MEDICAL CONDITIONS

FIELD OF THE INVENTION

The present invention relates to methods and compositions for diagnosing and/or monitoring non-infectious medical conditions, or a predisposition to develop such conditions. The methodological approach is based on the detection and/or (quantitative) analysis of one or more polyamines or salts or derivatives thereof.

BACKGROUND

Polyamines are alkylamine compounds with two or more (primary or secondary) amino groups. Well known types of polyamines occurring in the human body include putrescine (H ₂ N(CH2)4NH ₂ ), cadaverine (H ₂ N(CH2)5NH ₂ ), spermidine (H ₂ N(CH2)4NH(CH2)3NH ₂ ), and spermine (H2N(CH2) ₃ NH(CH2)4NH(CH2) ₃ NH ₂ ) as well as acetylated variants thereof.

Polyamines are synthesized in cells via highly-regulated pathways such as the ornithin carboxylase, arginine decarboxylase, lysine decarboxylase, spermidine synthase, spermidine synthase and spermine/spermidine acetyltransferase pathways (reviewed, e.g., in Wang and Casero (2006) J. Biochem. 139, 17-25; Flamigni et al. (2007) Amino Acids 33, 197-202; Pegg (2008) Am. J. Physiol. Endocrinol. Metab. 294, E995-E1010).

However, the actual functions polyamines exert in cells are still not fully unravelled. If cellular polyamine synthesis is inhibited, cell growth is stopped or severely retarded. The provision of exogenous polyamines restores the growth of these cells (see the references above). Most eukaryotic cells have a polyamine transporter system on their cell membrane that facilitates the internalization of exogenous polyamines. This system is highly active in rapidly proliferating cells and is the target of some chemotherapeutics currently under development (Wang et al. (2003) J. Med. Chem. 46, 5129-5138). Polyamines are also important modulators of a variety of ion channels, including NMDA receptors and AMPA receptors. They block inward-rectifier potassium channels so that the currents of the channels are inwardly rectified, and thereby the cellular energy is conserved. Recently, polyamines haven been shown to enhance the permeability of the blood-brain barrier (Zhang et al. (2009) J. Med. Chem. 52, 1514-1517).

It has thus been speculated that alterations of the polyamine metabolism is linked to the etiology and/or progression of various diseases, in particular to cancer. However, in most studies so far, the expression levels and/or the activities of the enzymes involved in polyamine metabolisms have been used as molecular markers for monitoring a disease (see, for example, the patent publications WO 2007/048076 A2, EP 0 558 338 A2, and WO 2006/038089 A2, all relating to tumor diagnostics).

European patent publication EP 1 729 129 A1 relates to a method for diagnosing stroke or asymptotic cerebral infarction, wherein also the total polyamine concentration present in a test sample is used as a marker. US patent publication US 2009/0263400 A2 relates to the use of total polyamines for diagnosing osteoporosis.

It is known in the art that metabolism of polyamines are activated in association with cell proliferation. In fact, polyamine contents tend to increase in various tumor tissues as compared to normal tissues. A kit which is capable of measuring the total polyamine content in urine or in blood by an enzymatic determination method has already been commercialized (Labo-Search PolyamineAuto; AIT Corporation). However, it has become clear that a relatively large number of false negative results are found in malignant tumor patients and that total polyamine significantly increases in association with not only malignant tumors but also with various diseases and conditions such as inflammatory diseases, cardiac infarction, cirrhosis, process of curing of wounds, etc.

Thus, it is considered that total polyamine cannot be evaluated as a specific biomarker. Furthermore, the sensitivity of the above enzymatic methods for determinant polyamine levels is limited, which becomes a critica factor when assessing samples having only very low polyamine levels, such as human serum. From the above it is immediately evident that a necessary prerequisite for a successful therapy of (potentially life threatening) diseases (such as, diabetes, cardiovascular diseases, immune- and inflammatory diseases, neuronal diseases, tumors, and the general state of critical illness) is the provision of accurate methods for diagnosing, staging and/or monitoring progression of such medical conditions which, in turn, enable a reliable prognosis and risk assessment, and thus the selection of an appropriate therapy.

Accordingly, there still remains a need for improved methods and compositions that enable the rapid, reliable and cost-saving diagnosis, staging, and monitoring of medical conditions or a predisposition to develop such conditions.

Thus, it is an object of the present invention to provide such methods and compositions. SUMMARY OF THE INVENTION

In one aspect, the present invention relates to a method for diagnosing and/or monitoring in a subject a non-infectious medical condition or a predisposition to develop a noninfectious medical condition, comprising: detecting and/or analyzing in a test sample derived from the subject one or more polyamines or one or more salts or derivatives thereof, wherein the presence or quantity of any one of the polyamines or the salts or derivatives thereof in the test sample is indicative of the non-infectious medical condition or the predisposition to develop a non-infectious medical condition.

In specific embodiments, the method further comprises: comparing the results obtained in the test sample with a reference. The reference may preferably be a control sample derived from the subject to be analyzed.

In further preferred embodiments, the method further comprises: quantifying the levels of the one or more polyamines or the one or more salts or derivatives thereof in the test sample, wherein an altered level of any one the polyamines or the salts or derivatives thereof in the test sample as compared to the reference is indicative of the non-infectious medical condition or a predisposition to develop such condition. The method may further comprise correlating with each other the quantified levels of the one or more polyamines or the one or more salts or derivatives thereof obtained. In other preferred embodiments, the one or more polyamines or the salts or derivatives thereof that have altered levels in the test sample as compared to the reference together represent a signature that is indicative of a medical condition or a predisposition to develop a medical condition.

In further specific embodiments, at least one of the one or more polyamines is a metabolisable polyamine.

In other specific embodiments at least one of the one or more polyamines is a substrate for the enzyme spermine/spermidine acetyltranferase.

Preferably, at least one of the one or more polyamines is not modified at one or more of the NH ₂ or NH groups.

In further preferred embodiments, the one or more polyamines are selected from the group consisting of 1 ,3-diamino-propane, 1 ,4-diamino-butane (putrescine), N-acetyl- putrescine, 1 ,5-diamino-pentane (cadaverine), 1 ,7-diamino-heptane, 1 ,8-diamino-octane, spermine, N-acetyl-spermine, cholesteryl spermine, spermine phosphate hexahydrate, spermidine, N-acetyl-spermidine, spermidine trihydrochloride, spermidine phosphate hexahydrate, L-arginyl-3,4-spermidine, and 1 ,4-butane-diamine N-(3-aminopropyl)- monohydrochloride.

In particularly preferred embodiments, the method further comprises determining any one or more values selected from the group consisting of: spermidine/putrescine ratio, spermine/puterscine ratio, spermine/spermidine ratio, spermine/N-acetyl-spermine ratio, and spermidine/N-acetyl spermidine ratio.

In further specific embodiments, the medical condition is selected from the group consisting of lactic acidosis, muscle weakening, hyperglycemia, multiple organ failure, failed or disturbed homeostasis, severe or multiple trauma, high risk or extensive surgery, cerebral trauma or bleeding, respiratory insufficiency, abdominal peritonitis, acute kidney injury, acute liver injury, severe burns, diabetes, cardiovascular diseases, immune diseases, inflammatory diseases, and polyneuropathy.

In other specific embodiments, the method is performed in vitro.

In further preferred embodiments, the one or more polyamines or the one or more salts or derivatives thereof are detected and/or analyzed in the test sample by one or more of the techniques selected from the group consisting of:

(i) detecting and/or analyzing the one or more polyamine or salts or derivatives thereof as such;

(ii) detecing and/or analyzing a degradation product of the one or more polyamine or salts or derivatives thereof;

(iii) detecting and/or analyzing spermine/spermidine acetyltranferase activity and/or polyamine oxidase activity;

and combinations thereof.

In another aspect, the present invention relates to a kit-of-parts for diagnosing and/or monitoring in a subject a non-infestious medical condition or a predisposition to develop a non-infesctious medical condition, comprising: means for detecting and/or analyzing one or more polyamines or one or more salts or derivatives thereof, as defined herein.

In still another aspect, the present invention relates to the use of one or more polyamines or one or more salts or derivatives thereof, as defined herein, as a panel of molecular markers for diagnosing a non-infectious medical condition or a predisposition to develop a non-infectious medical condition.

Other embodiments, features, characteristics, and advantages of the present invention will become apparent from the detailed description hereinafter.

DESCRIPTION OF THE DRAWINGS

Figure 1 : (A) Bar graph showing intracellular spermidine concentrations of aging wild type yeast cells; the data represent means ± SEM of 4 biological replicates. (B) Bar graph showing intracellular putrescine concentrations of aging wild type yeast cells; the data represent means ± SEM of 4 biological replicates.

Figure 2: (A) Bar graph showing relative intracellular spermidine concentrations normalized to day 1 values of aging wild type yeast cells; the data represent means ± SEM of 4 biological replicates. (B) Bar graph showing intracellular putrescine concentration normalized to day 1 values of aging wild type yeast cells; the data represent means ± SEM of 4 biological replicates.

Figure 3: Plasma spermidine levels drop in critically ill non-survivors.

A rabbit model of critical illness (herein also referred to as an acute life-threatening noninfectious disease or a predisposition to develop such disease) was developed that was shown to mimick the dynamic endocrine, immunological and metabolic changes characteristic of human critical illness (cf. also Example 3). Spermidine levels (ng/ml) were measured in last-day (i.e. day 7 for "Sick Survivors", see below) plasma samples obtained from test animals and healthy controls ("Healthy"). The test animals were split by survival ("Sick Survivor" versus "Sick Non-Survivor"). Data are given as box-whiskerplots (n = 55). Boxplot indicates median and interquartile range. Whiskers indicate the 10 ^th and 90 ^th percentile. P-values were calculated by means of the Mann-Whitney U test.

Figure 4: Putrescine levels increase in critically ill non-survivors.

Putrescine levels (ng/ml) were measured in the rabbit animal model according to Fig. 3 in last-day (i.e. day 7 for "ick Survivors", see below) plasma samples obtained from test animals and healthy controls ("Healthy"). The test animals were split by survival ("Sick Survivor" versus "Sick Non-Survivor"). Data are given as box-whiskerplots (n = 55). Boxplot indicates median and interquartile range. Whiskers indicate the 10 ^th and 90 ^th percentile. P-values were calculated by means of the Mann-Whitney U test.

Figure 5: Spermine levels differ in critically ill survivors and non-survivors.

Spermine levels (ng/ml) were measured in the rabbit animal model according to Fig. 3 in last-day (i.e. day 7 for "Sick Survivors") plasma samples obtained from test animals and healthy controls ("Healthy"). The test animals were split by survival ("Sick Survivor" versus "Sick Non-Survivor"). Data are given as box-whiskerplots (n = 16). Boxplot indicates median and interquartile range. Whiskers indicate the 10 ^th and 90 ^th percentile.

Figure 6: Spermidine/putrescine ratios are of diagnostic value in a model of critical illness.

Spermidine and putrescine levels were in the rabbit animal model according to Fig. 3 in last-day (i.e. day 7 for "Sick Survivors", see below) plasma samples obtained from test animals and healthy controls ("Healthy"). The test animals were split by survival ("Sick Survivor" versus "Sick Non-Survivor"), and respective ratios were calculated ("Spd/Put"). Data are given as box-whiskerplots (n = 55). Boxplot indicates median and interquartile range. Whiskers indicate the 10 ^th and 90 ^th percentile. P-values were calculated by means of the Mann-Whitney U test.

Figure 7: Spermidine/putrescine ratios inversely correlate with plasma lactate.

The correlation between the plasma spermidine/putrescine ratio on the last day (according to Fig. 6) and the plasma lactate level on that day was calculated be means of Pearson correlation. As both lactate levels and spermidine/putrescine ratios were not normally distributed, the Pearson correlation coefficient was calculated after square root transformation of both variables (n = 55).

Figure 8: Spermidine/putrescine ratios correlate with plasma pH.

The correlation between the plasma spermidine/putrescine ratio on the last day (according to Fig. 6) and the pH value on that day was calculated by means of Pearson correlation. As spermidine/putrescine ratios were not normally distributed, the Pearson correlation coefficient was calculated after square root transformation of this variable (n = 55).

Figure 9: Spermidine/putrescine ratios inversely correlate with plasma AST.

Plasma aspartate aminotransferase (AST) levels, a marker of liver failure, were determined in the rabbit animal model according to Fig. 3 (i.e. at day 7 for "Sick Survivors"), and a Pearson correlation was performed with plasma spermidine/putrescine ratios according to Fig. 6. As both AST levels and spermidine/putrescine ratios were not normally distributed, the Pearson correlation coefficient was calculated after square root transformation of both variables (n = 24).

DETAILED DESCRIPTION OF THE INVENTION

The present invention is based on the unexpected finding that the detection and/or (quantitative) analysis of one or more (individual) polyamines or one or more salts or derivatives thereof represents a reliable and efficient approach for diagnosing and/or monitoring non-infectious conditions or a predisposition of developing such conditions. The presence and/or amount of said polyamines correlate with biochemical and/or clinical data. Hence, the detection (and optionally quantitative correlation) of one or more (individual) polyamines in a sample represents a suitable measure for the rapid and accurate staging of said conditions as well as for the monitoring of their progression and their responsiveness to a particular therapy. In addition, the approach of the present invention is simple, does not require sophisticated equipment, and thus is cost-effective.

The present invention illustratively described in the following may suitably be practiced in the absence of any element or elements, limitation or limitations, not specifically disclosed herein.

Where the term "comprising" is used in the present description and the claims, it does not exclude other elements or steps. For the purposes of the present invention, the term "consisting of" is considered to be a preferred embodiment of the term "comprising". If hereinafter a group is defined to comprise at least a certain number of embodiments, this is also to be understood to disclose a group, which preferably consists only of these embodiments.

Where an indefinite or definite article is used when referring to a singular noun, e.g., "a", "an" or "the", this includes a plural of that noun unless specifically stated otherwise.

In case, numerical values are indicated in the context of the present invention the skilled person will understand that the technical effect of the feature in question is ensured within an interval of accuracy, which typically encompasses a deviation of the numerical value given of ± 10%, and preferably of ± 5%.

Furthermore, the terms first, second, third, (a), (b), (c), and the like, in the description and in the claims, are used for distinguishing between similar elements and not necessarily for describing a sequential or chronological order. It is to be understood that the terms so used are interchangeable under appropriate circumstances and that the embodiments of the invention described herein are capable of operation in other sequences than described or illustrated herein.

Further definitions of term will be given in the following in the context of which the terms are used. The following terms or definitions are provided solely to aid in the understanding of the invention. These definitions should not be construed to have a scope less than understood by a person of ordinary skill in the art.

In a first aspect, the present invention relates to a method for diagnosing and/or monitoring in a subject a non-infectious medical condition or a predisposition to develop a non-infectious medical condition, comprising:

detecting and/or analyzing in a test sample derived from the subject one or more polyamines or one or more salts or derivatives thereof,

wherein the presence or quantity of any one of the polyamines or the salts or derivatives thereof in the test sample is indicative of the medical condition or the predisposition to develop such a medical condition.

The method of the invention comprises the detection and/or analysis of one or more (i.e. at least one) polyamines or one or more salts or derivatives thereof. Typically, the present invention is directed to the evaluation of one or more individual species of polyamines or any combinations thereof. In some embodiments, the method of the invention also comprises the evaluation of the total polyamine concentration in a given sample without discriminating between particular species. The term "detecting" (or "detection"), as used herein, may be interpreted in the sense of "identifying" at least one polyamine, and optionally also in the sense of "selecting" any one or more of the polyamines identified for further consideration (e.g., for quantitative analysis). The selection may vary, for example, depending on treatment modalities, including therapeutic intervention, diagnostic criteria such as disease stages, and disease monitoring and surveillance in the subject to be treated.

The term "analyzing" (or "analysis"), as used herein, may be interpreted as also including a quantitative characterization of at least one polyamine detected. The term also refers to mathematical calculations using the quantities of two or more polyamines determined, such as correlating the quantities with each other, such as by determing the ratios of two or more polyamines detected in a sample.

Within the present invention, the terms "diagnosing" and "monitoring" are intended to encompass not only a diagnosis stricto sensu but also predictions and likelihood analysis (based on both the qualitative and quantitative measurements; including healthy subjects as well). The present method is intended to be used clinically in making decisions concerning treatment modalities, including therapeutic intervention, disease staging, and disease monitoring and surveillance. According to the present invention, an intermediate result for examining the condition of a subject may be provided. Such intermediate result may be combined with additional information to assist a physician, nurse, or other practitioner to diagnose that a subject suffers from the disease. Alternatively, the present invention may be used to detect cancerous cells in a subject-derived sample, and provide a doctor with useful information to diagnose that the subject suffers from the disease.

The terms "one or more" or "any one or more" or "at least one", as used herein, relate to any one, any subgroup of any two or more (i.e. any two, any three, any four, any five, any six, any seven, any eight, any nine, any ten, and so forth) or all polyamines or salts thereof present in a given sample. The term "polyamine", as used herein, refers to any basic, water soluble, low molecular weight aliphatic molecules having two or more primary amine groups -NH ₂ or secondary amine groups -NH-.

In specific embodiments, at least one of the one or more polyamines employed is a metabolizable polyamine, that is, it is accessible for the cellular polyamine metabolism. In particular, the term "metabolizable" denotes that the polyamines are a substrate for the acetylating enzyme spermidine/spermine N-acetyltransferase (SSAT). SSAT is a rate- limiting enzyme in the catabolic (i.e. degrading) pathway of polyamine metabolism. It catalyzes the N-acetylation of spermidine and spermine and, by the successive activity of polyamine oxidase, spermine can be converted to spermidine and spermidine to putrescine (reviewed, e.g., in Pegg (2008) Am. J. Physiol. Endocrinol. Metab. 294, E995- E1010). Accordingly, in further specific embodiments, at least one of the one or more polyamines is a substrate for the enzyme spermine/spermidine acetyltranferase.

Preferably, at least one of the one or more polyamines is not modified at one or more of the -NH ₂ or -NH- groups. In specific embodiments, at least one of the one or more polyamines is methylated at one or more -NH ₂ or -NH- groups. In other specific embodiments, at least one of the one or more polyamines is acetylated at one or more of -NH ₂ or -NH- groups

Particular polyamines employed in the present invention are diamines (i.e. polyamines having two primary amine groups) represented by the general formula NH ₂ -(CH2) ₂ . ₁₀ -NH ₂ . The carbon atoms may be unsubstituted or optionally substituted, for example, with a methylgroup, NH or oxygen. The diamine group of polyamines comprises inter alia ethylene diamine, 1 ,3 diaminopropane, 1 ,4 diaminobutane (also known as putrescine), N- acetyl-putrescine, 1 ,5 diaminopentane (also known as cadaverine), 1 ,6-diamino-hexane, 1 ,7-diamino-heptane and 1 ,8-diamino-octane. Other particular polyamines employed in the present invention have a general structure NH ₂ -((CH ₂ ) _m -NH) _n -H, wherein m and n are each independently integers from 2 to 6. These polyamines are typically unsubstituted at the carbon atoms. Optionally, one or more carbon atoms may be substituted, for example, with a methylgroup, and/or NH and/ or oxygen group. In specific embodiments, m is 3, 4 or 5. In specific embodiments, n is 2, 3 or 4. Examples include inter alia spermine having the formula H2N((CH ₂ )4-NH)3-H (i.e. m = 4 and n = 3), N-acetyl-spermine, spermidine having the formula NH2((CH2)4-NH) ₂ H (i.e. m = 4 and n = 2), and N-acetyl-spermidine.

In preferred embodiments, the one or more polyamines employed are selected from the group consisting of 1 ,3-diamino-propane, 1 ,4-diamino-butane (putrescine), N-acetyl- putrescine, 1 ,5-diamino-pentane (cadaverine), 1 ,7-diamino-heptane, 1 ,8-diamino-octane, spermine, N-acetyl-spermine, cholesteryl spermine, spermine phosphate hexahydrate, spermidine, N-acetyl-spermidine, spermidine trihydrochloride, spermidine phosphate hexahydrate, L-arginyl-3,4-spermidine, and 1 ,4-butane-diamine N-(3-aminopropyl)- monohydrochloride.

In particularly preferred embodiments, the one or more polyamines are selected from the group consisting of putrescine, N-acetyl-putrescine, spermine, N-acetyl-spermine, spermidine, and N-actely-spermidine. Hence, the method performed herein may include the detection and or analysis of (putrescine), (N-acetyl-putrescine), (spermine), (N-acetyl- spermine), (spermidine), (N-actely-spermidine), (putrescine and N-acetyl-putrescine), (putrescine and spermine), (putrescine and N-acetyl-spermine), (putrescine and spermidine), (putrescine and N-acetyl-spermidine), (N-acteyl-putrescine and spermine), (N-acetyl-putrescine and N-acetyl-spermine), (N-acteyl-putrescine and spermidine), (N- acetyl-putrescine and N-acetyl-spermidine), (spermine and N-acetyl-spermine), (spermine and spermidine), (spermine and N-acetyl-spermidine), (N-acetyl-spermine and spermidine), (N-acetyl-spermine and N-acetyl-spermidine), (spermidine and N-acetyl- spermidine), (putrescine, N-acetyl-putrescine, and spermine), (putrescine, N-acetyl- putrescine, and N-acetyl-spermine), (putrescine, N-acetyl-putrescine, and spermidine), (putrescine, N-acetyl-putrescine, and N-acetyl-spermidine), (putrescine, spermine, and N- acetyl-spermine), (putrescine, spermine, and spermidine), (putrescine, spermine, and N- acetyl-spermidine), (putrescine, N-acetyl-spermine, and spermidine) (putrescine, N-acetyl- spermine, and N-acetyl-spermidine), (putrescine, spermidine, and N-acetyl-spermidine), (N-acetyl-putrescine, spermine, and N-acetyl-spermine), (N-acetyl-putrescine, spermine, and spermidine), (N-acety-putrescine, spermine, and N-acetyl-spermidine), (N-acetyl- putrescine, N-acetyl-spermine, spermidine), (N-acetyl-putrescine, N-acetyl-spermine, and N-acetyl-spermine), (N-acteyl-putrescine, spermidine, and N-acetyl-spermidine), (spermine, N-acetyl-spermine, and spermidine), (spermine, N-acetyl-spermine, and N- acetyl-spermidine), (spermine, spermidine, and N-acetyl-spermidine), (N-acetyl-spermine, spermidine, and N-acetyl-spermidine), (putrescine, N-acetyl-putrescine, spermine, and N- acetyl-spermine), (putrescine, N-acetyl-putrescine, spermine, and spermidine), (putrescine, N-acetyl-putrescine, spermine, and N-acetyl-spermidine), (putrescine, N- acetyl-putrescine, N-acetyl-spermine, and spermidine), (putrescine, N-acetyl-putrescine, N-acetyl-spermine, and N-acetyl-spermidine), (putrescine, N-acetyl-putrescine, spermidine, and N-acetyl-spermidine), (putrescine, spermine, N-acetyl-spermine, and spermidine), (putrescine, spermine, N-acetyl-spermine, and N-acetyl-spermidine), (putrescine, spermine, spermidine, and N-acetyl-spermidine), (putrescine, N-acetyl- spermine, spermidine, and N-acetyl-spermidine), (N-acetyl-putrescine, spermine, N- acetyl-spermine, and spermidine), (N-acetyl-putrescine, spermine, N-acetyl-spermine, and N-acetyl-spermidine), (N-acetyl-putrescine, spermine, spermidine, and N-acetyl- spermidine), (N-acetyl-putrescine, N-acetyl-spermine, spermidine, and N-acetyl- spermidine), (spermine, N-acetyl-spermine, spermidine, and N-actely-spermidine), (putrescine, N-acetyl-putrescine, spermine, N-acetyl-spermine, and spermidine), (putrescine, N-acetyl-putrescine, spermine, N-acetyl-spermine, and N-actely-spermidine), (N-acetyl-putrescine, spermine, N-acetyl-spermine, spermidine, and N-actely-spermidine), (putrescine, spermine, N-acetyl-spermine, spermidine, and N-actely-spermidine) (putrescine, N-acetyl-putrescine, N-acetyl-spermine, spermidine, and N-actely- spermidine) (putrescine, N-acetyl-putrescine, spermine, spermidine, and N-actely- spermidine), and (putrescine, N-acetyl-putrescine, spermine, N-acetyl-spermine, spermidine, and N-actely-spermidine).

The term "polyamine salt", as used herein, denotes any physiologically acceptable salts being present in a test sample to be analyzed including inorganic and organic acids and bases, such as inter alia sulfuric, citric, maleic, acetic, oxalic, hydrochloriic, hydrobromic, hydroiodine, nitrate, sulfate, bisulfite, phosphate, acid phosphate, isonicotinate, acetate, lactate, salicylate, citrate, acid citrate, tartrate, oleate, tannate, pantothenate, bitartrate, ascorbate, succinate, maleate, gentisinate, fumarate, gluconate, glucaronate, saccharate, fornate, benzoate, glutamate, methanesulfonate, ethanesulfonate, benzenesulfonate, p- toluenesulfonate, and pamoate. Physiologically acceptable salts further include those formed with free amino groups such as inter alia those derived from hydrochloric, phosphoric, acetic, oxalic, and tartaric acids. Pharmaceutically acceptable salts also include those formed with free carboxyl groups such as inter alia those derived from sodium, potassium, ammonium, sodium lithium, calcium, magnesium, ferric hydroxides, isopropylamine, triethylamine, 2-ethylamino ethanol, histidine, and procaine.

The term "polyamine derivative", as used herein, denotes any chemical compounds having a similar chemical structure as a polyamine Generally, a specific functional group of a given polyamine is involved in a derivatization reaction and transforms the polyamine educt to a derivate of deviating reactivity, solubility, boiling point, melting point, aggregate state, chemical composition, or the like. Resulting new chemical properties can, for example, be used for quantification or separation of the educt. As used herein, the term also includes enantiomers or diastereomers.

The term "non-infectious medical condition" (commonly also referred to as "disease" or "illness") refers to any abnormal condition of an organism that impairs body functions not caused by the colonization with foreign pathogen, is associated with specific symptoms and signs, and may be caused by external factors or by internal disfunctions. Examples of such non-infectious medical conditions include inter alia cancer, immune diseases, cardiovascular diseases, neuronal diseases, and inflammatory diseases. In the context of the present invention, a non-infectious medical condition may also be an acute (potentially) life-threatening episode, commonly associated with single or multiple organ dysfunction or organ failure, that when remaining untreated likely result in the decease of the subject affected. The term "having a predisposition to develop a non-infectious medical condition", as used herein, denotes any cellular phenotype being indicative for a pre-disease state. In other words, the term denotes a state of risk of developing a noninfectious medical condition.

The term "cancer", as used herein, denotes any type or form of malignant neoplasm characterized by uncontrolled division of target cells based on genetic re-programming and by the ability of the target cells to spread, either by direct growth into adjacent tissue through invasion, or by implantation into distant sites by metastasis (where cancer cells are transported through the bloodstream or lymphatic system). Examples include inter alia breast cancer, colorectal cancer, prostate cancer, leukemia, lymphomas, neuroblastoma, glioblastoma, melanoma, liver cancer, and lung cancer.

The term "immune disease", as used herein, refers to any disorder of the immune system. Examples of such immune diseases include inter alia immunosenescence, immunodeficiencies (i.e. congenital or acquired conditions in which the immune system's ability to fight infectious diseases is compromised or entirely absent, such as AIDS or SCID), hypersensitivity (such as allergies or asthma), and autoimmune diseases. The term "autoimmune disease", as used herein, is to be understood to denote any disorder arising from an overactive immune response of the body against endogenic substances and tissues, wherein the body attacks its own cells. Examples of autoimmune diseases include inter alia multiple sclerosis, Crohn's disease, lupus erythematosus, myasthenia gravis, rheumatoid arthritis, and polyarthritis.

The term "cardiovascular disease", as used herein, refers to any disorder of the heart and the coronary blood vessels. Examples of cardiovascular diseases include inter alia coronary heart disease, angina pectoris, artheriosclerosis, cardiomyopathyies, myocardial infaction, ischemia, and myocarditis.

The term "neuronal disease" (or "neurological disorder"), as used herein, refers to any disorder of the nervous system including diseases of the central nervous system (CNS) (i.e. brain and spinal cord) and diseases of the peripheral nervous system. Examples of CNS diseases include inter alia stroke, Alzheimer's disease, Parkinson's disease, Huntington's disease, Locked-in syndrome, and Tourettes syndrome. Examples of diseases of the peripheral nervous system include, e.g., mononeuritis multiplex and polyneuropathy.

The term "inflammatory disease", as used herein, refers to any disorder associated with inflammation including, e.g., acne, asthma, hay fever, arthritis, inflammatory bowel disease, pelvic inflammatory disease, and transplant rejection. ln further preferred embodiments, the non-infectious medical condition is selected from the group consisting of lactic acidosis, muscle weakening, hyperglycemia, multiple organ failure, failed or disturbed homeostasis, severe or multiple trauma, high risk or extensive surgery, cerebral trauma or bleeding, respiratory insufficiency, abdominal peritonitis, acute kidney injury, acute liver injury, severe burns, diabetis, cardiovascular disease, immune disease, inflammatory disease, and polyneuropathy.

The term "lactic acidosis", as used herein, refers to a physiological condition characterized by low pH in body tissues and blood (i.e. acidosis) accompanied by the buildup of lactate, and is considered a distinct form of metabolic acidosis. The condition typically occurs when cells receive too little oxygen (that is, during hypoxia), for example during vigorous exercise. In this situation, impaired cellular respiration leads to lower pH levels. Simultaneously, cells are forced to metabolize glucose anaerobically, which leads to lactate formation.

The term "muscle weakening" (also referred to as "muscular dystrophy") refers to a group of (hereditary) muscle diseases that weaken the muscles that move the human body. These conditions are characterized by progressive skeletal muscle weakness, defects in muscle proteins, and the death of muscle cells and tissue. Examples include inter alia congenital muscular dystrophy, Duchenne muscular dystrophy, and Becker's muscular dystrophy.

The term "hyperglycemia", as used herein, refers to a condition of high blood sugar, that is, a condition in which an excessive amount of glucose circulates in the blood plasma. Hyperglycemia may be caused by diabetes mellitus, a critical illness such as stroke or myocardial infarction or by physiological stress such as during infection and inflammation.

The term "multiple organ failure", as used herein, denotes a descriptive clinical syndrome, defined by a dysfunction or failure of at least two vital organ systems. The vital organ systems that are uniformly and most specifically affected are the liver, the kidneys, the lungs, as well as the cardiovascular system, the nervous sytem and the hematological system. The term "polyneuropathy", as used herein, refers to neurological disorders that occur when many peripheral nerves throughout the body malfunction simultaneously. Polyneuropathy may be acute and appear without warning, or may be chronic and develop gradually over a longer period of time. Many polyneuropathies have both motor and sensory involvement; some also involve dysfunction of the autonomic nervous system.

In some embodiments, the non-infectious medical condition (or predisposition to develop such condition) does not include cancer.

In some other embodiments, the non-infectious medical condition (or predisposition to develop such condition) does not include acute liver disease and/or acute kidney disease.

In yet some other embodiments, the non-infectious medical condition (or predisposition to develop such condition) does not include diabetes and/or cerebral trauma.

A subject to be diagnosed and/or monitored by the present method is typically a mammal such as a mouse, rat, hamster, rabbit, cat, dog, pig, cow, horse or monkey. Preferably, the subject to be diagnosed is a human.

The test samples to be employed in the present invention are derived (i.e. collected) from the subject to be diagnosed and/or monitored for the presence or the predisposition to develop a medical condition (that is, a subject at least suspected to exhibit or develop such condition). The test samples may include body tissues (e.g., biopsies or resections) and body fluids, such as blood, sputum, saliva, cerebrospinal fluid, and urine. Furthermore, the test samples may contain a single cell, a cell population (i.e. two or more cells) or a cell extract derived from a body tissue. The test samples used in the method of the present invention should generally be collected in a clinically acceptable manner. The test samples may be used in unpurified form or subjected to any enrichment or purification step(s) prior to use, for example in order to isolate a particular fraction comprised in a given sample. The skilled person is well aware of various such purification methods (see, e.g., Sambrook, J., and Russel, D.W. (2001), Molecular cloning: A laboratory manual (3rd Ed.) Cold Spring Harbor, NY, Cold Spring Harbor Laboratory Press; Ausubel, F.M. et al. (2001) Current Protocols in Molecular Biology, Wiley & Sons, Hoboken, NJ, USA).

In specific embodiments, the test sample is a blood sample such as whole blood, blood cells, plasma, and serum. The term "whole blood", as used herein, refers to blood with all its constituents (i.e. both blood cells and plasma). The term "plasma", as used herein, denotes the blood's liquid medium. The term "serum", as used herein, refers to plasma from which the clotting proteins have been removed. Preferably, the test sample is a plasma sample.

In other specific embodiments, the test sample is a urine sample.

Typically, the method of the present invention is performed as an in vitro method.

In specific embodiments, the method further comprises: comparing the results obtained in the test sample with a reference. The reference may preferably be a control sample derived from the subject to be analyzed. The term "control sample", as used herein, refers to a sample derived from the subject to be diagnosed that is not at least suspected to exhibit a medical condition or to develop such condition (i.e. a healthy tissue, cell or fluid sample). Within the present invention, the term "reference" also refers to control values derived from databases, published in the scientific literature or based on large sample numbers of healthy subjects.

In other preferred embodiments, the method further comprises:

quantifying the levels of the one or more polyamines or the one or more salts or derivatives thereof in the test sample,

wherein an altered level of any one the polyamines or the salts or derivatives thereof in the test sample as compared to the reference is indicative of a medical condition or a predisposition to develop a medical condition.

The term "altered level" in the context of the present invention denotes an increase or a decrease of the level (i.e. the concentration) of any one of the polyamines or salts thereof present in the test sample. Polyamine levels are deemed to be "increased" (i.e. elevated) if they are increased in the test sample by, for example, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, or more than 50% as compared to the reference, or if they are at least 0.1 fold, at least 0.2 fold, at least 1 fold, at least 2 fold, at least 5 fold, at least 10 fold or more higher as compared to the reference. Vice versa, polyamine levels are deemed to be "decreased" (i.e. reduced) if they are decreased in the test sample by, for example, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, or more than 50% as compared to the reference, or if they are at least 0.1 fold, at least 0.2 fold, at least 1 fold, at least 2 fold, at least 5 fold, at least 10 fold or more lower as compared to the reference.

In some embodiments, the levels (i.e. quantities) of all of the one or more polyamines or the salts or derivatives thereof evaluated are reduced in the test sample as compared to the reference. In some other embodiments, the levels of all of the one or more polyamines or the salts or derivatives thereof evaluated are increased in the test sample as compared to the reference. In yet some other embodiments, the level of at least one of the one or more polyamines or the salts or derivatives thereof evaluated is reduced in the test sample as compared to the reference and at least one other of the one or more polyamines or the salts or derivatives thereof evaluated is increased in the test sample as compared to the reference.

In preferred embodiments, the term "quantifying" further comprises correlating with each other (e.g., by performing mathematical calculations) the quantified levels of the one or more polyamines or the one or more salts or derivatives thereof obtained. For example, the method of the invention may include the determination of the ratio (i.e. the portions with respect to each other) of any two or more polyamines present in a given sample, wherein an altered ratio of the two or more polyamines in the test sample as compared to the reference is indicative of a non-infectous medical condition or a predisposition to develop such condition.

In preferred embodiments, the one or more polyamines used for such correlations and/or mathematical calculations are selected from the group consisting of putrescine, N-acetyl- putrescine, spermine, N-acetyl-spermine, spermidine, and N-actely-spermidine. Hence, the method performed herein may inter alia include the determination of the following ratios: (putrescine versus N-acetyl-putrescine), (putrescine versus spermine), (putrescine versus N-acetyl-spermine), (putrescine versus spermidine), (putrescine versus N-acetyl- spermidine), (N-acteyl-putrescine versus spermine), (N-acetyl-putrescine versus N-acetyl- spermine), (N-acteyl-putrescine versus spermidine), (N-acetyl-putrescine versus N-acetyl- spermidine), (spermine versus N-acetyl-spermine), (spermine versus spermidine), (spermine versus N-acetyl-spermidine), (N-acetyl-spermine versus spermidine), (N-acetyl- spermine versus N-acetyl-spermidine), (spermidine versus N-acetyl-spermidine).

In particularly preferred embodiments, the method comprises determining any one or more values selected from the group consisting of: spermidine/putrescine ratio, spermine/ puterscine ratio, spermine/spermidine ratio, spermine/N-acetyl-spermine ratio, and spermidine/N-acetyl spermidine ratio.

The analogous correlation may also be performed for any three or more polyamines present in a given sample (e.g., determining the putrescine/spermine/spermidine ratio).

In other embodiments, in order to improve the diagnostic impact of the method performed it is also possible to combine any one or more of the "calculated" values obtained by correlating the levels of two or more polyamines present in a sample with the levels of one or more additional polyamines present in the sample. For example, it may be possible to calculate the spermidine/putrescine ratio in a given sample and to combine this result with the cadaverin level obtained in said sample.

In other preferred embodiments, the one or more polyamines or the salts or derivatives thereof that have altered levels in the test sample as compared to the reference together (i.e. in their totality) represent a signature that is indicative of a medical condition or a predisposition to develop a medical condition.

The term "signature", as used herein, denotes a set of two or more polyamines or salts or derivatives thereof, wherein the level of the individual polyamines or salts or derivatives thereof differs between the test sample and the reference. Herein, a signature is also referred to as a set of polyamine markers and represents a minimum number of (different) markers that is capable for identifying a medical condition in the test sample analyzed.

In further preferred embodiments, the one or more polyamines or the one or more salts or derivatives thereof are detected and/or analyzed in the test sample by one or more of the techniques selected from the group consisting of:

(i) detecting and/or analyzing the one or more polyamine or salts or derivatives thereof as such;

(ii) detecing and/or analyzing a degradation product of the one or more polyamine or salts or derivatives thereof;

(iii) detecting and/or analyzing spermine/spermidine acetyltranferase activity and/or polyamine oxidase activity;

and combinations thereof.

The detection and/or analysis of the one or more polyamine or salts or derivatives thereof as such may, for example, be performed by means of specific antibodies (either polyclonal or preferably monoclonal) antibodies being directed againt a particular polyamine species. For the detection of two or more polyamine species different antibodies may be employed in parallel. Anti-polyamine antibodies are well known in the art and commercially available from different suppliers. Instead of and/or additionally to the use of full-length antibodies antibody fragments (such as Fab fragments or single-chain antibodies) or other binding moieties (such as anticalins, haptens (i.e. a small molecule that can elicit an immune response only when attached to a larger carrier, such as hydralazine, urushiol, fluorescein, biotin, and digoxigenin) and aptamers) may be employed as well.

Polyamine detection may be carried out via any immunochemical detection techniques known in the art, for example, by the separation of the test sample (and optionally also of the control sample) on a polyacrylamide gel, followed by identification of a specific polyamine or salt thereof using appropriate antibodies in a Western blot analysis. For example, detection may be performed used a monoclonal antibody-based ELISA assay, as described (Garthwaite, I. et al. (1993) J. Immunol. Meth. 162, 175-178). Alternatively, proteins can be separated by two-dimensional gel electrophoresis systems. Two-dimensional gel electrophoresis is well known in the art and typically involves isoelectric focusing along a first dimension followed by SDS-PAGE electrophoresis along a second dimension. The analysis of two-dimensional (2D) SDS-PAGE gels may be performed by determining the intensity of protein spots on the gel or by using immune detection. In other embodiments, protein samples are analyzed by mass spectroscopy.

In order to facilitate immunodetection a primary antibody (i.e. an antibody directed against a polyamine or salt or derivative thereof) may be fused to one or more detectable labels which are detected, for example, by a secondary antibody. Labels that may be used herein include any compound, which directly or indirectly generates a detectable compound or signal in a chemical, physical or enzymatic reaction. Labeling and subsequent detection can be achieved by methods well known in the art (see, for example, Sambrook, J., and Russel, D.W. (2001), Molecular cloning: A laboratory manual (3rd Ed.) Cold Spring Harbor, NY, Cold Spring Harbor Laboratory Press; and Lottspeich, F., and Zorbas H. (1998) Bioanalytik, Spektrum Akademischer Verlag, Heidelberg/Berlin, Germany). The labels can be selected inter alia from fluorescent labels, enzyme labels, chromogenic labels, luminescent labels, radioactive labels, haptens, biotin, metal complexes, metals, and colloidal gold. All these types of labels are well established in the art and can be commercially obtained from various suppliers. An example of a physical reaction that is mediated by such labels is the emission of fluorescence or phosphorescence upon irradiation. Alkaline phosphatase, horseradish peroxidase, β- galactosidase, and β-lactamase are examples of enzyme labels, which catalyze the formation of chromogenic reaction products, and which may be used in the invention.

Alternatively, the polyamines in a given sample can also be measured by high- performance liquid chromatography (HPLC). Specific columns are available from various suppliers. For example, in cases where polyamine column commercially available from TOSO (Tokyo, Japan) are used, retention time of some specific polyamines (putrescine, spermidine and spermine) on the HPLC is 7 minutes, 12 minutes and 25 minutes, respectively. Further, other normal amino acid columns can be used ad libitum (cf., e.g., Karikasa, G.A. et al. (1997) J. Liquid Chromatography 20, 1789-1796; Glod, B.K. et al. (2000) Chem. Anal. 45, 27-33).

Instead of detecting and/or analyzing the one or more polyamine or salts thereof as such the method of the present invention may also be performed via detecing and/or analyzing a degradation product and/or derivative of the one or more polyamine or salts thereof (cf., e.g., Morgan, D. (1998) Polyamine Prot. 79, 13-18, Springer Press).

For example, polyamines are subjected to oxidative deamination by polyamine oxidase, resulting in the production of aldehyde compounds such as acrolein. The acrolein content in a sample may be determined by measuring the content of FDP-lysine (N-formyl- piperidino-lysine), which is an amino acid adder with acrolein. FDP-lysine content may be measured by using the ACR-Lysinadduct ELISA system (NOF Corporation, Japan). In addition, acrolein content could be measured in the form of derivatives other than FDP- lysine. Furthermore, it is also possible to measure acrolein content directly as described, e.g., by Alarcon et al. (Alarcon et al. (1968) Anal. Chem. 40, 1704-1708).

However, it is also possible to directly detemine the enzyme activity of polyamine oxidase, for example, by following the various protocols described (cf., e.g., Kubota, S. et al. (1983) Cancer Res. 43, 2363-2367; Sharmin et al. (2001) Biochem. Biophys. Res. Commun. 282, 228-235; Sakata et al. (2003) Biochem. Biophys Res. Commun. 305, 143-149; and Igarashi et al. (1986) J. Bacteriol. 166, 128-134; Takagia, K. et al. (2004) Clin. Chim. Acta 340, 219-227). Furthermore, protein content of polyamine oxidase can be measured by enzyme-linked immunosorbent assay (ELISA), western blotting analysis or immunoprecipitation method using a specific antibody for polyamine oxidase (see above).

In addition, detection and/or analysis of the one or more polyamines or the one or more salts may also be performed by measuring spermine/spermidine acetyltranferase activity (SSAT). Activity may be determined at the protein level, for example by means of an ELISA (cf. above) or via the analysis of the SSAT gene expression level. For example, SSAT expression levels may be assessed by separation of nucleic acid molecules (e.g. RNA or cDNA) obtained from the sample in agarose gels or polyacrylamide gels followed by hybridization with gene-specific oligonucleotide probes. Alternatively, the difference in expression level may be determined by the labeling of nucleic acid obtained from the sample followed by separation on a sequencing gel. Nucleic acid samples are placed on the gel such that patient and control or standard nucleic acids are in adjacent lanes. Comparison of expression levels is accomplished visually or by means of a densitometer. Methods for the detection of mRNA are known to the person skilled in the art or can be derived from standard textbooks, for example Sambrook, J., and Russel, D.W. (2001), supra. Typically, Northern blot analysis may be used for such a purpose. Preferably, mRNA may be detected in a microarray approach, e.g., sample nucleic acids derived from subjects to be tested are processed and labeled, preferably with a fluorescent label. Subsequently, such nucleic acid molecules are used in a hybridization approach with immobilized capture probes corresponding to the SSAT gene. Suitable means for carrying out microarray analyses are known to the person skilled in the art. Typically, microarray based expression profiling may be carried out, for example, by the method as disclosed in Microarray Biochip Technology (Schena M., Eaton Publishing, 2000).

In some other embodiments, the method is performed in a multiplex format. The term "multiplex format", as used herein, refers to the parallel detection analysis of two or more polyamines or salts thereof present in the same test sample within a single assay (for example, depending on the detection method employed by using separate reaction containers for each of the polyamines or salts thereof concerned) as well as two the parallel analysis of two or more test samples in parallel (wherein the one or more polyamines or one or more salts thereof analyzed in the two or more test samples may be the same or different). The term also includes high-throughput analyses, for example by employing microarray technology.

In another aspect, the present invention relates to a kit-of-parts for diagnosing and/or monitoring a non-infectious medical condition or a predisposition to develop a noninfectious medical condition, comprising

means for detecting and/or analyzing means for detecting and/or analyzing one or more polyamines or one or more salts or derivatives thereof, as defined herein above. Means for detecting and/or analyzing the one or more polyamines or salts or derivatives thereof may include inter alia one or more specific antibodies to be employed as capture or probe molecules for detecting and/or analyzing the expression levels and/or enzymatic activities of one or more components involved in anabolic (i.e. synthesizing) or catabolic (i.e. degrading) polyamine metabolism. The kit-of-part according to the invention may further comprise reagents for performing said assays such as enzymes, probes or labels as well as for isolating and/or purifying a test sample (and a control sample) to be analyzed.

The various components of the kit may be packaged in one or more containers such as one or more vials. For example, each component comprised in the kit may be packaged in a separate container.

The components of the kit may be provided in lyophilized or dry form or dissolved in a suitable buffer such as phosphate-buffered saline or Tris/EDTA (TE)-buffer. The kit may also comprise additional reagents including inter alia preservatives, buffers for storage and/or reconstitution of the above-referenced components, washing solutions, and the like. These reagents may be provided in combination with one or more of the components indicated above, that is, in the same container (e.g., an antibody dissolved in an appropriate buffer). Alternatively, at least some of these additional reagents may be provided in separate containers.

In another aspect, the present invention relates to the use of one or more polyamines or one or more salts or derivatives thereof, as defined herein above, as a panel of molecular markers for diagnosing a non-infectious medical condition or a predisposition to develop a non-infectious medical condition.

Within the present invention, the term "use" is to be understood as referring to both the qualitative and quantitative information obtained by performing the methods defined herein above (i.e. the "polyamine status" in a given test sample including the identification of any polyamines as well as the determination of their amounts). In other words, such "polyamine status" is used as a "signature" for the diagnosis and/or staging of a medical condition and/or for the monitoring of said condition or responsiveness to a given therapy.

The invention is further described by the figures and the following examples, which are solely for the purpose of illustrating specific embodiments of this invention, and are not to be construed as limiting the scope of the invention in any way.

EXAMPLES

Example 1 : Yeast model of aging post-mitotic cells

Yeast chronological aging serves as a model for the aging process of post-mitotic non- dividing cells of higher eukaryotes, including human cells. It is defined as the time a yeast culture remains viable in stationary phase and follows molecular pathways that are shared with those dictating longevity of higher organisms (Herker et al. (2004) J. Cell Biol. 164, 501-507; Longo and Finch (2003) Science 299, 1342-1346). With ongoing age, cells accumulate cellular damage, including, e.g., ROS-mediated protein oxidation and become prone to initiate programmed cell death pathways (e.g. apoptosis or necrosis). Importantly, age-related cellular damage, cellular dysfunction and dysregulation of cell death are associated with a plethora of human diseases, including cancer, cardiovascular diseases and immune disorders.

It was previously shown that depletion of polyamines by genetic means enhances cell death, oxidative stress and thus causes premature aging (Eisenberg et al. (2009) Nat. Cell Biol. 11 , 1305-1314). On the other hand, application of spermidine extends the life span of various organisms and suppresses cellular damage, oxidative stress and cell death (Eisenberg et al., supra). Thus, it was rendered feasible that intracellular concentrations of natural polyamines could reflect the cellular age (and therefore likely also the status of cellular damage and probability to undergo cell death). Example 2: Measurement of spermidine and putrescine in aging yeast

In order to test the hypothesis that intracellular concentrations of polyamines might indicate the "health status" of cells, the above described model system of chronological aging yeast was employed (cf. example 1). With progressing time, cells typically display enhanced levels of oxidative stress and cell death. Indeed, both spermidine and putrescine levels declined with progressing time in a chronological aging yeast culture (Figure 1).

Experiments were carried out in BY4741 (MATa his3A1 leu2A0 met15A0 ura3A0). Strains were grown at 28 °C on SC medium containing 0.17% yeast nitrogen base (Difco) 0.5% (NH ₄ ) ₂ S0 ₄ and 30 mg/l of all amino acids (except 80 mg/l histidine and 200 mg/l leucine), 30 mg/l adenine, and 320 mg/l uracil with 2% glucose (SCD) and aged until the indicated time points. After extraction using 5% TCA and 10 OD(600) cells for 1 h at 4 °C and neutralization with ammonium formiate (0.4 M final concentration), polyamines were determined according to the method described previously (Gianotti et al. (2008) J Chromatogr A, 1185, 296-300). All experiments were carried out on an Ultimate 3000 System (Dionex, LCPackings) coupled to a Quantum TSQ Ultra AM (ThermoFinnigan) using an APCI ion source. The system was controlled by Xcalibur Software 1.4. The stationary phase was a Sequant ZIC-HILIC column (150 x 2.1 mm, 3μηι, 100 A). The elution solvent A was 50 mM ammonium formiate in ultra pure water and solvent B was acetonitrile. Separation was performed with 15% acetonitrile for 2 min. Thereafter, the acetonitrile content was linearly decreased to 5% over 2 min. After 1 min, acetonitrile content was increased to 15% for column equilibration. Flow rate was set to 300 μΙ/min.

Polyamines were detected in MRM mode using following transitions: spermidine (m/z 146 -> 72, CE 34 eV), putrescine (m/z 89 -> 72, CE 28 eV), SAM (298 -> 136, CE 13 eV), bis(hexamethylene)-triamine as internal standard (m/z 216 -> 100, CE 36 eV). Calibration standards were prepared by spiking extraction buffer with specific concentrations of spermidine, putrescine or internal standard. 20μΙ of each sample were injected. Example 3: Animal model of critical illness

An animal model of critical illness (i.e. a (potentially) life-threatening disease as defined herein above) was developed previously that was shown to mimick the dynamic endocrine, immunological and metabolic changes characteristic of human critical illness (Weekers F, et al. (2003) Endocrinol. 144, 5329-5338). In this animal model, the effect of critical illness on polyamine levels (in particular, spermidine and putrescine) was investigated. Animals were treated according to the "Principals of Laboratory Animal Care" formulated by the U.S. National Society for Medical Research and the "Guide for the Care and Use of Laboratory Animals" prepared by the National Institutes of Health. The protocol was approved by the K.U.Leuven Ethical Review Board for Animal Research. Adult male New Zealand White rabbits, weighing approximately 3 kg, were purchased from a local rabbitry, were housed individually with free access to water, hay and regular rabbit chow, and were exposed to artificial light for 14 h per day.

In this animal model, critical illness was induced by placing intravascular catheters, selectively destroying pancreatic β-cells by alloxan, followed by burn injury. As mentioned, this model revealed the dynamic endocrine and metabolic changes characteristic of human critical illness, including hyperglycemia and endogenous insulin deficiency. Alloxan is a toxic glucose analogue, which selectively destroys insulin-producing cells in the pancreas. The administration of alloxan was necessary to control both blood glucose and plasma insulin levels independently. After imposing a burn wound, animals were brought to hyperinsulinaemia, because this reflects most the human situation of critical illness. Non-injured, healthy rabbits served as control.

At 09:00 ± 1 h of day -1 (i.e. morning of day -1), animals were weighed and general anesthesia was induced by an intramuscular injection of ketamine and medetomidine (30 mg/kg ketamine [Imalgene 1000; Merial, Lyon, France]; 0.15 ml/kg medetomidine [Orion, Espoo, Finland]). The animals were shaved in the back, the flanks, and the neck. Anesthesia was maintained by isoflurane inhalation (Isoba Vet.; Schering-Plough, Brussels, Belgium). Thereafter, intravascular catheters were placed in the right common jugular vein and the right carotid artery. Placement of these catheters allowed intravenous infusion of fluids, nutrients and insulin, respectively, and repetitive blood sampling. After having inserted the catheters, an ice-cold 10% solution of alloxan-monohydrate (150 mg/kg; Alloxan; Sigma-Aldrich, Bornem, Belgium) was injected slowly via the intravenous access. Animals were then fitted to a homemade jacket to secure catheters and immediately returned to their cages. During emergence from anesthesia, an opioid analgesic was injected intramuscularly (piritramide, Dipidolor 0.15 mg/kg, Janssen-Cilag, Beerse, Belgium). A basal fluid resuscitation (9ml/h Hartmann solution [Baxter, Lessines, Belgium], supplemented with 5% glucose) was administered via a volumetric pump using a homemade swivel device to allow free moving in the cage. In the first night, part of fluid resuscitation was replaced by glucose 50% infusion (Baxter), administered by a syringe pump to prevent transient hypoglycemia expected 12-24 h after alloxan-injection. The animals received regular rabbit chow, water and hay ad libitum.

At day 0, at 16:00h ± 1 h (i.e. afternoon of day 0), the animal was anesthetized again, and after a paravertebral block with 5ml lidocaine 1 % (Xylocaine, AstraZeneca, Brussels, Belgium), a third degree burn wound (painless because nerve endings were also destroyed), 15-20% body surface area, was applicated on both flanks. The animal was returned to its cage, and fluid resuscitation was given (16 ml/h Hartmann-glucose solution). All animals were targeted to hyperglycemia (target 300-315 mg/dl) with concomitant hyperinsulinemia. To this purpose, insulin (Actrapid; Novo Nordisk, Bagsvaerd, Denmark) was infused at a minimum rate of 2 U/kg/24h. In case, glycemia was under target level with this regimen, supplemental glucose 50% was administered by a syringe pump. The hyperglycemic-hyperinsulinemic condition is relevant to the human situation, as critically ill patients are hyperglycemic - unless treated with insulin -, and are hyperinsulinemic regardless of insulin treatment. While the animal emerged from anesthesia, an intramuscular injection of piritramide was given. The animals were deprived of regular rabbit chow and received water and hay ad libitum. In the evening, a supplementary dose of piritramide was given subcutaneously (0.2 mg/kg Dipidolor; Janssen-Cilag, Beerse, Belgium).

At 13:00 ± 1 h of day 1 , Hartmann solution was replaced by parenteral nutrition infused at 10 ml/h. Parenteral nutrition contained 35% Clinomel N7 (Baxter; Clinitec, Maurepas Cedex, France), 35% Hartmann solution, and 30% glucose 50%. All intravenous infusions were prepared daily under sterile conditions and weighed before and after administration for exact quantification of intake.

Parenteral nutrition was changed daily at 13:00 ± 1 h of days 2-7, at which time the amount of parenteral nutrition and supplementary glucose, and the amount of insulin given was recorded.

At 14:00 ± 1 h of day 7, animals were anesthetized intravenously using half of the above mentioned dose of anesthetics, and the animals were weighed. After tracheostomy, animals were normoventilated (small animal ventilator KTR4; Hugo Sachs, March- Hugstetten, Germany). Anesthesia was maintained with 1.5 volume-% isoflurane inhalation and 0.15 mg/kg piritramid intravenously. Vital organs were sampled and animals were sacrificed by cutting out the heart.

A 4cc blood sample was withdrawn daily (after instrumentation at day -1 , at day 0 before induction of anesthesia, and thereafter daily at 08:00 ± 1 h). Blood was centrifuged and plasma was stored at -80°C until further analysis. The term "last day" plasma samples, as used herein, refers to day 7 of "survivors" and healthy controls or to the last day of "non- survivors" (animals that died before the period of 7 days).

Throughout the study, glycemia was measured at fixed time points (08:00 ± 1 h, 12:00 ± 1 h, 17:00 ± 1 h and 22:00 ± 1 h), using a blood gas analyser (ABL 725, Radiometer, Copenhagen, Denmark), which also allowed to monitor pH, blood gases, lactate and electrolytes. If glycemia remained stable, the 12h and 22h measurement was performed on a glucometer (Hemocue Glucose 201 +; Hemocue, Angelholm, Sweden).

Urinary volume was recorded daily. Animals were thoroughly monitored clinically, and changes in clinical condition were recorded.

Animals entering a preterminal state before day 7, as evidenced clinically and by blood gas analysis, were anesthetized, organs were sampled in vivo, and the animal was sacrificed (same protocol as on day 7). In animals that deteriorated very rapidly and eventually died spontaneously, organs were sampled immediately post mortem. If the organs could not be harvested within 30 minutes of death, autopsy was performed without harvesting tissue samples.

For polyamine measurements "last day" plasma samples refer to day 7 of "survivors" and healthy controls or to the latest obtained plasma sample of "non-survivors" (animals that died or entered preterminal stage before the period of 7 days).

In order to establish a healthy reference range for all measured parameters, blood and tissue samples were also harvested from healthy control animals. Control rabbits underwent a daily blood sampling via the central ear artery, to withdraw a 4cc-blood sample, but were otherwise left undisturbed in the cage until day 7, at what time they were anesthetised and sampling of vital organs took place. The control animals had free access to regular rabbit chow, and received water and hay ad libitum.

Example 4: Polyamine measurements and sample preparation

4.1 Analytical method

The analytical method employed for the quantification of polyamines such as putrescine, N-acetylputrescine, cadaverin, spermine, N-acetylspermine, spermidine, N-acetyl- spermidine) is based on online solid-phase extraction (SPE) combined with liquid chromatography (LC) coupled to MS/MS mass spectrometry. Using this method simultaneous quantification of numerous polyamines and their ratios is feasible in one analytical run.

4.2 Sample pre-treatment

The internal standard was added to 100 μΙ serum/plasma, urine, any other body fluid or to polyamine containing extracts. For analysis of tissue or cellular polyamines, extracts are prepared by using 5% trichloroacetic acid extraction at 4°C for 1 h after sample homogenization. Stably isotope-labeled polyamines were used as internal standards. Trichloroacetic acid was used for protein precipitation. After centrifugation, the supernatant was transferred into low binding PCR tubes and derivatization was performed by using isopropylchloroformate. The final liquid was injected without any further preparation steps into the online-SPE-LC-MS/MS system.

4.3 Instrumental method

Analysis time was 3 minutes for one online SPE-LC- MS/MS run. Specifications were validated according to FDA guidelines.

Chromatography was performed according to the following parameters:

HPLC Parameter: Dionex 3000

Pre-column X-Bridge C18 Guard Cartridge, 2.5μηι 10mm x 2,1 mm

2x Strata-X Online Extraction, 25μηι 20mm x 2mm as online SPE

HPLC Krudcatcher Ultra Column In Line Filter with Ο,δμηι as gard column for SPE

Analytical column Kinetex 2,6μηι C18 100A 50mm x 2, 1 mm

Column temperatur Room temperature

Mobile Phases solvent A: 0,2% acetic acid in MilliQ

solvent B: 0,2% acetic acid in CAN

Gradient and column switching SPE-column (Loading Pump):

0.0 min: solvent B 90%; 1500μΙΛηίη

1.5 min: solvent B 5%; ΙδΟΟμΙ/min

2.3 min: inject sample

4.0 min: solvent B 5%; ΙδΟΟμΙ/min

Analytical column (Pump):

O.Omin: solvent B 80%; 250μΙΛηίη

0.1 min: solvent B 100%; 250μΙΛηίη (Flush)

0.5min: solvent B 80%; 250μΙΛηίη

4.0min: solvent B 80%; 250μΙΛηίη

AUTOSAMPLER Parameter:

Loop 500μΙ

Injection volume 250μΙ Injection mode partial loop

Temperature 5°C

Mass spectrometry was performed according to the following parameters:

Parent mass Product mass Colission energy Tube lens

231.145 1 15.230 17 55

275.153 101.220 18 58

289.236 1 15.259 15 59

297.200 123.256 15 59

303.160 129.270 16 49

317.165 143.190 17 57

388.140 201.120 15 39

446.378 298.139 15 69

454.370 306.100 15 69

545.282 471.300 15 87

603.247 155.200 39 49 Example 5: Results

Measurement of plasma spermidine levels in above-referenced animal model of critical illness revealed a totally different progression in rabbits that survived their illness until the day of sampling (day 7; "Survivor") as compared to animals that died because of their illness before that time point ("Non-Survivor"). Whereas plasma spermidine levels remained within the normal range in the surviving animals, the concentration significantly decreased in the non-surviving animals (cf. Fig. 3). Concomitantly, the plasma levels of putrescine, the breakdown product of spermidine, increased solely in the non-surviving animals (cf. Fig. 4). This result illustrates that an increased catabolism of spermidine (into putrescine) or otherwise disordered polyamine metabolism is detrimental for the development of a critical illness. Likewise, the spermidine/putrescine ratio, while within the normal range in surviving animals, strongly decreased in the non-surviving animals and thus represents a powerful prognostic and potentially diagnostic marker during conditions of critical illness (cf. Fig. 5).

Furthermore, the spermidine/putrescine ratios as determined also represent an important marker of organ (dys)function, as an inverse correlation between the spermidine/ putrescine ratio and the levels of plasma aspartate aminotransferase (AST), a marker of liver failure, could be observed (cf. Fig. 9). A higher spermidine/putrescine ratio in the plasma corresponds to lower levels of AST, which points to a better preservation of liver function. In addition, the spermidine/putrescine ratio also correlated inversely with plasma lactate levels (cf. Fig. 7) and directly (i.e. positively) with blood pH (cf. Fig. 8). Both latter parameters could be considered two other markers of illness severity. A higher plasma lactate and a lower blood pH point in general to an increased illness severity. Both conditions were found to be associated with lower spermidine/putrescine ratios in plasma.

Finally, plasma spermine levels appear to be an important marker of organ (dys)function as well, as there was a strong positive correlation between plasma spermine levels on the last day, and the levels of plasma aspartate aminotransferase (AST), a marker of liver failure (Spearman Rank correlation: Rho = 0.771 ; P = 0.0106). Higher spermine levels in the plasma correspond to higher AST levels, which points to a worsening of liver function. Furthermore, there was a nearly significant correlation between last-day spermine levels and the levels of another liver failure marker alanine aminotransferase (ALT) (Spearman Rank correlation: Rho = 0.529; P = 0.0809), which supports the result that the plasma spermine levels can reflect or predict liver function.

The present invention illustratively described herein may suitably be practiced in the absence of any element or elements, limitation or limitations, not specifically disclosed herein. Thus, for example, the terms "comprising", "including", "containing", etc. shall be read expansively and without limitation. Additionally, the terms and expressions employed herein have been used as terms of description and not of limitation, and there is no intention in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention claimed. Thus, it should be understood that although the present invention has been specifically disclosed by embodiments and optional features, modifications and variations of the inventions embodied therein may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention.

The invention has been described broadly and generically herein. Each of the narrower species and sub-generic groupings falling within the generic disclosure also form part of the invention. This includes the generic description of the invention with a proviso or negative limitation removing any subject matter from the genus, regardless of whether or not the excised material is specifically recited herein.

Other embodiments are within the following claims. In addition, where features or aspects of the invention are described in terms of Markush groups, those skilled in the art will recognize that the invention is also thereby described in terms of any individual member or subgroup of members of the Markush group.