WO2010060738A1

WO2010060738A1 - Rbp4 as a marker in type 1 diabetes patients

Info

Publication number: WO2010060738A1
Application number: PCT/EP2009/064431
Authority: WO
Inventors: Georg Hess; Dietmar Zdunek; Andrea Horsch
Original assignee: Roche Diagnostics Gmbh; F. Hoffmann-La Roche Ag
Priority date: 2008-11-03
Filing date: 2009-11-02
Publication date: 2010-06-03

Abstract

The present invention pertains to a method for assessing whether a subject with type 1 diabetes is eligible to an insulin-sensitizing therapy based on determining the amount of Retinol-binding protein 4 (RBP4) in a sample of said subject and comparing the, thus, determined amount with a reference amount. Moreover, the present invention relates to a method for monitoring a subject with type 1 diabetes who is on insulin-sensitizing therapy based on RBP4. Also encompasses by the present invention is a kit and a device adapted to carry out the method of the present invention.

Description

RBP4 as a marker in type 1 diabetes patients

The present invention pertains to a method for assessing whether a subject with type 1 diabetes is eligible to an insulin-sensitizing therapy. Said method is based on determining the amount of retinol-binding protein 4 (RBP4) in a sample of said subject and comparing the, thus, determined amount with a reference amount. Moreover, the present invention relates to a method for monitoring a subject with type 1 diabetes who is on insulin- sensitizing therapy based on RBP4. Also encompasses by the present invention is a kit and a device adapted to carry out the method of the present invention.

Diabetes mellitus is a disease of disordered metabolism resulting in abnormally high blood sugar levels. Patient with diabetes do not sufficiently produce or use the hormone insulin which is produced by the beta cells of the pancreas and which controls blood glucose levels.

Type 1 diabetes (previously also referred to as juvenile diabetes) develops due to an absent or reduced production of insulin leading to a deficiency of insulin. Generally, type 1 diabetes patients are treated with insulin (usually administered by an injection or by a pump). Moreover, patients with type 1 diabetes need to carefully monitor their blood sugar levels.

Type 2 diabetes develops as a consequence of a resistance to the effects of insulin. In early stages, type 2 diabetes is usually treated with lifestyle changes such as physical exercise and weight reduction. Later, type 2 diabetes patients may be treated with antidiabetic drugs, e.g. sulfonylureas or insulin-sensitizers.

Particularly, the administration of insulin-sensitizers such as metformin and thiazolinediones has been proven to be very beneficial for the therapy of subjects suffering from type 2 diabetes.

Metformin was approved for the treatment of patients with type 2 diabetes patients in 1995. Metformin improves insulin sensitivity and lowers blood glucose levels through various mechanisms including peripheral glucose uptake and storage in muscle, inhibition of gluconeogenesis in the liver and suppression of hepatic glycogenolysis (Khan et al, Diabetic medicine, 2006, 23: 1079-1084).

Thiazolidinediones are a drug class that lowers the blood sugar by increasing the sensitivity of cells to insulin. They increase the action of insulin in several tissues such as muscle and fat tissue and effectively reduce the HbAlc-level. They are thought to act by binding to peroxisome proliferator-activated receptors (PPARs). The only FDA approved use of thiazolidinediones is the treatment of type 2 diabetes.

Thus, metformin and thiazolidinediones are not approved for the treatment of type 1 diabetes patients. Metformin and thiazolidinediones were initially thought to be contraindicated for the treatment of type 1 diabetes since they require the presence of insulin.

However, there is recent evidence that metformin and thiazolidinediones also may be used for the treatment of type 1 diabetes. Since metformin and thiazolidinediones have been effective in improving blood glucose control in subjects with type 2 diabetes, it is thought that these drugs may also be of benefit in some, but not all, type 1 diabetic individuals (Strowig et al., Diabetes Care, 2005, Volume 28 (7), 1562-1567). For example, Strowig et al. (loc. cit.) showed that rosiglitazone in type 1 diabetic patients in combination with insulin resulted in an improved glycemic control and blood pressure without an increase in insulin requirement compared with insulin- and placebo treated subjects, whose improved glycemic control required an 11 % increase in insulin dose. In another study, Khan et al. (Diabetic medicine, 2006, 23: 1079-1084) showed that metformin can improve glycemic control and reduce the daily insulin dose in overweight people with type 1 diabetes.

However, Khan also suggests that type 1 diabetic patients receiving insulin sensitizers shall be carefully selected and supervised in order to avoid complications.

Insulin sensitizers have some adverse side effects. For example, troglitazone, the first thiazolidinedione to be approved by the FDA, can cause liver damage. Other known side effects of thiazolidinediones are elevations of LDL cholesterol levels and fluid retention. Thiazolidinediones are also suspected to cause oedema and are associated with an incidence of hepatocellular injury. Thiazolidinediones should not be administered to patients with heart disease and to patients who have abnormal hepatic function. Metformin is associated with a risk of lactic of lactic acidosis. Moreover, some subjects also experience significant gastrointestinal problems while using metformin, (see Strowig et al, Diabetes, Obesity and Metabolism. 2005 Nov;7(6):633-41).

Therefore, if insulin sensitizers shall be administered to type 1 diabetic subjects, it has to be considered whether the benefits of said therapy (particularly improved glycemic control and/or reduced insulin demand) outweigh the disadvantages (risk of adverse side effects and/or costs) or not. For example, if the administration of an insulin sensitizer would not improve glycemic control in an individual with type 1 diabetes, the administration of an insulin sensitizer is not recommended since i) it would put said individual at risk of adverse side effects ii) it would generate costs without any benefits for said subject. However, if said insulin sensitizer would improve glycemic control in an individual, then treatment with insulin sensitizers would be beneficial.

However, methods for reliably identifying those subjects with type 1 diabetes that will benefit from a therapy with insulin-sensitizers are still missing.

Thus, the technical problem underlying the present invention could be seen as the provision of means and methods for assessing whether a subject with type 1 diabetes is eligible to an insulin-sensitizing therapy. The technical problem is solved by the embodiments characterized in the accompanying claims and herein below.

Accordingly, the present invention relates to a method for assessing whether a subject with type 1 diabetes is eligible to an insulin-sensitizing therapy, comprising the steps of a) determining the amount of Retinol-Binding-Protein 4 (RBP4) in a sample of said subject, b) comparing the amount of RBP4 as determined in step a) with a reference amount, and c) assessing whether said subject is eligible to an insulin-sensitizing therapy.

The method of the present invention, preferably, is an in vitro method. Moreover, it may comprise steps in addition to those explicitly mentioned above. For example, further steps may relate to sample pre-treatments or evaluation of the results obtained by the method. The method may be carried out manually or assisted by automation. Preferably, step (a) and/or (b) may in total or in part be assisted by automation, e.g., by a suitable robotic and sensory equipment for the determination in step (a) or a computer-implemented comparison in step (b), or a computer-implemented assessment in step (c).

The term "assessing whether a subject is eligible to an insulin-sensitizing therapy" as used herein refers to determining whether a subject is susceptible/eligible to an insulin- sensitizing therapy or not. A subject who is eligible (is susceptible) to said therapy, preferably is a subject who will benefit from said therapy. Preferably, a subject benefits from an insulin-sensitizing therapy, if said therapy improves the glycemic control of said subject. More preferably, a subject benefits from an insulin-sensitizing therapy, if said therapy significantly improves the glycemic control of said subject. Preferably, a subject does not benefit from an insulin-sensitizing therapy, if said therapy does not improve glycemic control. If said therapy does not improve glycemic control of said subject, then said therapy would put said subject at risk of adverse side effects and/or said therapy would generate health care costs without any significant benefit to said subject (for adverse side effects of insulin sensitizers, see, e.g, Strowig et al., Diabetes, Obesity and Metabolism. 2005 Nov;7(6):633-41).

As mentioned above, the subject benefits from an insulin-sensitizing therapy, if glycemic control is improved. The term "improving glycemic control" is understood by the skilled person. Preferably, glycemic control is improved, if the insulin sensitizing therapy lowers postprandial and/or fasting blood glucose levels, reduces haemoglobin AIc levels, ameliorates glycosuria, reduces hepatic glucose outpout, and/or improves whole body glucose disposal. It is known in the art that the daily dose of insulin may be reduced, if glycemic control is improved. It is also known in the art that subjects with an improved glycemic control are at reduced risk of suffering from co-morbidities of diabetes such as neuropathy, nephropathy, fatty liver disease, retinopathy and diseases affecting the vacular system (e.g. microangiopathy).

As will be understood by those skilled in the art, the assessment whether a subject is eligible to an insulin-sensitizing therapy or not is usually not intended to be correct for 100% of the subjects to be assessed. The term, however, requires that assessment can be made for a statistically significant portion of subjects in a proper and correct manner. Whether a portion is statistically significant can be determined without further ado by the person skilled in the art using various well known statistic evaluation tools, e.g., determination of confidence intervals, p-value determination, Student's t-test, Mann- Whitney test etc.. Details are found in Dowdy and Wearden, Statistics for Research, John Wiley & Sons, New York 1983. Preferred confidence intervals are at least 90%, at least 95%, at least 97%, at least 98% or at least 99 %. The p-values are, preferably, 0.1, 0.05, 0.01, 0.005, or 0.0001. Preferably, the probability envisaged by the present invention allows that the assessment will be correct for at least 60%, at least 70%, at least 80%, or at least 90% of the subjects of a given cohort or population.

The term "subject" as used herein relates to animals, preferably mammals, and, more preferably, humans. However, it is envisaged by the present invention that the subject shall be suffering from diabetes mellitus, particularly type 1 diabetes mellitus. It is particularly contemplated that the subject is subjected to an insulin therapy (e.g. by injection or by a pump). Preferably, the subject has normal kidney function (with creatinine levels within the normal range. Moreover, the subject, preferably, shall not suffer from acute inflammatory disorders (when the sample is obtained and within the two weeks before the sample has been obtained). Is is also envisaged, that the patient, preferably, shall be clinically stable and asymptomatic, preferably, with respect to renal and cardiac disorders.

Type 1 diabetes (also known juvenile-onset or insulin-dependent), occurs when the pancreas does not produce enough insulin to properly control the blood sugar level. Insulin production is low or even absent because of autoimmune pancreatic beta-cell destruction possibly triggered by environmental exposure in genetically susceptible people. Destruction progresses subclinically over months or years until beta-cell mass decreases to the point that insulin concentrations are no longer adequate to control plasma glucose levels. The type 1 diabetes generally develops in childhood or adolescence and until recently was the most common form diagnosed before age 30; however, it can also develop in adults. Type 1 diabetes is thought to be an autoimmune disease.

How to diagnose type 1 diabetes as well as how to distinguish type 1 diabetes from type 2 diabetes is well known in the art. Generally, diabetes mellitus may be diagnosed by carrying out a blood sugar test (e.g. a fasting or a random blood sugar test). E.g., increased fasting blood sugar levels are indicative for diabetes in general, particularly a level larger than 125 mg/dL. Type 1 diabetes can be suspected if a subject has little or no ability to produce insulin, antibodies to insulin-producing cells are present in the blood of said subject, and/or if certain toxic acids that are a product the breakdown of fat (ketones) are increased in urine.

Type 1 diabetes is usually treated by administration of insulin (particularly by injection or by using an insulin pump). Administration of insulin allows for reducing ketosis, decreasing hyperglucagonemia, and normalizing lipid and protein metabolism.

As mentioned elsewhere herein, the method of the present invention allows for identifying those subjects with type 1 diabetes which are eligible to an insulin- sensitizing therapy.

The term "insulin-sensitizing therapy" as used herein, preferably, refers to a therapy that aims to improve the body's response to insulin. Preferably, said insulin-sensitizing therapy is by administration of an insulin-sensitizing drug. Preferably said insulin-sensitizing drug is metformin, more preferably, said insulin-sensitizing drug is a thiazolidinedione.

Thiazolidinediones are class a drug class that lowers the blood sugar by increasing the sensitivity of cells to insulin. They are thought to act by binding to peroxisome proliferator-activated receptors (PPARs). Preferred thiazolidinediones in the context of the present invention are selected from the group consisting of rosiglitazone (IUPAC name: 5- ((4-(2-(methyl-2-pyridinylamino) ethoxy)phenyl)methyl)- 2,4-thiazolidinedione), pioglitazone (IUPAC name: 5-((4-(2-(5-ethyl-2-pyridinyl)ethoxy)phenyl)methyl)-,(+-)-2,4- thiazolidinedion), troglitazone (IUPAC name: 5-(4-((6-hydroxy-2, 5,7,8- tetramethylchroman-2-yl-methoxy)benzyl)-2,4-thiazolidinedione)), MCC-555 (also know as netoglitazone) and rivoglitazone, and variants of the aforementioned compounds. Of the aforementioned thiazolidinedones, rosiglitazone and pioglitazones are the most preferred drugs.

Metformin (IUPAC name: N,N-dimethylimidodicarbonimidic diamide) is an oral antidiabetic drug from the biguanide class. Metformin was shown to improve hyperglycemia. Metformin is thought to act primarily through suppressing hepatic glucose production, especially hepatic gluconeogenesis (see e.g. Kirpichnikov D, McFarlane SI, Sowers JR (2002). "Metformin: an update". Ann Intern Med 137 (1): 25-33). Other insulin-sensitizing drugs contemplated by the present invention are disclosed in WO/2002/094744 which hereby is incorporated by reference with respect to its entire disclosure content.

The term "sample" refers to a sample of a body fluid, to a sample of separated cells or to a sample from a tissue or an organ. Samples of body fluids can be obtained by well known techniques and include, preferably, samples of blood, plasma, serum, or urine, more preferably, samples of blood, plasma or serum. Tissue or organ samples may be obtained from any tissue or organ by, e.g., biopsy. Separated cells may be obtained from the body fluids or the tissues or organs by separating techniques such as centrifugation or cell sorting. Preferably, cell-, tissue- or organ samples are obtained from those cells, tissues or organs which express or produce the peptides referred to herein.

The term "retinol binding protein 4", herein also referred to as RBP4 or as retinol binding protein, is well known in the art (see e.g. Newcomer ME, Ong DE (2000). "Plasma retinol binding protein: structure and function of the prototypic lipocalin.". Biochim. Biophys. Acta 1482 (1-2): 57-64). The term also encompasses variants of RBP4. Preferably, the term "RBP4" referred to in the context of the present invention relates to the human RBP4 polypeptide.

RBP4 is a polypeptide and a member of the lipocalins and is capable of binding to retinol. Specifically, RBP4 delivers retinol from the liver to the peripheral tissues. RBP4 is an adipokine, i.e. it is a cell-to-cell signalling molecule secreted by adipocytes. In plasma, the RBP-retinol complex interacts with transthyretin which prevents its loss by filtration through the kidney glomeruli. A deficiency of vitamin A blocks secretion of the binding protein posttranslationally and results in defective delivery and supply to the epidermal cells.

The sequence of RBP4 as well as methods for determining the amount of RBP4 are well known in the art (for the human sequence, e.g., see Genbank Ace. Number NM_006744.3 (nucleic acid sequence) / NP_006735.2 (protein sequence)).

RBP4 as used herein encompasses also variants of the aforementioned specific RBP4 polypeptide. Such variants have at least the same essential biological and immunological properties as the specific RBP4 polypeptide. In particular, they share the same essential biological and immunological properties if they are detectable by the same specific assays referred to in this specification, e.g., by ELISA assays using polyclonal or monoclonal antibodies specifically recognizing the said RBP4 polypeptide. Moreover, it is to be understood that a variant as referred to in accordance with the present invention shall have an amino acid sequence which differs due to at least one amino acid substitution, deletion and/or addition wherein the amino acid sequence of the variant is still, preferably, at least 50%, 60%, 70%, 80%, 85%, 90%, 92%, 95%, 97%, 98%, or 99% identical with the amino sequence of the specific RBP4 polypeptide (preferably over the entire length of said polypeptide). The degree of identity between two amino acid sequences can be determined by algorithms well known in the art. Preferably, the degree of identity is to be determined by comparing two optimally aligned sequences over a comparison window, where the fragment of amino acid sequence in the comparison window may comprise additions or deletions (e.g., gaps or overhangs) as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment. The percentage is calculated by determining the number of positions at which the identical amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity. Optimal alignment of sequences for comparison may be conducted by the local homology algorithm of Smith and Waterman Add. APL. Math. 2:482 (1981), by the homology alignment algorithm of Needleman and Wunsch J. MoI. Biol. 48:443 (1970), by the search for similarity method of Pearson and Lipman Proc. Natl. Acad Sci. (USA) 85 : 2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, BLAST, PASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group (GCG), 575 Science Dr., Madison, WI), or by visual inspection. Given that two sequences have been identified for comparison, GAP and BESTFIT are preferably employed to determine their optimal alignment and, thus, the degree of identity. Preferably, the default values of 5.00 for gap weight and 0.30 for gap weight length are used. Variants referred to above may be allelic variants or any other species specific homologs, paralogs, or orthologs. Moreover, the variants referred to herein include fragments or subunits of the specific RBP4 polypeptide or the aforementioned types of variants as long as these fragments have the essential immunological and biological properties as referred to above. Such fragments may be, e.g., degradation products of the RBP4 peptide. Further included are variants which differ due to posttranslational modifications such as phosphorylation or myristylation.

Preferably, a variant of RBP4 shall have the biological properties of RBP4, preferably, including its ability to bind to retinoids (preferably retinol) and/or its ability to bind to transthyretin. Whether a variant of RBP4 binds to retinoids or to transthyretin can be determined by well known methods. A particularly preferred method for determining whether a variant binds to retinoids or to transthyretin is described by Sharif et al. (2009, Time-resolved fluorescence resonance energy transfer and surface plasmon resonance- based assays for retinoid and transthyretin binding to retinol-binding protein 4, 2009, Analytical Biochemistry, Volume 392, Issue 2, Pages 162-168) which hereby is incorporated by reference with respect to its entire disclosure content.

Determining the amount of the polypeptides referred to in this specification relates to measuring the amount or concentration, preferably semi-quantitatively or quantitatively. Measuring can be done directly or indirectly. Direct measuring relates to measuring the amount or concentration of the polypeptide based on a signal which is obtained from the polypeptide itself and the intensity of which directly correlates with the number of molecules of the polypeptide present in the sample. Such a signal - sometimes referred to herein as intensity signal -may be obtained, e.g., by measuring an intensity value of a specific physical or chemical property of the polypeptide. Indirect measuring includes measuring of a signal obtained from a secondary component (i.e. a component not being the polypeptide itself) or a biological read out system, e.g., measurable cellular responses, ligands, labels, or enzymatic reaction products.

In accordance with the present invention, determining the amount of a polypeptide can be achieved by all known means for determining the amount of a polypeptide in a sample. Said means comprise immunoassay devices and methods which may utilize labeled molecules in various sandwich, competition, or other assay formats. Said assays will develop a signal which is indicative for the presence or absence of the polypeptide. Moreover, the signal strength can, preferably, be correlated directly or indirectly (e.g. reverse- proportional) to the amount of polypeptide present in a sample. Further suitable methods comprise measuring a physical or chemical property specific for the polypeptide such as its precise molecular mass or NMR spectrum. Said methods comprise, preferably, biosensors, optical devices coupled to immunoassays, biochips, analytical devices such as mass- spectrometers, NMR- analyzers, or chromatography devices. Further, methods include micro-plate ELISA-based methods, fully-automated or robotic immunoassays (available for example on Elecsys™ analyzers), CBA (an enzymatic Cobalt Binding Assay, available for example on Roche-Hitachi™ analyzers), and latex agglutination assays (available for example on Roche-Hitachi™ analyzers).

Preferably, determining the amount of a polypeptide comprises the steps of (a) contacting a cell capable of eliciting a cellular response the intensity of which is indicative of the amount of the polypeptide with the said polypeptide for an adequate period of time, (b) measuring the cellular response. For measuring cellular responses, the sample or processed sample is, preferably, added to a cell culture and an internal or external cellular response is measured. The cellular response may include the measurable expression of a reporter gene or the secretion of a substance, e.g. a peptide, polypeptide, or a small molecule. The expression or substance shall generate an intensity signal which correlates to the amount of the polypeptide.

Also preferably, determining the amount of a polypeptide comprises the step of measuring a specific intensity signal obtainable from the polypeptide in the sample. As described above, such a signal may be the signal intensity observed at an mass to charge (m/z) variable specific for the polypeptide observed in mass spectra or a NMR spectrum specific for the polypeptide.

Determining the amount of a polypeptide may, preferably, comprises the steps of (a) contacting the polypeptide with a specific ligand of RBP4, (b) (optionally) removing non- bound ligand, (c) measuring the amount of bound ligand. The bound ligand will generate an intensity signal. Binding according to the present invention includes both covalent and non-covalent binding.

A ligand according to the present invention can be any compound, e.g., a peptide, polypeptide, nucleic acid, or small molecule, binding to the RBP4 polypeptide. Preferred ligands include antibodies, nucleic acids, peptides or polypeptides such as receptors or binding partners for the polypeptide and fragments thereof comprising the binding domains for the peptides, and aptamers, e.g. nucleic acid or peptide aptamers. Methods to prepare such ligands are well-known in the art. For example, identification and production of suitable antibodies or aptamers is also offered by commercial suppliers. The person skilled in the art is familiar with methods to develop derivatives of such ligands with higher affinity or specificity. For example, random mutations can be introduced into the nucleic acids, peptides or polypeptides. These derivatives can then be tested for binding according to screening procedures known in the art, e.g. phage display. Antibodies as referred to herein include both polyclonal and monoclonal antibodies, as well as fragments thereof, such as Fv, Fab and F(ab)₂ fragments that are capable of binding antigen or hapten. The present invention also includes single chain antibodies and humanized hybrid antibodies wherein amino acid sequences of a non-human donor antibody exhibiting a desired antigen-specificity are combined with sequences of a human acceptor antibody. The donor sequences will usually include at least the antigen-binding amino acid residues of the donor but may comprise other structurally and/or functionally relevant amino acid residues of the donor antibody as well. Such hybrids can be prepared by several methods well known in the art. Preferably, the ligand or agent binds specifically to the polypeptide. Specific binding according to the present invention means that the ligand or agent should not bind substantially to ("cross-react" with) another peptide, polypeptide or substance present in the sample to be analyzed. Preferably, the specifically bound polypeptide should be bound with at least 3 times higher, more preferably at least 10 times higher and even more preferably at least 50 times higher affinity than any other relevant peptide or polypeptide. Non-specific binding may be tolerable, if it can still be distinguished and measured unequivocally, e.g. according to its size on a Western Blot, or by its relatively higher abundance in the sample. Binding of the ligand can be measured by any method known in the art. Preferably, said method is semi-quantitative or quantitative. Suitable methods are described in the following. First, binding of a ligand may be measured directly, e.g. by NMR or surface plasmon resonance.

Second, if the ligand also serves as a substrate of an enzymatic activity of the polypeptide of interest, an enzymatic reaction product may be measured (e.g. the amount of a protease can be measured by measuring the amount of cleaved substrate, e.g. on a Western Blot). Alternatively, the ligand may exhibit enzymatic properties itself and the "ligand/ polypeptide" complex or the ligand which was bound by the polypeptide, respectively, may be contacted with a suitable substrate allowing detection by the generation of an intensity signal. For measurement of enzymatic reaction products, preferably the amount of substrate is saturating. The substrate may also be labeled with a detectable lable prior to the reaction. Preferably, the sample is contacted with the substrate for an adequate period of time. An adequate period of time refers to the time necessary for a detectable, preferably measurable, amount of product to be produced. Instead of measuring the amount of product, the time necessary for appearance of a given (e.g. detectable) amount of product can be measured. Third, the ligand may be coupled covalently or non-covalently to a label allowing detection and measurement of the ligand. Labeling may be done by direct or indirect methods. Direct labeling involves coupling of the label directly (covalently or non-covalently) to the ligand. Indirect labeling involves binding (covalently or non-covalently) of a secondary ligand to the first ligand. The secondary ligand should specifically bind to the first ligand. Said secondary ligand may be coupled with a suitable label and/or be the target (receptor) of tertiary ligand binding to the secondary ligand. The use of secondary, tertiary or even higher order ligands is often used to increase the signal. Suitable secondary and higher order ligands may include antibodies, secondary antibodies, and the well-known streptavidin-biotin system (Vector Laboratories, Inc.). The ligand or substrate may also be "tagged" with one or more tags as known in the art. Such tags may then be targets for higher order ligands. Suitable tags include biotin, digoxygenin, His-Tag, Glutathion-S- Transferase, FLAG, GFP, myc-tag, influenza A virus haemagglutinin (HA), maltose binding protein, and the like. In the case of a peptide or polypeptide, the tag is preferably at the N-terminus and/or C-terminus. Suitable labels are any labels detectable by an appropriate detection method. Typical labels include gold particles, latex beads, acridan ester, luminol, ruthenium, enzymatically active labels, radioactive labels, magnetic labels ("e.g. magnetic beads", including paramagnetic and superparamagnetic labels), and fluorescent labels. Enzymatically active labels include e.g. horseradish peroxidase, alkaline phosphatase, beta-Galactosidase, Luciferase, and derivatives thereof. Suitable substrates for detection include di-amino-benzidine (DAB), 3,3'-5,5'-tetramethylbenzidine, NBT- BCIP (4-nitro blue tetrazolium chloride and 5-bromo-4-chloro-3-indolyl-phosphate, available as ready-made stock solution from Roche Diagnostics), CDP-Star™ (Amersham Biosciences), ECF™ (Amersham Biosciences). A suitable enzyme-substrate combination may result in a colored reaction product, fluorescence or chemoluminescence, which can be measured according to methods known in the art (e.g. using a light-sensitive film or a suitable camera system). As for measuring the enyzmatic reaction, the criteria given above apply analogously. Typical fluorescent labels include fluorescent proteins (such as GFP and its derivatives), Cy3, Cy5, Texas Red, Fluorescein, and the Alexa dyes (e.g. Alexa 568). Further fluorescent labels are available e.g. from Molecular Probes (Oregon). Also the use of quantum dots as fluorescent labels is contemplated. Typical radioactive labels include ³⁵S, ¹²⁵I, ³²P, ³³P and the like. A radioactive label can be detected by any method known and appropriate, e.g. a light-sensitive film or a phosphor imager. Suitable measurement methods according the present invention also include precipitation (particularly immunoprecipitation), electrochemiluminescence (electro-generated chemiluminescence), RIA (radioimmunoassay), ELISA (enzyme-linked immunosorbent assay), sandwich enzyme immune tests, electrochemiluminescence sandwich immunoassays (ECLIA), dissociation-enhanced lanthanide fluoro immuno assay (DELFIA), scintillation proximity assay (SPA), turbidimetry, nephelometry, latex- enhanced turbidimetry or nephelometry, or solid phase immune tests. Further methods known in the art (such as gel electrophoresis, 2D gel electrophoresis, SDS polyacrylamid gel electrophoresis (SDS-PAGE), Western Blotting, and mass spectrometry), can be used alone or in combination with labeling or other dectection methods as described above.

The amount of a polypeptide may be, also preferably, determined as follows: (a) contacting a solid support comprising a ligand for the polypeptide as specified above with a sample comprising the polypeptide and (b) measuring the amount of polypeptide which is bound to the support. The ligand, preferably chosen from the group consisting of nucleic acids, peptides, polypeptides, antibodies and aptamers, is preferably present on a solid support in immobilized form. Materials for manufacturing solid supports are well known in the art and include, inter alia, commercially available column materials, polystyrene beads, latex beads, magnetic beads, colloid metal particles, glass and/or silicon chips and surfaces, nitrocellulose strips, membranes, sheets, duracytes, wells and walls of reaction trays, plastic tubes etc. The ligand or agent may be bound to many different carriers. Examples of well-known carriers include glass, polystyrene, polyvinyl chloride, polypropylene, polyethylene, polycarbonate, dextran, nylon, amyloses, natural and modified celluloses, polyacrylamides, agaroses, and magnetite. The nature of the carrier can be either soluble or insoluble for the purposes of the invention. Suitable methods for fixing/immobilizing said ligand are well known and include, but are not limited to ionic, hydrophobic, covalent interactions and the like. It is also contemplated to use "suspension arrays" as arrays according to the present invention (Nolan 2002, Trends Biotechnol. 20(l):9-12). In such suspension arrays, the carrier, e.g. a microbead or microsphere, is present in suspension. The array consists of different microbeads or microspheres, possibly labeled, carrying different ligands. Methods of producing such arrays, for example based on solid-phase chemistry and photo-labile protective groups, are generally known (US 5,744,305).

The term "amount" as used herein encompasses the absolute amount of a polypeptide, the relative amount or concentration of the said polypeptide as well as any value or parameter which correlates thereto or can be derived therefrom. Such values or parameters comprise intensity signal values from all specific physical or chemical properties obtained from the said polypeptides by direct measurements, e.g., intensity values in mass spectra or NMR spectra. Moreover, encompassed are all values or parameters which are obtained by indirect measurements specified elsewhere in this description, e.g., response levels determined from biological read out systems in response to the peptides or intensity signals obtained from specifically bound ligands. It is to be understood that values correlating to the aforementioned amounts or parameters can also be obtained by all standard mathematical operations.

The term "comparing" as used herein encompasses comparing the amount of the polypeptide comprised by the sample to be analyzed with an amount of a suitable reference source specified elsewhere in this description. It is to be understood that comparing as used herein refers to a comparison of corresponding parameters or values, e.g., an absolute amount is compared to an absolute reference amount while a concentration is compared to a reference concentration or an intensity signal obtained from a test sample is compared to the same type of intensity signal of a reference sample. The comparison referred to in step (b) of the method of the present invention may be carried out manually or computer assisted. For a computer assisted comparison, the value of the determined amount may be compared to values corresponding to suitable references which are stored in a database by a computer program. The computer program may further evaluate the result of the comparison, i.e. automatically provide the desired assessment in a suitable output format. Based on the comparison of the amount determined in step a) and the reference amount, it is possible to assess whether a subject with type 1 diabetes is eligible to an insulin- sensitizing therapy. Therefore, the reference amount is to be chosen so that either a difference or a similarity in the compared amounts allows identifying those subjects are eligible to said therapy or which are not eligible to said therapy.

Accordingly, the term "reference amounts" as used herein refers to amounts of the polypeptides which allow assessing whether a subject with type 1 diabetes is eligible to an insulin-sensitizing therapy. Therefore, the reference may either be derived from (i) a subject with type 1 diabetes known to be eligible to said therapy or (ii) with type 1 diabetes known to be not eligible to said therapy. Moreover, the reference amounts, preferably, define thresholds. Suitable reference amounts or threshold amounts may be determined by the method of the present invention from a reference sample to be analyzed together, i.e. simultaneously or subsequently, with the test sample. A preferred reference amount serving as a threshold may be derived from the upper limit of normal (ULN), i.e. the upper limit of the physiological amount to be found in a population of subjects (e.g. patients enrolled for a clinical trial). The ULN for a given population of subjects can be determined by various well known techniques.

The inventors of the present invention surprisingly identified the following reference amounts and ULN, respectively.

The ULN for RBP4 referred to herein, preferably, varies between about 35 and about 100 mg/1 (preferably in a serum sample). Most preferably, it is about 46 mg/1.

Accordingly, a preferred reference amount for RBP4 in the context of the present invention defining a threshold amount for RBP4 as referred to in accordance with the present invention is about 38 mg/1 or about 75 mg/1 or, more preferably, about 46 mg/1. The term "about" as used herein encompasses a range of + and - 20% relative to the specific value, amount, concentration, level, etc, e.g. indication of a value of "about 100" is meant to encompass a value of a numerical range of 100 +/- 20%, i.e. a value range from 80 to 120. Preferably the term "about" encompasses a range of + and - 10% relative to the specific value, amount, concentration, level, etc, more preferably a range of + and - 5% relative to the specific value, amount, concentration, level, etc, and most preferably the exact value, amount, concentration, level etc.

Preferably, an amount of RBP4 in a sample of a subject larger than the reference amount is indicative for a subject (with type 1 diabetes) being eligible to an insulin-sensitizing therapy.

Preferably, an amount of RBP4 in a sample of a subject lower than the reference amount is indicative for a subject (with type 1 diabetes) not being eligible to an insulin-sensitizing therapy.

Reference amounts of a RBP4 can be easily confirmed or established, and the level of the marker in a patient sample can simply be compared to the reference amount. The sensitivity and specificity of a diagnostic and/or prognostic test depends on more than just the analytical "quality" of the test-they also depend on the definition of what constitutes an abnormal result. In practice, Receiver Operating Characteristic curves, or "ROC" curves, are typically calculated by plotting the value of a variable versus its relative frequency in a population that is eligible to an insulin-sensitizing therapy and a population that is not eligible to said therapy. For any particular marker, a distribution of marker levels for subjects will likely overlap. Under such conditions, a test does not absolutely distinguish "eligible" from "not eligible" with 100% accuracy, and the area of overlap indicates where the test cannot distinguish "eligible" from "not eligible". A threshold is selected, above which (or below which, depending on how a marker changes) the test is considered to be abnormal and below which the test is considered to be normal. The area under the ROC curve is a measure of the probability that the perceived measurement will allow correct identification of subject being eligible to an insulin-sensitizing therapy. ROC curves can be used even when test results do not necessarily give an accurate number. As long as one can rank results, one can create an ROC curve. For example, results of a test on "eligible" samples might be ranked according to degree (say l=low, 2=normal, and 3=high). This ranking can be correlated to results in the "not eligible" population, and a ROC curve created. These methods are well known in the art. See, e.g., Hanley et al, Radiology 143: 29-36 (1982). In certain embodiments, markers are selected to exhibit at least about 70% sensitivity, more preferably at least about 80% sensitivity, even more preferably at least about 85% sensitivity, still more preferably at least about 90% sensitivity, and most preferably at least about 95% sensitivity, combined with at least about 70% specificity, more preferably at least about 80% specificity, even more preferably at least about 85% specificity, still more preferably at least about 90% specificity, and most preferably at least about 95% specificity. In particularly preferred embodiments, both the sensitivity and specificity are at least about 75%, more preferably at least about 80%, even more preferably at least about 85%, still more preferably at least about 90%, and most preferably at least about 95%. The term "about" in this context refers to +/- 5% of a given measurement.

As set forth above, a reference may preferably, obtained from a sample from a subject known to be eligible to an insulin sensitizing therapy or a subject known not to be eligble therefor. The reference can also be the average or mean obtained from a group of such samples. The reference results may be obtained by applying the method of the present invention. The absolute or relative amounts of RBP4 of said individuals of the population can be determined as specified elsewhere herein. How to calculate a suitable reference value, preferably, the average or median, is well known in the art. The population of subjects referred to before shall comprise a plurality of subjects, preferably, at least 5, 10, 50, 100, 1,000 or 10,000 subjects. It is to be understood that the subject to be assessed by the method of the present invention and the subjects of the said plurality of subjects are of the same species.

It is further contemplated that a "reference" will be obtained by determining the amount of RBP4 in a group of reference subjects, i.e. a group of subjects known to be eligible to an insulin sensitizing therapy, a group of subjects known not to be eligible to an insulin sensitizing therapy, and calculating the reference by appropriate statistic measures including those referred to elsewhere herein, such as median, average, quantiles, PLS-DA, logistic regression methods, random forest classification or others that give a threshold value. The threshold value should take the desired clinical settings of sensitivity and specificity of the test into consideration.

It is also envisaged that the assessment whether a subject is eligible to an insulin sensitizing therapy can be carried out on the degree of identity or similarity between the test results obtained from the test sample and the aforementioned reference results, i.e. based on an identical or similar amount with respect to RBP4. The results of the test sample and the reference results are identical, if the values for the characteristic features and, in the case of quantitative determination, the intensity values are identical. Said results are similar, if the values of the characteristic features are identical but the intensity values are different. Such a difference is, preferably, not significant and shall be characterized in that the values for the intensity are within at least the interval between 1st and 99th percentile, 5th and 95th percentile, 10th and 90th percentile, 20th and 80th percentile, 30th and 70th percentile, 40th and 60th percentile of the reference value, the 50th, 60th, 70th, 80th, 90th or 95th percentile of the reference value.

It is also contemplated in the context of the method of the present invention, that the assessment may be based on differences between the test results obtained from the test sample and the aforementioned reference results. The same applies if a calculated reference as specified above is used. The difference, preferably, shall be an increase in the absolute or relative amount of a diagnostic marker according to present invention. Preferably, the increase in the relative or absolute amount is significant, i.e. outside of the interval between 45th and 55th percentile, 40th and 60th percentile, 30th and 70th percentile, 20th and 80th percentile, 10th and 90th percentile, 5th and 95th percentile, 1st and 99th percentile of the reference value.

The experiments carried out in the context of the present invention strongly suggest that determining the amount of RBP4 in a sample of a subject and comparing the, thus, determined amount with a suitable reference amount is required in order to reliably assess whether a subject suffering from type 1 diabetes is eligible to an insulin-sensitizing therapy (see, e.g., Examples). Particularly, an amount of RBP4 in a sample of said subject larger than the reference amount indicates that said subject is eligible to said therapy, and, thus, will benefit thereof. Particularly, an amount of RBP4 in a sample of said subject lower than the reference amount indicates that said subject is not eligible to said therapy, and, thus, will not benefit thereof. Specifically, in the context of the present invention the amount of RBP4 was determined in cohorts of subjects with type 1 diabetes, with type 2 diabetes, with a metabolic syndrome and in healthy subjects. It was shown that the median of RBP4 was significantly increased in subjects with type 2 diabetes and a metabolic syndrome compared with healthy subject and subjects with type 1 diabetes. Interestingly, in the cohort of type 1 diabetic patients, there was a subgroup of subjects that had significantly elevated RBP4 levels. These subjects will benefit from an insulin sensitizing therapy. Particularly, an insulin sensitizing therapy would improve the glycemic control in said subject. Type 1 diabetic subjects with amounts of RBP4 lower than the reference amounts, are not eligible to an insulin sensitizing therapy. Those subjects would not benefit from said therapy, since the therapy would not improve the glycemic control in said subject, but would put the subject at risk of adverse side effects (and would increase health care costs). Of course, subjects benefiting from an insulin-sensitizing therapy are also at risk of adverse side effects, but here the benefits outweigh the disadvantages of said therapy.

The definition and explanations given herein above apply mutatis mutandis to the following (except stated otherwise).

The present invention also relates to a method for monitoring a subject with type 1 diabetes on insulin-sensitizing therapy (preferably subjected to insulin-sensitizing therapy), comprising the steps of a) determining the amount of Retinol Binding Protein 4 (RBP4) in a first sample of said subject, b) determining the amount of Retinol Binding Protein 4 (RBP4) in a second sample of said subject, and c) comparing the amount of RBP4 in said first sample with the amount of RBP4 in said second sample, wherein a decrease of the amount in the second sample compared with the amount in the first sample indicates that said subject benefits from said therapy.

Said method, preferably, is an in vitro method. Moreover, it may comprise steps in addition to those explicitly mentioned above. For example, further steps may relate to sample pre- treatments or evaluation of the results obtained by the method. The method of the present invention preferably is used for monitoring a subject being on insulin-sensitizing therapy (and, thus, for assessing whether said subject benefits from said therapy or not). However, the method of the present invention may also be used for confirmation, and subclassification of said subject. The method may be carried out manually or assisted by automation. Preferably, step (a), (b) and/or (c) may in total or in part be assisted by automation, e.g., by a suitable robotic and sensory equipment for the determination in steps (a) and (b), or a computer-implemented comparison in step (c).

The subject in the context of the aforementioned method shall receive an insulin- sensitizing therapy as described elsewhere herein.

The term "monitoring" as used herein, preferably, relates to assessing the effects of the insulin-sensitizing therapy for a subject suffering from type 1 diabetes. Monitoring said subject allows for assessing whether said subject benefits from said therapy. Preferably, by carrying out the aforementioned method of the present invention decisions can be made whether the insulin-sensitizing therapy shall be continued, stopped or amended (for an explanation of the term "benefit", see elsewhere herein).

As will be understood by those skilled in the art, the assessment whether a subject is eligible to an insulin- sensitizing therapy or not, is usually not intended to be correct for all (i.e. 100%) of the subjects to be monitored. The term, however, requires that a statistically significant portion of subjects can be correctly monitored (e.g. a cohort in a cohort study). Whether a portion is statistically significant can be determined without further ado by the person skilled in the art using various well known statistic evaluation tools, e.g., determination of confidence intervals, p-value determination, Student's t-test, Mann- Whitney test etc.. Details are found in Dowdy and Wearden, Statistics for Research, John Wiley & Sons, New York 1983. Preferred confidence intervals are at least 90%, at least 95%, at least 97%, at least 98% or at least 99 %. The p-values are, preferably, 0.1, 0.05, 0.01, 0.005, or 0.0001. More preferably, at least 60%, at least 70%, at least 80% or at least 90% of the subjects of a population can be properly monitored by the method of the present invention.

Preferably, the first sample in the context of the present invention is obtained prior to the start of the insulin-sensitizing therapy. More preferably, the first sample is obtained shortly prior to said therapy. Even more preferably, the sample is obtained within about 1 hour, within about 12 hours, within about 24 hours, within about one week, or within about 2 weeks before the said therapy is started. Since the aforementioned method of the present invention comprises the assessment of changes of the amount of RBP4 that are caused by the treatment regimen, the first sample may also be obtained after the start of the therapy (but before the second sample is obtained).

Thus, the second sample, preferably, is obtained (i) after the first sample, and preferably (ii) after the start of the insulin-sensitizing therapy. Regarding (i), it is particularly contemplated that the second sample is obtained after a reasonable period of time after obtaining the first sample, preferably after about 1 hour, after about 12 hours, after about

24 hours, after about one week, or after about 2 weeks. Regarding (ii), it is particularly contemplated that the second sample is obtained after a reasonable period of time after the start of the insulin-sensitizing therapy, preferably after about 1 hour, after about 12 hours, after about 24 hours, after about one week, or after about 2 weeks. It is to be understood, that the amount of RBP4 referred herein, does not instantly change (e.g. within 1 minute or 1 hour) after the therapy is started. Therefore, "reasonable" in this context refers to intervals between obtaining the first and second sample which intervals allow the biomarker(s) to adjust. Therefore (with respect to (i)), the second sample, preferably, is obtained at least one month after said first sample, at least two months after said first sample, at least three months after said first sample, at least four months after said first sample, at least six months, or at least 12 months after the first sample was obtained (or after treatment initiation). It is particularly contemplated to obtain the second sample one month after said first sample (and/or after treatment initiation).

It is also envisaged to assess the time course of the amount of a RBP4. Accordingly, the aforementioned method may comprise the additional step of determining the amount of a said marker in at least one further sample from said subject (thus, in a third sample, in a fourth sample, in a fifth sample etc.) and comparing the, thus, determined amount with the amount of RBP4 in said first sample and/or in said second sample and/or in any sample that was obtained before said at least one further sample was obtained. For preferred time intervals for obtaining the samples, please see above.

As mentioned above, the assessment whether a subject with type 1 diabetes benefits from an insulin-sensitizing therapy is based on the comparison of the amount of RBP4 in one sample of a subject (preferably, in the first sample) with amount of the respective marker in another sample of that subject (preferably, in said second sample) that is obtained after a reasonable time interval after said one, preferably, said first sample.

Preferably, a decrease and, more preferably, a significant decrease, and, most preferably, a statistically significant decrease of the amount of RBP4 in the second sample compared with the first sample is indicative for a subject who benefits from an insulin-sensitizing therapy.

Particularly, a significant decrease is decrease of a size which is considered to be significant for diagnosis, particularly said decrease is considered statistically significant. The terms "significant" and "statistically significant" are known by the person skilled in the art. Thus, whether a decrease is significant or statistically significant can be determined without further ado by the person skilled in the art using various well known statistic evaluation tools. Preferred significant decreases of the amount of a RBP4, preferably in a blood, blood serum or blood plasma sample, which shall be indicative for a subject who benefits from an insulin- sensitizing therapy are given below.

According to the invention, a decrease of the amount of RBP4 in the second sample compared to the amount in the first sample (or in a sample compared with the amount in a sample that was obtained earlier), preferably, of at least about 7.5 mg/1 RBP4 polypeptide, more preferably of at least about 15 mg/1 and, even more preferably, of at least about 20 mg/1, and most preferably, of at least about 25 mg/1 is considered to be significant and, thus, to be indicative for a subject with type 1 diabetes who benefits from an insulin- sensitizing therapy. Preferably, the aforementioned decreases are drawn to an interval of about 8 weeks or, more preferably, of about 4 weeks of treatment (or to be more precisely to an interval of about 8 weeks or, more preferably, of about 4 weeks between the first and second sample).

It is to be understood that the preferred decreases of the absolute amount of RBP4, preferably, depend on the amount of RBP4 in the first sample of subject. Generally, the larger the amount of RBP4 in the first sample, the larger shall be the decrease of the absolute amount of RBP4.

Preferably, the percentage decrease is determined. If the percentage decrease is determined, a decrease of the amount of a RBP4 in the second sample compared to the amount in the first sample, preferably, of at least about 15 %, of at least about 20 %, more preferably of at least about 25 %, and even more preferably, of at least about 30 %, of at least about 40 %, and most preferably of at least about 50 % is considered to be significant and, thus, to be indicative for a subject who benefits from an insulin- sensitizing therapy. Preferably, the aforementioned decreases are drawn to an interval of about 8 weeks or, more preferably, of about 4 weeks of treatment (or to be more precisely to an interval of about 8 weeks or, more preferably, of about 4 weeks between the first and second sample).

It is to be understood that an increase of the amount of RBP4 in the second sample compared with the first sample (or unchanged amounts of the amount of RBP4 in the second sample compared with the first sample) is (are) indicative for a subject with type 1 diabetes who does not benefit from said therapy.

The experiments carried out in the context of the present invention strongly suggest that determining the amount of RBP4 in a first and a second sample of a subject who suffers from type 1 diabetes and who is on an insulin-sensitizing therapy, and comparing said amount of RBP4 in said first sample with the amount in said second sample, allows for monitoring the effect of an insulin-sensitizing therapy. Particularly, said therapy is effective, if the amount of RBP4 decreases in the course of the therapy. Said therapy is not effective, if the amount of RBP4 increases or remains unchanged in the course of the therapy. If the therapy is effective for an individual subject, an improvement of glycemic control can be assumed. Thus, a continuation of the insulin-sensitizing therapy is indicated. If the therapy is not effective for an individual subject, it can be assumed that the glycemic control was not improved by the therapy. In order not to put said subject at further risk of adverse side effects and in order to reduce costs, a stop of the therapy shall be considered.

Moreover the present invention envisages a device and a kit for carrying out the method of the present invention.

Accordingly, the present invention relates to a device for assessing whether a subject with type 1 diabetes is eligible to an insulin- sensitizing therapy, comprising means for determining the amount of retinol binding protein 4 in a sample of the subject and means for comparing said amount to a reference amount, whereby it is assessed whether said subject is eligible to an insulin- sensitizing therapy.

Moreover, the present invention relates to a device for monitoring a subject with type 1 diabetes being on insulin-sensitizing therapy, comprising means for determining the amount of a RBP4 in a first sample and in a second sample of said subject, and means for comparing the amount in said first sample with the amount in said second sample determined by said means, whereby it is assessed whether said subject benefits from said therapy.

The term "device" as used herein relates to a system of means comprising at least the aforementioned means operatively linked to each other as to allow the assessment according to the present invention. Preferred means for determining the amount of a one of the aforementioned polypeptides as well as means for carrying out the comparison are disclosed above in connection with the method of the invention. How to link the means in an operating manner will depend on the type of means included into the device. For example, where means for automatically determining the amount of the peptides are applied, the data obtained by said automatically operating means can be processed by, e.g., a computer program in order to obtain the desired results. Preferably, the means are comprised by a single device in such a case. Said device may accordingly include an analyzing unit for the measurement of the amount of the polypeptides in an applied sample and a computer unit for processing the resulting data for the evaluation. The computer unit, preferably, comprises a database including the stored reference amounts or values thereof recited elsewhere in this specification as well as a computer-implemented algorithm for carrying out a comparison of the determined amounts for the polypeptides with the stored reference amounts of the database. Computer-implemented as used herein refers to a computer-readable program code tangibly included into the computer unit. Alternatively, where means such as test stripes are used for determining the amount of the peptides or polypeptides, the means for comparison may comprise control stripes or tables allocating the determined amount to a reference amount. The test stripes are, preferably, coupled to a ligand which specifically binds to the peptides or polypeptides referred to herein. The strip or device, preferably, comprises means for detection of the binding of said peptides or polypeptides to the said ligand. Preferred means for detection are disclosed in connection with embodiments relating to the method of the invention above. In such a case, the means are operatively linked in that the user of the system brings together the result of the determination of the amount and the diagnostic or prognostic value thereof due to the instructions and interpretations given in a manual. The means may appear as separate devices in such an embodiment and are, preferably, packaged together as a kit. The person skilled in the art will realize how to link the means without further ado. Preferred devices are those which can be applied without the particular knowledge of a specialized clinician, e.g., test stripes or electronic devices which merely require loading with a sample. The results may be given as output of raw data which need interpretation by the clinician. Preferably, the output of the device is, however, processed, i.e. evaluated, raw data the interpretation of which does not require a clinician. Further preferred devices comprise the analyzing units/devices (e.g., biosensors, arrays, solid supports coupled to ligands specifically recognizing the polypeptides referred to herein, Plasmon surface resonace devices, NMR spectrometers, mass- spectrometers etc.) and/or evaluation units/devices referred to above in accordance with the method of the invention.

Moreover, the present invention relates to a kit for assessing whether a subject with type 1 diabetes is eligible to an insulin sensitizing therapy, said kit comprising instructions for carrying out the said assessment and means for determining the amount of retinol binding protein 4 in a sample of the subject and means for comparing said amount to a reference amount, allowing assessing whether a subject with type 1 diabetes is eligible to an insulin sensitizing therapy. Moreover, the present invention relates to a kit for monitoring a subject with type 1 diabetes being on insulin- sensitizing therapy, comprising means for determining the amount of a RBP4 in a first sample and in a second sample of said subject, and means for comparing the amount in said first sample with the amount in said second sample determined by said means, whereby it is assessed whether said subject benefits from said therapy.

The term "kit" as used herein refers to a collection of the aforementioned means, preferably, provided in separately or within a single container. The container, also preferably, comprises instructions for carrying out the method of the present invention.

Also, the present invention relates to the use of RBP4, in a sample of a subject with type 1 diabetes, for assessing whether said subject is eligible to an insulin-sensitizing therapy.

In addition, the present invention relates to the use of RBP4, in a sample of a subject with type 1 diabetes being on insulin-sensitizing therapy, for monitoring whether said subject benefits from said therapy.

The present invention also envisages the use of a ligand of RBP4 for assessing, in a sample of subject with type 1 diabetes, whether said subject is eligible to an insulin-sensitizing therapy.

Moreover, the present invention relates to the use of RBP4 for preparing a diagnostic composition for assessing whether a subject with type 1 diabetes is eligible to an insulin- sensitizing therapy.

Preferred ligands of RBP4 are described elsewhere herein.

In yet another aspect, it is provided a method for assessing whether a subject with type 1 diabetes is eligible to an insulin- sensitizing therapy, comprising the steps of a) determining the weight of said subject, b) comparing weight of step a) with a reference amount, and c) assessing whether said subject is eligible to an insulin- sensitizing therapy, wherein a subject having overweight or being obese is eligible to an insulin-sensitizing therapy. In yet another aspect the invention relates to a method for diagnosing whether a diabetes patient is suffering from a cardiovascular complication or is at risk of suffering from a cardiovascular complication, comprising the steps of a) measuring, preferably in vitro, the level(s) of at least one cardiac hormone (e.g. NT- proBNP) in a sample from the patient, b) diagnosing the cardiovascular complication or the risk of suffering from cardiovascular complication by comparing the measured level(s) to known level(s) associated with the cardiovascular complication or the risk.

Moreover, it is provided a method for diagnosing whether a diabetes patient is suffering from a microangiopathy or is at risk of suffering from a microangiopathy, comprising the steps of a) measuring in vitro the level(s) of PLGF or PLGF-I variant in blood serum, blood or blood plasma sample from the patient, b) diagnosing microangiopathy or the risk of suffering from microangiopathy by comparing the measured level(s) to known level(s) associated with microangiopathy or the risk.

All references cited in this specification are herewith incorporated by reference with respect to their entire disclosure content and the disclosure content specifically mentioned in this specification.

The following Examples shall merely illustrate the invention. They shall not be construed, whatsoever, to limit the scope of the invention.

Example 1

The amount of RBP4 was determined in serum samples in i) 49 subjects with type 2 diabetes, ii) 1011 subjects with type 1 diabetes, iii) 356 subjects with a metabolic syndrome and iv) 1907 subjects showing no symptoms of diabetes by using the "Retionol- binding (RBP)/RBP4 Elisa Kit" (Immundiagnostik, Bensheim, Germany). The medians for RBP4 for the cohorts are shown in table 1. Table 1

It was shown that the cohort including subjects with type 2 diabetes and the cohort including subjects with a metabolic syndrome had increased levels of RBP4, compared with the other two cohorts (subjects with type 1 diabetes and subjects showing no symptoms of diabetes). Moreover, the majority of subjects with type 1 diabetes had lower RBP4 levels than the other three groups. However, a subgroup of subjects with type 1 diabetes had RBP4 levels that were comparable with the levels in subjects with type 2 diabetes and in subjects with a metabolic syndrome (85^th percentile diabetes type 1 patients, 46.4 mg/1, some type 1 diabetes patients had even RBP4 amounts of larger than 100 mg/1). Most likely, patients belonging to said subgroup will benefit from an insulin- sensitizing therapy, whereas subjects with type 1 diabetes having low levels of RBP will not benefit from said therapy.

Thus, carrying out the method of the present invention would allow for identifying those patients with type 1 which will benefit from an insulin-sensitizing therapy.

Example 2

A male subject (34 years) with type 1 diabetes receives a daily amount of 80 IU insulin. Despite the administration of large amounts of insulin, the blood sugar level and the HbAIc level remain high (HbAIc: 8.5%). Moreover, the administration of even larger amounts of insulin does not improve glycemic control.

The amount of RBP4 is determined in a serum sample of the patient (72 mg/1) indicating that the RBP4 level is elevated above the reference amount. A therapy is initiated including the administration of both insulin and the insulin sensitizer metformin. After 2 months, the serum RBP4-level is 45 mg/1. After 3 months of therapy, the daily requirement of insulin is signifϊcantly reduced (48 IU per day), the blood sugar level is improved, and the HbAlC level of 6.5 % indicates a significantly improved glycemic control.

Example 3

A 37 years old male patient (HbAIc: 7.1%, without a pathological 24 h blood sugar profile) with type 1 diabetes requires a daily dosage of 84 units of insulin. A therapy with pioglitazone is initiated. However, the therapy does neither effect the required insulin dosage nor the 24 h blood glucose profile, nor the HbAIc level (7.0%). After two months of therapy the amount of RBP4 is determined in a serum sample of the patient (34 mg/1, i.e. a level below the reference amount). Moreover, RBP4 is determined in a sample that was obtained at the start of therapy with pioglitazone (35 mg/1).

After three months of therapy, the patient suffers from oedema, a common side effect of therapy with piogliazone. The therapy is stopped. A new therapy with metformin is started. This therapy is also stopped after three more months since the therapy did not improve of the condition of the subject.

Claims

1. A method for assessing whether a subject with type 1 diabetes is eligible to an insulin-sensitizing therapy, comprising the steps of a) determining the amount of Retinol Binding Protein 4 (RBP4) in a sample of said subject, b) comparing the amount of RBP4 as determined in step a) with a reference amount, and c) assessing whether said subject is eligible to an insulin-sensitizing therapy.

2. The method of claim 1, wherein said subject is human.

3. The method of claim 1 and 2, wherein said sample is a serum sample.

4. The method of any one of claims 1 to 3, wherein said insulin-sensitizing therapy is administration of at least one insulin-sensitizer.

5. The method of claim 4, wherein said insulin-sensitizer is selected from the group consisting of metformin and thiazolinediones.

6. The method of any one of claims 1 to 5, wherein an amount of RBP4 larger than the reference amount is indicative for a subject being eligible to an insulin-sensitizing therapy.

7. The method of any one of claims 1 to 5, wherein an amount of RBP4 lower than the reference amount is indicative for a subject not being eligible to an insulin- sensitizing therapy.

8. The method of any one of claims 1 to 7, wherein said reference amount is between about 35 and about 100 mg/1.

9. The method of claim 1 , wherein instead of RBP4 the weight of the subject is determined and said subject is considered to be eligible to insulin-sensitizing therapy if the subject has overweight.

10. A device for assessing whether a subject with type 1 diabetes is eligible to an insulin- sensitizing therapy, comprising means for determining the amount of RBP4 in a sample of the subject and means for comparing said amount to a reference amount.

11. A kit for carrying out the method of any one of claims 1 to 9, comprising instructions for carrying out the said method and means for determining the amount of RBP4 in a sample of the subject and means for comparing said amount to a reference amount.

12. Use of RBP4 in a sample of a subject with type 1 diabetes, or of a ligand of RBP4, for assessing whether said subject is eligible to an insulin-sensitizing therapy.

13. A method for monitoring a subject with type 1 diabetes on insulin- sensitizing therapy, comprising the steps of a) determining the amount of Retinol Binding Protein 4 (RBP4) in a first sample of said subject, b) determining the amount of Retinol Binding Protein 4 (RBP4) in a second sample of said subject, and c) comparing the amount of RBP4 in said first sample with the amount of RBP4 in said second sample, wherein a decrease of the amount in the second sample compared with the amount in the first sample indicates that said subject benefits from said therapy.