MXPA06003173A - Method for the diagnosis of diseases by determining apolipoprotein ci - Google Patents

Abstract

A method for the diagnosis, early detection, risk estimation and control of the course of diseases, wherein the amount of apolipoprotein CI and/or derivatives thereof is determined in a serum or plasma sample from a human patient and it is possible to conclude as to the presence of a disease, particularly a tumoral disease or sepsis, on the basis of a result deviating significantly from the value range determined for normally healthy persons. The invention also relates to the use of apolipoprotein CI in the therapy and prevention of diseases wherein the amount of or binding ability of apolipoprotein C1 in the blood of sick persons is significantly different from that of normal persons.

Description

METHOD FOR THE DIAGNOSIS OF DISEASES BY THE DETERMINATION OF APOLIPOPROTEIN C-I FIELD OF THE INVENTION The present invention relates to a method for the diagnosis, early detection, risk assessment and monitoring of the course of diseases, in which the apolipoprotein IC protein is determined by a biomarker in serum or plasma samples of human patients. and to the use of apolipoprotein CI as an active, therapeutic substance.

BACKGROUND OF THE INVENTION It should be noted in principle that when the term "diagnostic method" is generally used in the following description, this term is proposed as a general rule for reasons of simplicity to represent methods for diagnosis, early detection, risk assessment. and supervision of the course, including supervision of the course associated with the therapy, of diseases, unless it is evident from the specific context that only measurements with limited, specific objectives are referred to in the respective point. Furthermore, it should be noted that, in the context of this Application, the term "diseases" is used for, as a general rule, serious, chronic and in particular acute diseases and is not proposed to refer to disorders of fat metabolism, since which can be considered as risk factors for the development of atherosclerosis. In particular, cancerous diseases and sepsis should be singled out as "diseases". However, the determination according to the invention can also be used in association with diagnostic methods which are proposed to provide more detailed information about the state of a patient who is suffering from other diseases, in particular acute heart disease, such such as cardiac infarction, angina pectoris and an arterial occlusive disease, or diabetes, Graves' disease and Crohn's disease. The proteins that form lipoproteins together with lipids (triglycerides, cholesterol, phospholipids) are designated as "apolipoproteins". An important function of. Apolipoproteins is to allow the transport of water-insoluble lipids in serum and plasma. Based on their migration behavior in the density gradient of an ultracentrifuge, lipoproteins are assigned to four different density classes: chylomicrons, VLDL (from: very low density lipoproteins), LDL (from: low density lipoproteins) and HDL (from: high density lipoproteins). In addition, a group designated as lipoprotein (a) is distinguished. The lipid fraction of lipoproteins is smaller the higher the density of the same. The lipoproteins of different density classes differ not only based on the amount of lipid fraction present as a whole but also with respect to the composition thereof. In addition, it is possible to assign typical apolipoprotein profiles to the respective density classes. Apolipoproteins are proteins of different length and composition of amino acids, which are assigned to groups designated by A-H, a distinction that is made in turn within the individual groups between different proteins, which are characterized by a linked number. Instead of the complete designation of, for example, apolipoprotein A-1, it has also been usual to speak only of Apo A-I. The primary structures (amino acid sequences) of the various apolipoproteins are known and the specific data or concepts with respect to their three-dimensional structure also exist for a variety of different apolipoproteins, particularly in association with lipids. The most detailed information must be found in the relevant scientific literature. It is known that apolipoproteins can also be determined specifically in connection with obtaining the data measured in a patient's fat metabolism, in particular for the early detection of the risk of atherosclerosis or for monitoring therapy in the treatment of a patient with drugs to lower lipids. The use is made of the fact that certain apolipoproteins are found exclusively or at least predominantly in lipoproteins of the individual lipoprotein density classes between those mentioned above and that determine their specificity, so that when determining the apolipoprotein fraction of the lipoproteins it is also possible to determine the proportion of the latter in the serum or plasma of a patient. It is also known that the profiles of lipoproteins and also of apolipoproteins different from a normal case are found in individual patients due to considerable effects that influence the formation of apolipoproteins or the enzymes that cleave them or manifest as receptor defects. In addition, it is known that alterations in the metabolism of fats (secondary dyslipoproteinemias), which are temporary in nature and disappear again after curing the disease, can also occur as a result of other diseases (see (20), pages 186- 189).; the bibliographical references in the form of numbers in parentheses - refer to the attached list of bibliographic references. Where changes in fat metabolism also manifest as changes in apolipoprotein compositions, the scientific literature mainly documents those changes that refer to apolipoproteins AI and A-II and as a general rule that reflect abnormal forms and concentrations of apolipoproteins. HDL However, apolipoproteins play no practical, significant role as biomarkers for the diagnosis of diseases which are also known to be accompanied by alterations in fat metabolism. The present Application relates to the determination of apolipoprotein C-I for diagnostic purposes. Apolipoprotein C-I belongs to a group of apolipoproteins that have a comparatively low molecular weight, which are designated as the apolipoproteins Apo C-I, Apo C-II and Apo C-III. The primary structures of these three proteins are known (see, (1), (2) and, with respect to apolipoprotein C-I, in particular (3)). Relatively little is known about the function of apolipoproteins CI and C-III, and some documented knowledge concerning changes in the amounts or forms and forms of association of apolipoprotein IC in human serum or plasma as a function is not available. of certain pathological conditions. Publications according to (1) to (18) are representative of publications in the scientific literature that refer to group C apolipoproteins or apolipoprotein IC, particularly work according to (11), which provide good vision general in relation to what is known about the role of apolipoproteins C in the metabolism of lipoproteins. The biological effects described for apolipoprotein IC include mainly the inhibition of the cholesterol ester transfer protein (CETP), the inhibition of the protein (LRP) associated with the LDL receptor (LDLR, for its Acronyms in English), and LDLR itself and VLDL receptors (VLDLR, for its acronym in English), and activation of lecithin-cholesterol acyltransferase (LCAT, for its acronym in English). There is no knowledge that allows a determination of the apolipoprotein IC as a biomarker for the diagnosis of diseases, such as, for example, sepsis or cancer or that suggests that a determination of the apolipoprotein IC in diagnostic contexts could be derived from the content of the publications. The present invention is the result of systematic research work by the Applicant which is directed to the investigation of changes in the protein composition of patients with sepsis and to make the results obtained from the research specifically usable for the diagnosis of sepsis and inflammation. A starting point of these investigations was the discovery that, in the case of sepsis, the prohormone procalcitonin is dramatically increased and that the determination of procalcitonin as a biomarker for sepsis is of great practical value for diagnosis and for monitoring associated with therapy (see, for example, EP 656 121 Bl). Additional discoveries regarding the change in protein composition in the serum or plasma of patients with sepsis, which were obtained by the Applicant starting from different approaches to research, must be found, for example, in the published EP Patent Application. 1 121 600 A2 and the published, additional Patent Applications with more recent date, such as WO02 / 085937, WO03 / 005035, O03 / 002600, EP 1 355 158 Al, EP 1 318 406 Al, EP 1 318 407 Al and EP 1 355 159 Al and the Patent Requests not yet published, relevant, additional to the Applicant. Reference is made to the content of the Patent Applications, in particular with respect to explanations of the modern understanding of the term sepsis and the importance of the determination of biomarkers in the case of sepsis. Since some of the investigations have shown that a variety of biomarkers which were traditionally considered only as typical markers of cancer (see EP 1 318 402 A1, EP 1 318 403 A1, EP 1 318 404 A1 and EP 1 318 405 Al) are significantly increased in the case of sepsis as well, plasma and serum samples from patients who suffered from various diseases due to malignant tumors were increasingly taken into account throughout the investigation of samples from patients with sepsis. In many of the cases investigated, it was found that individual biomarkers that are at a high level in the case of sepsis also rise significantly in the case of cancer patients. However, numerous cases also revealed substantial differences that prevent septic diseases and diseases from malignant tumors from being treated for simplification reasons generally as if they were fundamentally similar. The invention described in the present Application is the result of an additional research approach to determine proteins that are potentially suitable as biomarkers for diagnostic purposes because their measurable amounts in patients with sepsis and possibly in patients who are suffering from other serious diseases , in particular inflammatory diseases or cancerous diseases, are significantly changed compared to healthy people. For this purpose, the CLAR profiles of serum or plasma fractions of patients with sepsis were compared with those of apparently normal healthy persons and the significant changes in the profiles of the HPLC chromatography were subjected to a more precise examination. These investigations gave the results that became the basis of the present invention, specifically that significant changes in the CLAR elution profiles of serum or plasma samples from patients are detectable when these samples were separated early on a column containing a chromatography material which is capable of interacting with the hydrophobic or lipophilic constituents of the sample and selectively binds to these constituents ("hydrophobic interaction chromatography"). If the constituents of the separated sample are bound to the chromatography material are subsequently eluted in a suitable manner, a subfraction of the original serum or plasma sample is obtained, in which the constituents, in particular those of a proteinaceous nature, which are capable of interacting with the hydrophobic surfaces are selectively enriched. A variant of this method applied to lipoproteins is described in (19). The separation of these fractions from the plasma or serum of patients with sepsis and other diseases by means of high performance liquid chromatography (HPLC) led to the surprising discovery, on which the present invention is based, that a constituent which is found at Substantial levels in the sera and plasmas of normal healthy people are greatly reduced or apparently completely absent in the sera of patients with sepsis and certain other patients. Further investigations on the nature of this constituent, which are described in more detail below, showed that the significantly reduced constituent is apolipoprotein C-I. Subsequent additional investigations, which are described in the same way in greater detail later, then led to the surprising, additional result that the findings which, in an initial interim evaluation, indicated a parallelism of the changes in patients with sepsis and cancer should in fact be clearly distinguished from each other and make it possible to clearly distinguish the septic diseases from the cancers through the measurement of apolipoprotein CI in the serum or plasma of human patients, which as such constitutes a discovery which can be used for diagnostic purposes.

SUMMARY OF THE INVENTION An object of the present invention is to find a novel protein biomarker that is suitable for diagnostic determinations and make it usable for diagnostic purposes, the quantitative presence of this biomarker in human serum or plasma differs significantly from the values found in apparently normal healthy people and the determination of this constitutes therefore a valuable diagnostic tool. This object is achieved by means of a method claimed in claim 1. Preferred developments, in particular with respect to diagnostic applications in the case of patients with sepsis, patients with cancer and patients with other diseases, are established in the -reclarations. Where, in the present Application or in the claims, in addition to the apolipoprotein IC, the "derivatives thereof" are also mentioned as species to be determined, they should be understood as meaning in particular fragments or aggregates, in particular those that are they behave the same as the free protein apolipoprotein CI in the respective test method, respectively. The "derivatives" can be, for example, apolipoprotein C-I molecules shortened by individual amino acids or amino acid sequences or complete molecules of apolipoprotein C-I modified in a steric or conformational manner - for example by means of aggregation. The novel findings that allow the use of apolipoprotein C-I as a biomarker indicate that, in patients who are suffering from relevant diseases, the detectable amount of apolipoprotein C-I is greatly reduced compared to apparently normal healthy people. From this, it is possible to conclude that apolipoprotein C-I is formed in insufficient amounts, consumed and / or abnormally bound in the case of diseases, which can be interpreted as a deficiency of apolipoprotein C-I compared to healthy people. According to a further aspect, the invention also relates therefore to the use of apolipoprotein IC as a therapeutic agent for the treatment of apolipoprotein Ic deficiency conditions related to diseases and for the preparation of medicaments for the treatment of diseases which, at the diagnostic level, are manifested through a significant reduction in the measurable amounts of the apolipoprotein IC. These aspects of the invention form the main subject matter of claim 10. All the facts and teachings to be protected which are reproduced in the introduction and in the claims to which reference is made are explained in more detail below with reference to the experimental data and the associated figures, the meaning of the individual terms used in the present description is further explained. Therefore, express reference is made to the following statements for the interpretation of the patent claims and for the more accurate interpretation of the general statements made up to now.

BRIEF DESCRIPTION OF THE DRAWINGS In the figures: Figure 1 shows the elution profiles of the reverse phase CLAR C18 of the fraction of serum and plasma that were eluted with acetic acid (50 mM) from the chromatography columns Octyl Sepharose "11 which had been loaded with serum or plasma from (a) apparently normal healthy persons, (b) patients with various diseases due to malignant tumors and (c) patients with sepsis, after intermediate washing of the columns loaded with phosphate-buffered saline (PBS) Figure 2 shows the relative amounts obtained from the integration of peaks from the elution profiles of the CLAR of the type shown in Figure 1, of the eluble apolipoprotein CI in samples from healthy persons apparently normal and patients who suffered from diseases of different types (sepsis, tumor, cardiac infarction, angina pectoris, arterial occlusive diseases, Crohn's disease, Graves' disease, autoimmune thyroiditis, type I diabetes, type II diabetes, rheumatoid arthritis, Alzheimer's disease, seropositive HIV, in each case the diagnoses were confirmed clinically independently). Figure 3 shows the standard curve of a double-phase immunoassay operating with two apolipoprotein polyclonal antibodies against apolipoprotein C-I, which were obtained using the synthetic apolipoprotein C-I as standard. Figure 4 shows the results of the direct determination of apolipoprotein IC in sera and plasma of apparently normal healthy people, patients with sepsis and patients with tumors using a double phase immunoassay of the apolipoprotein IC, of which a calibration curve is shown typical in figure 3; Y Figure 5 shows the results of a repeat of the measurements, the results of which are shown in Figure 4, after the respective samples have been treated with Octyl SepharoseMR DESCRIPTION OF THE INVENTION Description of the experiments carried out and the results obtained.

A. CHROMATOGRAPHIC INVESTIGATIONS 1. Obtaining serum or plasma fractions containing human serum / plasma constituents that bind to hydrophobic surfaces and can be eluted from them. Samples in each case of 0.5 ml serum / plasma of healthy persons apparently normal and from patients suffering from a variety of clinically diagnosed diseases independently were mixed with 0.5 ml of Octyl Sepharose ™ chromatography material (source: Pharmacia, 0.25 ml of packed material, washed in PBS) in PBS and incubated for 10 minutes. minutes at room temperature with gentle agitation. Then, the mixture was introduced into a polypropylene column (5 mm in diameter) and the unbound substances were separated from the substances bound to the Octyl Sepharose ™ material by washing with 20 ml of PBS. The desorption of the substances (proteins) bound to the Octyl Sepharose ™ material was effected by means of elution with 50 mM acetic acid (pH 2.5). 2 Reverse Phase CLAR C18 The acetic acid eluate obtained from the Octyl Sepharose MR columns was analyzed in each case directly by means of the reverse phase HPLC C18 on a C18 μ Bondapak ™ column (3.9 x 300 mm, Millipore, Waters). The gradients of a mobile phase A (mixture of 95 parts by volume of water, 5 parts by volume of acetonitrile, 0.1 part by volume of trifluoroacetic acid) and a mobile phase B (10 parts by volume of water, 90 parts by volume of acetonitrile, 0.1 part by volume trifluoroacetic acid) were used for HPLC chromatography. After application of the acetic acid eluate to the HPLC column, the analysis was carried out in the following gradients of mobile phase A / mobile phase B: t = 0-5 minutes from 100/0 (A / B) to 65 / 35 (A / B) t = 5-20 minutes from 65/35 (A / B) to 40/60 (A / B) t = 50-22 minutes from 40/60 (A / B) to 0/100 (A / B) The flow rate was 1 ml / minute. The effluent from the column was continuously measured by absorption at 214 nm. Figure 1 shows three typical elution profiles for fractions of samples from apparently normal healthy patients (a), from patients with different tumor diseases (b) and from patients with sepsis (c). It was surprisingly found that the CLAR elution profiles of groups of people differ substantially and systematically from each other. The band that is critical to the present Application is that which is eluted in 17.05 minutes. This band was relatively quantified by the integration of peaks for all serum and plasma samples investigated and the relative amounts of substance determined in this manner are shown graphically in Figure 2 for a large number of control and patient samples. Specifically, the following samples were investigated: 42 samples from patients with tumors (9 x intestinal cancer, 7 x bronchial carcinoma, 13 x breast cancer, 8 x ovarian carcinoma, 5 x pancreatic carcinoma), 22 samples from patients with sepsis, 7 samples from patients with cardiac infarction (Ci), 4 samples from patients with angina pectoris (Ape), - 5 samples from patients with arterial occlusive diseases (AOD), 6 samples from patients with Crohn's disease (CD), 11 samples from patients with Graves' disease, 4 samples from patients with autoimmune thyroiditis (AIT), 10 samples from patients with type I diabetes (D-type I), 6 samples from patients with type II diabetes (D-type II), 3 samples from patients with rheumatoid arthritis (RA), 2 samples from patients with Alzheimer's disease (AD) and 6 samples from patients with HIV positive. As is immediately apparent from Figure 2, the values substantially positive for the substance corresponding to the peak measured at 17.05 minutes were found for samples from healthy, normal people. In the samples of those people suffering from diseases, particularly very clearly in the samples of patients with sepsis, patients with tumors, patients with manifest / acute heart diseases (Ci, APe, AOD) and patients with Crohn's disease, are measured the significantly reduced proportions of the same substance. Also for patients with type I diabetes and type II diabetes, significantly reduced values are found, although not reduced to the same degree, compared to healthy people. For patients with Graves' disease, autoimmune thyroiditis, rheumatoid arthritis, Alzheimer's disease and HIV-positive patients, deviations from the corresponding concentration are smaller or substantially insignificant. The average values of the relative amounts found, which are obtained for the groups of individual samples, are summarized in the following table: 3. Identification of the band used for the characterization of samples (band that ßluye in 17.5 minutes) For identification, the eluted substance corresponding to this band was collected, dried (centrifuged "Speed Vac" 11") and subjected to the analysis of peptides In a sequence analysis of the N terminus, the TPDVS sequence was determined.The molecular weight determined by means of the mass spectrum (ESI MS) is 6630 ± 15 D. These results are ambiguously correlated with the known data for human apolipoprotein IC (see for example (3)), which has the sequence of peptides according to SEQ ID No. 1 (see also access of SWISS PROT No. 3660227) and has a theoretical molecular weight of 6627 D After the trypsin digestion of the substance and the subsequent mass spectroscopy (ESI MS, "digital trypsin printing") the assignment made was completely confirmed.The apolipoprotein CI which can be determined by means of the described chromatographic process is also referred to as "free apolipoprotein C-I" in the context of the present Application. It has the binding properties of a serum or plasma sample (in dilution with PBS) to hydrophobic molecular structures, for example the octyl radical of a hydrophobic chromatography material Octyl Sepharose ™, from which it can be eluted under typical conditions for the elution of proteins, in particular with dilute acetic acid. However, the use of the term "free apolipoprotein C-I" does not necessarily mean that the material that can be separated by means of hydrophobic interaction chromatography has to be present in a completely free form in the original samples. The associates or aggregates, also with lipids which are separated under the experimental conditions in contact with Octyl Sepharose ™ with the binding of the apolipoprotein CI to the chromatography material, should also be considered as "apolipoprotein free CI" in the context of the use of this term in the present Application. In this way, the "hydrophobic molecular structures" can also be parts of the surface of a hydrophobic chromatography material. However, they can also be individual molecules having hydrophobic structural regions through which the binding of this molecule with the apolipoprotein IC can take place, for example molecules having hydrophobic structures which can be labeled and, for example, can serve in homogeneous assays to label the apolipoprotein CI in a sample fluid. The ability to bind to "hydrophobic molecular structures" is present when the regions of the apolipoprotein C-I in the sample which are available for this link are not blocked by other substances, for example the lipids in the lipoproteins. Even though, for example, it is established that these fractions of the apolipoprotein IC are determined to be bound to the Octyl Sepharose ™, this does not mean that it is really necessary to make use of the binding to the Octyl Sepharose "11 in the determination. this statement must be understood as a characterization of the substance to be determined, even if it is determined by completely different methods, for example, the corresponding apolipoprotein CI may be the same as that which is also determined, as described below, by means of of an immunoassay.

B. DETERMINATION OF DIRECT IMMUNODIAGNOSIS OF APOLIPOPPROTEIN CI IN SERMS / PLASM 1. Immunoassay of apolipoprotein CI For the determination of direct immunodiagnosis of apolipoprotein IC in serum, a double phase type immunoassay was formed of the components described below : (a) Coated tubes: the polystyrene tubes (Greiner) were coated with a polyclonal affinity-purified antibody, commercially available against apolipoprotein CI (source: Acris Antibody, Bad Neuheim, Germany). According to the manufacturer's information, the antibody had been obtained by immunizing rabbits with human Apo C-I and purified on a Sepharose ™ affinity column with human apolipoprotein C-I. For the coating, 0.2 μg of the antibody in 300 μl of PBS was attached to the polystyrene tubes (Greiner, Germany) which had been coated with sheep anti-rabbit IgG antibodies (Sigma). The union was completed after 18 hours at room temperature. The tubes were then washed twice with 3 ml portions of 0.5% bovine serum albumin (BSA) in PBS. After they had dried in vacuo, the tubes were used as a solid phase for the immunoassay of apolipoprotein C-I. b) Antibody labeled with acridinium ester: 100 μg of another antibody against human apolipoprotein IC (from rabbit, source: Academie Bio-Medical Company, Texas, USA) in 100 μl of PBS were reacted with 10 μg of acridinium ester NHS (in 10 μl of acetonitrile). After incubation for 10 minutes at room temperature, the labeled antibody was purified by separating the unconverted constituents of the reaction mixture by means of HPLC on SW 300 (Waters). For use in the immunoassay, the labeled antibody in PBS with 0.5% BSA and 1 mg / ml rabbit IgG was sted to approximately 1 million RLU / 300 [mu] L (RLU = relative light units) for saturation of the walls of the tubes. 2. Performance of the immunodiagnostic determination of apolipoprotin C-I in serum or plasma samples Serum or plasma samples were diluted 1: 10,000 with PBS, BSA 0.5%. In each case, 300 μl of them were pipetted into the aforementioned tubes coated with the immobilized antibody and then incubated for 4 hours at room temperature with shaking (300 rpm on a Heidolph ™ rotary shaker). The content of the tubes was then washed with PBS (filling and decanting 4 times with 1 ml of PBS in each case) and the apolipoprotein CI attached to the wall of the tubes was reacted in the course of 20 hours at room temperature and 300 rpm with 300 μl per tube of anti-apolipoprotein CI antibody labeled with acridinium ester. Then, the unbound, labeled antibody was removed by washing 5 times with 1 ml portions of PBS and the remaining chemiluminescence was measured in a known manner on a Berthold 952 TMR luminometer. A synthetic C-I apolipoprotein was used to calibrate the assay. A typical standard curve that was obtained for the previous test is shown in Figure 3. 3. Results With the help of the described immunoassay of the double phase type, the controlled sera of apparently normal healthy persons, also used in the chromatographic investigations, and samples of sepsis and tumors were measured. Measurements of samples from patients with sepsis substantially confirm the results of the HPLC investigation, because significantly reduced measured values were obtained for apolipoprotein C-I compared to normal persons. In the measurement of samples from patients with tumors, for which reduced concentrations were also determined in the chromatographic investigations, however, there was a tendency to increase in the measured values that were obtained. The results are shown graphically in figure 4. To verify the question whether the different results for patients with tumors in the chromatographic determination on the one hand and the immunodiagnostic determinations on the other hand were due to the previous screening step by means of chromatography of hydrophobic interaction, the samples measured in the immunoassay were treated with Octyl Sepharose ™ before an additional determination for the binding of the "free" fractions of the apolipoprotein CI.

Treatment of samples with Octyl Sepharose ™ 150 μl of the same serum or plasma samples that had been measured as described in the immunoassay were mixed with 200 μl of Octyl Sepharose ™ 1 (Pharmacia, 50 μl of a gel in PBS) and they were incubated for one hour at room temperature with gentle agitation, after which Octyl Sepharose ™ with apolipoprotein CI fractions bound thereto were separated by centrifugation (15 minutes at 200 G). The supernatant obtained was diluted 1: 5000 in PBS / BSA 0.5% and then measured, as described above, in the immunoassay. The results obtained are shown in Figure 5. It is evident that, as a result of treatment with Octyl Sepharose ™, the total of "free" apolipoprotein IC that is detectable in the measurement by means of the described immunoassay of the double-phase type had been linked to Octyl Sepharose ™ so that the apolipoprotein CI was no longer detectable in the supernatant. Similar observations were made for the treated sepsis samples, since most of the determinable amounts of apolipoprotein IC that were already greatly reduced compared to the normal samples were removed from the sample by means of the Octyl Sepharose treatment. . Surprisingly, the C-I apolipoprotein determinable in an immunoassay of the type described was not removed, however, by treatment with Octyl Sepharose ™ from the samples of patients with tumors.

C. DISCUSSION The described findings show that at least a major part of the apolipoprotein IC in sera or plasmas in patients with tumors is present in a form in which it has lost its ability to bind to hydrophobic surfaces but can still be determined as immunoreactivity. in an immunoassay. One possible explanation for these observations would be that, in the case of the apolipoprotein IC found in sera from patients with tumors, the molecular regions available for hydrophobic interactions are saturated or blocked, in particular by binding partners which can not be displaced by Octyl SepharoseMR. The nature of these union partners is currently unknown. However, it can not be ruled out that they are substances specific for tumors which, as such or on the basis of the deactivation of the hydrophobic binding partners, as such, for example, of the apolipoprotein IC, play an important role for the development of tumors, for example by having an adverse effect on the natural immune response or by actively promoting the growth of tumors. Although a significant reduction of the apolipoprotein CI fraction in the samples investigated by the immunodiagnostic method is not detectable, parallel chromatographic investigations show that it is not "free" in the sense mentioned above but is present in another form with reduced capacity of binding to hydrophobic binding partners. The "free" apolipoprotein C-I, ie the apolipoprotein C-I which binds to hydrophobic binding partners, is on the other hand reduced in the sera of patients with tumors as in the case of other diseases such as, for example, sepsis. Based on the described findings, it can be assumed that it is advantageous for patients with tumors to compensate for their altered balance in the sense of a reduced proportion of the "free" lipoprotein IC through the external supply of the apolipoprotein IC and therefore have a positive influence on the pathological process, for example by saturating the typical binding partners for tumors by the external apolipoprotein IC and by providing free, additional apolipoprotein IC. A similar assumption, specifically that an external supply of the apolipoprotein C-I to compensate for an altered level of the "free" apolipoprotein C-I is advantageous, also applies to the other diseases, for example in the case of sepsis. If the production of apolipoprotein C-I is altered in the case of sepsis, an external supply can compensate for a deficiency caused by it. Whether the reduction of the "free" apolipoprotein CI in the serum / plasma of a patient in the case of sepsis is attributable to the removal of the apolipoprotein IC from the circulation by binding to pathological tissue structures or possibly insoluble bacterial structures, a External supply of the apolipoprotein IC can compensate for the resulting deficiency or effect saturation of the binding sites in a pathological tissue or in the bacterial structures. By monitoring the concentration of the free apolipoprotein increased by the external supply or the apolipoprotein determinable as immunoreactivity in the blood samples of the treated patient, the amount required in each case can be monitored in a simple manner. The present invention also allows in this way a novel therapeutic use of the apolipoprotein IC or of suitable derivatives or compounds that simulate its physiological behavior, for the treatment of cancer, sepsis and other diseases which are associated with a reduction of the free apolipoprotein IC. , detectable compared to healthy people.

List of references: 1. Erika Polz et al., Human Apolipoprotein C-I: Concentration in Blood Serum and Lipoproteins, Biochemical Medicine 24, 229-237 (1980) 2. Kane, J.P., in "Lipid Metabolism in Mammals" (F. Snyder, Ed.), Vol. 1, S. 209, Plenum, New York, 1977 3. Shulman, RS et al., The complete amino acid sequence of CI (apoLP-Ser ), an apolipoprotein from human low-density lipoproteins, J Biol Chem. 250, 182-190 (1975) 4. Xiao-Ren Pan et al., Abnormal Composition of Apolipoproteins CI, C-II, and C-II in Plasma and Very -Low-Density Lipoproteins of Non-Insulin-Dependent Diabetic Chine, Clin. Chem. 32, No. 10, 1914-1920 (1986) 5. Jutta Poensgen, Apolipoprotein C-I inhibits the hydrolysis by phospholipase A2 of phospholipids in liposomes and cell membranes, Biochim. Biophys. Acta, 1042 (1990), 188-192 6. Ming Liu et al., Activation of plasma lysolecithin acyl-transferase reaction by apolipoproteins A-I, C-I and E, Biochim. Biophys. Acta, 1168 (1993), 144-152 - 7. Nina D. Bren et al., Quantification of Human Plasma Ápolipoproteins C-I, C-II, and C-III b Radioimmunoassays, Mayo Clin Proc, July 1993, Vol. 68, 657-664 8. Rampratap. S. Kushwaha et al., Characterization of cholesteryl ester transfer protein inhibitor from plasma of baboons (Papio sp.), J. Lipid Res. 1993, 34: 1285-1297 9. N. Savion et al., Role of apolipoproteins A-I, A-II and CI in cholesterol efflux from endothelial - and smooth muscle cells, Eur Heart J (1993) 14, 930-935 10. Janine H. van Ree and collaborators, Inactivation of Apoe and Apocl by two consecutive rounds of gene targeting: effects on mRNA expression levéis of gene cluster members, Human Molecular Genetics, 1995, Vol. 4, No. 8, 1403-1409 11. Miek C. Jong et al., Role of ApoCs in Lipoprotein Metabolism - Functional differences Between ApoCl, ApoC2, and ApoC3, Arterioscler Thromb Vasc Biol. 1999; 19: 472-484 12. Thomas Gautier et al., Human Apolipoprotein CI Accounts for the Ability of Plasma High Density Liproproteins to Inhibit the Cholesteryl Ester Transfer Protein Activity, J. Biol. Chem. 275, No. 48, 37504-37509, ( 2000) 13. Jong MC et al., Insights into Apolipoprotein C Metabolism from Transgenic and Gene-Targeted Mice, Int. J. Tissue React. XXII (2/3) 59-66 (2000) 14. Neil S. Shachter, Apolipoproteins C-I and C-III as important modulators of lipoprotein metabolism, Current Opinion in Lipidology 2001, 12: 297-304 15. Benjamín W. Atcliffe et al., The interaction of human apolipoprotein C-I with sub-micellar phospholipid, Eur. J. Biochem. 268, 2838-2846 (2001) 16. Thomas Gautier et al., Apolipoprotein Cl Deficiency Markedly Augments Plasma Lipoprotein Changes Mediated by Human Cholesteryl Ester Transfer Protein (CETP) in CETP Transgenic / ApoCI-knocked Out Mice, J. Biol. Chem. 277 , No. 35, 31354-31363 (2002) 17. Puiying A. Mak et al., Regulated Expression of the Apolipoprotein E / CI / C-IV / C-II Gene Cluster in Murine and Human Macrophages, J. Biol. Chem. 277, No. 35, 31900-31908 (2002) 18. Johan Bjórkegren et al., Postprandial Enrichment of Remnant Lipoproteins With ApoC-I in Healthy Normolipi emic Men With Early Asymptomatic Atherosclerosis, Arterioscler Thromb Vasc Biol. 2002; 22: 1470-1474 19. John G. Raynes et al., Purification of Serum Amyloid A and Other High Density Apolipoproteins by Hydrophobic Interaction Chromatography, Analytical Biochemistry 173, 116-124 (1988) 20. Lothar Thomas (Editor): Labor und Diagnose, 5. erweiterte Auflage, Frankfurt 200, Kapitel 4 Fettstoffwechsel, S.177-190 21. Cesare R. Sitori, Evaluation of Lipoproteins / Apolipoproteins as Therapeutic Agents for the Treatment of Vascular and Nonvascular Disease, Am J Cardiol., Vol 81, 36F-39F (1998)

Claims (10)

1. CLAIMS 1. A method for the diagnosis, early detection, risk assessment and monitoring of the course of diseases, characterized in that the content of apolipoprotein IC and / or derivatives thereof is determined in a serum or plasma sample of a human patient and the presence of a disease is concluded based on a result of the determination that differs significantly from the range of value determined for normal healthy persons.
2. 2. The method according to claim 1, characterized in that the fraction of the total apolipoprotein C-I that is present in a sample which has the ability to bind to hydrophobic molecular structures ("free apolipoprotein C-I") is determined.
3. 3. The method according to claim 1, characterized in that the fraction of the total apolipoprotein CI that is present in a sample is determined which is detectable by direct determination in the sample using a double phase type immunoassay ( "Immunoreactivity of the apolipoprotein CI").
4. The method according to any of claims 1 to 3, characterized in that it is carried out for the diagnosis, early detection, risk assessment and monitoring of the course of diseases which are selected from the group consisting of cancerous diseases, sepsis , acute heart disease, diabetes, Graves' disease and Crohn's disease.
5. 5. The method according to any of claims 1 to 4, characterized in that it is carried out for the diagnosis, early detection, risk assessment and supervision of the course of sepsis and correlates a proportion of the apolipoprotein CI that is significantly reduced compared to normal healthy people and binds to hydrophobic molecular structures ("free C-I apolipoprotein") and / or a reduced apolipoprotein C-I immunoreactivity in a serum or plasma sample of the patient with a septic condition.
6. 6. The method according to any of claims 1 to 4, characterized in that it is carried out for the diagnosis, early detection, risk assessment and monitoring of the cause of a cancerous disease and correlates a proportion of the apolipoprotein IC that it is significantly reduced compared to normal healthy people and binds to hydrophobic molecular structures ("apolipoprotein free CI") and increased immunoreactivity of the apolipoprotein CI in a serum or plasma sample of the patient with a cancerous disease.
7. 7. The method according to claim 6, characterized in that when an increased lipoprotein IC immunoreactivity is found in a patient's serum or plasma sample, the measured value is verified by means of the determination of additional control in which a check is carried out to determine if the value for the determinable immunoreactivity in the sample is significantly changed by the treatment of the sample with an adsorbent material with hydrophobic surfaces and wherein the presence of a cancerous disease is concluded with high probability when the value does not deviate or does not deviate significantly from the original measured value.
8. 8. The method according to any of claims 1 to 7, characterized in that the apolipoprotein CI that binds to molecular, hydrophobic structures ("apolipoprotein free CI") is determined by subjecting a sample of serum or plasma to the interaction chromatography hydrophobic, in which the apolipoprotein CI binds to the chromatography material and then determine the apolipoprotein CI in the eluted protein fraction.
9. 9. The method according to claim 8, characterized in that Octyl Sepharose is used as the chromatography material, the unbound constituents of the sample are removed by washing the chromatography material, the bound proteins are eluted with a dilute acid , in particular acetic acid, and the amount of the apolipoprotein CI in the eluate is then determined by means of HPLC and / or by means of immunodiagnosis.
10. 10. The use of apolipoprotein C-I for the preparation of a drug to be administered by injection or proposed for the therapeutic treatment of sepsis or cancerous diseases.