Introduction

Early diagnosis of cancer remains an important goal in any treatment plan. Cancer that is diagnosed at an early stage is more likely to be treated successfully. If the cancer spreads, effective treatment becomes more difficult, and generally a person’s chances of surviving are much lower.

Lung cancer is the second most prevalent cancer in adult men and women around the world. The prognosis and treatment options of lung cancer patients depend directly on tumor size and its spread at the time of diagnosis; survival time decreases significantly as the disease is more progressive at detection. Therefore, early detection of lung cancer is paramount and there is a need for non-invasive and reliable screening techniques. More than 80% of lung cancer patients will survive for at least a year if diagnosed at the earliest stage compared to around 15% for people diagnosed with the most advanced stage of disease.

Other examples of the benefits of early detection apply to breast cancer, ovarian cancer and bowel cancer. More than 90% of women diagnosed with breast cancer at the earliest stage survive their disease for at least 5 years compared to around 15% for women diagnosed with the most advanced stage of disease.

Whilst early detection of cancer is paramount, there are few non-invasive test methods available which enable a reliable diagnosis whilst increasing patience compliance due to the convenient testing methods. Moreover, there is also a need for monitoring the progression of cancer in a reliable and non-invasive way to determine treatment options.

Bodily fluids such as breath, urine blood, contain low concentrations of various volatile organic compounds (VOCs) produced by the body. These are believed to reflect endogenous metabolic processes at the tissue level, such as inflammation and oxidative stress. When cancer develops, metabolic changes can create a unique endogenous VOC profile that is potentially reflected in the body fluids, including breath. Detection of cancer by analysing bodily fluids has therefore become an area of interest as it allows the development of non-invasive detection techniques. However, whilst a large number of exhaled VOCs have been identified, it is difficult to identify VOCs that are indeed specific to a particular cancer to provide a reliable diagnosis.

Endogenous VOC compounds are by-products of specific metabolic pathways that are likely to be affected by several physiological and pathological factors. Endogenous VOCs can be a reliable readout for the activity of internal metabolic processes - acetone and isoprene are good markers for ketosis and blood cholesterol, respectively. However, as these metabolic processes are altered by several pathological conditions and physiological factors, the association of endogenous breath VOCs to specific diseases is complicated. Moreover, breath secretion of endogenous VOCs is the cumulative result of metabolic reactions occurring in different tissues. While specific diseases may alter the function of individual tissues and organs, this effect can be challenging to detect when monitoring the sum contribution of healthy and diseased tissues in breath. This aspect significantly complicates the task of identifying the tissue of origin of specific breath VOCs.

Cancer cells are known to undergo profound metabolic changes to support survival, proliferation, immune escape and metastasis (Pavlova and Thompson 2016; Gaude and Frezza 2016). For instance, lung cancer cells have been shown to increase glucose and lactate consumption in order to feed carbon flow into mitochondria (Fan et al. 2009; Sellers et al. 2015; Faubert et al. 2017; Hensley et al. 2016). These metabolic changes can result in changes to the tumour microenvironment, in particular acidification of the extracellular space.

Acidic extracellular pH (pH _e ) is a key microenvironmental characteristic of solid tumours, along with hypoxia, glucose and energy deprivation, and high levels of lactate (Vaupel et al. 2004) (Kalliomake and Hill, 2004). Acidic pHe increases not only the activation of some lysosomal enzymes with acidic optimal pH, but also the expression of some genes involved with pro- metastatic factors (Kato et al, 2013). The acidification of the tumour microenvironment (TME) is (at least in part) produced by the Warburg effect (Mullapudi et al, 2020) In brief, the Warburg effect describes a process whereby cancerous cells preferentially (but not exclusively) use glycolysis as an energy production pathway over oxidative phosphorylation, regardless of the aerobic status of the tumoral environment. In glycolysis, for each glucose molecule processed, 2 pyruvate molecules are produced. The pyruvate then, instead of preferentially re-entering the citric acid cycle (as would occur under normal oxidative phosphorylation), predominantly becomes converted to lactic acid. In order to maintain intracellular homeostasis, the lactic acid is then exported from the cell by the proton-linked monocarboxylate transporter (MCT), particularly MCT113 and MCT4; and accumulates in the TME, thereby reducing the extracellular pH.

As glycolysis produces significantly less ATP per molecule of glucose than oxidative phosphorylation, cancerous cells must greatly increase their glucose uptake compared to regular cells. In general, hexose sugars are taken into cells via two transport mechanisms - facilitated diffusion via glucose facilitative transporters (GLUTs) and actively via sodium-coupled glucose transporters (SGLTs) based on the electrochemical gradient of sodium and potassium. GLUT1 is the most abundantly expressed hexose sugar transporter in the majority of cancer types, and so may be used as a fairly reliable marker of hypoxia and increased glucose metabolism associated with cancerous cells. However, it is worth noting that some cancer types (notably sarcomas, lymphomas, melanomas and hepatoblastomas) do not generally share the GLUT1 overexpression, and therefore likely have their glycolytic pathway supported by other GLUTs such as GLUT5, which has increased selectivity for fructose. Fructose, while not taken up by the heavily overexpressed GLUT1 , has been shown to contribute to the Warburg effect of decreased pHe (5), however this is primarily through the production of uric acid over lactic acid (although lactic acid is still produced).

The Warburg effect and the acidic tumour microenvironment has been taken advantage of for drug targeting to the tumour microenvironment and for drug delivery to cancer cells. For example; Luoping et al, 2021 demonstrated that a pH responsive liposome and administration of glucose could be used to enhance efficacy of doxorubicin. Doxorubicin release from the liposomes was tested under different pH conditions, the release rates and cellular uptake was found to be higher at low pH. In a mouse model a high dose of glucose was shown to significantly increase the uptake of the liposomes which persisted up to 72 hours post administration. Schem and Dahl 2021 evaluated the response to treatment of the nitrosurea nimustine, with localised hyperthermia, and hypertonic glucose prior to treatment. In a mouse model, tumour growth time (TGT; the time taken to double the size of the initial tumour) was calculated for several timepoints after treatment with 10 or 20mg/kg nimustine, nimustine with hyperthermia, nimustine with glucose, and nimustine with both hyperthermia and glucose. 20mg/kg of the drug had a TGT (tumour growth time) of 5.6±0.76, and glucose treatment had a minimal effect. Treatment with hyperthermia and drug significantly increased this TGT to >38.4. The response after this thermochemotherapy was further improved by a glucose bolus 2 hours prior to treatment.

Furthermore, it has been shown that specific enzymes that are found in the tumour microenvironment can be exploited to enhance drug targeting to tumours. For example, Devalapally et al, 2008, showed that a galactoside prodrug of doxorubicin could be used to enhance tumour targeting of the drug and reduce the incidence of serious side effects. The prodrug was designed for specific activation by p-galactosidase which was found to be present in the tumour microenvironment, this specific targeting improved safety and biodistribution of the prodrug.

As such the present inventors aim to exploit the acidic tumour microenvironment and the Warburg effect to enhance the sensitivity of cancer diagnostics that rely on the presence of specific enzymes in the tumour microenvironment

Summary of the Invention

The invention provides an improved method of detecting, diagnosing, prognosing, or screening for cancer in a subject. The method of the invention provides an exogenous substrate that is metabolised within a subject to produce a detectable metabolite. The detectable metabolite can be detected in a biological sample obtained from the subject. The substrate is specific for an enzyme that is indicative of cancer, as such release of the detectable marker from the exogenous substrate is indicative of cancer. There are a number of enzymes which are have been identified within the tumour microenvironment. For example, the micro-environment of tumors are known to have high levels of extracellular p-glucuronidase (FISHMAN & ANLYAN, 1947; Young et al., 1976) whilst in normal tissue this enzyme only resides in lysosomes in the cytoplasm (Bosslet et al., 1998). The present inventors have found that p-glucuronidase has an acidic pH optimum. As such, by further reducing the pH of the tumour microenvironment the activity of p-glucuronidase can be increased thereby leading to an increased turn-over of substrate.

By administering a dose of sugar, the pH of the tumour microenvironment will be lowered via the Warburg effect. Cancerous cells preferentially use glycolysis as an energy production pathway over oxidative phosphorylation, regardless of the aerobic status of the tumoral environment. The pyruvate that is produced via glycolysis is converted to lactic acid which is the exported from the cell, further lowering the pH of the tumour microenvironment. This lower pH can result in enhanced activity of cancer- specific enzymes such as p-glucuronidase. Therefore, the turn-over rate of the exogenous substrate and release of the detectable metabolite is increased. This will result in a more accurate detection of cancer.

In an aspect the present invention relates to a method for the detection or prognosis of cancer comprising; administering an exogenous substrate in combination with a sugar to a subject, measuring the concentration of a metabolite of said substrate in a biological sample that has been obtained from said subject.

In an aspect the present invention relates to a method for determining the activity of an enzyme comprising; administering an exogenous substrate for an enzyme in combination with a sugar to a subject, measuring the concentration of a metabolite of said substrate in a biological sample that has been obtained from said subject.

In an aspect the present invention relates to a method for enhancing the sensitivity of an assay for the detection or prognosis of cancer comprising administering an exogenous substrate in combination with a sugar to a subject, measuring the concentration of a metabolite of said substrate in a biological sample that has been obtained from said subject. In an aspect the present invention relates to a method for monitoring the progression of cancer in a subject diagnosed with cancer comprising: administering an exogenous substrate in combination with a sugar to a subject, assessing the activity of a cancer-specific enzyme by measuring the concentration of a metabolite of said substrate in a biological sample obtained from the subject.

In an aspect the present invention relates to a method for determining efficacy of a treatment comprising in a subject diagnosed with cancer: administering an exogenous substrate in combination with a sugar to a subject, assessing the activity of a cancer-specific enzyme by measuring the concentration of a metabolite of said substrate in a biological sample obtained from the subject wherein said subject has received anti- cancer treatment.

In an aspect the present invention relates to a kit comprising an exogenous substrate, a sugar and a device for obtaining a biological sample from a subject, and optionally instructions for use.

In an aspect the present invention relates to a composition comprising an exogenous compound and a sugar, wherein the exogenous substrate comprises a glycoside, wherein the glycoside comprises a glycosidic bond linked to a volatile organic compound.

In an aspect the present invention relates to a method for the detection or prognosis of cancer comprising; measuring the concentration of a metabolite of an exogenous substrate in a biological sample that has been obtained from a subject, wherein said subject has been administered said exogenous substrate in combination with a sugar.

In an aspect the present invention relates to an exogenous substrate in combination with a sugar for use in a method for the detection or prognosis of cancer comprising; measuring the concentration of a metabolite of an exogenous substrate in a biological sample that has been obtained from a subject, wherein said subject has been administered said exogenous substrate in combination with a sugar.

Figures

Figure 1. Metabolic pathways underpinning the utility of D5-ethyl-pD-glucuronide for detection of lung cancer. The first panel illustrates the probe cleavage. The second panel illustrates how the probe is not cleaved in the vicinity of normal cells. The third panel shows cleavage of the probe in the tumour micro- environment.

Figure 2. In vivo proof of concept data for D5-ethyl-pD-glucuronide probe. Panel A - With growing tumour volume (dashed line) the level of exhaled D5-ethanol increases (left side bars). No change can be observed in control mice (right side bars). Panel B - Injecting chemotherapy to reduce the tumour volume in mice is paralleled by a reduction in exhaled D5-ethanol upon injection of the D5-ethyl-pD-glucuronide probe.

Figure 3. p-glucuronidase activity in vitro at pH 5 at various concentrations.

Figure 4. Expected results with treatment with glucose and/or D5-ethyl-pD-glucuronide probe in health and subcutaneous lung cancer xenograft mice. Effect of time (tumor growth) and glucose in the detection of D5-EtOH. Probe dose 1 = D5-ethyl-pD-glucuronide 0.05 mg/kg. Probe dose 2 = OWL-EVO1 2 mg/kg. Glucose = 2 g/kg.

Figure 5. Mice breath sampler device design.

Figure 6. Kinetic curve for p-glucuronidase activity analysis. Standard curve plotted over time with pH ranging from 4 to 7; Mean and standard error absorbance are shown. All graphs were plotted in the same scale.

Figure 7. Effect of pH in p-glucuronidase activity over time. Standard curve plotted over time with pH ranging from 4 to 7. Each enzyme concentration tested is shown in individual plots, with concentration 1 being the highest concentration tested. Mean and standard error absorbance are shown.

Detailed Description

The present invention will now be further described. In the following passages, different aspects of the invention are defined in more detail. Each aspect so defined may be combined with any other aspect or aspects unless clearly indicated to the contrary. In particular, any feature indicated as being preferred or advantageous may be combined with any other feature or features indicated as being preferred or advantageous.

The present invention provides an improved method of detecting, diagnosing, prognosing, or screening for cancer in a subject. Cancer cells exhibit uniform metabolic abnormalities, termed the cancer metabolic phenotype. The mechanisms can be associated with (1) oncogenesis; (2) rapid reproduction; (3) stress responses; (4) invasiveness and metastasis; (5) resistance to host immune surveillance; (6) resistance to therapy. It is known that certain enzymes are cancerspecific. The present methods are therefore based on detecting the activity of a cancer-specific enzyme by providing an exogenous substrate which is metabolised by the cancer- specific enzyme. The presence of the substrate and/or its breakdown product (metabolite) in exhaled breath is detected, thereby detecting the presence of cancer. Provision of an exogenous substrate that is metabolised by a cancer-specific mechanism thus results in differential secretion in bodily fluids of the substrate itself and/or of its metabolites in breath of diseased subjects compared to healthy subjects. The improvement to the present method is achieved by administering a sugar in combination with an exogenous substrate to a subject. The exogenous substrate is metabolised within the subject to provide a detectable metabolite. It is hypothesised that provision of sugar will result in the exogenous substrate being metabolised at an increased rate and therefore releasing the detectable metabolite at an enhanced rate. Thereby allowing an improved detection of cancer.

The invention relates to a method for the detection or prognosis of cancer comprising; administering an exogenous substrate in combination with a sugar to a subject, measuring the concentration of a metabolite of said substrate in a biological sample that has been obtained from said subject.

In an embodiment the method for the detection or prognosis of cancer may comprise the steps of; measuring the concentration of a metabolite of an exogenous substrate in a biological sample that has been obtained from a subject, wherein said subject has been administered said exogenous substrate in combination with a sugar.

In an embodiment the invention relates to an exogenous substrate in combination with glucose for use in a method for the detection or prognosis of cancer comprising; measuring the concentration of a metabolite of an exogenous substrate in a biological sample that has been obtained from a subject, wherein said subject has been administered said exogenous substrate in combination with a sugar.

In an embodiment the invention relates to an exogenous substrate in combination with glucose for use in a method for the detection or prognosis of cancer comprising; administering an exogenous substrate in combination with a sugar to a subject, measuring the concentration of a metabolite of said substrate in a biological sample that has been obtained from said subject.

The present method is based on administration of an exogenous substrate to a subject. An “exogenous substrate” is any compound that can be administered to a subject that is metabolised by an enzyme within the subject. An exogenous substrate refers to a chemical compound that is recognized by the cancer-specific enzyme of interest and for which the enzyme catalyzes conversion of the substrate into a different chemical compound which is referred to herein as a "metabolite". The substrate used in the methods of the invention is an exogenous substance, i.e. a xenobiotic. The term xenobiotic refers to a substance that is foreign to the subject’s body and which is specifically and selectively metabolised by the cancer-specific enzyme. Preferably, the exogenous substance is converted into a metabolite by the cancer- specific enzyme is also a xenobiotic, that is it does not normally occur in the subject’s body. In an embodiment the exogenous substrate is a generally recognised as safe (GRAS) compound.

As mentioned above the present method uses an exogenous substrate that is metabolised by an enzyme within a subject. As the method are for the detection of cancer the enzyme may be a cancer-specific enzyme. A cancer-specific enzyme as used herein is an enzyme that is selected from one or more of the following: the enzyme is absent in cancer tissue, but present in non-cancer tissue; the enzyme is present in cancer tissue, but absent in non-cancer tissue; the enzyme is differentially expressed or in cancer tissue compared to non-cancer tissue or the enzyme is differentially active or in cancer tissue compared to non-cancer tissue. For example, the enzyme may be expressed at a higher level in cancer tissue or lower level compared to expression in non-cancer tissue. Expression can be measured by techniques known in the art, for example by mRNA quantification or measuring cDNA. Non-cancer tissue refers for example to healthy tissue. The tissue may be from a specific organ, e.g. lung, colon, breast, prostate etc.

The enzyme may be localised to a different location in cancer tissue compared to non-cancer tissue, for example the enzyme may be present in the extracellular space of cancer tissue, whereas in non-cancer tissue the enzyme is not present in the extracellular space. A combination of tumour necrosis and release of lysosomal enzymes from macrophages and neutrophils can result in the presence of certain enzymes being present in the extracellular space of the tumour microenvironment. In an embodiment the exogenous substrate is a substrate for an enzyme which is present in the extracellular space of solid tumours. The enzyme may be an enzyme which is an extracellular lysosomal enzyme. An “extracellular lysosomal enzyme” is an enzyme that has been released from the lysosome into the extracellular space. Specific example of enzymes that can be released from the lysosome into the extracellular space include p-glucuronidase, or p-galactosidase. In particular the presence of p-glucuronidase in the extracellular space is a hall mark of cancer. In an embodiment the cancer-specific enzyme is selected from p-glucuronidase, p-galactosidase a-L-arabinofuranosidase, N-acetyl-p-D- galactosaminidase, N-acetyl-p-D-glucosaminidase, Hexosaminidase, a-L-Fucosidase, a- galactosidase, a-glucosidase, p-glucosidase, a-L-iduronidase, a-mannosidase, p-mannosidase, Lipases, Phosphatases, Sulfatases. In a preferred embodiment the cancer-specific enzyme is selected from p-glucuronidase, p-galactosidase a-L-arabinofuranosidase, N-acetyl-p-D- galactosaminidase, N-acetyl-p-D-glucosaminidase, Hexosaminidase, a-L-Fucosidase, a- galactosidase, a-glucosidase, p-glucosidase, a-L-iduronidase, a-mannosidase, p-mannosidase.

Thus, methods described herein measure the activity of enzymes directly associated with a cancer disease state.

The method for the detection or prognosis of cancer may comprise the steps; administering an exogenous substrate for a cancer-specific enzyme in combination with a sugar to a subject, measuring the concentration of a metabolite of said substrate in a biological sample that has been obtained from said subject.

In an embodiment the method for the detection or prognosis of cancer may comprise the steps of; measuring the concentration of a metabolite of an exogenous substrate, for a cancerspecific enzyme, in a biological sample that has been obtained from a subject, wherein said subject has been administered said exogenous substrate in combination with a sugar.

In an embodiment the invention relates to an exogenous substrate in combination with glucose for use in a method for the detection or prognosis of cancer comprising; measuring the concentration of a metabolite of an exogenous substrate for a cancerspecific enzyme, in a biological sample that has been obtained from a subject, wherein said subject has been administered said exogenous substrate in combination with a sugar.

The exogenous substrate in combination with glucose may be for use in the in vivo detection or prognosis of cancer, wherein the in vivo detection or prognosis of cancer comprises; administering an exogenous substrate for a cancer-specific enzyme, in combination with a sugar to a subject, measuring the concentration of a metabolite of said substrate in a biological sample that has been obtained from said subject.

The present method provides an improved method for the detection of cancer by administering the exogenous substrate in combination with a sugar. The presence of a sugar results in a further lowering of the pH in the tumour microenvironment via the Warburg effect. This results in enhanced activity of enzymes with low/acidic pH optima. In an embodiment the cancer-specific enzyme has a low/acidic pH optimum. And acidic pH optimum may be a pH optimum below pH 7, in an embodiment the pH optimum is between pH 1 to pH 7, or pH 2 to pH 7, or pH 3 to pH 7, or pH 4 to pH 7, or pH 5 to pH 7, or pH 1 to pH 6.5, or pH 2 to pH 6.5, or pH 3 to pH 6.5, or pH 4 to pH 6.5, or pH 5 to pH 6.5, or pH 1 to pH 6, or pH 2 to pH 6, or pH 3 to pH 6, or pH 4 to pH 6, or pH 5 to pH 6. Example of cancer-specific enzymes with low pH optima include but are not limited to p-glucuronidase, p-galactosidase, a-mannosidase, p-N-acetylglucosaminidase. In particular p-glucuronidase is known to have a low pH optimum in the region of pH 5-6. The exogenous substrate is metabolised by a cancer-specific enzyme which leads to generation of a breakdown and/or cleavage product of the substrate, i.e. a metabolite. This metabolite is then detected and the presence of the metabolite is indicative of cancer. The metabolite of the substrate is detectable by any reasonable means. The detectable metabolite may be detected by any known detection methods. These methods may involve a single detection step of the detectable metabolite or may require a step of capturing the detectable metabolite followed by a step of detecting the detectable metabolite. A variety of methods may be used, depending on the nature of the detectable metabolite. Detectable metabolite may be directly detected, following capture, through optical density, radioactive emissions, nonradiative energy transfers, or detectable markers may be indirectly detected with antibody conjugates, affinity columns, streptavidin-biotin conjugates, PCR analysis, DNA microarray, and fluorescence analysis.

The capture assay in some embodiments involves a detection step selected from the group consisting of mass spectrometry, an ELISA, including fluorescent, colorimetric, bioluminescent and chemiluminescent ELIS As, a paper test strip or LFA, bead-based fluorescent assay, and label- free detection, such as surface plasmon resonance (SPR). The capture assay may involve, for instance, binding of the capture ligand to an affinity agent.

The analysis step may be performed directly on the biological sample or the detectable marker component may be purified to some degree first. For instance, a purification step may involve isolating or separating the detectable marker from other components in the biological sample.

As discussed above the exogenous substrate is metabolised to provide a detectable metabolite, as such the exogenous substrate may comprise a cleavable organic compound, wherein once the exogenous substrate is metabolised it results in the cleavage of the cleavable organic compound from the substrate. The cleavable organic compound therefore becomes the detectable metabolite. The term “cleavable” means that the organic compound can be cleaved from the exogenous substrate, cleavage may be effected by a cancer-specific enzyme. Since cleavage is enzymatically driven the exogenous substrate typically comprises an enzyme sensitive region i.e. a region which is recognised and cleaved by a cancer-specific enzyme. Therefore, the exogenous substrate may comprise an enzyme sensitive region linked to a cleavable organic compound, wherein when the enzyme sensitive region is recognised and cleaved by the target enzyme this results in release of the cleavable organic compound as a detectable metabolite.

In an embodiment the enzyme sensitive region may be protease sensitive or glycosidase sensitive. For example where the enzyme sensitive region is glycosidase sensitive the enzyme sensitive region may comprise a glycosidic bond. In an embodiment the cleavable organic compound is labelled. As such when the cleavable organic compound is released from the exogenous substrate this results in a labelled detectable metabolite. The label may improve detection of the detectable metabolite. For example, the label may be fluorescently labelled or isotopically labelled. The label may be selected from 12C, 13C, 14C, 2H, 14N or 180.

The cleavable organic compound may be a volatile organic compound (VOC). The term VOC refers to any compound of carbon, excluding carbon monoxide, carbon dioxide, carbonic acid, metallic carbides or carbonates and ammonium carbonate, which participates in atmospheric photochemical reactions but excluding carbon monoxide, carbon dioxide, carbonic acid, metallic carbides or carbonates, and ammonium carbonate. Generally, VOCs are defined as organic chemical compounds whose composition makes it possible for them to evaporate under normal indoor atmospheric conditions of temperature and pressure. Since the volatility of a compound is generally higher the lower its boiling point temperature, the volatility of organic compounds is sometimes defined and classified by their boiling points. Volatile compounds are compounds that are secreted by the human body into gas fluids, including for example breath, skin emanations and others. In one embodiment, a VOC is any organic compound having an initial boiling point less than or equal to about 250° C measured at a standard atmospheric pressure of about 101.3 kPa.

Where the cancer specific enzyme is an enzyme that is differentially located in cancer tissue, for example where the enzyme is found in the extracellular space in cancerous tissue and within cells in healthy tissue, it may be advantageous that the exogenous substrate is not taken up by cells. Alternatively, where the cancer specific enzyme is an enzyme that is upregulated in cancer tissue compared to healthy tissue, it may be advantageous that the exogenous substrate is taken up by cells.

In a specific embodiment the substrate comprises a glycoside, wherein the glycoside comprises a glycosidic bond linked to a volatile organic compound. Where the substrate comprises a glycoside this substrate may be targeted to a glycosidase enzyme. The glycoside substrate is metabolised by a glycosidase, resulting in cleavage of the glycosidic bond and release of the volatile organic compound.

As used herein the term “glycoside” has its usual meaning in the art and refers to a compound comprising a carbohydrate portion usually a sugar molecule or uronic acid molecule which is linked to a non-sugar molecule via a glycosidic bond. The sugar portion or uronic acid portion may be referred to as the “glycone” and the non-sugar portion may be referred to as the “aglycone”. Glycosides may comprise a six, five or four membered sugar or uronic acid. The glycoside may comprise a monosaccharide, disaccharide, or polysaccharide. Examples of suitable glycosides include but are not limited to glucoside, galactoside, rhamnoside, riboside, arabinoside, fructoside, xyloside. Other suitable glycosides include glycosides of uronic acids including glycosides of alluronic acid, altruronic acid, arabinuronic acid, fructuronic acid, glucuronic acid, galacturonic acid, guluronic acid, iduronic acid, lyxuronic acid, mannuronic acid, psicuronic acid, riburonic acid, ribuluronic acid, sorburonic acid, tagaturonic acid, taluronic acid, xyluluronic acid, xyluronic acid. Glycosides of uronic acids may also be referred to as iduronide, mannoside, glucosamide, galactosamide. In an embodiment the glycone is a glycoside of glucuronic acid also referred to as glucuronide.

A glycoside comprises a glycosidic bond linking the glycone (sugar or uronic acid) to an aglycone (non-sugar molecule). In an embodiment the aglycone is a cleavable organic compound. As such the glycoside may comprise a glycosidic bond linked to a cleavable organic compound. The glycoside may comprise a glycosidic bond linked to a cleavable volatile organic compound. Suitable volatile organic compounds include but are not limited to ketones, alcohols, carboxylic acids. The cleavable volatile organic compound when attached via the glycosidic bond to the glycone may not be volatile, however once the aglycone is released from the glycone it may become volatile. The aglycone may be released, for example by enzymatic cleavage.

Where the exogenous substrate comprises a volatile organic compound, the volatile organic compound is released from the substrate as a volatile detectable metabolite. As such in order to improve detection of the volatile functional group the group may be labelled. In an embodiment the volatile organic compound is selected from any compound that when cleaved from the glycoside produces a volatile hydroxyl containing group. For example the volatile organic compound may be selected from methyl, ethyl, propyl, isopropyl, butyl, methyl-D3, ethyl-D5, propyl-D7. When the volatile organic compound is cleaved from the glycoside it produces a volatile detectable metabolite which can be detected. The volatile detectable metabolite is a compound containing a hydroxyl group. The volatile detectable metabolite may be selected from methanol, ethanol, propanol, isopropyl alcohol, isobutyl alcohol, butyl alcohol, 2-methyl-3-buten- 2-ol, 1-penten-3-ol, isoamyl alcohol, amyl alcohol. The volatile reporter may be labelled as discussed above for example D3-methanol, D5-ethanol, D7-propanol, D7-isopropyl alcohol. The volatile reporter is produced when the volatile functional group is cleaved from the glycoside via cleavage of the glycosidic bond. For example, when the volatile functional group ethyl-D5 is cleaved from the glycoside it produces D5-ethanol.

As mentioned above the glycoside comprises a glycosidic bond. The glycosidic bond links the glycone molecule and the aglycone molecule of the glycoside. There are multiple types of glycosidic bond that may be present, for example the glycosidic bond may be selected from an O-, N-, S-, C- glycosidic bond. In an embodiment the glycosidic bond is an O- glycosidic bond. A glycosidic bond is formed between the hemiacetal or hemiketal group of the sugar or uronic acid molecule and the aglycone. The glycosidic bond may adopt an a or a p stereochemistry.

In an embodiment the exogenous substrate is D5-ethyl-pD-glucuronide and the metabolite is D5-ethanol. In the tumour micro-environment where glucuronidase is present, cleavage of D5- ethyl-pD-glucuronide produces glucuronate which may be excreted through the kidneys and D5- ethanol which may be exhaled and further metabolized by the liver. Presence of D5-ethanol in the extracellular space and biological samples such as breath, blood or urine would therefore be considered a marker of tumour presence. In healthy subjects glucuronidase is normally present intracellularly. D5-ethyl-pD-glucuronide is not taken up by cells and therefore in healthy subjects there is limited capability to cleave D5-ethyl-pD-glucuronide. Therefore, in a healthy subject little to no D5-ethnol would be detected

The present method results in production of enzyme-specific metabolic products production of enzyme-specific metabolic products. As opposed to washout curves of the substrate, metabolic products are excreted into breath over time, starting at low levels and increasing over time due to biotransformation of the substrate by the cancer-specific enzyme. Measurement of such a metabolic product can be applied as a probe for assessing the metabolic phenotype of the enzyme or enzymes responsible for the production of said product. Measuring a substrate and corresponding metabolic product(s) in exhaled breath can thus be used either alone or in combination to assess the activity of one or more cancer-specific enzyme.

As exogenous substrates can be targeted to various enzymes which may result in the production of different metabolic products, the method can be performed with one or more exogenous substrate. This allows testing for the presence of more than one type of cancer. Furthermore, multiple compounds can be measured in bodily fluids which are specific to a certain type of cancer thereby enabling a more accurate diagnosis due to multiple parameters that are assessed. In one embodiment, the invention therefore relates to a method for the detection of cancer comprising administering one or more exogenous substrate and glucose to a subject, measuring the concentration of one or more metabolite of said substrate in a sample of bodily fluid that has been obtained from said subject. The method can therefore be a multiplex method enabling the detection of cancer via multiple cancer-specific enzymatic activities which can be detected in a single biological sample obtained from a subject.

The present invention is based on the surprising finding that administration of a sugar with a disease specific probe (i.e the exogenous substrate) results in improved metabolism of the disease specific probe. The administration of sugar results in a lowering of the pH in tumour tissue. In an embodiment the sugar may be a heptose sugar, a hexose sugar, a pentose sugar, a tetrose sugar. The sugar may be a monosaccharide or a polysaccharide. In an embodiment the sugar is selected from one or more of glucose, fructose, sucrose, maltose, fucose, allose, altrose, gulose, talose, idose, ribose, xylose, arabinose, lyxose, erythrose, threose. The sugar may be administered as a mixture of sugars. In an embodiment the sugar is not mannose or galactose. In a preferred embodiment the sugar is selected from glucose or fructose or a combination thereof.

The biological sample may be a tissue sample such as adipose tissue, liver, brain, bone marrow, muscle or hair. In and embodiment the biological sample is a sample of bodily fluid. Methods are well known in the art for obtaining bodily fluid samples. In an embodiment the bodily fluid sample may be a sample of blood, urine or exhaled breath. The sample of blood may comprise one or more of blood plasma, red blood cells, white blood cells, platelets. The blood sample may comprise any combination of blood plasma, red blood cells, white blood cells, platelets.

Where the bodily fluid sample is a sample of exhaled breath, the breath sample can include air exhaled from one or more different parts of the subject’s body (e.g. nostrils, pharynx, trachea, bronchioles, alveoli etc.). For the collection of a breath sample and methods of measurement, the device and methods described in W02017/187120 or WO2017/187141 (both publications are hereby incorporated by reference) can be used.

The concentration of the substrate and/or metabolite can be measured using methods known in the art. The concentration as used herein means the content or mass of the substrate and/or metabolite in the biological sample as expressed, for example in grams/litre (g/l). In one embodiment, concentration is measured over time, for example by measuring the kinetics of the clearance. For example, concentration is measured by assessing the kinetic profile of the clearance of the substrate for example from breath which is then used as a readout. In addition or alternatively, secretion of metabolic products that can derive from the substrate can be measured over time. For example, clearance of the substrate from breath and secretion of metabolic products can both be measured in the same breath sample at the same time or at different times.

In one embodiment, the concentration or amount of the substrate and/or its metabolite may be determined in absolute or relative terms in multiple breath samples, e.g. in a first breath sample (collected at a first time period) and in a second and/or further breath sample (collected at a later, second or further time period), thus permitting analysis of the kinetics or rate of change of concentration thereof over time.

In some embodiments, the capture device comprises an adsorbent material in the form of a porous polymeric resin. Suitable adsorbent materials include Tenax® resins and Carbograph® materials. Tenax® is a porous polymeric resin based on a 2,6-diphenyl-p-propylene oxide monomer. Carbograph® materials are graphitized carbon blacks. In one embodiment, the material is Tenax GR, which comprises a mixture of Tenax® TA and 30% graphite. One Carbograph® adsorbent is Carbograph 5TD. In one embodiment, the capture device comprises both Tenax GR and Carbograph 5TD. The capture device is conveniently a sorbent tube. These are hollow metal cylinders, typically of standard dimensions (3% inches in length with a % inch internal diameter) packed with a suitable adsorbent material.

In one embodiment, the methods of the invention further comprise establishing a subject value for one or more substrate and/or metabolite concentration.

In one embodiment, the methods of the invention further comprise comparing the subject value to one or more reference value. In one embodiment, said reference value is from healthy subjects. In another embodiment, the reference value is from subjects diagnosed with cancer.

In one embodiment, the reference value is a healthy subject value corresponding to values calculated from healthy subjects. In one embodiment, the presence of one or more subject values at quantities greater than their respective range of healthy subject values indicates a substantial likelihood of a cancer disease state in the test subject.

In one embodiment, when an appropriate reference is indicative of a subject being free of lung cancer, a detectable difference (e.g., a statistically significant difference) between the value determined from a subject in need of characterization or diagnosis of cancer and the appropriate reference may be indicative of cancer in the subject. In one embodiment, when an appropriate reference is indicative of cancer, a lack of a detectable difference (e.g., lack of a statistically significant difference) between the value determined from a subject in need of characterization or diagnosis of cancer and the appropriate reference may be indicative of cancer in the subject. In an embodiment an increase in the concentration of the metabolite compared to the reference sample is indicative of cancer.

In the methods of the present invention a sugar is administered in order to enhance metabolism of the exogenous substrate. The sugar may be administered separately, sequentially or simultaneously with the exogenous substrate. The presence of sugar causes a reduction in the pH in the tumour microenvironment via the Warburg effect, in an embodiment in order for there to be sufficient time for the pH in the tumour microenvironment to lower the sugar may be administered prior to the exogenous substrate.

In an embodiment the sugar is provided at a dose of 50 to 500 g, or 50 to 400 g, or 50 to 350 g, or 50 to 300 g, or 50 to 200 g. The sugar may be provided in a single dose or multiple doses for example 2, 3, 4, or 5 doses. The sugar may be administered in one or more doses that are prior to administering the exogenous substrate. Further sugar may be administered simultaneously with the exogenous substate.

The exogenous substrate may be provided at a dose of 10 pg/kg to 2.5 mg/kg, or 20 pg/kg to 2.4 mg/kg, or 30 pg/kg to 2.3 mg/kg, or 40 pg/kg to 2.2 mg/kg or 50 pg/kg to 2 mg/kg.

In an embodiment the method may require administering the sugar to a subject. Administration of the sugar may be performed via any reasonable route including but not limited to oral, parenteral, sublingual, rectal, vaginal, ocular, intranasal, pulmonary, intradermal, intravitrial, intramuscular, intraperitoneal, intravenous, subcutaneous, intracerebral, transdermal, transmucosal. Parenteral administration includes, for example, intravenous, intramuscular, intraarterial, intraperitoneal, intranasal, rectal, intravesical, intradermal, topical or subcutaneous administration. Preferably the sugar is administered orally.

Administration of the exogenous substrate may be by any convenient route, including but not limited to oral, topical, parenteral, sublingual, rectal, vaginal, ocular, intranasal, pulmonary, intradermal, intravitrial, intramuscular, intraperitoneal, intravenous, subcutaneous, intracerebral, transdermal, transmucosal, by inhalation, or topical, particularly to the ears, nose, eyes, or skin or by inhalation. Parenteral administration includes, for example, intravenous, intramuscular, intraarterial, intraperitoneal, intranasal, rectal, intravesical, intradermal, topical or subcutaneous administration. Preferably, the compositions are administered orally.

The exogenous substrate and the sugar may be administered by the same or different routes. The skilled person would know that the route of administration depends on the enzyme and cancer tested. For instance, if the target enzyme is present in the gastrointestinal tract, oral administration is preferable, while in case of hepatic expression either oral or intravenous administration could constitute viable options.

In an embodiment the cancer is a solid tumour. The cancer may be selected from cancer, breast cancer, ovarian cancer, bowel cancer, prostate cancer, bladder cancer, colorectal cancer, pancreas carcinoma, kidney cancer, renal cancer, leukaemias, multiple myeloma, lymphomas (e.g. Hodgkin's disease and non-Hodgkin's Lymphoma), brain cancer and other CNS and intracranial tumours cancer, head and neck cancer, oesophageal cancers, solid tumors such as sarcoma and carcinomas, mesothelioma, osteosarcoma, endometrial cancer or melanoma. The cancer may be selected from a sarcoma, a carcinoma, or a lymphoma, preferably wherein the cancer is selected from non-small cell lung cancer, adenocarcinoma, squamous cell carcinoma, large cell carcinoma, small cell lung cancer. A “subject” as used herein refers to a test subject, e.g. a mammalian subject, preferably a human. In one embodiment, a sample of exhaled breath is obtained for the purpose of diagnosing or screening the presence/absence of a cancer disease state. The subject may be a male or female. The subject may be an infant, a toddler, a child, a young adult, an adult or a geriatric. The subject may be a smoker, a former smoker or a non-smoker. The subject may have a personal or family history of cancer. The subject may have a cancer-free personal or family history. The subject may exhibit one or more symptoms of cancer.

As used herein, “healthy subject” is defined as a subject that does not have a diagnosable cancer disease state.

As used herein, “reference value” means a value determined by performing the testing method on a plurality of reference subjects. A reference subject can be a healthy subject or a subject diagnosed with cancer.

A “likelihood of a cancer disease state” means that the probability that the cancer disease state exists in the subject specimen is about 50% or more, for example 60%, 70%, 80% or 90%.

In an aspect the invention relates to a method for determining the activity of an enzyme comprising; administering an exogenous substrate for an enzyme in combination with a sugar to a subject, measuring the concentration of a metabolite of said substrate in a sample of bodily fluid that has been obtained from said subject.

The method may be used to determine the activity of an enzyme in vitro or ex vivo. The method of determining the activity of an enzyme may comprise contacting a population of cells with an exogenous substrate in combination with a sugar, measuring the concentration of a metabolite of said substrate in a sample of the population of cells.

The population of cells may be a tissue sample or a tumour sample, for example the tissue sample may be a sample of adipose tissue, liver, brain, bone marrow, muscle or hair. The population of cell may be an artificial population of cells for example a tumour model or an organoid.

The use of a sugar in the method of the present invention is believed to enhance the metabolism of the exogenous substrate thereby increasing the rate of production of the detectable metabolite. As such the invention relates to a method for enhancing the sensitivity of an assay for the detection or prognosis of cancer comprising administering an exogenous substrate in combination with a sugar to a subject, measuring the concentration of a metabolite of said substrate in a sample of bodily fluid that has been obtained from said subject.

The invention also relates to a method for monitoring the progression of cancer in a subject diagnosed with cancer comprising: administering an exogenous substrate in combination with a sugar to a subject, assessing the activity of a cancer-specific enzyme by measuring the concentration of a metabolite of said substrate in a biological sample obtained from the subject.

The invention also relates to a method for determining efficacy of a treatment comprising in a subject diagnosed with cancer: administering an exogenous substrate in combination with a sugar to a subject, assessing the activity of a cancer-specific enzyme by measuring the concentration of a metabolite of said substrate in a biological sample obtained from the subject wherein said subject has received anti- cancer treatment.

The invention also relates to a kit comprising an exogenous substrate, a sugar and a device for obtaining a biological sample from a subject, and optionally instructions for use

The device for obtaining a biological sample may be a device for obtains a blood, urine or exhaled breath sample. The device may be as described herein. In one embodiment, the kit includes a device for capturing a breath sample as described in W02017/187120 or WO2017/187141. The device in W02017/187120 comprises a mask portion which, in use, is positioned over a subject’s mouth and nose, so as to capture breath exhaled from the subject. The exhaled breath samples are fed into tubes containing a sorbent material, to which the compounds of interest adsorb. After sufficient sample has been obtained, the sorbent tubes are removed from the sampling device and the adsorbed compounds desorbed (typically by heating) and subjected to analysis to identify the presence and/or amount of any particular compounds or other substances of interest. The preferred analytic technique is field asymmetric ion mobility spectroscopy (abbreviated as “FAIMS”). The method in WO2017/187141 refinement of the method described in W02017/187120 is disclosed in WO2017/187141. In that document, it is taught to use breath sampling apparatus substantially of the sort described in W02017/187120, but in a way such as to selectively sample desired portions of a subject’s exhaled breath, the rationale being that certain biomarkers or other analytes of interest are relatively enriched in one or more fractions of the exhaled breath, which fractions themselves are relatively enriched in air exhaled from different parts of the subject’s body (e.g. nostrils, pharynx, trachea, bronchioles, alveoli etc).

In the kit the exogenous substrate and the sugar may be formulated together or separately. Multiple doses of glucose may be provided in the kit. The exogenous substrate and/or the glucose in the kit may be formulated with pharmaceutically acceptable carrier or vehicle. This can be a particulate, so that the compositions are, for example, in tablet or powder form. The term "carrier" refers to a diluent, adjuvant or excipient, with which a substrate is administered. Such pharmaceutical carriers can be liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. The carriers can be saline, gum acacia, gelatin, starch paste, talc, keratin, colloidal silica, urea, and the like. In addition, auxiliary, stabilizing, thickening, lubricating and coloring agents can be used. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical carriers also include excipients such as starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like. The composition, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents. The composition can be in the form of a liquid, e.g., a solution, emulsion or suspension. The liquid can be useful for delivery by injection, infusion (e.g., IV infusion) or subcutaneously. As a solid composition for oral administration, the composition can be formulated into a powder, granule, compressed tablet, pill, capsule, chewing gum, wafer or the like form. There may be one or more doses of the exogenous substrate and or the glucose in the kit.

The invention also relates to a composition comprising an exogenous compound and a sugar, wherein the exogenous substrate comprises a glycoside, wherein the glycoside comprises a glycosidic bond linked to a volatile organic compound.

The composition can be for administration to a subject in the methods described herein. The composition may also include a pharmaceutically acceptable carrier or vehicle. This can be a particulate, so that the compositions are, for example, in tablet or powder form. The term "carrier" refers to a diluent, adjuvant or excipient, with which a substrate is administered. Such pharmaceutical carriers can be liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. The carriers can be saline, gum acacia, gelatin, starch paste, talc, keratin, colloidal silica, urea, and the like. In addition, auxiliary, stabilizing, thickening, lubricating and coloring agents can be used. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical carriers also include excipients such as starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like. The composition, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents. The composition can be in the form of a liquid, e.g., a solution, emulsion or suspension. The liquid can be useful for delivery by injection, infusion (e.g., IV infusion) or sub-cutaneously. As a solid composition for oral administration, the composition can be formulated into a powder, granule, compressed tablet, pill, capsule, chewing gum, wafer or the like form. Compositions can take the form of one or more dosage units.

Typically, the kit or compositions comprise the sugarin a dose of 50 to 200 g, preferably in a dose of 100mg. The kit may comprise one or more doses of said sugar.

Typically, the kit or compositions comprise the exogenous substrate in a dose of 50 pg/kg to 2 mg/kg. The kit may comprise one or more doses of the exogenous substrate

Unless otherwise defined herein, scientific and technical terms used in connection with the present disclosure shall have the meanings that are commonly understood by those of ordinary skill in the art. While the foregoing disclosure provides a general description of the subject matter encompassed within the scope of the present invention, including methods, as well as the best mode thereof, of making and using this invention, the following examples are provided to further enable those skilled in the art to practice this invention and to provide a complete written description thereof. However, those skilled in the art will appreciate that the specifics of these examples should not be read as limiting on the invention, the scope of which should be apprehended from the claims and equivalents thereof appended to this disclosure. Various further aspects and embodiments of the present invention will be apparent to those skilled in the art in view of the present disclosure.

All documents mentioned in this specification are incorporated herein by reference in their entirety.

"and/or" where used herein is to be taken as specific disclosure of each of the two specified features or components with or without the other. For example, "A and/or B" is to be taken as specific disclosure of each of (i) A, (ii) B and (iii) A and B, just as if each is set out individually herein. Unless context dictates otherwise, the descriptions and definitions of the features set out above are not limited to any particular aspect or embodiment of the invention and apply equally to all aspects and embodiments which are described.

Numbered embodiments

1 . A method for the detection or prognosis of cancer comprising; administering an exogenous substrate in combination with a sugar to a subject, measuring the concentration of a metabolite of said substrate in a biological sample that has been obtained from said subject.

2. The method according to embodiment 1 , wherein the exogenous substrate is a generally recognised as safe (GRAS) compound.

3. The method according to embodiment 1 or 2, wherein said substrate comprises a cleavable volatile organic compound.

4. The method according to embodiment 1 to 3, wherein the substrate is a substrate for an enzyme which is present in the extracellular space of solid tumours.

5. The method according to embodiment 1 to 4, wherein the substrate is a substrate for an enzyme with a low pH optimum.

6. The method according to embodiment 1 to 5, wherein said substrate is a substrate for an extracellular lysosomal enzyme.

7. The method according to embodiments 4 to 6, wherein the enzyme is p-glucuronidase, p-galactosidase a-L-arabinofuranosidase, N-acetyl-p-D-galactosaminidase, N-acetyl-p- D-glucosaminidase, Hexosaminidase, a-L-Fucosidase, a-galactosidase, a-glucosidase, p-glucosidase, a-L-iduronidase, a-mannosidase, p-mannosidase, Lipases, Phosphatases, Sulfatases.

8. The method according to any preceding embodiment, wherein the substrate is labelled.

9. The method according to any of embodiment 3 to 8, wherein the cleavable organic compound is labelled.

10. The method according to embodiment 8 or 9, wherein the label is selected from 12C, 13C, 14C, 2H, 14N or 180.

11. The method according to any preceding embodiment wherein the substrate comprises a glycoside, wherein the glycoside comprises a glycosidic bond linked to a volatile organic compound.

12. The method according to embodiment 1 1 , wherein the glycoside is glucuronide.

13. The method according to any preceding embodiment, wherein the substrate is D5-ethyl- pD-glucuronide and the metabolite is D5-ethanol.

14. The method according to any preceding embodiment wherein the sugar is selected from; glucose, fructose, sucrose, maltose, fucose, allose, altrose, gulose, talose, idose, ribose, xylose, arabinose, lyxose, erythrose, threose.

15. The method according to any preceding embodiment, wherein the biological sample is a sample of bodily fluid, preferably wherein the bodily fluid is selected from; blood, urine or exhaled breath.

16. The method according to any preceding embodiment, wherein the sugar is administered separately, sequentially or simultaneously with the exogenous substrate,

17. The method according to any of embodiment 1 to 15, wherein the sugar is administered prior to the exogenous substrate. 18. The method according to any preceding embodiment, wherein the sugar is provided at a dose of 50 to 200 g.

19. The method according to any preceding embodiment, wherein the exogenous substrate is provided at a dose of 50 pg/kg to 2 mg/kg.

20. The method according to any preceding embodiment, wherein the sugar is administered orally.

21 . The method according to any preceding embodiment, wherein the exogenous substrate is administered orally, sublingually, buccally, intravenously, intramuscularly, subcutaneously, rectally, or intranasally.

22. The method according to any preceding embodiment, wherein the cancer is a solid tumour.

23. The method according to any preceding embodiment, wherein the cancer is selected from a sarcoma, a carcinoma, or a lymphoma, preferably wherein the cancer is selected from non-small cell lung cancer, adenocarcinoma, squamous cell carcinoma, large cell carcinoma, small cell lung cancer.

24. A method for determining the activity of an enzyme comprising; administering an exogenous substrate for an enzyme in combination with a sugar to a subject, measuring the concentration of a metabolite of said substrate in a biological sample that has been obtained from said subject.

25. A method of determining the activity of an enzyme comprising; contacting a population of cells with an exogenous substrate in combination with a sugar, measuring the concentration of a metabolite of said substrate in a sample of the population of cells.

26. A method for enhancing the sensitivity of an assay for the detection or prognosis of cancer comprising administering an exogenous substrate in combination with a sugar to a subject, measuring the concentration of a metabolite of said substrate in a biological sample that has been obtained from said subject.

27. A method for monitoring the progression of cancer in a subject diagnosed with cancer comprising: administering an exogenous substrate in combination with a sugar to a subject, assessing the activity of a cancer-specific enzyme by measuring the concentration of a metabolite of said substrate in a biological sample obtained from the subject.

28. A method for determining efficacy of a treatment comprising in a subject diagnosed with cancer: administering an exogenous substrate in combination with a sugar to a subject, assessing the activity of a cancer-specific enzyme by measuring the concentration of a metabolite of said substrate in a biological sample obtained from the subject wherein said subject has received anti- cancer treatment.

29. A kit comprising an exogenous substrate, a sugar and a device for obtaining a biological sample from a subject, and optionally instructions for use

30. The kit according to embodiment 29, where the kit comprises a device for capturing, urine, blood or exhaled breath.

31. A composition comprising an exogenous compound and a sugar, wherein the exogenous substrate comprises a glycoside, wherein the glycoside comprises a glycosidic bond linked to a volatile organic compound.

32. The kit according to any one of embodiments 29 to 30, or a composition according to embodiment 31 wherein the sugar is provided at a dose of 50 to 200 g.

33. The kit according to any one of embodiments 29 to 30, or a composition according to embodiment 31 wherein the exogenous substrate is provided at a dose of 50 pg/kg to 2 mg/kg.

34. A method for the detection or prognosis of cancer comprising; measuring the concentration of a metabolite of an exogenous substrate in a biological sample that has been obtained from a subject, wherein said subject has been administered said exogenous substrate in combination with a sugar.

35. An exogenous substrate in combination with sugar for use in an in vivo method for the detection or prognosis of cancer comprising; administering an exogenous substrate in combination with a sugar to a subject, measuring the concentration of a metabolite of said substrate in a biological sample that has been obtained from said subject.

Examples

Example 1. Overview of the Nonclinical Testing Strategy

An extended single-dose intravenous toxicity study in Sprague Dawley Rats was performed in compliance with GLP. The purpose of this nonclinical study was to evaluate the toxicity of D5- ethyl-pD-glucuronide given as a single-dose intravenous, slow injection using an identical formulation as intended to be used in the Phase I study. Histopathology was performed following termination at day 2 and at day 14 for the recovery animals after dosing. In addition, to haematology and clinical chemistry, the toxicokinetic profile of D5-ethyl-pD-glucuronide was also assessed.

The strain and species selected is Sprague Dawley (Crl:CD) rats. Both sexes were used. The rat is the rodent model commonly utilized for toxicity testing of new chemicals. Extensive documentation is also available for susceptibility of rats to a wide range of toxic substances for comparisons if necessary.

An overview of the nonclinical study design can be seen in Table 1 . Table 1. Dose groups, dose levels, dose volumes, dose concentrations, and the numbers and sexes of animals assigned to respective groups and cohorts ^a : TA: Test Article (OWL-EVO1- D5-ethyl-pD-glucuronide); CA: Control Article/vehicle (normal saline); see Sec. 8 of protocol. ^b : The actual dosing volume will be adjusted based on the last scheduled animal body weight measurement.

The selected highest dose level (100 mg/kg) is the maximum feasible for rats with this test article and was confirmed in a pilot study. This dose is equivalent to approximately 16.13 mg/kg in human on the basis of body surface area.

The study is designed to establish a margin of safety by demonstrating that a large multiple of the proposed human dose does not induce adverse effects in the experimental animals.

Male and female Sprague-Dawley rats were administered D5-ethyl-pD-glucuronide up to 100 mg/kg via a single intravenous (slow) injection of 5 minutes on Day 1 followed by a 14-day recovery period, all survived the length of the study. There was no treatment-related gross, organ weight or microscopic changes in the study animals examined.

The toxicokinetics of D5-ethyl-pD-glucuronide following a single intravenous (IV) slow bolus of D5-ethyl-pD-glucuronide at doses of 10, 30, and 100 mg/kg were determined in male and female Sprague Dawley rats.

Example 2. Primary Pharmacodynamics

The micro-environment of the tumour is known to have high levels of p-glucuronidase extra- cellularly (Fishman and Anylan 1947, Young 1976) whilst in normal tissue this enzyme only occurs intra-cellularly (Bosslet 1998). Research in humans and mice has pointed towards a combination of tumour necrosis and release of glucuronidase from lysosomes of macrophages and neutrophils in the tumour microenvironment (Bosslet 1998, Juan 2009) as the origins of the glucuronidase in the tumour micro-environment. Interestingly, the presence of increased numbers of tumour infiltrating neutrophils and macrophages has been linked to poorer prognostic outcomes (Bingle 2002, Bellocq 1998). This mechanism appears to apply to squamous, adeno and adeno-squamous non-small-cell lung cancers providing wide coverage of lung tumours (Murdter 1997). Therefore, a conjugate of glucuronide with a volatile would be cleaved in the tumour micro-environment resulting in production of glucuronate and a readily exhalable volatile. Glucuronide-conjugates are not readily imported into the cell when present in the blood at low concentrations. Presence of D5-ethanol in the extracellular space and breath would therefore be considered a hallmark of tumour presence.

Cleavage of D5-ethyl-pD-glucuronide in the tumour micro-environment would produce glucuronate excreted through the kidneys and D5-ethanol which will be exhaled and further metabolized by the liver (see Figure 1). This pathway is attractive as it reflects a normal in human metabolic pathway with widely studied toxicity that is generally regarded as safe.

Historically, glucuronidation has been extensively studied for targeted delivery of chemotherapeutics to the tumour which would be toxic if not delivered in a targeted manner. As an example: by linking doxorubicin to glucuronate the systemic toxicity could be drastically reduced achieving better therapeutic effects whilst administering lower dose of drug (Bosslet 1998). This efficacy of the conversion by p-glucuronidase appears to be further enhanced by lowering the pH in the tumour micro-environment, with a 10-fold increase realized by dropping the pH from a neutral 7.2 to pH 6.0 (Murdter 1997). In humans such a pH decrease in the tumour micro-environment can be achieved by administering a bolus of glucose (Ashby 1996).

The D5-EG EVOC probe approach proposed in this study has been evaluated in an in vivo mouse model by Lange et al (2019). In this study D5-ethyl-pD-glucuronide was administered at increasing concentrations while measuring the release of D5-ethanol in the breath of the mice (Lange et al., 2019). They used five doses including a very low dose (20 ug/kg), two mid-range doses (50 and 100 ug/kg) and two high doses (500 and 2000 ug/kg body weight) to determine the optimum dose where background D5-ethanol is minimal in healthy mice. The authors found that 50 ug/kg was the optimum dose where they started to detect D5-ethanol in the breath of healthy mice but at minute levels. The D5-ethanol detected in breath increased with the higher doses used. This study demonstrated a clear correlation between the levels of exhaled D5- ethanol in breath and the tumour volume of a grafted human tumour in the mice (Panel A, figure 2). The utility of this compound as a biomarker was further underpinned by a clear reduction of exhaled probe levels in response to treatment as illustrated in Panel B of figure 2.

Example 3. Pharmacokinetics and Product Metabolism in Animals

The toxicokinetic profiles of D5-ethyl-pD-glucuronide following a single intravenous (IV) slow bolus of D5-ethyl-pD-glucuronide at doses of 10, 30, and 100 mg/kg were determined in male and female Sprague Dawley rats (Study number T20221002-GN).

Male and female Sprague Dawley rats were assigned to four groups and received the control article or D5-ethyl-pD-glucuronide via a single IV slow injection over five minutes. Rats in Group 1 (3/sex) received the control article and rats in Groups 2, 3, and 4 (3/sex/group) received D5- ethyl-pD-glucuronide at 10, 30, and 100 mg/kg, respectively. Approximate 0.2 to 0.3 mL of blood was collected via jugular vein cannula and/or superficial veins (e.g. lateral tail veins) from rats in Group 1 pre-dose and IV injection completion. Blood was sampled from rats in Groups 2 to 4 pre-dose, IV injection completion, and at 10, 20, and 30 minutes, and 1 , 2, 4, 8, and 12 hours after injection completion.

The plasma samples were analyzed for D5-ethyl-pD-glucuronide using a validated LC-MS/MS method with a lower limit of quantitation (LLOQ) of 50.0 ng/mL at an upper limit of quantitation (ULOQ) of 50000 ng/mL. Toxicokinetic parameters were determined using non-compartmental analysis on individual profiles using Phoenix® WinNonlin® version 6.3.

No quantifiable levels of D5-ethyl-pD-glucuronide were found in control article-treated group and pre-dose samples collected from D5-ethyl-pD-glucuronide -treated rats.

Following a single intravenous slow bolus of D5-ethyl-pD-glucuronide at doses of 10, 30, and 100 mg/kg, mean t1/2 and MRT0-« values of D5-ethyl-pD-glucuronide ranged from 0.264 to 0.466 h and 0.288 to 0.324 h, respectively. Mean CL and Vd values of D5-ethyl-pD-glucuronide ranged from 0.690 to 0.941 L/h/kg and 0.31 1 to 0.567 L/kg, respectively.

Ratios (male/female) of D5-ethyl-pD-glucuronide Cmax, AUCO-12, and AUC0-« ranged from 0.9 to 1.2 following a single intravenous administration of D5-ethyl-pD-glucuronide at doses of 10, 30, and 100 mg/kg. These results indicate that Cmax, AUCO-12, and AUC0-« of D5-ethyl- pD-glucuronide in males were similar to those in females.

Increase in D5-ethyl-pD-glucuronide dose generally resulted in approximately dose-proportional increases in Cmax and AUCO-12 over the dose range from 10 to 100 mg/kg, except for AUCO- 12 in females from 10 mg/kg to 30 mg/kg, which exhibited greater than dose-proportional increases. The 3- and 10-fold increases in doses from 10 mg/kg to 30 and 100 mg/kg resulted in 3.3- and 9.7-fold increases in D5-ethyl-pD-glucuronide Cmax, and 3.3- and 9.9-fold increases in D5-ethyl-pD-glucuronide AUCO-12 for males, respectively, and 3.9- and 9.0-fold increases in D5-ethyl-pD-glucuronide Cmax, and 4.1- and 9.8-fold increases in D5-ethyl-pD-glucuronide AUCO-12 for females, respectively.

Example 4. Analytical Methods

An LC-MS/MS assay for the determination of D5-ethyl-pD-glucuronide in sodium heparin rat plasma was validated.

Plasma samples were spiked with internal standard, processed by protein precipitation extraction, and analyzed using reversed-phase HPLC with electrospray ionization MS/MS detection. Negative (M-H)- ions for D5-ethyl-pD-glucuronide and ethyl-p-D-glucuronide (EtG) were monitored in MRM mode. Analyte-to-IS peak area ratios for the standards were used to create a linear calibration curve using 1/x2 weighted least-squares regression analysis.

The linearity, precision and accuracy, selectivity, dilution linearity, extraction recovery, matrix effect, hemolyzed plasma, batch size, post-preparative reinjection reproducibility, and injection carryover were evaluated. Experiments were conducted to evaluate the stability of D5-ethyl-pD- glucuronide in sodium heparin rat plasma at ambient temperature, through freeze/thaw cycles, and following long-term storage (-70°C). D5-ethyl-pD-glucuronide stability was assessed in water (ambient temperature and 4°C), in rat whole blood, and in processed samples. The LC-MS/MS bioanalytical method, QPS Taiwan T202-2108, to determine the concentration of D5-ethyl-pD-glucuronide in sodium heparin rat plasma, was validated successfully with respect to linearity, sensitivity, accuracy, precision, dilution, selectivity, hemolyzed plasma, batch size, post-preparative reinjection reproducibility, recovery, matrix effect, and carryover. D5-ethyl- pD-glucuronide stability in sodium heparin rat plasma was demonstrated for 20 hours at ambient temperature, four freeze/thaw cycles at -70°C, and 21 days of long-term storage at -70°C. D5- ethyl-pD-glucuronide stability was also demonstrated in rat whole blood in an ice bath for 2 hours, in stock solution and spiking solution at ambient temperature for 23 hours, in stock solution and spiking solution at 4pC for 23 days, post-preparative reinjection reproducibility samples for 72 hours at 4°C, and in processed samples for 50 hours at 4°C. The quantitation range was 50 to 50000 ng/mL using a 30-pL sample volume.

In addition to blood, detectable EG levels have been reported in several other tissues including adipose tissue, liver, brain, bone marrow, muscle and hair

Example 5. pH sensitivity of p-glucuronidase

To optimise p-glucuronidase activity assay in vitro, 4-methylumbelliferyl-p-D-glucuronide was used. When metabolised by p-glucuronidase 4-methylumbelliferyl-p-D-glucuronide releases a fluorescent signal. The highest activity i.e. release of the fluorescent signal, was found to be between pH 5 and 6. This supports the need to decrease the pH of tumour microenvironment to close to pH6. Activity of the p-glucuronidase at pH 5 was investigated at various concentrations, experiments at pH 5 were performed in 0.1 M ammonium acetate.

Example 6. Assessment of D5-ethyl- D-glucuronide as a as a lung cancer EVOC® Probe in mice model.

Feasibility study

Design of the assessment of D5-ethyl-pD-qlucuronide as a lung cancer EVOC® Probe in mice model

After confirming the feasibility of the breath sampling and detection of D5-EtOH in mice breath samples, we will assess whether D5-ethyl-pD-glucuronide levels are higher in the breath of animals with lung cancer and understand if glucose has an additive or synergetic effect in the D5-ethyl-pD-glucuronide metabolism.

In this phase, after one week of acclimatization in the mice breath sampler device, 30 NMRI nude mice will be allocated in six groups of 5 animals each as described below:

• Group 1 : healthy mice treated with 0.050 mg/kg (dose 1) of D5-ethyl-pD-glucuronide intravenously;

• Group 2: healthy mice treated with oral bolus dose of 2 g/kg glucose followed by 0.050 mg/kg (dose 1) of D5-ethyl-pD-glucuronide intravenously; • Group 3 (positive control): healthy mice treated with oral bolus dose of 2 g/kg glucose followed by 2 mg/Kg (dose 2) of D5-ethyl-pD-glucuronide intravenously;

• Group 4: subcutaneous lung cancer xenograft mice treated with 0.050 mg/kg (dose 1) of D5- ethyl-pD-glucuronide intravenously;

• Group 5: subcutaneous lung cancer xenograft mice treated with oral bolus dose of 2 g/kg glucose followed by 0.050 mg/kg (dose 1) of D5-ethyl-pD-glucuronide intravenously;

• Group 6 (positive control): subcutaneous lung cancer xenograft mice treated with oral bolus dose of 2 g/kg glucose followed by 2 mg/kg (dose 2) of D5-ethyl-pD-glucuronide intravenously. Therefore, half of the animals (N=15) will be inoculated with lung cancer cell lines subcutaneously. The cell line H460 will be used (Reaction Biology). The cell line was selected based on the following criteria:

• Be a human lung cancer cell line;

• Be representative of a common lung cancer subtype such as Non-Small Cell Lung Cancer (NSCL);

• Present high expression of p-glucuronidase in public databases (e.g. Deepmap);

• Be available in the CRO;

• Fast growth in in vivo subcutaneous xenograft model;

• Possible to be used in orthotopic lung cancer model (future studies).

The schedule of events outlined below describes the targeted sequence and timepoints for procedures (Table 2). Further details about the glucose and/or probe administration, breath sampling and tumour excision are described in the following sections. Briefly, an oral bolus of glucose will be given 15 min before D5-ethyl-pD-glucuronide intravenous administration according with the group definition. Breath sampling will start immediately after probe administration in the last animal of a group. Breath sampling will be carried out for 30 minutes. Animals will be monitored during the study for signs of stress and weight will be measured.

Table 2: Timeline of events for the assessment of D5-ethyl-pD-glucuronide as a lung cancer probe in mice model. a Half of the animals (N=15); groups 4, 5 and 6. H460 lung cancer cell line will be used. b Oral bolus of glucose will be given 15 minutes before administration of the probe. c Breath analysis will start 0 or 15 min after probe administration based on the results of feasibility phase.

Timeline maybe adapted to accommodate working hours. i.v.: intravenously.

The duration of the study and treatment/breath sampling timepoints were defined based on tumor growth curve provided by Reaction Biology with H460 cell lines. It is expected to have a clear tumor mass in the end of the study that will be excised for further analysis, such as immunohistochemistry to evaluate p-glucuronidase expression and other markers.

Expected result

The dose 1 (0.05 mg/kg) is expected to the effective dose to discriminate animals with and without lung cancer and so D5-EtOH should be detected in high abundance only in animals with subcutaneous lung cancer. Moreover, Owlstone Medical expect to observe a positive correlation between the tumor volume and the abundance of D5-EtOH in breath samples.

The dose 2 (2 mg/kg) is expected to be a positive control and therefore, with this treatment, we expect to detect D5-EtOH in the breath of both heathy and lung cancer animals because of the intracellular penetration of the probe and cleavage by intracellular p-glucuronidase (Figure 4).

If the selected dose of glucose can lower the pH in the tumour microenvironment, we expect to see a higher activity of p-glucuronidase and therefore the bolus of glucose may have an additive or synergetic effective for the detection of D5-EtOH in subcutaneous lung cancer xenograft mice.

Probe and glucose administration

The probe is manufactured in compliance with GMP guidelines. The probe is stored between 2 °C and 8 °C. Drug substance and drug product specifications have been established to ensure the safety and consistency of the product. All tests have been conducted according to the European Pharmacopeia.

The required amount of the probe is administered as a single intravenous bolus dose by infusion with saline solution.

In four of six mice groups the probe will be administered after an oral bolus dose of 2 g/kg glucose to lower the pH of the tumor microenvironment. In the present study, animals will not be fasted before glucose administration. However, as described above, the dose of 2 g/kg glucose administered orally is tolerated by non-fasted mice.

Time of glucose and probe administration in each animal will be recorded. It will be randomized the order of probe administration (inside each group).

Mice monitoring

The weight of animals will be measured 3x/week after randomization.

Tumour volume will be measure via callipering 2x/week after implantation (including days of breath biopsies).

Any increased sign of animal stress during breath sampling will be recorded. It is important to underscore that animals will be acclimatized with the mouse boxes from the mice breath sampler device for one week before the study start. Breath sampling methodology

After probe administration, mice will be put in individual boxes connected with sorbent tubes for breath analysis. Breath samples will be collected for 30 min.

After collection, the sorbent tubes are stored in a refrigerator at 4-8 °C until shipment. Sorbent tubes containing breath VOCs will be shipped to at room temperature.

Mice breath sampler device

In the mice breath samples device, clean air will be pulled through the mouse boxes with an external pump (100 ml/min). This will provide fresh air to the mice and push the VOC molecules from the exhaled breath of the mice through the sorbent tubes. The volatile molecules of interest will be trapped in the sorbent tubes (Figure 5).

Individual mouse will be put inside a one litre transparent plastic box (17.6 cm L x 12.5 cm Wx 7.9 cm H). Inside each box, a table with a fan will be added to increase the air flow in each box. The estimated free height in the box is 49.5 mm and we anticipate that one week of acclimatization will be enough to make the animals acguired with the environment of breath sampling. The space free floor (>22 in2) is in accordance with the Guide for Care and Use of Laboratory Animals for mice weighing more than 25 g (>15 in2; weight of H460 NMRI nude mouse is used 25 - 30 g; National Research Council (US), 2011).

The non-noise fan will be protected by a mesh. Noise wise a whisper is around 30db, and this fan is rated to emit only 20db. The fan will produce a flow rate of 40 L/min and the air speed underneath the fan will range from 0.5-2 m/s (different points in the box). Rodents run at variable speeds on the order of ~0.5 m/s including a female NMRI nu/nu mice that will be used in the present study. Therefore, the air speed applied in the box is comparable to a breeze experienced by a mouse when running.

Five mouse boxes will be aligned horizontally allowing visual communication between mice in different boxes during breath collection. To keep the animals warm during the period of breath sampling, the boxes will be warmed up with a warming pad positioned on the bottom.

Sorbent tubes, provided by Owlstone Medical, used in the mice device is the same used by Owlstone Medical for breath analysis in humans. These tubes allow the analysis of VOC with a higher sensitivity than the filters or other methods applied in previous studies aiming breath analysis in mice (Barbour et al, 2013; Szymczak et al, 2014; Lange et al, 2019, Liu et al, 2019). The number of the tubes will be recorded for each breath sample taken. A new tube is connected to a mouse box before the sampling.

The breath sampling will start when the pump is switched on and will stop when the pump is switched off (expected time for breath collection: 30 min). Once the pump is switched off, sampling has ended and the mice can be taken out without any rush. Noteworthy, each mice box has a total volume of 1 L and it is estimated that a mouse can inhale approximately 30 ml air / min. Therefore, a mouse could be in the box without switched on the pump for around 30 min. Owlstone Medical design an easy to set-up device for breath analysis and it is estimated that less than 5 minutes will be needed to set up each round of breath sampling with five animals.

Procedure for breath sampling

Steps for setting up mice breath sampler device and collection of breath sampling:

Remove the caps from the sorbent tubes. The caps just pull off;

Install tubes between mouse boxes and pump;

Place mouse in camber. Lift up the lid of the box, place the mice inside and close the lid;

Switch on the pump to start the airflow and the breath collection;

Collect the breath for 30 minutes;

Record time to switch on the pump;

Record experiment number, box and tube number in table;

Switch off the pump to stop the airflow and the breath collection;

Record time to switch off the pump;

Remove mice from the boxes;

Detach the sorbent tubes from mouse boxes and the pump;

Place caps on sorbent tubes. Caps are just pushed onto tubes;

Store tubes refrigerated until the end of the study;

All tubes will be shipped at room temperature.

Example 7. Effect of pH on p-glucuronidase activity

The purpose of this study was to confirm that p-glucuronidase activity is higher under a low pH. Paella vulgate limpets enzyme was used in the study with 4-Methylumbelliferyl p-D-glucuronide as substrate. Fluorescence was measured as a readout of enzyme activity for each condition tested.

Figure 6 shows the standard curves performed with reaction assay buffers in different pHs. A clear effect of pH in p-glucuronidase activity can be observed. The reaction with pH 4-5 saturated quickly with the highest concentration of the enzyme. However, reactions under pH 5 shows the lowest limit of detection (Figure 7). A clear exponential growth could be detected with ~0.03 units/well only at pH 5. This study confirms that pH 5 is the optimum for p-glucuronidase activity in vitro using Paella vulgate limpets. Moreover, our analysis reinforces the p-glucuronidase has high activity under low and limited pH. As expected, the pH in the tumour microenvironment (TME) may impact in the kinetics of a cancer specific probe such as D5-ethyl-pD-glucuronide, highlighting the need of development of strategies to modulate the pH in the TME.

References

Pavlova, Natalya N., and Craig B. Thompson. 2016. “The Emerging Hallmarks of Cancer Metabolism.” Cell Metabolism 23 (1): 27-47. https://doi.Org/10.1016/j.cmet.2015.12.006. Gaude, Edoardo, and Christian Frezza. 2016. “Tissue-Specific and Convergent Metabolic Transformation of Cancer Correlates with Metastatic Potential and Patient Survival.” Nature Communications 7 (October): 13041. https://doi.org/10.1038/ncomms13041.

Fan, Teresa WM, Andrew N Lane, Richard M Higashi, Mohamed A Farag, Hong Gao, Michael Bousamra, and Donald M Miller. 2009. “Altered Regulation of Metabolic Pathways in Human Lung Cancer Discerned by 13C Stable Isotope-Resolved Metabolomics (SIRM).” Molecular Cancer 8 (1): 41. https://doi.org/10.1186/1476-4598-8-41.

Sellers, Katherine, Matthew P. Fox, Michael Bousamra, Stephen P. Slone, Richard M. Higashi, Donald M. Miller, Yali Wang, et al. 2015. “Pyruvate Carboxylase Is Critical for Non-Small-Cell Lung Cancer Proliferation.” The Journal of Clinical Investigation 125 (2): 687-98. https://doi.Org/10.1172/JCI72873.

Faubert, Brandon, Kevin Y. Li, Ling Cai, Christopher T. Hensley, Jiyeon Kim, Lauren G. Zacharias, Chendong Yang, et al. 2017. “Lactate Metabolism in Human Lung Tumors.” Cell 171 (2): 358-371 . e9. https://doi.Org/10.1016/J.CELL.2017.09.019.

Hensley, Christopher T, Brandon Faubert, Qing Yuan, Naama Lev-Cohain, Eunsook Jin, Jiyeon Kim, Lei Jiang, et al. 2016. “Metabolic Heterogeneity in Human Lung Tumors.” Cell 164 (4): 681- 94. https://doi.Org/10.1016/j.cell.2015.12.034.

Tumor microenvironmental physiology and its implications for radiation oncology. Vaupel, Peter.

3, 2004, Seminars in Radiation Oncology, Vol. 14, pp. 198-206. Kalliomake and Hill, 2004

Kato, Y, et al. 2013, Acidic extracellular microenvironment and cancer. 89, s.l. : Cancer Cell International, Vol. 13.

Potentiating anti-cancer chemotherapeutics and antimicrobials via sugar-mediated strategies. Mullapudi, SS, et al. s.l. : Royal Society of Chemistry, 2020, Mol.Syst. Des. Eng., Vol. 5, pp. 772- 791.

Harikrishna Devalapally, Kombu Subramanian Rajan, RaghuRam Rao Akkinepally & Rama Krishna Devarakonda. 2008 Safety, Pharmacokinetics and Biodistribution Studies of a p- galactoside Prodrug of Doxorubicin for Improvement of Tumor Selective Chemotherapy. 8, s.l. : Taylor & Francis, Drug Development and Industrial Pharmacy, Vol. 34, pp. 789-795.

Adidala, Raghuramreddy, et al. 2012An improved synthesis of lysosomal activated mustard prodrug for tumor-specific activation and its cytotoxic evaluation. 9, s.l.: Taylor & Francis, Drug Development and Industrial Pharmacy, Vol. 38, pp. 1047-1053. Zhai, Luoping, et al. 2021A Dual pH-Responsive DOX-Encapsulated Liposome Combined with Glucose Administration Enhanced Therapeutic Efficacy of Chemotherapy for Cancer., s.l. : Dovepress, International Journal of Nanomedicine, Vol. 16, pp. 3185-3199.

Schem, Baard-Christian and Dahl, Olav. 1991 , Thermal Enhancement of ACNU and potentiation of thermochemotherapy with ACNU by hypertonic glucose in the BT4An rat glioma, s.l. : Kluwer Academic Publishers, Journal of Neuro-Oncology, Vol. 10, pp. 247-252.

Fishman, W. H. & Anlyan, A. J. The presence of high beta-glucuronidase activity in cancer tissue. J. Biol. Chem. 169, 449 (1947).

Young, C. W. et al. Therapeutic trial of aniline mustard in patients with advanced cancer. Comparison of therapeutic response with cytochemical assessment of tumor cell betaglucuronidase activity. Cancer 38, 1887-1895 (1976).

Bosslet, K. et al. Elucidation of the mechanism enabling tumor selective prodrug monotherapy. Cancer Res. 58, 1 195-1201 (1998).

Bellocq, A. et al. Neutrophil alveolitis in bronchioloalveolar carcinoma: induction by tumor- derived interleukin-8 and relation to clinical outcome. Am. J. Pathol. 152, 83-92 (1998).

Bingle, L., Brown, N. J. & Lewis, C. E. The role of tumour-associated macrophages in tumour progression: implications for new anticancer therapies. J. Pathol. 196, 254-265 (2002).

Murdter, T. E. et al. Enhanced Uptake of Doxorubicin into Bronchial Carcinoma : @ - Glucuronidase Mediates Release of Doxorubicin from a Glucuronide Prodrug ( HMR 1826 ) at the Tumor Sitel . (1997).

Lange, J. et al. Volatile Organic Compound Based Probe for Induced Volatolomics of Cancers. Angew. Chem. Int. Ed. Engl. 58, 17563-17566 (2019).

Barbour, A. G. et al. Elevated carbon monoxide in the exhaled breath of mice during a systemic bacterial infection. PLoS One 8:e69802 (2013).

Liu, Y. A device to collect exhaled breath to study biomarkers in small animal models. BioRxiv. doi: https://doi.org/10.1 101/511931 (2019). Szymczak, W. et al. Online breath gas analysis in unrestrained mice by hs-PTR-MS. Mamm.

Genome 25:129-40 (2014).