Novel probiotics and analgesic effect thereof on neuropathic pain

Abstract

The present invention relates to the therapeutic intervention of chronic pain, in particular to the treatment of chronic pain using selected probiotic strains having analgesic properties, in particular three probiotics, Roseburia intestinae, Lacnospiraceae bacteria and Roseburia hominis, and a sterile secretion filtrate of Roseburia intestinae, which when administered in sufficient doses exhibit significant analgesic properties in the sciatic nerve branch selective injury (SNI) model.

Description

Field of the Invention

[0001] The present invention relates to the fields of therapeutic agents and probiotics for treating chronic pain. More specifically, it involves the use of specific probiotic strains, particularly three probiotics—Roseburia intestinalis, Lachnospiraceaebacterium, and Roseburia hominis. These strains demonstrate significant analgesic effects in an animal model of neuropathic pain caused by selective sciatic nerve branch injury (SNI), offering a potential new therapeutic avenue for patients with chronic pain. Background Art

[0002] Chronic pain management is a major public health concern worldwide. Neuropathic pain is a common chronic pain condition that affects up to 10% of the global population. Neuropathic pain is caused by damage to the peripheral or central nerves and is observed in a variety of clinical conditions, such as trigeminal neuralgia, diabetic polyneuropathy, postherpetic neuralgia, radiculopathy, spinal cord injury, stroke, and multiple sclerosis. Neuropathic pain is also common in cancer patients because tumor compression or treatments (such as chemotherapy, radiation injury, or surgery) can cause pain.

[0003] In addition to neuropathic pain, patients with arthritis and fibromyalgia also suffer from chronic pain syndromes. Chronic pain is often accompanied by a range of comorbidities, such as sleep disturbances, depression, and anxiety, leading to poor work performance, high absenteeism, and social difficulties. Therefore, chronic pain is a recognized risk factor that can significantly affect the quality of life of patients, their families, and caregivers.

[0004] Chronic pain is a difficult and complex pathological condition whose management presents major challenges. Existing therapeutic interventions often have limited effectiveness and significant side effects. In clinical practice, anticonvulsants (such as carbamazepine and gabapentin), antidepressants (such as amitriptyline and duloxetine), or opioids are often prescribed for treatment. However, these drugs are only effective in less than 40% of patients with certain types of neuropathic pain (such as postherpetic neuralgia and diabetic neuropathy). In addition, due to their neurological side effects and addictive potential, these drugs are strictly regulated and difficult for patients to obtain. Therefore, further research is urgently needed to discover new and more accessible analgesics.

[0005] Emerging research highlights the gut-brain axis—the bidirectional communication channel between the gastrointestinal tract and the central nervous system—as a potential target for novel therapeutic interventions. Probiotics are live microorganisms that can promote health. In preclinical and clinical studies, probiotic use may improve symptoms of neurological disorders such as autism, anxiety, and depression. These studies suggest that probiotic supplementation has potential in treating neurological diseases.

[0006] The medical field has recognized the need for more effective and safer alternative treatments for chronic pain. Therefore, there is a need to develop new probiotic compositions and methods of use to meet the needs of the medical industry. Summary of the Invention

[0007] The present invention provides a therapeutic composition and method for preventing or treating chronic pain. By using metagenomic sequencing technology to analyze fecal samples from healthy individuals and patients with chronic pain such as herpes zoster or postherpetic neuralgia, the present invention identifies specific probiotic strains that are more abundant in healthy individuals. The present invention overcomes the limitations of the prior art by introducing specific probiotic strains that show analgesic effects in the sciatic nerve branch selective injury (SNI) neuralgia model. The abundance of these probiotic strains in healthy individuals is higher than that in patients with neuropathic pain, providing an innovative method for treating chronic pain by regulating the gut-brain axis.

[0008] The advantage of this invention is that it provides a safer, more natural alternative or complementary therapy, reducing reliance on traditional drug treatments and their associated side effects. By specifically targeting the gut-brain axis, this new approach may address an underlying mechanism of chronic pain, providing a more effective and targeted treatment strategy.

[0009] The present invention relates to therapeutic interventions for chronic pain, particularly the use of specific probiotic strains with analgesic properties to treat chronic pain. When administered at adequate doses, these strains exhibit significant analgesic properties. In certain embodiments, the probiotic strains are identified by metagenomic sequencing based on their individual and shared analgesic properties.

[0010] In certain embodiments, the present invention provides a composition for treating chronic pain, the composition comprising a plurality of probiotic strains, wherein each probiotic strain is more abundant in healthy individuals than in pain patients. In a preferred embodiment, the probiotic strain is selected from the following group of strains: Anaerostipes hadrus, Clostridium sp. SY8519, Anaerobutyricum hallii, Flintibacters sp. KGMB00164, Romboutsia hominis, Longibaculum sp. KGMB06250, Alistipes megaguti, Veillonella dispar, Lachnospiraceae bacterium Choco86, Faecalibacterium prausnitzii, Erysipelotrichaceae bacterium GAM147, Streptococcus dysgalactiae, Haemophilusparainfluenzae, Roseburia hominis, Ruminococcus champanellensis, Lachnospiraceae bacterium KM106.2, Lachnospiraceae bacterium, Roseburiaintestinalis, Muribaculaceae bacterium DSM 108610, Lachnospiraceae bacteriumGAM79, Prevotella intermedia, Prevotella scopos, Prevotellafusca, Lachnospiraeligens and Prevotella melaninogenica.

[0011] More specifically, three strains—Roseburia intestinalis, Lachnospiraceae bacteria, and Roseburia hominis—exhibited significant analgesic properties in a sciatic nerve branch selective injury (SNI) model of neuropathic pain. Mechanistically, the present invention reveals that administration of Roseburia intestinalis exerts its effects through the gut-brain axis, potentially modulating activity in the vagal ganglion and multiple brain regions.

[0012] In certain embodiments, the probiotic strain is selected from Roseburia intestinalis, Lachnospiraceaebacterium, and Roseburia hominis. These probiotic strains have analgesic properties and are capable of modulating communication along the gut-brain axis, thereby producing an analgesic effect. In certain embodiments, the compositions of the present invention are formulated for oral administration as capsules, tablets, powders, liquids, or gels.

[0013] The present invention provides a method for managing and treating chronic pain by using various probiotic strains that are more abundant in healthy individuals than in those with neuropathic pain. The invention also encompasses the fields of neurology and pain management, as it utilizes the gut-brain axis to modulate pain signaling, thereby alleviating chronic pain.

[0014] In certain embodiments, the present invention provides a method for preventing or treating chronic pain in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a plurality of probiotic strains that are more abundant in healthy subjects than in pain patients, or administering a therapeutically effective amount of a composition comprising a plurality of probiotic strains. In certain embodiments, the administering step is performed orally, topically, or by any other suitable route of administration.

[0015] In certain embodiments, the plurality of probiotic strains that are more abundant in healthy subjects compared to pain patients include: Anaerostipes hadrus, Clostridium sp.SY8519, Anaerobutyricum hallii, Flintibacter sp.KGMB00164, Romboutsia hominis, Longibaculum sp.KGMB06250, Alistipes megaguti, Veillonella dispar, Lachnospiraceae bacterium Choco86, Faecalibacterium prausnitzii, Erysipelotrichaceae bacterium GAM147, Streptococcus dysgalactiae, Haemophilus parainfluenzae, Roseburia hominis, Ruminococcus champanellensis, Lachnospiraceae bacterium KM106.2, Lachnospiraceae bacterium, and intestinalis, Muribaculaceae bacterium DSM108610, Lachnospiraceae bacterium GAM79, Prevotella intermedia, Prevotella scopos, Prevotellafusca, Lachnospira eligens, Prevotella melaninogenica, or any combination thereof. In a specific embodiment, the probiotic strain is selected from Roseburia intestinalis, Lachnospiraceae bacterium, and Roseburia hominis.

[0016] In certain embodiments, the method further comprises diagnosing the subject to determine whether the subject is deficient in or unbalanced with one or more probiotic strains in the composition. In certain embodiments, the method further comprises assessing the abundance of the one or more probiotic strains in the subject before and / or after the administering step.

[0017] In certain embodiments, the administering step results in an increase in the abundance of one or more probiotic strains in the subject, thereby alleviating symptoms of chronic pain.

[0018] In certain embodiments, the effectiveness of treatment is assessed by measuring the abundance of the probiotic strain in the subject and its pain behavior before and after administration.

[0019] In certain embodiments, the method is combined with existing chronic pain treatments including acupuncture, acetaminophen, exercise, physical therapy, breathing exercises, psychotherapy, stress reduction, and / or trigger point injections.

[0020] In certain embodiments, the chronic pain comprises pain caused by selective sciatic nerve branch injury (SNI) neuropathic pain.

[0021] In certain embodiments, the subject has been diagnosed with herpes zoster and / or postherpetic neuralgia (PHN).

[0022] In certain embodiments, the present invention provides a method for preventing or treating chronic pain in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a composition comprising Roseburia intestinalis, Lachnospiraceaebacterium and / or Roseburia hominis, or a sterile secretory filtrate of Roseburia intestinalis.

[0023] In certain embodiments, the present invention provides a method for preventing or treating chronic pain in a subject in need thereof, comprising administering to the subject an effective amount of a composition according to the present invention, wherein the subject is in a sciatic nerve branch selective injury (SNI) neuropathic pain model. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figures 1A-1F Alterations in the composition of the gut microbiota in patients with herpes zoster and postherpetic neuralgia. Figure 1A ) Metagenomic sequencing was used to analyze stool samples from healthy individuals (healthy group), patients with herpes zoster (herpes zoster group), and patients with postherpetic neuralgia (PHN group), and Shannon α diversity was measured to assess species richness. (Healthy group n = 63, herpes zoster group n = 115, PHN group n = 28; healthy group vs herpes zoster group P = 0.011, healthy group vs PHN group P = 0.0051). Figure 1B ) Based on the Bray-Curtis dissimilarity matrix, principal coordinates analysis (PCoA) was used to assess beta diversity in stool samples from healthy individuals, patients with herpes zoster, and patients with postherpetic neuralgia. (Healthy group n = 63, herpes zoster group n = 115, postherpetic neuralgia group n = 28; PERMANOVA test P = 0.001). Figure 1C) Wilcoxon rank sum test bar chart of intestinal microbiota in healthy individuals and herpes zoster patients. ( Figure 1D ) LEfSe analysis was used to identify differences in the gut microbiota between healthy individuals and patients with herpes zoster. Figure 1E ) Wilcoxon rank sum test bar chart of gut microbiota in healthy individuals and patients with postherpetic neuralgia. ( Figure 1F ) Identification of differences in the gut microbiota between healthy individuals and patients with postherpetic neuralgia by LEfSe analysis.

[0025] Figures 2A-2F Analgesic effect of Roseburia intestinalis supplementation on chronic pain induced by SNI surgery. Figures 2A-2C ) Paw withdrawal threshold (PWT) to von Frey filament stimulation, response rate to pinprick stimulation, and paw withdrawal latency (PWL) to acetone stimulation in male mice. ( Figures 2D-2F ) Paw withdrawal threshold (PWT) to von Frey filament stimulation, response rate to pinprick stimulation, and paw withdrawal latency (PWL) to acetone stimulation in female mice. * or # P < 0.05; ** or ## P < 0.01; *** or ### P < 0.001. Statistical significance was tested by repeated-measures analysis of variance (ANOVA) with pairwise comparisons. Asterisks indicate statistically significant differences between the SNI + medium group and the SNI + RI group. Pound signs indicate statistically significant differences between the sham-operated group and the SNI + medium group. Error bars represent standard error of the mean (SEM).

[0026] Figures 3A-3D Roseburia intestinalis supplementation enhanced the neural activity of the prefrontal cortex (PrL), dentate gyrus (DG), bed nucleus of the striatum terminalis (BNST), and nucleus tractus solitarius (NTS) in the chronic pain animal model. In the SNI+medium group (SNI+md, n=6) and SNI+Roseburia intestinalis group (SNI+RI, n=6) mice, PrL ( Figure 3A )、DG( Figure 3B )、BNST( Figure 3C ) and NTS( Figure 3D ) c-Fos expression levels in . *p<0.05, **p<0.01, Student's t-test, mean ± SEM.

[0027] Figures 4A-4CRoseburia intestinalis supplementation reduced the neural excitability of the agranular insular cortex (AI), paraventricular nucleus (PVN) of the hypothalamus, and central amygdala (CeA) in a chronic pain animal model. In the SNI+medium group (SNI+md, n=6) and SNI+Roseburia intestinalis group (SNI+RI, n=6) mice, AI ( Figure 4A )、PVN( Figure 4B ) and CeA( Figure 4C ) c-Fos expression levels in . *p<0.05, **p<0.01, Student's t-test, mean ± SEM.

[0028] Figures 5A-5F Vagotomy, but not chemical sympathectomy, reversed the analgesic-like effects of Roseburia intestinalis supplementation. Figures 5A-5C ) showed that the analgesic effect of Roseburia intestinalis supplementation on neuropathic pain depends on the integrity of the vagus nerve. ( Figure 5D ) Paw withdrawal threshold (PWT) to von Frey filament stimulation; ( Figure 5E )Response rate to acupuncture stimulation; Figure 5F ) Paw withdrawal latency (PWL) to acetone stimulation. ( Figures 5E-5F Effects of 6-hydroxydopamine-induced chemical sympathectomy on the analgesic efficacy of supplementary drugs in Roseburia intestinalis. Figure 5A ) Paw withdrawal threshold (PWT) to von Frey filament stimulation; ( Figure 5B )Response rate to acupuncture stimulation; ( Figure 5C ) Acetone-stimulated paw withdrawal latency (PWL). * or # P < 0.05; ** or ## P < 0.01; *** or ### P < 0.001. Statistical significance was tested by repeated-measures analysis of variance (ANOVA) with pairwise comparisons. Asterisks indicate statistically significant differences between the SNI + medium group and the SNI + RI group. Pound signs indicate statistically significant differences between the sham-operated group and the SNI + medium group. Error bars represent standard error of the mean (SEM).

[0029] Figures 6A-6G Structural projections of the vagus nerve-NTS-CeA pathway. Figure 6A )PRV virus was injected into the CeA; the image shows PRV virus expression at the injection site. ( Figure 6B )NTS projects signals to CeA. Figure 6C )HSV virus was injected into the NTS; the image shows PRV virus expression at the injection site. ( Figure 6D)NTS projects signals to CeA. Figure 6E )PRV virus was injected into the NTS; the image shows PRV virus expression at the injection site. ( Figure 6F )Vagus nerve projection signals to the NTS. ( Figure 6G ) Schematic diagram of the structural projections of the vagus nerve-NTS-CeA pathway.

[0030] Figures 7A-7H Chemical genetic silencing of CeA-projecting NTS neurons reversed the analgesic effect of Roseburia intestinalis supplementation. Figure 7A ) Schematic diagram of virus injection for unilateral NTS chemical genetic inhibition (200 nL virus mixture: AAV2 / 9-EF1a-DIO-hM4D(Gi)-mCherry-WPREs and rAAV-hSyn-Cre-WPRE-hGH-pA = 1:1). ( Figure 7B ) Paw withdrawal threshold (PWT) to von Frey filament stimulation. ( Figure 7C )Response rate to acupuncture stimulation. ( Figure 7D ) Paw withdrawal latency (PWL) to acetone stimulation. ( Figure 7E ) Schematic diagram of NTS injection of virus (rAAV-EF1a-DIO-hM4D(Gi)-mCherry-WPREs, AAV9) and unilateral CeA injection of virus (rAAV-hSyn-Cre-WPRE-hGH retroAAV). ( Figure 7F ) Paw withdrawal threshold (PWT) to von Frey filament stimulation. ( Figure 7G )Response rate to acupuncture stimulation. ( Figure 7H ) Acetone-stimulated paw withdrawal latency (PWL). * or # or & P < 0.05; ** or ## or && P < 0.01; *** or ### or &&& P < 0.001. Statistical significance was determined by repeated-measures analysis of variance (ANOVA) with pairwise comparisons. Asterisks indicate statistically significant differences between the SNI + medium group and the SNI + RI group. Pounds indicate statistically significant differences between the sham-operated group and the SNI + medium group. & symbols indicate statistically significant differences between the sham-operated group and the SNI + medium group. Error bars represent standard error of the mean (SEM).

[0031] Figures 8A-8D Chemogenetic activation of NTS-projecting CeA neurons reversed the analgesic effect of Roseburia intestinalis supplementation. Figure 8A) Schematic diagram of NTS injection virus (rAAV-hSyn-Cre-WPRE-Hgh, AAV1) and CeA injection virus (rAAV-EF1a-DIO-hM3D(Gq)-mCherry-WPREs, AAV9). ( Figure 8B ) Paw withdrawal threshold (PWT) to von Frey filament stimulation. ( Figure 8C )Response rate to acupuncture stimulation. ( Figure 8D ) Acetone-stimulated paw withdrawal latency. * or # or & P < 0.05; ** or ## or && P < 0.01; *** or ### or &&& P < 0.001. Statistical significance was determined by repeated-measures analysis of variance (ANOVA) with pairwise comparisons. Asterisks indicate statistically significant differences between the SNI + medium group and the SNI + RI group. Pounds indicate statistically significant differences between the sham-operated group and the SNI + medium group. & symbols indicate statistically significant differences between the sham-operated group and the SNI + medium group. Error bars represent standard error of the mean (SEM).

[0032] Figures 9A-9D Analgesic effect of Roseburia hominis supplementation on chronic pain induced by SNI surgery. Figure 9A ) Paw withdrawal threshold (PWT) to von Frey filament stimulation. ( Figure 9B ) Paw withdrawal latency (PWL) to acetone stimulation. ( Figures 9C-9D ) Acupuncture response rate and hyperalgesia score. **P < 0.01, ***P < 0.001. Statistical significance was tested by repeated-measures analysis of variance (ANOVA) with pairwise comparisons. Error bars represent standard error of the mean (SEM).

[0033] Figures 10A-10D Analgesic effect of Lachnospiraceae bacteria supplementation on chronic pain induced by SNI surgery. Figure 10A ) Paw withdrawal threshold (PWT) to von Frey filament stimulation. ( Figure 10B ) Paw withdrawal latency (PWL) to acetone stimulation. ( Figures 10C-10D ) Acupuncture response rate and hyperalgesia score. **P < 0.01, ***P < 0.001. Statistical significance was tested by repeated-measures analysis of variance (ANOVA) with pairwise comparisons. Error bars represent standard error of the mean (SEM).

[0034] Figure 11 Effects of Roseburia intestinalis sterile secretion filtrate on the pain response of SNI mice to different pain stimuli (Von Frey, Pinkprick, Acetone). DETAILED DESCRIPTION

[0035] The present invention provides a novel therapeutic composition and method for preventing or treating chronic pain. Specifically, by using metagenomic sequencing technology to analyze stool samples from healthy individuals and patients with chronic pain conditions such as herpes zoster or postherpetic neuralgia (PHN), specific probiotic strains that are more abundant in healthy individuals were identified.

[0036] In certain embodiments, the probiotic strains provided herein are more abundant in healthy individuals than in chronic pain patients. In specific embodiments, these probiotic strains include: Anaerostipes hadrus, Clostridium sp. SY8519, Anaerobutyricum hallii, Flintibacters sp. KGMB00164, Romboutsia hominis, Longibaculum sp. KGMB06250, Alistipes megaguti, Veillonella dispar, Lachnospiraceae bacterium Choco86, Faecalibacterium prausnitzii, Erysipelotrichaceae bacterium GAM147, Streptococcus dysgalactiae, Haemophilusparainfluenzae, Roseburia hominis, Ruminococcus champanellensis, Lachnospiraceae bacterium KM106.2, Lachnospiraceae bacterium, Roseburiaintestinalis, Muribaculaceae bacterium DSM 108610, Lachnospiraceae bacteriumGAM79, Prevotella intermedia, Prevotella scopos, Prevotellafusca, Lachnospiraeligens and Prevotella melaninogenica.

[0037] In certain embodiments, three strains—Roseburia intestinalis, Lachnospiraceaebacterium, and Roseburia hominis—as well as the sterile secretion filtrate of Roseburia intestinalis, demonstrated significant analgesic effects in animal models of chronic pain. These probiotic strains are more abundant in healthy individuals than in patients with neuropathic pain, providing an innovative, safe, and effective approach to treating chronic pain by modulating the gut-brain axis.

[0038] In certain embodiments, the present invention provides a therapeutic composition comprising a probiotic strain identified herein. In certain embodiments, the present invention provides a method of treating chronic pain by administering a therapeutically effective amount of a probiotic strain identified herein, a composition of the present invention, or a sterile secretion filtrate of Roseburia intestinalis.

[0039] The present invention overcomes the limitations of existing technologies by introducing a specific probiotic strain that demonstrates analgesic effects in a model of neuropathic pain caused by selective sciatic nerve branch injury (SNI). The present invention offers the advantage of providing a safer, more natural alternative or adjunctive therapy, reducing reliance on traditional medications and their associated side effects. By specifically targeting the gut-brain axis, this novel probiotic strain may address a potential mechanism underlying chronic pain, providing a more effective and targeted treatment strategy.

[0040] In certain embodiments, the present invention utilizes the power of metagenomic sequencing technology to identify specific probiotic strains with analgesic properties. By analyzing stool samples from healthy individuals and patients with PHN, the present invention identified probiotic strains that are more abundant in healthy individuals.

[0041] In certain embodiments, the present invention provides a novel approach to chronic pain management by utilizing specific probiotic strains identified using metagenomic sequencing technology. For example, stool samples are collected from healthy individuals and patients with herpes zoster or postherpetic neuralgia. These samples are then subjected to metagenomic sequencing, which enables the identification and quantification of all bacterial species present in the gut microbiota in each sample group. After sequencing is complete, the data are compared and analyzed. Significant differences in the abundance of certain bacteria were found between healthy individuals and patients. This difference suggests a possible correlation between these bacteria and the manifestations of herpes zoster or postherpetic neuralgia.

[0042] Among them, three specific bacterial strains were discovered that not only showed significant abundance differences between healthy and diseased states, but also exhibited analgesic effects in the sciatic nerve branch selective injury (SNI) neuropathic pain model.

[0043] To uncover the mechanisms underlying the analgesic effects of these bacteria, researchers investigated the vagus nerve-brain pathway, which plays a key role in the analgesic effects of these bacteria. To validate these findings, they manipulated this pathway using various methods, including chemical sympathectomy, vagotomy, and in vivo chemogenetic manipulations, and observed corresponding changes in analgesia, establishing a direct link.

[0044] In certain embodiments, the methods of the present invention can be used as a supplement to existing chronic pain treatment options, such as acupuncture, acetaminophen, exercise, physical therapy, breathing exercises, psychotherapy, stress reduction, and trigger point injections.

[0045] Selected Definitions

[0046] As used herein, the singular forms "a," "an," and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise. Furthermore, the terms "include," "comprising," "having," "with," or variations thereof, when used in the detailed description and / or claims, are intended to be inclusive in a manner similar to the word "comprising." The transitional terms / phrases (and any grammatical variations thereof) "comprise," "comprising," and "including" may be used interchangeably.

[0047] "Consisting essentially of" or "consisting essentially of" means that the claim covers embodiments that include the specified materials or steps, as well as embodiments that do not materially affect the basic and novel characteristics of the claim.

[0048] The transitional terms "comprising," "including," or "including" are inclusive or open-ended and do not exclude additional elements or method steps not recited. In contrast, the transitional phrase "consisting of" excludes any element, step, or ingredient not specified in the claim. The term "consisting of" or "consisting essentially of" indicates that the claim covers embodiments that include the specified materials or steps, as well as those that do not materially affect the basic and novel characteristics of the claim. Other "consisting of" or "consisting essentially of" embodiments are also contemplated by the use of the word "comprising."

[0049] The term "about" or "approximately" indicates an acceptable range of error for a particular value as determined by one of ordinary skill in the art, which depends to some extent on the manner in which the measurement is made, i.e., the limitations of the measurement system. In the context of using the word "about" to describe compositions containing an amount of an ingredient, these compositions contain the stated amount of the ingredient with a variation (error range) of ±10% (X±10%) of that value. In other contexts, the word "about" indicates a variation (error range) of ±10% (X±10%) around a given value. Obviously, this range of variation means a range of up to 10% above or below a given value, for example, X±1%, X±2%, X±3%, X±4%, X±5%, X±6%, X±7%, X±8%, X±9%, or X±10%.

[0050] In the present disclosure, scope is described in abbreviated form to avoid lengthily listing each value in the scope. Where appropriate, any appropriate value in the scope can be chosen as the endpoint of upper limit, lower limit or scope. For example, scope 0.1-1.0 represents endpoint values ​​0.1 and 1.0, and intermediate value 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, also includes all intermediate ranges within the 0.1-1.0 scope, such as 0.2-0.5, 0.2-0.8, 0.7-1.0 etc. The value within the scope has at least two significant figures, such as, scope 5-10 represents all values ​​between 5.0 to 10.0 and 5.00 to 10.00, including endpoint values. When using scope in this article, clearly include the combination and subcombination of scope (such as, the subrange in the open range) and specific embodiment wherein.

[0051] "Decrease" refers to a negative change of at least 1%, 5%, 10%, 25%, 50%, 75%, or 100%.

[0052] By "increase" is meant a positive change of at least 1%, 5%, 10%, 25%, 50%, 75%, or 100%.

[0053] As used herein, a "subject" refers to an animal in need of or desiring to receive the benefits provided by a therapeutic compound. The animal can be a human, pig, horse, goat, cat, mouse, rat, dog, ape, fish, chimpanzee, gorilla, guinea pig, hamster, cow, sheep, bird, chicken, or any other vertebrate or invertebrate. These benefits include, but are not limited to, treating a health condition, disease, or disorder; preventing a health condition, disease, or disorder; enhancing immune health; or enhancing the function of a bodily organ, tissue, or system. In the context of the present invention, the preferred subject is a human. The subject can be of any age or developmental stage, including infants, toddlers, adolescents, teenagers, adults, or the elderly.

[0054] As used herein, "therapeutically effective amount," "therapeutically effective dose," "effective amount," and "effective dose" are used to refer to an amount or dose of a compound or composition that, when administered to a subject, is capable of treating or ameliorating a condition, disease, or disorder in the subject, or of enhancing the health or function of an organ, tissue, or body system. In other words, the amount is "therapeutically effective" when administered to a subject. The actual amount will vary depending on a variety of factors, including, but not limited to: the specific condition, disease, or disorder being treated or ameliorated; the severity of the condition; the specific organ, tissue, or body system whose health or function is to be enhanced; the patient's weight, height, age, and health; and the route of administration.

[0055] "Treatment" or "treatment" (and grammatical variations of these terms) as used herein refers to an approach for obtaining beneficial or desired results, including but not limited to a therapeutic effect. A therapeutic effect is achieved when one or more physiological symptoms associated with an underlying condition are observed to be eliminated or improved in a subject, even though the subject may still be suffering from the underlying condition.

[0056] As used herein, the term "treat" means to eliminate, reduce, ameliorate, or reverse to any extent the signs or symptoms of a health condition, disease, or disorder, including, but not limited to, complete cure of the condition, disease, or disorder. Treatment can be a cure, amelioration, or partial alleviation of a disorder. "Treatment" can also include improving or enhancing a condition or characteristic, for example, by achieving a healthier or more balanced function of a particular body system.

[0057] As used herein, the term "prevent" or any grammatical variant thereof (e.g., prevent, preventing, etc.) includes, but is not limited to, at least reducing the likelihood of acquiring the risk (or susceptibility) of a disease or disorder (i.e., preventing the development of at least one clinical symptom in a patient who may be exposed to or susceptible to the disease but who has not yet shown symptoms of the disease). The term "prevent" can refer to avoiding, delaying, postponing, or minimizing undesirable features associated with a disease or disorder, and / or completely or nearly completely preventing the development of a disease or disorder and its symptoms. Prevention can also include, but is not limited to, absolute or complete prevention, meaning that the disease or disorder may still develop at a later time and / or its severity is less than it would have been if the preventive measures had not been taken.

[0058] As used herein, a "synergistically effective" therapeutic amount or a "synergistically effective" amount is an amount that, when used in combination with an effective or subtherapeutic amount of another drug or therapy, produces a greater effect than the effect of either drug or therapy when used alone. A "synergistically effective" therapeutic amount of a drug or therapy, optionally in combination with another drug or therapy, produces a greater effect than the additive effect of the two drugs or therapies when used alone. A "greater effect" includes not only a reduction in symptoms of the condition being treated, but also includes a reduction in side effects, improved tolerability, improved subject compliance, improved efficacy, or any other improved clinical outcome.

[0059] As used herein, "pharmaceutically acceptable carriers" or "pharmaceutically acceptable excipients" include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic agents, absorption delaying agents, and the like. The use of such media and excipients is well known in the pharmaceutical art. Unless any conventional media or excipients are incompatible with the active ingredient, their use in the therapeutic compositions of the present invention is acceptable. Other active ingredients may also be incorporated into these compositions.

[0060] In this article, “selective sciatic nerve branch injury” and “SNI” are used interchangeably to refer to a chronic pain model that can induce symptoms of chronic pain, including mechanical hyperalgesia.

[0061] In this article, "PHN" refers to postherpetic neuralgia, the most common complication of shingles.

[0062] In this article, the terms are defined as follows:

[0063] In this article, "RI" is used to abbreviate Roseburia intestinalis.

[0064] In this article, “PrL” refers to a brain region called the prefrontal cortex.

[0065] In this article, "DG" refers to a brain region called the dentate gyrus.

[0066] In this article, “BNST” refers to the brain region called the bed nucleus of the striatum terminalis.

[0067] In this article, “NTS” refers to a brain region called the nucleus tractus solitarius.

[0068] In this article, “AI” refers to a brain region called the agranular insular cortex.

[0069] In this article, "PVN" refers to a brain region called the paraventricular nucleus of the hypothalamus.

[0070] In this article, “CeA” refers to a brain region called the central amygdala.

[0071] "Analgesic" or "analgesic" has its conventional meaning herein and refers to a compound, agent, drug or substance that relieves pain in the broadest sense.

[0072] Any composition or method provided herein can be used in combination with any other provided composition or method.

[0073] Any description of a specific embodiment of a variable or aspect mentioned herein includes that embodiment as a single embodiment or in combination with any other embodiment or portion thereof.

[0074] Therapeutic compositions

[0075] The present invention uses the power of metagenomic sequencing technology to identify specific probiotic strains with analgesic properties. By analyzing stool samples from healthy individuals and patients with postherpetic neuralgia, the present invention identified probiotic strains that are more abundant in healthy individuals compared to those with chronic pain.

[0076] The present invention discloses a therapeutic composition for treating chronic pain. In certain embodiments, the composition may include multiple probiotic strains. In certain embodiments, the probiotic strains are more abundant in healthy individuals than in pain patients.

[0077] In certain embodiments, the compositions of the present invention comprise a probiotic strain selected from the group consisting of Anaerostipes hadrus, Clostridium sp.SY8519, Anaerobutyricum hallii, Flintibacter sp.KGMB00164, Romboutsia hominis, Longibaculum sp.KGMB06250, Alistipes megaguti, Veillonella dispar, Lachnospiraceae bacterium Choco86, Faecalibacterium prausnitzii, Erysipelotrichaceae bacterium GAM147, Streptococcus dysgalactiae, Haemophilus parainfluenzae, Roseburia hominis, Ruminococcus champanellensis, Lachnospiraceae bacterium KM106.2, Lachnospiraceae bacterium, Roseburia intestinalis, and Muribaculaceae bacterium. DSM 108610, Lachnospiraceae bacterium GAM79, Prevotella intermedia, Prevotella scopos, Prevotellafusca, Lachnospiraeligens, Prevotella melaninogenica, and any combination thereof. In certain embodiments, these probiotic strains are identified by metagenomic sequencing based on their individual and shared analgesic properties.

[0078] In certain embodiments, the compositions of the present invention comprise one or more probiotic strains selected from Roseburia intestinalis, Lachnospiraceae bacterium, and Roseburia hominis. In specific embodiments, the compositions of the present invention comprise Roseburia intestinalis, Lachnospiraceae bacterium, and / or Roseburia hominis.

[0079] In certain embodiments, compositions comprising Roseburia intestinalis, Lachnospiraceae bacterium, and Roseburia hominis can be combined in any ratio to provide collective and / or synergistic analgesic properties.

[0080] In certain embodiments, the probiotic composition may be present in an amount of at least 10 5 CFU, 10 6 CFU, 10 7 CFU, 10 8 CFU, 10 9 CFU, 10 10 CFU, 10 11 CFU or any dose therebetween is administered to a subject.

[0081] In certain embodiments, the probiotic composition may be taken as 10 9 A dose of CFU is administered to the subject.

[0082] In certain embodiments, the sterile secretion filtrate of Roseburia intestinalis is a filtrate obtained by culturing Roseburia intestinalis for 24 hours and then filtering to remove all microorganisms.

[0083] In certain embodiments, the compositions of the present invention also include pharmaceutically acceptable carriers, adjuvants and / or diluents. Carriers and / or diluents can generally be any suitable medium that can achieve the intended purpose. In particular, carriers and / or diluents should not reduce the pharmacological efficacy of the active ingredient and the ability of the component to be directed to the target site in the animal body or on the surface of the body. Preferably, the carriers and / or diluents are selected from water, saline and / or buffered physiologically acceptable aqueous solutions, and any other solution that can be used for animal administration. These carriers and diluents are well known to those skilled in the art, for example, can be distilled water, deionized water, pure water or ultrapure water, physiological saline, phosphate buffered saline (PBS), solutions containing conventional buffers compatible with other drug targeting system components, etc.

[0084] Carriers and / or excipients according to the present invention may include any and all solvents, diluents, buffers (e.g., neutral buffered saline, phosphate buffer, or alternatively Tris-HCl, acetate or phosphate buffer), water-in-oil or oil-in-water emulsions, aqueous formulations with or without organic co-solvents suitable for intravenous injection, etc., solvating agents (e.g., polysorbate 65, polysorbate 80), colloids, dispersion media, carriers, fillers, chelating agents (e.g., EDTA or glutathione), amino acids (e.g., glycine), proteins, disintegrants, binders, lubricants, wetting agents, emulsifiers, sweeteners, colorants, flavorings, flavoring agents, thickeners (e.g., carbomer, gelatin or sodium alginate), coating agents, preservatives (e.g., thimerosal, benzyl alcohol, polyquaternium), antioxidants (e.g., ascorbic acid, sodium metabisulfite), isotonicity regulators, absorption delaying agents, adjuvants, fillers (e.g., lactose, mannitol), etc. The use of carriers and / or excipients is well known in the pharmaceutical and health product fields. Unless any conventional media or excipients are incompatible with the target health-promoting substance or composition, carriers or excipients may be considered for use in the compositions of the present invention.

[0085] In certain embodiments, the compositions of the present invention are formulated into products that can be taken orally, such as food, capsules, pills, or drinkable liquids. Oral medications are any physiologically active substances that are initially absorbed through the gastrointestinal tract or enter the oral mucosa. These compositions can also be formulated into solutions that can be administered by injection (including intravenous, intraperitoneal, intramuscular, spinal, or subcutaneous injections).

[0086] In other embodiments, the compositions of the present invention are formulated for administration through the skin (e.g., transdermal patch) or directly on the skin to achieve local or systemic effects. These compositions can be administered sublingually, buccally, rectally, or vaginally. Additionally, these compositions can be absorbed through the nasal mucosa, atomized, inhaled through the mouth or nose, or administered in the eyes or ears.

[0087] An orally consumable product according to the present invention is any preparation or composition suitable for consumption, nutrition, oral hygiene or recreation, which is intended to be introduced into the oral cavity of a human or animal, to remain there for a period of time and then either to be swallowed (e.g., ready-to-eat food or pills) or to be removed from the oral cavity again (e.g., chewing gum or oral hygiene or medical mouthwash). Although oral medications can be formulated as orally consumable products, and oral consumable products can contain oral medications, the two terms should not be used interchangeably herein.

[0088] Products for oral consumption include all substances or products intended for ingestion by humans or animals in a processed, semi-processed or unprocessed state. This also includes substances that are added to products for oral consumption (especially foods and pharmaceuticals) during production, treatment or processing and are intended to be introduced into the oral cavity of humans or animals.

[0089] Orally consumable products may also include substances intended to be swallowed by humans or animals and digested in an unmodified, prepared or processed state; therefore, orally consumable products according to the present invention also include shells, coatings or other coatings that are intended to be swallowed with the product or are expected to be swallowed.

[0090] In certain embodiments, the oral consumable product is a capsule, pill, syrup, emulsion or liquid suspension containing a desired oral medication. In certain embodiments, the oral consumable product can include an oral medication in powder form that can be mixed with water or other liquids to produce a drinkable oral consumable product.

[0091] In certain embodiments, orally consumable products according to the present invention may contain one or more formulations intended for nutrition or pleasure. These include, in particular, bakery products (e.g., bread, dry biscuits, cakes and other pastries), confectionery (e.g., chocolate, chocolate bar products, other bar products, fruit gum, coated tablets, hard caramels, toffees and caramels, and chewing gum), alcoholic or non-alcoholic beverages (e.g., cocoa, coffee, green tea, black tea, black tea or green tea beverages enriched with green tea or black tea extracts, Rooibos tea, other herbal teas, fruit-containing lemonades, isotonic beverages, soft drinks, fruit and vegetable juices and preparations thereof), instant beverages (e.g., instant cocoa beverages, instant tea beverages and instant coffee beverages), meat products (e.g., ham, fresh or raw sausage products and seasoned or cured fresh meat or bacon products), eggs or egg products (e.g., whole egg powder, egg whites and egg yolks), cereal products (e.g., breakfast cereals, granola bars and precooked instant rice products), dairy products (e.g., full-fat or low-fat or skim milk beverages, rice pudding, yogurt, kefir, cream cheese, soft cheese, hard cheese, skim milk powder, whey, butter, buttermilk and partially or completely hydrolyzed milk protein-containing products), soy protein or other soy ingredient products (e.g., soy milk and its products, beverages containing isolated or enzyme-treated soy protein, beverages containing soy flour, preparations containing soy lecithin, fermented products such as tofu or tempeh products and mixtures thereof with fruit preparations, optionally containing flavoring substances), fruit preparations (e.g., jams, fruit ice creams, fruit spreads and fruit fillings), vegetable preparations (e.g., ketchup, sauces, dried vegetables, frozen vegetables, precooked vegetables and cooked vegetables), snacks (e.g., baked or fried potato chips (French fries) or corn or peanut-based potato dough products and extrudates), products based on fats and oils or their emulsions (e.g., mayonnaise, sauces and salad dressings), other prepared meals and soups (e.g., dry soups, instant soups and precooked soups), seasonings (e.g., sprinkle seasonings), sweetener compositions (e.g., tablets, sachets and other preparations for sweetening or whitening beverages or other foods). The composition of the invention may also serve as a semi-finished product for the production of other compositions intended for nutritional or recreational use.

[0092] The composition of the present invention can be formulated into various dosage forms, for example, solid, semisolid, liquid or gaseous forms, such as tablets, capsules, powders, granules, ointments, solutions, suppositories, injections, inhalants and aerosols.

[0093] In certain embodiments, the composition is in powder form. A pharmaceutically acceptable carrier is a finely dispersed solid mixed with particles of the active ingredient. In another embodiment, the composition is in tablet form. The active ingredient is mixed with a pharmaceutically acceptable carrier having the necessary binding capacity in appropriate proportions and pressed into the desired shape and size. Suitable carriers include magnesium carbonate, magnesium stearate, talc, sucrose, lactose, pectin, dextrin, starch, gelatin, gum arabic, methylcellulose, sodium carboxymethylcellulose, low melting point wax, cocoa butter, and the like.

[0094] In certain embodiments, the compositions of the present invention can be made into aerosol form so that atomization or inhalation can be performed. Pharmaceutical preparations suitable for administration in aerosol or spray form include powders, particles, solutions, suspensions or emulsions. Aerosol preparations for oral or nasal inhalation can also include carriers, such as normal saline, polyethylene glycol or glycol, DPPC, methylcellulose, or mixed with powder dispersants or freons. Aerosol preparations can be loaded with pressurized propellants, such as dichlorodifluoromethane, propane, nitrogen, freons, or other solvating or dispersing agents known in the art. For example, the administration method can use any of disposable dosing devices, nebulizers, breath-activated powder inhalers, aerosol metered dose inhalers (MDIs) or the numerous nebulizer dosing devices available in this area. In addition, atomization tents or direct administration through endotracheal intubation can also be used.

[0095] In certain embodiments, the compositions of the present invention can be formulated into preparations for topical application to the skin, such as douches, sprays, drops, emulsions, gels, ointments, creams, foams, powders, solids, sponges, patches, steam, pastes, tinctures, or use transdermal patches. Except pharmaceutically active carriers, suitable preparations for topical application can also include emollients such as palm wax, cetyl alcohol, cetyl ester wax, emulsifying wax, hydrated lanolin, lanolin, lanolin alcohol, microcrystalline wax, paraffin, vaseline, polyethylene glycol, stearic acid, stearyl alcohol, white beeswax or yellow beeswax. In addition, the compositions can also include wetting agents such as glycerol, propylene glycol, polyethylene glycol, sorbitol solution and 1,2,6-hexanetriol, or penetration enhancers such as ethanol, isopropyl alcohol or oleic acid.

[0096] The effective amount of the pharmaceutical composition can be administered, for example, orally, rectally, bronchially, nasally, topically, buccally, sublingually, transdermally, vaginally, intramuscularly, intraperitoneally, intravenously, intraarterially, intracerebrally, intraocularly, or in a form suitable for inhalation or infusion, including powder and liquid aerosol administration, or by a sustained-release system, such as a semipermeable membrane solid hydrophobic polymer matrix containing the compounds of the invention. Administration can also be carried out by other carriers or carriers, such as micelles, liposomes, vesicles, implants (e.g., microimplants), synthetic polymers, microspheres, nanoparticles, etc.

[0097] In certain embodiments, the compositions of the present invention can be formulated for administration by injection, such as solutions or suspensions. The solution or suspension may contain a suitable, non-toxic, acceptable diluent or solvent for parenteral administration, such as mannitol, 1,3-butanediol, water, Ringer's solution, or isotonic sodium chloride solution, or a suitable dispersant, wetting agent, and suspending agent, such as a sterile, non-irritating fixed oil, including synthetic mono- or diglycerides, and fatty acids, including oleic acid. An example of a carrier for intravenous injection includes 10% United States Pharmacopoeia Standard (USP) ethanol, 40% USP propylene glycol, or polyethylene glycol 600, with the remainder being water for injection (WFI). Other examples of carriers for intravenous injection include 10% USP ethanol and USP WFI; 0.01-0.1% triethanolamine in USP WFI; or 0.01-0.2% dipalmitoyl diphosphatidylcholine in USP WFI; and 1-10% squalene or a vegetable oil-water emulsion for parenteral use. Water or physiological saline solutions and aqueous solutions containing glucose and glycerol are preferred as carriers, particularly for injectable solutions. Examples of carriers for subcutaneous or intramuscular injection include phosphate buffered saline (PBS) solution, 5% glucose in WFI, and 5% glucose or 0.9% sodium chloride in USP WFI, 0.01-0.1% triethanolamine, or a 1:2 or 1:4 mixture of 10% USP ethanol, 40% propylene glycol, and an isotonic solution (such as 5% glucose or 0.9% sodium chloride); or 0.01-0.2% dipalmitoyl diphosphatidylcholine and 1-10% squalene in USP WFI or a vegetable oil-water emulsion for parenteral use.

[0098] In certain embodiments, the composition is formulated for parenteral administration (e.g., by injection). In addition, the composition can be presented in single-dose form, such as in ampoules, prefilled syringes, and small-volume infusions or in multi-dose containers, with or without added preservatives. The composition can be in the form of a suspension, solution, or emulsion in an oily or aqueous vehicle. The composition can further comprise formulating agents, such as suspending agents, stabilizers, and / or dispersants.

[0099] In certain embodiments, the composition can be directly applied to the nasal cavity, by conventional means, for example, using a dropper, a pipette or a spray. The composition can be in single dose or multiple dose form. Administration by the respiratory tract can also be achieved by aerosol formulations, wherein the active ingredient is provided in a pressurized package with a suitable propellant, for example a chlorofluorocarbon (CFC), such as dichlorodifluoromethane, trichlorofluoromethane or dichlorotetrafluoroethane, carbonic acid gas or other applicable gases.

[0100] The compositions of the present invention can also be administered in the form of a controlled release formulation, such as a sustained release or immediate release formulation. Such controlled release formulations of the compositions of the present invention can be prepared using methods well known to those skilled in the art. The method of administration will be determined by the attending physician or other professional after evaluating the subject's condition and needs.

[0101] In certain embodiments, the pharmaceutical composition is provided in unit dosage form, wherein the composition of the desired form is divided into unit doses containing an appropriate amount of active ingredient. The unit dosage form can be a packaged preparation containing discrete quantities, such as packaged tablets, capsules, and powders in vials or ampoules. In addition, the unit dosage form can be a capsule, tablet, sachet, or lozenge itself, or an appropriate number of these in packaged form. In a preferred embodiment, tablet or capsule form is used for oral administration, and liquid form is used for intravenous administration and continuous infusion.

[0102] In certain embodiments, the pharmaceutical composition of the present invention may further comprise one or more (co-) analgesics, which may be selected from, for example: muscle relaxants: baclofen, dantrolene, tizanidine, carisoprodol or cyclobenzaprine; tricyclic antidepressants: amineptine, amitriptyline, amitriptyline oxide, butryline, demetriptyline, dibenzodiazepine; Dimethoprim, dosulpyrine, fluoxetine, isopentindole, clomipramine, desipramine, nortriptyline, doxepin, imipramine, imipramine oxide, trimipramine, lofepramine, maprotiline, metapramine, metactracin, norclomipramine, nortriptyline, noxetine, opipramol, perapram, pizotifen, propiazepine Protriptyline, quinpiramine, reboxetine or tianeptine, or derivatives, prodrugs, stereoisomers and / or salts thereof; Tetracyclic antidepressants: amoxapine, aptazapine, cyclosindole, esmitazapine, loxapine, mazindole, metrindole, maprotiline, mianserin, mirtazapine, oxaprotiline, pyrrolidinole, cetipridin, or derivatives, prodrugs, stereoisomers and / or salts thereof; Alpha-2 adrenergic agonists: amide epinephrine, amitraz, anisodamine, apraclonidine, brimonidine, cilazoline, clonidine, dexmedetomidine, epinephrine, ergotamine, etilefrine, faldomidine, guanabenzyl, guanethidine, guanfacine, guanoxabenzyl, indanidine, lofexidine, medetomidine, mefentermine, metaraminol, Methoxamine, methyldopa, mivacyclidine, naphazoline, norepinephrine, norephedrine, octopamine, oxymetazoline, phenylpropanolamine, rilmenidine, romifidine, synephrine, talipexol, xylazine, or derivatives, prodrugs, stereoisomers and / or salts thereof; selective serotonin reuptake inhibitors: paroxetine, celecoxib, citalopram, dapoxetine, escitalopram, fluoxetine, fluvoxamine, indalpine, paroxetine, sertraline, zimelidine, and diastereomers, derivatives, prodrugs, stereoisomers or salts thereof; serotonin-norepinephrine reuptake inhibitors**: duloxetine, desvenlafaxine, levomilnacipran, milnacipran, venlafaxine, or derivatives, prodrugs, stereoisomers and and / or salts thereof; nonsteroidal anti-inflammatory drugs: diclofenac, duloxetine, desvenlafaxine, levomilnacipran, milnacipran, venlafaxine, or derivatives, prodrugs, stereoisomers, and / or salts thereof; opioid receptor agonists: loperamide, alfentanil, allylprodine, alfaprodine, anileridine, benzylmorphine, bezitamide, buprenorphine, butorphanol, clonitrazine, codeine, cyclazocine, normorphine, dextromethorphan, dextromorphamide, dezocine, diaminopropionamide, dimorphone, dihydrocodeine, dihydromorphine, dimenoxadol, dimepeptol, dimethylthiobutene, diphenylethyl ester, diphenylpiperazine, etazocin, ethoxyheptazine, ethylmethylthiobutene, ethylmorphine, etonizine, fentanyl, heroin, hydrocodone, hydromorphine ketone, hydroxymethidine, isomethadone, ketobemidone, levallorphanol, levorphanol, levophenylmorphan, lofentanil, loperamide, meperidine, meptazocine, pentazocine, methadone, methoprene, morphine, melamine, nalbuphine, nalbuphine, nalcein, nicotinoylmorphine, norlevorphanol, normethadone, nalorfen, normorphine, norperirone, opium, oxycodone, oxymorphone, poppy fruit extract, pentazocine, phenadrine, phenadrine, phenammonium, phenazocine, phenoperidine, piminodine, pirithromide, proheptazine, prometazine, propamdol, propiperidine, propipram, propoxyphene, sufentanil, tilidine, tramadol, tapentadol, axomadol, fasiladol, and diastereomers thereof, or derivatives, prodrugs, stereoisomers and / or salts thereof;Local anesthetics: tetracaine, articaine, benzocaine, bupivacaine, chloroprocaine, cinchocaine, cocaine, cyclomethicaine, dibucaine, dimethicaine, etidocaine, ethyl aminobenzoate, eugenol, levobupivacaine, lidocaine, menthol, mepivacaine, neosand anemone toxin, oxycaine, oxybuprocaine, piperocaine, prilocaine, propoxycaine, procaine, proparacaine, ropivacaine, anemone toxin, tetracaine, tetrodotoxin, trimecaine, and diastereomers thereof, or derivatives, prodrugs, stereoisomers and / or salts thereof; benzodiazepines; Class: Adizalam, alprazolam, benzazepam, bretanib, bromazepam, brotizolam, carbamazepam, cildiazepam, ciproazepam, chlordiazepoxide Clomazolam, clobazam, clonazepam, clonazolam, chlordiazepoxide Acid, chlorine nitrogen Acid, clotiazepam, cloxazolam, norclorazepam, diazepam, diclorazepam, fluchloride Ethyl ester, estazolam, etizolam, chlorofluoro Ethyl ester, flubrozepam, flubrozolam, flunitrazepam, flurazepam, flutazepam, flutazolane, halazepam, ketozolam, loprazolam, lorazepam, lormetazepam, medazepam, metazepam, midazolam, nifozepam, nimetazepam, noradizolam, nitrazepam, nordiazepam, oxazepam, phenazepam, pineazepam, prazepam, pramazepam, pyrazole Amobarbital, quazepam, rimazafone, temazepam, tetrazepam, thiophene alprazolam, triazolam, derivatives flumazenil, eszopiclone, zaleplon, zolpidem, zopiclone, or their prodrugs, stereoisomers and / or salts; barbiturates: amobarbital, isobutabarbital, aprobarbital, afenal, barbiturate, barbexalon, bralobarbital, butyral Barbiturate, butalbital, butenylbarbital, butobarbital, barbital, cyclobarbital, cyclopropylbarbital, deoxyphenobarbital, diphenylbarbituric acid, ethylammobarbital, nonobarbital esters, heptylbarbital, hexylbarbital, methylbarbital, methohexital, methylhexenylbarbital, methylphenobarbital, analbital, nibarbital, pentobarbital, phenobarbital, propanol, propanol, propanol bromide, propanol bromide, ribosalfate, sec-butabarbital, secobarbital, sigmodal, talbutabarbital, thioammobarbital, thioammobarbital, thiobutabarbital, thiopental sodium, valprofen, vinylbarbital, vinylbarbital, or derivatives, prodrugs, stereoisomers and / or salts thereof; dimethyl sulfoxide;N-methyl-D-aspartate (NMDA) receptor antagonists: (levorotatory, dextrorotatory, or racemic) ketamine, amantadine, atiganib, caroverine, dextrorphan, dextromethorphan, fullerene, ibogaine, ketamine, lidocaine, memantine, dizocilpine, neramexane, norketamine, dilucimine, 3-fluoro-γ-(3-fluorophenyl)-N-methyl-amphetamine hydrochloride, phencyclidine, tiletamine, remacemide, decahydro-6-(phosphonomethyl)-3-isoquinolinecarboxylic acid, (3S,4aR,6S,8aR)-decahydro-6-(phosphonomethyl)-3-isoquinolinecarboxylic acid, (2R,4S)-rel-4-(1H-tetrazol-5-yl-methyl)-2-piperidinecarboxylic acid, α-Amino-2-(2-phosphonoethyl)-cyclohexanepropionic acid, glutathione, aminomethyl mercaptan, 5-phosphononorvaline, 4-(3-phosphonopropyl)-2-piperazinecarboxylic acid, safortyl, cis-4(phosphonomethyl)-2-piperidinecarboxylic acid, (3E)-2-amino-4-methyl-5-phosphono-3-pentenoic acid, (3E)-2-amino-4-methyl-5-phosphono-3-pentenoic acid 1-ethyl ester, (αS)-α-amino-2′-chloro-5-(phosphonomethyl)-[1,1′-biphenyl]-3-propionic acid, S-nitrosoglutathione, camprostic acid, acamin, conotoxin-G, elitodil, haloperidol, ifenprodil, troxaprid, (R,S)-α-(4-hydroxy)- phenyl)-β-methyl-4-(phenylmethyl)-1-piperidinol, aminocyclopropanecarboxylic acid, 7-chlorokynurenine, D-cycloserine, gavistinide, 4,6-dichloro-3-[(E)-(2-oxo-1-phenyl-3-pyrrolidinyl)methylene]-1H-indole-2-carboxylic acid monosodium salt, lisofylline, 8-chloro-2,3-dihydropyridazino[4,5-b]quinoline-1,4-dione 5-oxide 2-hydroxy-N,N,N-trimethyl-ethylammonium salt, 7-chloro-4-hydroxy-3-(3-phenoxyphenyl)-2(1H)-quinolinone, 3-amino-1-hydroxy-2-pyrrolidinone, 7-chloro-4-hydroxy-2-(4-methoxy-2-methylphenyl)-1, 2,5,10-tetrahydropyrido[4,5-b]quinoline-1,10-dione sodium salt, or derivatives, prodrugs, stereoisomers and / or salts thereof; N-acylethanolamines: N-arachidonoylethanolamine, docosahexaenoylethanolamine, oleoylethanolamine, palmitoylethanolamine, stearoylethanolamine, or derivatives, prodrugs, stereoisomers and / or salts thereof; cannabinoids: cannabinol, cannabidiol, Δ9-tetrahydrocannabinol, Δ8-tetrahydrocannabinol, 11-hydroxy-tetrahydrocannabinol, 11-hydroxy-Δ9-tetrahydrocannabinol, levo-anthraquinone, Δ11-tetrahydrocannabinol, tetrahydrocannabinol, dronabinol, anandamide, nabilone, or derivatives, prodrugs, stereoisomers and / or salts thereof;Antiepileptic compounds: acetazolamide, beclamide, brivaracetam, carbamazepine, valproate hemisodium, eslicarbazepine acetate, ethosuximide, felbamate, gabapentin, lamotrigine, levetiracetam, methsuximide, methazolamide, oxcarbazepine, paramethadione, phenylacetamide, phenylbutyrylamide, phensuximide, potassium bromide, pregabalin, perampanel, primidone, pregabalin, celecoxib, sodium valproate, sultiame, tiagabine, topiramate, trimethadione, valproamide, valproylmorpholine, valproic acid, vigabatrin, zonisamide, or derivatives, prodrugs, stereoisomers or salts thereof, and combinations thereof;

[0103] How to use

[0104] In certain embodiments, the present invention provides an innovative approach to managing chronic pain by utilizing specific probiotic strains identified using metagenomic sequencing technology.

[0105] In certain embodiments, the present invention provides a novel approach to managing and treating chronic pain by using multiple probiotic strains that are more abundant in healthy individuals than in patients with neuropathic pain. Central to this approach is the gut microbiome, the complex community of bacteria that inhabits our gastrointestinal tract, which has been shown to influence the brain and, in turn, our perception of pain.

[0106] The present invention represents a significant advancement in the field of pain management, particularly chronic pain management, by leveraging the gut-brain axis and the therapeutic potential of specific probiotic strains identified through metagenomic sequencing of stool samples from healthy individuals and patients with postherpetic neuralgia (PHN), a chronic pain condition associated with shingles.

[0107] In certain embodiments, the strains identified include, for example: Anaerostipes hadrus, Clostridium sp. SY8519, Anaerobutyricum hallii, Flintibacter sp. KGMB00164, Romboutsiahominis, Longibaculum sp. KGMB06250, Alistipes megaguti, Veillonella dispar, Lachnospiraceae bacterium Choco86, Faecalibacteriumprausnitzii, Erysipelotrichaceae bacterium GAM147, Streptococcus dysgalactiae, Haemophilusparainfluenzae, Roseburia hominis, Ruminococcus champanellensis, Lachnospiraceae bacterium KM106.2, Lachnospiraceae bacterium, Roseburiaintestinalis, Muribaculaceae bacterium DSM 108610, Lachnospiraceae bacteriumGAM79, Prevotella intermedia, Prevotella scopos, Prevotellafusca, Lachnospiraeligens and Prevotella melaninogenica.

[0108] In certain embodiments, the probiotic strains were identified as Roseburia intestinalis, Lachnospiraceae bacteria, and Roseburia hominis, which exhibited significant analgesic properties in a sciatic nerve branch selective injury (SNI) model of neuropathic pain. Mechanistically, the present invention reveals that administration of Roseburia intestinalis acts through the gut-brain axis, potentially modulating activity in the vagal ganglion and multiple brain regions. This approach may address a potential mechanism of chronic pain and provide a more effective and targeted treatment strategy.

[0109] The present invention relates to therapeutic interventions for chronic pain, particularly the use of selected probiotic strains with analgesic properties, particularly three probiotics—Roseburia intestinalis, Lachnospiraceaebacterium, and Roseburia hominis—and a sterile secretory filtrate of Roseburia intestinalis that exhibit significant analgesic properties when administered in adequate doses. The invention encompasses the fields of neurology and pain management, as it utilizes the gut-brain axis to modulate pain signaling, thereby alleviating chronic pain.

[0110] In certain embodiments, the present invention provides a method for preventing or treating chronic pain in a subject, comprising administering to the subject an effective amount of a probiotic strain, a composition comprising the probiotic strain, or an effective amount of a sterile secretion filtrate of Roseburia intestinalis.

[0111] In certain embodiments, the present invention provides a method for preventing or treating chronic pain in a subject, the method comprising administering to the subject an effective amount of a composition comprising one or more probiotic strains selected from the group consisting of Anaerostipes hadrus, Clostridium sp.SY8519, Anaerobutyricum hallii, Flintibacter sp.KGMB00164, Romboutsia hominis, Longibaculum sp.KGMB06250, Alistipes megaguti, Veillonella dispar, Lachnospiraceae bacterium Choco86, Faecalibacterium prausnitzii, Erysipelotrichaceae bacterium GAM147, Streptococcus dysgalactiae, Haemophilus parainfluenzae, Roseburia hominis, Ruminococcus champanellensis, Lachnospiraceae bacterium KM106.2, Lachnospiraceae bacterium, Roseburia intestinalis, Muribaculaceae bacterium DSM108610, Lachnospiraceae bacterium GAM79, Prevotella intermedia, Prevotellascopos, Prevotellafusca, Lachnospira eligens and Prevotella melaninogenica.

[0112] In certain embodiments, a method for preventing or treating chronic pain in a subject comprises administering to the subject an effective amount of a composition comprising one or more probiotic strains selected from the group consisting of Roseburia intestinalis, Lachnospiraceaebacterium, and Roseburia hominis.

[0113] In certain embodiments, the present invention provides a method of reducing or inhibiting one or more symptoms of chronic pain in a subject, comprising administering to the subject an effective amount of a probiotic strain of the present invention, an effective amount of a composition comprising a probiotic strain of the present invention, or a sterile secretion filtrate of Roseburia intestinalis.

[0114] Reducing chronic pain and / or symptoms associated with chronic pain means reducing the severity (including reducing the need and / or dosage of other medications and / or therapies typically used for pain relief), duration, or frequency. Improving chronic pain and / or symptoms associated with chronic pain means alleviating or improving one or more chronic pain and / or related symptoms compared to not using the therapy. Improving also includes shortening or reducing the duration of symptoms.

[0115] In certain embodiments, the present invention provides a method for delaying the progression of chronic pain in a subject, comprising administering to the subject an effective amount of a probiotic strain of the present invention or an effective amount of a composition comprising a probiotic strain of the present invention.

[0116] As used herein, "delaying" the development of chronic pain means postponing, hindering, slowing, delaying, stabilizing and / or postponing the progression of chronic pain and / or symptoms associated with chronic pain. This delay can last for different lengths of time depending on the medical history and / or individual circumstances of the subject. For those skilled in the art, a sufficient or significant delay may actually encompass prevention, i.e., the individual does not develop chronic pain. A method of "delaying" the progression of a symptom is a method that reduces the probability of the symptom occurring and / or reduces the severity of the symptom within a given time frame, compared to not using the method. Such comparisons are typically based on clinical studies using a statistically significant number of subjects.

[0117] In certain embodiments, chronic pain includes one or more of the following types: chronic nociceptive pain, chronic neuropathic pain, chronic inflammatory pain, arthritic pain, fibromyalgia, breakthrough pain, persistent pain, hyperalgesia, allodynia, central sensitization, peripheral sensitization, disinhibition and facilitation, and cancer pain.

[0118] In certain embodiments, the chronic pain is cancer pain, preferably cancer pain caused by a malignant tumor, or preferably pain caused by one or more of the following cancers: adenocarcinoma in glandular tissue, blastoma in embryonic tissue of an organ, carcinoma in epithelial tissue, leukemia in blood cell forming tissue, lymphoma in lymphoid tissue, myeloma in bone marrow, sarcoma in connective tissue or supporting tissue, adrenal cancer, AIDS-related lymphoma, anemia, bladder cancer, bone cancer, brain cancer, breast cancer, carcinoid tumors, cervical cancer, chemotherapy, colon cancer, cytopenia, ovarian cancer, cervical cancer, uterine cancer, cervical cancer, uterine fibroids ... Endometrial cancer, esophageal cancer, gastric cancer, head cancer, neck cancer, hepatobiliary cancer, kidney cancer, leukemia, liver cancer, lung cancer, lymphoma, Hodgkin's disease, non-Hodgkin's lymphoma, nervous system tumors, oral cancer, ovarian cancer, pancreatic cancer, prostate cancer, rectal cancer, skin cancer, gastric cancer, testicular cancer, thyroid cancer, urethral cancer, bone cancer, connective tissue sarcoma, bone tissue cancer, blood cell-forming cell cancer in the bone marrow, bone marrow cancer, multiple myeloma, leukemia, primary or secondary bone cancer, tumors that metastasize to bone, tumors that invade nerves and hollow organs, tumors near neural structures. Further preferred cancer pain includes visceral pain, in particular visceral pain caused by pancreatic cancer and / or abdominal metastasis. Further preferred cancer pain includes somatic pain, in particular somatic pain caused by one or more of the following: bone cancer, bone metastasis, postoperative pain, connective tissue sarcoma, bone tissue cancer, blood cell-forming cell cancer in the bone marrow, multiple myeloma, leukemia, primary or secondary bone cancer.

[0119] In preferred embodiments, the chronic pain is chronic neuropathic pain. In particular embodiments, the chronic pain is associated with herpes zoster and / or postherpetic neuralgia (PHN).

[0120] As used herein, "neuropathic pain" has its traditional meaning and refers to pain that is a direct or indirect consequence of a lesion or disease affecting the somatosensory system (central and / or peripheral). Neuropathic pain as used herein includes all types of neuropathic pain, such as peripheral neuropathy caused by type 1 or type 2 diabetes, neuropathy caused by various noxious substances (such as alcohol), neuropathy caused by various deficiencies (such as vitamin B1, B6 and / or B12 deficiency), various poisonings (such as vitamin B6 excess, hypothyroidism), neuropathy caused by chemotherapy drugs (such as paclitaxel or other taxane derivatives, vinblastine or other vinca alkaloids, cisplatin or other platinum derivatives), drug-induced neuropathy, neuropathy caused by compounds used to treat infections (such as streptomycin, didanosine or zalcitabine), or neuropathy caused by any other chemical toxic compound. Other peripheral neuropathies include, for example, trigeminal neuralgia, postherpetic neuralgia, intercostal neuralgia, compression neuropathies (such as carpal tunnel syndrome, tarsal tunnel syndrome, and abdominal cutaneous nerve compression syndrome), small fiber neuropathies, hereditary motor and sensory neuropathies, chronic inflammatory demyelinating polyneuropathy, sciatica, chronic idiopathic sensory neuropathy, infectious diseases (such as post-polio syndrome, AIDS or HIV-related, Lyme disease-related, Sjögren's syndrome-related, lymphomatous neuropathy, myelomatous neuropathy, cancerous neuropathy, acute panautonomic neuropathy, vasculitic / ischemic neuropathy, and other mono- and polyneuropathies). In addition, neuropathic pain also includes: complex regional pain syndrome type I and type II (reflex sympathetic dystrophy), central neuropathic pain (such as thalamic neuropathy, spinal cord injury neuropathy, post-stroke pain, multiple sclerosis neuropathy, syringomyelia, spinal cord tumors), phantom limb pain, restless genital syndrome (pain), and postoperative scar pain (including cardiac surgery and mastectomy).

[0121] In this context, the term "inflammatory pain" has its traditional meaning, referring to pain caused by inflammation, which may be caused by, but is not limited to, trauma, burns, extreme cold, fractures, (osteo)arthritis, rheumatoid arthritis, chronic strain, surgery, infections and autoimmune diseases, overstretching, infection, and vasoconstriction. Various inflammatory mediators can directly affect nociceptors or make them more sensitive to touch or movement, even outside the area of ​​inflammation.

[0122] In certain embodiments, the methods of the present invention further comprise diagnosing the subject to determine whether the subject has a deficiency or imbalance in one or more probiotic strains. In a preferred embodiment, the one or more probiotic strains are selected from the group consisting of: Anaerostipes hadrus, Clostridium sp.SY8519, Anaerobutyricum hallii, Flintibacter sp.KGMB00164, Romboutsia hominis, Longibaculum sp.KGMB06250, Alistipes megaguti, Veillonella dispar, Lachnospiraceae bacterium Choco86, Faecalibacterium prausnitzii, Erysipelotrichaceae bacterium GAM147, Streptococcus dysgalactiae, Haemophilus parainfluenzae, Roseburia hominis, Ruminococcus champanellensis, Lachnospiraceae bacterium KM106.2, Lachnospiraceae bacterium, Roseburia intestinalis, Muribaculaceae bacterium DSM 108610, Lachnospiraceae bacteriumGAM79, Prevotella intermedia, Prevotella scopos, Prevotellafusca, Lachnospiraeligens and Prevotella melaninogenica.

[0123] In certain embodiments, the method of the present invention further comprises evaluating the pain condition of the subject before and / or after the administration step. In certain embodiments, chronic pain can be evaluated by a reduction in muscle pressure pain threshold (an indicator of chronic muscle pain) and / or skin pain sensation threshold (an indicator of tactile hyperalgesia). In addition, chronic pain can also be evaluated in the following ways: a reduction in the response threshold to mechanical stimulation (such as Randall-Selitto plantar stimulation and clamping stimulation), a reduction in the response threshold to thermal stimulation, a reduction in the response threshold to cold stimulation, a reduction in the response threshold to electrical stimulation, an increase in pain behavior to chemical stimulation, and an increase in spontaneous pain behavior in the absence of stimulation (e.g., foot lifting behavior), etc. However, the method for evaluating chronic pain is not limited thereto.

[0124] For example, chronic pain can be assessed by measuring one or more of: muscle pressure pain threshold (an indicator of chronic muscle pain); a decrease in the skin pain sensation threshold (an indicator of tactile hyperalgesia); cold sensitivity (an indicator of cold hyperalgesia); and heat sensitivity (an indicator of heat hyperalgesia).

[0125] The muscle pressure pain threshold (MPT) is the minimum pressure stimulus that elicits an avoidance response in a subject when gradually increasing pressure is applied to the muscle. Various conventional methods can be used to measure this threshold, depending on the animal species. For example, in rats, the MPT can be measured by applying gradually increasing pressure to the gastrocnemius muscle of the rat's right hind leg. The minimum pressure stimulus that elicits an avoidance response is the MPT.

[0126] The skin pain threshold (TP) is the minimum tactile stimulus magnitude that elicits an avoidance response in a subject when gradually increasing tactile stimuli are applied to the skin. Various conventional methods can be used to measure TP, depending on the subject. For example, in rats, von Frey hair testing is used: von Frey filaments of varying diameters are applied to the plantar surface of the rat's right hind paw. The minimum filament diameter that elicits an avoidance response is the TP.

[0127] Cold sensitivity refers to a subject's reactivity to cold stimulation. Various conventional methods can be used to measure cold sensitivity, depending on the animal species. For example, in rats, cold sensitivity can be measured using the acetone method, which involves dripping acetone onto the rat's hind legs and counting the number of responses.

[0128] Thermal sensitivity refers to a subject's reactivity to thermal stimulation, and can be measured using various conventional methods depending on the animal species. For example, in rats, it can be measured by stimulating the rat's hind paw with infrared heat and observing the hind paw's avoidance reaction.

[0129] On the other hand, depressive symptoms can be assessed using an increase in immobility time in the forced swim test. However, the measurement of depressive symptoms is not limited to this indicator; immobility time in the tail suspension test and spontaneous locomotor activity can also be used as assessment indicators.

[0130] In certain embodiments, the methods of the present invention further comprise assessing the abundance of one or more probiotic strains in the subject before and / or after the administering step. Specifically, the assessing step comprises: collecting a stool sample from the subject, and optionally collecting a stool sample from a healthy subject, before and / or after the administering step; comparing the abundance of the one or more probiotic strains in the subject's stool sample with that in a healthy subject or control sample by metagenomic sequencing; and assessing / determining whether there is a difference in the abundance of the one or more probiotic strains between the subject and the control or healthy subject.

[0131] In certain embodiments of the present invention, the method comprises multiple administrations of the composition of the present invention. The method may comprise administering 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40 or more therapeutically effective doses of a composition comprising the probiotics of the present invention. In certain embodiments, the dosage is administered over a period of 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 10 days, 14 days, 21 days, 30 days or more than 30 days. In addition, the use of the probiotics of the present invention to treat a subject may comprise a single treatment or a series of treatments. It should be understood that the effective dose for treatment may increase or decrease during a specific treatment process. The variation in dosage may manifest according to diagnostic test results known in the art. In certain embodiments of the present invention, the method comprises administering the composition multiple times a day, including but not limited to 2 times, 3 times and 4 times a day.

[0132] In certain embodiments, the present invention provides a method for improving / curing a deficiency or imbalance of one or more probiotic strains in a subject, the method comprising administering to the subject an effective amount of the probiotic strain of the present invention or an effective amount of a composition comprising the probiotic strain. In certain embodiments, the one or more probiotic strains are selected from the group consisting of: Anaerostipes hadrus, Clostridium sp.SY8519, Anaerobutyricum hallii, Flintibacter sp.KGMB00164, Romboutsia hominis, Longibaculum sp.KGMB06250, Alistipes megaguti, Veillonella dispar, Lachnospiraceae bacterium Choco86, Faecalibacterium prausnitzii, Erysipelotrichaceae bacterium GAM147, Streptococcus dysgalactiae, Haemophilus parainfluenzae, Roseburia hominis, Ruminococcus champanellensis, Lachnospiraceae bacterium KM106.2, Lachnospiraceae bacterium, Roseburia intestinalis, Muribaculaceae bacterium DSM108610, Lachnospiraceae bacterium GAM79, Prevotella intermedia, Prevotella scopos, Prevotellafusca, Lachnospira eligens, and Prevotella melaninogenica. In a preferred embodiment, the one or more probiotic strains are selected from Roseburia intestinalis, Lachnospiraceae bacterium, and Roseburia hominis. In a specific embodiment, the subject has been diagnosed with, is suffering from, or is at risk of developing chronic pain. In a specific embodiment, the subject has been diagnosed with or is suffering from a disease associated with chronic pain.

[0133] The probiotics of the present invention or compositions comprising the probiotics can be administered to the treated subject by standard routes, including topical, oral, buccal, bronchial, ocular, sublingual, nasal, topical, intratumoral, transdermal, intraarticular, parenteral (e.g., intravenous, intraperitoneal, intradermal, subcutaneous or intramuscular), intracranial, intracerebral, intraspinal, intravaginal, intrauterine or rectal routes. Administration can also be carried out by other carriers or instruments, such as patches, micelles, liposomes, vesicles, implants (e.g., microimplants), synthetic polymers, microspheres, nanoparticles, etc. Depending on the disease being treated, one approach may be superior to other approaches, as can be determined by those skilled in the art.

[0134] Administration can be performed at different times, such as once or multiple times. In certain embodiments, administration is performed regularly or substantially regularly, such as daily, weekly, monthly, multiples thereof, fractions thereof, or combinations thereof. In certain embodiments, administration is performed daily, multiples thereof, fractions thereof, or combinations thereof. In certain embodiments, administration is performed weekly, multiples thereof, fractions thereof, or combinations thereof. In certain embodiments, administration can be regular, such as once a week during the treatment period, or irregular, such as once a week for a few weeks, then twice a week for a few weeks, or no administration at all. Similarly, in certain embodiments, there may be a rest period between administrations in which no administration is performed. Rest periods can be regular or irregular.

[0135] In certain embodiments, the subject has been diagnosed with chronic nociceptive pain, chronic neuropathic pain, chronic inflammatory pain, arthritis pain, fibromyalgia, breakthrough pain, persistent pain, hyperalgesia, allodynia, central sensitization, peripheral sensitization, disinhibition and facilitation, and / or cancer pain. In specific embodiments, the subject has been diagnosed with herpes zoster or postherpetic neuralgia (PHN), or is experiencing or is at risk for developing chronic pain.

[0136] In certain embodiments, the present invention provides a method for diagnosing a deficiency or imbalance of one or more probiotic strains in a subject, the method comprising: collecting a stool sample from the subject, and optionally collecting a stool sample from a healthy subject who does not have a deficiency or imbalance of one or more probiotic strains; comparing the distribution of all bacteria in the subject's stool sample with that of a control sample by metagenomic sequencing; and identifying and quantifying one or more probiotic strains that are significantly differently abundant between the subject and the control or healthy subjects.

[0137] In certain embodiments, the present invention provides a method for modulating neural activity in one or more brain regions of a subject, the method comprising administering to the subject an effective amount of a probiotic strain of the present invention or an effective amount of a composition comprising the probiotic strain. In preferred embodiments, the one or more brain regions are selected from, for example, the prefrontal cortex (PrL), the dentate gyrus (DG), the bed nucleus of the striatum terminalis (BNST), and the nucleus tractus solitarius (NTS).

[0138] In certain embodiments, the methods of the present invention can be used in conjunction with existing chronic pain treatment options, such as acupuncture, acetaminophen, exercise, physical therapy, breathing exercises, psychotherapy, stress reduction, and trigger point injections.

[0139] In certain embodiments, the pharmaceutical compositions of the present invention have a very fast onset of action, typically within 1 to 180 minutes, 1 to 120 minutes, 1 to 60 minutes, typically within 60 minutes, 50 minutes, 40 minutes, 30 minutes, 20 minutes, 10 minutes or 5 minutes.

[0140] In certain embodiments, the methods of the present invention may further comprise co-administering to the subject one or more (adjuvant) analgesics, which may be selected from, for example: muscle relaxants: baclofen, dantrolene, tizanidine, carisoprodol or cyclobenzaprine; tricyclic antidepressants: amineptine, amitriptyline, amitriptyline oxide, butryline, demetriptyline, dibenzodiazepine; , dimethoprim, dosulpyrine, fluoxetine, isopentanol, clomipramine, desipramine, nortriptyline, doxepin, imipramine, imipramine oxide, trimipramine, lofepramine, maprotiline, metapramine, metactracin, norclomipramine, nortriptyline, noxetine, opipramol, perapram, pizotifen, propiazepine , protriptyline, quinpiramine, reboxetine or tianeptine, or derivatives, prodrugs, stereoisomers and / or salts thereof; tetracyclic antidepressants: amoxapine, aptazapine, cyclosindole, esmitazapine, loxapine, mazindole, metrindole, maprotiline, mianserin, mirtazapine, oxaprotiline, pyrrolidinole, cetipridin, or derivatives, prodrugs, stereoisomers and / or salts thereof; alpha-2 adrenergic agonists: amide epinephrine, amitraz, anisodamine, apraclonidine, brimonidine, cilazoline, clonidine, dexmedetomidine, epinephrine, ergotamine, etilefrine, faldomidine, guanabenzyl, guanethidine, guanfacine, guanoxabenzyl, indanidine, lofexidine, medetomidine, mefentermine, metaraminol , methoxamine, methyldopa, mivacyclidine, naphazoline, norepinephrine, norephedrine, octopamine, oxymetazoline, phenylpropanolamine, rilmenidine, romifidine, synephrine, talipexol, xylazine, or derivatives, prodrugs, stereoisomers and / or salts thereof; selective serotonin reuptake inhibitors: paroxetine, celecoxib, citalopram, dapoxetine, escitalopram, fluoxetine, fluvoxamine, indalpine, paroxetine, sertraline, zimelidine, and diastereomers, derivatives, prodrugs, stereoisomers or salts thereof; serotonin-norepinephrine reuptake inhibitors: duloxetine, desvenlafaxine, levomilnacipran, milnacipran, venlafaxine, or derivatives, prodrugs, stereoisomers and and / or salts thereof; nonsteroidal anti-inflammatory drugs: diclofenac, duloxetine, desvenlafaxine, levomilnacipran, milnacipran, venlafaxine, or derivatives, prodrugs, stereoisomers, and / or salts thereof; opioid receptor agonists: loperamide, alfentanil, allylprodine, alfaprodine, anileridine, benzylmorphine, bezitamide, buprenorphine, butorphanol, clonitrazine, codeine, cyclazocine, normorphine, dextromethorphan, dextromorphamide, dezocine, diaminopropionamide, dimorphone, dihydrocodeine, dihydromorphine, dimenoxadol, dimepeptol, dimethylthiobutene, diphenylethyl ester, diphenylpiperazine, etazocin, ethoxyheptazine, ethylmethylthiobutene, ethylmorphine, etonizine, fentanyl, heroin, hydrocodone, hydromorphine ketone, hydroxymethidine, isomethadone, ketobemidone, levallorphanol, levorphanol, levophenylmorphan, lofentanil, loperamide, meperidine, meptazocine, pentazocine, methadone, methoprene, morphine, melamine, nalbuphine, nalbuphine, nalcein, nicotinoylmorphine, norlevorphanol, normethadone, nalorfen, normorphine, norperirone, opium, oxycodone, oxymorphone, poppy fruit extract, pentazocine, phenadrine, phenadrine, phenammonium, phenazocine, phenoperidine, piminodine, pirithromide, proheptazine, prometazine, propamdol, propiperidine, propipram, propoxyphene, sufentanil, tilidine, tramadol, tapentadol, axomadol, fasiladol, and diastereomers thereof, or derivatives, prodrugs, stereoisomers and / or salts thereof;Local anesthetics: tetracaine, articaine, benzocaine, bupivacaine, chloroprocaine, cinchocaine, cocaine, cyclomethicaine, dibucaine, dimethicaine, etidocaine, ethyl aminobenzoate, eugenol, levobupivacaine, lidocaine, menthol, mepivacaine, neosand anemone toxin, oxycaine, oxybuprocaine, piperocaine, prilocaine, propoxycaine, procaine, proparacaine, ropivacaine, anemone toxin, tetracaine, tetrodotoxin, trimecaine, and diastereomers thereof, or derivatives, prodrugs, stereoisomers and / or salts thereof; benzodiazepines; Class: Adizalam, alprazolam, benzazepam, bretanib, bromazepam, brotizolam, carbamazepam, cildiazepam, ciproazepam, chlordiazepoxide , clomazolam, clobazam, clonazepam, clonazolam, chlordiazepoxide Acid, chlorine nitrogen Acid, clotiazepam, cloxazolam, norclorazepam, diazepam, diclorazepam, fluchloride Ethyl ester, estazolam, etizolam, chlorofluoro Ethyl ester, flubrozepam, flubrozolam, flunitrazepam, flurazepam, flutazepam, flutazolane, halazepam, ketozolam, loprazolam, lorazepam, lormetazepam, medazepam, metazepam, midazolam, nifozepam, nimetazepam, noradizolam, nitrazepam, nordiazepam, oxazepam, phenazepam, pineazepam, prazepam, pramazepam, pyrazole Amobarbital, quazepam, rimazafone, temazepam, tetrazepam, thiophene alprazolam, triazolam, derivatives flumazenil, eszopiclone, zaleplon, zolpidem, zopiclone, or their prodrugs, stereoisomers and / or salts; barbiturates: amobarbital, isobutabarbital, aprobarbital, afenal, barbiturate, barbexalon, bralobarbital, butyral Barbiturate, butalbital, butenylbarbital, butobarbital, barbital, cyclobarbital, cyclopropylbarbital, deoxyphenobarbital, diphenylbarbituric acid, ethylammobarbital, nonobarbital esters, heptylbarbital, hexylbarbital, methylbarbital, methohexital, methylhexenylbarbital, methylphenobarbital, analbital, nibarbital, pentobarbital, phenobarbital, propanol, propanol, propanol bromide, propanol bromide, ribosalfate, sec-butabarbital, secobarbital, sigmodal, talbutabarbital, thioammobarbital, thioammobarbital, thiobutabarbital, thiopental sodium, valprofen, vinylbarbital, vinylbarbital, or derivatives, prodrugs, stereoisomers and / or salts thereof; dimethyl sulfoxide;N-methyl-D-aspartate (NMDA) receptor antagonists: (levorotatory, dextrorotatory, or racemic) ketamine, amantadine, atiganib, caroverine, dextrorphan, dextromethorphan, fullerene, ibogaine, ketamine, lidocaine, memantine, dizocilpine, neramexane, norketamine, dilucimine, 3-fluoro-γ-(3-fluorophenyl)-N-methyl-amphetamine hydrochloride, phencyclidine, tiletamine, remacemide, decahydro-6-(phosphonomethyl)-3-isoquinolinecarboxylic acid, (3S,4aR,6S,8aR)-decahydro-6-(phosphonomethyl)-3-isoquinolinecarboxylic acid, (2R,4S)-rel-4-(1H-tetrazol-5-yl-methyl)-2-piperidinecarboxylic acid, α-Amino-2-(2-phosphonoethyl)-cyclohexanepropionic acid, glutathione, aminomethyl mercaptan, 5-phosphononorvaline, 4-(3-phosphonopropyl)-2-piperazinecarboxylic acid, safortyl, cis-4(phosphonomethyl)-2-piperidinecarboxylic acid, (3E)-2-amino-4-methyl-5-phosphono-3-pentenoic acid, (3E)-2-amino-4-methyl-5-phosphono-3-pentenoic acid 1-ethyl ester, (αS)-α-amino-2′-chloro-5-(phosphonomethyl)-[1,1′-biphenyl]-3-propionic acid, S-nitrosoglutathione, camprostic acid, acamin, conotoxin-G, elitodil, haloperidol, ifenprodil, troxaprid, (R,S)-α-(4-hydroxy)- phenyl)-β-methyl-4-(phenylmethyl)-1-piperidinol, aminocyclopropanecarboxylic acid, 7-chlorokynurenine, D-cycloserine, gavistinide, 4,6-dichloro-3-[(E)-(2-oxo-1-phenyl-3-pyrrolidinyl)methylene]-1H-indole-2-carboxylic acid monosodium salt, lisofylline, 8-chloro-2,3-dihydropyridazino[4,5-b]quinoline-1,4-dione 5-oxide 2-hydroxy-N,N,N-trimethyl-ethylammonium salt, 7-chloro-4-hydroxy-3-(3-phenoxyphenyl)-2(1H)-quinolinone, 3-amino-1-hydroxy-2-pyrrolidinone, 7-chloro-4-hydroxy-2-(4-methoxy-2-methylphenyl)-1, 2,5,10-tetrahydropyrido[4,5-b]quinoline-1,10-dione sodium salt, or derivatives, prodrugs, stereoisomers and / or salts thereof; N-acylethanolamines: N-arachidonoylethanolamine, docosahexaenoylethanolamine, oleoylethanolamine, palmitoylethanolamine, stearoylethanolamine, or derivatives, prodrugs, stereoisomers and / or salts thereof; cannabinoids: cannabinol, cannabidiol, Δ9-tetrahydrocannabinol, Δ8-tetrahydrocannabinol, 11-hydroxy-tetrahydrocannabinol, 11-hydroxy-Δ9-tetrahydrocannabinol, levo-anthraquinone, Δ11-tetrahydrocannabinol, tetrahydrocannabinol, dronabinol, anandamide, nabilone, or derivatives, prodrugs, stereoisomers and / or salts thereof;Antiepileptic compounds: acetazolamide, beclamide, brivaracetam, carbamazepine, valproate hemisodium, eslicarbazepine acetate, ethosuximide, felbamate, gabapentin, lamotrigine, levetiracetam, methsuximide, methazolamide, oxcarbazepine, paramethadione, phenylacetamide, phenylbutyrylamide, phensuximide, potassium bromide, pregabalin, perampanel, primidone, pregabalin, celecoxib, sodium valproate, sultiame, tiagabine, topiramate, trimethadione, valproamide, valproylmorpholine, valproic acid, vigabatrin, zonisamide, or derivatives, prodrugs, stereoisomers or salts thereof, and combinations thereof;

[0141] In certain embodiments, kits for practicing the above methods are provided. The provided kits may include the necessary components for practicing one or more of the above methods. In certain embodiments, the present invention provides a kit for treating chronic pain. The kit may include at least one composition of the present invention and instructions for use.

[0142] Exemplary embodiments of the present invention include, but are not limited to:

[0143] Example 1. A composition for treating chronic pain, comprising a plurality of probiotic strains that are more abundant in healthy individuals than in pain patients.

[0144] Example 2. The composition of Example 1, wherein the probiotic strain is selected from the group consisting of Anaerostipeshadrus, Clostridium sp.SY8519, Anaerobutyricum hallii, Flintibacter sp.KGMB00164, Romboutsia hominis, Longibaculum sp.KGMB06250, Alistipes megaguti, Veillonella dispar, Lachnospiraceae bacterium Choco86, Faecalibacterium prausnitzii, Erysipelotrichaceae bacterium GAM147, Streptococcus dysgalactiae, Haemophilus parainfluenzae, Roseburia hominis, Ruminococcus champanellensis, Lachnospiraceae bacterium KM106.2, Lachnospiraceae bacterium, Roseburia intestinalis, and Muribacula ovata. bacterium DSM 108610, Lachnospiraceae bacteriumGAM79, Prevotella intermedia, Prevotella scopos, Prevotellafusca, Lachnospiraeligens and Prevotella melaninogenica.

[0145] Example 3. The composition of Example 1, wherein the probiotic strain is selected from Roseburia intestinalis, Lachnospiraceae bacterium and Roseburia hominis.

[0146] Example 4. The composition of Example 1, wherein the probiotic strain has analgesic properties.

[0147] Example 5. The composition of Example 1, formulated as a capsule, tablet, powder, liquid or gel for oral administration.

[0148] Example 6. The composition of Example 1, wherein the probiotic strain is capable of modulating communication in the gut-brain axis, thereby producing an analgesic effect.

[0149] Example 7. The composition of Example 1, comprising a concentration of 10 9 CFU of probiotic strains.

[0150] Example 8. A method for treating chronic pain in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of the composition of Example 1.

[0151] Example 9. The method of Example 8, wherein the administering step is performed orally, topically, parenterally, transdermally, nasally, or intraspinal.

[0152] Example 10. The method of Example 8, further comprising diagnosing the subject to determine whether the subject has a deficiency or imbalance in one or more probiotic strains in the composition.

[0153] Example 11. The method of Example 8, wherein the administering step results in an increase in the abundance of one or more probiotic strains in the subject.

[0154] Example 12. The method of Example 8, wherein the effectiveness of the treatment is assessed by measuring the abundance of the probiotic strain in the subject and the pain symptoms before and after administration.

[0155] Example 13. The method of Example 8, wherein the method is combined with an existing chronic pain treatment.

[0156] Example 14. The method of Example 13, wherein the existing chronic pain treatment includes acupuncture, acetaminophen, exercise, physical therapy, breathing exercises, psychotherapy, stress reduction, and / or trigger point injections.

[0157] Example 15. The method of Example 8, wherein the probiotic strain is selected from the group consisting of Anaerostipeshadrus, Clostridium sp.SY8519, Anaerobutyricum hallii, Flintibacter sp.KGMB00164, Romboutsia hominis, Longibaculum sp.KGMB06250, Alistipes megaguti, Veillonella dispar, Lachnospiraceae bacterium Choco86, Faecalibacterium prausnitzii, Erysipelotrichaceae bacterium GAM147, Streptococcus dysgalactiae, Haemophilus parainfluenzae, Roseburia hominis, Ruminococcus champanellensis, Lachnospiraceae bacterium KM106.2, Lachnospiraceae bacterium, Roseburia intestinalis, Muribacula ovata bacterium DSM 108610, Lachnospiraceae bacteriumGAM79, Prevotella intermedia, Prevotella scopos, Prevotellafusca, Lachnospiraeligens and Prevotella melaninogenica.

[0158] Example 16. The method of Example 8, wherein the probiotic strain is selected from Roseburia intestinalis, Lachnospiraceae bacterium, and Roseburia hominis.

[0159] Example 17. The method of Example 8, wherein the chronic pain comprises pain caused by selective sciatic nerve branch injury (SNI).

[0160] Example 18. The method of Example 8, wherein the subject has been diagnosed with herpes zoster and / or postherpetic neuralgia (PHN).

[0161] Example 19. The method of Example 8, further comprising assessing the abundance of one or more probiotic strains in the subject before and / or after the administering step.

[0162] Example 20. A method for treating chronic pain in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a composition comprising Roseburia intestinalis, Lachnospiraceae bacteria, and / or Roseburia hominis.

[0163] Example 21. A method for treating chronic pain in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a sterile secretion filtrate of Roseburia intestinalis.

[0164] Example

[0165] All patents, patent applications, provisional applications, and publications cited herein are incorporated by reference in their entirety, including all figures and tables, unless not inconsistent with the explicit teachings of this specification.

[0166] The following are examples of procedures for implementing the present invention. These examples should not be construed as limiting. Unless otherwise indicated, all percentages are by weight and all solvent mixing ratios are by volume.

[0167] Materials and Methods

[0168] Subject recruitment and sample collection

[0169] From January 2020 to December 2021, a total of 115 patients with herpes zoster, 28 patients with postherpetic neuralgia (PHN), and 63 healthy subjects were recruited for fecal metagenomic analysis at Tongji Shenzhen Hospital, Huazhong University of Science and Technology. All fecal samples were collected, aliquoted into cell culture cryovials, and immediately stored at -80°C. Patients with herpes zoster or PHN were diagnosed by physicians at the clinic. Age- and sex-matched participants who did not report any acute or persistent pain in the past 3 months were considered healthy controls. Exclusion criteria were as follows: (1) use of antibiotics in the past 3 months; (2) use of probiotics or yogurt in the past 3 months; and (3) vegetarian diet. All participants signed written informed consent. The study protocol was approved by the Clinical Research Ethics Committee of Tongji Shenzhen Hospital, Huazhong University of Science and Technology.

[0170] Fecal DNA extraction

[0171] Using Quick-DNA TM Fecal DNA was extracted using a fecal / soil microbial cartridge kit (Zymo Research, Irvine, CA, USA) according to the manufacturer's instructions. Approximately 150 mg of fecal sample was added to a 750 μl BashingBead TM ZR BashingBead BufferTM The supernatant was collected and added to a Zymo-Spin TM The mixture was pre-washed with 200 μl DNA pre-wash buffer and 500 μl g-DNA wash buffer and then transferred to a Zymo-Spin TM The DNA was then eluted by centrifugation at 10,000 × g for 1 minute. Finally, 100 μl of DNA elution buffer was added directly to the column matrix and centrifuged at 10,000 × g for 30 seconds to elute the DNA.

[0172] Metagenomic sequencing and taxonomic annotation

[0173] Whole-genome shotgun sequencing of samples was performed on the Novasek 6000 platform of Novogene Co., Ltd (Guangzhou, China). An average of 54 ± 3.6 million reads (15 GB of data) were obtained for each sample. The raw reads were first trimmed using Trimmomatic (Aachen, Germany, version 0.39) to remove low-quality reads and adapters, and then the reads were aligned to the host genome (human genome assembly GRCh38) using Bowtie2 (San Diego, USA, version 2.4.4) to remove host reads, using the "very-sensitive" default parameter setting. The filtered reads were then annotated and classified using Kraken2 (Columbia, USA, version 2.0.8). Bracken was used to generate accurate genus and species-level abundance estimates based on the results of Kraken2.

[0174] Analysis of differentially abundant bacteria

[0175] The Shannon diversity index was used to estimate the species richness (observed index) and Pielou evenness of the microbial communities and was calculated using the R packages phyloseq and vegan. The differences in microbial community composition between the different groups were visualized based on the Bray-Curtis dissimilarity matrix. Permutational multivariate analysis of variance (PERMANOVA, Molndal, Sweden) was performed using the adonis function in the R package vegan to test the differences between the healthy group and the other groups. After FDR correction, an adjusted P value of less than 0.05 was considered statistically significant. To identify biomarkers with statistically significant differences between the groups, a linear discriminant analysis effect size (LEfSe) analysis was performed. The statistical significance of the different bacteria was determined using the Mann-Whitney test, with an adjusted P value of less than 0.05.

[0176] Bacterial strains and culture conditions

[0177] Roseburia intestinalis [DSM 14610] (RI), Lachnospiraceae bacteria [DSM 24404], and Roseburia hominis [DSM 16839] were purchased from the German Collection of Microorganisms and Cell Cultures (DSMZ). Roseburia intestinalis [DSM 14610] and Roseburia hominis [DSM 16839] were cultured in modified YCFA medium (DSMZ medium 1611), while Lachnospiraceae bacteria [DSM 24404] were cultured in minced meat medium containing carbohydrates (DSMZ medium 110) at 37°C under aerobic conditions for 24 h.

[0178] Selective sciatic nerve injury (SNI) surgery

[0179] C57BL / 6 mice underwent SNI surgery to induce behaviors associated with chronic pain. The left sciatic nerve was exposed at the level where the sciatic nerve bifurcates into the sural, tibial, and common peroneal nerves. The tibial and common peroneal nerves were tightly ligated and completely severed using 6.0 silk sutures, leaving the sural nerve intact. In the sham-operated group, only the sciatic nerve was exposed. After surgery, mice were placed in a clean cage with a paper towel and a heating pad under the cage to recover from anesthesia in a comfortable position.

[0180] Oral gavage

[0181] 1×10 9 Colony-forming units (CFU) of Roseburia intestinalis [DSM 14610], Roseburia hominis [DSM 16839], or Lachnospiraceae bacteria [DSM 24404] were resuspended in 100 μL of saline and administered to mice by oral gavage. A separate group used modified minced meat medium as a control medium.

[0182] Oral administration of Roseburia intestinalis sterile secretion filtrate

[0183] Roseburia intestinalis was inoculated into a modified minced meat medium and cultured under anaerobic conditions for 24 hours. The culture was then centrifuged at 5000 rpm for 10 minutes. The supernatant was filtered through a 0.22 μm filter and used as the Roseburia intestinalis sterile secretion filtrate. The supernatant was administered to mice via oral gavage (100 μl / time).

[0184] von Frey test

[0185] Mice are placed individually in a Plexiglas cube with a metal grid floor and adapted for at least 30 minutes until the mice are quiet. Mechanical hyperalgesia is measured using a series of 10 von Frey filaments (Bioseb, bending forces are 0.008, 0.02, 0.07, 0.16, 0.4, 0.6, 1, 1.4, 2 and 4 grams respectively). Von Frey filaments are applied to the distal region of the paw. A positive reaction is defined as the retraction of the test paw associated with the stimulus. Paw retraction threshold (PWT) is determined by the Dixon up-and-down method. PWT is calculated as the mean value of at least 3 measurements, with a 5-minute interval between each measurement.

[0186] Acupuncture test

[0187] Mice were individually placed in a Plexiglas cube with a metal grid floor and allowed to acclimate for at least 30 minutes, until they were quiet. A blunt-tipped steel needle was gently applied to the plantar surface of the mouse's hind paw without piercing the skin. Positive responses associated with the stimulus (e.g., paw lifting, shaking, or licking) were recorded. The test was repeated 10 times every 10 minutes, and paw withdrawal responses were calculated as the percentage of positive responses after the stimulus.

[0188] Acetone evaporation test

[0189] Mice were individually placed in a Plexiglas cube with a metal grid floor and allowed to acclimate for at least 30 minutes until the mice were quiet. Using a 500 μL syringe tip, 50 μL of acetone (100012, Merck, New Jersey, USA) was dropped onto the surface of the left hind limb of the mouse, and the latency to paw withdrawal (in seconds) was recorded. The paw withdrawal latency (PWL) was calculated as the average of three measurements recorded at 10-minute intervals. The minimum and maximum cutoff values ​​were set to 1 second and 30 seconds, respectively.

[0190] Mechanical stimulation induces c-Fos expression

[0191] c-Fos expression was induced in mice by dynamic brush stimulation. Mice were anesthetized with 2%-3% isoflurane using an isoflurane anesthesia machine (R520, RWD Life Sciences). The surface of the mouse hind paw was gently brushed 150 times in 5 minutes, repeated 3 times, with a 1 minute interval between each time (a total of 450 times). Mice were sacrificed 1.5 hours after stimulation, and the spinal cord and brain were collected.

[0192] Tissue sample collection and frozen sections

[0193] Mice were anesthetized by intraperitoneal injection of a mixture of 100 mg / kg ketamine and 15 mg / kg xylazine. Forceps and dissecting scissors were used to completely expose the chest cavity of the mouse without damaging organs such as the heart, liver, and blood vessels. The ribs were cut to expose the heart, and then the needle was inserted into the tip of the left ventricle. After fixing the needle inserted into the heart with hemostats, the right atrium was immediately cut off. The heart was perfused at a slow rate, first with 50 mL of 0.9% saline, and then with 50 mL of 4% paraformaldehyde solution. When the perfusion fluid flowing out of the heart became clear and the liver and limbs of the mouse became pale, the perfusion was stopped. The skin and skull cap on the skull were removed to collect the entire brain. The brain was post-fixed in 4% paraformaldehyde solution for 24 hours, then dehydrated in 15% sucrose solution for 24 hours, and then dehydrated in 30% sucrose solution for 24 hours (4°C). After dehydration, the brains were embedded in OCT embedding medium (Tissue-Tek OCT embedding medium, catalog number 4583, Sakura Fintech, Torrance, CA, USA) and frozen at −20° C. The frozen brains were cut into 20 μm thick sections using a freezing microtome (Cryostat NX70, Thermo Scientific, Waltham, MA, USA) for immunofluorescence staining.

[0194] Immunofluorescence staining

[0195] The brain slices were placed in a 12-well plate (Nunc TMCell culture treated multiwell plates (Cat. No. 150628, ThermoScientific) were placed in a PBS-treated plate and washed three times with PBS for 5 minutes each. Subsequently, the sections were incubated with 400 μL of blocking solution (10% goat serum + 0.003% Triton X-100 in PBS) at room temperature for 60-90 minutes. The blocking solution was carefully removed with a pipette tip, and 400 μL of c-Fos antibody solution (diluted to 1:1000 with blocking solution, c-Fos antibody - Cat. No. 226017, Synaptic Systems) was added to each well and incubated overnight at 4°C. The primary antibody solution was removed the next day, and the sections were washed three times with PBS for 5 minutes each. Then, the sections were incubated in the dark with secondary antibody solution (diluted to 1:1000 in blocking solution, goat anti-rat IgG (H+L), cross-adsorbed secondary antibody, AlexaFluor 555, Cat. No.: A-21434, Invitrogen) at room temperature for 60-90 minutes. The sections were washed three times with PBS for 5 minutes each and then carefully transferred to microscope slides (Menzel Plus white adhesive microscope slides, 90° chamfered, Cat. No. J1800AMNZ, Epidia (Kalamazoo, MI, USA). Finally, the sections were covered with a cover slip and then left horizontally in the dark to dry for 24 h before imaging.

[0196] Chemical sympathectomy

[0197] This study used 6-hydroxydopamine (6-OHDA, Catalog No. HY-B1081A, MedChemExpress (South Brunswick, NJ, USA)) for sympathetic denervation. Three days before RI administration, mice were treated with an intraperitoneal injection of 80 mg / kg of 6-OHDA (dissolved in sterile saline containing 0.1% L-ascorbic acid, Catalog No. HY-B0166, MedChemExpress). A control group received sterile saline containing 0.1% L-ascorbic acid as a vehicle. Thereafter, mice received an injection of 80 mg / kg of 6-OHDA every 10 days to maintain sympathetic denervation until the end of the experiment.

[0198] Vagotomy

[0199] Mice were anesthetized by intraperitoneal injection of a mixture of 100 mg / kg ketamine and 12.5 mg / kg xylazine. The anesthetized mice were placed in the supine position, and the hair from the chest to the bladder area was shaved to prepare the surgical field. After disinfecting the surgical field, a 1 cm longitudinal incision was made to expose the stomach and esophagus without damaging the liver. The anterior and posterior vagus nerve branches of the esophagus were separated and resected. The connective tissue around the esophagus was removed to ensure that the vagus nerve branches were completely severed. Finally, the abdominal muscle layer and skin were sutured with 4-0 sutures. The mice were placed on a clean electric heating blanket after surgery until they recovered from anesthesia.

[0200] Stereotaxic tracer virus injection

[0201] Mice were anesthetized by intraperitoneal injection of a mixture of 100 mg / kg ketamine and 12.5 mg / kg xylazine. The anesthetized mice were fixed in a stereotaxic apparatus and their eyes were moistened with ophthalmic ointment. The hair between the ears was shaved. An approximately 1.5 cm anterior-posterior incision was made on the top of the mouse's head. A cotton swab was used to clean the skull surface to allow visualization of the Bregma and Lambda. The tip of the syringe was fixed to the stereotaxic apparatus, pointing toward the Bregma, and the three axes (anteroposterior, left-right, and dorsoventral) were set to zero when the Bregma and Lambda were in the horizontal plane. Injections were performed in the following two brain regions: CeA (anteroposterior axis -1.37 mm, left-right axis ±3 mm, dorsoventral axis 4.75 mm) and NTS (anteroposterior axis -7.32 mm, left-right axis ±0.5 mm, dorsoventral axis 4.3 mm). The virus was injected at a rate of 50 nanoliters per minute using a Hamilton syringe with a glass capillary. After injection, the syringe was kept in place for 10 minutes to prevent leakage. Finally, the skin was closed with 4-0 sutures and the mouse was placed on a clean electric heating blanket until recovery from anesthesia.

[0202] In vivo chemical genetic manipulation

[0203] To inhibit neuronal activity in the nucleus tractus solitarius (NTS), AAV2 / 9-EF1a-DIO-hM4D(Gi)-mCherry-WPREs (Pt0043, BrainVTA, China) and AAV2 / 1-hSyn-Cre-WPRE-hGH-pA (Pt0010, BrainVTA, China) were mixed at a 1:1 ratio in a total volume of 200 nL and then injected into the NTS. To inhibit neuronal activity in the CeA-NTS pathway, 200 nL of rAAV-EF1a-DIO-hM4D(Gi)-mCherry-WPREs (AAV9, Pt0043, BrainVTA, China) were injected into the NTS, and 200 nL of rAAV-hSyn-Cre-WPRE-hGH retroAAV (Pt 0136, BrainVTA, China) were injected into the CeA. To activate neuronal activity in the NTS-CeA, 200 nanoliters of rAAV-hSyn-Cre-WPRE-hgh (AAV1, PT 0136, BrainVTA, China) were injected into the NTS, and 200 nanoliters of rAAV-EF1a-DIO-hM3D(Gq)-mCherry-WPREs (AAV9, PT 0891, BrainVTA, China) were injected into the CeA. On the same day, mice received RI or vehicle treatment by oral gavage until the end of the experiment. After a 2-week recovery period, SNI surgery was performed. On days 21 (day 7 after SNI surgery) and 28 (day 14 after SNI surgery) after viral injection, pain behavior tests (von Frey test, pinprick test, and acetone vaporization test) were performed 1 hour after intraperitoneal injection of 2 mg / kg clozapine N-oxide (CNO, BrainVTA, China).

[0204] Statistical analysis

[0205] The independent-sample Student's t-test was used to compare differences between two groups. Two-way analysis of variance (ANOVA, San Francisco, USA) followed by multiple comparisons was used to compare differences between multiple groups. A P value of less than 0.05 was considered statistically significant.

[0206] Example 1 - Significantly different bacterial strains between healthy individuals and patients with neuropathic pain

[0207] Metagenomic sequencing was performed on stool samples from healthy individuals (n = 63), patients with herpes zoster (n = 115), and patients with postherpetic neuralgia (PHN) (n = 28). The alpha diversity of the microbial community, as assessed by the Shannon diversity index, was significantly higher in healthy individuals than in patients with herpes zoster or PHN (healthy individuals vs. herpes zoster, P = 0.011; healthy individuals vs. PHN, P = 0.0051; see Figure 1A ). Principal coordinate analysis (PCoA) was also performed to explore the differences in microbiome composition between the groups. Further PERMANOVA tests confirmed that the gut microbiome composition of healthy individuals was significantly different from that of patients with herpes zoster or PHN (PERMANOVA test based on the Bray-Curtis difference matrix P = 0.001; post hoc test, healthy individuals vs. herpes zoster P = 0.0015; healthy individuals vs. PHN P = 0.0015; see Figure 1B ). The Wilcoxon rank sum test was then applied to identify species with different abundances between the groups. Compared with healthy individuals, patients with herpes zoster showed a depletion of 25 bacterial species and an enrichment of 8 bacterial species (adjusted P values ​​< 0.05; see Figure 1C LEfSe analysis also determined that these bacteria with different abundances contributed to the between-group differences between healthy individuals and patients with herpes zoster (adjusted P value < 0.05; see Figure 1D ). PHN patients showed enrichment of 3 and depletion of 11 bacterial species compared with healthy individuals. Interestingly, these bacteria with different abundances also differed between healthy individuals and herpes zoster patients (adjusted P values ​​< 0.05; see Figures 1E-1F These bacteria are key to explaining the differences in the gut microbiome between healthy individuals and PHN patients. Taken together, these analyses highlighted alterations in the gut microbiome in patients with herpes zoster or PHN compared with healthy individuals.

[0208] Example 2 – Roseburia intestinalis treatment alleviates SNI-induced pain hypersensitivity in male and female mice

[0209] The effects of Roseburia intestinalis supplementation on SNI-induced pain hypersensitivity were investigated in male and female mice. The mice were randomly divided into three groups: a sham surgery group that only underwent sciatic nerve exposure (Sham group), a culture medium-treated group that underwent SNI surgery (SNI+culture medium group), and a group that underwent SNI surgery followed by Roseburia intestinalis supplementation (SNI+RI group). Figures 2A-2FAs shown, mice showed significant increases in mechanical and thermal sensitivity 7 days after SNI surgery, and Roseburia intestinalis treatment increased the paw withdrawal threshold (PWT) and paw withdrawal latency (PWL) in male and female mice in the von Frey test and acetone vaporization test, respectively. Furthermore, Roseburia intestinalis supplementation also reduced the response rate to pinprick stimulation. This analgesic-like effect persisted for at least 21 days after SNI surgery.

[0210] Example 3 – Roseburia intestinalis treatment modulates neuronal activity in multiple brain regions

[0211] The mice were divided into two groups: a culture medium-treated group that underwent SNI surgery (SNI group) and a group that underwent SNI surgery followed by Roseburia intestinalis supplementation (SNI+RI group). Figures 3A-3D As shown, compared with the SNI group, the SNI+RI group had increased numbers of c-Fos-positive neurons in multiple brain regions, including the prefrontal cortex (PrL) ( Figure 3A )、dentate gyrus (DG) ( Figure 3B )、BNST ( Figure 3C ) and nucleus tractus solitarius (NTS) ( Figure 3D On the other hand, RI supplementation decreased agranular insular cortex (AI) ( Figure 4A ), paraventricular nucleus of the hypothalamus (PVN) ( Figure 4B ) and the central nucleus of the amygdala (CeA) ( Figure 4C ) of c-Fos expression.

[0212] Example 4 – Vagotomy, but not chemical sympathectomy, abolishes the analgesic effect of Roseburia intestinalis

[0213] Notably, the nucleus tractus solitarius (NTS), a center for the transmission of information from the autonomic nervous system to higher-order central nervous system regions, was activated after Roseburia intestinalis supplementation. This result suggests that neural circuits involving the vagus nerve, NTS, and other central nervous system regions may be involved in the analgesic effects mediated by Roseburia intestinalis.

[0214] To evaluate the role of the vagus nerve in Roseburia intestinalis-mediated analgesia, mice underwent vagotomy before Roseburia intestinalis treatment. The mice were randomly divided into three groups: a group that underwent SNI surgery followed by medium treatment (SNI+medium), a group that underwent SNI surgery followed by Roseburia intestinalis administration (SNI+RI), and a group that underwent vagotomy and Roseburia intestinalis treatment after SNI surgery (SNI+RI+vagotomy).

[0215] Consistent with the above test results, SNI surgery induced pain hypersensitivity, while Roseburia intestinalis supplementation completely abolished the development of chronic pain-like behaviors. Notably, the analgesic effect of Roseburia intestinalis was abolished after vagotomy, as evidenced by comparable paw withdrawal thresholds (von Frey test, Figure 5A ), acupuncture reaction rate ( Figure 5B ) and paw withdrawal latency (acetone test, Figure 5C ) was confirmed. As a control, 6-hydroxydopamine (6-OHDA) was also used to eliminate the sympathetic nervous system. The mice were divided into 4 groups, namely the group that underwent SNI surgery (SNI), the group that underwent SNI surgery followed by Roseburia intestinalis supplementation (SNI+RI), the group that underwent 6-OHDA-induced sympathectomy followed by SNI surgery (SNI+6-OHDA), and the group that underwent 6-OHDA-induced sympathectomy and SNI surgery followed by Roseburia intestinalis treatment (SNI+RI+6-OHDA). The study found that chemical elimination of the sympathetic nervous system did not affect the analgesic effect mediated by Roseburia intestinalis ( Figures 5E-5F Taken together, these results suggest that Roseburia intestinalis-induced gut-brain pain-relief signaling is mediated through the vagus nerve rather than the sympathetic nervous system.

[0216] Example 5—Structural Projections of the Vagus-NTS-CeA Pathway

[0217] Next, we investigated the downstream brain regions of the vagus nerve-NTS pathway. The anterograde neural tracer HSV-EGFP (BrainVTA, China) was injected into the NTS, and brain tissue was collected 24 hours after tracer injection. The EGFP tracer signal was detected at the NTS injection site ( Figure 6C ) and CeA( Figure 6D On the other hand, after the retrograde tracer PRV-CMV-EGFP (PT01001, BrainVTA, China) was injected into the CeA, the injection site ( Figure 6A ) and EGFP markers were observed in NTS ( Figure 6B ), which confirmed the direct projection of NTS to CeA. At the same time, after unilateral injection of PRV-EGFP into NTS, strong expression of EGFP signal was observed in the vagal ganglion ( Figures 6E-6F ), confirming that the vagus nerve projects to structures in the NTS. These findings delineate a vagus-NTS-CeA neural circuit ( Figure 6G ).

[0218] Example 6 – In vivo chemogenetic inhibition of NTS projections reverses the analgesic effect of Roseburia intestinalis

[0219] To further determine the functional role of the proposed vagus nerve-NTS-CeA circuit, a series of chemical genetic experiments were performed in the NTS and CeA. AAV2 / 9-EF1a-DIO-hM4D(Gi)-mCherry-WPREs (Pt0043, BrainVTA, China) and AAV2 / 1-hSyn-Cre-WPRE-hGH-pA (Pt0010, BrainVTA, China) were mixed at a 1:1 ratio in a total volume of 200 nL and then injected into the unilateral NTS ( Figure 7A ). The activity of NTS neurons was silenced by chemical genetics to rule out the role of the NTS in transmitting information from the vagus nerve to the anterior brain. Similar to the effect of vagotomy, chemical genetic silencing of NTS neurons reversed the relief of RI-induced mechanical and thermal sensitivity ( Figures 7B-7D ).

[0220] In addition, to silence NTS neurons projecting to the CeA, 200 nL of rAAV-EF1a-DIO-hM4D(Gi)-mCherry-WPREs (AAV9, Pt0043, BrainVTA, China) was injected into the unilateral NTS, and simultaneously 200 nL of rAAV-hSyn-Cre-WPRE-hGH retro AAV (Pt 0136, BrainVTA, China) was injected into the unilateral CeA ( Figure 7E Selective inhibition of NTS neurons projecting to the CeA reversed the relief of mechanical and thermal sensitivity induced by RI supplementation ( Figures 7F-7H ).

[0221] Example 7 – Chemogenetic activation of NTS-projecting CeA neurons reverses the analgesic effect of Roseburia intestinalis administration

[0222] To chemically activate NTS-projecting CeA neurons, 200 nL of rAAV-hSyn-Cre-WPRE-Hgh (AAV1, Pt 0136, BrainVTA, China) was injected into the unilateral NTS, and simultaneously 200 nL of rAAV-EF1a-DIO-hM3D(Gq)-mCherry-WPREs (AAV9, Pt 0891, BrainVTA, China) was injected into the unilateral CeA ( Figure 8A Activation of CeA neurons projecting from the NTS abolished the analgesic function of Roseburia intestinalis, as evidenced by a significant decrease in the paw withdrawal threshold (PWT) and paw withdrawal latency (PWL) in the von Frey test and acetone vaporization test, as well as an increase in the response frequency after acupuncture stimulation in the SNI+RI+hM3D-CNO group compared with the SNI+RI group ( Figures 8B-8D ).

[0223] Example 9 - Treatment with sterile secretion filtrate of Roseburia intestinalis alleviates SNI-induced pain hypersensitivity in mice

[0224] The sterile secretion filtrate of Roseburia intestinalis was administered using the same dosing regimen as that for Roseburia intestinalis. Compared to the control culture medium, the sterile secretion filtrate of Roseburia intestinalis significantly reduced hyperalgesia in SNI mice. This therapeutic effect was abolished in vagotomized mice, suggesting that the analgesic active ingredient of Roseburia intestinalis is secreted into the filtrate. This also demonstrates that the sterile secretion filtrate of Roseburia intestinalis has the same analgesic effect and mechanism. Figure 11 ).

[0225] Example 8 - Roseburia hominis treatment alleviates SNI-induced pain hypersensitivity in mice

[0226] Roseburia hominis was administered using the same dosing regimen as Roseburia intestinalis. Roseburia hominis is one of the microorganisms that is significantly reduced in patients with neuropathic pain (e.g., herpes zoster or PHN) compared with healthy individuals (see Figure 1D and Figure 1FCompared with the culture medium control group, Roseburia hominis significantly reduced the paw withdrawal threshold (PWT) in the von Frey test, as well as the response rate and hyperalgesia score in the pinprick test on days 7, 14, and 21 after SNI. However, Roseburia hominis had no effect on SNI-induced cold hyperalgesia ( Figures 9A-9D ).

[0227] Example 9 - Lachnospiraceae bacteria treatment alleviates SNI-induced pain hypersensitivity in mice

[0228] Lachnospiraceae bacteria were administered using the same dosing regimen as Roseburia intestinalis. Lachnospiraceae bacteria were also among the microorganisms significantly reduced in patients with neuropathic pain compared with healthy individuals (see Figure 1D and Figure 1F Compared with the culture medium control group, Lachnospiraceae bacteria significantly reduced the paw withdrawal threshold (PWT) in the von Frey test, as well as the response rate and hyperalgesia score in the pinprick test on days 7, 14, and 21 after SNI surgery. As for cold hyperalgesia, Lachnospiraceae bacteria supplementation showed an analgesic-like effect on days 14 and 21 after SNI surgery ( Figures 10A-10D ).

[0229] It should be understood that the examples and embodiments described herein are for illustrative purposes only, and that those skilled in the art may propose various modifications or changes based on these contents, and that these modifications or changes should be included in the spirit and scope of this application and protected by the appended claims. In addition, any element or limitation in any invention or embodiment disclosed herein may be combined with any other element or limitation disclosed herein (alone or in any combination) or any other invention or embodiment, and all such combinations are within the scope of the present invention without limitation.

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Claims

1. A composition for treating chronic pain, comprising a plurality of probiotic strains that are more abundant in healthy individuals than in pain patients.

2. The composition of claim 1 , wherein the probiotic strain is selected from the group consisting of Anaerostipes hadrus, Clostridium sp.SY8519, Anaerobutyricum hallii, Flintibacter sp.KGMB00164, Romboutsia hominis, Longibaculum sp.KGMB06250, Alistipes megaguti, Veillonella dispar, Lachnospiraceae bacterium Choco86, Faecalibacterium prausnitzii, Erysipelotrichaceae bacterium GAM147, Streptococcus dysgalactiae, Haemophilus parainfluenzae, Roseburia hominis, Ruminococcus champanellensis, Lachnospiraceae bacterium KM106.2, Lachnospiraceae bacterium, Roseburia intestinalis, Muribacula ovata, bacterium DSM 108610, Lachnospiraceae bacteriumGAM79, Prevotella intermedia, Prevotella scopos, Prevotellafusca, Lachnospiraeligens and Prevotella melaninogenica.

3. The composition of claim 1, wherein the probiotic strain is selected from the group consisting of Roseburia intestinalis, Lachnospiraceae bacterium and Roseburia hominis.

4. The composition of claim 1, wherein the probiotic strain has analgesic properties.

5. The composition of claim 1, formulated as a capsule, tablet, powder, liquid or gel for oral administration.

6. The composition of claim 1, wherein the probiotic strain is capable of modulating communication of the gut-brain axis, thereby producing an analgesic effect.

7. The composition of claim 1, comprising a concentration of 10 9 CFU of probiotic strains.

8. A method for treating chronic pain in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of the composition of claim 1.

9. The method of claim 8, wherein the administering step is performed by an oral, topical, parenteral, transdermal, nasal or intraspinal route.

10. The method of claim 8, further comprising diagnosing the subject to determine if the subject has a deficiency or imbalance in one or more probiotic strains in the composition.

11. The method of claim 8, wherein the administering step results in an increase in the abundance of the one or more probiotic strains in the subject.

12. The method of claim 8, wherein the effectiveness of the treatment is assessed by measuring the abundance of the probiotic strain in the subject and the pain symptoms before and after administration.

13. The method of claim 8, wherein the method is combined with an existing chronic pain treatment.

14. The method of claim 13, wherein the existing chronic pain treatment comprises acupuncture, acetaminophen, exercise, physical therapy, breathing exercises, psychotherapy, stress reduction, and / or trigger point injections.

15. The method of claim 8, wherein the probiotic strain is selected from the group consisting of Anaerostipes hadrus, Clostridium sp.SY8519, Anaerobutyricum hallii, Flintibacter sp.KGMB00164, Romboutsia hominis, Longibaculum sp.KGMB06250, Alistipes megaguti, Veillonella dispar, Lachnospiraceae bacterium Choco86, Faecalibacterium prausnitzii, Erysipelotrichaceae bacterium GAM147, Streptococcus dysgalactiae, Haemophilus parainfluenzae, Roseburia hominis, Ruminococcus champanellensis, Lachnospiraceae bacterium KM106.2, Lachnospiraceae bacterium, Roseburia intestinalis, Muribacula ovata, bacterium DSM 108610, Lachnospiraceae bacteriumGAM79, Prevotella intermedia, Prevotella scopos, Prevotellafusca, Lachnospiraeligens and Prevotella melaninogenica.

16. The method of claim 8, wherein the probiotic strain is selected from the group consisting of Roseburia intestinalis, Lachnospiraceae bacterium, and Roseburia hominis.

17. The method of claim 8, wherein the chronic pain comprises pain caused by selective sciatic nerve branch injury (SNI).

18. The method of claim 8, wherein the subject has been diagnosed with herpes zoster and / or postherpetic neuralgia (PHN).

19. The method of claim 8, further comprising assessing the abundance of one or more probiotic strains in the subject before and / or after the administering step.

20. A method for treating chronic pain in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a composition comprising Roseburia intestinalis, Lachnospiraceae bacteria, and / or Roseburia hominis.

21. A method for treating chronic pain in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a sterile secretory filtrate of Roseburia intestinalis.

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