Use of IDO1 inhibitor as sepsis therapeutic agent

Abstract

Provided is use of an IDO1 inhibitor as a sepsis therapeutic agent, and specifically, use of the IDO1 inhibitor in preparing a pharmaceutical composition for treating sepsis. The IDO1 inhibitor can effectively ameliorate cytokine storm and immunosuppression associated with sepsis. In addition, the IDO1 inhibitor can improve the survival rate in mice with sepsis experiencing secondary infection under immunosuppression, thereby providing new insights for the treatment of sepsis.

Description

Use of IDO1 inhibitors as sepsis treatment agents Technical Field

[0001] This invention belongs to the field of medicinal chemistry technology, specifically relating to the use of IDO1 inhibitors as therapeutic agents for sepsis. Background Technology

[0002] Sepsis is a life-threatening syndrome of organ dysfunction caused by a dysregulation of the host's response to infection. Causes include infections from bacteria, fungi, viruses, and parasites, leading to an imbalance in the body's inflammatory response and immune regulation. Sepsis includes a prodromal cytokine storm phase and a later immunosuppressive phase. The cytokine storm refers to an excessive immune response triggered by a massive surge in cytokines.

[0003] Indoleamine 2,3-dioxygenase 1 (IDO1) is the primary rate-limiting enzyme catalyzing the catabolism of tryptophan (Trp) along the kynurenine pathway (KP). Tryptophan 2,3-dioxygenase (TDO) is an isoenzyme of IDO1.

[0119] Kyn is an endogenous ligand of the transcription factor aryl hydrocarbon receptor (AHR). Kyn binds to and activates AHR to mediate CD4. + T cells differentiate into Th1, Th2, Th17, and Treg cells, participating in the regulation of pro-inflammatory responses and immunosuppression. Activation of the AHR (Avoidance, Influence, and Response) also significantly induces the expression of the pro-inflammatory cytokine IL-6. IL-6 activates STAT3, further inducing IDO1 expression, forming an IDO1-AHR-IDO1 positive feedback loop. This disrupts the self-regulation of the host immune response, impairs the host's inflammatory repair capacity, and leads to the persistence of inflammation. IDO1 has been reported to be closely related to the risk of sepsis; increased IDO1 activity in sepsis patients is associated with an increased risk of death. IDO1 activity can predict the prognosis of sepsis patients and is an independent prognostic biomarker.

[0004] Cytokine storm is a major contributing factor to the morbidity and mortality of sepsis patients. In the early stages of sepsis, the host immune system monitors pathogens by recognizing damage-associated molecular patterns (DAMPs). The immune system then initiates an inflammatory response (such as promoting immune cell activation and producing and releasing inflammatory cytokines) to eliminate pathogens. IDO1 is strongly activated by inflammatory signals, but its role in the cytokine storm phase of sepsis remains unclear. On the one hand, blocking IDO1 plays a beneficial role in host protection. Studies by Jung et al. found that pharmacological blocking or gene deletion of IDO1 led to decreased levels of pro-inflammatory cytokines TNF-α, IL-6, and IL-12 in the plasma of LPS-induced septic mice, while increasing levels of the anti-inflammatory cytokine IL-10. Blocking IDO1 improved the survival rate of LPS-induced septic mice, and the molecular mechanism lies in restoring the balance between IL-12 and IL-10. Furthermore, Wu et al. found that blocking IDO1 alleviates kidney inflammation, apoptosis, and oxidative stress by inhibiting the TLR4 / NF-κB signaling pathway, thereby preventing LPS-induced kidney damage in septic mice. Moreover, IDO1 blockade can reduce sepsis mortality by decreasing the production of chemokines in mouse peritoneal cells and reducing the recruitment of neutrophils and monocytes into the infection focus. On the other hand, altered IDO1 activity also plays a role in controlling inflammation. IDO1 activated by inflammatory cytokines defends by degrading Trp, as many microorganisms require Trp for replication. Furthermore, the IDO1 metabolite Kyn has antibacterial activity and can directly inhibit pathogen replication. Lee et al. found that IDO is a key regulator of acute lung inflammation; histone deacetylase inhibitors promote IDO expression and can improve acute lung inflammation because Kyn inhibits CD4 through the AHR pathway. + Inflammatory activity of T cells. In summary, IDO1 is involved in septic cytokine storms, but the mechanism is complex.

[0005] Persistent immunosuppression is a major cause of death, secondary infection, and poor prognosis in sepsis patients. Studies have shown that prolonged elevation of IDO1 activity during persistent sepsis increases the risk of poor prognosis by inducing chronic immunosuppression, reducing immune reactivity, and weakening host defense. While the importance of IDO1 in the pathological progression of sepsis is well-established, its role and mechanism in sepsis-induced immune dysregulation remain unclear. Hoshi et al. found that in a mouse model of sepsis-induced immunosuppression induced by cecal ligation and perforation (CLP), the IDO inhibitor 1-MT reduced mortality and blood bacterial levels, while Kyn reversed the IDO1 knockout phenotype. Inhibition or knockout of IDO1 also improved the survival rate of Staphylococcus aureus-induced sepsis in mice. Furthermore, studies by Gao et al. found that plasma IDO1 activity was elevated in CLP sepsis model mice, and the levels of pro-inflammatory cytokines TNF-α, IL-6, and IL-1β were upregulated. Treatment with the IDO1 inhibitor 1-MT not only reduced TNF-α, IL-1β, and IL-6 levels in the model mice, thus decreasing inflammation, but also improved immunosuppression and promoted bacterial clearance. This indicates that targeting IDO1 can improve immunosuppression in sepsis and plays an important role in host protection against sepsis.

[0006] In summary, there is an urgent need in this field to develop the application of IDO1 inhibitors in the treatment of sepsis, especially to evaluate the role of IDO1 inhibitors in the septic cytokine storm and the immunosuppressive phase of sepsis. Summary of the Invention

[0007] The purpose of this invention is to provide an application of IDO1 inhibitors in the treatment of sepsis, particularly to evaluate the role of IDO1 inhibitors in the cytokine storm and immunosuppressive phase of sepsis, to evaluate the intervention effect of independently developed IDO1 inhibitors on sepsis, and to provide new insights into the treatment of sepsis.

[0008] In a first aspect of the invention, there is provided the use of an IDO1 inhibitor for preparing a pharmaceutical composition for treating sepsis.

[0009] In another preferred embodiment, the pharmaceutical composition is used to improve cytokine storm caused by sepsis.

[0010] In another preferred embodiment, the IDO1 inhibitor is a compound selected from the group consisting of 1-MT, N-aryltryptamine derivatives, epacadostat, or N-benzyltryptamine derivatives.

[0011] In another preferred embodiment, the IDO1 inhibitor has the structure shown in formula A1:

[0012] Wherein: R 1Selected from the group consisting of hydrogen, fluorine, and -(substituted or unsubstituted C1-C6 alkyl)-substituted or unsubstituted 5-12 membered heterocyclic groups; wherein the heterocyclic group comprises 1-3 heteroatoms selected from the group consisting of N, O, or S;

[0013] R 2 Selected from the following group: H, Cl, Br, substituted or unsubstituted C1-C4 alkyl groups, -NR 3 R 4 , or -(substituted or unsubstituted C1-C6 alkyl)-NR 3 R 4 ;

[0014] The R mentioned 3 R 4 Each is independently selected from the group consisting of: H, substituted or unsubstituted C1-C4 alkyl, substituted or unsubstituted C2-C4 alkenyl, substituted or unsubstituted C2-C4 alkynyl, and substituted or unsubstituted C3-C6 cycloalkyl.

[0015] Or R 3 R 4 Together with adjacent nitrogen atoms, they form a substituted or unsubstituted 5-12 membered heterocyclic group, wherein the 5-12 membered heterocyclic group has 1-2 nitrogen atoms and 0-2 heteroatoms selected from the group consisting of O and S.

[0016] The substitution refers to the substitution of one or more hydrogen atoms (preferably hydrogen atoms on nitrogen atoms) on the group by a substituent selected from the group consisting of: C1-C4 alkyl, C1-C4 haloalkyl, amino protecting group (preferably tert-butyloxycarbonyl), halogen, and phenyl.

[0017] In another preferred embodiment, the -NR 3 R 4 Selected from the following group:

[0018] In another preferred embodiment, the derivative is a compound selected from the group consisting of:

[0019] In another preferred embodiment, the IDO1 is human IDO1.

[0020] In another preferred embodiment, the pharmaceutical composition is also used to improve or reverse apoptosis caused by septic cytokine storm.

[0021] In another preferred embodiment, the pharmaceutical composition is also used to improve or reverse cytokine storm caused by sepsis cytokine storm.

[0022] In another preferred embodiment, the pharmaceutical composition is also used to reduce the high expression of inflammatory cytokines caused by septic cytokine storm.

[0023] In another preferred embodiment, the pharmaceutical composition is also used to downregulate the increased proportion of cells selected from the group consisting of neutrophils and M1 macrophages in the spleen caused by septic cytokine storm.

[0024] In another preferred embodiment, the pharmaceutical composition is also used to downregulate AHR-CYP1A1 expression in lung and kidney tissues induced by septic cytokine storm, and / or upregulate STAT3 phosphorylation.

[0025] In another preferred embodiment, the pharmaceutical composition is also used to reduce mortality caused by septic cytokine storm.

[0026] In another preferred embodiment, the pharmaceutical composition is also used to improve organ damage caused by septic cytokine storm.

[0027] In another preferred embodiment, the pharmaceutical composition is also used to improve the immunosuppressive state caused by sepsis.

[0028] In another preferred embodiment, the pharmaceutical composition is also used to reduce the elevated ratio of MDSCs to Tregs in tissues caused by sepsis-induced immunosuppression; preferably, the tissues are selected from the group consisting of spleen and blood.

[0029] In another preferred embodiment, the pharmaceutical composition is also used to downregulate elevated blood IL-10 levels caused by sepsis and sepsis-related infections.

[0030] In another preferred embodiment, the pharmaceutical composition is also used to reduce the elevated proportion of M2 macrophages in the peritoneal cavity caused by sepsis-induced immunosuppression.

[0031] In another preferred embodiment, the pharmaceutical composition is also used to increase the proportion of reduced M1 macrophages in the peritoneal cavity caused by sepsis-induced immunosuppression.

[0032] In another preferred embodiment, the pharmaceutical composition is also used to reduce the elevated IDO1 activity caused by sepsis.

[0033] In another preferred embodiment, the pharmaceutical composition is also used to improve secondary infections (bacterial or viral) of sepsis and reduce mortality from secondary infections.

[0034] In another preferred embodiment, the bacterial infection is an infection caused by bacteria selected from the group consisting of Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, non-spore-forming anaerobic bacteria, or a combination thereof.

[0035] In another preferred embodiment, the viral infection is COVID-19 infection.

[0036] In another preferred embodiment, the pharmaceutical composition is also used to reduce the mortality rate of sepsis.

[0037] In another preferred embodiment, the pharmaceutical composition is also used to improve symptoms of sepsis selected from the group consisting of: weight loss, abnormal body temperature, organ and / or tissue damage.

[0038] In another preferred embodiment, the pharmaceutical composition is also used to downregulate the increase in the proportion of M1 macrophages in the spleen and the increase in the level of inflammatory cytokines in the blood caused by sepsis.

[0039] In another preferred embodiment, the pharmaceutical composition further includes a second therapeutic agent; preferably, the second therapeutic agent is an antibiotic.

[0040] In another preferred embodiment, the antibiotic is selected from the group consisting of ceftriaxone, imipenem, or combinations thereof.

[0041] In another preferred embodiment, the pharmaceutical composition is more effective than antibiotics under equal dose administration conditions; more preferably, the pharmaceutical composition is more effective than antibiotics under administration conditions where the mass ratio of the IDO inhibitor to the antibiotic in the pharmaceutical composition is 1:4-5.

[0042] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described in detail here. Attached Figure Description

[0043] Figure 1 shows the differential expression analysis of inflammatory cytokines, IDO1, AHR, and C1P1A1 in the blood transcriptome of healthy subjects (n=9) and sepsis patients (n=30) using the GEO dataset (GSE137342).

[0044] Figure 2 shows the correlation between IDO1 and inflammatory cytokines, as well as the expression of AHR and CYP1A1 genes in the blood transcriptome of sepsis patients (n=30) using linear regression analysis based on the GEO dataset GSE137342.

[0045] Figure 3 shows a Pearson correlation heatmap analysis of the expression of IDO1, AHR, CYP1A1 and inflammatory cytokines in the blood transcriptome of healthy individuals (left, n=9) and sepsis patients (right, n=30) using the GEO dataset (GSE137342).

[0046] Figure 4 shows a Pearson correlation heatmap analysis of the expression of AHR, CYP1A1 and classical cytokine storm signaling pathway genes in the blood transcriptome of healthy individuals (left, n=9) and sepsis patients (right, n=30) using the GEO dataset (GSE137342).

[0047] Figure 5 shows that IDO1 promotes cytokine storm in the THP-1 cytokine storm model through the AHR-CYP1A1 axis.

[0048] Figure 6 shows how the IDO1 inhibitor downregulated the expression of the AHR-CYP1A1 axis and inflammatory cytokines in the THP-1 cytokine storm model.

[0049] Figure 7 shows how IDO1 promotes cytokine storm in the Raw264.7 cytokine storm model via the AHR-CYP1A1 axis.

[0050] Figure 8 shows how the IDO1 inhibitor downregulated the expression of the AHR-CYP1A1 axis and inflammatory cytokines in the Raw264.7 cytokine storm model.

[0051] Figure 9 shows how the IDO1 inhibitor prevented the activation of the classical cytokine storm pathway in the THP-1 cytokine storm model.

[0052] Figure 10 shows how the IDO1 inhibitor prevented the activation of the classical cytokine storm pathway in the Raw264.7 cytokine storm model.

[0053] Figure 11 shows how IDO1 promotes apoptosis in THP-1 and Raw264.7 cytokine storm models via the AHR-CYP1A1 axis.

[0054] Figure 12 shows the results of Kyn reversing the reduction in apoptosis rate in THP-1 and Raw264.7 cytokine storm models by IDO1 inhibitor.

[0055] Figure 13 shows that the IDO1 inhibitor improves the inflammatory response in mice with a sepsis cytokine storm model.

[0056] Figure 14 shows the effect of IDO1 inhibitors in downregulating the proportion of neutrophils and upregulating the proportion of monocytes in the spleen of mice with a sepsis cytokine storm model.

[0057] Figure 15 shows the effect of IDO1 inhibitors on downregulating the proportion of M1 macrophages in the spleen of mice with a sepsis cytokine storm model.

[0058] Figure 16 shows the effect of IDO1 inhibitors on inhibiting AHR-CYP1A1 expression and STAT3 phosphorylation in lung and kidney tissues of mice with sepsis cytokine storm model.

[0059] Figure 17 shows the results of pre-dose administration of IDO1 inhibitors reducing mortality in mice with a sepsis cytokine storm model.

[0060] Figure 18 shows the results of IDO1 inhibitor pre-dose improving organ damage in a mouse model of septic cytokine storm.

[0061] Figure 19 shows the decrease in body weight and increase in spleen weight in septic mice established by CLP modeling.

[0062] Figure 20 shows the changes in the levels of pro-inflammatory and anti-inflammatory cytokines in the serum of septic mice established by CLP modeling.

[0063] Figure 21 shows the changes in the proportion of MDSCs in the spleen and blood of septic mice established by CLP modeling.

[0064] Figure 22 shows the changes in the ratio of leukocytes to Tregs in the spleen of septic mice established by CLP modeling.

[0065] Figure 23 shows the changes in IDO1 activity in the serum of septic mice established by CLP modeling.

[0066] Figure 24 shows the effects of IDO1 inhibitors on body weight and spleen weight in CLP septic mice.

[0067] Figure 25 shows how the IDO1 inhibitor reduced IDO1 activity in the serum of CLP septic mice.

[0068] Figure 26 shows the effect of IDO1 inhibitors on reducing the serum IL-10 concentration in CLP septic mice.

[0069] Figure 27 shows how the IDO1 inhibitor increased the proportion of M1 macrophages and decreased the proportion of M2 macrophages in the peritoneal cavity of CLP septic mice.

[0070] Figure 28 shows how the IDO1 inhibitor reduced the number of bacteria in the peritoneal cavity of CLP septic mice.

[0071] Figure 29 shows how the IDO1 inhibitor reduced the proportion of MDSCs in the spleen and blood of CLP septic mice.

[0072] Figure 30 shows how IDO1 inhibitors can reduce the proportion of Tregs in the spleen of CLP septic mice.

[0073] Figure 31 shows how the IDO1 inhibitor reduced the mortality rate of CLP septic mice after secondary infection.

[0074] Figure 32 shows how IDO1 inhibitors reduce IDO1 activity in the serum of CLP septic mice after secondary infection.

[0075] Figure 33 shows how the IDO1 inhibitor reduced the serum IL-10 concentration in CLP septic mice after secondary infection.

[0076] Figure 34 shows how the IDO1 inhibitor reduced the proportion of Treg cells in the spleen of CLP septic mice after secondary infection.

[0077] Figure 35 shows how IDO1 inhibitors reduced the proportion of MDSCs in the spleen and blood of CLP septic mice after secondary infection.

[0078] Figure 36 shows that IDO1 inhibitors are more effective than antibiotics in reducing mortality in septic mice, and the effect is even better when used in combination with antibiotics.

[0079] Figure 37 shows that IDO1 inhibitors can improve weight loss and body temperature reduction in septic mice, and the effect is even better when used in combination with antibiotics.

[0080] Figure 38 shows that IDO1 inhibitors reduce IDO1 activity and inflammatory cytokine levels in the serum of septic mice, and the effect is better when used in combination with antibiotics.

[0081] Figure 39 shows that IDO1 inhibitors downregulate the proportion of M1 macrophages in the spleen of septic mice, and the effect is better when used in combination with antibiotics.

[0082] Figure 40 shows that IDO1 inhibitors can improve tissue damage in a mouse model of sepsis. Detailed Implementation

[0083] Through long-term and in-depth research, the inventors unexpectedly discovered that IDO1 is highly expressed in animal models of sepsis, and this high expression is associated with a series of cytokine storm processes. Administration of IDO1 inhibitors can effectively improve the aforementioned sepsis and the corresponding cytokine storm. Furthermore, IDO1 activity is upregulated in septic mice under immunosuppression, and inhibiting IDO1 activity can improve the survival rate of septic mice when subjected to secondary infections under immunosuppression. Based on these findings, the inventors completed this invention.

[0084] IDO1 inhibitors

[0085] Human IDO1 (hIDO1) is a monomeric oxidase expressed at low levels in most tissues, containing a large C-terminal domain (CTD) and a small N-terminal domain (NTD). The CTD, associated with the catalytic activity of IDO1, consists of several α-helices. A heme cofactor binds to the C-terminal domain, and a dynamic equilibrium exists between heme-containing IDO1 (holo-IDO1) and heme-free IDO1 (apo-IDO1). Heme exhibits a typical porphyrin macrocyclic planar conformation, and the iron ion in heme is complexed with the imidazole N of the His residue, thereby stabilizing heme. The NTD contains two small domains involved in signal transduction functions and contains two immunoreceptor tyrosine inhibitory motifs (ITIMs).

[0086] Current IDO1 inhibitors are classified into four types (I-IV) based on their inhibitory kinetics. Type I, II, and III inhibitors bind to holo-IDO1 at the Sa site, while type IV inhibitors (such as BMS986205) bind to apo-IDO1. Specifically, type I inhibitors (such as L-1MT) are Trp-competitive inhibitors, binding to IDO1-Heme(Fe3+)-O2; type II inhibitors (such as epacadostat) interact with IDO1-Heme(Fe2+) and compete with O2 for the sixth coordination binding site of heme; type III inhibitors (such as 4-benzimidazole (4PI)) bind to IDO1-Heme(Fe3+) and inhibit the reductive activation of the enzyme. Furthermore, compounds with inhibitory activity act by interfering with the redox cycle, particularly quinones, which are not specifically targeted at IDO1 enzymes and are therefore defined as type X inhibitors.

[0087] In this invention, the three preferred IDO1 inhibitors are 1-MT, epacadostat, and tryptophan analogues. 1-Methyltryptophan (1-MT) is a derivative of the IDO1 substrate Trp and is a commonly used IDO inhibitor in in vitro and in vivo experiments. RY103 is a dual inhibitor of tryptophan series IDO1 and its isoenzyme TDO. The chemical formulas of 1-MT, epacadostat, and N-benzyl derivatives (such as RY103) are shown below:

[0088] Wherein, the R 1 Selected from the group consisting of hydrogen, fluorine, and -(substituted or unsubstituted C1-C6 alkyl)-substituted or unsubstituted 5-12 membered heterocyclic groups; wherein the heterocyclic group comprises 1-3 heteroatoms selected from the group consisting of N, O, or S; R 2’ For NR 3 R 4 The definitions of the remaining groups are as described above.

[0089] The IDO1 and TDO inhibitory activities of the representative compound RY103 are shown in Table 1:

[0090] Table 1: Inhibitory Activity and Chemical Formula

[0091] (RY103 is a representative compound in this series, and its various biological activities are shown in the table above.)

[0092] The main advantages of this invention include:

[0093] 1. This invention provides the use of IDO1 inhibitors, which inhibit cytokine storm-related pathways such as STAT3 through AHR-CYP1A1, reduce the level of inflammatory cytokines and the apoptosis rate of model cells, restore immune homeostasis in model mice, and reverse sepsis cytokine storm.

[0094] 2. This invention provides the use of IDO1 inhibitors. In septic mice under immunosuppression, IDO1 activity is increased, the concentration of anti-inflammatory cytokines is increased, the number of immunosuppressive cells is increased, the proportion of M2 macrophages is increased, and the body's ability to clear infection is reduced. Therefore, treating immunosuppressed septic mice with IDO1 inhibitors can reverse the above changes and improve the survival rate of septic mice when they are infected secondary to infection under immunosuppression.

[0095] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions or as recommended by the manufacturer. Percentages and parts are by weight unless otherwise stated.

[0096] Example 1: IDO1 is closely related to septic cytokine storm in sepsis patients:

[0097] 1.1 Compared with healthy individuals, patients with sepsis showed upregulated expression of inflammatory cytokines and IDO1, AHR, and CYP1A1.

[0098] The gene expression levels of TNF-α, IL-6, IL-10, IFN-γ, IL-4, CXCL10, IDO1, AHR, and CYP1A1 in the blood transcriptomes of healthy individuals (n=9) and sepsis patients (n=30) were analyzed using the GEO dataset (GSE137342). Data were analyzed using t-tests, and values ​​are expressed as mean ± SD. *P < 0.05, **P < 0.01, ****P < 0.0001. The results, as shown in Figure 1, indicate that the expression of inflammatory cytokines and IDO1, AHR, and CYP1A1 was upregulated in sepsis patients, with statistically significant differences.

[0099] 1.2 The expression of inflammatory cytokines in sepsis patients was positively correlated with the expression of IDO1, AHR, and CYP1A1.

[0100] Using the GEO dataset (GSE137342), linear regression analysis was performed to analyze the correlation of gene expression in the blood transcriptome of sepsis patients. IDO1 was correlated with cytokines IL-6, CXCL10, and IL-4; IDO1 was correlated with AHR and C1P1A1; and AHR was correlated with C1P1A1, n=30. The results, shown in Figure 2, indicate that the expression of inflammatory cytokines in sepsis patients was positively correlated with IDO1 expression, and IDO1 was positively correlated with the expression of AHR and CYP1A1.

[0101] Using the GEO dataset (GSE137342), Pearson correlation analysis was performed on the expression of IDO1, AHR, CYP1A1, and inflammatory cytokines in the blood transcriptomes of healthy individuals (left, n=9) and patients with septic shock (right, n=30), and heatmaps were generated. Data were analyzed using t-tests, and values ​​are expressed as mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. The results, shown in Figure 3, indicate a positive correlation between the expression of inflammatory cytokines and the expression of IDO1, AHR, and CYP1A1 in patients with septic shock.

[0102] 1.3 In sepsis patients, the expression of AHR and CYP1A1 was positively correlated with the expression of the classical cytokine storm pathways STAT3, NF-κB / STAT1, and JNK / p38 MAPK.

[0103] Using the GEO dataset (GSE137342), Pearson correlation analysis was performed on the expression of AHR, CYP1A1, and genes of the classical cytokine storm pathway in the blood transcriptomes of healthy individuals (left, n=9) and sepsis patients (right, n=40), and a heatmap was generated. Data were analyzed using a t-test, and values ​​are expressed as mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. The results are shown in Figure 4, indicating a positive correlation between the expression of genes of the classical cytokine storm pathway and the expression of AHR and CYP1A1 in sepsis patients.

[0104] Based on the above study using bioinformatics analysis to analyze the expression levels of inflammatory cytokines and IDO1, AHR, and CYP1A1 in sepsis patients, it can be inferred that IDO1 and its mediated AHR-CYP1A1 pathway are closely related to the cytokine storm in sepsis.

[0105] Example 2: Validation of cytokine storm in an in vitro model by which IDO1 inhibitors block cytokine storm:

[0106] 2.1 IDO1 promotes cytokine storm in the THP-1 cytokine storm model via the AHR-CYP1A1 axis

[0107] After THP-1 cells reached 70% confluence, they were treated with 25 nM PMA for 24 h to induce cell adhesion and differentiation into M0 macrophages. Then, 10 μg / mL LPS was added for 24 h to establish a THP-1 cytokine storm model. The results are shown in Figure 5: IDO1 and AHR protein expression (5A) and IDO1 and CYP1A1 mRNA expression (5B) were increased in the THP-1 cytokine storm model. Kyn, an intermediate product of the kynurenine pathway catalyzed by IDO1, increased the expression of AHR, CYP1A1 (5C), and classic inflammatory cytokines TNF-α and IL-6 (5D) in the THP-1 cytokine storm model. Figure 5A shows the protein expression levels of IDO1 and AHR in cells detected by Western blot, with quantitative analysis using ImageJ (n=3). Figure 5B shows the mRNA levels of AHR and CYP1A1 in cells detected by qPCR. Figure 5C shows the expression of IDO1, AHR, and CYP1A1 in cells detected by Western blot. Figure 5D shows the mRNA levels of TNF-α and IL-6 in cells detected by qPCR, n=3.

[0108] 2.2 IDO1 inhibitors inhibit AHR-CYP1A1 and reduce inflammatory cytokine expression in the THP-1 cytokine storm model

[0109] After THP-1 cells reached 70% confluence, they were treated with 25 nM PMA for 24 h to induce cell adhesion and differentiation into M0 macrophages. Then, 10 μg / mL LPS was added for 24 h to establish a THP-1 cytokine storm model. Simultaneously with LPS stimulation, the cells were treated with either the IDO1 inhibitor 1-MT (100 μM) or RY103 (400 nM) for 24 h. The results are shown in Figure 6. The expression of IDO1, AHR, and CYP1A1 in the model cells (Figure 6A) and the expression of inflammatory cytokines (Figure 6C) were all upregulated, and IDO1 activity was increased (Figure 6B). The addition of the IDO1 inhibitor reduced these changes. Figure 6A shows the protein expression levels of IDO1, AHR, and CYP1A1 in cells detected by Western blot, with quantitative analysis using Image J (n=3). Figure 6B shows the Trp and Kyn content in the cell culture supernatant detected by HPLC, with (Kyn / Trp) × 100 calculated as IDO1 activity (n=3). Figure 6C shows the mRNA expression levels of IL-1β and IL-10 in cells detected by qPCR (n=3); the concentrations of TNF-α (n=3) and IL-6 (n=4) in cell culture supernatant were detected by ELISA. In the figure, Ctrl: untreated dTHP-1 cells; Model: THP-1 cytokine storm model group; 1-MT / RY103: 1-MT / RY103 treated model cells. Data were analyzed using one-way ANOVA. Values ​​are expressed as mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

[0110] 2.3 IDO1 promotes cytokine storm in the Raw 264.7 cytokine storm model via the AHR-CYP1A1 axis.

[0111] After Raw264.7 cells reached 70% confluence, a cytokine storm model was established by simultaneously stimulating them with 100 ng / mL IFN-γ and 10 μg / mL LPS for 24 h. The results are shown in Figure 7: In the Raw264.7 cytokine storm model, the expression of IDO1 and AHR proteins (Figure 7A) and the mRNA expression of IDO1 and C1P1A1 (Figure 7B) were increased, and IDO1 activity was increased (Figure 7C). A siRNA that could knock down IDO1 was designed (Figure 7D). Knocking down IDO1 downregulated the mRNA expression of IDO1, AHR, and CYP1A1 in the model (Figure 7E) and the expression of cytokines (Figure 7F). Kynine, an intermediate product of the kynurenine pathway catalyzed by IDO1, increased the expression of AHR, CYP1A1 (Figure 7G), and classic inflammatory cytokines TNF-α and IL-6 (Figure 7H) in the model cells. Figure 7A shows the protein expression levels of IDO1 and AHR in cells detected by Western blot, with quantitative analysis using Image J (n=3). Figure 7B shows the mRNA levels of AHR and CYP1A1 in cells detected by qPCR. Figure 7C shows the Trp and Kyn content in cell culture supernatant detected by HPLC, with (Kyn / Trp)×100 calculated as IDO1 activity (n=3). Figure 7D shows the expression of IDO1 in cells detected by Western blot. Figure 7E shows the mRNA levels of IDO1, AHR, and CYP1A1 in cells detected by qPCR (n=3). Figure 7F shows the mRNA levels of inflammatory factors in cells detected by qPCR (n=3).

[0112] Figure 7G shows the expression of IDO1, AHR, and CYP1A1 in cells detected by Western blot, n=3. Figure 7H shows the mRNA levels of TNF-α and IL-6 in cells detected by qPCR, n=3.

[0113] 2.4 IDO1 inhibitors inhibit AHR-CYP1A1 and reduce inflammatory cytokine expression in the Raw 264.7 cytokine storm model.

[0114] After Raw264.7 cells reached 70% confluence, a cytokine storm model was established by simultaneously stimulating them with 100 ng / mL IFN-γ and 10 μg / mL LPS for 24 h. During LPS stimulation, the cells were treated with either the IDO1 inhibitor 1-MT (100 μM) or RY103 (400 nM) for 24 h. The results are shown in Figure 8. The expression of IDO1, AHR, and CYPA1 in the model cells (Figure 8A), as well as the expression of inflammatory cytokines (Figure 8C), were all upregulated, and IDO1 activity was increased (Figure 8B). The addition of IDO1 inhibitors reduced these changes. Furthermore, in addition to the IDO1 inhibitors (1-MT, RY103), the CYP1A1 inhibitor Rhapontigenin (Rha) and the AHR antagonist CH-223191 also reduced IL-6 levels, while Kyn could reverse the inhibitory effect of RY103 (Figure 8D), suggesting that the mechanism of action of RY103 may be partly related to the downregulation of Kyn levels. Figure 8A shows the Western blot analysis of protein expression levels of IDO1 (n=3), AHR (n=4), and CYP1A1 (n=3) in cells, followed by Image J quantitative analysis. Figure 8B shows the HPLC analysis of Trp and Kyn content in cell culture supernatant, with (Kyn / Trp)×100 calculated as IDO1 activity (n=3). Figure 8C shows the qPCR analysis of mRNA expression levels of TNF-α, IL-6, IL-1β, MCP-1, and IL-10 in cells (n=3). Figure 8D shows ELISA analysis of IL-6 in cell supernatant (n=4). In the figures, Ctrl: untreated Raw264.7 cells; Model: Raw264.7 cytokine storm model group; 1-MT / RY103: 1-MT / RY103 treated model cells. Data were tested using a one-way ANOVA. Values ​​are expressed as mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

[0115] 2.5 IDO1 inhibitors inhibit the activation of the classical cytokine storm pathway in the THP-1 cytokine storm model

[0116] After THP-1 cells reached 70% confluence, they were treated with 25 nM PMA for 24 h to induce cell adhesion and differentiation into M0 macrophages. Then, 10 μg / mL LPS stimulation was added for 24 h to establish a THP-1 cytokine storm model. Simultaneously with LPS stimulation, the cells were treated with either the IDO1 inhibitor 1-MT (100 μM) or RY103 (400 nM) for 24 h. The results are shown in Figure 9. The IDO1 inhibitor prevented phosphorylation activation of the classical cytokine storm pathway (Figure 9A). The CYP1A1 inhibitor Rhapontigenin (Rha) reduced STAT3 and JNK phosphorylation (Figure 9B), while the NF-κB inhibitor BAY 11–7082 and the STAT3 inhibitor Static downregulated IDO1 and CYP1A1 expression (Figure 9C). The IDO1-AHR-CYP1A1 axis can activate STAT3 and JNK, while STAT3 and NF-κB can also activate the IDO1-AHR-CYP1A1 axis, forming a positive feedback loop. Figure 9 shows the protein expression levels of STAT3, P-STAT3, NF-κB-P65, P-NF-κB-P65, STAT1, P-STAT1, JNK, P-JNK, p38 MAPK, and P-p38 MAPK in cells detected by Western blot, and quantitative analysis using Image J. n=3. Ctrl: Untreated dTHP-1 cell group; Model: THP-1 cytokine storm model group; 1-MT / RY103: 1-MT / RY103 treated model cells. Data were analyzed using a one-way ANOVA test. Values ​​are expressed as mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001.

[0117] 2.6 IDO1 inhibitors inhibit activation of the classic cytokine storm pathway in the Raw264.7 model

[0118] After Raw264.7 cells reached 70% confluence, a cytokine storm model was established by simultaneously stimulating them with 100 ng / mL IFN-γ and 10 μg / mL LPS for 24 h. During LPS stimulation, the cells were treated with either the IDO1 inhibitor 1-MT (100 μM) or RY103 (400 nM) for 24 h. The results are shown in Figure 10. The IDO1 inhibitor prevented the activation (phosphorylation) of the classical cytokine storm pathway. Figure 10 shows the protein expression levels of STAT3, P-STAT3, NF-κB-P65, P-NF-κB-P65, STAT1, P-STAT1, JNK, P-JNK, p38 MAPK, and P-p38 MAPK in cells using Western blot analysis, followed by Image J quantitative analysis. n = 3-4. In the figure, Ctrl: untreated Raw264.7 cells; Model: Raw264.7 cytokine storm model group; 1-MT / RY103: 1-MT / RY103 treated model cells. Data were tested using a one-way ANOVA. Values ​​are expressed as mean ± SD. *P < 0.05, **P < 0.01, ****P < 0.0001.

[0119] 2.7 IDO1 promotes apoptosis in THP-1 and Raw264.7 cytokine storm model cells via the AHR-CYP1A1 axis.

[0120] Using the Raw264.7 cytokine storm model (Fig. 11A, B) and the THP-1 cytokine storm model (Fig. 11C, D), we investigated the effects of kynurenine (Kyn), an intermediate product of the IDO1-catalyzed kynurenine pathway, on apoptosis in model cells, as well as the intervention effects of the CYP1A1 inhibitor Rhapontigenin (Rha) and the AHR antagonist CH-223191.

[0121] In the figure, Kyn: cells were first incubated with 0–150 μM Kyn before modeling; Model+Kyn+Rha: cells were first incubated with 50 μM Kyn for 24 h, then treated with LPS (10 μg / mL) or IFN-γ (100 ng / mL) + LPS (10 μg / mL) and Rhapontigenin (20 μM) for 24 h; Model+Kyn+CH-223191: cells were first incubated with 50 μM Kyn for 24 h, then treated with LPS (10 μg / mL) or IFN-γ (100 ng / mL) + LPS (10 μg / mL) and CH-223191 (3 μM) for 24 h. Flow cytometry was used to detect the percentage of Annexin V+ cells (early apoptotic cells + late apoptotic cells).

[0122] The results are shown in Figure 11. Kyn promotes apoptosis in model cells, while inhibiting the AHR-CYP1A1 axis can interfere with Kyn-promoted apoptosis.

[0123] 2.8 IDO1 inhibitors reduced the apoptosis rate in THP-1 and Raw264.7 model cells, which could be reversed by Kyn.

[0124] Using the Raw264.7 and THP-1 cytokine storm models, we investigated the effects of IDO1 inhibitors on apoptosis in model cells, their impact on ROS levels generated by apoptosis, and the intervention effect of IDO1 on kynurenine (Kyn), an intermediate product of the kynurenine pathway catalyzed by the cytokinine pathway.

[0125] The THP-1 cytokine storm model (Figure 12A) and the Raw264.7 cytokine storm model (Figure 12B) were used. During modeling, the IDO1 inhibitor 1-MT (100 μM) or RY103 (400 nM) was added. Annexin V was detected by flow cytometry using Annexin V / PI double staining. + The percentage of cells (early apoptotic cells + late apoptotic cells) and the ROS content in cells are shown in Figure 12A and B (Figure 12C). In the figures, Ctrl: untreated dTHP-1 cells or Raw264.7 cells; Model: THP-1 or Raw264.7 cytokine storm model group; 1-MT / RY103: model cells treated with IDO1 inhibitor 1-MT (100 μM) or RY103 (400 nM) during modeling; 1-MT / RY103+Kyn: model cells treated with 1-MT / RY103 for 4 h followed by treatment with Kyn (50 μM) for 20 h during modeling. Data were analyzed using a one-way ANOVA test. Values ​​are expressed as mean ± SD. *P < 0.05, **P < 0.01, ****P < 0.0001. As shown in Figure 12, the effect of IDO1 inhibitors in reducing apoptosis rates in THP-1 and Raw264.7 models could be reversed by Kyn, suggesting that the effect of IDO1 inhibitors in improving apoptosis rates is related to the downregulation of Kyn.

[0126] Example 3: Verification of the effect of IDO1 inhibitor on reversing cytokine storm in septic mice:

[0127] 3.1 IDO1 inhibitors reduce serum IDO1 activity and inflammatory cytokine levels in mice with a septic cytokine storm model.

[0128] C57BL / 6 mice were intraperitoneally injected with 1-MT (100 mg / kg) or RY103 (6 mg / kg) every 24 hours for three consecutive days. Six hours after the third administration, 20 mg / kg LPS was injected intraperitoneally to establish a septic cytokine storm mouse model. Mice were sacrificed 18 hours after modeling. Figure 13A shows the HPLC detection of Trp and Kyn levels in mouse serum, with (Kyn / Trp) × 100 calculated as IDO1 activity (n = 6). Figure 13B shows the ELISA detection of the concentrations of inflammatory cytokines TNF-α, IL-6, IL-10, and IFN-γ in mouse serum (n = 5). In the figures, Ctrl represents the control group (mice injected intraperitoneally with an equal volume of saline), Model represents the septic cytokine storm model mouse group, and 1-MT / RY103 represents the model mouse group after three consecutive days of 1-MT / RY103 administration. Data were analyzed using a one-way ANOVA test. Values ​​are expressed as mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. The results, as shown in Figure 13, indicated that administration of the IDO1 inhibitor 1-MT / RY103 significantly reduced the levels of inflammatory cytokines in mice, suggesting that IDO1 inhibitor administration helps improve the inflammatory response.

[0129] 3.2 IDO1 inhibitors downregulated the percentage of neutrophils and upregulated the percentage of monocytes in the spleen of mice with a sepsis cytokine storm model.

[0130] C57BL / 6 mice were intraperitoneally injected with 1-MT (100 mg / kg) or RY103 (6 mg / kg) every 24 hours for three consecutive days. Six hours after the third administration, 20 mg / kg LPS was injected intraperitoneally to establish a mouse model of septic cytokine storm. All mice were sacrificed 18 hours after modeling. Figure 14 shows the flow cytometry analysis of Ly6C in the spleen of mice. - Ly6G + Cells (neutrophils), Ly6C + Ly6G - Cells (monocytes) account for 5% of CD45 + CD11b + Cell proportion changes, n=6. In the figure, Ctrl: control group, mice injected intraperitoneally with an equal volume of physiological saline. Model: septic cytokine storm model mouse group. 1-MT / RY103: model mouse group after three consecutive days of administration of the IDO1 inhibitor 1-MT / RY103. Data were analyzed by one-way ANOVA, and values ​​are expressed as mean ± SD, *P<0.05, ***P<0.001.

[0131] The results are shown in Figure 14 (the left is a representative flow cytometry plot, and the right is the statistical analysis). After administration of the IDO1 inhibitor 1-MT / RY103, the proportion of neutrophils in the spleen of mice was significantly reduced and the proportion of monocytes was increased, suggesting that administration of the IDO1 inhibitor helps to improve the inflammatory response.

[0132] 3.3. IDO1 inhibitors downregulate the proportion of M1 macrophages in the spleen of mice with a sepsis cytokine storm model.

[0133] C57BL / 6 mice were intraperitoneally injected with 1-MT (100 mg / kg) or RY103 (6 mg / kg) every 24 hours for three consecutive days. Six hours after the third administration, 20 mg / kg LPS was injected intraperitoneally to establish a mouse model of septic cytokine storm. All mice were sacrificed 18 hours after modeling. Figure 15 shows the flow cytometry analysis of CD86 in mouse spleen. + Cells (M1 type macrophages) account for 5% of CD45 + CD11b + F4 / 80 + Cell proportion changes, n=6. Ctrl: Control group, mice injected intraperitoneally with an equal volume of physiological saline. In the figure, Model: septic cytokine storm model mouse group. 1-MT / RY103: Model mouse group after three consecutive days of administration of the IDO1 inhibitor 1-MT / RY103. Data were analyzed using a one-way ANOVA test, and values ​​are expressed as mean ± SD, *P < 0.05. The results are shown in Figure 15 (left is a representative flow cytometry plot, right is the statistical analysis). The results show that after administration of the IDO1 inhibitor 1-MT / RY103, the proportion of M1 macrophages in the spleen of mice was significantly reduced, suggesting that administration of the IDO1 inhibitor helps to improve the inflammatory response.

[0134] 3.4. IDO1 inhibitors inhibit AHR-CYP1A1 expression and STAT3 phosphorylation in lung and kidney tissues of mice with septic cytokine storm model.

[0135] C57BL / 6 mice were intraperitoneally injected with 1-MT (100 mg / kg) or RY103 (6 mg / kg) every 24 hours for three consecutive days. Six hours after the third administration, 20 mg / kg LPS was injected intraperitoneally to establish a mouse model of septic cytokine storm. Mice were sacrificed 18 hours after modeling. Figures 16A-D show the protein expression levels of IDO1, AHR, CYP1A1, STAT3, and P-STAT3 in the lungs (A, C) and kidneys (B, D) of mice detected by Western blot analysis. Image J quantitative analysis was performed (n = 3-4). In the figures, Ctrl represents the control group (mice injected intraperitoneally with an equal volume of saline), Model represents the septic cytokine storm model mouse group, and 1-MT / RY103 represents the mouse group that developed the model after three consecutive days of 1-MT / RY103 administration. Data were analyzed using a one-way ANOVA test. Values ​​are expressed as mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001.

[0136] As shown in Figure 16, after administration of the IDO1 inhibitor 1-MT / RY103, the expression levels of AHR, CYP1A1 and phosphorylation level of STAT3 in the lung and kidney tissues of mice were significantly reduced, suggesting that administration of the IDO1 inhibitor helps to improve the inflammatory response.

[0137] 3.5 Pre-administration of IDO1 inhibitors reduced mortality in mice with a sepsis cytokine storm model.

[0138] C57BL / 6 mice were intraperitoneally injected with 1-MT (100 mg / kg) or RY103 (30 mg / kg) every 24 hours for three consecutive days. Six hours after the third injection, mice were intraperitoneally injected with 10 mg / kg LPS to establish a septic cytokine storm mouse model. Survival rates were recorded every 24 hours for seven consecutive days after LPS injection. In the figure, Ctrl: control group (mice injected intraperitoneally with saline); Model: model group (mice injected intraperitoneally with 10 mg / kg LPS); 1-MT and RY103 groups: mouse groups that developed the septic cytokine storm after three consecutive days of 1-MT / RY103 administration. Data were analyzed using a one-way ANOVA test. Values ​​are expressed as mean ± SD. *P < 0.05, ***P < 0.001.

[0139] As shown in Figure 17, pre-administration of the IDO1 inhibitor 1-MT / RY103 reduced the mortality rate of septic mice.

[0140] 3.6 Pre-dose administration of IDO1 inhibitors improves organ damage in a mouse model of septic cytokine storm.

[0141] C57BL / 6 mice were intraperitoneally injected with 1-MT (100 mg / kg) or RY103 (6 mg / kg) every 24 hours for three consecutive days. Six hours after the third administration, 20 mg / kg LPS was injected intraperitoneally to establish a septic cytokine storm mouse model. All mice were sacrificed 18 hours after modeling. The results, as shown in Figure 18, indicate that pretreatment with the IDO1 inhibitors 1-MT / RY103 improved organ damage in septic mice.

[0142] Example 4: IDO1 is closely related to immunosuppression in sepsis:

[0143] 4.1. CLP septic mice showed decreased body weight and increased spleen weight.

[0144] For 10 consecutive days, a sepsis mouse model was established using cecal ligation and puncture (CLP) on a group of 3-5 week old male C57BL / 6 mice. On the day after the 10th group's surgery, all mice were sacrificed and their organs were collected and weighed. Figure 19 shows the change in body weight before sacrifice relative to the body weight before CLP surgery in each group (Figure 19A); and a comparison of spleen weight and spleen index at sacrifice in each group (Figure 19B). In the figures, 0d: mice that did not undergo CLP surgery; 1d-10d: mice that underwent CLP treatment and developed sepsis 1-10 days later. n=4. Data were analyzed using one-way ANOVA. Compared with the 0d group, the mice showed decreased body weight and increased spleen weight, suggesting that they exhibited immune dysregulation after the establishment of the sepsis model.

[0145] 4.2. Changes in serum concentrations of IL-6, TNF-α, and IL-10 in CLP-infected mice

[0146] For 10 consecutive days, a sepsis mouse model was established in a group of 3-5 week old male C57BL / 6 mice via cecal ligation and puncture (CLP). Blood was collected from all mice in the 10th group the day after the surgery, and all mice were sacrificed. Figure 20 shows the serum concentrations of IL-6, TNF-α, and IL-10 in each group. In the figure, 0d: mice that did not undergo CLP treatment; 1d-10d: mice that received CLP treatment and developed sepsis 1-10 days later. n=4. Data were analyzed using one-way ANOVA. Comparisons with the 0d group showed ***P<0.001; ****P<0.0001. The results, as shown in Figure 20, indicate that mice exhibited an inflammatory response after CLP modeling. The levels of IL-6, TNF-α, and IL-10 were significantly upregulated initially, then gradually decreased. One week after CLP surgery, the concentrations of pro-inflammatory cytokines IL-6 and TNF-α decreased to stable levels, while the concentration of anti-inflammatory cytokine IL-10 increased significantly again, suggesting that septic mice began to enter an immunosuppressive phase.

[0147] 4.3. Changes in the ratio of MDSCs in the spleen and blood of CLP septic mice

[0148] For 10 consecutive days, a sepsis mouse model was established in a group of 3-5 week old male C57BL / 6 mice via cecal ligation (CLP). All mice were sacrificed the day after the 10th group's surgery, and their spleens and blood were collected. Figure 21A shows the ratio of M-MDSCs to PMN-MDSCs in the spleen of each group of mice. Figure 21B shows the ratio of M-MDSCs to PMN-MDSCs in the blood of each group of mice. In the figures, 0d: mice that did not undergo CLP surgery; 1d-10d: mice that received CLP treatment and developed sepsis 1-10 days later. n=4. Data were analyzed using one-way ANOVA. Comparisons with the 0d group showed **P<0.01; ***P<0.001; ****P<0.0001. The results, as shown in Figure 21, indicated that one week after CLP modeling, the proportion of MDSCs in the spleen and blood of mice increased, suggesting that sepsis mice began to enter an immunosuppressive phase.

[0149] 4.4. Changes in the ratio of leukocytes to Treg cells in the spleen of CLP-septic mice

[0150] For 10 consecutive days, a sepsis mouse model was established by cecal ligation (CLP) in a group of 3-5 week old male C57BL / 6 mice. All mice in the 10th group were sacrificed the day after surgery, and their spleens were collected. Figure 22 shows the ratio of leukocytes to Treg cells in the spleen of each group of mice. In the figure, 0d: mice that did not undergo CLP surgery; 1d-10d: mice that underwent CLP treatment and developed sepsis 1-10 days later. n=4. Data were analyzed using one-way ANOVA. Comparisons with the 0d group showed *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001. The results, as shown in Figure 22, indicated that the proportion of leukocytes in the spleen began to decrease on day 5 after CLP surgery, with the most significant decrease on days 6-7; the proportion of immunosuppressive Treg cells significantly increased again starting on day 7. This suggests that septic mice begin to enter an immunosuppressive phase one week after CLP surgery.

[0151] 4.5. Changes in serum IDO1 activity in CLP-septic mice

[0152] For 10 consecutive days, a sepsis mouse model was established in a group of 3-5 week old male C57BL / 6 mice via cecal ligation (CLP). Blood was collected from all mice in the 10th group the day after surgery, and all mice were sacrificed. Figure 23 shows the serum IDO1 activity in each group. In the figure, 0d: mice that did not undergo CLP surgery; 1d-10d: mice that underwent CLP treatment and developed sepsis 1-10 days later. n=4. Data were analyzed using one-way ANOVA. Compared with the 0d group, *P<0.05; **P<0.01; ***P<0.001. The results, as shown in Figure 23, showed a significant increase in serum IDO1 activity in mice from day 1-3 after CLP surgery; a decrease starting on day 4, continuing until day 7; and a significant increase again starting on day 8.

[0153] In summary, about one week after CLP surgery, mice exhibited increased levels of anti-inflammatory cytokines in serum, an increased proportion of MDSCs in the spleen and blood, and an increased proportion of Treg cells in the spleen. This confirms that CLP-induced septic mice entered an immunosuppressive state at this time. With the onset of immunosuppression, the activity of IDO1 in CLP-induced septic mice also increased, indicating that IDO1 activity is closely related to immunosuppression in CLP-induced septic mice.

[0154] Example 5: IDO1 inhibitor treatment improves the immune status of CLP septic mice

[0155] 5.1. IDO1 inhibitors had no significant effect on body weight and spleen weight in CLP-septic mice.

[0156] Male C57BL / 6 mice aged 3-5 weeks were used to establish a septic mouse model via cecal ligation (CLP). IDO1 inhibitor intervention was initiated 12 hours after surgery and continued for 7 days. Mice were weighed every 24 hours post-surgery. Eight days after surgery, mice were sacrificed, and organs were collected and weighed. Figure 24 shows the changes in body weight in each group (Figure 24A); body weight, spleen weight, and spleen index at sacrifice in each group (Figure 24B). In the figures, sham: sham-operated group; CLP: septic mice treated with 10% HPBCD; CLP+1-MT: septic mice treated with 1-MT (100 mg / kg); CLP+RY103: septic mice treated with RY103 (6 mg / kg). The drugs were administered intraperitoneally every 36 hours, all dissolved in 10% HPBCD (2-hydroxypropyl-β-cyclodextrin), n=6. Data were analyzed using one-way ANOVA and compared with the CLP group; **P<0.01; ***P<0.001. The results are shown in Figure 24. Administration of the IDO1 inhibitor had no significant effect on mouse body weight or spleen weight.

[0157] 5.2. IDO1 inhibitors can reduce IDO1 activity in the serum of CLP-septic mice.

[0158] Male C57BL / 6 mice aged 3-5 weeks were used to establish a septic mouse model via cecal ligation (CLP). IDO1 inhibitor intervention was initiated 12 hours after surgery and continued for 7 days. Eight days after surgery, mice were sacrificed after blood collection. Figure 25 shows the HPLC detection of IDO1 activity in mouse serum. In the figure, sham: sham-operated group; CLP: septic mice treated with 10% HPBCD; CLP+1-MT: septic mice treated with 1-MT (100 mg / kg); CLP+RY103: septic mice treated with RY103 (6 mg / kg). The drugs were administered intraperitoneally every 36 hours, all dissolved in 10% HPBCD (2-hydroxypropyl-β-cyclodextrin), n=6. Data were analyzed using one-way ANOVA. Comparison with the CLP group showed *P<0.05; **P<0.01; ***P<0.001. As shown in Figure 25, the IDO1 activity in the serum of septic mice decreased after administration of an IDO1 inhibitor.

[0159] 5.3. IDO1 inhibitors reduce serum IL-10 concentration in CLP-secreting mice.

[0160] Male C57BL / 6 mice aged 3-5 weeks were used to establish a septic mouse model via cecal ligation (CLP). IDO1 inhibitor intervention was initiated 12 hours after surgery and continued for 7 days. Eight days after surgery, mice were sacrificed after blood collection. Figure 26 shows the IL-10 concentration in mouse serum as detected by ELISA. In the figure, sham: sham-operated group; CLP: septic mice treated with 10% HPBCD; CLP+1-MT: septic mice treated with 1-MT (100 mg / kg); CLP+RY103: septic mice treated with RY103 (6 mg / kg). The drugs were administered intraperitoneally every 36 hours, all dissolved in 10% HPBCD (2-hydroxypropyl-β-cyclodextrin), n=6. Data were analyzed using one-way ANOVA. Comparison with the CLP group showed **P<0.01; ***P<0.001; ****P<0.0001. As shown in Figure 26, the concentration of IL-10 in the serum of septic mice decreased after administration of IDO1 inhibitors.

[0161] 5.4. IDO1 inhibitors increased the proportion of M1 macrophages and decreased the proportion of M2 macrophages in the peritoneal cavity of CLP-secreting mice.

[0162] Male C57BL / 6 mice aged 3-5 weeks were used to establish a septic mouse model via cecal ligation and puncture (CLP). IDO1 inhibitor intervention was initiated 12 hours after surgery and continued for 7 days. Eight days after surgery, the mice were sacrificed, and 3 mL of PBS was injected intraperitoneally. After 5 minutes of massage, the peritoneal lavage fluid was aspirated using a pipette and analyzed by flow cytometry. Figure 27 shows the proportion of macrophages in the peritoneal cavity of septic mice as detected by flow cytometry. In the figure, sham: sham-operated group; CLP: septic mice treated with 10% HPBCD; CLP+1-MT: septic mice treated with 1-MT (100 mg / kg); CLP+RY103: septic mice treated with RY103 (6 mg / kg). The drugs were administered intraperitoneally every 36 hours, all dissolved in 10% HPBCD (2-hydroxypropyl-β-cyclodextrin), n=5. Data were analyzed using one-way ANOVA and compared with the CLP group; *P<0.05; **P<0.01; ****P<0.0001. The results, shown in Figure 27, indicate that after administration of IDO1 inhibitors, the proportion of M1 macrophages and the proportion of M2 macrophages in the peritoneal cavity of septic mice increased. This suggests that IDO1 inhibitors reverse immunosuppression.

[0163] 5.5. IDO1 inhibitor treatment can reduce the number of bacteria in the peritoneal cavity of CLP septic mice.

[0164] Male C57BL / 6 mice aged 3-5 weeks were used to establish a septic mouse model via cecal ligation (CLP). IDO1 inhibitor intervention was initiated 12 hours after surgery and continued for 7 days. Eight days after surgery, the mice were sacrificed, and 3 mL of PBS was injected intraperitoneally. After massaging for 5 minutes, the PBS was aspirated with a pipette. 100 μL of the peritoneal lavage fluid from each mouse was spread onto LB (Luria-Bertani) solid medium and cultured for 24 hours. In the figure, sham: sham-operated group; CLP: septic mice treated with 10% HPBCD; CLP+1-MT: septic mice treated with 1-MT (100 mg / kg); CLP+RY103: septic mice treated with RY103 (6 mg / kg). The drugs were administered intraperitoneally every 36 hours, all dissolved in 10% HPBCD (2-hydroxypropyl-β-cyclodextrin), n=3. Data were analyzed using one-way ANOVA and compared with the CLP group; P < 0.0001. The results, shown in Figure 28, indicate that administration of the IDO1 inhibitor reduced the number of bacteria in the peritoneal cavity of CLP septic mice.

[0165] 5.6. IDO1 inhibitors reduce the proportion of MDSCs in the spleen and blood of CLP-secreting mice.

[0166] Male C57BL / 6 mice aged 3-5 weeks were used to establish a septic mouse model via cecal ligation (CLP). IDO1 inhibitor intervention was initiated 12 hours after surgery and continued for 7 days. Mice were sacrificed 8 days after surgery, and blood and spleens were collected. Flow cytometry was used to detect the proportion of MDSC cells in the spleen (Figure 29A, left is a representative flow cytometry plot, right is the statistical analysis) and blood (Figure 29B, left is a representative flow cytometry plot, right is the statistical analysis) of septic mice. In the figures, sham: sham-operated group; CLP: septic mice treated with 10% HPBCD; CLP+1-MT: septic mice treated with 1-MT (100 mg / kg); CLP+RY103: septic mice treated with RY103 (6 mg / kg). The drugs were administered intraperitoneally every 36 hours, all dissolved in 10% HPBCD (2-hydroxypropyl-β-cyclodextrin), n=4. Data were analyzed using one-way ANOVA and compared with the CLP group. *P<0.05; ***P<0.001; ****P<0.0001. The results are shown in Figure 29. After administration of IDO1 inhibitors, the proportion of MDSCs in the spleen (Figure 29A) and blood (Figure 29B) of CLP septic mice decreased.

[0167] 5.7. IDO1 inhibitors reduce the proportion of Treg cells in the spleen of CLP-septic mice.

[0168] Male C57BL / 6 mice aged 3-5 weeks were used to establish a septic mouse model via cecal ligation (CLP). IDO1 inhibitor intervention was initiated 12 hours after surgery and continued for 7 days. Mice were sacrificed 8 days after surgery, and their spleens were collected. Treg cells in the spleens of septic mice were detected by flow cytometry (Figure 30, left is a representative flow cytometry plot, right is the statistical analysis). In the figure, sham represents the sham-operated group; CLP represents septic mice treated with 10% HPBCD; CLP+1-MT represents septic mice treated with 1-MT (100 mg / kg); and CLP+RY103 represents septic mice treated with RY103 (6 mg / kg). The drugs were administered intraperitoneally every 36 hours, all dissolved in 10% HPBCD (2-hydroxypropyl-β-cyclodextrin), n=4. Data were analyzed using one-way ANOVA and compared with the CLP group. *P<0.05; **P<0.01; ***P<0.001. The results are shown in Figure 30. After administration of the IDO1 inhibitor, the proportion of Tregs in the spleen of CLP septic mice decreased.

[0169] The above studies show that IDO1 inhibitors can reverse immunosuppression in septic mice, including reducing IL-10 concentration, decreasing the proportion of immunosuppressive M2 macrophages, MDSCs, and Treg cells, and reducing the number of bacteria in the peritoneal cavity of septic mice.

[0170] Example 6: IDO1 inhibitor treatment improves survival in CLP septic mice after secondary infection.

[0171] 6.1. IDO1 inhibitors reduce mortality in CLP-secreting mice following secondary infection.

[0172] Male C57BL / 6 mice aged 3-5 weeks were used to establish a sepsis mouse model via cecal ligation (CLP). IDO1 inhibitor treatment was initiated 12 hours post-surgery and continued for 7 days. On the 8th day post-surgery, 1×10⁻⁶ IDO1 inhibitors were administered via tail vein injection. 7CFU of Staphylococcus aureus was used to assess mouse survival every 24 hours until the end of the experiment. In the figure, sham+S. aureus: sham-operated group infected with Staphylococcus aureus; CLP+S. aureus: septic mice given 10% HPBCD and infected with Staphylococcus aureus; CLP+1-MT+S. aureus: septic mice given 1-MT and infected with Staphylococcus aureus; CLP+RY103+S. aureus: septic mice given RY103 and infected with Staphylococcus aureus. RY103 (6 mg / kg) and 1-MT (100 mg / kg) were administered intraperitoneally every 36 hours, both dissolved in 10% HPBCD (2-hydroxypropyl-β-cyclodextrin). n=10. The results, as shown in Figure 31, showed that administration of the IDO1 inhibitor significantly reduced the mortality rate of CLP-treated septic mice following secondary infection.

[0173] 6.2. IDO1 inhibitors reduce serum IDO1 activity in septic mice after secondary infection.

[0174] Male C57BL / 6 mice aged 3-5 weeks were used to establish a sepsis mouse model via cecal ligation (CLP). IDO1 inhibitor treatment was initiated 12 hours after surgery and continued for 7 days. On the 8th day after surgery, 1×10 7 CFU of Staphylococcus aureus was used to collect blood from mice one day later. After standing, the serum was centrifuged and the IDO1 activity in the serum was detected by HPLC. Figure 32 shows the calculation of IDO1 activity using Kyn and Trp concentrations. In the figure, sham: sham-operated group; sham+S. aureus: sham-operated group infected with Staphylococcus aureus; CLP+S. aureus: septic mice given 10% HPBCD and infected with Staphylococcus aureus; CLP+1-MT+S. aureus: septic mice given 1-MT and infected with Staphylococcus aureus; CLP+RY103+S. aureus: septic mice given RY103 and infected with Staphylococcus aureus. RY103 (6 mg / kg) and 1-MT (100 mg / kg) were administered intraperitoneally every 36 hours. Both drugs were dissolved in 10% HPBCD (2-hydroxypropyl-β-cyclodextrin). n=4. Data were analyzed using one-way ANOVA and compared with the CLP+S. aureus group. *P<0.05; **P<0.01; ***P<0.001. The results are shown in Figure 32. After administration of IDO1 inhibitors, IDO1 activity in the serum of mice with sepsis secondary infection decreased.

[0175] 6.3. IDO1 inhibitors reduce serum IL-10 concentration in CLP-secreting mice after secondary infection.

[0176] Male C57BL / 6 mice aged 3-5 weeks were used to establish a sepsis mouse model via cecal ligation (CLP). IDO1 inhibitor treatment was initiated 12 hours after modeling and continued for 7 days. On the 8th day post-surgery, mice were injected with 1×10⁻⁶ IDO1 inhibitors. 7 CFU of Staphylococcus aureus was administered to mice. Blood was collected from the mice one day later, and the serum was collected by centrifugation after standing for ELISA detection. In the figure, sham: sham-operated group; sham+S. aureus: sham-operated group infected with Staphylococcus aureus; CLP+S. aureus: septic mice given 10% HPBCD and infected with Staphylococcus aureus; CLP+1-MT+S. aureus: septic mice given 1-MT and infected with Staphylococcus aureus; CLP+RY103+S. aureus: septic mice given RY103 and infected with Staphylococcus aureus. RY103 (6 mg / kg) and 1-MT (100 mg / kg) were administered intraperitoneally every 36 hours. Both drugs were dissolved in 10% HPBCD (2-hydroxypropyl-β-cyclodextrin). n=4. Data were analyzed using one-way ANOVA and compared with the CLP+S. aureus group. **P<0.01; ***P<0.001; ****P<0.0001. The results are shown in Figure 33. After administration of IDO1 inhibitors, the serum IL-10 concentration in CLP septic mice with secondary infection decreased.

[0177] 6.4. IDO1 inhibitors reduce the proportion of Treg cells in the spleen of CLP-secreting mice after secondary infection.

[0178] Male C57BL / 6 mice aged 3-5 weeks were used to establish a sepsis mouse model via cecal ligation (CLP). IDO1 inhibitor treatment was initiated 12 hours after surgery and continued for 7 days. On the 8th day post-surgery, mice were injected with 1×10⁻⁶ IDO1 inhibitors. 7CFU of Staphylococcus aureus was used. One day later, mice were sacrificed, and spleens were collected, ground, and stained with schizoaflavins. The proportion of Treg cells in the spleen was analyzed by flow cytometry. In the figure, sham: sham-operated group; sham+S. aureus: sham-operated group infected with Staphylococcus aureus; CLP+S. aureus: septic mice given 10% HPBCD and infected with Staphylococcus aureus; CLP+1-MT+S. aureus: septic mice given 1-MT and infected with Staphylococcus aureus; CLP+RY103+S. aureus: septic mice given RY103 and infected with Staphylococcus aureus. RY103 (6 mg / kg) and 1-MT (100 mg / kg) were administered intraperitoneally every 36 hours. Both drugs were dissolved in 10% HPBCD (2-hydroxypropyl-β-cyclodextrin). n=4. Data were analyzed using one-way ANOVA and compared with the CLP+S. aureus group. *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001. The results are shown in Figure 34 (left is a representative flow cytometry plot, right is the statistical analysis). After administration of IDO1 inhibitors, the proportion of Treg cells in the spleen of CLP septic mice after secondary infection decreased.

[0179] 6.5. IDO1 inhibitors reduce the proportion of MDSCs in the spleen and blood of CLP-secreting mice after secondary infection.

[0180] Male C57BL / 6 mice aged 3-5 weeks were used to establish a sepsis mouse model via cecal ligation (CLP). IDO1 inhibitor intervention was initiated 12 hours after modeling and continued for 7 days. On the 8th day post-surgery, 1×10⁻⁶ IDO1 inhibitors were injected. 7One day after ingesting Staphylococcus aureus with CFU, the spleen and blood of mice were collected, and erythrocytes were lysed. The changes in the proportion of MDSCs in the spleen and blood were analyzed by flow cytometry. Figure 35 shows the proportion of MDSCs in the spleen (A, left: representative flow cytometry plot; right: statistical analysis) and blood (B, left: representative flow cytometry plot; right: statistical analysis) of each group of mice. In the figure, sham: sham-operated group; sham+S. aureus: sham-operated group infected with Staphylococcus aureus; CLP+S. aureus: septic mice given 10% HPBCD and infected with Staphylococcus aureus; CLP+1-MT+S. aureus: septic mice given 1-MT and infected with Staphylococcus aureus; CLP+RY103+S. aureus: septic mice given RY103 and infected with Staphylococcus aureus. RY103 (6 mg / kg) and 1-MT (100 mg / kg) were administered intraperitoneally every 36 hours. Both drugs were dissolved in 10% HPBCD (2-hydroxypropyl-β-cyclodextrin). n = 4. Data were analyzed by one-way ANOVA and compared with the CLP+S. aureus group. *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001. The results are shown in Figure 35. After administration of the IDO1 inhibitor, the proportion of MDSCs in the spleen and blood of mice with sepsis secondary infection decreased.

[0181] The above studies show that IDO1 inhibitors have a therapeutic effect on secondary infections in septic mice, including prolonging survival and reversing immunosuppression such as reducing IL-10 concentration and decreasing the proportion of MDSCs.

[0182] Example 7: Pharmacodynamic study of IDO1 inhibitors in mice with sepsis

[0183] 7.1. IDO1 inhibitors are more effective than antibiotics in reducing mortality in septic mice, and their efficacy is even better when used in combination with antibiotics.

[0184] Sepsis mice were constructed using LPS and treated with antibiotics (Ceftriaxone, Imipenem), IDO1 inhibitors (epacadostat, RY103), and combinations of RY103 and antibiotics (RY103+Imip, RY103+Ceft). The efficacy was compared.

[0185] A sepsis mouse model was established by intraperitoneal injection of LPS (10 mg / kg) into male C57BL / 6 mice aged 3-5 weeks. Two hours later, the model mice were divided into groups and treated with the drug, which was administered intraperitoneally every 24 hours for 3 consecutive days. All drugs were dissolved in 10% HPBCD (2-hydroxypropyl-β-cyclodextrin). The survival rate was recorded every 24 hours for 6 consecutive days after the establishment of the sepsis model. In the figure, the Ctrl group consisted of wild-type mice injected intraperitoneally with an equal volume of physiological saline; the Model group consisted of septic mice given 10% HPBCD; the Ceftriaxone group consisted of septic mice given Ceftriaxone (25 mg / kg); the Imipenem group consisted of septic mice given Imipenem (25 mg / kg); the epacadostat group consisted of septic mice given epacadostat (6 mg / kg); the RY103 group consisted of septic mice given RY103 (6 mg / kg); the RY103+Ceft group consisted of septic mice given RY103 (6 mg / kg) and Ceftriaxone (25 mg / kg); and the RY103+Imip group consisted of septic mice given RY103 (6 mg / kg) and Imipenem (25 mg / kg). Data were analyzed using a one-way ANOVA test. Values ​​are expressed as mean ± SD. *P < 0.05, ***P < 0.001.

[0186] As shown in Figure 36, sepsis significantly increased the mortality rate of mice. Deaths occurred within the first three days after sepsis modeling, with the highest mortality rate in the Model group. The mortality rate was lower in the IDO1 inhibitor-only group than in the antibiotic-only group. All mice in the RY103 combined with the antibiotic imipenem group survived. Antibiotic treatment appeared to increase the acute mortality rate in mice, resulting in a higher mortality rate on the first day of sepsis, but it still reduced the overall mortality rate. IDO1 inhibitor-only treatment was more effective at reducing the mortality rate of septic mice than antibiotic-only treatment. The combination of RY103 and imipenem showed the most significant effect. This suggests that IDO1 inhibitors can reduce the mortality rate of septic mice, and the combination of RY103 and the antibiotic imipenem is more effective.

[0187] 7.2. IDO1 inhibitors can improve weight loss and hypothermia in septic mice, and their effects are even better when used in combination with antibiotics.

[0188] Sepsis mice were constructed using LPS and treated with antibiotics (Ceftriaxone, Imipenem), IDO1 inhibitors (epacadostat, RY103), and combinations of RY103 and antibiotics (RY103+Imip, RY103+Ceft). The efficacy was compared.

[0189] A septic mouse model was established by intraperitoneal injection of LPS (7 mg / kg) into male C57BL / 6 mice aged 3-5 weeks. Two hours later, the model mice were divided into groups and treated with the drug once every 24 hours via intraperitoneal injection for 3 consecutive days. All drugs were dissolved in 10% HPBCD (2-hydroxypropyl-β-cyclodextrin). In the figure, the Ctrl group consisted of wild-type mice injected intraperitoneally with an equal volume of physiological saline; the Model group consisted of septic mice given 10% HPBCD; the Ceftriaxone group consisted of septic mice given Ceftriaxone (25 mg / kg); the Imipenem group consisted of septic mice given Imipenem (25 mg / kg); the epacadostat group consisted of septic mice given epacadostat (6 mg / kg); the RY103 group consisted of septic mice given RY103 (6 mg / kg); the RY103+Ceft group consisted of septic mice given RY103 (6 mg / kg) and Ceftriaxone (25 mg / kg); and the RY103+Imip group consisted of septic mice given RY103 (6 mg / kg) and Imipenem (25 mg / kg). Data were analyzed using a one-way ANOVA test. Values ​​are expressed as mean ± SD. *P < 0.05, ***P < 0.001. Figure 37 shows that one day after modeling, the body weight and body temperature of all mice in the modeling group decreased. After drug treatment, the body weight and body temperature of the mice rebounded, indicating that the drug treatment changed the condition of the mice. As shown in Figure 37A, the combined treatment of RY103 and antibiotics could restore the body weight of mice to the level of non-septic mice in the Ctrl group. As shown in Figures 37B-C, before modeling (day 0), the body temperature of mice in each group was similar; one day after modeling and drug administration (day 1), the body temperature of mice in the modeling group was significantly lower than that of the non-modeling Ctrl group; two days after modeling and drug administration (day 2), the body temperature of mice in the drug administration group began to rebound and was significantly higher than that of the Model group; three days after modeling and drug administration (day 3), the body temperature of mice continued to rise, and the body temperature of mice in the RY103 combined with antibiotics group returned to normal levels, especially the RY103 combined with Imipenem group, whose body temperature was the same as that of the non-modeling Ctrl group. The results show that IDO1 inhibitors can improve weight loss and body temperature reduction in septic mice, and the effect is even better when used in combination with antibiotics.

[0190] 7.3. IDO1 inhibitors reduce elevated IDO1 activity and cytokine levels in septic mice, with better efficacy when used in combination with antibiotics.

[0191] Sepsis mice were constructed using LPS and treated with antibiotics (Ceftriaxone, Imipenem), IDO1 inhibitors (epacadostat, RY103), and combinations of RY103 and antibiotics (RY103+Imip, RY103+Ceft). The efficacy was compared.

[0192] A septic mouse model was established by intraperitoneal injection of LPS (7 mg / kg) into male C57BL / 6 mice aged 3-5 weeks. Two hours later, the model mice were divided into groups and treated with the drug once every 24 hours via intraperitoneal injection for 3 consecutive days. All drugs were dissolved in 10% HPBCD (2-hydroxypropyl-β-cyclodextrin). In the figure, the Ctrl group consisted of wild-type mice injected intraperitoneally with an equal volume of physiological saline; the Model group consisted of septic mice given 10% HPBCD; the Ceftriaxone group consisted of septic mice given Ceftriaxone (25 mg / kg); the Imipenem group consisted of septic mice given Imipenem (25 mg / kg); the epacadostat group consisted of septic mice given epacadostat (6 mg / kg); the RY103 group consisted of septic mice given RY103 (6 mg / kg); the RY103+Ceft group consisted of septic mice given RY103 (6 mg / kg) and Ceftriaxone (25 mg / kg); and the RY103+Imip group consisted of septic mice given RY103 (6 mg / kg) and Imipenem (25 mg / kg). Data were analyzed using a one-way ANOVA test. Values ​​are expressed as mean ± SD. *P < 0.05, ***P < 0.001. Figure 38A shows the HPLC detection of Trp and Kyn levels in mouse serum. (Kyn / Trp) × 100 was calculated as IDO1 activity. The results showed decreased Trp and increased Kyn levels in the serum of sepsis-affected mice, indicating a significant increase in IDO1 activity. Antibiotic treatment did not reduce IDO1 activity in the serum of sepsis-affected mice, but treatment with the IDO1 inhibitors epacadostat and RY103 both reduced IDO1 activity in the serum of sepsis-affected mice. ELISA was used to detect the concentrations of inflammatory cytokines CCL2, IL-6, and IL-10 in mouse serum. Figure 38B shows that the levels of cytokines CCL2, IL-6, and IL-10 in the serum of sepsis-affected mice were significantly increased. Antibiotics alone did not significantly reduce the concentrations of these cytokines, but the use of IDO1 inhibitors alone or in combination with antibiotics significantly reduced CCL2 and IL-6 levels. The combination of RY103 and Imipene also significantly reduced IL-10 levels.

[0193] 7.4. IDO1 inhibitors downregulate the proportion of M1 macrophages in the spleen of septic mice, and their effect is better when used in combination with antibiotics.

[0194] Sepsis mice were constructed using LPS and treated with antibiotics (Ceftriaxone, Imipenem), IDO1 inhibitors (epacadostat, RY103), and combinations of RY103 and antibiotics (RY103+Imip, RY103+Ceft). The efficacy was compared.

[0195] A septic mouse model was established by intraperitoneal injection of LPS (7 mg / kg) into male C57BL / 6 mice aged 3-5 weeks. Two hours later, the model mice were divided into groups and treated with the drug once every 24 hours via intraperitoneal injection for 3 consecutive days. All drugs were dissolved in 10% HPBCD (2-hydroxypropyl-β-cyclodextrin). In the figure, the Ctrl group consisted of wild-type mice injected intraperitoneally with an equal volume of physiological saline; the Model group consisted of septic mice given 10% HPBCD; the Ceftriaxone group consisted of septic mice given Ceftriaxone (25 mg / kg); the Imipenem group consisted of septic mice given Imipenem (25 mg / kg); the epacadostat group consisted of septic mice given epacadostat (6 mg / kg); the RY103 group consisted of septic mice given RY103 (6 mg / kg); the RY103+Ceft group consisted of septic mice given RY103 (6 mg / kg) and Ceftriaxone (25 mg / kg); and the RY103+Imip group consisted of septic mice given RY103 (6 mg / kg) and Imipenem (25 mg / kg). Data were analyzed using a one-way ANOVA test. Values ​​are expressed as mean ± SD. *P < 0.05, ***P < 0.001. The change in the proportion of CD86+ cells (M1 macrophages) to CD45+CD11b+F4 / 80+ cells in the spleen of mice was detected by flow cytometry. Figure 39 (left is a representative flow cytometry plot, right is the statistical analysis) shows that the proportion of M1 macrophages in the spleen tissue of septic model mice is increased. Antibiotics failed to effectively reduce the proportion of M1 macrophages. Treatment with the IDO1 inhibitor epacadostat and RY103 can reduce the proportion of M1 macrophages. The combination of RY103 and antibiotics has the best effect, especially the combination of RY103 and Imipenem.

[0196] 7.5. IDO1 inhibitors can improve tissue damage in septic model mice, and their effect is even better when used in combination with antibiotics.

[0197] Sepsis mice were constructed using LPS and treated with antibiotics (Ceftriaxone, Imipenem), IDO1 inhibitors (epacadostat, RY103), and combinations of RY103 and antibiotics (RY103+Imip, RY103+Ceft). The efficacy was compared.

[0198] A septic mouse model was established by intraperitoneal injection of LPS (7 mg / kg) into male C57BL / 6 mice aged 3-5 weeks. Two hours later, the model mice were divided into groups and treated with the drug once every 24 hours via intraperitoneal injection for 3 consecutive days. All drugs were dissolved in 10% HPBCD (2-hydroxypropyl-β-cyclodextrin). In the figure, the Ctrl group consisted of wild-type mice injected intraperitoneally with an equal volume of physiological saline; the Model group consisted of septic mice given 10% HPBCD; the Ceftriaxone group consisted of septic mice given Ceftriaxone (25 mg / kg); the Imipenem group consisted of septic mice given Imipenem (25 mg / kg); the epacadostat group consisted of septic mice given epacadostat (6 mg / kg); the RY103 group consisted of septic mice given RY103 (6 mg / kg); the RY103+Ceft group consisted of septic mice given RY103 (6 mg / kg) and Ceftriaxone (25 mg / kg); and the RY103+Imip group consisted of septic mice given RY103 (6 mg / kg) and Imipenem (25 mg / kg). Data were analyzed using a one-way ANOVA test. Values ​​are expressed as mean ± SD. *P < 0.05, ***P < 0.001. Elevated levels of inflammatory factors in the lungs and kidneys often lead to acute lung injury (ALI), respiratory failure, and acute kidney injury (AKI). These inflammatory factors can also spread through circulation, exacerbating liver, heart, and brain damage. Figure 40 shows that in sepsis-induced mouse models, the expression of cytokines IL-6 and CCL2 in the lungs and kidneys was significantly elevated. Treatment with IDO1 inhibitors and antibiotics reduced the expression levels of these cytokines. Treatment with the IDO1 inhibitor epacadostat and RY103 alone was more effective than antibiotic treatment alone, with the combination of RY103 and antibiotics showing the best therapeutic effect. This indicates that the combination of RY103 and antibiotics can effectively reduce the levels of inflammatory factors in vital organs of sepsis-induced mouse models, reducing the risk of organ damage and secondary infections. HE staining results also suggest that the combination of RY103 and antibiotics can better improve organ damage in sepsis-induced mice.

[0199] in conclusion:

[0200] As described in the background section, the course of sepsis includes a cytokine storm phase and an immunosuppressive phase. The inventors have discovered that IDO1 participates in and mediates both the cytokine storm and immunosuppression phases. Administering an IDO1 inhibitor during either the cytokine storm or immunosuppression phase can reverse the course of the disease. Therefore, IDO1 inhibitors have excellent potential applications in the treatment of sepsis.

[0201] IDO1 is closely associated with cytokine storm in sepsis patients. IDO1 inhibitors inhibit cytokine storm-related pathways such as STAT3 through AHR-CYP1A1, reducing inflammatory cytokine levels and apoptosis rate in model cells, restoring immune homeostasis in model mice, and reversing the cytokine storm in sepsis. This reversal effect was observed whether the drug was administered before or after modeling, suggesting that IDO1 inhibitors not only have a therapeutic effect on cytokine storm caused by sepsis but also have a certain preventive effect.

[0202] In septic mice under immunosuppression, IDO1 activity is upregulated, anti-inflammatory cytokine concentrations increase, the number of immunosuppressive cells increases, macrophage activation is inhibited, and the body's ability to clear infection decreases. Treatment of septic mice with IDO1 inhibitors can reverse these changes and improve the survival rate of septic mice when subjected to secondary infections under immunosuppression.

[0203] Furthermore, the inventors discovered that, under equal-dose administration, IDO1 inhibitors alone were more effective than antibiotics alone in improving sepsis-induced cell infections. Unexpectedly, a further improvement was observed when IDO1 inhibitors were used in combination with antibiotics, suggesting a synergistic effect between IDO1 inhibitors and antibiotics, which could potentially be used to develop new drugs for treating sepsis.

[0204] All documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference. Furthermore, it should be understood that after reading the foregoing teachings of this invention, those skilled in the art can make various alterations or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims.

Claims

1. The use of an IDO1 inhibitor, characterized in that, Used to prepare pharmaceutical compositions for the treatment of sepsis.

2. The use as described in claim 1, characterized in that, The IDO1 inhibitor is a compound selected from the group consisting of 1-MT, N-aryltryptamine derivatives, epacadostat, or N-benzyltryptamine derivatives.

3. The use as described in claim 1, characterized in that, The IDO1 inhibitor has the structure shown in formula A1: Wherein: R 1 Selected from the group consisting of hydrogen, fluorine, and -(substituted or unsubstituted C1-C6 alkyl)-substituted or unsubstituted 5-12 membered heterocyclic groups; wherein the heterocyclic group comprises 1-3 heteroatoms selected from the group consisting of N, O, or S; R 2 Selected from the following group: H, Cl, Br, substituted or unsubstituted C1-C4 alkyl groups, -NR 3 R 4 , or -(substituted or unsubstituted C1-C6 alkyl)-NR 3 R 4 ; The R mentioned 3 R 4 Each is independently selected from the group consisting of: H, substituted or unsubstituted C1-C4 alkyl, substituted or unsubstituted C2-C4 alkenyl, substituted or unsubstituted C2-C4 alkynyl, and substituted or unsubstituted C3-C6 cycloalkyl. or R 3 R 4 Together with adjacent nitrogen atoms, they form a substituted or unsubstituted 5-12 membered heterocyclic group, wherein the 5-12 membered heterocyclic group has 1-2 nitrogen atoms and 0-2 heteroatoms selected from the group consisting of O and S. The substitution refers to the substitution of one or more hydrogen atoms (preferably hydrogen atoms on nitrogen atoms) on the group by a substituent selected from the group consisting of: C1-C4 alkyl, C1-C4 haloalkyl, amino protecting group (preferably tert-butyloxycarbonyl), halogen, and phenyl.

4. The use as described in claim 1, characterized in that, The pharmaceutical composition is also used to improve or reverse apoptosis caused by septic cytokine storm.

5. The use as described in claim 1, characterized in that, The pharmaceutical composition is also used to improve or reverse cytokine storm caused by sepsis; preferably, the pharmaceutical composition is also used to reduce the high expression of inflammatory cytokines caused by sepsis cytokine storm.

6. The use as described in claim 1, characterized in that, The pharmaceutical composition is also used to downregulate the increased proportion of cells selected from the following groups in the spleen caused by septic cytokine storm: neutrophils and M1 macrophages.

7. The use as described in claim 1, characterized in that, The pharmaceutical composition is also used to downregulate AHR-CYP1A1 expression in lung and kidney tissues and / or upregulate STAT3 phosphorylation caused by septic cytokine storm; preferably, the pharmaceutical composition is also used to reduce mortality caused by septic cytokine storm or to improve organ damage caused by septic cytokine storm.

8. The use as described in claim 1, characterized in that, The pharmaceutical composition is also used to improve the immunosuppressive state caused by sepsis; preferably, the pharmaceutical composition is also used to reduce the elevated ratio of MDSCs to Tregs in tissues caused by sepsis immunosuppression; more preferably, the tissues are selected from the group consisting of spleen and blood.

9. The use as described in claim 1, characterized in that, The pharmaceutical composition is also used to downregulate elevated blood IL-10 levels caused by sepsis and sepsis-related infections.

10. The use as described in claim 1, characterized in that, The pharmaceutical composition is also used to reduce the proportion of elevated M2 macrophages in the peritoneal cavity caused by sepsis-induced immunosuppression; preferably, the pharmaceutical composition is also used to increase the proportion of decreased M1 macrophages in the peritoneal cavity caused by sepsis-induced immunosuppression; preferably, the pharmaceutical composition is also used to reduce the elevated IDO1 activity caused by sepsis.

11. The use as described in claim 1, characterized in that, The pharmaceutical composition is also used to improve secondary infections (bacterial or viral) in sepsis and reduce the mortality rate of secondary infections; preferably, the bacterial infection is an infection caused by bacteria selected from the group consisting of Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, non-spore-forming anaerobic bacteria, or a combination thereof; preferably, the viral infection is SARS-CoV-2 infection.

12. The use as described in claim 1, characterized in that, The pharmaceutical composition described above is also used to reduce the mortality rate of sepsis.

13. The use as described in claim 1, characterized in that, The pharmaceutical composition is also used to improve symptoms of sepsis selected from the group consisting of: weight loss, abnormal body temperature, organ and / or tissue damage; preferably, the pharmaceutical composition is also used to downregulate the increase in the proportion of M1 macrophages in the spleen and the increase in the level of inflammatory cytokines in the blood caused by sepsis.

14. The use as described in claim 1, characterized in that, The pharmaceutical composition further includes a second therapeutic agent; preferably, the second therapeutic agent is an antibiotic.

15. The use as described in claim 14, characterized in that, The antibiotics mentioned are selected from the group consisting of ceftriaxone, imipenem, or combinations thereof.

Images