Composition for diagnosing sepsis and medicine for preventive treatment of sepsis

Abstract

The invention belongs to the technical field of medicines, and particularly relates to a composition for diagnosing sepsis and a medicine for preventive treatment of sepsis, by detecting the content of tryptophan and the composition of 32 metabolites of tryptophan of a patient infected with sepsis in the abdominal cavity, the composition is divided into seven combinations, the invention relates to a method for diagnosing / predicting sepsis by using at least one of tryptophan, a combination of 32 tryptophan metabolites, a combination of tryptophan-Indole / AhR pathway metabolites, a combination of tryptophan-5-HT pathway metabolites, a combination of tryptophan-Kynin / IDO1 pathway metabolites, a combination of differential metabolites in plasma and a combination of differential metabolites in excrement. And tryptophan is applied to the mice infected with sepsis in the abdominal cavity for prevention and treatment, so that early diagnosis and treatment of sepsis are realized, and the survival rate of the mice infected with sepsis is remarkably improved.

Description

Technical Field

[0001] The present invention belongs to the technical field of pharmaceuticals, and particularly relates to a composition for diagnosing sepsis and a drug for prophylactic treatment of sepsis. Background Art

[0002] Sepsis is a life-threatening acute organ dysfunction syndrome caused by a dysregulated host response to infection. Due to the "high clinical heterogeneity" of sepsis, its diagnosis and early standardized treatment face many challenges.

[0003] Tryptophan (Trp) is an essential amino acid for humans and can only be obtained through diet. It plays an important role in various physiological processes such as affecting neuronal function, metabolism, inflammatory response, oxidative stress, immune response, and intestinal homeostasis. The level of human tryptophan depends on food intake and the activity of three tryptophan metabolic pathways. The tryptophan metabolic pathway mainly includes three metabolic pathways, namely the kynurenine (Kyn / IDO1) pathway and the 5-hydroxytryptamine (5-HT) pathway of the host, and the indole / AhR pathway regulated by the microbiota.

[0004] Studies have shown that there are disorders in the tryptophan metabolism of the microbiota and the host in sepsis patients, and the significant reduction of tryptophan metabolites is closely related to more severe clinical outcomes. Tryptophan plays a pro-inflammatory or anti-inflammatory role through three metabolic pathways. 90% of 5-HT in the human body is catalyzed and synthesized by tryptophan hydroxylase 1 (TPH1) in enterochromaffin cells. Studies suggest that the level of 5-hydroxyindoleacetic acid (5-HIAA), a downstream product of the 5-HT pathway in plasma, is significantly positively correlated with the disease severity index, and 5-HIAA / 5-HT is an important potential biomarker for sepsis. Tryptamine, as one of the metabolites of the tryptophan-indole / AhR pathway, has been shown to reduce lipopolysaccharide-induced inflammatory responses. However, there is no report on the role of direct tryptophan intervention in the diagnosis and prophylactic treatment of sepsis, and the specific application of tryptophan in sepsis still needs further research and verification. Summary of the Invention

[0005] The purpose of the present invention is to provide a composition for diagnosing sepsis and a drug for prophylactic treatment of sepsis to solve the problems existing in the above background art.

[0006] To achieve the above object, the present application is implemented through the following technical solutions:

[0007] A composition for diagnosing sepsis, wherein the composition is one or more combinations of tryptophan and its metabolites.

[0008] Further, the composition is tryptophan, picolinic acid, 5-hydroxyanthranilic acid, nicotinic acid, quinolinic acid, o-aminophenol, nicotinamide, DL-3-hydroxykynurenine, 5-hydroxytryptophan, serotonin, kynurenine, 3-hydroxyanthranilic acid, xanthurenic acid, kynurenic acid, tryptamine, 5-hydroxyindole-3-acetic acid, 5-methoxytryptamine, N-acetyl-serotonin, indole-3-acetamide, indole-3-lactic acid, N-(3-indolylacetyl)-L-alanine, indole-3-carboxylic acid, melatonin, indole-3-acetic acid, tryptophol, cinnabarinic acid, indole-3-acrylic acid, indole-3-propionic acid, 3-indoleacetonitrile, indole, 3-methylindole, indole-3-carboxaldehyde, indoxyl sulfate.

[0009] Further, the diagnosis for sepsis includes seven combinations, namely:

[0010] Combination 1: Tryptophan;

[0011] Combination 2: The combination of the above 32 tryptophan metabolites except tryptophan;

[0012] Combination 3: The combination of tryptophan-Indole / AhR pathway metabolites, including N-(3-indolylacetyl)-L-alanine, indoxyl sulfate, indole-3-carboxylic acid, indole-3-acrylic acid, indole-3-carboxaldehyde, indole-3-lactic acid, tryptophol, indole-3-propionic acid, tryptamine, indole-3-acetic acid, indole-3-acetamide, indole, 3-methylindole, 3-indoleacetonitrile;

[0013] Combination 4: The combination of tryptophan-5-HT pathway metabolites, including 5-hydroxytryptophan, serotonin, 5-hydroxyindole-3-acetic acid, 5-methoxytryptamine, N-acetyl-serotonin, melatonin;

[0014] Combination 5: The combination of tryptophan-Kyn / IDO1 pathway metabolites, including picolinic acid, 5-hydroxyanthranilic acid, nicotinic acid, quinolinic acid, o-aminophenol, nicotinamide, DL-3-hydroxykynurenine, kynurenine, 3-hydroxyanthranilic acid, xanthurenic acid, kynurenic acid, cinnabarinic acid;

[0015] Combination 6: The combination of differential metabolites in plasma, including tryptophan, kynurenine, quinolinic acid, N-(3-indolylacetyl)-L-alanine, 3-hydroxyanthranilic acid, DL-3-hydroxykynurenine, indoxyl sulfate, indole-3-carboxylic acid, picolinic acid, nicotinic acid, N-acetyl-serotonin, melatonin, serotonin, indole-3-acrylic acid;

[0016] Combination Seven: The differential metabolite combination in feces, including kynurenic acid, tryptamine, N-acetyl-5-hydroxytryptamine, tryptophol, N-(3-indolylacetyl)-L-alanine, 5-hydroxyanthranilic acid, indole-3-propionic acid, picolinic acid, o-aminophenol, indole-3-acetic acid, indole-3-carboxaldehyde, serotonin, xanthurenic acid, luteoskyrinic acid, 3-indoleacetonitrile.

[0017] A drug for the preventive treatment of sepsis is tryptophan, and the drug is provided to patients with intra-abdominal infection before the diagnosis of sepsis in any of the above.

[0018] Furthermore, the drug is used for the preventive treatment of sepsis.

[0019] Furthermore, the drug also includes pharmaceutically acceptable excipients.

[0020] Furthermore, the drug is in a dosage form that can be used pharmaceutically.

[0021] Furthermore, the dosage of the drug is 9 - 27 mg / kg / day.

[0022] Furthermore, it is determined whether to use the drug and the dosage of the drug by the content of tryptophan and its metabolites in human plasma and / or feces.

[0023] Furthermore, tryptophan in the drug achieves the preventive treatment of sepsis by reducing the expression of TPH1 in the intestine.

[0024] The beneficial effects of the present invention are:

[0025] 1. Through targeted metabolomics, this technical solution discovers that there are disorders in tryptophan metabolism in the plasma and feces of sepsis patients, and the content of tryptophan metabolites in the plasma and feces can achieve early diagnosis of sepsis.

[0026] 2. Tryptophan pretreatment significantly improves the survival rate of sepsis mice.

[0027] 3. Tryptophan pretreatment reduces the systemic inflammation level (TNF-a / IL-6 / HMGB1) in sepsis mice.

[0028] 4. Tryptophan effectively prevents intestinal pathological damage in sepsis.

[0029] 5. Tryptophan pretreatment restores the content of Tryptamine in the feces of sepsis mice.

[0030] 6. Tryptophan pretreatment reduces the expression of intestinal TPH1 in sepsis mice. Description of the Drawings

[0031] Figure 1 It is a schematic diagram of the animal experiment process of the present invention.

[0032] Figure 2 Figure for the content of plasma tryptophan and its metabolites for the early diagnosis of sepsis. Among them, A shows that targeted metabolomics indicates extensive tryptophan metabolic disorders in the plasma of sepsis patients, with enhanced tryptophan catabolism; B shows that PCA analysis shows that the content of tryptophan-related metabolites can distinguish between infected and sepsis patients; C shows the differential metabolites in the plasma of abdominal infection and sepsis patients; D shows that all three metabolic pathways of tryptophan are significantly dysregulated (analysis of metabolites with an average content > 0.1 ng / ml is selected); E shows plasma tryptophan and all metabolites; F shows the metabolites of each of the three pathways of tryptophan; G shows that the level of differential metabolites is used to diagnose sepsis, and the area under the curve (AUC) is above 0.7 in all cases, S = 15, N = 15.

[0033] Figure 3 Figure for the content of tryptophan and its metabolites in mouse feces for the early diagnosis of sepsis. Among them, A shows that targeted metabolomics indicates extensive tryptophan metabolic disorders in the feces of sepsis mice; B shows that PCA analysis shows that the content of tryptophan-related metabolites can distinguish between Sham and CLP group mice; C shows that both tryptophan and its metabolites are significantly correlated with the levels of plasma inflammatory factors (TNF-α, IL-6, HMGB1) and the degree of intestinal pathological damage in mice; D shows the differential metabolites in the feces of Sham group and CLP group mice; E shows tryptophan and all metabolites in mouse feces; F shows the metabolites of each of the three pathways of tryptophan; G shows that the level of differential metabolites is used to diagnose sepsis, and the area under the curve (AUC) is above 0.75 in all cases. Among them, Sham = 6, CLP = 8, CLP+Trp = 8.

[0034] Figure 4 Figure for tryptophan pretreatment to improve the phenotype of sepsis mice. Among them, A shows that tryptophan pretreatment significantly improves the survival rate of CLP mice (CLP group: 63.33% at 24 hours, 13.33% at 48 hours; CLP+Trp group: 96.77% at 24 hours, 40% at 48 hours, Log-rank test p < 0.001); B shows the levels of plasma TNF-a, IL-6, HMGB1 in each group of mice; C shows the representative HE pathological staining of the intestine and the intestinal pathological damage score in each group of mice, N ≥ 8 in each group, *p < 0.05, **p < 0.01, ***p < 0.001, ***p < 0.0001.

[0035] Figure 5Tryptamine content in the feces of septic mice was restored by tryptophan pretreatment. Among them, A shows the PCA analysis of the contents of tryptophan and its metabolites as feature vectors; B shows the common differential metabolites in the feces of mice in the Sham group, CLP group, and CLP+Trp group; C shows that Tryptamine is significantly correlated with the levels of plasma inflammatory factors (TNF-α, IL-6, HMGB1) and the degree of intestinal pathological damage in mice. Among them, Sham = 6, CLP = 8, CLP+Trp = 8.

[0036] Figure 6 Tryptophan pretreatment reduced the expression of TPH1 in the intestine of septic mice. Western Blot was used to detect the expression of key molecules in the three pathways of tryptophan metabolism in the intestinal tissues of mice in each group. N≥3 in each group, **p<0.01, ****p<0.0001. Specific implementation manners

[0037] The technical solutions of the present invention will be described in detail below with reference to the accompanying drawings. The following embodiments are only exemplary and can only be used to explain and illustrate the technical solutions of the present invention, and cannot be construed as a limitation of the technical solutions of the present invention.

[0038] The present application provides a composition for diagnosing sepsis. The composition of the present application includes tryptophan and 32 of its metabolites. The composition for diagnosing sepsis includes a total of 33 tryptophan and its metabolites, including L-Tryptophan (Trp), Picolinic acid (PA), 5-Hydroxyanthranilic Acid (5-HAA), Nicotinic Acid (NA), Quinolinic acid (QA), 2-Aminophenol (2-AP), Nicotinamide (Nam), 3-Hydroxy-DL-kynurenine (HK), 5-hydroxytryptophan (5-HTP), Serotonin (5-HT), rac-Kynurenine (Kyn), 3-hydroxyanthranilic acid (3-HAA), Xanthurenic acid (XA), Kynurenic Acid (KynA), Tryptamine, 5-Hydroxyindole-3-acetic Acid (5-HIAA), 5-Methoxytryptamine (MeOTA), N-Acetyl-serotonin (NAS), Indole-3-acetamide (IAM), Indole-3-lactic Acid (ILA), N-(3-Indolylacetyl)-L-alanine (IAA-L-Ala), Indole-3-carboxylic acid (I3CA), Melatonin, Indole-3-acetic acid (IAA), Tryptophol, Cinnabarinic Acid (CA), 3-Indoleacrylic acid (IArA), 3-Indolepropionic acidIndole-3-acetic acid (IAA), 3-Indoleacetonitrile (IAN), Indole, 3-Methylindole (3MI), Indole-3-carboxaldehyde (IAld), Indoxyl Sulfate Potassium Salt (3IS).

[0039] The above tryptophan and its 32 metabolites are divided into seven combinations, namely:

[0040] Combination 1: Tryptophan;

[0041] Combination 2: The combination of the above 32 tryptophan metabolites except tryptophan;

[0042] Combination 3: Tryptophan-Indole / AhR pathway metabolite combination, including N-(3-Indolylacetyl)-L-alanine, Indoxyl Sulfate Potassium Salt, Indole-3-carboxylic acid, Indole-3-acrylic acid, Indole-3-carboxaldehyde, Indole-3-lactic acid, Tryptophol, Indole-3-propionic acid, Tryptamine, Indole-3-acetic acid, Indole-3-acetamide, Indole, 3-Methylindole, 3-Indoleacetonitrile;

[0043] Combination 4: Tryptophan-5-HT pathway metabolite combination, including 5-Hydroxytryptophan, Serotonin, 5-Hydroxyindole-3-acetic acid, 5-Methoxytryptamine, N-Acetyl-serotonin, Melatonin;

[0044] Combination 5: Tryptophan-Kyn / IDO1 pathway metabolite combination, including Picolinic acid, 5-Hydroxyanthranilic acid, Nicotinic acid, Quinolinic acid, o-Aminophenol, Nicotinamide, DL-3-Hydroxykynurenine, Kynurenine, 3-Hydroxyanthranilic acid, Xanthurenic acid, Kynurenic acid, Cinnabarinic acid;

[0045] Combination 6: Plasma differential metabolite combination, including Tryptophan, Kynurenine, Quinolinic acid, N-(3-Indolylacetyl)-L-alanine, 3-Hydroxyanthranilic acid, DL-3-Hydroxykynurenine, Indoxyl Sulfate Potassium Salt, Indole-3-carboxylic acid, Picolinic acid, Nicotinic acid, N-Acetyl-serotonin, Melatonin, Serotonin, Indole-3-acrylic acid;

[0046] Combination 7: Fecal differential metabolite combination, including Kynurenic acid, Tryptamine, N-Acetyl-serotonin, Tryptophol, N-(3-Indolylacetyl)-L-alanine, 5-Hydroxyanthranilic acid, Indole-3-propionic acid, Picolinic acid, o-Aminophenol, Indole-3-acetic acid, Indole-3-carboxaldehyde, Serotonin, Xanthurenic acid, Cinnabarinic acid, 3-Indoleacetonitrile.

[0047] The above composition is used for the diagnosis of sepsis in abdominal infections such as appendicitis, peptic ulcer, gastrointestinal perforation (gastric, duodenal, small intestine, colorectal, appendiceal perforation), biliary tract infection, infectious complications of intestinal obstruction, liver abscess, severe pancreatitis, etc. If the detection content of one of the above seven combinations reaches the sepsis determination standard, it is diagnosed as sepsis.

[0048] In this application, there is also a drug for the preventive treatment of sepsis, and the drug can also have only one component, tryptophan.

[0049] In this application, the drug is used for the preventive treatment of sepsis, and moreover, the drug can also be used as one of the drugs or drug combinations for the treatment of patients already diagnosed with sepsis.

[0050] In this application, the drug also includes pharmaceutically acceptable excipients. The specific excipients are any components that can be used as pharmaceutical excipients in the prior art, and no specific limitations are imposed here. Those skilled in the art can arbitrarily select according to the dosage form, materials and sources at the production site, etc., without affecting the implementation of the technical solution of this application.

[0051] In this application, the drug can currently be used in pharmaceutically acceptable dosage forms, including but not limited to powder, tablet, capsule, sustained-release agent and other dosage forms.

[0052] In this application, the dosage of the drug is 100 - 300 mg / kg / day. For those already diagnosed with sepsis, the dosage can be appropriately increased. Specifically, it is still necessary to determine whether to use the drug and the dosage of the drug based on the content of tryptophan metabolites in human plasma and / or feces.

[0053] In this application, the drug achieves the preventive treatment of sepsis by reducing the systemic inflammation level (TNF-a / IL-6 / HMGB1).

[0054] In this application, tryptophan in the drug restores the content of Tryptamine in the feces of sepsis patients.

[0055] In this application, tryptophan in the drug achieves the preventive treatment of sepsis by reducing the expression of TPH1 in the intestine.

[0056] Clinical samples:

[0057] Sample source: Plasma on the first day after diagnosis was collected from patients with abdominal infections (n = 15) and patients with confirmed sepsis induced by abdominal infections (n = 15) for tryptophan-targeted metabolomics analysis.

[0058] Animal experiments are as Figures 1 to 6 shown:

[0059] 1. Experimental grouping:

[0060] Sham group (sham operation group): n = 8

[0061] CLP group (sepsis group): n = 30 (15 were sacrificed at 24 hours for subsequent analysis, and 15 were sacrificed at 48 hours to observe the survival rate).

[0062] CLP + Trp group (tryptophan intervention group): n = 31 (16 were sacrificed at 24 hours for subsequent analysis, and 15 were sacrificed at 48 hours to observe the survival rate).

[0063] 2. Experimental procedures:

[0064] (1) Tryptophan intervention:

[0065] In the tryptophan intervention group, mice were gavaged with Trp (200 mg / kg / day) 7 days in advance, and the Sham and CLP groups were gavaged with the same volume of PBS. A mouse sepsis model was established by cecal ligation and puncture (CLP).

[0066] (2) Sample collection at 24 hours:

[0067] At 24 hours after modeling, the survival rate of mice was counted, and plasma, intestinal tissues, and feces of the Sham group (n = 8), CLP group (n = 15), and CLP + Trp group (n = 16) were collected.

[0068] ① Plasma: ELISA was used to quantify cytokines.

[0069] ② Intestinal tissues: Histological changes were evaluated by HE staining. Western blot was used to analyze the expression of key molecules in the tryptophan metabolic pathway in intestinal tissues, including tryptophan hydroxylase 1 (TPH1), indoleamine 2,3-dioxygenase 1 (IDO1), and aryl hydrocarbon receptor (AhR).

[0070] ③ Feces: Metabolomics analysis was performed to determine tryptophan metabolic changes.

[0071] (3) Calculation of the survival rate at 48 hours:

[0072] At 48 hours after modeling, the survival rates of the CLP group (n = 15) and the CLP + Trp group (n = 15) were calculated.

[0073] The above are only embodiments of the present application and do not impose any form of limitation on the present application. Although the present application is disclosed as above with preferred embodiments, it is not intended to limit the present application. Any person skilled in the art, without departing from the scope of the technical solution of the present application, making some changes or modifications using the disclosed technical content above is equivalent to equivalent implementation cases and all fall within the scope of the technical solution.

Claims

1. A composition for diagnosing sepsis, characterized in that: The composition is a combination of more than one tryptophan and its metabolites.

2. The composition for diagnosing sepsis according to claim 1, characterized in that: The composition comprises tryptophan, pyridine carboxylic acid, 5-hydroxyanthranilic acid, nicotinic acid, quinolinic acid, o-aminophenol, nicotinamide, DL-3-hydroxykynurenine, 5-hydroxytryptophan, serotonin, kynurenine, 3-hydroxyanthranilic acid, xanthurenic acid, kynurenic acid, tryptamine, 5-hydroxyindole-3-acetic acid, 5-methoxytryptamine, N-acetyl-serotonin, indole-3-acetamide, indole-3-lactic acid, N-(3-indolylacetyl)-L-alanine, indole-3-carboxylic acid, melatonin, indole-3-acetic acid, tryptol, cinnabarinic acid, indole-3-acrylic acid, indole-3-propionic acid, 3-indoleacetonitrile, indole, 3-methylindole, indole-3-carboxaldehyde, and indoxyl sulfate.

3. The composition for diagnosing sepsis according to claim 2, characterized in that: There are seven combinations for sepsis diagnosis, namely: Combination 1, tryptophan; Combination 2: a combination of the above 32 tryptophan metabolites except tryptophan; Combination 3: Tryptophan-Indole / AhR pathway metabolite combination, including N-(3-indolylacetyl)-L-alanine, indoxyl sulfate, indole-3-carboxylic acid, indole-3-acrylic acid, indole-3-carboxaldehyde, indole-3-lactic acid, tryptol, indole-3-propionic acid, tryptamine, indole-3-acetic acid, indole-3-acetamide, indole, 3-methylindole, and 3-indoleacetonitrile; Combination 4: Tryptophan-5-HT pathway metabolite combination, including 5-hydroxytryptophan, serotonin, 5-hydroxyindole-3-acetic acid, 5-methoxytryptamine, N-acetyl-serotonin, and melatonin; Combination 5, tryptophan-Kyn / IDO1 pathway metabolite combination, including pyridine carboxylic acid, 5-hydroxyanthranilic acid, nicotinic acid, quinolinic acid, o-aminophenol, nicotinamide, DL-3-hydroxykynurenine, kynurenine, 3-hydroxyanthranilic acid, xanthuric acid, kynurenic acid, and cinnabarinic acid; Combination VI: Combination of differential metabolites in plasma, including tryptophan, kynurenine, quinolinic acid, N-(3-indolylacetyl)-L-alanine, 3-hydroxyanthranilic acid, DL-3-hydroxykynurenine, indoxyl sulfate, indole-3-carboxylic acid, pyridine carboxylic acid, nicotinic acid, N-acetyl-5-hydroxytryptamine, melatonin, serotonin, and indole-3-acrylic acid; Combination seven: Combination of differential metabolites in feces, including kynurenic acid, tryptamine, N-acetyl-5-hydroxytryptamine, tryptol, N-(3-indolylacetyl)-L-alanine, 5-hydroxyanthranilic acid, indole-3-propionic acid, pyridinecarboxylic acid, o-aminophenol, indole-3-acetic acid, indole-3-carboxaldehyde, 5-hydroxytryptamine, xanthurenic acid, cinnabarinic acid, and 3-indoleacetonitrile.

4. A drug for the preventive treatment of sepsis, characterized in that: The drug is tryptophan, and the drug is provided to the patient with abdominal infection before sepsis is diagnosed according to any one of claims 1 to 3.

5. The drug for preventive treatment of sepsis according to claim 4, characterized in that: The medicament is used for the preventive treatment of sepsis.

6. The drug for preventive treatment of sepsis according to claim 4, characterized in that: The medicine also includes pharmaceutically acceptable excipients.

7. The drug for preventive treatment of sepsis according to claim 4, characterized in that: The drug is in a dosage form that can be used for pharmaceutical purposes.

8. The preventive treatment drug for sepsis according to claim 4, characterized in that: The dosage of the drug is 9-27 mg / kg / day.

9. The drug for preventive treatment of sepsis according to claim 8, characterized in that: Whether to use the drug and the dosage of the drug are determined by the content of tryptophan and its metabolites in human plasma and / or feces.

10. The drug for diagnosis or preventive treatment of sepsis according to claim 4, characterized in that: Tryptophan in the drug achieves preventive treatment of sepsis by reducing the expression of TPH1 in the intestine.

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