New application of S-methyl-5-thioadenosine

Abstract

The invention discloses a new application of S-methyl-5-thioadenosine, and the S-methyl-5-thioadenosine can be used as a hepatic encephalopathy biomarker. The invention relates to application of S-methyl-5-thioadenosine, a pharmaceutically acceptable salt thereof and / or a prodrug thereof as an active ingredient in preparation of drugs for preventing and / or treating hepatic encephalopathy, MHE and neuroinflammation and application of S-methyl-5-thioadenosine, a pharmaceutically acceptable salt thereof and / or a prodrug thereof as an active ingredient in preparation of drugs for preventing and / or treating hepatic encephalopathy, MHE and neuroinflammation. The invention also relates to application in medicines for improving cognition. Clinical data, animal experiments and cell model researches find that MTA can effectively relieve neuroinflammatory response of MHE patients, inhibit neuronal apoptosis and improve cognition, so that learning and memory ability is improved, and the application prospect is wide.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to the role of S-methyl-5-thioadenosine in neural remodeling and improving cognitive function. Background Technology

[0002] Hepatic encephalopathy (HE) is a brain dysfunction caused by acute or chronic liver dysfunction or portosystemic shunting. It is mainly manifested as neurological and mental abnormalities, cognitive and motor function changes [Rose CF, Amodio P, Bajaj JS, Dhiman RK, Montagnese S, Taylor-Robinson SD, Vilstrup H, Jalan R: Hepatic encephalopathy: Novel insights into classification, pathophysiology and therapy. J Hepatol 2020, 73(6):1526-1547]. Patients with liver disease are prone to various cognitive disorders [Sun T, DuH, Li Z, Xiong J, Liu Y, Li Y, Zhang W, Liang F, He J, Liu Y, Xiang H, Xiong J, Bao S: The Role of GutMicrobiota in Mice With Bile Duct Ligation-Evoked Cholestatic Liver Disease-Related Cognitive Dysfunction. Front Microbiol 2022,13:909461]. Even in healthy individuals, plasma transaminase levels are significantly negatively correlated with memory function [Kamada Y, Hashimoto R, Yamamori H, Yasuda Y, Takehara T, Fujita Y, Hashimoto K, Miyoshi E: Impact of plasma transaminase levels on the peripheral blood glutamate levels and memory functions in healthy subjects. BBA Clin 2016, 5: 101-107].Hepatic encephalopathy (HE) is a common and potentially disabling complication of chronic liver disease. It encompasses a range of neuropsychiatric abnormalities, from discrete impairment of executive function to deep coma. The development of HE affects patients' daily activities, reduces their quality of life, and has a significant negative impact on survival. Nevertheless, there remains no "gold standard" for diagnosing this syndrome. Because the cognitive impairments in HE are often not universal, the diagnosis of minimal hepatic encephalopathy (MHE) remains challenging. MHE is a very insidious stage in the pathogenesis of HE [American Association for the Study of Liver Diseases, European Association for the Study of the Liver. Hepatic encephalopathy in chronic liver disease: 2014 practice guideline by the European Association for the Study of the Liver and the American Association for the Study of Liver Diseases[J]. J Hepatol, 2014, 61(3):642-659.DOI:10.1016 / j.jhep.2014.05.042]. The incidence rate is not related to age, sex, smoking, or education level, but it is clearly related to Child-Pugh classification. The prevalence of MHE is 48% in patients with a MELD score ≥20. Therefore, the clinical focus is on screening for MHE in patients with end-stage liver diseases such as cirrhosis and providing early intervention and treatment.

[0003] S-methyl-5-thioadenosine (MTA) is a derivative of purine nucleosides. MTA is a byproduct of intracellular polyamine metabolism and participates in multiple metabolic regulation processes, including adenosine metabolism, methyl cycle, and sulfur cycle. Previous studies have found that 5'-methylthioadenosine inhibits tumor cell proliferation, invasion, and apoptosis, while also regulating the inflammatory microenvironment of tumor tissue, thereby exerting a tumor-inhibiting effect. It has a significant regulatory effect on tumorigenesis [Yaofeng Li, et al. 5'-Methylthioadenosine and Cancer: old molecules, new understanding. J Cancer. 2019; 10(4): 927-936.; Tang Y, et al. 5'-Methylthioadenosine attenuates ischemia reperfusion injury after liver transplantation in rats. Inflammation. 2014; 37(5): 1366-1373.].

[0004] Patent application number 202111091735.6 discloses an Alzheimer's disease biomarker, S-methyl-5'-thioadenosine, and its applications. The Alzheimer's disease biomarker is S-methyl-5'-thioadenosine. This invention is the first to detect that the level of S-methyl-5'-thioadenosine in fecal samples from Alzheimer's patients is significantly lower than that in normal fecal samples. Using S-methyl-5'-thioadenosine in feces as a biomarker for Alzheimer's disease can assist in the early diagnosis of Alzheimer's disease by detecting its level. It facilitates non-invasive and rapid detection and is timely, convenient, highly specific, and highly sensitive.

[0005] Patent application number 201080035593.5 relates to the use of MTA, its pharmaceutically acceptable salts, and / or prodrugs as active ingredients in the preparation of drugs for preventing or treating nerve cell death or damage, neuroprotective drugs, drugs for regenerating nerve cells, and drugs for preventing or treating neurological or mental illnesses. This invention also relates to methods for preventing or treating nerve cell death or damage, neuroprotective methods, methods for regenerating nerve cells, and methods for preventing or treating neurological or mental illnesses. However, the uses disclosed in the prior art are relatively limited, mainly focusing on the preparation of neuroprotective drugs. Summary of the Invention

[0006] The purpose of this invention is to provide a novel use for S-methyl-5-thioadenosine (MTA), specifically its application in the preparation of drugs for improving cognitive function in MHE patients. This application, through clinical data, animal experiments, and cell model studies, has found that MTA can effectively reduce neuroinflammatory responses in MHE patients, inhibit neuronal apoptosis, improve cognition, and thus enhance learning and memory abilities.

[0007] This invention discloses the use of S-methyl-5-thioadenosine, its pharmaceutically acceptable salt and / or its prodrug as an active ingredient in the preparation of drugs for the prevention and / or treatment of hepatic encephalopathy, MHE, and neuroinflammatory diseases.

[0008] The present invention also discloses the use of S-methyl-5-thioadenosine, the use of S-methyl-5-thioadenosine, its pharmaceutically acceptable salt and / or its prodrug as active ingredients in the preparation of medicaments for neuroprotection, neural remodeling and cognitive improvement.

[0009] Preferably, the neural remodeling is neural remodeling in inflammation-related neural injury.

[0010] The present invention also discloses a biomarker for hepatic encephalopathy, wherein the biomarker is S-methyl-5-thioadenosine, its pharmaceutically acceptable salt and / or its prodrug.

[0011] The present invention also discloses an MHE biomarker, wherein the MHE biomarker is S-methyl-5-thioadenosine, its pharmaceutically acceptable salt and / or its prodrug.

[0012] The present invention also discloses the application of the above-mentioned S-methyl-5-thioadenosine, its pharmaceutically acceptable salt and / or its prodrug as biomarkers in constructing early diagnostic models of hepatic encephalopathy and MHE and / or preparing early diagnostic devices for hepatic encephalopathy and MHE.

[0013] The present invention also discloses an early diagnostic model for hepatic encephalopathy and MHE, wherein the input variables of the early diagnostic model include the peak intensity value of S-methyl-5-thioadenosine mass spectrometry.

[0014] This invention discloses a novel use of S-methyl-5-thioadenosine (MTA), specifically its application in the preparation of a product for improving cognitive function in MHE patients. Through clinical data, animal experiments, and cell model studies, this application has found that MTA can effectively reduce neuroinflammatory responses in MHE patients, inhibit neuronal apoptosis, improve cognition, and thus enhance learning and memory abilities. Attached Figure Description

[0015] Figure 1 This is a comparison of MTA expression in healthy individuals and the MHE group in Example 1;

[0016] Figure 2 MTA was used as a biomarker to distinguish between MHE and healthy individuals.

[0017] Figure 3 MTT assay results for HT22 and SH-SY5Y cells;

[0018] Figure 4 Results of ROS detection in HT22 and SH-SY5Y cells;

[0019] Figure 5 Comparison of neuronal excitability in the CA1 region of the hippocampus in mouse brain slices under LPS stimulation and ACSF (artificial cerebrospinal fluid) treatment between the control group (Ctrl) and the MTA treatment group.

[0020] Figure 6 For MTA current testing;

[0021] Figure 7 The experimental results show the frequency variation of spontaneous excitatory postsynaptic currents (sEPSC) in neurons.

[0022] Figure 8 Behavioral trajectory experiment in rat novel object recognition;

[0023] Figure 9 To explore the time-based experiment in the rat novel object recognition experiment. Detailed Implementation

[0024] The following embodiments are further illustrations of the present invention and serve as explanations of the technical content of the present invention. However, the essence of the present invention is not limited to the embodiments described below. Those skilled in the art can and should know that any simple changes or substitutions based on the spirit of the present invention should fall within the protection scope claimed by the present invention.

[0025] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0026] Example 1

[0027] This invention provides a novel use for S-methyl-5-thioadenosine (MTA): its application in the preparation of medications for improving cognitive function in MHE patients. Through clinical data, animal experiments, and cell model studies, this invention has found that MTA can effectively reduce neuroinflammatory responses in MHE patients, inhibit neuronal apoptosis, improve cognition, and thus enhance learning and memory abilities.

[0028] Experimental methods:

[0029] 1. Clinical Data: Eighteen clinically diagnosed patients with myocardial hyperplasia (MHE) were collected, along with a healthy control group. Neuropsychological assessments were performed on both groups. Peripheral blood (2 ml) was collected, centrifuged, and plasma was used for non-targeted metabolomics sequencing. Results showed that MTA metabolites were significantly elevated in the healthy group compared to the MHE group. MTA can serve as a biomarker to distinguish between MHE and healthy individuals.

[0030] 2. Cell experiments: The experimental subjects were primary cultured neurons or neuron-like cells, which were randomly divided into the following four groups:

[0031] (1) Control group;

[0032] (2) LPS treatment group (inducing inflammatory damage);

[0033] (3) MTA pretreatment group (MTA added separately);

[0034] (4) MTA+LPS group (LPS stimulation was given after MTA pretreatment).

[0035] 2.1 Cell viability assay (MTT)

[0036] Cells from each group were seeded in 96-well plates and treated for 24 hours according to their respective groups. MTT reagent was added to each well, and after incubation, DMSO was added to dissolve the cells. The absorbance at 570 nm was measured to assess cell viability.

[0037] 2.2 Reactive Oxygen Species Detection (ROS)

[0038] After treatment, the cells in each group were incubated with the DCFH-DA fluorescent probe for 30 minutes, washed with PBS, and the fluorescence intensity was measured to reflect the intracellular ROS apoptosis level.

[0039] 2.3 Electrophysiological recording of ex vivo brain slices (patch clamp)

[0040] The electrophysiological characteristics of neurons in each group were recorded using whole-cell patch-clamp technique. Action potentials were induced in current-clamp mode to measure excitability changes; Na+ was recorded in voltage-clamp mode. + and K +Current was used to assess ion channel function; spontaneous excitatory postsynaptic currents (sEPSCs) were recorded by fixing membrane potentials to analyze changes in synaptic activity. The electrophysiological data acquisition system was a standard patch-clamp workstation with uniform operating conditions, and the data were processed using specialized software.

[0041] 3. Animal experiments: The animals were divided into three groups for new object recognition behavioral experiments:

[0042] (1) Control group;

[0043] (2) Intraperitoneal injection of LPS treatment group (inducing inflammatory damage);

[0044] (3) MTA+LPS group (LPS stimulation was given after MTA pretreatment).

[0045] Experimental Results: The results of the neuropsychological scale assessment are shown in Table 1.

[0046] Table 1

[0047]

[0048] Table 1 shows the differences in performance on a series of neuropsychological scales between patients with mild hepatic encephalopathy (MHE) and healthy controls. All data are expressed as mean ± standard deviation (mean ± SD), and values ​​marked with an asterisk (*) indicate statistically significant differences compared to the MHE group (p < 0.05).

[0049] In the Digit Span (DS) test, the MHE group scored significantly lower than the healthy group (25.0±9.9 vs. 42.2±10.7, p<0.05), indicating impaired attention and working memory. In the Number Connection Test-A and B (NCT-A / B), the MHE group took significantly longer to complete the tasks than the healthy group (67.4±25.3 seconds and 213.2±116.6 seconds, respectively), while the healthy group took 37.1±13.7 seconds and 98.7±55.5 seconds (both p<0.05), reflecting a decline in processing speed and executive function.

[0050] Similarly, the Symbol Digit (SD) test and Line Tracing Test (LTT) results also showed that the MHE group had significant disadvantages in visual-motor coordination and reaction speed (SD: 77.3±21.3 vs. 54.6±17.2, LTT: 117.4±43.4 vs. 82.2±35.1, both p<0.05). In addition, the total PHES score of the MHE group was also significantly lower than that of the healthy group (-2.4±0.6 vs. -0.7±0.8), indicating that their overall cognitive function was significantly impaired.

[0051] The above findings indicate that MHE patients exhibit significant deficits across multiple cognitive dimensions, including attention, executive function, reaction speed, and visual-motor coordination. The PHES (Psychometric Hepatic Encephalopathy Score) is a composite score comprised of the five sub-tests: NCT-A, NCT-B, LTT, SD, and DS. The significance of the score: Normal individuals score 0 or higher; a more negative score indicates greater severity of cognitive impairment. The average score for MHE patients was -2.4 ± 0.6, while that for healthy individuals was only -0.7 ± 0.8, demonstrating a statistically significant difference.

[0052] The expression of MTA in healthy individuals and the MHE group was compared, for example... Figure 1 As shown, the results indicate that MTA expression is significantly elevated in healthy individuals. Figure 1 This represents a violin plot illustrating the abundance differences of MTA metabolites between groups in metabolomics. The horizontal axis represents the different groups, and the vertical axis represents the range of metabolite signal values. Significant differences between groups are expressed using t-tests or p-values ​​calculated from analysis of variance. In the figure, ****, **, **, and * represent p < 0.0001, 0.001, 0.01, and 0.05, respectively.

[0053] MTA is used as a biomarker to distinguish MHE from healthy individuals, such as Figure 2 As shown, MTA can be used as a biomarker to distinguish between MHE and healthy individuals, with an AUC value of 0.793.

[0054] The results of MTT assay for HT22 and SH-SY5Y cells are as follows: Figure 3 As shown, 1 mM LPS significantly reduced cell viability in HT22 and SH-SY5Y cells, suggesting a significant neurotoxic effect. 100 μM MTA alone had no significant effect on cell viability. Co-treatment with MTA after LPS induction significantly increased cell viability, approaching the control group level, indicating that MTA has a neuroprotective effect.

[0055] Figure 4For ROS detection results, LPS treatment significantly increased ROS levels in HT22 and SH-SY5Y cells, suggesting that it induced a significant oxidative stress response. MTA co-treatment significantly reduced LPS-induced ROS levels, indicating its anti-oxidative stress function. The results in this section show that S-methyl-5-thioadenosine (MTA) can effectively alleviate LPS-induced neurotoxicity and oxidative stress, exhibiting good neuroprotective effects in both mouse and human neural cells. This suggests that MTA, as a natural metabolite, has potential neuroprotective and anti-inflammatory therapeutic value.

[0056] like Figure 5 As shown, the comparison of neuronal excitability in the CA1 region of the hippocampus in mouse brain slices under LPS stimulation and ACSF (artificial cerebrospinal fluid) treatment is illustrated in the control group (Ctrl) and the MTA treatment group, respectively. The LPS group (red line) showed a significantly higher number of action potentials fired at medium-to-high current injection levels than the ACSF group, indicating that LPS enhances neuronal excitability, making it easier to generate action potentials. This suggests that neurons are more prone to overactivation under inflammatory conditions, possibly related to neurotoxicity or synaptic damage. MTA treatment significantly inhibited LPS-induced neuronal excitability toxicity. MTA has the effect of resisting or reversing inflammation-induced neuronal overexcitation, demonstrating potential neural remodeling function. These patch-clamp experimental results indicate that LPS treatment enhances neuronal excitability, manifested as more action potentials fired; MTA pretreatment can significantly inhibit this enhancement of excitability, exerting a protective effect; supporting the therapeutic potential of MTA in neural remodeling, possibly by modulating the excitability-inhibition balance to reduce neurotoxicity.

[0057] like Figure 6 As shown, MTA enhances the repolarization current after the action potential by stimulating K+ current channels, thereby increasing the efficiency of intersynaptic potential transmission. MTA also indirectly promotes synaptic transmission stability by stabilizing sodium channel function, thus playing a role in neural remodeling in inflammation-related neurological injuries.

[0058] like Figure 7As shown, to evaluate the regulatory effect of MTA on LPS-induced neurotoxicity at the synaptic level, this example examined the frequency changes of spontaneous excitatory postsynaptic currents (sEPSCs) in neurons. In the control group, LPS treatment led to a significant increase in sEPSC frequency, suggesting that it induced increased presynaptic glutamate release and enhanced synaptic excitability. This abnormal enhancement of synaptic activity may reflect excitotoxicity under inflammatory stimulation, easily leading to neuronal over-discharge and calcium overload, ultimately resulting in neuronal injury. In the MTA pretreatment group, LPS failed to induce an increase in sEPSC frequency, indicating that MTA can effectively inhibit LPS-induced enhancement of synaptic excitability, thereby alleviating synaptic dysfunction and potential neurotoxic responses. This result further supports the multidimensional protective role of MTA in inflammation-related neurological injury, especially its potential in regulating synaptic excitability and preventing neuronal overactivation.

[0059] like Figure 8 The behavioral trajectories of rats in the new object recognition experiment are used to assess cognitive function and the effects of intervention drugs: The movement trajectories of rats in each group during the new object recognition experiment are shown. Black lines represent exploration paths, green circles represent the locations of familiar objects, and blue boxes represent the locations of new objects. The left image is the Sham group, the middle image is the LPS treatment group, and the right image is the LPS+MTA intervention group. The LPS group showed a significant reduction in activity and a decreased willingness to explore new objects.

[0060] like Figure 9 The figure shows the exploration time of rats in each group during the new object recognition experiment. Compared with the Sham group, the LPS group had a significantly reduced exploration time for new objects (P<0.01), indicating a decline in cognitive ability; MTA intervention significantly improved exploration time (P<0.01), indicating that it has a role in improving cognitive function.

[0061] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. The use of S-methyl-5-thioadenosine, characterized in that, The use of the S-methyl-5-thioadenosine, its pharmaceutically acceptable salt and / or its prodrug as an active ingredient in the preparation of drugs for the prevention and / or treatment of hepatic encephalopathy, MHE, and neuroinflammatory diseases.

2. The use of S-methyl-5-thioadenosine, characterized in that, Use of the S-methyl-5-thioadenosine, its pharmaceutically acceptable salt and / or its prodrug as an active ingredient in the preparation of medicaments for neuroprotection, neural remodeling and cognitive improvement.

3. The use of S-methyl-5-thioadenosine, characterized in that, The neural remodeling refers to neural remodeling in inflammation-related nerve damage.

4. A biomarker for hepatic encephalopathy, characterized in that, The biomarker for hepatic encephalopathy is S-methyl-5-thioadenosine, its pharmaceutically acceptable salt, and / or its prodrug.

5. An MHE biomarker, characterized in that, The MHE biomarker is S-methyl-5-thioadenosine, its pharmaceutically acceptable salt, and / or its prodrug.

6. The use of S-methyl-5-thioadenosine as described in claim 4 or 5, its pharmaceutically acceptable salt and / or its prodrug as a biomarker in constructing early diagnostic models of hepatic encephalopathy and MHE and / or preparing early diagnostic devices for hepatic encephalopathy and MHE.

7. An early diagnostic model for hepatic encephalopathy and MHE, characterized in that, The input variables for the early diagnostic model include the peak intensity values ​​of the S-methyl-5-thioadenosine mass spectrum.

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