WO2017077120A1

WO2017077120A1 - Diagnosis of diabetes

Info

Publication number: WO2017077120A1
Application number: PCT/EP2016/076867
Authority: WO
Inventors: Alexander Braun; Martin Elsner; Christian GRIEBLER
Original assignee: Alexander Braun; Martin Elsner; Griebler Christian
Priority date: 2015-11-06
Filing date: 2016-11-07
Publication date: 2017-05-11

Abstract

The present invention relates to a composition comprising or consisting of: (a) one or more inorganic substrates that carry a label and can be fed into the tricarboxylic acid cycle and/or the gluconeogenesis pathway selected from (i) heavy water; and (ii) a mono-carbon substrate; and (b) glucose, a source of glucose, and/or an inhibitor of gluconeogenesis.

Description

Diagnosis of diabetes

In this specification, a number of documents including patent applications and manufacturer's manuals is cited. The disclosure of these documents, while not considered relevant for the patentability of this invention, is herewith incorporated by reference in its entirety. More specifically, all referenced documents are incorporated by reference to the same extent as if each individual document was specifically and individually indicated to be incorporated by reference.

Diabetes mellitus is the most common metabolic disease in the world (Lowell and Shulman (2005), Science, 307, 384-387) and has reached epidemic proportions (Patti and Corvera (2010), Endocrine Reviews, 31 , 364-395). The estimated number of diagnosed cases has risen steadily over the past decade - from 151 to 285 million cases and is expected to rise to 552 million by 2030 (Whiting et al. (2011), Diabetes Research and Clinical Practice, 94, 311- 321 ). Suffering from Diabetes mellitus implies higher risk of heart disease, stroke, nephropathy, retinopathy, neuropathy, amputations, peripheral arterial disease and pregnancy-related complications and finally leads to death (Patti and Corvera (2010) loc. cit.; Madsen-Bouterse and Kowluru (2008), Reviews in Endocrine & Metabolic Disorders, 9, 315- 327). In particular, diabetes is considered responsible for a death every 10 seconds, with the mortality rate of approximately 4 million per year (Silink (2007), International Journal of Clinical Practice, 61, 5-8). Next to the decrease of the patient's life time and quality, the resulting health care costs sum up to ~ $ 170 billion alone in the US, making diabetes a major human health care issue (Patti and Corvera (2010) loc. cit.). Consequently, the United Nations have declared diabetes as a "global threat" in a recent landmark resolution (UN Resolution 61/225). For the first time, a non-infectious disease has been seen as challenging as infectious epidemics such as HIV/AIDS. In light of these findings, one of the most important public health goals lies in the development of a diagnostic tool that (i) allows diagnosing diabetes even before the onset of serious health complications (ii) and simultaneously has non-invasive sampling character. A non-invasive diagnosis procedure would reduce harm to test persons, in particular compared to the current "gold standard", i.e. invasive blood sampling, and could thus contribute to a higher acceptance among potentially affected people.

Based on etiology, diabetes can be divided into two main forms: type 1 Diabetes mellitus (T1 DM); and type 2 Diabetes mellitus (T2DM). T1 DM is an autoimmune disease resulting from destruction of the insulin secreting pancreatic β cells leading to a complete absence of insulin in the body (Sparre et al. (2005), Molecular & Cellular Proteomics, 4, 441-457), and accounts for 5%-10% of all diabetic cases. T2DM is characterized by relative insulin deficiency due to decreased insulin secretion by the β-cells and/or the decreased effect of insulin in the target tissues (i.e. liver, muscle and lipid tissue), also known as insulin resistance (Zimmet et al. (2001), Nature, 414, 782-787). T2DM is the most common form of diabetes and affects over 90% of diabetic patients (Chen et al. (2012), Protein & Cell, 3, 648-660). As insulin mediates the transport of glucose from blood plasma into cells, the absolute or relative lack of insulin leads to two distinguishing features of the two diabetes types: on the one hand fasting and postprandial hyperglycemia in blood plasma, but on the other hand glucose limitation in the cells (Basu et al. (2005), Diabetes, 54, 1942-1948; Chen et al. (2012) loc. cit.). The cellular response of hepatocytes is the de novo synthesis of glucose, i.e. gluconeogenesis, which further exaggerates the plasma glucose concentration (Magnusson et al. (1992), The Journal of clinical investigation, 90, 1323-7; Lowell and Shulman (2005), loc. cit.; Giaccari et al. (1998), Diabetologia, 41 , 307-314; Basu et al. (2005), loc. cit.).

State-of-the-art diabetes diagnostics therefore focus primarily on glucose plasma concentrations. A downside thereof, as noted above, is the need for invasive sample taking. Non-invasive glucose monitoring is described in So et al. (2012) (Medical Devices: Evidence and Research, 5, 45-52) and Zhang et al. (2015) (Sensing and Bio-Sensing Research, 4, 23- 29). The latter methods determine the overall concentration of glucose and do not permit to identify glucose of gluconeogenetic origin.

In diabetes, gluconeogenesis is up-regulated; see, e.g. Gastaldelli et al. (2000) (Diabetes, 49, 1367-1373). One way to prove gluconeogenic activity is the application of stable isotope tracers or radioactive tracers. Isotope tracers are compounds that are chemically and functionally identical to the naturally occurring compound of interest but are distinct in molecular mass and/or radioactivity to allow for their unique detection by mass spectrometry or other analytical methods, respectively. Mass spectrometry is applicable for any type of isotope labeled tracers including stable isotopes as well as radioactive isotopes. Radioactive tracers can furthermore be measured with a Geiger counter or liquid scintillation spectrometry. Further analytical methods exploiting isotope effects are well-known in the art and include ultracentrifugation, densimetry, chromatography, IR spectroscopy, emission spectroscopy, polarimetry, optical rotatory dispersion, circular dichroism, NMR, electron resonance spectroscopy and Mossbauer spectroscopy.

Following the fate of the tracer in the body through different metabolites yields information on the metabolic regulation (Aleman (2008), PhD thesis at the Massachusetts Institute Of Technology). For example, by subcutaneous infusion of [¹³C, ²H]-glycerol and mass spectrometry analysis of plasma glucose, gluconeogenic activity could be proven in diabetic rats (Aleman (2008), loc. cit.). However, because of its late entry point into the gluconeogenic pathway at the triose phosphate isomerase reaction, glycerol usually contributes only about 3% of the total glucose produced (Bortz et al. (1972), The Journal of clinical investigation, 51, 1537-46; Wolfe et al. (1987), The American journal of physiology, 252, E189-96). In contrast, pyruvate and molecules that can be directly transformed into pyruvate, like alanine from protein breakdown and lactate from cellular respiration, constitute >90% of gluconeogenic sources (Aleman (2008), loc. cit.). Simultaneous labeling of all pyruvate precursors, e.g. ¹³C lactate has been described (Marsch et al. (2015), Isotopes in Environmental and Health Studies, 51 , 11-23).

The technical problem underlying the present invention can be seen in the provision of alternative or improved means and methods for diagnosing diabetes and metabolic syndrome, especially non-invasive means and methods as well as of means and methods of personalized medicine in the field of diabetes.

The technical problem is solved by the subject-matter of the claims.

Accordingly, in a first aspect, the present invention relates to a composition comprising or consisting of: (a) one or more inorganic substrates that carry a label and can be fed into the tricarboxylic acid cycle and/or the gluconeogenesis pathway selected from (i) heavy water; and (ii) a mono-carbon substrate; and (b) glucose, a source of glucose, and/or an inhibitor of gluconeogenesis. Related thereto, the invention provides a composition comprising or consisting of: (a) one or more mono-carbon substrates that carry a label and can be fed into the tricarboxylic acid cycle and/or the gluconeogenesis pathway; and (b) glucose, a source of glucose, and/or an inhibitor of gluconeogenesis.

The term "substrate" designates compounds which can be processed by an enzyme as well as compounds which are precursors of compounds which can be processed by an enzyme.

A mono-carbon substrate is a compound comprising exactly one carbon atom. Preferably, the mono-carbon substrate according to the present invention can be directly processed by an enzyme. As described further below, a preferred enzyme is pyruvate carboxylase. More generally speaking, the mentioned enzyme is preferably an enzyme of the tricarboxylic acid cycle and/or the gluconeogenesis pathway. Being a substrate for one of these enzymes is a preferred means of rendering the substrate amenable to being fed into the tricarboxylic acid cycle or the gluconeogenesis pathway.

The mono-carbon substrate according to the invention carries a label. The label is preferably an isotope label. Also envisaged, albeit less preferred, are the labels which are of chemical nature and may be conjugated to the substrate. Preferred isotope labels are disclosed further below.

The term "heavy water" has its art-established meaning and relates to water, wherein at least one hydrogen atom is replaced with deuterium (²H). Also heavy water is a substrate in accordance with the invention. Preferred enzymes capable of processing it include phosphopyruvate hydratase, triose-phosphate isomerase and fumarate hydratase.

The terms "tricarboxylic acid cycle" and "gluconeogenesis" are well-established in the art. They designate central parts of the carbohydrate metabolism of the vast majority of living organisms. For example, they are found in organisms as different from each other as mammals, especially humans on the one hand and members of the genus Bacillus, especially Bacillus subtilis, on the other hand. Gluconeogenesis is the anabolic pathway corresponding to the catabolic glycolysis pathway, wherein starting materials of the one pathway are products of the other and vice versa.

Bacillus subtilis is in widespread use as a model of the central carbohydrate metabolism including gluconeogenesis; see, for example, Fischer and Sauer (2005) (Nature Genetics, 37, 636-640), Sauer and Eikmanns (2005) (FEMS Microbiology Reviews, 29, 765-794), Dauner et al. (2001 ) (Journal of Bacteriology, 183, 7308-7317), Chubukov et al. (2013) (Molecular Systems Biology 9, 709), Buescher et al. (2012) (Science, 335, 1099) and Kohlstedt et al. (2014) (Environmental Microbiology, 16, 1898-1917). As such, Bacillus subtilis is not only the major model organism of the prokaryotic kingdom beside E. coli, but is furthermore of utmost and general importance in the field of carbohydrate metabolism.

Bacillus subtilis has been used as a model system for poof of principle purposes, see Example 3. Further model systems in accordance with the present invention are mammals, especially mice. Mouse models of diabetes are established and known in the art; see, for example, King, British Journal of Pharmacology (2012), 166, 877-894 and Fujiwara et al., Metabolism (1995), 44, 486-490. Table 1 below presents rodent models of type 2 diabetes which are useful for the present invention.

Table 1

Summary of rodent models of type 2 diabetes

Induction mechanism Model Main features Possible uses

Obese models (monogenic) Lep mice Obesity-induced hyperglycaemia Treatments to improve insulin resistance

Lepr^{db d} mice Treatments to improve beta cell function ZDF Rats

Obese models (polygenic) KK mice Obesity-induced hyperglycaemia Treatments to improve insulin resistance

OLETF rat Treatments to improve beta cell function NZO mice Some models show diabetic complications TallyHo/Jng mice

NoncNZO10/LtJ mice

Induced obesity High fat feeding Obesity-induced hyperglycaemia Treatments to improve insulin resistance

(mice or rats)

Desert gerbil Treatments to improve beta cell function

Nile grass rat Treatments to prevent diet-induced obesity

Non-obese models GK rat Hyperglycemia induced by insufficient Treatments to improve beta cell function beta cell function/mass Treatments to improve beta cell survival

Genetically indcued models hIAPP mice Amyloid deposition in islets Treatments to prevent amyloid deposition of beta cell dysfunction Treatments to improve beta cell survival

AKITA mice Beta cell destruction due to ER stress Treatments to prevent ER stress

Treatments to improve beta cell survival

The common denominator of the compounds listed in item (b) of the first aspect of the invention is that they have an inhibitory effect on gluconeogenesis. To give an example, if there is an abundance of glucose, gluconeogenesis is down-regulated because there is no need for the production of further glucose. Alternatives to glucose which also have an inhibitory effect on gluconeogenesis are known to the skilled person. Preferred examples thereof are also given in item (b) and preferred embodiments thereof.

In a second aspect, the present invention provides a diagnostic composition comprising or consisting of: (a) one or more inorganic substrates that carry a label and can be fed into the tricarboxylic acid cycle and/or the gluconeogenesis pathway selected from (i) heavy water; and (ii) a mono-carbon substrate; and optionally (b) glucose, a source of glucose, and/or an inhibitor of gluconeogenesis.

Related thereto, the invention provides a diagnostic composition comprising or consisting of: (a) one or more mono-carbon substrates that carry a label and can be fed into the tricarboxylic acid cycle and/or the gluconeogenesis pathway; and optionally (b) glucose, a source of glucose, and/or an inhibitor of gluconeogenesis.

The composition according to the second aspect differs from the composition according to the first aspect in that (i) it is diagnostic in nature, and (ii) item (b) is optional.

As mentioned above, glucose as well as other compounds in accordance with item (b) cause down-regulation of gluconeogenesis. This applies to healthy persons, but not to diabetes patients or individuals having a prediabetic condition. Rather, characteristic for the latter groups of individuals is concomitant presence of hyperglycemia and active gluconeogenesis.

It has to be appreciated that the diabetic and the prediabetic condition entail a metabolic status where carbon fixation occurs in humans. Carbon fixation is the uptake of a mono- carbon substrate, for example carbon dioxide. Carbon fixation is a process normally associated with autotrophic organisms. The present inventors realized the surprising possibility of exploiting carbon fixation in humans for the purpose of diagnosing diabetes or prediabetes.

Gluconeogenesis can also be quantified by measuring the incorporation of ²H from ²H₂0 into glucose. This method measures gluconeogenesis from glycerol as well as from pyruvate-level precursors. Briefly, the principle is as follows: Obviously, all of the H on the OH groups of glucose is readily exchangeable. Additionally, the H covalently bound to carbon 2 on glucose is readily exchangeable with the extensive isomerization of glucose-6-phosphate with fructose-6-phosphate (glucose cycling). Enrichment of deuterium on carbon 2 thus has good agreement with the enrichment of the labeled body water pool. Therefore, deuterium labeling on carbon 2 is a useful marker of total body water but not of gluconeogenesis. In contrast, deuterium labeling of the other carbons 1 , 3, 4, 5, and 6 reflects gluconeogenesis, e.g. the interconversion of malate and fumarate in liver mitochondria results in ²H being bound to the C-3 of malate, which subsequently becomes the C-2 of phosphoeno/pyruvate and eventually the C-5 of glucose. Gluconeogenesis from glycerol also results in labeling of the C-5 of glucose, via incorporation of the label into the C-2 of glyceraldehyde-3-phosphate during its equilibration with dihydroxyacetone phosphate.

Accordingly, and to the extent use is made of heavy water, it is preferred to determine presence and/or amount of deuterium label at one, more or all carbon atoms 1 , 3, 4, 5, and 6 of glucose or one, more or all corresponding carbon atoms of metabolites, metabolites being gluconeogenetic metabolites downstream of pyruvate such as glucose-6 phosphate, fructose- 6 phosphate, ribose-5 phosphate and sedoheptulose-7 phosphate.

Table 2 below provides a comparison of inorganic tracers to quantify gluconeogenesis. Figure 6 depicts the pathways involved in ²H₂0 incorporation.

An advantage of the use of inorganic tracers like H¹³C0₃ ^" and ²H₂0 over the use of organic tracers is their rapid diffusion across membranes, and thus the better equilibration across the arterial, portal venous and intrahepatic precursor pools. The use of compounds according to item (b) further aids in distinguishing the healthy from the diseased state. While not being compulsory, the compounds in accordance with item (b) suppress gluconeogenesis in healthy individuals, noting that gluconeogenesis may occur in healthy individuals who are hungry. In diabetic and prediabetic persons such inhibitory mechanism is not available any more or it functions to a lesser degree.

In diabetic and prediabetic individuals, the label of the inorganic substrate, including the mono-carbon substrate, is incorporated into glucose. Surprisingly, the inventors found that the amount of label found in glucose or metabolites as further detailed below in patient samples scales with the rate of gluconeogenesis and accordingly is a measure of the severity of disease.

Additionally, the rate of gluconeogenesis can be determined by multiple non-invasive samplings of the (labeled) glucose (e.g. in in time intervals of ~10 min over a period of -90 min, like in Example 4) and analyzing the change in the amount of label in glucose over time (which is indicative for the gluconeogenic rate).

The rate of gluconeogenesis scales with the severity of the disease and is hence a measure of the severity of the disease. Determining the rate of gluconeogenesis via multiple sampling is more sensitive, more accurate and more precise than determination via single sampling. This enhanced sensitivity is particularly advantageous for individual optimization of anti- diabetes agent dosage in the context of personalized medicine.

Surprisingly, it turns out that the non-invasive diagnostic method of the invention (the substrate of the invention is preferably administrated via non-invasive routes, for details and preferred embodiments see below; and taking a sample from an individual is preferably noninvasive as well, for example by taking saliva) allows for a more precise analysis and diagnosis than art-established methods which throughout are invasive, at least with regard to one aspect out of administration and sample taking.

Having regard to the examples, the present invention uses isotopically labeled bicarbonate (or carbonate or C0₂) as a tracer, since the fixation of bicarbonate to pyruvate by the enzyme pyruvate carboxylase, which yields oxaloacetate, is the initial step of gluconeogenesis and integrates all pyruvate precursors. The inventors found that gluconeogenesis is qualitatively and quantitatively measureable via the administration of labeled bicarbonate (H¹³C0₃ ) following mass spectrometry of glucose. The fully sequenced microorganism Bacillus subtilis 168 strain was used as a model organism for mammalian metabolism, because the gluconeogenic enzymes are consistent, except for the last step from glucose-6-phosphate to glucose mediated by glucokinase, which is missing in Bacillus subtilis; see examples.

Advantageously, the present invention can be implemented as a non-invasive test for diabetes and prediabetes. Owing to the fact that the test may also be exploited quantitatively (the degree of incorporation of label is indicative of the severity of disease), it follows that the invention also aids in proper adjustment of dosage of antidiabetic drugs. These preferred uses of the invention will be described in more detail further below.

Furthermore, since a subtle increase of glucose concentrations stemming from the gluconeogenesis pathway can be detected by using the current invention, the state of pre- diabetic disease can be monitored in a highly accurate way.

In fact, and as compared to the prior art, especially Aleman, loc. cit. and Marsch, loc. cit., the invention surprisingly provides for a significantly higher degree of incorporation of the label into glucose or the mentioned metabolites.

A further advantage of the present invention (which is exploited by methods and uses disclosed further below) is the possibility to fine-tune and optimize the anti-diabetic treatment. Typical treatments include the administration of insulin, the administration of metformin and the administration of both insulin and metformin. For all these treatment regimens, fine-tuning and optimization of the respective dosage regimens, including the dosage regimen of the combination therapy with metformin and insulin, are rendered possible by the present invention, notably in a non-invasive manner.

In a third aspect, the present invention provides a kit comprising (a) one or more inorganic substrates that carry a label and can be fed into the tricarboxylic acid cycle and/or the gluconeogenesis pathway selected from (i) heavy water; and (ii) a mono-carbon substrate; and (b) glucose, a source of glucose and/or an inhibitor of gluconeogenesis.

Related thereto, the invention provides a kit comprising (a) one or more mono-carbon substrates that carry a label and can be fed into the tricarboxylic acid cycle and/or the gluconeogenesis pathway; and (b) glucose, a source of glucose and/or an inhibitor of gluconeogenesis. The kit according to the invention typically provides compounds in accordance with items (a) and (b) in separate vessels, respectively. The kit may also comprise further constituents, said further constituents including chemical compounds, wherein such chemical compounds are generally provided in further separate vessels. Preferred further constituents are described below and include those constituents which may be used to prepare final preferred compositions in accordance with the present invention, such final compositions including mineral water, lemonade and compositions confectioned for inhalation.

In preferred embodiments of the first, second and third aspect of the present invention the (i) heavy water is selected from ²H₂0, 0²Η^{~ 2}H₃0⁺, H²HO, H²H₂0⁺ and H₂ ²HO⁺; and/or (ii) the mono-carbon substrate is a substrate of pyruvate carboxylase and/or selected from HC0₃ ^~,

Pyruvate carboxylase is an enzyme which catalyzes the carboxylation of pyruvate to yield oxaloacetate. Accordingly, it is a ligase. It has EC number 6.4.1.1. HC0₃ ^~, also known as bicarbonate, and C0₃ ^2~ are anions of carbonic acid H₂C0₃. C0₂ is carbon dioxide, the anhydrate of carbonic acid.

In further preferred embodiments of the first, second and third aspect, said label (i) deviates from the naturally occurring distribution of isotopes; and/or (ii) is a stable isotope label such as ¹³C or a radioactive label such as ¹⁴C and ¹¹C.

Preference is given stable isotope labels.

A preferred analytical means is mass spectrometry; see further below. Especially preferred is isotopic ratio mass spectrometry (IRMS). IRMS is described, for example in Krummen et al. (Rapid Commun. Mass Spectrom. 2004; 18: 2260-2266) and Godin et al. (Mass Spectrometry Reviews, 2007, 26, 751-774).

Preference is given to compositions and kits where the mono-carbon substrate is 100% labeled. This is not the requirement, though. Any distribution of isotopes in the mono-carbon substrate of the invention which deviates from the naturally occurring isotope distribution permits the tracing of the gluconeogenetic process. This is expressed in item (i) of this preferred embodiment. C, owing to its radioactivity, can in principle be detected using a Geiger counter or by scintillation spectroscopy. Alternatively, ¹⁴C may also be detected by mass spectrometry. A preferred carbon isotope for radioactive detection schemes is ¹ C.

While for diagnostic purposes in humans preference will be given to the use of stable isotopes, especially ³C, it is understood that for tests in animals and in the pre-clinical phase, radioactive labels are advantageous.

In a further preferred embodiment of the first, second and third aspect of the invention, said source of glucose is selected from (a) disaccharides, oligosaccharides and polysaccharides which comprise glucose; and (b) glucose phosphates; or said inhibitor of gluconeogenesis is a metabolite, a hormone or a drug.

Exemplary disaccharides in accordance with (a) are saccharose and lactose. An exemplary polysaccharide is starch. Glucose phosphates include glucose-6-phosphate.

Preferred hormones are glucocorticoids, insulin, and insulin precursors, for example proinsulin. Preferred drugs are antihypertonica such as beta-blocking agents, thiazide diuretics such as hydrochlorothiazide, chlorthalidone, angiotensin converting enzyme (ACE) inhibitors, and metformin.

Accordingly, particularly preferred compositions, diagnostic compositions and kits according to the invention comprise or consist of one or more mono-carbon substrates, preferably HCO₃ ^", preferably the ¹³C labeled form thereof; and insulin or proinsulin.

A further particularly preferred composition, diagnostic composition or kit of the invention comprises or consists of one or more mono-carbon substrates, preferably HC0₃ ^", preferably the ¹³C labeled form thereof; and metformin.

A yet further particularly preferred composition, diagnostic composition or kit of the invention comprises or consists of one or more mono-carbon substrates, preferably HC0₃ ^", preferably the ¹³C labeled form thereof; and insulin and metformin.

In a further preferred embodiment of compositions, including diagnostic compositions or of the kit according to the present invention, said composition, diagnostic composition or kit further comprises one or more of the following: (c) water; (d) salts; (e) colorants; (f) flavorings; (g) preservatives; (h) fruit juice and/or fruit extracts; (i) whey and/or milk; and (j) propellants.

These are preferred further constituents which can be used to manufacture ready-to-use compositions in accordance with the present invention. Ready-to-use compositions include compositions for the intake by patients. Intake may be by drinking, eating or inhalation.

Accordingly, particularly preferred compositions and diagnostic compositions are mineral water, lemonade and compositions confectioned for inhalation.

A mineral water in accordance with the present invention may comprise or consist of one or more mono-carbon substrates in accordance with item (a), preferably HC0₃ ^~ optionally a compound in accordance with item (b) as disclosed above, preferably glucose, water, and optionally salts, such as chlorides and sulfates of sodium, potassium, calcium and/or magnesium. One or more preservatives are furthermore envisaged as ingredients of a mineral water in accordance with the present invention.

A preferred lemonade according to the invention may comprise or consist of a mineral water in accordance with the invention where fruit juice and/or fruit extracts, flavorings and/or colorants have been added.

Further envisaged are drinks containing whey. As compared to mineral water, whey may be used to substitute water or may be mixed with water.

A composition confectioned for inhalation in accordance with the present invention may comprise one or more mono-carbon substrates in accordance with item (a), preferably HC0₃ ^~; optionally a compound in accordance with item (b) such as glucose; water; salts, preferably as defined above and/or preferably for the purpose of rendering the composition isotonic and, to the extent said composition confectioned for inhalation is a spray, propellants.

Another composition for inhalation comprises or consists of labeled C0₂, e.g. ¹³C0₂.

One or more preservatives are further preferred constituents of any of the compositions in accordance with the present invention.

Mineral waters and lemonades may be filled into bottles or containers which are equipped with a device, e.g. a valve, preventing loss of gas. In a further preferred embodiment of the composition, the diagnostic composition or the kit of any one of the preceding aspects of the invention, said composition, diagnostic composition or kit is for (a) diagnosing diabetes, a predisposition to develop diabetes or metabolic syndrome, preferably type 2 diabetes; and/or (b) dosage adjustment of diabetes treatment.

The term "predisposition to develop diabetes" includes the condition of prediabetes as well as metabolic syndrome. The relevance of gluconeogenesis in metabolic syndrome is described in, for example, Bagby (2004) (J Am Soc Nephrol, 15, 2775-2791 ) and Moller (2001) (Nature, 414, 821-827).

Throughout this specification, type 2 diabetes is the preferred form of diabetes.

In a particularly preferred embodiment and in relation to item (b) of the preceding preferred embodiment, said treatment is the administration of metformin and said adjustment is the establishing of an adequate dosage regimen.

In a fourth aspect, the present invention provides an inorganic substrate that carries a label and can be fed into the tricarboxylic acid cycle and/or the gluconeogenesis pathway selected from (i) heavy water; and (ii) a mono-carbon substrate; and optionally glucose, a source of glucose and/or an inhibitor of gluconeogenesis; for use in an in vivo method of diagnosing diabetes or metabolic syndrome or a predisposition to develop diabetes or metabolic syndrome.

Said in vivo method of diagnosing includes a method of diagnosing practised on the human or animal body.

Related thereto, the invention provides a labeled mono-carbon substrate that can be fed into the tricarboxylic acid cycle and/or the gluconeogenesis pathway, and optionally glucose, a source of glucose and/or an inhibitor of gluconeogenesis, for use in method of diagnosing diabetes, a predisposition to develop diabetes or metabolic syndrome and/or for dosage adjustment of diabetes treatment.

In a preferred embodiment, said method comprises determining presence and/or amount of label in glucose or a metabolite, said metabolite being as defined herein below, wherein presence and/or a statistically significantly elevated amount of label in glucose or said metabolite as compared to a healthy reference is indicative of diabetes, metabolic syndrome or a predisposition to develop diabetes or metabolic syndrome.

A healthy reference may be a single person or a population average. In either case, for an individual to be considered as healthy reference, preferably to said individual a composition in accordance with the first aspect is administered and preferably no labelled glucose or labelled metabolite as defined herein is subsequently found in said individual. Such individual would be characterized in that is does not suffer from diabetes or metabolic syndrome nor does it have a predisposition therefor.

As an alternative, and preferably, the reference state is the same individual to which the label substrate in accordance with the present invention is to be administered. In fact, the present invention allows for a subject-specific or patient-specific reference state. This is described in more detail further below.

Related thereto, a patient suffering from diabetes, in particular type 2 diabetes, and receiving optimal anti-diabetes treatment, such as optimal dosage of an anti-diabetic agent such as metformin or insulin, can be identified based on the teaching of the present invention. Such patient would be characterized in that no labelled glucose or labelled metabolite as defined herein is found in said patient upon administration of a composition in accordance with the first aspect of this invention.

The term "metabolic syndrome" has its art-established meaning. It relates to a condition where at least three of the following symptoms occur: abdominal obesity, elevated blood pressure, elevated fasting serum glucose, high serum triglycerides and low high-density lipoprotein levels. Metabolic syndrome is characterized by an elevated risk to develop type 2 diabetes.

As noted above, means and methods of the present invention are characterized by particularly high sensitivity. As a consequence, early stages of a disease or predisposition thereto can be determined. To the extent a subject exhibits no clinical symptoms of diabetes or metabolic syndrome, presence and/or amount of label is indicative of the above mentioned predisposition.

In a fifth aspect, the invention relates to an inorganic substrate that carries a label and can be fed into the tricarboxylic acid cycle and/or the gluconeogenesis pathway selected from (i) heavy water; and (ii) a mono-carbon substrate; and optionally glucose, a source of glucose and/or an inhibitor of gluconeogenesis; for use in a method of treating diabetes in a patient, wherein (a) said method comprises treating said patient with an anti-diabetic agent such as insulin or metformin; and (b) the amount of label determined in glucose or a metabolite in a sample taken from said patient, said metabolite being a gluconeogenetic metabolite downstream of pyruvate such as glucose-6-phosphate, fructose-6-phosphate, ribose-5- phosphate and sedoheptulose-7-phosphate, is used for adjusting the dosage of said antidiabetic agent.

In the context of personalized medicine, the aim of adjusting the dose is to prevent a shortcoming of the anti-diabetic agent in order to maximize its effect and at the same time to prevent an overdose of the anti-diabetic agent, in order not to cause hypoglycemia.

Samples may be taken from said patient prior to and after or only after administration of said anti-diabetic agent. It is expected that administration of the anti-diabetic agent entails a decrease of label in glucose or a metabolite as defined above. If said decrease is not sufficient, dosage of the anti-diabetic agent may be increased. An increased dosage which entails an improved treatment can be determined by a (further) decrease or disappearance of label.

Preferred embodiments of the composition, the diagnostic composition and the kit according to the present invention also define preferred embodiments of the fourth aspect. The same applies mutatis mutandis to the further aspects of the present invention as disclosed further below.

Having regard to the recited dosage adjustment of diabetes treatment, it has to be understood that, as noted above, the degree and/or the rate of incorporation of the label into glucose by the gluconeogenetic process is reflective of the severity of the disease.

Accordingly, the higher the degree and/or the rate of incorporation, the higher the dosage of the anti-diabetic treatment to be administered. The precise correlation between the two parameters can be determined by the skilled person without further ado when provided with the teaching of the present invention.

Having regard to the diagnosis of diabetes or a predisposition to develop diabetes, said predisposition including prediabetes, the following applies. In the healthy individual, no or very small amounts of labeled glucose are formed upon administration of compositions in accordance with the present invention. This is because gluconeogenesis is not or hardly active in such healthy individual. As a consequence, already the mere presence of labeled glucose or another labeled metabolite as disclosed herein is indicative of a disorder of glucose metabolism. An elevated amount of glucose formed in the individual, especially a statistically significantly elevated amount of glucose or of a metabolite as disclosed below is (i) indicative of the disorder, and (ii) a measure of the severity of the disease.

In a preferred embodiment of the substrate for use, administration of said substrate and, where applicable of glucose, a source of glucose and/or an inhibitor of gluconeogenesis is to be effected non-invasively, and taking of said sample is to be effected non-invasively.

Preferred formulations for non-invasive administration are disclosed herein as are noninvasive means and methods for taking samples.

In a sixth aspect, the present invention provides a method of diagnosing in a subject diabetes or metabolic syndrome or a predisposition to develop diabetes or metabolic syndrome, said method comprising: (a) administering to said subject an inorganic substrate that carries a label and can be fed into the tricarboxylic acid cycle and/or the gluconeogenesis pathway selected from (i) heavy water; and (ii) a mono-carbon substrate; and optionally glucose, a source of glucose and/or an inhibitor of gluconeogenesis; (b) taking a sample from said subject; (c) determining presence of and/or amount of said label in glucose or a metabolite comprised in said sample, said metabolite being a gluconeogenetic metabolite downstream of pyruvate such as glucose-6-phosphate, fructose-6-phosphate, ribose-5-phosphate and sedoheptulose- 7-phosphate, wherein presence of and/or a statistically significantly elevated amount of said label in said glucose or said metabolites as compared to a healthy reference or prior to administering in accordance to step (a) is indicative of diabetes or metabolic syndrome or said predisposition. Preferred or typical values of a significantly elevated amount of said label are disclosed further below.

The present invention also provides a method of monitoring treatment with an anti-diabetic agent, said method comprising: (a) administering to a subject receiving said treatment an inorganic substrate that carries a label and can be fed into the tricarboxylic acid cycle and/or the gluconeogenesis pathway selected from (i) heavy water; and (ii) a mono-carbon substrate; and optionally glucose, a source of glucose and/or an inhibitor of gluconeogenesis; (b) taking a sample from said subject; (c) determining presence of and/or amount of said label in glucose or a metabolite comprised in said sample, said metabolite being a gluconeogenetic metabolite downstream of pyruvate such as glucose-6-phosphate, fructose-6-phosphate, ribose-5- phosphate and sedoheptulose-7-phosphate, wherein presence of and/or a statistically significantly elevated amount of said label in said glucose or said metabolites as compared to a healthy reference, compared to a subject suffering from diabetes and receiving optimal treatment, or prior to administering in accordance to step (a) is indicative of insufficient treatment. Preferred or typical values of a significantly elevated amount of said label characteristic of insufficient treatment are comparable to those typical of the diseased state.

The present invention also provides a method for dosage adjustment of treatment with an antidiabetic agent, said method comprising said method of monitoring treatment, wherein in case of a finding of insufficient treatment the dosage of the anti-diabetic agent is increased.

Related to the sixth aspect, the present invention provides an in vitro method of diagnosing diabetes or metabolic syndrome or a predisposition to develop diabetes or metabolic syndrome in a subject to which an inorganic substrate that carries a label and can be fed into the tricarboxylic acid cycle and/or the gluconeogenesis pathway selected from (i) heavy water; and (ii) a mono-carbon substrate; and optionally glucose, a source of glucose and/or an inhibitor of gluconeogenesis have been administered; said method comprising determining presence of and/or amount of said label in glucose or a metabolite comprised in a sample taken from said subject, said metabolite being a gluconeogenetic metabolite downstream of pyruvate such as glucose-6-phosphate, fructose-6-phosphate, ribose-5-phosphate and sedoheptulose-7-phosphate, wherein presence of and/or a statistically significantly elevated amount of said label in said glucose or said metabolites as compared to a healthy reference or prior to said substrate having been administered is indicative of diabetes or metabolic syndrome or said predisposition.

The present invention also provides an in vitro method of monitoring treatment with an antidiabetic agent of a subject to which an inorganic substrate that carries a label and can be fed into the tricarboxylic acid cycle and/or the gluconeogenesis pathway selected from (i) heavy water; and (ii) a mono-carbon substrate; and optionally glucose, a source of glucose and/or an inhibitor of gluconeogenesis have been administered; said method comprising determining presence of and/or amount of said label in glucose or a metabolite comprised in a sample taken from said subject, said metabolite being a gluconeogenetic metabolite downstream of pyruvate such as glucose-6-phosphate, fructose-6-phosphate, ribose-5-phosphate and sedoheptulose-7-phosphate, wherein presence and/or a statistically significantly elevated amount of said label in said glucose or said metabolites as compared to a healthy reference, compared to a subject suffering from diabetes and receiving optimal treatment, or prior to said substrate having been administered is indicative of insufficient treatment.

In a preferred embodiment of said in vitro method of monitoring treatments, said method further comprises the step of providing the information that the dosage of said treatment is to be increased.

Also related thereto, the invention provides a method of diagnosing diabetes, a predisposition to develop diabetes or metabolic syndrome and/or for dosage adjustment of diabetes treatment, said method comprising: (a) administering a mono-carbon substrate that carries a label and can be fed into the tricarboxylic acid cycle and/or the gluconeogenesis pathway, or the composition or kit as defined in any one of the preceding claims, and optionally glucose, a source of glucose and/or an inhibitor of gluconeogenesis, to a subject; (b) taking a sample from said subject; (c) determining presence and/or amount of said label in glucose or a metabolite comprised in said sample, said metabolite being a gluconeogenetic metabolite downstream of pyruvate such as glucose-6-phosphate, fructose-6-phosphate, ribose-5- phosphate and sedoheptulose-7-phosphate, wherein presence and/or a statistically significantly elevated amount of glucose or of said metabolites as compared to a healthy reference is indicative of diabetes, said predisposition and/or of the severity of diabetes, the degree of severity providing for said dosage adjustment.

To explain further, depending on the degree of severity, response to an anti-diabetic agent will differ from patient to patient. Hence, the optimal dose of the anti-diabetic agent may vary from patient to patient caused by different rates of gluconeogenesis. The optimal dosage may be established as follows, possibly in several steps beginning with low anti-diabetic agent doses. In those patients, where upon administration of these low doses, the label is still incorporated in unacceptably high amounts into glucose or a metabolite as defined herein above, the dosage will be deemed as too low and the dosage needs to be increased until the amount of label in glucose is reduced to an acceptably small amount or the label in glucose completely disappears, indicating the complete inhibition of gluconeogenesis. In such a case, it is the incorporated label (which in turn reflects severity of the disease or insufficient dosage) which provides for dosage adjustment in the sense that it provides guidance for increasing dosage of the anti-diabetic agent.

In a preferred embodiment, the line between unacceptably high amount or statistically significantly elevated amount of label and acceptably small amount or not statistically significantly elevated amount of label may be drawn as follows: (Unacceptably) high refers to a δ¹³0 value of glucose that is at least about 5 %o higher, at least about 10 %o higher, at least about 20 %o higher, at least about 50 %o higher, at least about 100 %o higher, at least about 200 %o higher, at least about 300 %o higher, or at least about 500 %₀ higher than the 5¹³C value of unlabeled endogenous glucose that naturally ranges from ~-27 %o to ~-10 %o, according to whether the diet is primarily based on plants with C3 and C4 photosynthesis, respectively. The parameter 6¹³C is defined in Example 4. To explain further, for example in Europe diet is largely based on wheat, typical 5 ³C values are in the range from ~-27 %o to ~-15 %o. On the other hand, for example in the United States, where diet is generally based on maze, typical δ¹³0 values are in the range from ~-20 %o to ~-10 %o. The 5¹³C value of the unlabeled endogenous glucose (reference state) can be retrieved through sampling the saliva and analyzing glucose before administration of the label or can be estimated after administration of the label from the 5 ³C value of other metabolites (from saliva), preferably carbohydrates, that are not subject to gluconeogenesis and thus intrinsically do not carry a label after label administration and active gluconeogenesis. Such metabolites could be acetate, propionic acid, urea, fructose, galactose, fucose or mannose. These metabolites are not or substantially not effected by label incorporation and are accordingly distinct from the above disclosed gluconeogenetic metabolites. In other words, the sample(s) defining the reference state do not even have to be taken prior to administration of the labelled substrate.

(Acceptably) small amount refers to a 5¹³C value of glucose that lies within the range of ~5 %o around the 6¹³C value of unlabeled endogenous glucose.

Notably, the reference value can be determined within the same subject or patient. For that reason, whenever reference is made to reference state in this disclosure, instead of a healthy individual or a subject suffering from diabetes and receiving optimal treatment, a sample from the subject under investigation (possibly suffering from disease and/or possibly receiving suboptimal treatment) may be taken prior to administering the labelled inorganic substrate and optionally glucose, a source of glucose and/or any inhibitor of gluconeogenesis to said substrate.

Related to the above, the present invention also permits to monitor the treatment of diabetes. Optimal treatment is characterized by absence of gluconeogenesis or a minimal level thereof upon administration of the compositions of the present invention. The present invention allows to determine when the optimal dosage has been established. Similarly, non-optimal dosages in the course of diabetes therapy can be monitored and detected by the methods of the invention and suggestions for improvement can be made.

Preferred embodiments of the preceding aspects also define mutatis mutandis preferred embodiments of the sixth aspect. Accordingly, HC0₃ ^~ is a particularly preferred mono-carbon substrate. ¹³C is a particularly preferred label. A particularly preferred compound for the purpose of inhibiting gluconeogenesis, to the extent such compound is to be used in the course of the method in accordance with the invention, is glucose.

The label contained in the mono-carbon substrate can not only be retrieved in glucose, but also in further metabolites. Further metabolites are preferably metabolites of the gluconeogenetic pathway or the pentose phosphate pathway. Exemplary or preferred metabolites are mentioned above.

In preferred embodiments of the method according to the sixth aspect, (a) said administering is non-invasive, preferably oral; (b) said taking a sample is non-invasive, preferably by means of a swab and/or taking saliva; (c) a given time, preferably selected from at least 5 min, at least 10 min, at least 15 min, at least 30 min, at least 1 h, at least 2 h, at least 3 h, at least 4h, at least 6h or at least 8h, is allowed to elapse between steps (a) and (b); (d) step (b) is effected at more than one time after step (a), preferably in regular time intervals of, for example, 5 min, 10 min, 15 min or 30 min; and/or (d) said determining is by means of mass spectrometry, preferably by isotope ratio mass spectrometry.

Preferred formulations for non-invasive administration (lemonade, spray etc.) are disclosed herein above.

Particularly preferred is the combination of preferred embodiments (a) and (b), thereby providing for completely non-invasive diagnosis, treatment monitoring and dosage adjustment.

Figure 1 contains a scheme illustrating a particularly preferred implementation of the invention.

Advantageously, the present invention allows for a diagnostic test which in its entirety is noninvasive.

Alternative analytical methods for isotope detection and quantification are known in the art and disclosed in the introductory part of this specification. Preferred is mass spectrometry (MS). Particularly preferred is isotopic ratio mass spectrometry (IRMS). The latter permits particularly sensitive detection, for example of labeled glucose and/or the abovementioned metabolites.

The swab is preferably for the purpose of collecting a sample of saliva. Detection of glucose in saliva is well-established (see, for example, Abikshyeet et al. (2012), Diabetes Metab Syndr Obes., 5, 149-154; Gupta et al. (2015), Journal of Clinical and Diagnostic Research, 9, ZC106-ZC109; Mascarenhas et al. (2014), PLOS ONE, 9, e101706; Patel et al. (2015), Journal of International Oral Health, 7, 70-76; and Satish et al. (2014) Journal of International Oral Health, 6, 114-117).

Related to the fourth, fifth and sixth aspect, the present invention provides, in a seventh aspect, the use of an inorganic substrate that carries a label and can be fed into the tricarboxylic acid cycle and/or the gluconeogenesis pathway selected from (i) heavy water; and (ii) a mono-carbon substrate; and optionally glucose, a source of glucose and/or an inhibitor of gluconeogenesis, for (a) diagnosing diabetes or metabolic syndrome or a predisposition to develop diabetes or metabolic syndrome; (b) monitoring diabetes treatment; and/or (c) dosage adjustment of diabetes treatment.

Related thereto, the invention provides the use of a mono-carbon substrate that carries a label and can be fed into the tricarboxylic acid cycle and/or the gluconeogenesis pathway, and optionally glucose and/or a source of glucose, for diagnosing diabetes, a predisposition to develop diabetes or metabolic syndrome and/or for dosage adjustment of diabetes treatment.

In a preferred embodiment of the use of the invention, said substrate and, where applicable, glucose, a source of glucose and/or an inhibitor of gluconeogenesis are administered noninvasive^, and wherein any sample to be taken from a subject for any of purposes (a), (b) or (c) is taken non-invasively.

In a preferred embodiment of the seventh and related aspects, said diagnosing is not practised on the human or animal body.

As regards the embodiments characterized in this specification, in particular in the claims, it is intended that each embodiment mentioned in a dependent claim is combined with each embodiment of each claim (independent or dependent) said dependent claim depends from. For example, in case of an independent claim 1 reciting 3 alternatives A, B and C, a dependent claim 2 reciting 3 alternatives D, E and F and a claim 3 depending from claims 1 and 2 and reciting 3 alternatives G, H and I, it is to be understood that the specification unambiguously discloses embodiments corresponding to combinations A, D, G; A, D, H; A, D, I; A, E, G; A, E, H; A, E, I; A, F, G; A, F, H; A, F, I; B, D, G; B, D, H; B, D, I; B, E, G; B, E, H; B, E, I; B, F, G; B, F, H; B, F, I; C, D, G; C, D, H; C, D, I; C, E, G; C, E, H; C, E, I; C, F, G; C, F, H; C, F, I, unless specifically mentioned otherwise.

Similarly, and also in those cases where independent and/or dependent claims do not recite alternatives, it is understood that if dependent claims refer back to a plurality of preceding claims, any combination of subject-matter covered thereby is considered to be explicitly disclosed. For example, in case of an independent claim 1 , a dependent claim 2 referring back to claim 1 , and a dependent claim 3 referring back to both claims 2 and 1 , it follows that the combination of the subject-matter of claims 3 and 1 is clearly and unambiguously disclosed as is the combination of the subject-matter of claims 3, 2 and 1. In case a further dependent claim 4 is present which refers to any one of claims 1 to 3, it follows that the combination of the subject-matter of claims 4 and 1 , of claims 4, 2 and 1 , of claims 4, 3 and 1 , as well as of claims 4, 3, 2 and 1 is clearly and unambiguously disclosed.

The figures show:

Figure 1 : HPLC chromatogram of an unlabeled Glucose-6-Phosphate (G6P) solution. The dotted line represents the unlabeled G6P and the solid line represents the labeled G6P. The fraction of labeling represents natural abundance.

Figure 2: HPLC chromatogram of a Bacillus subtilis extract. The dotted line represents the unlabeled Glucose-6-Phosphate (G6P) and the solid line represents the labeled G6P. The Bacillus extract shown here was sampled before H¹³C0₃ ^" administration. The fraction of labeling hence represents natural abundance.

Figure 3: Scheme illustrating a preferred implementation of the use of the invention.

Figure 4: Experimental setup for oral administration of H¹³C0₃ ^" followed by mass spectrometry as a Non-Invasive Diabetes Test - Proof of principle in mammals.

Figure 5: Isotopic ratio mass spectrometry distinguishes active from inactive gluconeogenesis following the herein proposed non-invasive diagnostic approach. Figure 6: Incorporation of ²H from ²H₂0 into glucose during gluconeogenesis from pyruvate. PEP, phosphoenolpyruvate; G-3-P, glyceraldehydes-3-phophate; DHAP, dihydroxyacetone phosphate; F-1 ,6-bisP, fructose-1 ,6-biphosphate. (Taken from Coggan, A. R. (1999) Use of stable isotopes to study carbohydrate and fat metabolism at the whole-body level. Proceedings of the Nutrition Society, 58, 953-961 )

The examples illustrate the invention: Example 1

Materials and methods

Cultivation, labeling, sampling, quenching and extraction

The wild type Bacillus subtilis 168 strain (DSM No.: 23778) purchased from DSMZ (Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures, Braunschweig) was used for the experiments. Cryocultures were stored at -80°C in liquid CASO medium containing 50% (v/v) glycerol. Precultivation was performed overnight (12 h, 150 rpm, 30 °C) in culture tubes (10 mL) containing 5 mL precultivation medium inoculated with 1 mL cryoculture. The preculture solution was used to inoculate Erlenmeyer flasks (250 mL). Cultivation media contained per liter 0.3 g lactate, 8.5 g Na₂HP04 ^* 2H₂0, 3 g KH₂P0₄, 1 g NH₄CI, 0.5 g NaCI, 1 mL 0.1 M CaCI₂, 1 mL 1 M MgS0₄, 1 mL 50 mM FeCI₃, 170 mg ZnCI₂, 100 mg MnCI₂ * 4H₂0, 60.0 mg CoCI₂ ^* 6H₂0, 60.0 mg Na₂Mo0₄ * 2H₂0 and 43.0 mg CuCI₂ * 2H₂0. Cultivation was performed at 30°C. When the medium became visually turbid, indicating bacterial growth, 20 mg of the isotopic tracer H¹³C0₃ ^" (isotopic purity 99%, Sigma Aldrich) was added to the culture and time-resolved sampling was initiated. Samples were taken right before tracer administration (i.e. time at 0 min) and at 5, 10, 15 and 60 min after tracer administration, respectively. To this end, 5 mL of cell suspension were rapidly vacuum- filtrated. The filter was immediately put into a Falcon tube filled with 15 mL aqueous ethanol solution (-20 °C, 60%, v/v) to stop cell metabolism quickly. Directly after quenching, the Falcon tubes were sonicated for 15 min on ice to destruct cell membranes. To separate the metabolites from the cell debris, the Falcon tubes were centrifuged for 15 min at 10,000*g and 4 °C and the supernatant containing the metabolites in aqueous ethanol solution was collected. Samples were lyophilized and resuspended in 2 mL MilliQ water. The aqueous metabolite solution was transferred into 2 mL glass vials for LC-MS/MS analysis. LC-MS/MS measurements

The experiment was carried out on an Agilent HP 1200 HPLC system coupled with a Q-Trap MS/MS system (Applied Biosystems, Toronto, Canada), lonisation was accomplished by electrospray ionization in the negative ion mode. Mass spectrometry was carried out in the multiple reaction monitoring mode. To determine the MS/MS fragment pattern for Glucose-6- Phosphate (hence G6P), a single analyte standard containing G6P (purity 98%, Sigma- Aldrich) was dissolved in a mixture of water and methanol 50:50 (v/v) at a concentration of 1 mg/L. The standard was infused at a flow rate of 200 L/min for tuning the compound dependent MS parameters. The infusion experiment was performed using a syringe pump directly connected to the interface. Optimal detection was provided by scanning for the mass pair 258.9/96.9, where the first mass represents the intact G6P molecule and the second represents its P0₄H₂ ^" group. Since the intended isotopic labeling should introduce a mass shift in the mother ion of G6P but not in the phosphate group, further MS analysis was extended to the mass pair 259.9/96.9. Declustering potential, collision energy, collision cell exit potential and entrance potential were optimized to -21 , -24, 0 and -6 V. To finely tune the source dependent parameters 50 μΙ_ standard mixture solution was injected by the autosampler into the mobile phase flow (50% A:50% B; A: 10 mM tributylamine adjusted to pH 4.9 with 15 mM acetic acid; B: methanol) at a flow rate of 200 pLJmin. The optimized values for the parameters ion-spray voltage, nebulizer gas, auxiliary gas, curtain gas, collision gas and auxiliary gas temperature were -4500, 50, 50, 25, 8 and 550 °C, respectively. The curtain, collision, turbo and nebulizer gas was nitrogen generated from pressurized air in a nitrogen generator. To obtain adequate selectivity and sensitivity, the mass spectrometer was set to unit resolution and the dwell time for each mass pair was 150 ms. The chromatographic separation was achieved on a Synergi Hydro-RP (C18) 150 mmx2.1 mm I.D., 4 pm 80 °A particles column (Phenomenex, Aschaffenburg, Germany) at room temperature (20°C) with eluent A (10 mM tributylamine aqueous solution adjusted pH to 4.9 with 15 mM acetic acid) and eluent B (methanol). The gradient profile is displayed in Table 3. Before each run, the column was equilibrated for 15 min. The injected volume was 50 μΙ_. Mobile phase at a flow rate of 200 pL/min was directly introduced into the mass spectrometer via the Turbo ion-spray source. The experimental method thus followed the standard procedure of detecting G6P on a LC-MS/MS system (Luo et al. (2007), Journal of Chromatography A, 147, 153-164). Table 3: Gradient profile of the LC-MS/MS method

Total time Eluent A Eluent B

Step (min) (vol.%) (vol.%)

1 1 5 100 0

2 25 80 20

3 55 80 20

4 60 65 35

5 65 65 35

6 70 40 60

7 75 40 60

8 75.1 10 90

9 80 10 90

1 0 81 1 00 0

1 1 1 00 1 00 0

Data analysis

Data were acquired and chromatographic peak areas were integrated via Analyst software (version 1 .4.2, AB/MDS Sciex, Concord, Canada). In the following, G6P_uni_abeied represents the peak area of unlabeled G6P, as quantified by scanning for the mass pair 258.9/96.9 and G6Piabeied represents the peak area of labeled G6P, as quantified by the mass pair 269.9/96.9.

The formula

^labeled = (G6P|abeled/G6P_unlabeled) ^β 1 00 (Eqn 1 ) gives the fraction of labeled G6P in the sample. To account for the non-linear reaction progress during the time course of fG6P_labeled after administration of H¹³C0₃ ^", the first order kinetic model ^fG6Plabeled ⁼ ^labeled; final ⁺ (^f(36Pnatural abundance ^{" fG6P}labeled; final)*³ ' ' (^{Ε< Π 2}) was fitted to the data, where the parameter fG6P_{natural abundance} represents the fraction of labeled G6P before label administration, i.e. at natural abundance, and fG6P_labeled. _final represents the fraction of labeled G6P after complete turnover. The parameter r is the reaction rate constant and f is the time. The turnover time is denoted as half-life (λ) and calculated as λ= ln(2)/r (Eqn3)

Example 2

Results

The chromatography peaks of G6P in the standard solution appeared after 18 min (Fig. 1). The f_G6P labeled was 1%, indicating ³C labeling at natural abundance.

In all Bacillus subtilis extracts, the peaks of G6P appeared likewise after 18 min. However, a second peak, representing the G6P isomer Fructose-6-Phosphate appeared two minutes later.

The f_G6P labeled in the Bacillus subtilis extracts was 1 % before label administration. It was thus equal to the G6P standard solution and equal to natural abundance of ³C. During the time course, the fG6P labeled increased exponentially to a maximum value of 10% after 60 min of label administration (Fig. 2). This reaction progress was represented by the model l_abeled = 10.1 + (1 - 10.1 )β-⁰⁰⁴⁹·¹ (Eqn 4) and the resulting half-life was 14 min. Example 3

Oral administration of H¹³COg^" followed by mass spectrometry as a Non-Invasive Diabetes Test - Proof of principle in mammals

After the successful proof of principle in the model organism Bacillus subtilis, the next step is to conduct experiments with model organisms that are closer to humans, in particular with mice. To this end, healthy and diabetic mice (e.g. KK mice) are treated by oral administration of (un)labeled bicarbonate and unlabeled glucose. Following non-invasive saliva sampling 10, 20, 30 and 60 minutes after administration, respectively, the ¹³C label should only appear in the glucose of diabetic mice treated with labeled bicarbonate and unlabeled glucose (here Group 4) after mass spectrometric analysis, because of their up-regulated gluconeogenesis.

An exemplary experimental setup is depicted in Figure 4.

Example 4

Sensitivity of H CCV administration

To proof the feasibility of a non-invasive method for the detection of gluconeogenesis, the following experiments were conducted in humans. In the first experiment, gluconeogenesis was activated through a starvation period of 32 h. In the second experiment, gluconeogenesis was inactivated through a carbohydrate rich diet. Both experiments were followed by the oral administration of 2 g of the stable isotope tracer H¹³C0₃ ^". Saliva samples were taken every 15 min for 130 min and processed as follows: An aliquot of 1 mL saliva was spit into a beaker. A aliquot of 1 mL methanol was added. The sample was centrifuged (15 min, 1400 rpm, 4°C), filtrated (0.22 pm), freeze-dried and resolved in 50 μΐ_ mQ water. An aliquot of 10pL was used for liquid chromatography hyphenated to an isotopic ratio mass spectrometer (IRMS). The isotopic enrichment of glucose in ¹³C was quantified as δ¹³0 against an internal reference C0₂ standard. 5¹³C is an isotopic signature, a measure of the ratio of stable isotopes ¹³C : ¹²C, reported in parts per thousand (per mil, %o). The definition is, in per mil:

X standard where the standard is an established reference material.

In the experiment where gluconeogenesis was activated, the ¹³C of glucose was still within normal limits directly after the oral administration of the tracer, but reached a peak enrichment of more than 100%o after 10min, followed by an exponential decrease of the enrichment in glucose (Fig. 5). In sharp contrast, the 5¹³C of glucose in the experiment where gluconeogenesis was inactivated remained within the normal range of ~-24%o for the whole sampling period. The IRMS thus enabled to distinguish active from inactive gluconeogenesis.

Claims

A composition comprising or consisting of:

(a) one or more inorganic substrates that carry a label and can be fed into the tricarboxylic acid cycle and/or the gluconeogenesis pathway selected from

(i) heavy water; and

(ii) a mono-carbon substrate; and

(b) glucose, a source of glucose, and/or an inhibitor of gluconeogenesis.

A diagnostic composition comprising or consisting of:

(i) heavy water; and

(ii) a mono-carbon substrate; and optionally

(b) glucose, a source of glucose, and/or an inhibitor of gluconeogenesis.

A kit comprising

(i) heavy water; and

(ii) a mono-carbon substrate; and

(b) glucose, a source of glucose and/or an inhibitor of gluconeogenesis.

The composition of claim 1 or 2, or the kit of claim 3, wherein

(i) heavy water is selected from ²H₂0, 0²hT, ²H₃0⁺, H²HO, H H₂0⁺ and H₂ ²HO⁺; and/or

(ii) the mono-carbon substrate is a substrate of pyruvate carboxylase and/or selected from HC0₃ ^~ C0₃ ^2~ C0₂ and H₂C0₃.

The composition or the kit of any one of the preceding claims, wherein said label

(i) deviates from the naturally occurring distribution of isotopes; and/or

(ii) is a stable isotope label such as ¹³C or a radioactive label such as ¹ C and ¹¹C.

The composition or kit of any one of the preceding claims, wherein

said source of glucose is selected from

(a) disaccharides, oligosaccharides and polysaccharides which comprise glucose; and

(b) glucose phosphates;

or

said inhibitor of gluconeogenesis is a metabolite, a hormone or a drug.

The composition or kit of any one of the preceding claims, further comprising more of the following:

(c) water;

(d) salts;

(e) colorants;

(f) flavorings;

(g) preservatives;

(h) fruit juice and/or fruit extracts;

(i) whey and/or milk; and

(j) propellants.

8. The composition of any one of the preceding claims which is a mineral water, a lemonade, or a composition confectioned for inhalation.

9. The composition or kit of any one of the preceding claims which is for

(a) diagnosing diabetes, preferably type 2 diabetes, a predisposition to develop diabetes or metabolic syndrome; and/or

(b) dosage adjustment of diabetes treatment.

10. The composition or kit of claim 9(b), wherein said treatment is metformin.

11. An inorganic substrate that carries a label and can be fed into the tricarboxylic acid cycle and/or the gluconeogenesis pathway selected from

(i) heavy water; and

(ii) a mono-carbon substrate;

and optionally glucose, a source of glucose and/or an inhibitor of gluconeogenesis; for use in an in vivo method of diagnosing diabetes or metabolic syndrome or a predisposition to develop diabetes or metabolic syndrome.

12. An inorganic substrate that carries a label and can be fed into the tricarboxylic acid cycle and/or the gluconeogenesis pathway selected from (i) heavy water; and

(ii) a mono-carbon substrate;

and optionally glucose, a source of glucose and/or an inhibitor of gluconeogenesis; for use in a method of treating diabetes in a patient, wherein

(a) said method comprises treating said patient with an anti-diabetic agent such as insulin or metformin; and

(b) the amount of label determined in glucose or a metabolite in a sample taken from said patient, said metabolite being a gluconeogenetic metabolite downstream of pyruvate such as glucose-6-phosphate, fructose-6-phosphate, ribose-5-phosphate and sedoheptulose-7-phosphate, is used for adjusting the dosage of said anti-diabetic agent.

13. The substrate for use of claim 11 or 12, wherein administration of said substrate and, where applicable of glucose, a source of glucose and/or an inhibitor of gluconeogenesis is to be effected non-invasively, and/or wherein taking of said sample is to be effected non-invasively.

14. A method of diagnosing in a subject diabetes or metabolic syndrome or a predisposition to develop diabetes or metabolic syndrome, said method comprising:

(a) administering to said subject an inorganic substrate that carries a label and can be fed into the tricarboxylic acid cycle and/or the gluconeogenesis pathway selected from

(i) heavy water; and

(ii) a mono-carbon substrate;

and optionally glucose, a source of glucose and/or an inhibitor of gluconeogenesis;

(b) taking a sample from said subject;

(c) determining presence of and/or amount of said label in glucose or a metabolite comprised in said sample, said metabolite being a gluconeogenetic metabolite downstream of pyruvate such as glucose-6-phosphate, fructose-6-phosphate, ribose-5-phosphate and sedoheptulose-7-phosphate,

wherein presence of and/or a statistically significantly elevated amount of said label in said glucose or said metabolite as compared to a healthy reference or prior to administering in accordance to step (a) is indicative of diabetes or metabolic syndrome or said predisposition.

15. A method of monitoring treatment with an anti-diabetic agent, said method comprising:

(a) administering to a subject receiving said treatment an inorganic substrate that carries a label and can be fed into the tricarboxylic acid cycle and/or the gluconeogenesis pathway selected from

(i) heavy water; and

(ii) a mono-carbon substrate;

(b) taking a sample from said subject;

wherein presence of and/or a statistically significantly elevated amount of said label in said glucose or said metabolites as compared to a healthy reference, compared to a subject suffering from diabetes and receiving optimal treatment, or prior to administering in accordance to step (a) is indicative of insufficient treatment.

16. A method for dosage adjustment of treatment with an anti-diabetic agent, said method comprising the method of claim 15, wherein in case of a finding of insufficient treatment the dosage of the anti-diabetic agent is increased.

17. An in vitro method of diagnosing diabetes or metabolic syndrome or a predisposition to develop diabetes or metabolic syndrome in a subject to which

an inorganic substrate that carries a label and can be fed into the tricarboxylic acid cycle and/or the gluconeogenesis pathway selected from

(i) heavy water; and

(ii) a mono-carbon substrate;

and optionally glucose, a source of glucose and/or an inhibitor of gluconeogenesis have been administered;

said method comprising determining presence of and/or amount of said label in glucose or a metabolite comprised in a sample taken from said subject, said metabolite being a gluconeogenetic metabolite downstream of pyruvate such as glucose-6-phosphate, fructose-6-phosphate, ribose-5-phosphate and sedoheptulose- 7-phosphate, wherein presence of and/or a statistically significantly elevated amount of said label in said glucose or said metabolite as compared to a healthy reference or prior to said substrate having been administered is indicative of diabetes or metabolic syndrome or said predisposition.

18. An in vitro method of monitoring treatment with an anti-diabetic agent of a subject to which

(i) heavy water; and

(ii) a mono-carbon substrate;

said method comprising determining presence of and/or amount of said label in glucose or a metabolite comprised in a sample taken from said subject, said metabolite being a gluconeogenetic metabolite downstream of pyruvate such as glucose-6-phosphate, fructose-6-phosphate, ribose-5-phosphate and sedoheptulose- 7-phosphate,

wherein presence and/or a statistically significantly elevated amount of said label in said glucose or said metabolites as compared to a healthy reference, compared to a subject suffering from diabetes and receiving optimal treatment, or prior to said substrate having been administered is indicative of insufficient treatment.

19. The method of claim 18, further comprising the step of providing the information that the dosage of said treatment is to be increased.

20. The method of any one of claims 14 to 19, wherein

(a) said administering is non-invasive, preferably oral;

(b) said taking a sample is non-invasive, preferably by means of a swab and/or taking saliva;

(c) a given time, preferably selected from at least 5 min, at least 10 min, at least 15 min, at least 30 min, at least 1 h, at least 2 h, at least 3 h, at least 4 h, at least 6 h or at least 8 h is allowed to elapse between steps (a) and (b);

(d) step (b) is effected at more than one time after step (a), preferably in regular time intervals of, for example, 5 min, 10 min, 15 min or 30 min; and/or (e) said determining is by means of mass spectrometry, preferably by isotope ratio mass spectrometry.

21. Use of an inorganic substrate that carries a label and can be fed into the tricarboxylic acid cycle and/or the gluconeogenesis pathway selected from

(i) heavy water; and

(ii) a mono-carbon substrate;

and optionally glucose, a source of glucose and/or an inhibitor of gluconeogenesis, for

(a) diagnosing diabetes or metabolic syndrome or a predisposition to develop diabetes or metabolic syndrome;

(b) monitoring diabetes treatment; and/or

(c) dosage adjustment of diabetes treatment.

22. The use of claim 21 , wherein said substrate and, where applicable, glucose, a source of glucose and/or an inhibitor of gluconeogenesis are administered non-invasively, and wherein any sample to be taken from a subject for any of purposes (a), (b) or (c) is taken non-invasively.

23. The use of claim 21 or 22, wherein said diagnosing is not practised on the human or animal body.