/mL).

Results and Discussion

The inventors have developed a novel method for measuring nine thiopurine metabolites simultaneously from as few as 50,000 isolated PBMC or erythrocytes. The method was linear in the range from 0.781 nM to 50 nM for all metabolites assayed (MeTGMP, MeTGDP, MeTGTP, MeTIMP, MeTIDP, MeTITP, TGMP, TGDP and TGTP) with an R ² value of >0.99 (n=10) for the standard curves (Table 2). Three QC-relevant samples were included in all runs (n=10) with coefficient of variation for intra-day variation of <8.3% and inter-day variation of <11.6%. The limit of detection (LOD) for all metabolites was 0.195 nM and the limit of quantification (LOQ) for MeTGMP, MeTGTP, TGDP and TGTP was 0.781 nM and for MeTGDP, MeTIMP, MeTIDP, MeTITP, TGMP 0.391 nM (Table 2).

Table 2

a Limit of detection (S/N = 3; n = 5)

f _t Lower Limit of Quantification (S/N = 5 + replicate CV<15%; n = 5) c Calibration curves (y=ax+b)

a Intra-day, n = 10

e Inter-day, n =5

Table 3 Quantification of azathioprine metabolites in PBMC and erythrocytes (ERYTH) from patients treated with low-dose azathioprine an allopurinol.

MEAN 0.357 0.024 0.009 0.197 0.000 0.000 0.587 0.916 0.044 0.000 0.959

SD 0.209 0.030 0.025 0.253 0.000 0.000 0.356 0.318 0.115 0.000 0.355

MEAN 0.006 0.371 0.485 0.638 0.951 1.703 4.154 0.261 1.081 1.696 3.038

SD 0.017 0.234 0.269 0.801 0.611 0.860 2.236 0.301 0.484 0.928 1.354

Using the new method, the inventors determined azathioprine metabolite concentrations in PBMC and erythrocytes from a small cohort of IBD patients (n=7) treated with azathioprine and low dose allopurinol (Table 3); all patients in this cohort had no therapeutic response complications. These data show a statistically significant difference between erythrocytes and PBMC in the concentrations of methylated metabolites (4.15 vs 0.59 pmol/mg protein for erythrocytes and PBMC, respectively, paired Wilcoxon test, P=0.016) and TGNs (3.04 vs 0.96 pmol/mg protein, paired Wilcoxon test P=0.03); notably most TGNs in PBMC were TGMP whereas in erythrocytes all three were present (Table 3). Furthermore, the majority of methylated metabolites in PBMCs were MeTGMP, but MeTGMP accounted for none, or in one case only a very small proportion, of the methylated thiopurine metabolites in erythrocytes. These results demonstrate marked differences in the thiopurine metabolite profiles between PBMCs and erythrocytes from the same patients, highlighting the potential drawbacks with currently used methods. This is the first time these metabolites have been measured without extensive sample pre-processing. These results therefore give a precise reflection of TGNs present in the various cellular compartments analysed providing improved assay accuracy and sensitivity.

The first assay to measure TGNs and 6-methylmercaptopurine (6-MMP) was developed by Lennard ¹² in washed erythrocytes by HPLC. Since then, the various assays developed to measure TGNs in different cellular compartments of patient samples have reported the data using different measurement units: pmol/0.8 X 10 ⁸ erythrocytes ^{19, 42} , nmol/mmol of haemoglobin ⁴³ , pmol/100 erythrocytes ²² , pmol/25 mg of haemoglobin ^{20, 44} , pmol/8 X 10 ⁸ erythrocytes -0.2 ml_ with haemoglobin of 120g/L ²³ , and, most recently, ng/mL ⁴⁵ . None of these measurement units are ideal, given variation in cell size, both between different cell types and within cell types between patients and treatment cycles or disease state. The inventors have therefore expressed their data relative to the protein content of lysates before LC-MS/MS analysis. It is felt that this approach offers the most robust benchmark for comparisons between samples, facilitating rapid sample processing while minimising error- prone cell counting methods and obviating the need for assumptions about cell size.

The detail of sample preparation for these assays is critical when evaluating the methodology and the reported metabolites. The Lennard method ¹⁹ relied on the formation of mercury adducts and organic solvent extraction which is not ideal for use in a routine laboratory. A modification by Dervieux ⁴⁶ involved de-proteinisation by perchloric acid and the inclusion of DTT with subsequent hydrolysis of the nucleotides back to their bases by heating to 100 °C for 45 min, resulted in the formation and measurement of 6-MMP free base and the nucleotides. In 2003 Shipkova ⁴⁴ reported a comparison of these two assays to determine target or threshold levels of 6-TGNs required to achieve a response in patients, and what units would be most useful. A consistent 2.6 fold difference in 6-TGN concentrations between the two methods was evident, highlighting the effect of differences in sample processing prior to analysis. The authors concluded that the difference was at least in part due to a more complete hydrolysis of thioguanine nucleotides using the Dervieux method. A similar result was reported by Lowry ⁴⁷ who found a 1.6-fold difference between the Lennard ¹² and Erdmann ¹⁴ methods. These studies highlight the problems with TGN assays involving inconsistency between methodological threshold ranges and preanalysis sample processing. Furthermore, most TGN measurements in established erythrocyte assays relate not to the thiopurine nucleotide but to the thiopurine base due to the acid hydrolysis step; this can be contaminated by the parent 6-TG drug and recovery efficiency is method dependent ⁴⁴ . The acid hydrolysis step also produces methyl mercaptopurine, which is often reported as methyl mercaptopurine riboside (MMPR) or methyl-thio-IMP. A method incorporating the enzymatic conversion of the TGNs to the thioguanine nucleosides has been reported ²⁵ but individual thioguanine nucleoside mono, di and tri phosphates are indistinguishable.

Despite methodological limitations, significant correlations have been reported between clinical response and 6-TGN levels and a therapeutic range for 6-TGN concentrations in erythrocytes of 235-450 pmol/8x 10 ⁸ erythrocytes is used ^48"51 with a concentration of over 235 pmol/8x 10 ⁸ erythrocytes being associated with clinical response ^{48, 49} . The suggested maximum before withdrawal of treatment is above 450 pmol/8x 10 ⁸ erythrocytes based on an increased risk of side effects (myelotoxicity and nodular regenerative hyperplasia of the liver) without an increase in efficacy ^{50, 52} . Patients with erythrocyte 6-MMP concentrations above 5,700 pmol/8x 10 ⁸ erythrocytes are at increased risk of hepatotoxicity and are unlikely to respond to treatment by increasing the drug dose ^{48, 53, 54} .

Meta-analyses ³⁸ have suggested a strong association between 6-TGN levels and induction of remission among patients with IBD. However, Mayer ⁵⁵ commented on the real problem of what to do for the 33% of thiopurine-treated patients in remission but with TGN levels below the suggested threshold of 230 pmol/8x 10 ⁸ erythrocytes. Equally, the question arises if a patient has TGN levels higher than the suggested upper limit, but is clinically well and responding. A recent review of data from paediatric patients on thiopurines ³⁷ concluded that metabolite testing does not safely predict clinical outcome, but may facilitate toxicity surveillance and treatment optimisation in poor responders. These analyses highlight controversy in the utility of thiopurine metabolite monitoring, but given the cumbersome methodology and the cell types in which these have been measured it is perhaps not surprising that there is no clear-cut correlation. Previous studies using LCMS/MS technology ^{16, 23, 56} have not measured thiopurine metabolites isolated from PBMCs. Dervieux and Hofmann ^{16, 56} both used washed erythrocytes and Kirchherr ²³ used whole blood. Even though the most recent study measured metabolites in isolated PBMCs, levels were only reported for 6-mercaptoguanosine and 6-methylmercaptopurine riboside and, again, extensive sample pre-processing was involved ⁴⁵ . The inventors believe that their method is superior to previous methods in that it is applicable to isolated PBMCs in which thiopurine metabolites can be identified individually without extensive sample preprocessing.

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