Methods for synthesizing EVOC probes

Abstract

A method for synthesizing glycosides comprising hydrolyzing a methyl ester glycoside in the presence of a base in water to form the glycoside, wherein the tendency of the methyl ester glycoside to reform is reduced. The invention also extends to a method for the detection or prognosis of cancer using the glycoside obtained by said method.

Description

[Technical field]

[0001] The present invention relates to a method for synthesizing EVOC probes. [Background technology]

[0002] Introduction Lung cancer is the leading cause of cancer-related deaths worldwide. Early detection of lung cancer is crucial for patient survival but remains extremely challenging due to the absence of clinical symptoms in the early stages, and better screening methods are needed. Despite decades of dedicated research efforts, the 5-year survival rate of lung cancer remains dismal at 19% (Siegel et al., 2019). The main reason is that over 75% of patients are diagnosed with advanced stage disease that is not amenable to treatment with curative intent (Walters et al., 2013). For stage I, the 1-year survival rate is approximately 80%, whereas for stage IV, it is only 20%. Therefore, early detection of lung cancer is of utmost importance to improve survival rates. Tumor cells are characterized by metabolic alterations at the earliest stages of their development. Therefore, measuring biochemicals associated with these metabolic changes can yield diagnostic biomarkers useful for early cancer detection (Muthu & Nordstrom, 2019). These biomarkers can be detected in biological matrices such as breath, urine, and blood. An attractive matrix for detecting these metabolites is breath, which can be accessed completely non-invasively at the point of care, lowering the barrier to participation in screening (Hakim et al., 2012).

[0003] Recently, exogenous volatile organic compound (EVOC) probe methods have been pioneered to actively interrogate disease-specific pathways, in which exogenous metabolic probe compounds are administered to patients, metabolized by disease-specific pathways, and release volatile products in the patient's breath ( Gaude et al., 2018 ).

[0004] Therefore, there is a need to develop methods for consistent production of EVOC probes that can be used in non-invasive therapeutic settings. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] WO2017 / 187120 [Patent Document 2] WO2017 / 187141 [Non-patent literature]

[0006] [Non-Patent Document 1] Czalgoszerski, Edward J., "Reactions of Synthesis of Methyl D-Glucuronide" (1934) Summary of the Invention [Means for solving the problem]

[0007] Recently, it has been demonstrated that EVOC probes can be used to detect tumors in mice by breath analysis. In this study, healthy and tumor mice were administered the metabolic probe D5-ethyl-βD-glucuronide, which is metabolized by tumor-specific extracellular β-glucuronidase to release D5-ethanol, and a discernible D5-ethanol signal was detected in the breath of tumor mice (Lange et al., 2019). It is known that high levels of β-glucuronidase are present extracellularly in the tumor microenvironment (FISHMAN & ANLYAN, 1947; Young et al., 1976), whereas in normal tissues this enzyme is only present in lysosomes in the cytoplasm (Bosslet et al., 1998). Studies in humans and mice point to a combination of tumor necrosis and release of β-glucuronidase from lysosomes of macrophages and neutrophils in the tumor microenvironment as the source of extracellular β-glucuronidase in the tumor microenvironment (Bosslet et al., 1998; Juan et al., 2009). Thus, glucuronide conjugates with volatile molecules such as D5-ethanol are cleaved in the tumor microenvironment, resulting in the production of glucuronate and the easily excreted volatile molecule D5-ethanol. Glucuronide conjugates are not readily taken up into cells when present in blood at low concentrations. Thus, the presence of D5-ethanol in the extracellular space or in biological matrices is considered a hallmark of tumor presence. The lack of intracellular diffusion of D5-ethyl-βD-glucuronide (and other glucuronide conjugates) at low concentrations is essential for successful discrimination of healthy and tumor tissues.

[0008] However, it is demonstrated herein that standard methods for preparing glucuronide-based probes produce a methyl ester impurity (D5-ethyl-βD-glucuronide-methyl ester). This methyl ester alters cell permeability, resulting in intracellular uptake of the probe. Inside the cell, the methyl ester can be cleaved, resulting in the release of D5 ethanol that is not the result of the presence of tumor. This results in high background levels of D5 ethanol, which can lead to false positives.

[0009] Therefore, the present inventors have developed a method for producing glucuronides that reduces the formation of methyl ester impurities.

[0010] Thus, one aspect of the present invention relates to a method for synthesizing a glycoside, the method comprising: The method includes hydrolyzing a methyl ester glycoside in water in the presence of a base to form a glycoside, Here, the tendency to reform the methyl ester glycoside is reduced.

[0011] One aspect of the present invention relates to a glycoside obtainable by the method of the present invention.

[0012] One aspect of the invention relates to a composition comprising a glycoside comprising a glycosidic bond linked to a volatile functional group, wherein the composition comprises less than 1% methyl ester glycoside comprising a glycosidic bond linked to a volatile functional group.

[0013] One aspect of the present invention relates to a kit comprising a glycoside obtained by the method of the present invention or a composition comprising a glycoside linked to a volatile functional group via a glycosidic bond, wherein the composition comprises less than 1% methyl ester glycosides linked to a volatile functional group via a glycosidic bond, and optionally instructions for use.

[0014] One aspect of the present invention relates to a method for reducing the cell permeability of a glycoside-based exogenous volatile organic compound (EVOC) probe, comprising: applying conditions that promote conversion of methyl ester glycosides to glycosides, Here, conditions that promote the formation of glycosides include hydrolysis of methyl ester glycosides in the presence of a base in water.

[0015] One aspect of the invention relates to the use of a glycoside obtained by the method of the invention or a composition comprising a glycoside linked to a volatile functional group via a glycosidic bond, wherein the composition comprises less than 1% glycoside methyl esters linked to a volatile functional group via a glycosidic bond, in a diagnostic breath test.

[0016] In one aspect, the present invention relates to a method for the detection or prognosis of cancer, comprising a step of assessing the activity of a cancer-specific enzyme by measuring the concentration of an exogenous substrate for said enzyme and / or the concentration of a metabolite of said substrate in a biological matrix of a subject, wherein said exogenous substrate is a glycoside obtainable according to the invention or a composition according to the invention.

[0017] In one aspect, the present invention relates to a composition comprising a glycoside comprising a glycosidic bond linked to a volatile functional group, wherein the composition comprises less than 1% methyl ester glycoside comprising a glycosidic bond linked to a volatile functional group, for use in a method for cancer detection or prognosis comprising measuring a concentration of an exogenous substrate for a cancer-specific enzyme in a biological matrix of a subject and / or assessing the activity of said enzyme by measuring a concentration of a metabolite of said substrate, wherein said exogenous substrate is a composition comprising a glycoside comprising a glycosidic bond linked to a volatile functional group, wherein the composition comprises less than 1% methyl ester glycoside comprising a glycosidic bond linked to a volatile functional group.

[0018] In another aspect, the invention relates to a method for the detection or prognosis of cancer, comprising a step of assessing the activity of a cancer-specific enzyme by measuring the concentration of an exogenous substrate for said enzyme and / or the concentration of a metabolite of said substrate in a biological matrix of a subject, wherein said exogenous substrate is a glycoside, said glycoside being administered at a concentration of 0.05-10 mg / kg, and wherein the concentration of the metabolite is measured up to 300 minutes after administration of the glycoside.

[0019] In another aspect, the invention relates to a glycoside for use in the diagnosis or prognosis of cancer, comprising the step of administering to a subject the glycoside or a composition comprising the glycoside, wherein the glycoside is administered at a concentration of 0.05-10 mg / kg.

[0020] The invention is further illustrated in the following non-limiting figures. [Brief description of the drawings]

[0021] [Figure 1] Schematic of the assay to determine the levels of D5 ethanol release in blood samples exposed to D5-ethyl-βD-glucuronide (D5-EG) or D5-ethyl-βD-glucuronide-methyl ester (D5-EG-ME). [Diagram 2] D5-ethanol production from blood cells incubated with D5-EG or D5-EG-ME. [Diagram 3] 1H NMR of the product produced under standard conditions. The singlet peak at 3.7 is assigned to the methyl ester group. [Figure 4] 1H NMR of the product produced under conditions that reduced the methyl ester content: 1H NMR (396MHz, DO, ppm), δH 4.54 (d, J=7.9Hz,1H), 4.00 (d,J=9.7Hz,1H), 3.59 (t, J=9.1Hz,1H), 3.54 (t,J=9.1Hz,1H), 3.32 (t, J=8.8Hz,1H). [Diagram 5] Schematic diagram of the metabolism of D5-ethyl-βD-glucuronide by β-glucuronidase. [Figure 6] Peak area of ​​D5 ethanol detected in breath. [Figure 7-1] Expression of β-glucuronidase in human lung cancer and normal lung tissue. (A) Representative images of tissue microarray core samples stained for β-glucuronidase expression. β-glucuronidase expression in the tumor microenvironment (TME) was observed in all stages of lung cancer, including stage 1. No extracellular expression of β-glucuronidase was observed in healthy tissue. [Figure 7-2] (B+C) Quantification of β-glucuronidase expression in lung cancer and normal lung cancer tissues by tumor microarray. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0022] The present invention is further described below. In the following clauses and phrases, different aspects of the invention are defined in more detail. Each aspect thus defined can be combined with any other aspect or aspects, unless expressly indicated to the contrary. In particular, any feature indicated as being preferred or advantageous can be combined with any other feature or features indicated as being preferred or advantageous.

[0023] The present invention relates to a method for synthesizing glycosides, The method includes a step of hydrolyzing a methyl ester glycoside in water in the presence of a base to form a glycoside.

[0024] As described herein, the inventors have found that hydrolysis of methyl ester glycosides in the presence of base in water reduces the tendency to reform methyl ester glycosides. Glycoside compounds can be used as metabolic probes in cancer detection because the glycosides can be metabolized by tumor-specific extracellular β-glucuronidase to release detectable aglycone molecules. However, conventional methods for preparing glycosides from methyl ester glycosides include hydrolysis in methanol in the presence of base. It is shown herein that the glycosides produced by this hydrolysis method can reform methyl esters. The presence of methyl ester glycosides as impurities increases the cellular permeability of the final glycoside. Therefore, the methyl ester glycosides enter the cells and are cleaved by intracellular β-glucuronidase. This cleavage is not tumor-specific and may cause false positives in cancer detection. Therefore, the reliability of glycosides as metabolic probes can be increased by developing a production method that does not reform methyl ester glycosides.

[0025] The hydrolysis of the methyl ester glycoside is preferably carried out in the absence of methanol.

[0026] The hydrolysis of the methyl ester glycoside is carried out in water in the presence of a base. The base may be selected from NaOH, KOH, or LiOH, and preferably the base is NaOH. The hydrolysis may be carried out at a temperature between 18-30°C, or 20-29°C, or 22-28°C, or 23-27°C, or 24-26°C. The hydrolysis may be carried out at about 25°C. About 25°C may include a temperature between 23-27°C, or 24-26°C, or 24.5-25.5°C. The hydrolysis may be carried out at a pressure between 95-105 kPa, or 98-102 kPa, or 100-102 kPa. The hydrolysis may be carried out at about 101 kPa, where about 101 kPa may include a pressure between 100-102 kPa, or 101-102 kPa, or 100.5-101.5 kPa. The hydrolysis may be carried out at about 101.325 kPa, where about 101.325 kPa includes pressures between 101.0 and 101.5 kPa. The hydrolysis step may be carried out under ambient conditions, where ambient conditions include a temperature of 25° C. (298.15 K) and a pressure of 101.325 kPa.

[0027] The term "glycoside" as used herein has its usual meaning in the art and refers to a compound that contains a carbohydrate moiety, usually consisting of a sugar molecule or a uronic acid molecule, linked to a non-sugar molecule via a glycosidic bond. The sugar or uronic acid moiety may be referred to as a "glycone" and the non-sugar moiety may be referred to as an "aglycone." A glycoside may be composed of a six-, five-, or four-membered sugar or uronic acid. A glycoside may include a monosaccharide, a disaccharide, or a polysaccharide. In one embodiment, a glycoside includes a carboxylic acid group. Examples of suitable glycosides include, but are not limited to, glucosides, galactosides, rhamnosides, ribosides, arabinosides, fructosides, xylosides, and fucosides. Other suitable glycosides include uronic acid glycosides, including those 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, and xyluronic acid. Glycosides of uronic acid may also be called iduronides, mannosides, glucosamides, galactosamides, etc. In a preferred embodiment, the glycone is a glycoside of glucuronic acid, also called a glucuronide.

[0028] A glycoside contains a glycosidic bond linking a glycocon (a sugar or uronic acid) to an aglycon (a molecule other than a sugar). A methyl ester glycoside also contains a glycosidic bond linking a glycocon to an aglycon. In one embodiment, the aglycon is a volatile compound. Such glycosides may contain a glycosidic bond linked to a volatile functional group. A methyl ester glycoside may contain a glycosidic bond linked to a volatile functional group. Suitable volatile functional groups include, but are not limited to, ketones, alcohols, and carboxylic acids. The functional group attached to the glycocon is not volatile, but becomes volatile when the aglycon is released from the glycocon. The aglycon can be released, for example, by enzymatic or acid cleavage.

[0029] The term "methyl ester glycoside" refers to a glycoside that contains a methyl ester substituent in place of a hydroxyl or carboxylic acid group of the glycone molecule. In a preferred embodiment, when the glycone molecule is a glucuronide, the methyl ester group is attached to C6 of the glucuronide molecule.

[0030] As mentioned above, glycosides contain a glycosidic bond. The glycosidic bond connects the glycocon and aglycon molecules of the glycoside. There can be various types of glycosidic bonds, for example, the glycosidic bond is selected from O-, N-, S-, and C-glycosidic bonds. In one embodiment, the glycosidic bond is an O-glycosidic bond. The glycosidic bond is formed between a hemiacetal or hemiketal group of a sugar or uronic acid molecule and the aglycon. The glycosidic bond can have an α or β stereochemistry. Methyl ester glycosides contain a glycosidic bond.

[0031] Under certain conditions, such as in the presence of certain enzymes or under environmental conditions such as acidic pH, the volatile functional group can be released from the glycoside. When the glycoside is used as a cancer detection probe, it is the volatile functional group that is detected. Therefore, the volatile functional group can be labeled to improve the detection of the volatile functional group. The volatile functional group can label a single position in the compound or multiple positions in the compound. Examples of suitable labels include, but are not limited to, 12C, 13C, 14C, 2H, 14N, or 18O. In one embodiment, the volatile functional group is selected from any compound that generates a volatile hydroxyl-containing group when cleaved from the glycoside. For example, the volatile functional group is selected from methyl, ethyl, propyl, isopropyl, butyl, methyl-D3, ethyl-D5, and propyl-D7. When the volatile functional group is cleaved from the glycoside, a detectable volatile reporter is generated. The volatile reporter is a compound that contains a hydroxyl group. The volatile reporter is 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 can be labeled as described above, for example, D3-methanol, D5-ethanol, D7-propanol, D7-isopropyl alcohol, etc. The volatile reporter is generated when a volatile functional group is cleaved from a glycoside via cleavage of a glycosidic bond. For example, D5-ethanol is generated when the volatile functional group ethyl-D5 is cleaved from a glycoside.

[0032] The method according to the invention may include further steps. For example, the method may further include an ion exchange step. The ion exchange may be carried out after the hydrolysis step is completed. The ion exchange may be carried out using an acidic cation exchange resin, preferably a strong cation exchange resin, suitable resins are known in the art, for example Dowex® 50WX8. The resin exchange may be carried out in a solvent, such as NaOH in water. The method may include a solvent evaporation step. The solvent evaporation step may be carried out after the hydrolysis is completed. The solvent evaporation step may be carried out after the resin exchange step. In one embodiment, the method may further include carrying out both the resin exchange step and the solvent evaporation step. In one embodiment, the method may include carrying out hydrolyzing the methyl ester glycoside in water in the presence of a base to form a glycoside; applying the glycoside obtained by hydrolysis to an ion exchange resin to carry out ion exchange; evaporating the solvent from the glycoside obtained after ion exchange to obtain a solid; Includes.

[0033] To determine whether the hydrolysis is complete, various techniques can be used to monitor the reaction. For example, techniques such as 1H NMR can be used to monitor the hydrolysis reaction. By monitoring the hydrolysis of the methyl ester glycoside to form the glycoside, it is possible to determine when the conversion to the glycoside is complete, e.g., when it is no longer possible to detect the presence of the methyl ester glycoside. Thus, the method according to the present invention may include a step of monitoring the hydrolysis of the methyl ester glycoside to form the glycoside using 1H NMR.

[0034] The methyl ester glycosides used in the method according to the invention can be obtained by any reasonable route. When the methyl ester glycoside contains a glycosidic bond linked to a volatile functional group, the methyl ester glycoside can be obtained by conjugating the methyl ester glycoside to the volatile functional group. For example, the methyl ester can be obtained by conjugation of hexuronic acid with methyl alcohol or by reaction of unsubstituted aldohexuronic acid with methyl alcohol containing dry hydrogen chloride. Suitable methods are outlined in Czalgoszerski, Edward J., "Reactions of Synthesis of Methyl D-Glucuronide" (1934). Master's Thesis. 21. In one embodiment, the methyl ester glycoside is glucuronidation of D6 ethanol with bromo sugar 1 using silver carbonate to give glucopyranoside 2;

[0035] [ka]

[0036] deacetylation of glucopyranoside 2 using sodium methoxide to give D5-ethyl-βD-glucuronide methyl ester 3;

[0037] [ka]

[0038] is obtained by

[0039] In one embodiment, the method for synthesizing a glycoside comprises: Providing a methyl ester glycoside by any suitable route; hydrolyzing the methyl ester glycoside in water in the presence of a base to form a glycoside; Includes.

[0040] According to the present invention, it has been shown that hydrolysis of methyl ester glycosides produces a final product containing glycosides and less than 1% of methyl ester glycosides. In one embodiment, after hydrolysis, methyl ester glycosides are present in less than 2%, less than 1.9%, less than 1.8%, less than 1.7%, less than 1.6%, less than 1.5%, less than 1.4%, less than 1.3%, less than 1.2%, less than 1.1%, less than 1%, less than 0.9%, less than 0.8%, less than 0.7%, less than 0.6%, less than 0.5%, less than 0.4%, less than 0.3%, less than 0.2%, less than 0.1% of the glycosides. Preferably, methyl ester glycosides are present in less than 1% of the glycosides. The amount of methyl ester glycosides present in the final product after hydrolysis can be determined using routine laboratory techniques, such as 1H NMR. In one embodiment, methyl ester glycosides are present at less than 1%, less than 0.9%, less than 0.8%, less than 0.7%, less than 0.6%, less than 0.5%, less than 0.4%, less than 0.3%, less than 0.2%, or less than 0.1% of the glycosides as determined by 1H NMR. The % of methyl ester glycosides may refer to the weight % of the composition. The % of methyl ester glycosides may refer to the relative % to the amount of glycosides.

[0041] In a preferred embodiment, the methyl ester glycoside is D5-ethyl-βD-glucuronide-methyl ester. In a preferred embodiment, the glycoside is D5-ethyl-βD-glucuronide. In such a preferred embodiment, the present invention relates to a method for synthesizing D5-ethyl-βD-glucuronide, the method comprising: Hydrolysis of D5-ethyl-βD-glucuronide methyl ester in water in the presence of a base to form the glycoside. Includes.

[0042] In one aspect, the present invention relates to glycosides obtained by the method as defined above. The glycosides obtained from the method of the present invention show a reduced tendency to reform the methyl ester glycoside intermediate. Preferably, the glycosides obtained by the method of the present invention contain a glycosidic bond linked to a volatile functional group / compound.

[0043] In one aspect, the present invention relates to a composition comprising a glycoside comprising a glycosidic bond linked to a volatile functional group, wherein the composition comprises less than 2% of a methyl ester glycoside comprising a glycosidic bond linked to a volatile functional group.Preferably, the composition comprises less than 1% of a methyl ester glycoside comprising a glycosidic bond linked to a volatile functional group.

[0044] The glycosides present in the composition may have any of the characteristics of a glycoside as defined herein.The methyl ester glycosides present in the composition may have any of the characteristics of a methyl ester glycoside as defined herein.

[0045] In one embodiment, the composition comprises D5-ethyl-βD-glucuronide, wherein the composition contains less than 1% D5-ethyl-βD-glucuronide-methyl ester.

[0046] The composition contains less than 2%, less than 1.9%, less than 1.8%, less than 1.7%, less than 1.6%, less than 1.5%, less than 1.4%, less than 1.3%, less than 1.2%, less than 1.1%, less than 1%, less than 0.9%, less than 0.8%, less than 0.7%, less than 0.6%, less than 0.5%, less than 0.4%, less than 0.3%, less than 0.2%, less than 0.1% methyl ester glycosides comprising a glycosidic bond linked to a volatile functional group. Methyl ester glycosides that contain glycosidic bonds linked to volatile functional groups are less than 2%, less than 1.9%, less than 1.8%, less than 1.7%, less than 1.6%, less than 1.5%, less than 1.4%, less than 1.3%, less than 1.2%, less than 1.1%, less than 0.9%, less than 0.8%, less than 0.7%, less than 0.6%, less than 0.5%, less than 0.4%, less than 0.3%, less than 0.2%, less than 0.1% can be determined using any suitable technique, for example, via 1H NMR. The % of methyl ester glycosides may refer to the weight % of the composition. The % of methyl ester glycosides may refer to the relative % to the amount of glycosides.

[0047] The composition of the present invention or the glycosides described herein can be used as a probe in the detection of cancer. Such compositions are suitable for administration by any reasonable route, for example, any parenteral or enteral route. For example, any convenient route includes, but is not limited to, oral, topical, parenteral, sublingual, rectal, vaginal, ocular, intranasal, pulmonary, intradermal, intravitreal, intramuscular, intraperitoneal, intravenous, subcutaneous, intracerebral, transdermal, transmucosal, or by inhalation. Parenteral administration includes, for example, intravenous, intramuscular, intraarterial, intraperitoneal, intranasal, intrarectal, intravesical, intradermal, topical, or subcutaneous administration. In one embodiment, the composition can be formulated for administration by oral route or by inhalation. In one embodiment, the composition can be formulated for administration by intravenous route.

[0048] The composition may include a pharma- ceutically acceptable carrier or vehicle. This may be particulate, for example, so that the composition is in the form of a tablet or powder. The term "carrier" refers to a diluent, adjuvant, or excipient for administration of the glycoside. Such pharmaceutical carriers include 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. Carriers include saline, gum acacia, gelatin, gelatinized starch, talc, keratin, colloidal silica, urea, and the like. In addition, auxiliary agents, stabilizers, thickeners, lubricants, colorants, and the like may be used. Saline, aqueous solutions of dextrose, and glycerol may 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, etc. The composition can optionally contain small amounts of wetting or emulsifying agents, or pH buffering agents. The composition can also be in the form of a liquid, such as a solution, emulsion, or suspension. Liquids can be useful for delivery by injection, infusion (e.g., intravenous infusion), or subcutaneous administration. As a solid composition for oral administration, the composition can be formulated into the form of a powder, granules, compressed tablets, pills, capsules, chewing gum, wafers, etc.

[0049] The composition may be in the form of one or more dosage units. A variety of oral dosage forms may be used, including solids such as tablets, capsules, liquids, granules, powders, etc. Tablets may be compressed, powder tablets, enteric coated, sugar coated, film coated, or multiple compressed, and contain suitable binders, lubricants, diluents, disintegrants, colorants, flavorings, flow inducers, and melting agents. Liquid oral dosage forms include aqueous solutions, emulsions, suspensions, solutions and / or suspensions reconstituted from non-effervescent granules, and effervescent preparations reconstituted from effervescent granules, and contain suitable solvents, preservatives, emulsifiers, suspending agents, diluents, sweeteners, melting agents, colorants, and flavorings.

[0050] The composition may be in the form of a liquid, for example, a solution, an emulsion or a suspension. Whether in a solution, a suspension or other similar form, the liquid composition may also contain one or more of the following: water, saline, preferably saline, Ringer's solution, a sterile diluent such as isotonic sodium chloride, fixed oils such as synthetic mono- or diglycerides, polyethylene glycols, glycerin or other solvents; antibacterial agents such as benzyl alcohol or methylparaben; and a tonicity adjuster such as sodium chloride or glucose. The composition may be enclosed in an ampoule, a disposable syringe or a multiple dose vial made of glass, plastic or other material.

[0051] The composition may be formulated for inhalation, so that the composition may be formulated with suitable particle size and aerodynamic diameter. The glycoside probe of the present invention usually has a particle size small enough to allow it to be formulated for inhalation. In other words, the small particle size of the glycoside probe of the present invention means that it may be suitable for administration by inhalation. The composition may be optimized for aerosolization. Techniques such as spray drying and spray freeze drying may be used to generate a composition with sufficient powder dispersibility and low aggregation to be used as an inhalation composition.

[0052] The method may be performed by the subject or by a third party, such as a clinician. In one embodiment, the method may be performed by the subject (or a third party) in a non-clinical environment. By "non-clinical environment" is meant that a person who is not a trained medical professional performs some or all of the steps of the method. For example, one or more steps of the method may be performed by the subject at home. For example, the steps of administering the composition and obtaining the biological sample may be performed by the subject (or a third party) in a non-clinical environment, such as at home. For example, the biological sample may be sent for external analysis (such as by a trained medical professional, as appropriate) in a clinical environment. In such an embodiment, the composition may be formulated for inhalation.

[0053] In one embodiment, the method may be performed by a clinician (or other appropriately trained medical professional). In such an embodiment, the composition may be formulated for intravenous administration.

[0054] One aspect of the invention relates to a kit comprising a glycoside obtainable by the method of the invention or a composition as described herein, and optionally instructions for use.

[0055] In one embodiment, the kit comprises an apparatus for capturing a biological matrix sample from a subject. The biological matrix may then be analyzed for metabolites of the glycosides obtained by the method of the invention. The apparatus for capturing the biological matrix sample may be an apparatus for capturing a blood sample, a urine sample or a breath sample. Suitable apparatus are known in the art.

[0056] In one embodiment, the kit further comprises a device for capturing a breath sample from the patient. The device for capturing breath may be as described in WO2017 / 187120 or WO2017 / 187141. The device of WO2017 / 187120 comprises a mask portion that, in use, is placed over the subject's mouth and nose to capture the breath exhaled by the subject. The breath sample is fed into a tube containing an adsorbent material, which adsorbs compounds of interest. After a sufficient sample is obtained, the adsorbent tube is removed from the sampling device, and the adsorbed compounds are desorbed (usually by heating) and subjected to analysis to identify the presence and / or amount of specific compounds or other substances of interest. A preferred analytical technique is field asymmetric ion mobility spectrometry (abbreviated as "FAIMS"). An improved method of the method described in WO2017 / 187120 is disclosed in WO2017 / 187141. This document teaches the use of a breath sampling device substantially of the type described in WO2017 / 187120, but in such a way as to selectively sample desired portions of a subject's exhaled breath, the rationale being that particular biomarkers or other analytes of interest are relatively concentrated in one or more fractions of the exhaled breath which are themselves relatively concentrated in air exhaled from different parts of the subject's body (e.g. the nostrils, pharynx, trachea, bronchioles, alveoli, etc.).

[0057] As shown herein, standard conditions used to hydrolyze methyl ester glycosides to glycosides result in end products that have a tendency to reform methyl ester glycosides, and such products have been shown to have higher cell permeability. Standard conditions include hydrolysis in methanol in the presence of a base. In contrast, the inventors have identified conditions under which methyl ester glycosides are hydrolyzed to glycosides that reduce the tendency of the glycosides to reform methyl ester glycosides. The end products obtained by hydrolyzing methyl ester glycosides in water in the presence of a base have reduced cell permeability compared to glycosides obtained under standard conditions.

[0058] Thus, in one aspect, the present invention relates to a method for reducing the cell permeability of a glycoside-based exogenous volatile organic compound (EVOC) probe, comprising: applying conditions that promote conversion of methyl ester glycosides to glycosides, Here, conditions that promote the formation of glycosides include hydrolysis of methyl ester glycosides in water in the presence of a base.

[0059] The term "exogenous volatile organic compounds" refers to compounds that are administered to a subject via various routes, metabolized and distributed within the body, and excreted via breathing. In addition, metabolism of EVOCs by cancer-specific enzymes can produce volatile compounds that are also detected in breath.

[0060] In one aspect, the present invention relates to the use of a glycoside obtained by the method of the present invention or a composition as described herein in a diagnostic respiratory test. In one embodiment, the present invention relates to the use of a glycoside obtained by the method of the present invention or a composition as described herein in a respiratory test that can detect or prognose a disease state in a subject. In one embodiment, the present invention relates to a glycoside obtained by the method of the present invention or a composition as described herein for use in a respiratory test that can detect or prognose a disease state in a subject. The disease state may be selected from cancer, preferably lung cancer, or lung inflammation.

[0061] The glycoside or a composition comprising the glycoside can be administered at a concentration of 0.05 to 10 mg / kg, for example 0.5 to 10 mg / kg, for example 0.5 to 5 mg / kg, for example 1 to 3 mg / kg, for example about 2 mg / kg. In one embodiment, the administration is intravenous administration, inhalation or oral administration. In one embodiment, the administration is intravenous administration, and is 0.5 to 10 mg / kg, for example 0.5 to 5 mg / kg, for example 1 to 3 mg / kg, for example about 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 mg / kg. In one embodiment, the dosage is 2 mg / kg. In one embodiment, the administration is inhalation or oral administration, and is 0.05 to 10 mg / kg, for example 0.5 to 5 mg / kg, for example 1 to 3 mg / kg, for example about 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 mg / kg. In one embodiment, the dosage is 2 mg / kg.

[0062] The breath test involves administering to a subject a glycoside obtained by the method of the invention or a composition described herein, where the administered glycoside is biotransformed by disease-specific enzymes. The biotransformation of the glycoside involves cleavage of the glycosidic bond and thus release of a volatile compound, which can be detected in the breath of the subject.

[0063] As used herein, "subject" refers to a test subject, e.g., a mammalian subject, preferably a human. In one embodiment, a breath sample is obtained for the purpose of diagnosing or screening for the presence or absence of disease. The subject may be male or female. The subject may be an infant, toddler, child, young adult, adult, or geriatric. In one embodiment, the subject is a cancer patient, e.g., a lung cancer patient, e.g., a stage IIA lung cancer patient.

[0064] The disease-specific enzyme may be a cancer-specific enzyme. As used herein, a "cancer-specific enzyme" is an enzyme selected from one or more of the following: the enzyme is absent in cancer tissue but present in non-cancerous tissue; the enzyme is present in cancer tissue but absent in non-cancerous tissue; the enzyme is differentially expressed in cancer tissue compared to non-cancerous tissue; or the enzyme is differentially active in cancer tissue compared to non-cancerous tissue. For example, the enzyme may be expressed at a higher level or at a lower level in cancer tissue compared to expression in non-cancerous tissue. Expression can be measured by techniques known in the art, such as, for example, quantifying mRNA or measuring cDNA. Non-cancerous tissue refers to, for example, healthy tissue. The tissue may be derived from a specific organ, such as, for example, lung, colon, breast, prostate, etc.

[0065] For example, an enzyme may be localized in a different location in cancer tissue than in non-cancerous tissue, e.g., an enzyme is present in the extracellular space of cancer tissue, whereas in non-cancerous tissue, the enzyme is not present in the extracellular space. Due to a combination of tumor necrosis and the release of lysosomal enzymes from macrophages and neutrophils, certain enzymes are present in the extracellular space of the tumor microenvironment. In one embodiment, the exogenous substrate is a substrate for an enzyme present in the extracellular space of a solid tumor. The enzyme may be an extracellular lysosomal enzyme. An "extracellular lysosomal enzyme" refers to an enzyme released from lysosomes into the extracellular space. Specific examples of enzymes that may be released from lysosomes into the extracellular space include β-glucuronidase, β-galactosidase, and the like. In particular, the presence of β-glucuronidase in the extracellular space is an indication of cancer. In one embodiment, the cancer specific enzyme is selected from β-glucuronidase, β-galactosidase, α-L-arabinofuranosidase, N-acetyl-β-D-galactosaminidase, N-acetyl-β-D-glucosaminidase, hexosaminidase, α-L-fucosidase, α-galactosidase, α-glucosidase, β-glucosidase, α-L-iduronidase, α-mannosidase, β-mannosidase, lipase, phosphatase, and sulfatase. In a preferred embodiment, the cancer specific enzyme is selected from β-glucuronidase, β-galactosidase, α-L-arabinofuranosidase, N-acetyl-β-D-galactosaminidase, N-acetyl-β-D-glucosaminidase, hexosaminidase, α-L-fucosidase, α-galactosidase, α-glucosidase, β-glucosidase, α-L-iduronidase, α-mannosidase, β-mannosidase.

[0066] In one embodiment, the respiratory test may be a test to detect cancer, optionally lung cancer.

[0067] In one aspect, the present invention relates to a method for the detection or prognosis of cancer, comprising a step of assessing the activity of a cancer-specific enzyme by measuring the concentration of an exogenous substrate for said enzyme and / or the concentration of a metabolite of said substrate in a biological matrix of a subject, wherein said exogenous substrate is a glycoside obtainable according to the invention or a composition according to the invention.

[0068] In one aspect, the invention relates to a method for the detection or prognosis of cancer, comprising a step of assessing the activity of a cancer-specific enzyme by measuring the concentration of an exogenous substrate for said enzyme and / or the concentration of a metabolite of said substrate in a biological matrix of a subject, wherein said exogenous substrate is a glycoside, said glycoside being administered at a concentration of 0.05-10 mg / kg, and wherein the concentration of the metabolite is measured up to 300 minutes after administration of the glycoside.

[0069] The glycoside may be administered at a concentration of 0.5-5 mg / kg, for example 1-3 mg / kg, about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mg / kg. In one embodiment, the dosage is 2 mg / kg. In one embodiment, the administration is intravenous, inhaled, or oral.

[0070] In one embodiment, the administration is intravenous and is 0.5 to 10 mg / kg, for example 0.5 to 5 mg / kg, for example 1 to 3 mg / kg, for example about 2 mg / kg. In one embodiment, the administration is by inhalation or oral administration and is 0.05 to 10 mg / kg, for example 0.5 to 5 mg / kg, for example 1 to 3 mg / kg, for example about 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 mg / kg. In one embodiment, the dosage is 2 mg / kg.

[0071] In one embodiment, the metabolite concentration is measured 10 to 30 minutes, 10 to 120 minutes, 10 to 180 minutes, 30 to 120 minutes, or 30 to 180 minutes after administration of the glycoside. In one embodiment, the metabolite concentration is measured 5 minutes, 10 minutes, 20 minutes, 30 minutes, 40 minutes, 50 minutes, 60 minutes, 70 minutes, 80 minutes, 90 minutes, 100 minutes, 110 minutes, 120 minutes, 130 minutes, 140 minutes, 150 minutes, 160 minutes, 170 minutes, 180 minutes, 190 minutes, 200 minutes, 210 minutes, 220 minutes, 230 minutes, 240 minutes, 250 minutes, 260 minutes, 270 minutes, 280 minutes, 29 minutes, or 300 minutes after administration of the glycoside.

[0072] In one embodiment, the method comprises a step of correlating the result with β-glucoronidase expression in the sample, e.g. as measured by immunohistochemistry. The sample may be derived from the subject tested or from a subject forming a reference sample, e.g. an individual diagnosed with cancer or a healthy subject.

[0073] The glycoside may be provided as a composition comprising a glycoside comprising a glycosidic bond linked to a volatile functional group, wherein the composition comprises less than 1% of a methyl ester glycoside comprising a glycosidic bond linked to a volatile functional group, as described herein. In one embodiment, the glycoside is obtained by a method for synthesizing a glycoside, the method comprising: The method includes hydrolyzing a methyl ester glycoside in water in the presence of a base to form a glycoside, Here, the tendency of the methyl ester glycosides to reform is reduced as described herein.

[0074] In one aspect, the present invention relates to a composition comprising a glycoside comprising a glycosidic bond linked to a volatile functional group, wherein the composition comprises less than 1% of a methyl ester glycoside comprising a glycosidic bond linked to a volatile functional group, for use in a method for the detection or prognosis of cancer, comprising measuring the concentration of an exogenous substrate for a cancer-specific enzyme in a biological matrix of a subject and / or assessing the activity of the enzyme by measuring the concentration of a metabolite of the substrate, wherein the exogenous substrate is a composition comprising a glycoside comprising a glycosidic bond linked to a volatile functional group, wherein the composition comprises less than 1% of a methyl ester glycoside comprising a glycosidic bond linked to a volatile functional group. In one embodiment, the glycoside may be administered at a concentration of 0.05-10 mg / kg, 0.5-10 mg / kg, 0.5-5 mg / kg, 1-3 mg / kg, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mg / kg. In one embodiment, the dosage is 2 mg / kg.

[0075] In one embodiment, the administration is intravenous administration, and is 0.5 to 10 mg / kg, for example 0.5 to 5 mg / kg, for example 1 to 3 mg / kg, for example about 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 mg / kg. In one embodiment, the dosage is 2 mg / kg. In one embodiment, the administration is inhalation administration, and is 0.05 to 10 mg / kg, for example 0.5 to 5 mg / kg, for example 1 to 3 mg / kg, for example about 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 mg / kg. In one embodiment, the dosage is 2 mg / kg. In one embodiment, the concentration of the metabolite is measured 10 to 30 minutes, 10 to 120 minutes or 10 to 180 minutes after administration of the glycoside.

[0076] In one embodiment, the metabolite concentration is measured 10 to 30 minutes, 10 to 120 minutes, 10 to 180 minutes, 30 to 120 minutes, or 30 to 180 minutes after administration of the glycoside. In one embodiment, the metabolite concentration is measured 5 minutes, 10 minutes, 20 minutes, 30 minutes, 40 minutes, 50 minutes, 60 minutes, 70 minutes, 80 minutes, 90 minutes, 100 minutes, 110 minutes, 120 minutes, 130 minutes, 140 minutes, 150 minutes, 160 minutes, 170 minutes, 180 minutes, 190 minutes, 200 minutes, 210 minutes, 220 minutes, 230 minutes, 240 minutes, 250 minutes, 260 minutes, 270 minutes, 280 minutes, 29 minutes, or 300 minutes after administration of the glycoside.

[0077] The disease-specific enzyme may be an inflammation-specific enzyme. In a particular embodiment, the enzyme may be specific for pulmonary inflammation. The respiratory test may thereby be used for the detection or prognosis of pulmonary inflammation. In one aspect, the present invention relates to a method for the detection or prognosis of pulmonary inflammation, comprising a step of assessing the activity of said enzyme by measuring the concentration of an exogenous substrate for said pulmonary inflammation-specific enzyme and / or the concentration of a metabolite of said substrate in a biological matrix of a subject, wherein said exogenous substrate is a glycoside obtained according to the present invention or a composition according to the present invention.

[0078] In one aspect, the present invention relates to a composition comprising a glycoside comprising a glycosidic bond linked to a volatile functional group, wherein the composition comprises less than 1% methyl ester glycoside comprising a glycosidic bond linked to a volatile functional group, for use in a method for detection or prognosis of pulmonary inflammation, the method comprising a step of assessing activity of a pulmonary inflammation-specific enzyme by measuring a concentration of an exogenous substrate for said enzyme and / or measuring a concentration of a metabolite of said substrate in a biological matrix of a subject, wherein said exogenous substrate comprises a glycoside comprising a glycosidic bond linked to a volatile functional group, wherein the composition comprises less than 1% methyl ester glycoside comprising a glycosidic bond linked to a volatile functional group.

[0079] The term "pulmonary inflammation" may refer to chronic or acute lung inflammation, which may be caused by an infectious disease, such as a respiratory infection, e.g., influenza, or a respiratory disease, such as asthma, bronchitis, chronic obstructive pulmonary disease, or lung cancer.

[0080] The term biological matrix refers to a biological sample that contains the analyte of interest. In one embodiment, the biological matrix is ​​selected from blood, urine, and exhaled air. In one embodiment, the biological matrix is ​​exhaled air. When the biological matrix is ​​blood, it may refer to plasma or blood cells. The sample of the biological matrix may be obtained by any reasonable means.

[0081] As mentioned above, glycosidic bonds can be cleaved, thereby releasing volatile functional groups from the glycoside. Thus, the metabolic products of the substrate (glycoside or composition of the present invention) can be volatile functional groups. The concentration of the substrate and / or metabolites can be measured using methods known in the art. Concentration as used herein means the content or mass of the substrate and / or metabolite in the exhaled breath, for example expressed in grams per liter (g / l). In one embodiment, the concentration is measured over time, for example by measuring the kinetics of clearance. For example, the concentration is measured by evaluating the kinetic profile of the clearance of the substrate from a biological matrix sample, which profile is then used as a readout. Additionally or alternatively, the secretion of metabolites that can be derived from the substrate can be measured over time. For example, the clearance of the substrate from a biological matrix sample and the secretion of metabolites can both be measured at the same time or at different times in the same biological matrix sample.

[0082] The method may include a step of providing an exogenous substrate, i.e., a glycoside or composition according to the invention, to the subject. Thus, in one embodiment, the method includes a step of administering the substrate to the subject. Administration may be by any convenient route, including but not limited to oral, topical, parenteral, sublingual, rectal, vaginal, ocular, intranasal, pulmonary, intradermal, intravitreal, intramuscular, intraperitoneal, intravenous, subcutaneous, intracerebral, transdermal, transmucosal, by inhalation, or locally, in particular to the ear, nose, eye, or skin, or by inhalation. Parenteral administration may include, for example, intravenous, intramuscular, intraarterial, intraperitoneal, intranasal, intrarectal, intravesical, intradermal, topical, or subcutaneous administration. Preferably, the composition is administered orally. The skilled artisan will know that the route of administration depends on the enzyme and the disease being tested. For example, if the target enzyme is present in the gastrointestinal tract, oral administration is preferred, whereas in the case of hepatic expression, either oral or intravenous administration is a viable option.

[0083] In one embodiment, the concentration or amount of a substrate and / or its metabolites may be determined absolutely or relatively in multiple biological matrix samples, for example when determining the concentration in exhaled breath samples, in a first breath sample (collected at a first time period) and a second breath sample (collected at a later second time period), thus allowing analysis of the kinetics or rate of change of concentration over time.

[0084] Breath samples are obtained by collecting breath from a subject, for example by asking the subject to exhale into a gas sampling container, such as a bag, bottle, or any other suitable gas sampling product. Preferably, the gas sampling container is resistant to gas transmission into and out of the bag and / or is chemically inert, thereby ensuring the integrity of the sample. Breath can also be collected using a breath collection device. Preferably, collection of the breath sample is performed in a minimally or non-invasive manner.

[0085] Determining the amount of substrate (i.e., glycoside or composition according to the present invention) and / or metabolite in a breath sample from a subject can be performed by using at least one technique, including, but not limited to, gas chromatography (GC), gas chromatography mass spectrometry (GC / MS), liquid chromatography mass spectrometry (LC / MS), ion mobility spectrometry / mass spectrometry (IMS / MS), proton transfer reaction mass spectrometry (PTR-MS), an electronic nose device, a quartz crystal microbalance, or a chemically sensitive sensor.

[0086] The amount of substrate (i.e., glycoside or composition according to the invention) and / or metabolite in a sample of breath from a subject can be determined using thermal desorption-gas chromatography time-of-flight mass spectrometry (GC-Tof-MS). In certain embodiments, the breath of a subject is collected in an inert bag, the contents of the bag are transported under standardized conditions to a desorption tube, and the contents of the tube are thermally desorbed to analyze the VOCs and separate them by capillary gas chromatography. Volatile organic peaks are then detected by MS and identified using a library, such as the National Institute of Standards and Technology. Thermal desorption can be performed at the GC inlet, for example at a temperature of about 200-350°C. In all chromatography, separation occurs when the sample mixture is introduced (injected) into the mobile phase. In gas chromatography (GC), an inert gas, such as helium, is typically used as the mobile phase. GC / MS allows for the separation, identification, and / or quantification of individual components from a biological sample. MS techniques that can be used in the present invention include, but are not limited to, electron ionization, electrospray ionization, glow discharge, field desorption (FD), fast atom bombardment (FAB), thermospray, desorption / ionization on silicon (DIOS), direct analysis in real time (DART), atmospheric pressure chemical ionization (APCI), secondary ion mass spectrometry (SIMS), spark ionization, thermal ionization (TIMS), etc. Matrix assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF-MS) is one example of a mass spectrometry technique that can be used to determine one or more VOCs from a subject's breath sample.

[0087] In one embodiment, the method comprises collecting different selected breath samples or fractions thereof with a single breath sample capture device, the method comprising: (a) collecting a first breath sample by contacting the sample with a capture device comprising an adsorbent material; (b) collecting a second breath sample by contacting the second sample with the capture device, wherein the first and second breath samples are spatially separated and captured on the capture device; Includes.

[0088] In some embodiments, the capture device includes a sorbent material in the form of a porous polymeric resin. Suitable sorbent materials include Tenax® resin and Carbograph® material. Tenax® is a porous polymeric resin based on 2,6-diphenyl-p-propylene oxide monomer. Carbograph® material is graphitized carbon black. In one embodiment, the material is Tenax GR, which contains a mixture of Tenax® TA and 30% graphite. One type of Carbograph® sorbent is Carbograph 5TD. In one embodiment, the capture device includes both Tenax GR and Carbograph 5TD. The capture device is conveniently a sorbent tube. It is a hollow metal cylinder, usually of standard dimensions (3 1 / 2 inches long, 1 / 4 inch inside diameter), filled with a suitable sorbent material.

[0089] In one embodiment, the method of the invention further comprises defining target values ​​for one or more substrate and / or metabolite concentrations.

[0090] In one embodiment, the method further comprises comparing the subject value to one or more reference values. In one embodiment, the reference values ​​are from healthy subjects. In another embodiment, the reference values ​​are from subjects diagnosed with a disease.

[0091] In one embodiment, the reference values ​​are healthy subject values ​​corresponding to values ​​calculated from healthy subjects. In one embodiment, the presence of one or more subject values ​​in an amount greater than each of the ranges of healthy subject values ​​indicates a substantial likelihood of a disease state in the test subject.

[0092] In one embodiment, if the appropriate standard indicates that the subject does not have a disease, a detectable difference (e.g., a statistically significant difference) between the value determined from the subject in need of disease characterization or diagnosis and the appropriate standard may be indicative of disease in the subject. In one embodiment, if the appropriate standard indicates that the subject does not have a disease, a lack of a detectable difference (e.g., a statistically significant difference) between the value determined from the subject in need of disease characterization or diagnosis and the appropriate standard may be indicative of disease in the subject.

[0093] Thus, in one embodiment, the method comprises detecting the concentration of a substrate and / or metabolite in a biological matrix sample from a subject, and diagnosing the subject as having a possible disease state if the level of one or more of the substrates and / or metabolites differs from a healthy subject value.

[0094] The method of the invention may further comprise the step of selecting a treatment for said disease.The method may further comprise the step of administering said treatment to said subject.

[0095] Unless otherwise defined herein, scientific and technical terms used in connection with this disclosure shall have the meanings commonly understood by those skilled in the art. The above disclosure provides a general description of the subject matter encompassed within the scope of the present invention, including how to make and use the invention, and the best mode thereof, but the following examples are provided to further enable those skilled in the art to practice the invention and to provide a complete written description. However, those skilled in the art will understand that the specific content of these examples should not be read as limiting the invention, the scope of which should be understood from the claims appended to this disclosure and their equivalents. Various further aspects and embodiments of the present invention will be apparent to those skilled in the art in light of the present disclosure.

[0096] All documents mentioned herein are incorporated herein by reference in their entirety.

[0097] As used herein, "and / or" is considered to be a specific disclosure of each of the two specified features or components, with or without the other. For example, "A and / or B" is considered to be a specific disclosure of (i) A, (ii) B, and (iii) each of A and B, as if each were individually described herein. Unless the context dictates otherwise, the descriptions and definitions of features set forth above are not limited to any particular aspect or embodiment of the present invention, but apply equally to all aspects and embodiments described. EXAMPLES

[0098] The invention is further illustrated in the following non-limiting examples.

[0099] Example 1 Dose ranges for D5-ethyl-βD-glucuronide and D5-ethyl-βD-glucuronide-methyl ester in blood experimental protocols Objectives - The following experiments were performed to compare the levels of D5-ethanol produced by blood cells from both D5-ethyl-βD-glucuronide and D5-ethyl-βD-glucuronide-methyl ester impurities; to understand how the probe works in human blood; to determine background cleavage in blood at different concentrations of the probe (D5-ethyl-βD-glucuronide) and the methyl ester (D5-ethyl-βD-glucuronide-methyl ester). The cleaved D5-ethanol was extracted from the headspace of blood and measured with the D5-ethanol assay. Method-6.1 Dilute D5-Ethyl-βD-glucuronide to 1ug / ul in PBS. · Dilute this 10μl with 90μl of PBS to 100ng / ul and use for small doses. · Blood drawn on the day will be sent to OML after screening. Aliquot 600 μl of blood into ITEX headspace vials: 10 for the probe samples and 10 for the methyl ester samples. Add D5-ethyl-βD-glucuronide (Table 1) and D5-ethyl-βD-glucuronide methyl ester (Table 2) to each vial at the concentrations indicated. Incubate all samples in a 37°C water bath for 2 hours. After 2 hours, add 100 μM D-saccharic acid-1,4-lactone via syringe through the septum of each ITEX vial, mix gently to inhibit β-glucuronidase activity, and place on ice (4°C). Place the sample in the ITEX autosampler and measure D5-ethanol using the D5-ethanol analysis method.

[0100] [Table 1]

[0101] [Table 2]

[0102] Results - β-glucuronidase is expected to be present only in blood cells. D5-ethyl-βD-glucuronide (D5-EG) is not cell permeable and therefore cannot enter cells to be cleaved by β-glucuronidase to produce D5-ethanol. Therefore, the concentration of D5-ethanol in blood is expected to be low when incubated with D5-ethyl-βD-glucuronide.

[0103] D5-ethyl-βD-glucuronide-methyl ester (D5-EG-ME) is expected to be highly cell permeable, and if so, it would enter cells and be cleaved by intracellular β-glucuronidase, resulting in increased production of D5-ethanol.

[0104] Indeed, when blood cells were incubated with D5-EG, only small amounts of D5-ethanol were produced at the highest concentration tested (Figure 2). In contrast, when blood cells were incubated with D5-EG-ME, much higher levels of D5-ethanol were produced at the highest and second highest concentrations tested (Figure 2).

[0105] Therefore, the presence of the methyl ester would result in the production of D5-ethanol by normal cells, which is not a suitable probe for detecting lung cancer.

[0106] Example 2 Identifying conditions that reduce methyl ester content. Conditions for converting D5-EG-ME to D5-EG include hydrolysis of D5-EG-ME in the presence of base in methanol as shown below:

[0107] [ka]

[0108] The reaction was monitored via proton NMR to ensure that no residual D5-EG-ME remained in the product (D5-EG). After cation exchange and a series of co-evaporations of water and methanol, D5-EG was isolated as a white solid. It was found that under these method conditions, the reverse reaction was possible and the methyl ester could be reformed. Thus, the presence of the methyl ester as an impurity in D5-EG would result in higher release levels of D5 ethanol when used as a probe, due to the high cell permeability of D5-EG-ME. Therefore, when this product was used as an EVOC probe for lung cancer detection, it would result in higher background levels of D5 ethanol, which could cause false positives.

[0109] 1HNMR was used to characterize the product after hydrolysis, resin exchange, and co-evaporation of water and methanol. The results showed that D5-EG-ME was present in the final product at a level of 1%. The NMR spectrum in Figure 3 shows a singlet peak at 3.7, indicative of the methyl ester.

[0110] Therefore, conditions were sought to identify which would suppress the modification of D5-EG-ME, and it was surprisingly found that hydrolysis of D5-EG-ME in the presence of base in water under ambient conditions resulted in the following:

[0111] [ka]

[0112] The reaction was monitored by proton NMR to ensure that no residual INT_005_0012 remained in the product. After resin exchange and evaporation of the water solvent, the product is isolated as a white solid. No methanol is present under the process conditions, and the methyl ester does not reform during product isolation. This allows the methyl ester impurity to be controlled to a level of less than 1% by proton NMR. 1 The H NMR spectrum shows the absence of a singlet peak at 3.7 indicative of a methyl ester.

[0113] Example 3 Large scale hydrolysis procedure D5-EG-ME was suspended in water (22 mL) and stirred at room temperature for 5 min to form a clear pale yellow solution. NaOH in water (22 mL) was then added dropwise to form a deep yellow solution. The reaction mixture was stirred for about 3 hours. The reaction mixture was analyzed by 1H NMR. 1H NMR: 50 μL of sample was taken and added to water (100 μL) along with approximately 25 mg of Dowex. The sample was allowed to sit for 15 minutes and then concentrated. There is no evidence of D5-EG-ME as there is no methyl peak at approximately 3.73 ppm. Prewashed Dowex® 50WX8 50-100 (H) (18 g) was added to the reaction mixture. Stirred at room temperature for 2.5 hours and filtered. Filtrate pH: 3.8, yellow solution. Dowex was washed with water (2 x 30 mL). Pre-washed Dowex® 50WX8 50-100(H) (18 g) was added to the filtrate. Stirred at room temperature for 90 minutes and filtered. Filtrate pH: 2.2 (pH meter pH still dropping) pale yellow solution. The Dowex was washed with water (2×30 mL). The filtrate was concentrated to dryness (up to 55°C, up to 2 hours) to give a cream solid. Yield = 7.94g (95%)

[0114] Example 4 Preparation of D5-ethyl-βD-glucuronide-methyl ester The starting material D5-ethyl-βD-glucuronide-methyl ester was prepared according to the following method.

[0115] Step 1 - D6 ethanol is glucuronidated with the bromo sugar 1 using silver carbonate to give the glucopyranoside 2.

[0116] [ka]

[0117] Step 2 - Deacetylation of glucopyranoside 2 using sodium methoxide gives D5-ethyl-βD-glucuronide-methyl ester 3.

[0118] [ka]

[0119] Once the methyl ester has been prepared, hydrolysis of the methyl ester can be carried out in accordance with the present invention.

[0120] Example 5 Probe administration D5-ethyl-βD-glucuronide was administered intravenously at a concentration of 2 mg / kg to healthy subjects and lung cancer patients.

[0121] Metabolite concentrations were then measured in the breath samples. Breath samples were collected using a ReCIVA® breath sampler at the designated times after probe administration. After breath collection, the adsorbent tubes were removed from the ReCIVA®, capped, and sent to Owlstone Medical for analysis. At Owlstone Medical, the adsorbent tubes were analyzed for D5-ethanol using TD-GC-MS.

[0122] After probe administration, no D5-ethanol was detected in healthy controls. A D5-ethanol signal was detected in lung cancer patients (stage IIA). As shown in Figure 6, metabolites were detected as early as 10 min after probe administration.

[0123] Example 6 Using immunohistochemical staining, the expression of β-glucuronidase was measured in human lung cancer tissues and normal lung tissues, and the results are shown in Figure 7. (References) TIFF2024542305000010.tif174152

Claims

1. 1. A method for synthesizing a glycoside, comprising: hydrolyzing the methyl ester glycoside in water in the presence of a base to form a glycoside; wherein the tendency to reform the methyl ester glycoside is reduced.

2. 2. The method of claim 1, wherein the base is NaOH, LiOH, or KOH.

3. 2. The method of claim 1, wherein the glycoside is selected from glucuronide, iduronide, mannoside, glucosamide, galactosamide, glucoside, galactoside, rhamnoside, riboside, arabinoside, fructoside, xyloside, and fucoside.

4. 2. The method of claim 1, wherein the glycoside comprises a glycosidic bond linked to a volatile functional group.

5. 5. The method of claim 4, wherein the glycosidic bond is selected from O-, N-, S-, and C-glycosidic bonds.

6. 5. The method of claim 4, wherein the volatile functional group is labeled, and the label can be selected from 12C, 13C, 14C, 2H, 14N, or 18O.

7. 5. The method of claim 4, wherein the volatile functional group is selected from methyl, ethyl, propyl, isopropyl, butyl, methyl-D3, ethyl-D5, or propyl-D7.

8. further comprising an ion exchange step after the hydrolysis step; The ion exchange may be carried out using an acidic cation exchange resin; and / or and / or may further comprise a solvent evaporation step after the hydrolysis step. monitoring the hydrolysis of the methyl ester glycoside to form the glycoside using 1 H NMR; and / or after the hydrolysis step, the methyl ester glycosides may be present at less than 1% of the glycosides; and / or The hydrolysis may be carried out at a temperature of 18 to 30°C, and / or The hydrolysis may be carried out under ambient conditions at a temperature of 25°C (298.15K) and a pressure of 101.325kPa; and / or 2. The method of claim 1, wherein the methyl ester glycoside is D5-ethyl-βD-glucuronide-methyl ester, and the glycoside can be D5-ethyl-βD-glucuronide.

9. A glycoside obtained by the method according to any one of claims 1 to 8.

10. A composition comprising the glycoside of claim 9.

11. A composition comprising a glycoside comprising a glycosidic bond linked to a volatile functional group, the composition comprising less than 1 wt% of a methyl ester glycoside comprising a glycosidic bond linked to a volatile functional group.

12. Less than 1 wt. % of methyl ester glycosides containing glycosidic bonds linked to volatile functional groups 1 11. The composition of claim 10, as determined via H NMR.

13. A composition for use in the diagnosis or prognosis of cancer, comprising the glycoside of claim 9, the use comprising administering the glycoside to a subject; The glycoside may be administered at a concentration of 0.05 to 10 mg / kg; and / or The administration may be intravenous and at a concentration of 0.5-10 mg / kg, or the administration may be by inhalation or oral administration and at a concentration of 0.05-10 mg / kg; and / or A composition in which the concentration of a metabolite of a glycoside can be measured up to 300 minutes after administration of said glycoside.

14. 1. A method for reducing the cell permeability of a glycoside-based exogenous volatile organic compound (EVOC) probe, comprising: applying conditions that promote conversion of methyl ester glycosides to glycosides; wherein the conditions promoting the formation of the glycoside comprise hydrolysis of the methyl ester glycoside in the presence of a base in water.

15. 11. The composition of claim 10 for use in a breath test for the detection or prognosis of a disease state.

16. 16. The composition of claim 15, wherein the respiratory test is for the detection of cancer, and the cancer may be lung cancer.

17. A composition for use in a method for detecting or prognosing cancer, comprising the glycoside described in claim 9, said method comprising a step of assessing the activity of a cancer-specific enzyme by measuring the concentration of an exogenous substrate for the cancer-specific enzyme and / or measuring the concentration of a metabolite of said substrate in a biological matrix of a subject, wherein said exogenous substrate is said glycoside.

18. The composition of claim 17, wherein the method comprises administering the glycoside to a subject.

19. 1. A method for determining the activity of an enzyme, comprising: administering to a subject an exogenous substrate for the enzyme; measuring the concentration of a metabolite of said substrate in a biological sample obtained from said subject; Including, wherein the exogenous substrate is the glycoside described in claim 9.

20. A composition for use in a method for monitoring the progression of cancer in a subject diagnosed with cancer, comprising the glycoside of claim 9, The method comprises: administering an exogenous substrate to a subject; assessing the activity of the cancer-specific enzyme by measuring the concentration of a metabolite of the substrate in a biological sample obtained from the subject; Including, wherein the exogenous substrate is the glycoside.

21. A composition for use in a method for determining the efficacy of treatment of a subject diagnosed with cancer, comprising the glycoside of claim 9, The method comprises: administering an exogenous substrate to a subject; assessing the activity of the cancer-specific enzyme by measuring the concentration of a metabolite of the substrate in a biological sample obtained from the subject who has undergone anti-cancer treatment; Including, wherein the exogenous substrate is the glycoside.

22. 18. The composition of claim 17, wherein the glycoside is D5-ethyl-βD-glucuronide and the metabolite can be D5 ethanol.

23. the cancer-specific enzyme is β-glucuronidase, and / or the biological matrix is ​​selected from blood, urine or exhaled air, e.g. exhaled air; and / or The glycoside is administered at a concentration of 0.05 to 10 mg / kg, and / or measuring the concentration of metabolites up to 300 minutes after administration of the glycoside; and / or The administration is intravenous at a concentration of 0.5-10 mg / kg, or by inhalation or oral administration at a concentration of 0.05-10 mg / kg, and / or the concentration is between 1 mg / kg and 5 mg / kg, for example about 2 mg / kg; and / or 18. The composition of claim 17, wherein the concentration of the metabolite is measured 10 to 30 minutes after administration of the glycoside.

24. A composition described in any one of claims 13, 17, 18, 20, and 21, or a method described in claim 19, further comprising a step of correlating the result with the expression of β-glucoronidase.

25. The glycoside according to claim 9, may further include instructions for use and / or a device for capturing a biological matrix sample from a patient; The kit, wherein the device may be suitable for capturing blood, urine or breath samples.

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