CN109868312B - IL-1ra and olanzapine induced metabolic adverse reaction - Google Patents

Abstract

The invention provides IL-1ra and application of a detection reagent thereof. In particular, the invention provides the use of an interleukin 1 antagonist (IL-1 ra) or a detection reagent thereof for the preparation of a diagnostic reagent or diagnostic kit for determining whether a patient suffering from schizophrenia is suitable for treatment with olanzapine. The invention also provides a corresponding detection kit and a detection method.

Description

IL-1ra and olanzapine induced metabolic adverse reaction

Technical Field

The invention relates to the fields of pharmacology and molecular detection, in particular to IL-1ra and application thereof in predicting olanzapine-induced metabolic adverse reactions.

Background

Schizophrenia (SZ) is a severe, highly ill, recurrent chronic disabling mental disorder, which is a major illness in young and young years with a lifetime incidence of about 1% of the world population. The patients suffering from the schizophrenia not only affect the life quality and family relationship of the patients, but also cause serious burden on society and economy. The death rate is 2-3 times higher than that of common people, and the service life is shortened by 10-20 years compared with that of common people. Of the mortality observed in schizophrenia, which is higher than that observed in the general population, approximately 60% are due to physiological co-diseases, especially cardiovascular diseases (cardiovascular disease, CVD).

The second generation antipsychotics, also known as atypical antipsychotics (atypical antipsychotics, AAP), are widely used clinically because they have a remarkable effect on positive symptoms such as hallucinations, delusions, etc. of schizophrenia, and are also effective on negative and cognitive symptoms. Furthermore, although atypical antipsychotics have a much lower risk of developing extrapyramidal symptoms (extrapyramidal syndrome, EPS) than typical antipsychotics, they cause a different degree of metabolic side effects such as: weight gain, abnormal carbohydrate metabolism, dyslipidemia, in turn leading to more serious cardiovascular disease. These side reactions severely limit the clinical use of the drug. In addition to causing long-term cardiovascular health risks, these side effects can also reduce compliance with the medication, ultimately leading to exacerbation of the patient's clinical symptoms.

Olanzapine is one of the representatives of atypical antipsychotics, as its stable therapeutic effect is often the therapeutic of choice in the clinic. Olanzapine has a faster onset of action in the acute phase as a first drug for the treatment of schizophrenia. In addition, a systematic review also shows that olanzapine compares to other antipsychotics in randomized clinical trials, such as: aripiprazole, quetiapine, risperidone, ziprasidone and perphenazine have lower all-factor withdrawal rates (all-cause treatment discontinuation). In observational studies, the total cause withdrawal rate of olanzapine was lower than for other antipsychotics, such as: amisulpride, risperidone, haloperidol and perphenazine. However, the risk of metabolic side effects is similar to that of clozapine, which is a second-line drug, and is far higher than that of other first-line antipsychotics.

Currently, there are many hypotheses about how olanzapine causes metabolic side reactions, but the pathophysiological processes and intrinsic mechanisms of olanzapine-induced metabolic side reactions are not well defined, the reasons for which may be related to the H1,5-HT, D2, M3 receptors. In recent years, the metabolic adverse reaction induced by cytokines and second-generation antipsychotics is attracting more and more attention, but most of the researches are carried out at present by one or a few factors, and most of the medicines are mixed and rarely single medicine. Thus, there is a need in the art to further explore whether cytokines are involved in the mechanisms by which olanzapine induces an adverse metabolic response, and whether cytokines can be predicted to cause an adverse metabolic response by olanzapine treatment.

Disclosure of Invention

The invention aims to provide IL-1ra and application thereof in predicting metabolic adverse reactions induced by olanzapine.

The invention also aims to provide a feasible detection method for detecting IL-1ra in serum of patients with schizophrenia.

In a first aspect of the invention there is provided the use of an IL-1ra gene sequence, protein (interleukin 1 antagonist), or detection reagent for the preparation of a diagnostic reagent or diagnostic kit for determining whether a patient suffering from schizophrenia is suitable for treatment with olanzapine.

In another preferred embodiment, said determining whether a patient suffering from schizophrenia is suitable for treatment with olanzapine comprises:

(a) Judging the risk (susceptibility) of metabolic abnormalities in schizophrenic patients after treatment with olanzapine, and/or

(b) The risk (susceptibility) of cardiovascular disease in schizophrenic patients after treatment with olanzapine is judged.

In another preferred embodiment, the metabolic abnormality comprises abnormal carbohydrate and lipid metabolism.

In another preferred embodiment, the lipid comprises cholesterol, low Density Lipoprotein (LDL), apolipoprotein B (ApoB).

In another preferred embodiment, the metabolic abnormality comprises a leptin metabolic abnormality.

In another preferred embodiment, the determination includes an auxiliary determination and/or a pre-treatment determination.

In another preferred embodiment, the determination is comparing IL-1ra content A1 of the sample from the subject with corresponding IL-1ra content A0 of the normal population, and if A1 is significantly higher than A0, then the subject is indicated to be unsuitable for treatment with olanzapine.

In another preferred embodiment, the subject is not suitable for treatment with olanzapine:

(a) The test subjects are at higher risk of developing metabolic abnormalities following olanzapine treatment, and/or

(b) The risk of cardiovascular disease is higher in the test subjects after olanzapine treatment.

In another preferred embodiment, the term "significantly higher" means that A1/A0 is 1.35 or more, preferably A1/A0 is 1.5 or more, more preferably A1/A0 is 2.0 or more.

In another preferred embodiment, the subject is a schizophrenic patient.

In another preferred embodiment, the number of normal people is at least 100; preferably at least 300 people; more preferably at least 500, most preferably at least 1000.

In another preferred embodiment, the diagnostic reagent or diagnostic kit is used for detecting a blood sample, a plasma sample, or a serum sample, preferably a peripheral blood sample.

In another preferred embodiment, the detection reagent comprises a protein chip, a nucleic acid chip, or a combination thereof.

In another preferred embodiment, the detection reagent comprises an IL-1ra specific antibody.

In another preferred embodiment, the IL-1ra specific antibody is conjugated or provided with a detectable label.

In another preferred embodiment, the detectable label is selected from the group consisting of: chromophores, chemiluminescent groups, fluorophores, isotopes or enzymes.

In another preferred embodiment, the IL-1ra specific antibody is a monoclonal antibody or a polyclonal antibody.

In another preferred embodiment, the IL-1ra gene sequence, protein is used as a standard.

In a second aspect of the invention, there is provided a diagnostic kit comprising a container containing a detection reagent for detecting IL-1 ra; and a label or instructions stating that the kit is for determining whether a schizophrenic patient is suitable for treatment with olanzapine.

In another preferred embodiment, the kit further comprises the detection reagent and instructions for use spotted on a test plate.

In another preferred embodiment, the kit further comprises a sample pretreatment reagent and instructions for use.

In another preferred embodiment, the specification describes a detection method and a method for determining based on the A1 value.

In another preferred embodiment, the kit further comprises IL-1ra gene sequences, protein standards.

In a third aspect of the invention, there is provided a method of determining whether a patient suffering from schizophrenia is suitable for treatment with olanzapine comprising the steps of:

(a) Providing a sample of the test subject;

(b) Determining the content of IL-1ra in the sample as A1;

(c) Comparing step (b) with the IL-1ra content A0 of the normal population sample, if A1 is significantly higher than A0, then the test subject is indicated to be unsuitable for treatment with olanzapine.

In another preferred embodiment, the subject is a human or non-human mammal.

In another preferred embodiment, the test sample is a blood sample and/or a serum sample.

In another preferred embodiment, the method is non-diagnostic and non-therapeutic.

It is understood that within the scope of the present invention, the above-described technical features of the present invention and technical features specifically described below (e.g., in the examples) may be combined with each other to constitute new or preferred technical solutions. And are limited to a space, and are not described in detail herein.

Drawings

FIG. 1 shows the effect of cytokines. Among them, fig. 1A, 1B and 1C show that cytokines are involved in the signal transduction processes of autocrine (a), paracrine (B) and endocrine (C), respectively, as immunomodulators.

Figure 2 shows a comparison of cytokine levels before olanzapine treatment in schizophrenic patients with healthy controls. FIG. 2A shows a comparison of IL-1ra levels at baseline for schizophrenic patients with healthy controls (P < 0.0001); figure 2B shows a comparison of schizophrenia patient at baseline with healthy control MCP-1 levels (p=0.0029); figure 2C shows a comparison of schizophrenic patients at baseline with healthy control VEGF levels (p=0.0141).

Figure 3 shows that metabolic index is significantly elevated after olanzapine treatment for 4 weeks, 8 weeks in schizophrenic patients. Figure 3A shows a significant increase in BMI (p=0.0023) following olanzapine treatment in schizophrenic patients; fig. 3B shows serum insulin elevation (p=0.00003); fig. 3C illustrates a significant increase in cholesterol (p=0.0035); fig. 3D is a triglyceride (p=0.0008); fig. 3E is low density lipoprotein (p=0.034); fig. 3F is apolipoprotein B (p=0.0051).

Figure 4 shows that leptin levels were significantly elevated (P < 0.0001) after olanzapine treatment for 4 weeks in schizophrenic patients.

Fig. 5 shows that IL-1ra levels at baseline correlate significantly with changes in cholesterol, low density lipoprotein, and fig. 5A shows IL-1ra baseline levels with changes in cholesterol (r=0.576, p=0.004); fig. 5B shows IL-1ra levels and low density lipoprotein variation (r=0.567, p=0.005) at baseline; fig. 5C shows IL-1ra levels at baseline versus apolipoprotein B variability (r=0.490, p=0.018); fig. 5D shows IL-1ra levels at baseline with leptin change (r=0.524, p=0.010).

Fig. 6 shows that baseline VEGF levels are significantly correlated with ApoA1, apoE changes, and fig. 6A shows VEGF baseline levels with ApoA1 changes (r=0.537, p=0.008); fig. 6B shows baseline VEGF levels versus ApoE change (r=0.419, p=0.047).

Detailed Description

The inventors have conducted extensive and intensive studies and have unexpectedly found for the first time that IL-1ra levels are significantly associated with olanzapine-induced metabolic adverse effects. Experiments show that IL-1ra levels before treatment are significantly positively correlated with elevated levels of cholesterol, LDL, apoB and leptin in patients after 8 weeks olanzapine treatment. The results suggest that IL-1ra may predict olanzapine-induced lipid and leptin metabolism abnormalities and may serve as a potential biomarker for cardiovascular risk assessment prior to olanzapine administration. IL-1ra and its detection reagents can be used to determine whether a schizophrenic patient is suitable for treatment with olanzapine. The present invention has been completed on the basis of the above.

In addition, the present invention also found that the levels of IL-1ra, MCP-1 and VEGF were significantly elevated in schizophrenic patients compared to the normal control group, and that there was no significant change in the levels of the three after treatment with the atypical antipsychotic olanzapine. The results suggest that elevated IL-1ra, MCP-1 and VEGF levels may be involved in the mechanism of schizophrenia occurrence through neuroinflammation, and that they may be potential biomarkers of schizophrenia status.

Cytokines and methods of use

Cytokines a well-defined group of small cell signaling proteins (5-20 kDa) were originally found in experiments in which interferon-alpha interfered with viral replication. Cytokines found in the same period also include macrophage migration inhibitory factor (macrophage migration inhibitory factor, MIF).

Dudley Dumonde in 1969 proposed the naming of proteins secreted by lymphocytes as "lymphokines", and hereinafter proteins produced by macrophages and monocytes are referred to as "monokines". As research is advanced, scientists recognize that these small molecule proteins should belong to a large class of proteins with self-defense functions, and therefore these small molecule proteins are named "cytokines".

Cytokines are synthesized by a variety of cells, the most major sources being immune cells such as macrophages, B lymphocytes, T lymphocytes and mast cells. Endothelial cells, fibroblasts and various stromal cells are also capable of synthesizing cytokines. A particular cytokine may be produced by different cell types. Cytokine secretion by cells and subsequent production by surrounding cells. Cytokines are involved as immunomodulators in the processes of autocrine, paracrine, and endocrine signal transduction (fig. 1).

Cytokines include chemokines, interferons, interleukins, lymphokines, tumor necrosis factors, and growth factors. Hormones do not belong to cytokines, but there is some conceptual overlap with cytokines. Cytokines differ from hormones in that, although also important cell signaling molecules, the concentration of the hormone in the blood circulation does not vary much, and the hormone is often produced by a specific class of cells.

In the immune system, the function of cytokines is extremely important. Cytokines regulate the balance between humoral and cellular immune responses by activating receptors; cytokines can also regulate maturation, growth, and immune responses of specific cell types. Some cytokines potentiate or inhibit the effects of other cytokines in a more complex manner.

Cytokines are involved not only in physiological processes in healthy humans, but also in the pathological response of many diseases, especially infections, immune responses, inflammation, wounds, sepsis, cancer, etc.

Monocyte chemotactic protein (monocyte chemo-attractant protein, MCP-1)

MCP-1 belongs to the C-C subfamily of chemokine superfamily, consisting of 76 amino acids, and is produced by a variety of cells in vivo, such as: endothelial cells, smooth muscle cells, monocytes, macrophages, adipocyte nuclear lineage membrane cells, and the like. MCP-1 can reduce sugar intake of fat cells after insulin action, and can influence sugar intake of fat tissues by changing endocrine function of fat cells, thereby causing insulin resistance of fat tissues. It has been shown that MCP-1 overexpression may be associated with NF-kB activation, which promotes TNF- α expression, thereby further promoting up-regulation of MCP-1 expression, and promoting the occurrence and development of inflammatory processes. The receptor C-C motif of MCP-1, chemokine receptor 2 (C-C motif chemokine receptor-2, CCR 2), is involved in the development of mononuclear/macrophage inflammation and atherosclerosis, fatty liver, and insulin resistance. Mice lacking the CCR2 gene are fed with high fat, are less obese than wild type control mice, and the inflammation of adipose tissue is correspondingly reduced. Short term administration of CCR2 antagonists in obese mice reduced macrophage content in adipose tissue and improved insulin resistance. Indicating that metabolic disorders may be associated with interactions between chemokines and chemokine receptors.

Tumor necrosis factor alpha (tumor necrosis factor-alpha, TNF-alpha)

TNF- α is a multifunctional cytokine that can mediate many biological effects, such as: can regulate lipid balance in organism metabolism. Studies have found that in vivo TNF- α in diabetic (diabetes mellitus, DM) patients is significantly higher than normal controls. TNF- α is distributed and expressed in adipose tissue in vivo, inhibits adipocyte differentiation and is involved in fat loss in many wasting diseases. Obesity can promote expression of fat cells and release TNF-alpha, and further control proliferation and differentiation of fat cells through an autocrine or paracrine pathway, but the specific mechanism is unknown so far.

Interleukin 1 antagonists (Interleukin-1receptor antagonist,IL-1 ra) and Interleukin 1 beta (Interleukin-1 beta, IL-1 beta)

Interleukin 1 (IL-1) was originally found to be an endogenous pyrogen and to have lymphocyte activating effects, mainly produced by monocytes and macrophages, and a small fraction derived from neutrophils, keratinocytes, epithelium, endothelium, lymphocytes, and smooth and fibroblasts. IL-1 consists of two structurally similar substructures, IL-1α and IL-1β, respectively. Under normal physiological conditions, IL-1 alpha is difficult to detect in human blood or body fluid due to its low content. However, in severe inflammatory reactions, dead cells released IL-1α in large amounts, which could be detected. IL-1β is produced and secreted extracellularly by cells, and therefore is a commonly detected indicator in peripheral blood. It has been found that the production of IL-1 beta can impair glucose tolerance and cause insulin resistance.

IL-1ra is a natural antagonist of anti-inflammatory factors, IL-1 beta, produced primarily by adipocytes. The only IL-1ra detectable in blood is secreted IL-1ra (secreted IL-1 ra) among the 4 subtypes. IL-1ra is significantly elevated in obese patients and is associated with BMI and insulin resistance. The IL-1ra genotype of type 2 diabetics may increase cardiovascular risk.

In the present invention, the terms "protein of the present invention", "IL-1ra protein", "IL-1ra polypeptide", "IL-1ra" are used interchangeably and refer to a protein or polypeptide having the amino acid sequence of a human IL-1ra protein (GeneBank accession No. 3557). They include IL-1ra proteins with or without an initiating methionine. In addition, the term also includes full-length IL-1ra and fragments thereof, particularly secretory fragments (or secreted proteins). The IL-1ra proteins referred to in the present invention include their complete amino acid sequences, their secreted proteins, their mutants, and functionally active fragments thereof.

Furthermore, the IL-1ra proteins of the invention include glycosylated and non-glycosylated proteins.

In the present invention, the terms "IL-1ra gene", "IL-1ra polynucleotide" are used interchangeably and refer to a nucleic acid sequence having a human IL-1ra nucleotide sequence (GeneBank accession No. 3557). It is understood that substitution of nucleotides in the codon is acceptable when encoding the same amino acid. It is further understood that nucleotide substitutions are also acceptable when conservative amino acid substitutions are made by the nucleotide substitutions.

In the case of an amino acid fragment of IL-1ra, a nucleic acid sequence encoding it can be constructed therefrom, and a specific probe can be designed based on the nucleotide sequence. The full-length nucleotide sequence or a fragment thereof can be obtained by PCR amplification, recombinant methods or artificial synthesis. For PCR amplification, primers can be designed based on the IL-1ra nucleotide sequences disclosed in the present invention, particularly the open reading frame sequences, and amplified to obtain the relevant sequences using a commercially available cDNA library or a cDNA library prepared by conventional methods known to those skilled in the art as a template. When the sequence is longer, it is often necessary to perform two or more PCR amplifications, and then splice the amplified fragments together in the correct order.

Once the relevant sequences are obtained, recombinant methods can be used to obtain the relevant sequences in large quantities. This is usually done by cloning it into a vector, transferring it into a cell, and isolating the relevant sequence from the propagated host cell by conventional methods.

Furthermore, the sequences concerned, in particular fragments of short length, can also be synthesized by artificial synthesis. In general, fragments of very long sequences are obtained by first synthesizing a plurality of small fragments and then ligating them.

Interleukin 8 (IL-8)

IL-8 is a multifunctional chemokine that is produced by macrophages, epithelial cells, airway smooth muscle cells and endothelial cells, the primary function of which is to induce chemotaxis and phagocytosis of immune cells. Oxidative stress stimulates interleukin-8 secretion to increase the stimulus, thereby causing recruitment of inflammatory cells, and induces further increase of oxidative stress mediators, which is an important index of local inflammation. IL-8 has been shown to be associated with insulin resistance and obesity. In an endo-secretory pancreatic study, it was found that IL-8 can be produced in situ in the pancreas and blocking secretion of pancreatic cells IL-8 can result in reduced monocyte/macrophage migration. But the specific mechanism is not known. In addition, IL-8 shares a common pathway with vascular endothelial growth factor (vascular endothelial growth factor, VEGF), a strong contributor to the stimulation of angiogenesis.

Interferon-gamma, IFN-gamma

Interferon is the earliest cytokine found. All cells can produce interferon alpha and interferon beta, while interferon gamma can only be produced by activated T cells and natural killer cells (NK cells). Interferon gamma has antiviral, immunomodulatory and antitumor properties.

Obesity is closely related to inflammation. Past studies have found that obese patients are elevated compared to normal controls, as demonstrated by animal experimentation. Furthermore, mice with knockdown interferon gamma gene produce insulin sensitization effects. The mechanism may therefore be the mediation of the occurrence of metabolic adverse reactions in patients through insulin resistance.

Leptin (leptin) and vascular endothelial growth factor (vascular endothelial growth factor, VEGF)

Leptin and vascular endothelial growth factor both belong to the family of growth factors.

Leptin is produced mainly by adipocytes and achieves energy homeostasis by acting on receptors in the arciform nucleus of the hypothalamus to regulate appetite, inhibit hunger, and regulate energy balance. Leptin action in the brain is inhibited by the hormone ghrelin (ghrelin). In obesity, the body's sensitivity to leptin is reduced, resulting in the body not feeling full. Exogenous leptin can promote capillary angiogenesis by increasing VEGF levels. Leptin has also been shown to be involved in immune responses and is a specific inflammatory cytokine of fat for inflammatory responses. Leptin regulates T cell immune responses in atherosclerosis in mice.

Vascular endothelial growth factor is an important signaling protein involved in embryonic angiogenesis and angiogenesis. Vascular endothelial growth factor, also known as vascular permeability factor (vascular permeability factor, VPF), is a signaling protein that stimulates angiogenesis and microvasculature. Vascular Endothelial Growth Factor (VEGF) is a signaling protein produced by cells that stimulate angiogenesis and vasculogenesis. VEGF stimulates angiogenesis to rapidly restore oxygen supply to tissues in the hypoxic state of cells when blood circulation is inadequate. VEGF is significantly elevated in the blood of patients with bronchial asthma and diabetes. VEGF acts primarily on vascular endothelial cells, but for other cell types, for example: stimulating monocyte/macrophage migration also plays a role.

Method for detecting cytokine

The method adopted by the invention is a liquid-phase suspension chip technology, and can detect a large number of factors on the same chip by utilizing the fluorescence of different microbeads. The method was developed by Bio-Rad corporation of the united states, agency of the Bio-pharmaceutical technology limited of the Shanghai warfarin company (Wayen Biotechnologies Shanghai, inc.). The suspension microbead chip platform was Bio-Plex MAGPIX System. This method has been successfully applied in the study of many immune diseases and neuropsychiatric diseases.

Specifically, the invention uses the liquid-phase suspension chip technology to detect the peripheral blood of schizophrenic patients before and after treatment for 6 months or more and healthy controls for 4 weeks and 8 weeks.

Table 1 Main functions and Gene positions of cytokines studied

Detection reagent

The detection reagents of the invention include protein chips, nucleic acid chips, or combinations thereof. In another preferred embodiment, the detection reagent of the present invention further comprises an IL-1ra specific antibody.

Protein chips are a high throughput monitoring system that monitors interactions between protein molecules by interaction of target molecules and capture molecules. Capture molecules are generally pre-immobilized on the chip surface and are widely used as capture molecules due to the high specificity of antibodies and their strong binding properties to antigens. The study of protein chips is very critical to the efficient immobilization of antibodies on the chip surface, and in particular to the enhancement of the sensitivity of protein chips in terms of immobilized antibody consistency. The G protein is an antibody binding protein which specifically binds to the FC fragment of an antibody and thus has been widely used for immobilization of different types of antibodies. The protein chip for detecting IL-1ra of the present invention can be prepared by various techniques known to those skilled in the art.

The nucleic acid chip, also called DNA chip, gene chip or gene microarray, refers to in situ synthesis of oligonucleotides on a solid support or direct microscopic printing of a large number of DNA probes on the surface of the support, hybridization with a labeled sample, and detection and analysis of hybridization signals to obtain genetic information of the sample. In other words, the gene chip is obtained by fixing DNA fragments (gene probes) of specific sequences in a regular array of tens of thousands or even millions to 2cm by a micro-processing technique ² A two-dimensional DNA probe array is formed on a support such as a silicon wafer or a glass slide, and is called a gene chip because it is very similar to an electronic chip on an electronic computer.

The present invention relates to polyclonal and monoclonal antibodies, in particular monoclonal antibodies, specific for human IL-1 ra. Here, "specific" refers to antibodies that bind to human IL-1ra gene products or fragments. Preferably, those antibodies that bind to human IL-1ra gene products or fragments but do not recognize and bind to other non-related antigenic molecules. Antibodies of the invention include those molecules that bind to and inhibit the human IL-1ra protein, as well as those that do not affect the function of the human IL-1ra protein. The invention also includes those antibodies that bind to the modified or unmodified form of the human IL-1ra gene product.

The invention includes not only intact monoclonal or polyclonal antibodies, but also immunologically active antibody fragments, such as Fab' or(Fab) ₂ Fragments; antibody heavy chain; an antibody light chain; genetically engineered single chain Fv molecules (Ladner et al, U.S. Pat. No.4,946,778); or chimeric antibodies, such as antibodies having murine antibody binding specificity but retaining antibody portions derived from humans.

Antibodies of the invention may be prepared by various techniques known to those skilled in the art. For example, a purified human IL-1ra gene product, or an antigenic fragment thereof, may be administered to an animal to induce the production of polyclonal antibodies. Similarly, cells expressing the human IL-1ra protein or antigenic fragment thereof can be used to immunize animals to produce antibodies. The antibodies of the invention may also be monoclonal antibodies. Such monoclonal antibodies can be prepared using hybridoma technology (see Kohler et al, nature 256;495,1975; kohler et al,Eur.J.Immunol.6:511,1976; the composition of Kohler et al,Eur.J.Immunol.6:292,1976; hammerling et al,InMonoclonalAntibodiesandTCellHyb ridomaselsevier, n.y., 1981). The antibodies of the invention include antibodies that block the function of human IL-1ra protein and do not affect the function of human IL-1ra protein. The various antibodies of the invention can be obtained by conventional immunization techniques using fragments or functional regions of the human IL-1ra gene product. These fragments or functional regions may be prepared by recombinant methods or synthesized by a polypeptide synthesizer. Antibodies that bind to an unmodified form of the human IL-1ra gene product can be produced by immunizing an animal with the gene product produced in a prokaryotic cell (e.g., e.coli); antibodies (e.g., glycosylated or phosphorylated proteins or polypeptides) that bind to post-translational modifications can be obtained by immunizing an animal with a gene product produced in a eukaryotic cell (e.g., a yeast or insect cell).

Detection method and detection kit

The invention provides a detection method and a detection kit using IL-1ra and a detection reagent thereof.

Specifically, the invention provides a kit, which comprises a container, wherein the container contains a detection reagent for detecting IL-1 ra; and a label or instructions stating that the kit is for determining whether a schizophrenic patient is suitable for treatment with olanzapine.

The invention also provides a method of determining whether a patient suffering from schizophrenia is suitable for treatment with olanzapine, comprising the steps of:

(a) Providing a sample of the test subject;

(b) Determining the content of IL-1ra in the sample as A1;

(c) Comparing step (b) with the IL-1ra content A0 of the normal population sample, if A1 is significantly higher than A0, then the test subject is indicated to be unsuitable for treatment with olanzapine.

Specifically, the invention can detect a plurality of cytokine concentrations simultaneously by adopting a liquid phase suspension protein chip technology. Kits (Human Premixed ulti-ANAlyte Kit panel; bio-Rad, austin, TX, USA) were purchased from Bio-Rad Inc. of America (cat. No.: LXSAHM-13). Cytokine analysis was performed according to the instructions of the xMAP technique of multiplex beads. Sample plates were measured using a Bio-Plex MagPix system and analyzed by Bio-Plex Manager software version 6.1 of the authorized Shanghai interference biotechnology Co. The Bio-Plex software calculated cytokine concentrations based on fluorescence values obtained from recombinant cytokine standards in 96-well plates. The software uses a nonlinear least square method minimization algorithm to generate a single curve fitted by a five-parameter logarithmic equation for the 8 collected standard concentration points, and determines the detection range. The levels of IFN-gamma, IL-1ra, IL-1 beta, IL-8, TNF-alpha, MCP-1, VEGF and leptin concentrations were within the detection range. Results are expressed in micrograms per milliliter.

The main advantages of the invention include:

(a) IL-1ra predicts olanzapine-induced lipid and leptin metabolic abnormalities and has potential biomarkers for cardiovascular risk assessment prior to olanzapine administration;

(b) IL-1ra and its detection reagents can be used to determine whether a schizophrenic patient is suitable for treatment with olanzapine;

(c) The invention can obtain a plurality of target protein concentrations at the same time, and can intuitively reflect the protein expression condition;

(d) The detection method has the advantages of small blood consumption and convenient sampling;

the invention will be further illustrated with reference to specific examples. It is to be understood that these examples are illustrative of the present invention and are not intended to limit the scope of the present invention. The experimental methods, in which specific conditions are not noted in the following examples, are generally conducted under conventional conditions or under conditions recommended by the manufacturer. Percentages and parts are by weight unless otherwise indicated.

Universal materials and methods

1. Design of experiment

The study adopts two experimental design methods of cross section study and prospective natural observational study to explore study hypothesis. We used a cross-sectional study design to detect and analyze whether or not the levels of peripheral blood cytokines Interferon gamma (Interferon-gamma, IFN-gamma), interleukin 1 antagonist (Intereukin-1receptor antagonist,IL-1 ra), interleukin 1 beta (Intereukin-1 beta, IL-1 beta), interleukin 8 (IL-8), tumor necrosis factor alpha (tumor necrosis factor-alpha, TNF-alpha), monocyte chemotactic protein (monocomp-attractant protein, MCP-1), leptin (leptin), vascular endothelial growth factor (vascular endothelial growth factor, VEGF) were altered compared to the normal control group, thereby investigating whether or not these cytokines were associated with the occurrence of schizophrenia. On the other hand, we used a natural observational prospective study design, followed up for 8 weeks for a schizophrenic patient, in which 8 weeks the patient was treated with olanzapine with a single antipsychotic drug. It was explored whether these cytokines mediate olanzapine-induced metabolic adverse effects and whether these factors could predict olanzapine-induced metabolic adverse effects by detecting whether changes in the levels of these cytokines' expression levels at concentrations before and after olanzapine treatment or at levels at the baseline stage of the cytokines are correlated with the presence of olanzapine-induced metabolic adverse effects.

2. Study object

The study was approved by the ethical committee of the Shanghai city mental health center, and all subjects signed informed consent. The study included 23 schizophrenic patients, from 2015, 4 to 2016, 11 to Shanghai city mental health center. In addition, 52 healthy controls were included, the ages and sexes of which matched those of schizophrenia, and the enrolled healthy volunteers were from the staff and students of the Shanghai city mental health center.

2.1 group of schizophrenic patients

Group entry criteria:

1) Is diagnosed as Schizophrenia (SZ) according to ICD-10 diagnostic criteria;

2) Age of group entering is 18-65 years old, han nationality;

3) The first patient or at least 6 months had no psychotropic medication (excluding benzodiazepines);

4) Written signed informed consent.

Exclusion criteria:

1) Meets ICD-10 diagnosis except for schizophrenia;

2) History of major somatic diseases such as: cancer, autoimmune diseases, infections, neurological diseases, and the like;

3) Suffering from metabolic diseases such as diabetes; dyslipidemia, etc.;

4) Has a history of use of hypoglycemic agents, hypolipidemic agents or special diets and immunosuppressants to reduce blood glucose or blood lipid levels;

5) History of past alcohol or other substance abuse;

6) Women in lactation or pregnant women;

7) The first generation antipsychotic drugs are taken together in the treatment.

2.2 Health Control group (Health Control, HC)

Age and sex are matched with the group of schizophrenic patients, excluding those with severe somatic or metabolic disease, those with prior personal and family history of mental disease, and lactating women or pregnant women.

3. Sample and data collection

52 healthy controls were treated with EDTA anticoagulant tubes at baseline with 5ml of peripheral venous blood, and 23 schizophrenic patients were treated with 5ml of each peripheral venous blood with EDTA anticoagulant tubes after baseline, 4 weeks and 8 weeks of antipsychotic medication. Venous blood (5 ml) was collected at 6:00 am to 7:00 am in order to avoid circadian fluctuations in the measured parameters. Centrifugation was performed at 4℃by passage at 3000rpm for 20 minutes within 2 hours after blood collection. And after centrifugation, the cell factor is stored in a refrigerator at the temperature of minus 80 ℃ for measurement, so that repeated freezing and thawing are avoided.

4. Mental examination and metabolic index evaluation of patients

Positive and negative symptom scales (Positive and Negative Syndrome Scale, PANSS) were used for patient groups before treatment and for each follow-up to assess patient disease severity. The evaluator is trained by a professional PANSS scale, and the consistency of the evaluator is good.

The metabolic index of the patient is recorded as follows:

body weight (kg) and height (cm) were recorded and body mass index (BMI = body weight/height) was calculated ² )。

Sugar metabolism index: fasting blood glucose concentration, glycosylated hemoglobin and serum insulin

Lipid metabolism index: cholesterol, triglycerides, high density lipoproteins, low density lipoproteins, apolipoprotein A1 (ApoA 1), apolipoprotein B (ApoB), apolipoprotein E, lipoprotein a

The data is independently input by two persons by using EpiData3.1, and is checked, so that the data quality is ensured.

5. Primary reagents and apparatus

5.1 major reagents

5.2 Main Equipment

6. Experimental method

The experiment adopts a liquid phase suspension chip technology, a suspension microbead chip platform is Bio-Plex MAGPIX System, and the used analysis software is Bio-Plex Manager version 6.1.6.1, which is developed by Bio-Rad company (Luminex, austin, TX, USA) in the United states and is proxied by Shanghai Ying biomedical science and technology Co., ltd (Wayen Biotechnologies Shanghai, inc.).

6.1 sample and Standard preparation

RD6-52Diluent was added to the standard vials as required, vortexed for 30s, and ice-bath for 15min. Then diluted as follows:

sample preparation: (operation on ice)

Serum samples: centrifuging at 10,000rpm for 10min, and collecting supernatant; after centrifugation, the samples were diluted 2-fold with the sample dilutions (RD 6-52) and tested.

6.2 chip detection

Numbered sample secondary antibody PE

LXSAHM-13 2hr 1hr 30min

6.2.1 sample incubation

(1) Taking the microbeads, oscillating for 30s at 1,400rpm on an oscillator, prompting according to instructions, and diluting the microbeads by using an Assay Buffer;

(2) According to the requirements of the specification, the diluent is used for suspending the standard substance, gently mixed and placed on ice for 30min so as to ensure that the standard substance is fully dissolved;

(3) Diluting the standard substance by RD6-52Diluent, wherein the experiment totally dilutes 8 gradients, S1-S8;

(4) The diluted microbeads were shaken for 30s with a shaker at 1,400rpm and added to 96-well plates in an amount of 50. Mu.L per well;

(5) Adding diluted standard substance and sample, sealing with sealing film at 50 μl per well, and incubating at 850rpm on flat-plate shaker at room temperature under dark condition for 2hr.

6.2.2 incubation of detection antibodies

(1) Discarding the sample, and washing 3 times by using a plate washer;

(2) Dilution Detection Antibody was performed using Detection Antibody Dilute Buffer as instructed by the specification;

(3) Adding diluted Detection Antibody, attaching sealing film to 50 μl of each well, and standing on flat-plate shaker at 850rpm to shake at dark room temperature for 1hr.

6.2.3 color development

(1) Discarding the detection antibody, and washing 3 times by using a plate washer;

(2) Diluting strepavidin-PE with Wash Buffer as the specification requires;

(3) 50. Mu.L of diluted strepitavidin-PE per well was added; attaching sealing film to the plate

Shaking at 850rpm on a shaking table, and incubating for 30min at room temperature in a dark place;

(4) Washing 3 times by using a plate washing machine;

(5) Resuspension with 100 μl of Wash Buffer per hole, attaching sealing film, keeping away from light at room temperature for 850rpm, and oscillating for 2min;

(6) Read into corrected Bio-Plex machine.

6.2.4 fluorescence data results

After the detected sample and standard substance are detected by a Bio-Plex detector, the fluorescence obtained by detection is automatically calculated and optimized by software to form an output file in an Excel format.

Excel file illustrates the following table:

6.2.5 chip detection quality control

Standard curve and sample case:

1. the standard curve and sample of this experiment were repeated, and the repeated CV conditions were as follows: % CV

Type	Well	IL-8	IFN-gamma	IL-1ra	VEGF	MCP-1	IL-1beta	Leptin	TNF-a
										S1	A1,A2	1.98	3.86	2.15	1.27	0.87	0.79	2.87	2.07
S2	B1,B2	2	1.42	1.74	2.02	1.31	1.44	4.73	1.21
										S3	C1,C2	1.72	4.12	3.51	1.38	5.89	1.77	8.34	3.91
S4	D1,D2	2.24	1.12	2.44	2.04	3.45	2.68	0.56	2.14
										S5	E1,E2	1.7	2.95	1.05	2.54	5.15	2.24	8.42	3.63
S6	F1,F2	3.54	6.43	4.42	3.95	5.89	0	17.68	2.67
										S7	G1,G2	2.57	0	0	0	1.99	0	0	0
S8	H1,H2	NA	0	0	NA	2.4	NA	NA	NA

Note that: NA is the value automatically discarded by the system.

From the CV case, it is known that: the standard curve CV% is less than 20% and the repetition is good.

2. From the fluorescence detection values (Fluorescence intensity, FI) obtained for the standards, the Standard Curve (Standard Curve) was obtained by fitting the Standard Curve using the 5-parameter model (5 PL), and the Standard Curve equation was set under the Standard Curve in pg/mL in the experimental results (Standard Curve of Table 2). In standard curve fitting, software automatically corrects some deviation points and fits effective points. In the standard curve, the ratio of the standard detection value (calculated from the standard curve, observed concentrations) to the expected value (standard concentration, expected concentrations) reflects the quality of the standard curve. The range of (Obs/Exp) 100 can float between 60-140%. In this experiment, (Obs/Exp) 100 of the standard curve is mostly between 80-120, indicating that the standard curve works well.

Type	Well	IL-8	IFN-γ	IL-1ra	VEGF	MCP-1	IL-1beta	Leptin	TNF-a
										S1	A1,A2	100	100	100	100	93	100	103	100
S2	B1,B2	100	100	100	100	104	100	97	100
										S3	C1,C2	100	99	100	101	98	101	99	100
S4	D1,D2	100	101	100	99	100	99	104	100
										S5	E1,E2	101	102	100	98	103	100	97	102
S6	F1,F2	98	97	98	107	96	103	106	98
										S7	G1,G2	102	92	103	90	103	96	71	101
S8	H1,H2	--	123	97	--	114	--	--	--

7. Statistical method

Data analysis was performed using SPSS 20.0 software, and results were plotted using GraphPad Prism 6.0 software. Classification variables such as gender, family history, education age, etc., were tested using chi-square. For continuous variables, a normalization test was first performed on the data using the Shan Yangben Kelmogorov-Seminov test (one-sample Kolmogorov-Smirnov test). According to the results of the normalization test of the data, the age and BMI of the patient group and the control group are compared by adopting two independent sample T tests, and metabolic indexes before and after treatment of the patient, total scores of the PANSS scale and various scores are repeatedly measured and analyzed by variance (repeated measured-ANOVA, RM-ANOVA). The three cytokines IL-1. Beta., IFN- γ, and IL-8 levels of VEGF, and those of schizophrenia, were all non-normal profiles in the healthy control group compared to the patient group. When the patient groups were compared before and after self-treatment, the IL-8 baseline levels and the follow-up levels of IFN-gamma and leptin were non-normal, while the remaining factor levels were in line with normal before and after treatment. Thus, we compared IL-1β, IL-8, IFN- γ, and VEGF levels in the normal control group with those in the patient prior to treatment using a rank-sum test (Mann-Whitney U-test) of two independent samples; comparing the expression of IFN-gamma, IL-8, leptin before and after treatment in the patient group using a full randomized block design rank sum test (Friedman two-way analysis) of the plurality of groups of related samples; the levels of remaining cytokines were compared before and after treatment with RM-ANOVA in the patient group. The correlation of the different metabolic index changes (post-treatment and pre-treatment differences) with the cytokine changes or cytokine baseline levels was screened using Pearson correlation analysis (normal distribution variables) or Spearman correlation analysis (non-normal distribution variables), as applicable. The relationship between cytokine levels and metabolic index changes before and after treatment or between cytokine levels and metabolic index changes was explored by multiplex linear regression analysis (multiple linear regression analysis, MLR).

All data except cytokines are expressed as mean ± standard deviation, P-values < 0.05 are considered statistically significant.

Example 1

General data and clinical characterization of patient groups and healthy controls

In this study, there were no statistical differences in age, sex in both schizophrenic patients and healthy controls, and detailed demographics and clinical features are shown in table 2. In 8 weeks of antipsychotic treatment, the antipsychotics during the follow-up period for 23 schizophrenic patients were olanzapine single drug (23 cases). Patients all received olanzapine single drug therapy, with only 2 patients taking zopiclone, 1 patient taking clonazepam, and 1 patient taking lorazepam. The total score of PANSS before treatment was 75.64 ±18.32 for schizophrenic patients, and the total score of PANSS after treatment was reduced to 44.77± 11.98 (p=0.000). The PANSS reduction was defined as (pre-treatment baseline total score-post-treatment total score)/(baseline total score-30). Times.100%. The PANSS percent reduction is more than or equal to 25 percent as a treatment effective standard and is less than 25 percent as an ineffective standard, 21 patients with treatment effective rate and 2 patients with treatment ineffective rate are treated with treatment effective rate of 91.30 percent.

TABLE 2 demographic data and clinical characteristics of schizophrenic patients

a chi-square test

b two independent sample t-test

*(p<0.05)

Example 2

Comparison of cytokine levels in healthy controls and pre-treatment patients with schizophrenia

The levels of pre-treatment cytokines were compared in healthy controls and schizophrenic patients using the method described in the general methods.

The results are shown in FIG. 2 and Table 3.

TABLE 3 comparison of factor levels at baseline and healthy controls for schizophrenic patients

The variables are represented by median (min-max)

a T test of two independent samples

b rank sum test of two independent samples

*(p<0.05)

Fig. 2, in combination with the data of table 3, shows that IL-1ra (p=0.0001), MCP-1 (p=0.003) and VEGF (p=0.014) levels were significantly elevated before treatment in schizophrenic patients compared to normal control groups. The remaining cytokine levels did not see significant differences between SZ and HC groups.

Example 3

Comparison of metabolic index and cytokine levels before and after olanzapine treatment in schizophrenic patients

The metabolic index and cytokine levels before and after olanzapine treatment in schizophrenic patients were compared using the method described in the general methods.

The results are shown in tables 4 and 5.

TABLE 4 metabolic index changes in patients with schizophrenia before and after olanzapine treatment

a repeated measurement analysis of variance

b Friedman rank sum test

*(p<0.05)

TABLE 5 comparison of cytokine levels before and after treatment of schizophrenic patients

a repeated measurement analysis of variance

b Friedman test

*(p<0.05)

Tables 4 and 5, in combination with the data of figures 3 and 4, show that after 8 weeks olanzapine treatment in schizophrenic patients, BMI, fasting insulin, cholesterol, triglycerides, LDL and ApoB levels in the metabolic index and leptin levels in the cytokines were significantly increased. Leptin is also used herein as an indicator of elevated metabolism. The average elevation of BMI, insulin, cholesterol, triglycerides, LDL, apoB and leptin was 0.86.+ -. 1.15kg/m2, 0.72.+ -. 0.58mmol/L, 0.61.+ -. 1.00mmol/L, 54.9.+ -. 55.2pmol/L, 0.50.+ -. 0.87mmol/L, 0.135.+ -. 0.240g/L and 7.45.+ -. 8.67ng/ml, respectively. Pearson correlation analysis showed that IL-1ra changes were significantly correlated with changes in fasting blood glucose (r= -0.485, p=0.019), TNF- α changes with HbA1c changes (r= -0.559, p=0.006), but no significant correlation with changes in elevated metabolic indicators (BMI, fasting insulin, cholesterol, triglycerides, LDL, apoB and leptin). The remaining cytokine level changes were not correlated with changes in metabolic index levels.

In addition, figures 5 and 6 show that pre-treatment IL-1ra levels are significantly correlated with elevated cholesterol, LDL, apoB, leptin levels (r=0.576, p=0.004; r=0.567, p=0.005; r=0.490, p=0.048; r=0.524, p=0.010); VEGF baseline levels were significantly correlated with cholesterol, apoA1, apoE changes (r=0.436, p=0.038; r=0.537, p=0.008; r=0.419, p=0.047). The baseline levels of all 8 cytokines were analyzed by multiple linear regression with the elevated metabolic index, and the elevated metabolic index changes were used as dependent variables that also included the patient gender, age, and BMI at baseline.

As shown in tables 6 and 7, the present study found that IL-1ra pre-treatment levels were significantly correlated with elevated amounts of cholesterol (adjustment R-party = 0.300, β = 0.576, p = 0.004), LDL (adjustment R-party = 0.289, β = 0.567, p = 0.005), apoB (adjustment R-party = 0.203, β = 0.490, p = 0.018), and leptin (adjustment R-party = 0.550, β = 0.540, p = 0.0012); pre-VEGF treatment levels were significantly correlated with elevated amounts of ApoA1 (adjustment R = 0.254, β = 0.537, p = 0.008) and ApoE (adjustment R = 0.136, β = 0.419, p = 0.047). In addition, leptin elevation was sexually different (adjustment R-square=0.254, β=0.562, p=0.008).

TABLE 6 Metabolic dependent variables associated with cytokine baseline levels after multiple regression analysis

*P<0.05,**P<0.01,***P<0.005

TABLE 7 independent variables associated with leptin level changes after multiple regression analysis

***P<0.005,****P<0.001

Discussion of the invention

The schizophrenia has high disability and high recurrence rate, and is extremely important for patients with schizophrenia, while the drug treatment is effective, the side effects of the drug are reduced, the life quality of the patients is improved, and the compliance of the drug administration is improved. There have been many studies that have found that schizophrenic patients are more prone to overweight and obesity than the general population. There are more obesity co-morbidities in patients who take antipsychotics, especially atypical antipsychotics, for long periods of time.

Antipsychotics can cause problems with glycolipid metabolism in psychotic patients, especially second generation antipsychotics. A large retrospective study found that 38,632 schizophrenic patients received first-or second-generation antipsychotic medication, after control of age, schizophrenic patients taking atypical antipsychotics had an increased probability of 9% of diabetes than patients using first-generation antipsychotics, olanzapine belonging to the atypical antipsychotic line most prone to metabolic disorders. In addition, dyslipidemia such as cholesterol caused by olanzapine is more severe than other atypical antipsychotics. Atypical antipsychotics may also induce patients to develop abnormal elevation of leptin levels and leptin resistance. The present study also found that olanzapine was able to significantly affect glycolipid metabolism in schizophrenic patients, causing an increase in insulin, cholesterol, triglycerides, LDL and ApoB, body weight, reducing patient compliance. Thus, there is a need to address the metabolic adverse effects caused by treatment of schizophrenic patients. At present, researches show that mental diseases, metabolic problems and inflammation are closely related, but the internal influence mechanism of the mental diseases, metabolic problems and inflammation are not yet defined, and the relationship among the mental diseases, metabolic problems and inflammation is still to be elucidated.

The study used a prospective longitudinal follow-up design, and was performed on patients with acute schizophrenia who did not take the drug for 6 months for a period of 8 weeks. The metabolic indexes of patients with schizophrenia after olanzapine single drug treatment are observed, the change of a plurality of cytokines in blood is detected simultaneously by a protein chip method, whether the metabolic adverse reaction induced by olanzapine is related to the change of the cytokine level is explored, the metabolic risk of SZ patients can be predicted by the cytokine level, the possible mechanism of the metabolic adverse reaction induced by olanzapine is explored, and potential drug targets are provided for patients with metabolic disorder caused by olanzapine treatment.

The study was mainly found to have 3 main findings: serum IL-1ra, vegf, and MCP-1 levels were significantly higher in schizophrenic patients than in healthy controls after the drug eluting period; the levels of IL-1ra prior to treatment correlated significantly with elevated cholesterol, LDL, apoB and leptin after 8 weeks of olanzapine treatment, indicating that levels of IL-1ra prior to treatment could have a predictive effect on lipid and leptin metabolism; pre-treatment VEGF levels correlated significantly with the amount of change in ApoA1 and ApoE after 8 weeks of olanzapine treatment.

The study found that baseline levels of IL-1ra were higher in schizophrenic patients than HC and that there was a positive correlation with elevated cholesterol, LDL, apoB and leptin. Notably, all pre-treatment glycolipid metabolism indicators were within the normal range for schizophrenic patients. After multiple regression analysis we could determine that IL-1ra could be an independent predictor of cholesterol, LDL, apoB and leptin elevation following olanzapine treatment.

The risk factors for traditional prediction of cardiovascular metabolic disease are: total cholesterol, HDL, triglycerides, LDL, apoA1, apoB, blood pressure, blood glucose, hbA1c, BMI, etc. Cholesterol, LDL, apoB are all risk factors for traditional cardiovascular metabolic diseases, and therefore, pre-treatment IL-1ra levels can be used as independent predictors of cardiovascular risk for olanzapine treatment SZ.

IL-1ra is a relatively important factor in the metabolic diseases and rheumatic immune diseases. The drug anakinra developed according to the function of IL-1ra has been FDA approved and used in the treatment of immune diseases such as rheumatoid arthritis. IL-1ra has also been studied in the field of mental diseases, but studies have focused mainly on the mild inflammatory states of psychotic patients and the pathogenesis of mental diseases, and there are few studies on metabolic problems in mental diseases and metabolic disorders induced by psychotic drugs.

IL-1ra is produced mainly by visceral fat and is a very sensitive indicator of the low-grade inflammatory status of obesity. IL-1ra has the effect of antagonizing IL-1 beta to protect insulin beta cells as an anti-inflammatory factor. The lower levels of glycosylated hemoglobin and C-peptide in the 13 week diabetic patients treated with Anakinra compared to the control group indicate that the artificially synthesized Anakinra can protect against lowering blood glucose and insulin resistance. Animal studies have also found that mice following knockout of the IL-1RN gene encoding IL-1ra develop obesity, leptin resistance, insulin resistance and impaired glucose tolerance.

IL-1ra can also predict insulin-resistant diabetes. The existence of positive correlation of IL-1ra with leptin elevation at baseline stage found in this study also supports the predictive role of IL-1 ra.

This finding was repeated in a large case control study that compares diabetics with healthy controls and found that elevated IL-1ra was associated with a higher risk of type 2 diabetes. The case control study results were again validated in a large cohort study. The cohort studies found that IL-1ra had been elevated 6 years prior to diagnosis of type 2 diabetes, suggesting that IL-1r is likely to have early predictive significance, and that olanzapine is not known to be a factor in accelerating the progression of metabolic adverse reactions. Subsequent genetic studies also demonstrated that IL-1ra can predict the occurrence of metabolic syndrome. Diabetes is also one of the clinically recognized important risk factors for coronary heart disease.

Persistent inflammation can not only cause insulin resistance, but also increase cardiovascular risk. Studies are directed to increased signaling of IL-1 in islet cells of type 2 diabetes. Some genotypes of IL-1ra have long been found to be associated with coronary atherosclerosis in type 2 diabetes. Freitag et al review the past studies and found that cardiovascular risk was linked to IL-1 signaling. In addition, populations carrying 4 IL-1 ra-increasing alleles (IL-1 ra-serving alleles) have a higher risk of coronary heart disease than populations carrying IL-1 ra-increasing alleles, with a ratio (od ratio, OR) of 1.15 (1.08-1.22; p=1.8X10 (-6)).

There were studies showing that IL-1ra and leptin levels were sex-different in diabetic patients, but no difference in IL-1ra levels was found between men and women in this study. Only men and women of leptin were found in this study, and no inter-sex differences of IL-1ra were found. The reasons for this may be related to the fact that our study population is schizophrenic.

IL-1ra may be used as an independent predictor of olanzapine-induced elevated cholesterol, LDL, apoB and leptin levels, probably because IL-1ra is a very sensitive protein, and adipocytes are stimulated during the prophase of metabolic disorder, thus secreting more IL-1ra early, and IL-1ra is stimulated to secrete earlier than other cytokines. Too high IL-1ra may also suggest decompensation of the body against the metabolic side effects, and thus more susceptibility to metabolic side effects or even metabolic diseases.

All documents mentioned in this application are incorporated by reference as if each were individually incorporated by reference. Further, it will be appreciated that various changes and modifications may be made by those skilled in the art after reading the above teachings, and such equivalents are intended to fall within the scope of the claims appended hereto.

Claims (13)

1. Use of a detection reagent for an IL-1ra gene sequence or a detection reagent for an IL-1ra protein for the preparation of a diagnostic reagent or a diagnostic kit for determining whether a patient suffering from schizophrenia is suitable for treatment with olanzapine, said determining whether a patient suffering from schizophrenia is suitable for treatment with olanzapine comprising:

(a) Judging the risk of metabolic abnormality after treatment with olanzapine in patients with schizophrenia, and/or

(b) Judging the risk of cardiovascular diseases of patients suffering from schizophrenia after treatment with olanzapine.

2. The use according to claim 1, wherein the metabolic abnormality comprises abnormal carbohydrate, lipid metabolism.

3. The use according to claim 2, wherein the lipid comprises cholesterol, low density lipoprotein, apolipoprotein B.

4. The use of claim 1, wherein the metabolic abnormality comprises a leptin metabolic abnormality.

5. The use according to claim 1, wherein said judgment comprises an auxiliary judgment and/or a pre-treatment judgment.

6. The use of claim 1, wherein the determination is that the IL-1ra content A1 of the sample from the test subject is compared to the corresponding IL-1ra content A0 of the normal population, and if A1 is significantly higher than A0, the test subject is indicated to be unsuitable for treatment with olanzapine, and the test subject is indicated to be unsuitable for treatment with olanzapine:

(a) The test subjects are at higher risk of developing metabolic abnormalities following olanzapine treatment, and/or

(b) The risk of cardiovascular diseases is higher in the test subjects after olanzapine treatment, and the expression "significantly higher" means that A1/A0 is more than or equal to 1.35.

7. The use according to claim 6, wherein "significantly higher" means A1/A0. Gtoreq.1.5.

8. The use according to claim 6, wherein "significantly higher" means A1/A0.gtoreq.2.0.

9. The use of claim 6, wherein the test subject is a schizophrenic patient.

10. The use according to claim 1, wherein the diagnostic reagent or diagnostic kit is used for detecting a blood sample, a plasma sample, or a serum sample.

11. The use of claim 1, wherein the detection reagent comprises a protein chip, a nucleic acid chip, or a combination thereof.

12. The use of claim 1, wherein the detection reagent comprises an IL-1ra specific antibody.

13. The use of claim 12, wherein the IL-1ra specific antibody is conjugated or provided with a detectable label.