WO2025043142A1

WO2025043142A1 - Compositions comprising lactobacillus strains, and methods of treating osteoarthritis and osteoarthritis co-morbidities

Info

Publication number: WO2025043142A1
Application number: PCT/US2024/043547
Authority: WO
Inventors: Hee-Jeong Im SAMPEN; InSug O-SULLIVAN; Gurjit Singh
Original assignee: The United States Government As Represented By The Department Of Veterans Affairs; The Board Of Trustees Of The University Of Illinois
Priority date: 2023-08-23
Filing date: 2024-08-23
Publication date: 2025-02-27

Abstract

Disclosed herein are compositions comprising or consisting of Lactobacillus acidophilus and one or more prebiotics. Also disclosed herein are methods for treating osteoarthritis, inhibiting or preventing cartilage degeneration, and reducing or inhibiting pain-associated depression in subjects with joint pain with compositions comprising or consisting of Lactobacillus acidophilus and one or more prebiotics.

Description

COMPOSITIONS COMPRISING LACTOBACILLUS STRAINS, AND METHODS OF TREATING OSTEOARTHRITIS AND OSTEOARTHRITIS CO-MORBIDITIES

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of the filing date of U.S. Provisional Application No. 63/578,275, filed on August 23, 2023. The content of this earlier filed application is hereby incorporated by reference in its entirety.

BACKGROUND

Osteoarthritis (OA) is the most common form of arthritis. It is a leading cause of debilitating pain and disability, affecting more than 32 million in the U.S. Worse, the prevalence and incidence of OA have been increasing due to increases in lifespan and obesity and are expected to rise to more than 78 million in the U.S. by 2040. The association of an elevated OA incidence with the military population is particularly significant as a leading cause of disability among the military population.

The annual economic burden of $185 billion caused by OA disease is alarming. Unfortunately, clinically accepted treatment strategies do not cure OA, are often ineffective, and there is over-reliance on opioids for pain. Recent insurance claim data indicate that over 50% of OA patients have been treated with opioids, and many have become opioiddependent. These high rates of chronic pain and subsequent opioid over-treatment have become a significant health concern in this society among military and civilian populations in the U.S. Many cases eventually require joint replacement with a prosthesis, which is costly, and the limited functional life of prostheses (~10 y) can make a second replacement necessary’. These factors increase the overall cost of treatment and the risk for associated morbidity. Importantly, surgical procedures to address OA typically do not result in a pain- free cure. Despite the major negative impact of chronic pain on quality of life and health care management, there is no way as yet to cure or prevent its progression. Also, there is no OA- specific anti -pain medication. Thus, there is an urgent, unmet need to identify OA targets and develop OA disease-modifying drugs to control chronic joint pain and halt OA pathological progression.

More recently, serious concerns have been raised regarding OA-related chronic comorbid health conditions. For example, individuals with OA have a 2.5-times greater risk of having three or more other chronic conditions. Compared to civilians, veterans are far more vulnerable to a chronic comorbid health condition (Richardson LM, et al., SAGE Open Medicine, 2016. 4: 1-118; and Clark-Raymond A and Halaris A. J Psychiatric Research, 2013 47: 1080) more than a third of veterans suffer from at least two such chronic conditions (e.g.. OA and depression) (Nowacka-Chmielewska MM, et al., Experimental and Therapeutic Medicine 2017, 13:723). In particular, OA patients show a higher prevalence of the devastating symptoms of depression and anxiety than those without OA. Having depression showed magnified and persistent joint pain sensitization in OA patients, which, in turn, worsens the depression. As OA is the most prevalent form of arthritis, depression is the most prevalent psychiatric disorder, ranking in the top five leading causes of disability worldwide (Zheng S, et al., BMC Musculoskeletal Disorders 2021 22:4013). Every day, 17.6 U.S. veterans commit suicide, primarily due to the ramifications of untreated depression (2020 National Veteran Suicide Prevention Annual Report. Office of Mental Health and Suicide Prevention. VA). These reports indicate an urgent unmet need to improve treatment strategies to manage OA symptoms and prevent depression, a comorbid chronic disease in OA patients.

SUMMARY

Disclosed herein are compositions comprising or consisting of Lactobacillus acidophilus and one or more prebiotics.

Disclosed herein are compositions comprising or consisting of Lactobacillus acidophilus and fructooligosaccharide.

Disclosed herein are compositions comprising or consisting of Lactobacillus acidophilus, fructooligosaccharide, and butyrate.

Disclosed herein are methods of inhibiting cartilage degeneration in a subject, the methods can comprise administering to the subject in need thereof a therapeutically effective amount of any of the compositions described herein or any of the pharmaceutical compositions described herein.

Disclosed herein are methods of reducing pain or reducing joint pain in a subject, the methods can comprise administering to the subject in need thereof a therapeutically effective amount of any of the compositions described herein or any of the pharmaceutical compositions described herein.

Disclosed herein are methods of protecting cartilage or preventing cartilage degeneration in a subject, the methods can comprise administering to the subject in need thereof a therapeutically effective amount of any of the compositions described herein or any of the pharmaceutical compositions described herein. Disclosed herein are methods of preventing or reducing or inhibiting pain-associated depression in a subject, the methods can comprise administering to the subject in need thereof a therapeutically effective amount of any of the compositions described herein or any of the pharmaceutical compositions described herein.

Disclosed herein are methods of reducing MMP13 levels in the cartilage or synovium of a subject, the methods can comprise administering to the subject in need thereof a therapeutically effective amount of any of the compositions described herein or any of the pharmaceutical compositions described herein.

Disclosed herein are methods of increasing expression of glutamate transporter- 1 (GLT-1) in a subject, the methods can comprise administering to the subject in need thereof a therapeutically effective amount of any of the compositions described herein or any of the pharmaceutical compositions described herein.

Disclosed herein are methods of preserving or maintaining expression of glutamate transporter-1 (GLT-1) in a subject, the methods can comprise administering to the subject in need thereof a therapeutically effective amount of any of the compositions described herein or any of the pharmaceutical compositions described herein.

Disclosed herein are methods of preventing a decrease in expression of glutamate transporter-1 (GLT-1) in a subject, the methods can comprise administering to the subject in need thereof a therapeutically effective amount of any of the compositions described herein or any of the pharmaceutical compositions described herein.

Disclosed herein are methods of reducing VEGF levels or cortisone levels in the blood of a subject, the methods can comprise administering to the subject in need thereof a therapeutically effective amount of any of the compositions described herein or any of the pharmaceutical compositions described herein.

Disclosed herein are methods of treating osteoarthritis in a subject in need thereof, the methods can comprise administering to the subject in need thereof a therapeutically effective amount of any of the compositions described herein or any of the pharmaceutical compositions described herein.

Disclosed herein are methods of reducing or ameliorating one or more symptoms of osteoarthritis in a subject, the methods can comprise administering to the subject in need thereof a therapeutically effective amount of any of the compositions described herein or any of the pharmaceutical compositions described herein. Disclosed herein are methods of treating ajoint disease in a subject in need thereof, the methods can comprise administering to the subject in need thereof a therapeutically effective amount of any of the compositions described herein or any of the pharmaceutical compositions described herein.

Disclosed herein are methods of treating ajoint condition in a subject in need thereof, the methods can comprise administering to the subject in need thereof a therapeutically effective amount of any of the compositions described herein or any of the pharmaceutical compositions described herein.

Disclosed herein are methods of reducing inflammation in a subject in need thereof, the methods can comprise administering to the subject in need thereof a therapeutically effective amount of any of the compositions described herein or any of the pharmaceutical compositions described herein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows pharmacological effect of probiotic, prebiotics or synbiotics on OA pain for 12 wees using Von Frey (n=8/group). Vehicle or the other pharmacological interventions were administered orally twice/week after PTOA induction in the left knee of mice. The values were expressed as mean ± SEM, n=8 mice/group. ****p<0.0001, **p<0.01 (compared to PBS); #p<0.05, ##p<0.01, ###p<0.001 (compared to LA).

FIG. 2 shows that treatment with L. acidophilus (LA) and fructooligosaccharide (FOS) protect cartilage in joints.

FIGS. 3A-D show the pharmacological effect of administering probiotics, prebiotics, and synbiotics on MMP-13 expression level induced by OA surgery in knee cartilage and synovium following 12 weeks of treatment. Pharmacological interventions were administered orally twice per week after PTOA for 12 weeks. Histopathological sections of the knee joint were harvested after 12 weeks upon OA induction by PMM and the effect of different pharmacological interventions on MMP-13 in knee cartilage and synovium (FIGS. 3A and 3B, respectively). For quantification, the MMP-13 expression data from FIG. 3A was subjected to quantitative analysis (FIG. 3C), while the MMP-13 expression data from FIG. 3B were similarly quantitatively analyzed (FIG. 3D). The values are presented as the mean ± standard deviation (SD). Statistical distinctions were established employing a two-way analysis of variance (ANOVA) and subsequently subjected to Tukey's test for post hoc analysis. Significance levels are represented as ****p < 0.0001 in comparison to the intact group, and ####p <0.0001, ###p < 0.001, ##p < 0.01, and #p < 0.05 in comparison to the PMM group. The study utilized a sample size of n=3 for immunohistology purposes.

FIGS. 4A-B show the pharmacological effect of administering probiotics, prebiotics. and synbiotics on GLT-1 expression induced by OA surgery in the ipsilateral lumbar region of the spinal cord (L3/L5) following a 12-week treatment twice per week. After inducing OA using PMM surgery, histological sections of spinal cords at the L3/L5 level were collected at the end of the 12-week. Immunofluorescence staining of GLT-1 in the ipsilateral lumbar region of the spinal cord was performed after PMM surgery treatment with various interv entions (FIG. 4 A). For quantification, GLT-1 expression data from FIG. 4A underwent quantitative analysis. The values are presented as the mean ± standard deviation (SD). Statistical differences were evaluated using a two-way analysis of variance (ANOVA). followed by Tukey's test for post hoc analysis. Significance levels are denoted as ****p < 0.0001, ***p < 0.001, **p < 0.01, and *p < 0.05 compared to the intact group, and ###p < 0.001, ##p < 0.01, and #p < 0.05 compared to the PMM group. The study utilized a sample size of n=3 for immunohistology purposes.

FIGS. 5A-B show that treatment with L. acidophilus (LA) and fructooligosaccharide (FOS) have anti -depression effects using behavioral tests in an OA-induced animal model. FIG. 5A shows that the number of squares crossed in an open field task is increased in the LA-FOS treatment group compared to control in an OA-induced animal model. FIG. 5B shows that the number of entries in open arm in the elevated plus maze is increased in the LA-FOS treatment group compared to control in an OA-induced animal model. N = 8 mice of each experimental group.

****p < 0.0001 intact vs PBS Control, #p < 0.05, ##p < 0.01, ###p < 0.001, ####p < 0.0001 vs PBS control.

FIGS. 6A-B show that behavioral depression correlates with depression biomarkers in serum, and that L. acidophilus (LA) coadministered with a prebiotic reduces depression biomarkers in serum in an animal of OA. FIG. 6A shows serum levels of VEGF. FIG. 6B shows serum levels corticosterone. The values are expressed as mean ± S.E.M. Statistical differences were determined by one-way ANOVA followed by Tukey’s test. ****p<0.0001. **p<0.01 vs. intact group and ##p<0.01, #<0.05 vs. PBS control. DETAILED DESCRIPTION

The present disclosure can be understood more readily by reference to the following detailed description of the invention, the figures and the examples included herein.

Before the present compositions and methods are disclosed and described, it is to be understood that they are not limited to specific synthetic methods unless otherwise specified, or to particular reagents unless otherwise specified, as such may, of course, vary. It is also to be understood that the terminology⁷ used herein is for the purpose of describing particular aspects only and is not intended to be limiting. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, example methods and materials are now described.

Moreover, it is to be understood that unless otherwise expressly stated, it is in no way intended that any method set forth herein be construed as requiring that its steps be performed in a specific order. Accordingly, where a method claim does not actually recite an order to be followed by its steps or it is not otherwise specifically stated in the claims or descriptions that the steps are to be limited to a specific order, it is in no way intended that an order be inferred, in any respect. This holds for any possible non-express basis for interpretation, including matters of logic with respect to arrangement of steps or operational flow, plain meaning derived from grammatical organization or punctuation, and the number or type of aspects described in the specification.

All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the publications are cited. The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided herein can be different from the actual publication dates, which can require independent confirmation.

As used in the specification and the appended claims, the singular forms “a,” "an” and “the” include plural referents unless the context clearly dictates otherwise.

The w ord “or” as used herein means any one member of a particular list and also includes any combination of members of that list.

Throughout the description and claims of this specification, the word “comprise” and variations of the word, such as “comprising” and “comprises,” means “including but not limited to,” and is not intended to exclude, for example, other additives, components, integers or steps. In particular, in methods stated as comprising one or more steps or operations it is specifically contemplated that each step comprises what is listed (unless that step includes a limiting term such as “consisting of’), meaning that each step is not intended to exclude, for example, other additives, components, integers or steps that are not listed in the step.

Ranges can be expressed herein as from “about’' or “approximately” one particular value, and/or to “about” or “approximately” another particular value. When such a range is expressed, a further aspect includes from the one particular value and/or to the other particular value. Similarly, when values are expressed as approximations, by use of the antecedent “about,” or “approximately,” it will be understood that the particular value forms a further aspect. It will be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint and independently of the other endpoint. It is also understood that there are a number of values disclosed herein and that each value is also herein disclosed as “about” that particular value in addition to the value itself. For example, if the value “10” is disclosed, then “about 10” is also disclosed. It is also understood that each unit between two particular units is also disclosed. For example, if 10 and 15 are disclosed, then 11, 12, 13, and 14 are also disclosed.

As used herein, the terms “optional” or “optionally” mean that the subsequently described event or circumstance may or may not occur and that the description includes instances where said event or circumstance occurs and instances where it does not.

As used herein, the term “subject” refers to the target of administration, e.g., a human. Thus, the subject of the disclosed methods can be a vertebrate, such as a mammal, a fish, a bird, a reptile, or an amphibian. The term “subject” also includes domesticated animals (e.g., cats, dogs, etc.), livestock (e.g., cattle, horses, pigs, sheep, goats, etc.), and laboratory animals (e.g., mouse, rabbit, rat, guinea pig, fruit fly, etc.). In one aspect, a subject is a mammal. In another aspect, the subject is a human. The term does not denote a particular age or sex. Thus, adult, child, adolescent and newborn subjects, as well as fetuses, whether male or female, are intended to be covered.

As used herein, the term “patient” refers to a subject afflicted with a disease or disorder. The term "patient" includes human and veterinary subjects. In some aspects of the disclosed methods, the “patient” has been diagnosed with a need for treatment for osteoarthritis or depression or anxiety associated with osteoarthritis, a joint injury, or a joint disease, such as, for example, prior to the administering step. As used herein, the term “treating’' refers to partially or completely alleviating, ameliorating, relieving, delaying onset of, inhibiting or slowing progression of. reducing severity of. and/or reducing incidence of one or more symptoms or features of a particular disease, disorder, and/or condition. Treatment can be administered to a subject who does not exhibit signs of a disease, disorder, and/or condition and/or to a subject who exhibits only early signs of a disease, disorder, and/or condition for the purpose of decreasing the risk of developing pathology associated with the disease, disorder, and/or condition. For example, the disease, disorder, and/or condition can be osteoarthritis or depression or anxiety associated with osteoarthritis.

As used herein, the term “prevent’' or “preventing” refers to precluding, averting, obviating, forestalling, stopping, or hindering something from happening, especially by advance action. It is understood that w here reduce, inhibit or prevent are used herein, unless specifically indicated otherwise, the use of the other two words is also expressly disclosed.

As used herein, the term “diagnosed” means having been subjected to a physical examination by a person of skill, for example, a physician, and found to have a condition that can be diagnosed or treated by the compounds, compositions, or methods disclosed herein.

As used herein, the terms “administering” and “administration” refer to any method of providing a pharmaceutical preparation to a subject. Such methods are well knowor to those skilled in the art and include, but are not limited to, oral administration, transdermal administration, administration by inhalation, nasal administration, topical administration, intravaginal administration, ophthalmic administration, intra-aural administration, intracerebral administration, rectal administration, sublingual administration, buccal administration, and parenteral administration, including inj ectable such as intravenous administration, intra-arterial administration, intramuscular administration, and subcutaneous administration. Administration can be continuous or intermittent. In various aspects, a preparation can be administered therapeutically; that is, administered to treat an existing disease or condition. In further various aspects, a preparation can be administered prophylactically; that is, administered for prevention of a disease or condition. In some aspects, administration can be oral administration.

As used herein, the terms “effective amount” and “amount effective” refer to an amount that is sufficient to achieve the desired result or to have an effect on an undesired condition. For example, a “therapeutically effective amount” refers to an amount that is sufficient to achieve the desired therapeutic result or to have an effect on undesired symptoms, but is generally insufficient to cause adverse side effects. The specific therapeutically effective dose level for any particular patient will depend upon a variety of factors including the disorder being treated and the severity of the disorder; the specific composition employed; the age, body weight, general health, sex and diet of the patient; the time of administration; the route of administration; the rate of excretion of the specific compound employed; the duration of the treatment; drugs used in combination or coincidental with the specific compound employed and like factors well known in the medical arts. For example, it is well within the skill of the art to start doses of a compound at levels lower than those required to achieve the desired therapeutic effect and to gradually increase the dosage until the desired effect is achieved. If desired, the effective daily dose can be divided into multiple doses for purposes of administration. Consequently, single dose compositions can contain such amounts or submultiples thereof to make up the daily dose. The dosage can be adjusted by the individual physician in the event of any contraindications. Dosage can var ⁷, and can be administered in one or more dose administrations daily, for one or several days. Guidance can be found in the literature for appropriate dosages for given classes of pharmaceutical products. In further various aspects, a preparation can be administered in a “prophylactically effective amount”; that is, an amount effective for prevention of a disease or condition.

As used herein, “kit” means a collection of at least two components constituting the kit. Together, the components constitute a functional unit for a given purpose. Individual member components may be physically packaged together or separately. For example, a kit comprising an instruction for using the kit may or may not physically include the instruction with other individual member components. Instead, the instruction can be supplied as a separate member component, either in a paper form or an electronic form which may be supplied on computer readable memory device or downloaded from an internet website, or as recorded presentation.

As used herein, “instruction(s)” means documents describing relevant materials or methodologies pertaining to a kit. These materials may include any combination of the following: background information, list of components and their availability information (purchase information, etc.), brief or detailed protocols for using the kit, trouble-shooting, references, technical support, and any other related documents. Instructions can be supplied with the kit or as a separate member component, either as a paper form or an electronic form, which may be supplied on computer readable memory⁷ device or downloaded from an internet website, or as recorded presentation. Instructions can comprise one or multiple documents, and are meant to include future updates.

The term '‘pharmaceutically acceptable” describes a material that is not biologically or otherwise undesirable, i.e., without causing an unacceptable level of undesirable biological effects or interacting in a deleterious manner.

The term “stable,” as used herein, refers to compositions that are not substantially- altered when subjected to conditions to allow for their production, detection, and, in certain aspects, their recovery, purification, and use for one or more of the purposes disclosed herein.

“Inhibit,” “inhibiting” and “inhibition” mean to diminish or decrease an activity', level, response, condition, disease, or other biological parameter. This can include, but is not limited to, the complete ablation of the activity, response, condition, or disease. This may also include, for example, a 10% inhibition or reduction in the activity, response, condition, or disease as compared to the native or control level. Thus, in some aspects, the inhibition or reduction can be a 10, 20, 30, 40, 50, 60, 70, 80, 90, 100%, or any amount of reduction in between as compared to native or control levels. In some aspects, the inhibition or reduction is 10-20, 20-30, 30-40. 40-50, 50-60, 60-70. 70-80, 80-90, or 90-100% as compared to native or control levels. In some aspects, the inhibition or reduction is 0-25, 25-50, 50-75, or 75- 100% as compared to native or control levels.

As used herein, the term “culture” refers to any sample or item that contains one or more microorganisms. “Pure cultures” are cultures in which the organisms present are only of one strain of a particular genus and species. This is in contrast to “mixed cultures,” which are cultures in which more than one genus and/or species of microorganism are present. In some embodiments of the present invention, pure cultures find use. For example, in some particularly preferred embodiments, pure cultures of Lactobacillus (e g., L. acidophilus') find use.

Comorbid joint pain and depression make clinical management extraordinarily challenging and complicated. For example, patients with comorbid OA pain and depression showed significantly reduced benefits from taking anti-depressant drugs compared to those without joint pain. The mechanisms that drive this reduction are unknown. Thus, there is an urgent unmet need to understand the underlying mechanisms to address depressive symptoms, manage OA disease better, and develop an effective treatment strategy for OA- associated comorbid conditions. OA-associated depression has often resulted from chronic joint pain affecting mobility and physical limitations, which in turn stirs feelings of depression. Researchers have pointed out that OA-induced social impairment (e.g., social isolation, difficulty walking around the block and talking to neighbors, shopping with friends, cooking dinner for friends, and social stresses, etc.) is linked to increased depression among individuals with OA. Anti-depressant medication is prescribed for moderate and severe depression. The most popular anti-depressants are selective serotonin reuptake inhibitors (SSRIs). SSRIs include paroxetine (Paxil) and fluoxetine (Sarafem, Prozac). These drugs reduce depression and may alleviate certain types of pain. However, there is no evidence that SSRIs can reduce OA-associated joint pain. Worse, patients with comorbid OA pain and depression show minimal or no benefit from taking anti-depressant drugs compared to patients without joint pain.

Described herein are compositions and methods comprising pre-/probiotic combinations that are safe and reliable OA disease-modifying drugs (OADMDs) that can prevent and/or reverse OA (e.g., knee OA) and another comorbid chronic conditions, including depression. The compositions and methods disclosed herein can be used for the management of other diarthrodial j oints. including the hip, shoulder, finger, and ankle joints, and synarthrodial joints (for low back pain).

COMPOSITIONS

Disclosed herein are compositions comprising or consisting of Lactobacillus acidophilus and one or more prebiotics. In some aspects, the Lactobacillus acidophilus can be Lactobacillus acidophilus (ATCC 4356). In some aspects, the one or more prebiotics can be inulin or fructooligosaccharide. In some aspects, the composition comprises or consists of Lactobacillus acidophilus and fructooligosaccharide. In some aspects, the compositions disclosed herein can further comprise a postbiotic. In some aspects, the postbiotic can be butyrate.

Disclosed herein are compositions comprising or consisting of Lactobacillus acidophilus and one or more postbiotics. In some aspects, the Lactobacillus acidophilus can be Lactobacillus acidophilus (ATCC 4356). In some aspects, the one or more postbiotics can be butyrate. In some aspects, the composition comprises or consists of Lactobacillus acidophilus and butyrate.

In some aspects, the compositions disclosed herein can further comprise a pharmaceutically acceptable carrier. In some aspects, the compositions may also include additives. Suitable additives include substances known in the art that may support growth, production of specific metabolites by the microorganism, alter pH, enrich for target metabolites, enhance insecticidal effects, and combinations thereof. Exemplary additives include carbon sources, nitrogen sources, phosphorous sources, inorganic salt, organic acid, growth media, vitamins, minerals, acetic acid, amino acids and the like.

Examples of suitable carbon sources include, without limitation, starch, peptone, yeast extract, amino acids, sugars such as sucrose, glucose, arabinose, mannose, glucosamine, maltose, sugar cane, alfalfa extracts, molasses, rum, and the like; salts of organic acids such as acetic acid, fumaric acid, adipic acid, propionic acid, citric acid, gluconic acid, malic acid, pyruvic acid, malonic acid and the like; alcohols such as ethanol, glycerol, and the like; oil or fat such as soybean oil, rice bran oil, olive oil, com oil, and sesame oil. The amount of the carbon source added varies according to the kind of carbon source and is typically between 1 to 100 grams per liter of medium. The weight fraction of the carbon source in the composition may be about 98% or less, about 95% or less, about 90% or less, about 85% or less, about 80% or less, about 75% or less, about 70% or less, about 65% or less, about 60% or less, about 55% or less, about 50% or less, about 45% or less, about 40% or less, about 35% or less, about 30% or less, about 25% or less, about 20% or less, about 15% or less, about 10% or less, about 5% or less, about 2%, or about 1% or less of the total weight of the composition.

Examples of suitable nitrogen sources include, without limitation, amino acids, yeast extract, alfalfa extract, tryptone, beef extract, peptone, potassium nitrate, ammonium nitrate, ammonium chloride, ammonium sulfate, ammonium phosphate, ammonia or combinations thereof. The amount of nitrogen source varies according to the nitrogen source, typically between 0. 1 to 30 grams per liter of medium. The weight fraction of the nitrogen source in the composition may be about 98% or less, about 95% or less, about 90% or less, about 85% or less, about 80% or less, about 75% or less, about 70% or less, about 65% or less, about 60% or less, about 55% or less, about 50% or less, about 45% or less, about 40% or less, about 35% or less, about 30% or less, about 25% or less, about 20% or less, about 15% or less, about 10% or less, about 5% or less, about 2%, or about 1% or less of the total weight of the composition.

Examples of suitable inorganic salts include, without limitation, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, disodium hydrogen phosphate, magnesium sulfate, magnesium chloride, ferric sulfate, ferrous sulfate, ferric chloride, ferrous chloride, manganous sulfate, manganous chloride, zinc sulfate, zinc chloride, cupric sulfate, calcium chloride, sodium chloride, calcium carbonate, sodium carbonate, and combinations thereof. The weight fraction of the inorganic salt in the composition may be about 98% or less, about 95% or less, about 90% or less, about 85% or less, about 80% or less, about 75% or less, about 70% or less, about 65% or less, about 60% or less, about 55% or less, about 50% or less, about 45% or less, about 40% or less, about 35% or less, about 30% or less, about 25% or less, about 20% or less, about 15% or less, about 10% or less, about 5% or less, about 2%, or about 1% or less of the total weight of the composition.

In some aspects, the compositions of the present disclosure may further comprise acetic acid or carboxylic acid. Suitable acetic acids include any known in the art including, without limitation, formic acid, acetic acid, propionic acid, butanoic acid, isobutyric acid, 3- methyl butanoic acid, methyl acetate ethyl acetate, propyl acetate, butyl acetate, isobutyl acetate, and 2-methyl butyl acetate. In some aspects, the acetic acid is included by using vinegar. The weight fraction of the acetic acid in the composition may be about 98% or less, about 95% or less, about 90% or less, about 85% or less, about 80% or less, about 75% or less, about 70% or less, about 65% or less, about 60% or less, about 55% or less, about 50% or less, about 45% or less, about 40% or less, about 35% or less, about 30% or less, about 25% or less, about 20% or less, about 15% or less, about 10% or less, about 5% or less, about 2%, or about 1 % or less of the total weight of the composition.

In some aspects, the composition can be frozen, cryopreserved or lyophilized. In some aspects, the composition can be a solid or liquid.

In some aspects, the compositions disclosed herein can be frozen. The compositions of the present disclosure may be in liquid or dry form. In some aspects, the compositions disclosed herein can be a solid. In some aspects, the compositions disclosed herein can be a liquid. In some aspects, the composition may comprise an aqueous suspension of components. This aqueous suspension may be provided as a concentrated stock solution which is diluted prior to application or as a diluted solution ready-to-use. Also, the composition may be a powder, granules, dust, pellet or colloidal concentrate. Such dry forms may be formulated to dissolve immediately upon wetting or dissolve in a controlled-release, sustained-release, or other time-dependent manner. Also, the composition may be in a dry form that does not depend upon wetting or dissolving to be effective. In some aspects, the composition can be in a powder, granule, a ready-to-use beverage, food bar, an extruded form, capsule, gel cap, or dispersible tablet form.

In some aspects, the composition of the present disclosure may comprise at least one optional excipient. Non-limiting examples of suitable excipients include antioxidants, additives, diluents, binders, fillers, buffering agents, mineral salts, pH modifying agents, disintegrants, dispersing agents, flavoring agents, nutritive agents, oncotic and osmotic agents, stabilizers, preservatives, palatability enhancers and coloring agents. The amount and types of excipients utilized to form the combination may be selected according to known principles of science.

In some aspects, the excipient may include at least one diluent. Non-limiting examples of suitable diluents include microcrystalline cellulose (MCC), cellulose derivatives, cellulose powder, cellulose esters (i.e., acetate and butyrate mixed esters), ethyl cellulose, methyl cellulose, hydroxypropyl cellulose, hydroxypropyl methylcellulose, sodium carboxymethylcellulose, com starch, phosphated com starch, pregelatinized com starch, rice starch, potato starch, tapioca starch, starch-lactose, starch-calcium carbonate, sodium starch glycolate, glucose, fructose, lactose, lactose monohydrate, sucrose, xylose, lacitol, mannitol, malitol, sorbitol, xylitol, maltodextrin, and trehalose.

In some aspects, the excipient may comprise a binder. Suitable binders include, but are not limited to, starches, pregelatinized starches, gelatin, polyvinylpyrrolidone, cellulose, methylcellulose, sodium carboxymethylcellulose, ethylcellulose, polyacrylamides, polyvinyloxoazolidone, pofyvinylalcohols, C12-C18 fatty⁷ acid alcohol, polyethylene glycol, polyols, saccharides, oligosaccharides, polypeptides, oligopeptides, and combinations thereof.

In some aspects, the excipient may include a filler. Suitable fillers include, but are not limited to, carbohydrates, inorganic compounds, and polyvinylpyrrolidone. By way of nonlimiting example, the filler may be calcium sulfate, both di- and tri-basic, starch, calcium carbonate, magnesium carbonate, microcrystalline cellulose, dibasic calcium phosphate, magnesium carbonate, magnesium oxide, calcium silicate, talc, modified starches, lactose, sucrose, mannitol, or sorbitol.

In some aspects, the excipient may⁷ comprise a buffering agent. Representative examples of suitable buffering agents include, but are not limited to, MOPS, HEPES, TAPS, Bicine, Tricine, TES, PIPES, MES, Tris buffers or buffered saline salts (e.g., Tris buffered saline or phosphate buffered saline).

In some aspects, the excipient may include a disintegrant. Suitable disintegrants include, but are not limited to, starches such as cornstarch, potato starch, pregelatinized and modified starches thereof, sweeteners, clays, such as bentonite, microcrystalline cellulose, alginates, sodium starch glycolate, gums such as agar, guar, locust bean, karaya, pecitin, and tragacanth.

In some aspects, the excipient may include a dispersion enhancer. Suitable dispersants may include, but are not limited to, starch, alginic acid, polyvinylpyrrolidones, guar gum, kaolin, bentonite, purified wood cellulose, sodium starch glycolate, isoamorphous silicate, and microcrystalline cellulose.

In some aspects, the excipient may include a lubricant. Non-limiting examples of suitable lubricants include minerals such as talc or silica; and fats such as vegetable stearin, magnesium stearate or stearic acid.

The weight fraction of the excipient(s) in the combination may be about 98% or less, about 95% or less, about 90% or less, about 85% or less, about 80% or less, about 75% or less, about 70% or less, about 65% or less, about 60% or less, about 55% or less, about 50% or less, about 45% or less, about 40% or less, about 35% or less, about 30% or less, about 25% or less, about 20% or less, about 15% or less, about 10% or less, about 5% or less, about 2%, or about 1% or less of the total weight of the combination.

In some aspects, the compositions of the present disclosure are stable at room temperature.

In some aspects, the Lactobacillus acidophilus may be kept at a reduced temperature for storage and transportation without significantly compromising the viability of the live microorganisms. The compositions comprising the same may be refrigerated, frozen, or lyophilized. The compositions may be refrigerated at between 32°F to 44°F.

In some aspects, the compositions comprising the same can be stored and transported in a frozen state. The live beneficial microorganisms can be reinvigorated quickly once the compositions are thawed and brought to ambient temperature, preferably with aeration and/or agitation.

In some aspects, the Lactobacillus acidophilus can be lyophilized. The Lactobacillus acidophilus can be first frozen. Water is then removed amendments under vacuum. This process further reduces the weight of the composition for storage and transportation. The compositions comprising the same can be reconstituted and reinvigorated prior to application or administration.

In some aspects, the concentrated Lactobacillus acidophilus, or compositions comprising the same can be diluted with water before application or administration. Diluted compositions can be stored for a prolonged period of time, e.g., as long as 30 days, without losing viability. To maintain the live beneficial microorganism in a substantially aerobic state, dissolved oxygen in the diluted compositions of the present disclosure are preferably kept at an optimal level. It is preferable to supply optimal amounts of oxygen to the diluted composition though slow aeration.

In some aspects, any of the composition disclosed herein can be administered in a form selected from the group consisting of powder, granules, a ready-to-use beverage, food bar, an extruded form, capsules, gel caps, and dispersible tablets.

In some aspects, the compositions disclosed herein can comprise around Ix lO³, Ixl O⁴, IxlO⁵, IxlO⁶, IxlO⁷, IxlO⁸, IxlO⁹, IxlO¹⁰, or IxlO¹¹ cells of the Lactobacillus acidophilus .

In some aspects, the compositions disclosed herein can comprise around 2xl0³, 2xl0⁴, 2xl0⁵, 2xl0⁶, 2xl0⁷, 2xl0⁸, 2xl0⁹, 2xlO¹⁰, or 2xlOⁿ cells of the Lactobacillus acidophilus.

In some aspects, the compositions disclosed herein can comprise around 3xl0³, 3x10⁴, 3xlO⁵, 3xl0⁶, 3xl0⁷, 3xl0⁸, 3xl0⁹, 3xlO¹⁰, or 3xlOⁿ cells of the Lactobacillus acidophilus.

In some aspects, the compositions disclosed herein can comprise around 4xl0³, 4xl0⁴, 4xl0⁵, 4xl0⁶, 4xl0⁷, 4xl0⁸, 4xl0⁹, 4xlO¹⁰, or xlO¹¹ cells of the Lactobacillus acidophilus.

In some aspects, the compositions disclosed herein can comprise around 5xl0³. 5xl0⁴, 5x10⁵, 5x10⁶, 5x l 0⁷, 5x10⁸, 5x10⁹, 5x10¹⁰, or 5x10¹¹ cells of the Lactobacillus acidophilus .

In some aspects, the compositions disclosed herein can comprise at least IxlO⁹ cells of the Lactobacillus acidophilus.

In some aspects, the compositions disclosed herein can comprise at least 4x10⁹ cells of the Lactobacillus acidophilus. In some aspects, a single dosage of any of the compositions disclosed herein can comprise between lx 10³ and 4x10⁹ cells of the Lactobacillus acidophilus.

In some aspects, the cells of the composition are active.

In some aspects, the dosage of the Lactobacillus acidophilus is about 10⁸ to about 10¹² CFU/day, while in other preferred embodiments, the dosage of the Lactobacillus acidophilus is about 10⁹to about 10¹¹ CFU/day.

In some aspects, the culture of the Lactobacillus acidophilus can be administered to the subject using a daily dose of from about 10⁸ to about 10¹² CFU/day.

Disclosed herein are also methods for the use of Lactobacillus acidophilus alone or in combination with one or more prebiotics for the preparation of compositions suitable for inhibiting cartilage degeneration in a subject. Disclosed herein are also methods for the use of Lactobacillus acidophilus alone or in combination with one or more prebiotics for the preparation of compositions suitable for reducing pain or reducing joint pain in a subject.

Disclosed herein are also methods for the use of Lactobacillus acidophilus alone or in combination with one or more prebiotics for the preparation of compositions suitable for protecting cartilage or preventing cartilage degeneration in a subject.

Disclosed herein are also methods for the use of Lactobacillus acidophilus alone or in combination with one or more prebiotics for the preparation of compositions suitable for preventing or reducing or inhibiting pain-associated depression in a subject.

Disclosed herein are also methods for the use of Lactobacillus acidophilus alone or in combination with one or more prebiotics for the preparation of compositions suitable for reducing MMP13 levels in the cartilage or synovium of a subject.

Disclosed herein are also methods for the use of Lactobacillus acidophilus alone or in combination with one or more prebiotics for the preparation of compositions suitable for increasing expression of glutamate transporter-1 (GLT-1) in a subject.

Disclosed herein are also methods for the use of Lactobacillus acidophilus alone or in combination with one or more prebiotics for preventing a decrease in expression of glutamate transporter- 1 (GLT-1) in a subject.

Disclosed herein are also methods for the use of Lactobacillus acidophilus alone or in combination with one or more prebiotics for reducing VEGF levels or cortisone levels in the blood of a subject.

Disclosed herein are also methods for the use of Lactobacillus acidophilus alone or in combination with one or more prebiotics for treating osteoarthritis in a subject in need thereof.

Disclosed herein are also methods for the use of Lactobacillus acidophilus alone or in combination with one or more prebiotics for reducing or ameliorating one or more symptoms of osteoarthritis in a subject.

Disclosed herein are also methods for the use of Lactobacillus acidophilus alone or in combination with one or more prebiotics for treating a joint disease in a subject in need thereof.

Disclosed herein are also methods for the use of Lactobacillus acidophilus alone or in combination with one or more prebiotics for treating a joint condition in a subject in need thereof. Disclosed herein are also methods for the use of Lactobacillus acidophilus alone or in combination with one or more prebiotics for reducing inflammation in a subject in need thereof.

PHARMACEUTICAL COMPOSITIONS

As disclosed herein, are pharmaceutical compositions, comprising or consisting of Lactobacillus acidophilus and one or more prebiotics as described herein. Also disclosed herein are compositions comprising one or more of the compositions described herein. Further disclosed herein are pharmaceutical compositions, comprising or consisting of Lactobacillus acidophilus and one or more postbiotics as described herein. As disclosed herein, are pharmaceutical compositions, comprising or consisting of Lactobacillus acidophilus and fructooligosaccharide. Further disclosed herein are compositions comprising or consisting of Lactobacillus acidophilus and one or more prebiotics or Lactobacillus acidophilus and fructooligosaccharide and a pharmaceutical acceptable carrier or excipient described herein. As also disclosed herein, are pharmaceutical compositions, comprising or consisting of Lactobacillus acidophilus, fructooligosaccharide, and butyrate. Further disclosed herein are compositions comprising or consisting of Lactobacillus acidophilus, one or more prebiotics, and one or more postbiotics or Lactobacillus acidophilus, fructooligosaccharide, and butyrate, and a pharmaceutical acceptable carrier or excipient described herein. As disclosed herein, are pharmaceutical compositions, comprising or consisting of Lactobacillus acidophilus and butyrate. Further disclosed herein are compositions comprising or consisting of Lactobacillus acidophilus and one or more postbiotics or Lactobacillus acidophilus and butyrate and a pharmaceutical acceptable carrier or excipient described herein.

In some aspects, compositions can be formulated for oral or parental administration. In some aspects, the compositions can be formulated as a liquid, a lyophilized powder, a cream, or an ointment. In some aspects, the parental administration can intravenous, subcutaneous, intramuscular or direct injection. The compositions can be formulated for administration by any of a variety of routes of administration, and can include one or more physiologically acceptable excipients, which can vary depending on the route of administration. As used herein, the term “excipient” means any compound or substance, including those that can also be referred to as “carriers” or “diluents.” Preparing pharmaceutical and physiologically acceptable compositions is considered routine in the art, and thus, one of ordinary skill in the art can consult numerous authorities for guidance if needed.

The compositions can be administered directly to a subject. Generally, the compositions can be suspended in a pharmaceutically acceptable carrier (e.g., physiological saline or a buffered saline solution) to facilitate their delivery⁷. Encapsulation of the compositions in a suitable delivery vehicle (e.g., implantable devices) may increase the efficiency of delivery.

The compositions can be formulated in various ways for parenteral or nonparenteral administration. Where suitable, oral formulations can take the form of tablets, pills, capsules, or powders, which may be enterically coated or otherwise protected. Sustained release formulations, suspensions, elixirs, aerosols, and the like can also be used.

Pharmaceutically acceptable carriers and excipients can be incorporated (e.g., water, saline, aqueous dextrose, and glycols, oils (including those of petroleum, animal, vegetable or synthetic origin), starch, cellulose, talc, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, magnesium stearate, sodium stearate, glycerol monosterate, sodium chloride, dried skim milk, glycerol, propylene glycol, ethanol, and the like). The compositions may be subjected to conventional pharmaceutical expedients such as sterilization and may contain conventional pharmaceutical additives such as preservatives, stabilizing agents, wetting or emulsifying agents, salts for adjusting osmotic pressure, buffers, and the like. Suitable pharmaceutical carriers and their formulations are described in "‘Remington's Pharmaceutical Sciences” by E.W. Martin, which is herein incorporated by reference. Such compositions will, in any event, contain an effective amount of the compositions together with a suitable amount of carrier so as to prepare the proper dosage form for proper administration to the patient.

The pharmaceutical compositions as disclosed herein can be prepared for oral or parenteral administration. The pharmaceutical compositions as disclosed herein can be prepared for oral administration. Pharmaceutical compositions prepared for parenteral administration include those prepared for intravenous (or intra-arterial), intramuscular, subcutaneous, intraperitoneal, transmucosal (e.g., intranasal, intravaginal, or rectal), or transdermal (e.g., topical) administration. Thus, compositions can be prepared for parenteral administration that includes Lactobacillus acidophilus and one or more prebiotics dissolved or suspended in an acceptable carrier, including but not limited to an aqueous carrier, such as water, buffered water, saline, buffered saline (e.g., PBS), and the like. One or more of the excipients included can help approximate physiological conditions, such as pH adjusting and buffering agents, tonicity adjusting agents, wetting agents, detergents, and the like. Where the compositions include a solid component (as they may for oral administration), one or more of the excipients can act as a binder or filler (e.g., for the formulation of a tablet, a capsule, and the like).

The pharmaceutical compositions can be sterile and sterilized by conventional sterilization techniques or sterile filtered. Aqueous solutions can be packaged for use as is, or lyophilized, the lyophilized preparation, which is encompassed by the present disclosure, can be combined with a sterile aqueous carrier prior to administration. The pH of the pharmaceutical compositions typically will be between 3 and 11 (e.g., between about 5 and 9) or between 6 and 8 (e.g.. between about 7 and 8). The resulting compositions in solid form can be packaged in multiple single dose units, each containing a fixed amount of the above- mentioned agent or agents, such as in a sealed package of tablets or capsules.

In some aspects, the pharmaceutical composition can be formulated for Lactobacillus acidophilus 4x10⁹ per dose. In some aspects, the pharmaceutical composition can be formulated for Lactobacillus acidophilus 4xl0⁹ per dose, and administered to a subject twice per week.

In some aspects, the pharmaceutical composition can be formulated for Lactobacillus acidophilus 4x10⁹ per dose and fructooligosaccharide, 20 mg/kg per dose. In some aspects, the pharmaceutical composition can be formulated for Lactobacillus acidophilus 4x10⁹ per dose and fructooligosaccharide, 20 mg/kg per dose, and administered to a subject twice per week.

In some aspects, the pharmaceutical composition can be formulated for Lactobacillus acidophilus 4x10⁹ per dose and sodium butyrate, 400 mg/kg per dose. In some aspects, the pharmaceutical composition can be formulated for Lactobacillus acidophilus 4x10⁹ per dose and administered to a subject twice per week, and sodium butyrate, 400 mg/kg per dose, and administered once daily.

In some aspects, the pharmaceutical composition can be formulated for Lactobacillus acidophilus 4x10⁹ per dose, fructooligosaccharide, 20 mg/kg per dose, and sodium butyrate 400 mg/kg per dose. In some aspects, the pharmaceutical composition can be formulated for Lactobacillus acidophilus 4x10⁹ per dose and fructooligosaccharide, 20 mg/kg per dose, and administered to a subject twice per week; and sodium butyrate 400 mg/kg per dose, and administered once daily. In some aspects, Lactobacillus acidophilus (LA) can be formulated, for example, dose 2~4xl0⁹, volume 200 pL. In some aspects, Lactobacillus acidophilus (LA) can be formulated, for example, dose 2~4xl0⁹. volume 200 pL, plus FOS 20 mg/kg (100 pL). for administration twice per week. In some aspects, Lactobacillus acidophilus (LA) can be formulated, for example, dose 2~4xl0⁹, volume 200 pL), twice per week plus sodium butyrate (dose 400 mg/kg) (100 pL), administered orally daily. In some aspects, Lactobacillus acidophilus (LA) can be formulated, for example, dose 2-4x10⁹. volume 200 pL, plus, FOS, 20 mg/kg twice per week, plus sodium butyrate (dose 400 mg/kg), administered orally daily.

METHODS OF TREATMENT

Disclosed herein are methods of inhibiting cartilage degeneration in a subject. In some aspects, the methods can comprise administering to the subject in need thereof a therapeutically effective amount of any of the compositions described herein or any of the pharmaceutical compositions described herein. In some aspects, the composition comprises or consists of Lactobacillus acidophilus and one or more prebiotics. In some aspects, disclosed herein are methods of inhibiting cartilage degeneration in a subject comprising administering to the subject in need thereof a therapeutically effective amount of a composition comprising or consisting of Lactobacillus acidophilus and one or more prebiotics. In some aspects, disclosed herein are methods of inhibiting cartilage degeneration in a subject comprising administering to the subject in need thereof a therapeutically effective amount of a composition comprising or consisting of a culture of Lactobacillus acidophilus and one or more prebiotics.

Disclosed herein are methods of reducing pain or reducing joint pain in a subject. In some aspects, the methods can comprise administering to the subject in need thereof a therapeutically effective amount of any of the compositions described herein or any of the pharmaceutical compositions described herein. In some aspects, the composition comprises or consists of Lactobacillus acidophilus and one or more prebiotics. In some aspects, disclosed herein are methods of reducing pain or reducing joint pain in a subject comprising administering to the subject in need thereof a therapeutically effective amount of a composition comprising or consisting of Lactobacillus acidophilus and one or more prebiotics. In some aspects, disclosed herein are methods of reducing pain or reducing joint pain in a subject comprising administering to the subject in need thereof a therapeutically effective amount of a composition comprising or consisting of a culture of Lactobacillus acidophilus and one or more prebiotics.

Disclosed herein are methods of protecting cartilage or preventing cartilage degeneration in a subject. In some aspects, the methods can comprise administering to the subj ect in need thereof a therapeutically effective amount of any of the compositions described herein or any of the pharmaceutical compositions described herein. In some aspects, the composition comprises or consists of Lactobacillus acidophilus and one or more prebiotics. In some aspects, disclosed herein are methods of protecting cartilage or preventing cartilage degeneration in a subject comprising administering to the subject in need thereof a therapeutically effective amount of a composition comprising or consisting of Lactobacillus acidophilus and one or more prebiotics. In some aspects, disclosed herein are methods of protecting cartilage or preventing cartilage degeneration pain in a subject comprising administering to the subject in need thereof a therapeutically effective amount of a composition comprising or consisting of a culture of Lactobacillus acidophilus and one or more prebiotics.

Disclosed herein are methods of preventing or reducing or inhibiting pain-associated depression in a subject. In some aspects, the methods can comprise administering to the subj ect in need thereof a therapeutically effective amount of any of the compositions described herein or any of the pharmaceutical compositions described herein. In some aspects, the depression can be a depression condition or anxiety. In some aspects, the composition comprises or consists of Lactobacillus acidophilus and one or more prebiotics. In some aspects, disclosed herein are methods of preventing or reducing or inhibiting pain- associated depression in a subject comprising administering to the subject in need thereof a therapeutically effective amount of a composition comprising or consisting of Lactobacillus acidophilus and one or more prebiotics. In some aspects, disclosed herein are methods of preventing or reducing or inhibiting pain-associated depression in a subject comprising administering to the subject in need thereof a therapeutically effective amount of a composition comprising or consisting of a culture of Lactobacillus acidophilus and one or more prebiotics.

Disclosed herein are methods of reducing MMP13 levels in the cartilage or synovium of a subject. In some aspects, the methods can comprise administering to the subject in need thereof a therapeutically effective amount of any of the compositions described herein or any of the pharmaceutical compositions described herein. In some aspects, the depression can be a depression condition or anxiety. In some aspects, the composition comprises or consists of Lactobacillus acidophilus and one or more prebiotics. In some aspects, disclosed herein are methods of reducing MMP13 levels in the cartilage or synovium in a subject comprising administering to the subject in need thereof a therapeutically effective amount of a composition comprising or consisting of Lactobacillus acidophilus and one or more prebiotics. In some aspects, disclosed herein are methods of reducing MMP13 levels in the cartilage or synovium in a subject comprising administering to the subject in need thereof a therapeutically effective amount of a composition comprising or consisting of a culture of Lactobacillus acidophilus and one or more prebiotics.

Disclosed herein are methods of increasing expression of glutamate transporter- 1 (GLT-1) in a subject. In some aspects, the methods can comprise administering to the subject in need thereof a therapeutically effective amount of any of the compositions described herein or any of the pharmaceutical compositions described herein. In some aspects, the expression of glutamate transporter-1 (GLT-1) can be GLT-1 expression in the spinal cord of the subject. In some aspects, the composition comprises or consists of Lactobacillus acidophilus and one or more prebiotics. In some aspects, disclosed herein are methods of increasing expression of glutamate transporter-1 (GLT-1) in a subject comprising administering to the subject in need thereof a therapeutically effective amount of a composition comprising or consisting of Lactobacillus acidophilus and one or more prebiotics. In some aspects, disclosed herein are methods of increasing expression of glutamate transporter- 1 (GLT-1) in a subject comprising administering to the subject in need thereof a therapeutically effective amount of a composition comprising or consisting of a culture of Lactobacillus acidophilus and one or more prebiotics.

Disclosed herein are methods of preserving or maintaining expression of glutamate transporter- 1 (GLT-1) in a subject In some aspects, the methods can comprise administering to the subject in need thereof a therapeutically effective amount of any of the compositions described herein or any of the pharmaceutical compositions described herein. In some aspects, the expression of glutamate transporter-1 (GLT-1) can be GLT-1 expression in the spinal cord of the subject. In some aspects, the composition comprises or consists of Lactobacillus acidophilus and one or more prebiotics. In some aspects, disclosed herein are methods of preserving or maintaining expression of glutamate transporter- 1 (GLT-1) in a subject comprising administering to the subject in need thereof a therapeutically effective amount of a composition comprising or consisting of Lactobacillus acidophilus and one or more prebiotics. In some aspects, disclosed herein are methods of preserving or maintaining expression of glutamate transporter-1 (GLT-1) in a subject comprising administering to the subject in need thereof a therapeutically effective amount of a composition comprising or consisting of a culture of Lactobacillus acidophilus and one or more prebiotics.

Disclosed herein are methods of preventing a decrease in expression of glutamate transporter-1 (GLT-1) in a subject. In some aspects, the methods can comprise administering to the subject in need thereof a therapeutically effective amount of any of the compositions described herein or any of the pharmaceutical compositions described herein. In some aspects, the expression of glutamate transporter-1 (GLT-1) can be GLT-1 expression in the spinal cord of the subject. In some aspects, the composition comprises or consists of Lactobacillus acidophilus and one or more prebiotics. In some aspects, disclosed herein are methods of preventing a decrease in expression of glutamate transporter- 1 (GLT-1) in a subject comprising administering to the subject in need thereof a therapeutically effective amount of a composition comprising or consisting of Lactobacillus acidophilus and one or more prebiotics. In some aspects, disclosed herein are methods of preventing a decrease in expression of glutamate transporter-1 (GLT-1) in a subject comprising administering to the subject in need thereof a therapeutically effective amount of a composition comprising or consisting of a culture of Lactobacillus acidophilus and one or more prebiotics.

Disclosed herein are methods of reducing VEGF levels or cortisone levels in the blood of a subject. In some aspects, the methods can comprise administering to the subject in need thereof a therapeutically effective amount of any of the compositions described herein or any of the pharmaceutical compositions described herein. In some aspects, the composition comprises or consists of Lactobacillus acidophilus and one or more prebiotics. In some aspects, disclosed herein are methods of reducing VEGF levels or cortisone levels in the blood of a subject comprising administenng to the subject in need thereof a therapeutically effective amount of a composition comprising or consisting of Lactobacillus acidophilus and one or more prebiotics. In some aspects, disclosed herein are methods of reducing VEGF levels or cortisone levels in the blood of a subject comprising administering to the subject in need thereof a therapeutically effective amount of a composition comprising or consisting of a culture of Lactobacillus acidophilus and one or more prebiotics.

Disclosed herein are methods of treating osteoarthritis in a subject in need thereof. In some aspects, the methods can comprise administering to the subject in need thereof a therapeutically effective amount of any of the compositions described herein or any of the pharmaceutical compositions described herein. In some aspects, the composition comprises or consists of Lactobacillus acidophilus and one or more prebiotics. In some aspects, disclosed herein are methods of treating osteoarthntis in a subject comprising administering to the subject in need thereof a therapeutically effective amount of a composition comprising or consisting of Lactobacillus acidophilus and one or more prebiotics. In some aspects, disclosed herein are methods of treating osteoarthritis in a subject comprising administering to the subject in need thereof a therapeutically effective amount of a composition comprising or consisting of a culture of Lactobacillus acidophilus and one or more prebiotics.

Disclosed herein are methods of reducing or ameliorating one or more symptoms of osteoarthritis in a subject. In some aspects, the methods can comprise administering to the subject in need thereof a therapeutically effective amount of any of the compositions described herein or any of the pharmaceutical compositions described herein. In some aspects, the one or more symptoms of osteoarthritis can be pain. Examples of symptoms of osteoarthritis include but are not limited to joint stiffness, decreased range of motion (flexibility) and swelling. Osteoarthritis occurs when the protective cartilage that cushions the ends of bones wears down overtime. In some aspects, osteoarthritis can damage or effect any joint. In some aspects, the joint can be in the hands, knees, hips, spine, feet, neck or shoulder. In some aspects, the composition comprises or consists of Lactobacillus acidophilus and one or more prebiotics. In some aspects, disclosed herein are methods of reducing or ameliorating one or more symptoms of osteoarthritis in a subject comprising administering to the subject in need thereof a therapeutically effective amount of a composition comprising or consisting of Lactobacillus acidophilus and one or more prebiotics. In some aspects, disclosed herein are methods of reducing or ameliorating one or more symptoms of osteoarthritis in a subject comprising administering to the subject in need thereof a therapeutically effective amount of a composition comprising or consisting of a culture of Lactobacillus acidophilus and one or more prebiotics.

Disclosed herein are methods of treating a joint disease in a subject in need thereof. In some aspects, the methods can comprise administering to the subject in need thereof a therapeutically effective amount of any of the compositions described herein or any of the pharmaceutical compositions described herein. In some aspects, the joint disease can be a degenerative joint disease. In some aspects, the degenerative joint disease can be osteoarthritis. In some aspects, the composition comprises or consists of Lactobacillus acidophilus and one or more prebiotics. In some aspects, disclosed herein are methods of treating a joint disease in a subject comprising administering to the subject in need thereof a therapeutically effective amount of a composition comprising or consisting of Lactobacillus acidophilus and one or more prebiotics. In some aspects, disclosed herein are methods of treating a joint disease in a subject comprising administering to the subject in need thereof a therapeutically effective amount of a composition comprising or consisting of a culture of Lactobacillus acidophilus and one or more prebiotics.

Disclosed herein are methods of treating a joint condition in a subject in need thereof. In some aspects, the methods can comprise administering to the subject in need thereof a therapeutically effective amount of any of the compositions described herein or any of the pharmaceutical compositions described herein. In some aspects, the joint condition can be a joint injury. In some aspects, the joint injury can be a traumatic injury’ or a post-operative injury. In some aspects, the joint injury can be a repetitive strain injury. In some aspects, the composition comprises or consists of Lactobacillus acidophilus and one or more prebiotics. In some aspects, disclosed herein are methods of treating a joint condition in a subject comprising administering to the subject in need thereof a therapeutically effective amount of a composition comprising or consisting of Lactobacillus acidophilus and one or more prebiotics. In some aspects, disclosed herein are methods of treating a joint condition in a subject comprising administering to the subject in need thereof a therapeutically effective amount of a composition comprising or consisting of a culture of Lactobacillus acidophilus and one or more prebiotics.

Disclosed herein are methods of reducing inflammation in a subject in need thereof. In some aspects, the methods can comprise administering to the subject in need thereof a therapeutically effective amount of any of the compositions described herein or any of the pharmaceutical compositions described herein. In some aspects, the inflammation can be inflammation in a peripheral tissue of the subject. In some aspects, the peripheral tissue can be tissue in the gut, knee joint, dorsal root ganglion (DRG) sensory neurons or spinal cord of the subj ect. In some aspects, the composition comprises or consists of Lactobacillus acidophilus and one or more prebiotics. In some aspects, disclosed herein are methods of reducing inflammation in a subject comprising administering to the subject in need thereof a therapeutically effective amount of a composition comprising or consisting of Lactobacillus acidophilus and one or more prebiotics. In some aspects, disclosed herein are methods of reducing inflammation in a subject comprising administering to the subject in need thereof a therapeutically effective amount of a composition comprising or consisting of a culture of Lactobacillus acidophilus and one or more prebiotics.

In any of the methods disclosed herein, the subject has or was diagnosed with osteoarthritis prior to the administering step.

In any of the methods disclosed herein, the subject has or was diagnosed with depression prior to the administering step.

In any of the methods disclosed herein, the subject has or was diagnosed with diabetes to the administering step.

In any of the methods disclosed herein, the administration of any of the compositions described herein or any of the pharmaceutical compositions described herein can be via oral administration.

In some aspects, the methods disclosed herein can comprise administering to a subject, a pharmaceutical composition formulated for Lactobacillus acidophilus (e.g., 4x10⁹ per dose). In some aspects, the pharmaceutical composition can be formulated for Lactobacillus acidophilus 4x10⁹ per dose, and administered to a subject twice per week. In some aspects, the pharmaceutical composition can be formulated tor Lactobacillus acidophilus 4x 10⁹ per dose, and administered to a subject twice per week.

In some aspects, the methods disclosed herein can comprise administering to a subject, a pharmaceutical composition formulated for Lactobacillus acidophilus (e.g., 4x10⁹ per dose) and fructooligosaccharide (e.g., 20 mg/kg per dose). In some aspects, the pharmaceutical composition can be formulated for Lactobacillus acidophilus (e.g., 4xl0⁹ per dose) administered to a subject twice per day and/or twice per week, and fructooligosaccharide (e.g., 20 mg/kg per dose), and administered to a subject twice per week.

In some aspects, the methods disclosed herein can comprise administering to a subject, a pharmaceutical composition formulated for Lactobacillus acidophilus (e.g., 4x10⁹ per dose) and sodium butyrate (e.g., 400 mg/kg per dose). In some aspects, the pharmaceutical composition can be formulated for Lactobacillus acidophilus (e.g., 4xl0⁹ per dose) and administered to a subject twice per day and/or twice per week, and sodium butyrate (e.g., 400 mg/kg per dose), and administered once daily.

In some aspects, the methods disclosed herein can comprise administering to a subject, a pharmaceutical composition formulated for Lactobacillus acidophilus (e.g., 4x10⁹ per dose), fructooligosaccharide (e.g., 20 mg/kg per dose), and sodium butyrate (e.g., 400 mg/kg per dose). In some aspects, the pharmaceutical composition can be formulated for Lactobacillus acidophilus (e.g., 4xl0⁹) per dose, administered to a subject twice per day and/or twice per week, fructoohgosaccharide (e.g., 20 mg/kg per dose), administered to a subject twice per week; and sodium butyrate (e.g., 400 mg/kg per dose), and administered once daily.

Dosage regimens are adjusted to provide the desired response (e.g., a therapeutic response). For example, a single bolus may be administered, several divided doses may be administered over time or the dose may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. It is especially advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the subjects to be treated; each unit contains a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specification for the dosage unit forms of the invention are dictated by and directly dependent on (a) the unique characteristics of the active compound and the particular therapeutic effect to be achieved, and (b) the limitations inherent in the art of compounding such an active compound for the treatment of sensitivity in individuals.

In some aspects, a treatment regimen entails administration once per day, twice per day, once per week, twice per week, once every two weeks, once every three weeks, once every four weeks, once a month, once every 3 months or once every three to 6 months or once every 6 months to a year. In some aspects, different treatment regimens can be used for different agents.

Actual dosage levels of the active ingredients in the pharmaceutical compositions of the present invention may be varied so as to obtain an amount of the active ingredient which is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient. The selected dosage level will depend upon a variety of pharmacokinetic factors including the activity of the particular compositions disclosed herein employed, the route of administration, the time of administration, the rate of excretion of the particular compound being employed, the duration of the treatment, other drugs, compounds and/or materials used in combination with the particular compositions employed, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors well known in the medical arts. Any of the compositions disclosed herein can be administered via one or more routes of administration using one or more of a variety of methods know n in the art. As will be appreciated by the skilled artisan, the route and/or mode of administration will vary depending upon the desired results. In some aspects, the routes of administration for antibodies disclosed herein include but are not limited to intravenous, intramuscular, intradermal, intraperitoneal, subcutaneous, or other parenteral routes of administration, for example by injection or infusion. The phrase “parenteral administration” as used herein means modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular injection and infusion.

The methods and compositions, including combination therapies, enhance the therapeutic or protective effect, and/or increase the therapeutic effect of another antiinflammatory agent, therapeutic agent or therapy. Therapeutic and prophylactic methods and compositions can be provided in a combined amount effective to achieve the desired effect, such as inhibiting cartilage degeneration, reducing pain or reducing joint pain, protecting cartilage or preventing cartilage degeneration, preventing or reducing or inhibiting pain- associated depression and/or reducing or ameliorating one or more symptoms of osteoarthritis, reducing VEGF levels or cortisone levels in the blood, preventing a decrease in expression of glutamate transporter- 1 (GLT-1), joint disease, preserving or maintaining expression of glutamate transporter-1 (GLT-1), joint condition or joint injury, reducing MMP13 levels in the cartilage or synovium of a subject, and reducing inflammation.

The compositions disclosed herein can be administered before, during, after, or in various combinations relative to a second therapeutic agent or therapy. The administrations may be in intervals ranging from concurrently to minutes to days to weeks. In aspects where the compositions are provided to a patient separately from a second therapeutic agent or therapy, one would generally ensure that a significant period of time did not expire between the time of each delivery⁷, such that the two compounds would still be able to exert an advantageously combined effect on the patient. In such instances, it is contemplated that one may provide a patient with the compositions described herein and the second therapeutic agent or therapy within about 0-60 minutes, 1 to 12 h, 12 to 24 or 72 h of each other and, more particularly, within about 6-12 h of each other. In some situations it may be desirable to extend the time period for treatment significantly where several days (2, 3, 4, 5, 6, or 7) to several weeks (1. 2, 3, 4, 5. 6, 7, or 8) lapse between respective administrations. In some aspects, a course of treatment can last between 1-90 days or more (this such range includes intervening days). It is contemplated that one agent may be given on any day of day 1 to day 90 (this such range includes intervening days) or any combination thereof, and another agent is given on any day of day 1 to day 90 (this such range includes intervening days) or any combination thereof. Within a single day (24-hour period), the patient may be given one or multiple administrations of the agent(s). Moreover, after a course of treatment, it is contemplated that there can be a period of time at which no a second therapeutic agent or therapy is administered. This time period may last 1 -7 days, and/or 1 -5 weeks, and/or 1 -12 months or more (this such range includes intervening days), depending on the condition of the patient, such as their prognosis, strength, health, etc. It is expected that the treatment cycles would be repeated as necessary. In some aspects, a single treatment may be needed to achieve the desired outcome.

In some aspects, compositions disclosed herein can be administered 1, 2, 3, 4, 5, 6, 7,

8, 9, 10 or more days after surgery'. In some aspects, the surgery' can be destabilization of the medial meniscus surgery. In some aspects, the surgery can be a type of surgery for the treatment of osteoarthritis. In some aspects, the surgery can be a cartilage replacement surgery' (e g., artificial endoprosthesis) or joint arthroplasty. In some aspects, the surgery can be a tendon repair surgery'. In some aspects, the surgery' can be a joint replacement. In some aspects, the compositions disclosed herein can be administered one or more times separated by one or more weeks. In some aspects, a second, third, fourth, fifth, and so on, administration of the compositions thereof disclosed herein can be separated by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 weeks, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months, or 1 or more years. In some aspects, the compositions disclosed herein can be administered one or more times separated by one or more months. In some aspects, a second, third, fourth, fifth, and so on, administration of the compositions disclosed herein can be separated by 1, 2. 3, 4, 5, 6. 7, 8,

9, 10, 11 or 12 months. In some aspects, the compositions disclosed herein can be administered one or more times separated by a variety' of intervals, days, weeks, months, years or any combination thereof.

ARTICLES OF MANUFACTURE

The composition described herein can be packaged in a suitable container labeled, for example, for use as a therapy to treat osteoarthritis or any of the methods disclosed herein. Accordingly, packaged products (e.g., sterile containers containing the composition described herein and packaged for storage, shipment, or sale at concentrated or ready-to-use concentrations) and kits, including at least Lactobacillus acidophilus and one or more prebiotics, one or more postbiotics, or a combination thereof as described herein and instructions for use, are also within the scope of the disclosure. A product can include a container (e.g., a vial, jar, bottle, bag, or the like) containing the composition described herein. In addition, an article of manufacture further may include, for example, packaging materials, instructions for use, syringes, buffers or other control reagents for treating or monitoring the condition for which prophylaxis or treatment is required. The product may also include a legend (e.g., a printed label or insert or other medium describing the product's use (e.g., an audio- or videotape)). The legend can be associated with the container (e.g., affixed to the container) and can describe the manner in which the compound therein should be administered (e.g., the frequency and route of administration), indications therefor, and other uses. The compositions can be ready for administration (e.g., present in dose- appropriate units), and may include a pharmaceutically acceptable adjuvant, carrier or other diluent. Alternatively, the compounds can be provided in a concentrated form with a diluent and instructions for dilution.

The amount of active ingredient which can be combined with a carrier material to produce a single dosage form will vary depending upon the subject being treated, and the particular mode of administration. The amount of active ingredient w hich can be combined with a carrier material to produce a single dosage form will generally be that amount of the composition which produces a therapeutic effect. Generally, out of one hundred percent, this amount will range from about 0.01 percent to about ninety -nine percent of active ingredient, preferably from about 0. 1 percent to about 70 percent, most preferably from about 1 percent to about 30 percent of active ingredient in combination with a pharmaceutically acceptable carrier.

EXAMPLES

Example 1: Combination of Synbiotic - L. acidophilus (LA) and Fructooligosaccharide (FOS) -Show the Powerful Therapeutic Effects for OA Treatment with Enhanced Reduction in Joint Pain and Depression-Like Symptoms in a Preclinical Animal Model.

Synbiotics, a mixture of probiotics and prebiotics, act synergistically to promote the growth and survival of beneficial microorganisms in the gut. By comparison studies, the most effective prebiotic, FOS, w as identified and was shown to markedly enhance the therapeutic power when combined with LA (called ^L‘LA-synbiotic”). LA-synbiotic demonstrated that long-term treatments with LA-synbiotic (i) eliminated joint pain (no statistical significance compared to “No Pain baseline,” FIG. 1) and (ii) reversed depression-like behaviors. Other pain assessments, including thermal hyperalgesia, also showed similar pain response patterns.

Over 95% of OA cases in military sendee members is attributed to posttraumatic OA (PTOA) from combat injury' and demanding physical activities. These are the primary' reasons for joint damage in veterans. The therapeutic results described herein were obtained using a PTOA animal model. Thus, the results are more meaningful as they represent veterans' health conditions that can apply to clinical settings. For the data shown in FIG. 1), the treatments were started immediately after traumatic injury' (inflammatory' pain stage, see a green arrow) and continued for 12 wks. In some aspects, LA-synbiotic can be used in comorbid conditions of OA and OA- associated depression, targeting treatment starting points at different OA phases: early- and advanced OA, for example.

Generation of mouse OA models. A PTOA animal model for knee OA pain was carried out using surgical procedures in mice (C57BL/6J). A partial medial meniscectomy (PMM) was done under anesthesia and sterile conditions using a medial para-patellar arthrotomy. Sham surgery (skin incision and medial capsulotomy) was done on control animals. In the PMM model, the animals show inflammatory pain right after surgery for a week. The gradual development of cartilage degeneration occurs over 16 weeks, representing early OA (~4 weeks) and advanced OA (8-weeks after PMM).

Longitudinal behavioral pain measurements. Structural, morphologic, histologic and biochemical changes in knee joints after joint injury can be correlated with behavioral and imaging changes in response to pain as determined by longitudinal pain measurements.

Spontaneous movement-evoked pain behavior: OA pain is associated with joint movement. Movement-induced joint pain is assessed by locomotive behaviors (distance traveled, climbing, horizontal and vertical activity) using a Photobeam Activity Monitoring System (San Diego Instruments) (4 control, 4 experimental mice per session, n=10/group).

Mechanical hyperalgesia: Von Frey testing can be done weekly as indirect pain tests. The threshold force required to elicit withdrawal of the paw (median 50% of paws withdrawn) is determined twice on each hind paw on each test day.

Hot or cold hyperalgesia: Responses to noxious heat or cold stimuli can be determined using either a hot plate at 55 °C or acetone spray-on plantar surface, the following are recorded: licking of the hind paw, shaking of the hind paw in the air, and the frequency of jumping that indicates a sign of hyperalgesia. Preparation of LA-synbiotic and treatment of animals. LA (ATCC 4357) is grown overnight in MRS broth at 37°C in an incubator without shaking. The next day, bacteria are spun down by centrifuging at 3,000 rpm for 10 min. The pellet is rinsed with 1 X PBS twice and spun again. The final pellet is diluted in 1 X PBS (vehicle). FOS (Meiji, Tokyo, Japan) is added into the resuspended in the LA pellet solution. Mice will receive twice/week gavage of 3x 109 colony-forming units (CFUs) in 200 mL and FOS 20mg/kg in 0.3 mL volume/animal. The control animals will receive 2x/wk oral gavage with PBS (vehicle) of the same volume. Twice/wk treatment will be done: immediate post-OA induction (Gp: 1 , within a week), early- (Gp:2, at 4wks post-surgery), and advanced OA (Gp:3, at 8wks post-surgery). An intact control group (t=0) will be included. After 16 wks (surgery model), animals will be humanely euthanized, and tissues are harvested for biochemical and histopathological analyses.

FIG. 2 shows that behavioral pain scored by histopatholgy correlated with the severity of cartilage destruction assessed using OARSI scoring criteria.

Traditional joint pathology score by histopathology. Behavioral pain scores can be correlated with the severity of cartilage destruction assessed using OARSI scoring criteria. Scoring is done by two blinded observers. The intra-observer and inter-observer reliability of this scoring system can be assessed by calculating the intra-class coefficient and the Bland- Altman bias. Hematoxylin and eosin (H&E) and/or Safranin-0 staining were done to assess changes in general morphology, proteoglycan loss, and infiltration of inflammatory cells.

FIG. 3 shows no statistical difference between intact cartilage or synovium when treated with L. acidophilus (LA)-synbiotic when targeting MMP-13.

FIGS. 4A-B show the pharmacological effect of administering probiotics, prebiotics, and synbiotics on GLT-1 expression induced by OA surgery' in the ipsilateral lumbar region of the spinal cord (L3/L5) following a 12-week treatment twice per week. FIG. 4A shows IF staining of GLT-1 in the spinal cord dorsal hom with different interventions (n=3/gp). FIG. 4B shows the quantitative analysis of GLT-1 expression.

Methodology. Immunohistochemical analyses were conducted following the methods (Ma, K., et al., 2023. International Journal of Biological Sciences, 19(2), p.675; and O'Sullivan, I., et al., 2022. International Journal of Molecular Sciences, 23(20), p. 12076). At 12 weeks post-PMM or post-sham surgery’, animals were humanely anesthetized and subjected to pre-perfusion with a solution of 4% paraformaldehyde in phosphate-buffered saline (PFA/PBS, pH 7.4). Subsequently, knee joint and spinal cord samples were further fixed using 4% PFA/PBS (pH 7.4) for a period of 48 hours at a temperature of 4 °C. For knee joint sections, to remove calcification, samples were decalcified over a 14-day span in chelation buffer (0.5M EDTA. pH 8.0, Invitrogen, AM9262) maintained at 4 °C. Whole knee joints and lumber region of spinal sections were then serially sectioned into 5-micron sections, either in the sagittal or coronal plane, to facilitate subsequent immunofluorescence microscopy. These sections were then incubated overnight at a temperature of 4 °C with primary antibodies targeting MMP-13 (1:500, Sigma- Aldrich, MAB 13424) for knee joint sections, and GLT1 (1:500, Sigma- Aldrich, ZRB1630) for spinal cord sections. Following three rinses with PBS, the sections were subjected to a 2-hour incubation with goat antimouse Alexa Fluor 488 IgG (1 :500, Invitrogen, A-11029). Once again, PBS rinses were employed before attaching the sections to slides with a mounting medium containing 4', 6- diamidino-2-phenylindole (DAPI; Southern Biotech) and subsequently covered with coverslips. Fluorescent images were captured using a Nikon Eclipse NiE upright microscope (Nikon Instruments Inc., Melville, NY) with consistent exposure settings and the associated software.

FIGS. 5A-B show that treatment with L. acidophilus (LA) and fructooligosaccharide (FOS) have anti-depression effects using behavioral tests in an OA-induced animal model.

Depression Tests Elevated plus-maze test (EPM). The EPM consisted of two open arms (5 cm x 30 cm) and two enclosed arms (5 cm x 30 cm) with 10 cm high open roof, connected with each other by a central square platform (5 cm x 5 cm). Each mouse is placed in the central square platform facing one of the open arms. The number of entries into open and enclosed arms are then be monitored for 5 minutes. The apparatus is wiped with an alcohol solution before every trial to exclude any bias related to olfaction.

Open-field test: The open field (40 cm x 40 cm x 40 cm) has white walls and floors. The 'center' field is defined as the central 20 cm x 20 cm area of the open field, one-fourth of the total area. A mouse is be placed inside an isolation chamber with dim illumination and a fan, and its activity is monitored for 5 min. The apparatus is wiped with an alcohol solution before every trial to exclude any bias related to olfaction.

Sucrose preference test can also be used to assess depression in rodents. Mice will be deprived of water and food for 24 hrs. Then, mice will be housed individually in each cage with free access to 2 bottles containing 100 ml of sucrose solution (1% w/v) and 100 ml of water for 2 h. The bottle position will be changed every hour to avoid side preference by the mice. After that, the volume of consumed sucrose solution and water will be recorded, and the sucrose preference will be calculated as follows: % sucrose consumption = sucrose consumption/ (water + sucrose consumption) x 100.

FIGS. 6A-B show that behavioral depression correlates with depression biomarkers in serum, and that L. acidophilus (LA) coadministered with a prebiotic reduces depression biomarkers in serum in an animal of OA.

Detection of depression markers. VEGF, and corticosterone in serum. Blood samples are collected from euthanized animals by cardiac puncture. Samples are centrifuged at 5.000 rpm at 4°C for 10 min, and plasma is collected and stored at -80°C. Serum level of VEGF is detected by following the manufacturer's instruction for mouse VEGF-A ELISA kit (Invitrogen). Serum level of corticosterone is detected by following the manufacturer's instruction of the Corticosterone ELISA kit (ABOR ASSAYS). Results are expressed as pg/mL (duplicate) and final optimum density are measured at 450 nm using SpectroMax iD3 ELISA plate reader.

In sum, the results described herein demonstrate that the combination of LA with FOS (referred to as LA-synbiotic) has beneficial effects on joint pain, cartilage protection and depressive disorder, and. thus can be used as a co-treatment strategy for OA as well as in subjects with OA that show signs of depression.

Claims

CLAIMS We claim:

1. A composition comprising or consisting of Lactobacillus acidophilus and one or more prebiotics.

2. The composition of claim 1, wherein the Lactobacillus acidophilus is Lactobacillus acidophilus (ATCC 4356).

3. The composition of any of claims 1 or 2, wherein the one or more prebiotics are inulin or fructooligosaccharide.

4. The composition of any of claims 1 to 3. wherein the composition comprises or consists of Lactobacillus acidophilus and fructooligosaccharide.

5. The composition of any of claims 1 to 4, further comprising butyrate.

6. The composition of any of the preceding claims, further comprising a pharmaceutically acceptable carrier.

7. The composition of any of the preceding claims wherein the composition is frozen, cryopreserved or lyophilized.

8. The composition of any of the preceding claims, wherein the composition is a solid or liquid.

9. The composition of any of the preceding claims, w herein the composition comprises at least 2x10⁹ cells of the Lactobacillus acidophilus .

10. The composition of any of the preceding claims, w herein the composition in is a powder, granule, a ready -to-use beverage, food bar, an extruded form, capsule, gel cap, or dispersible tablet form.

11. A pharmaceutical composition comprising one or more of the compositions of claims 1 to 10.

12. The pharmaceutical composition of claim 11, further comprising a pharmaceutically acceptable carrier or excipient.

13. The pharmaceutical composition of any of claims 1 1-12, wherein the pharmaceutical composition is formulated as a liquid, a lyophilized powder, a cream, or an ointment.

14. A method of inhibiting cartilage degeneration in a subject, the method comprising administering to a subject in need thereof a therapeutically effective amount of the composition of any of claims 1-10 or the pharmaceutical composition of any of claims 11-13.

15. A method of reducing pain or reducing joint pain in a subject, the method comprising administering to a subject in need thereof a therapeutically effective amount of the composition of any of claims 1-10 or the pharmaceutical composition of any of claims 11-13.

16. A method of protecting cartilage or preventing cartilage degeneration in a subject, the method comprising administering to a subject in need thereof a therapeutically effective amount of the composition of any of claims 1-10 or the pharmaceutical composition of any of claims 11-13.

17. A method of preventing or reducing or inhibiting pain-associated depression in a subject, the method comprising administering to a subject in need thereof a therapeutically effective amount of the composition of any of claims 1-10 or the pharmaceutical composition of any of claims 1 1 -13.

18. A method of reducing MMP13 levels in the cartilage or synovium of a subject, the method comprising administering to a subject in need thereof a therapeutically effective amount of the composition of any of claims 1-10 or the pharmaceutical composition of any of claims 11-13.

19. A method of increasing expression of glutamate transporter-1 (GLT-1) in a subject, the method comprising administering to a subject in need thereof a therapeutically effective amount of the composition of any of claims 1-10 or the pharmaceutical composition of any of claims 11-13.

20. A method of preserving or maintaining expression of glutamate transporter- 1 (GLT-1) in a subject, the method comprising administering to a subject in need thereof a therapeutically effective amount of the composition of any of claims 1-10 or the pharmaceutical composition of any of claims 11-13.

21. A method of preventing a decrease in expression of glutamate transporter-1 (GLT-1) in a subject, the method comprising administering to a subject in need thereof a therapeutically effective amount of the composition of any of claims 1-10 or the pharmaceutical composition of any of claims 11-13.

22. The method of any one of claims 19 to 21, wherein the expression of glutamate transporter-1 (GLT-1) is GLT-1 expression in the spinal cord of the subject.

23. A method of reducing VEGF levels or cortisone levels in the blood of a subject, the method comprising administering to a subject in need thereof a therapeutically effective amount of the composition of any of claims 1-10 or the pharmaceutical composition of any of claims 11-13.

24. A method of treating osteoarthritis in a subject in need thereof, the method comprising administering to a subject in need thereof a therapeutically effective amount of the composition of any of claims 1-10 or the pharmaceutical composition of any of claims 11-13.

25. A method of reducing or ameliorating one or more symptoms of osteoarthritis in a subject, the method comprising administering to a subject in need thereof a therapeutically effective amount of the composition of any of claims 1-10 or the pharmaceutical composition of any of claims 11-13.

26. A method of treating a joint disease in a subject in need thereof, the method comprising administering to a subject in need thereof a therapeutically effective amount of the composition of any of claims 1 -10 or the pharmaceutical composition of any of claims 11-13.

27. The method of claim 26, wherein the joint disease is a degenerative j oint disease.

28. The method of claim 27, wherein the degenerative joint disease is osteoarthritis.

29. A method of treating a joint condition in a subject in need thereof, the method comprising administering to a subject in need thereof a therapeutically effective amount of the composition of any of claims 1-10 or the pharmaceutical composition of any of claims 11-13.

30. The method of claim 29, wherein the joint condition is a joint injury.

31. The method of claim 30, wherein the joint injury' is a traumatic injury' or a post-operative injury.

32. The method of claim 29, wherein the joint injury is a repetitive strain injury.

33. A method of reducing inflammation in a subject in need thereof, the method comprising administering to a subject in need thereof a therapeutically effective amount of the composition of any of claims 1-10 or the pharmaceutical composition of any of claims 11-13.

34. The method of claim 33, wherein the inflammation is inflammation in a peripheral tissue of the subject.

35. The method of claim 34, wherein the peripheral tissue is tissue in the gut, knee joint, dorsal root ganglion (DRG) sensory neurons or spinal cord of the subject.

36. The method of any of claims 14-35, wherein the subject has or was diagnosed with osteoarthritis prior to the administering step.

37. The method of any of claims 14-35, wherein the subject has or was diagnosed with depression prior to the administering step.

38. The method of any of claims 14-35, wherein the subject has or was diagnosed with diabetes.

39. The method of any of claims 14-38, wherein the administration is via oral administration.