Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
  • A61K31/715—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
  • A61K31/739—Lipopolysaccharides
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
  • A61P1/04—Drugs for disorders of the alimentary tract or the digestive system for ulcers, gastritis or reflux esophagitis, e.g. antacids, inhibitors of acid secretion, mucosal protectants
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P11/00—Drugs for disorders of the respiratory system
  • A61P11/06—Antiasthmatics
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P17/00—Drugs for dermatological disorders
  • A61P17/02—Drugs for dermatological disorders for treating wounds, ulcers, burns, scars, keloids, or the like
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P19/00—Drugs for skeletal disorders
  • A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P25/00—Drugs for disorders of the nervous system
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P3/00—Drugs for disorders of the metabolism
  • A61P3/04—Anorexiants; Antiobesity agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P3/00—Drugs for disorders of the metabolism
  • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
  • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P37/00—Drugs for immunological or allergic disorders
  • A61P37/02—Immunomodulators
  • A61P37/06—Immunosuppressants, e.g. drugs for graft rejection
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• • C—CHEMISTRY; METALLURGY
  • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
  • C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
  • C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
  • C08B37/0003—General processes for their isolation or fractionation, e.g. purification or extraction from biomass
• • C—CHEMISTRY; METALLURGY
  • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
  • C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
  • C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
  • C08B37/006—Heteroglycans, i.e. polysaccharides having more than one sugar residue in the main chain in either alternating or less regular sequence; Gellans; Succinoglycans; Arabinogalactans; Tragacanth or gum tragacanth or traganth from Astragalus; Gum Karaya from Sterculia urens; Gum Ghatti from Anogeissus latifolia; Derivatives thereof
• • C—CHEMISTRY; METALLURGY
  • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
  • C08L—COMPOSITIONS OF MACROMOLECULAR COMPOUNDS
  • C08L5/00—Compositions of polysaccharides or of their derivatives not provided for in groups C08L1/00 or C08L3/00
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
  • A61K35/66—Microorganisms or materials therefrom
  • A61K35/74—Bacteria

Definitions

• the invention relates to compositions of capsular polysaccharide A (PSA) isolated from B. fragilis, and methods of isolation and purification and/or use thereof.
• PSA capsular polysaccharide A
• Capsular polysaccharide A (PSA) of Bacteroides fragilis (B. fragilis) NCTC9343 has been reported to be an immunomodulator with therapeutic and preventative applications.
• PSA Capsular polysaccharide A
• PSA Polysaccharide A
• Bacterioides fragilis B. fragilis
• the invention is based on the discovery of a significantly more potent form of PSA, made by B. fragilis, that comprises a lipid.
• This lipid is likely used by the microbe to anchor PSA in its outer membrane. Extraction techniques used heretofore do not yield this lipid- conjugated PSA.
• the lipid typically comprises less than 2% (w/w) of the native PSA-LT molecule. Proton NMR analysis of PSA does not distinguish the lipidated and non-lipidated forms. Rather, it has been found in accordance with the invention that presence of the lipidated from can only be visualized using specific gel staining techniques.
• the lipid may be removed from PSA-LT using mild hydrolysis (e.g., with dilute acetic acid) and subsequent extraction with an organic solvent (e.g., chloroform).
• This hydrolyzed form of PSA has an activity that is markedly reduced compared to the lipidated form of PSA (i.e., PSA-LT), and that its activity is at a level comparable to the earlier described PSA (i.e., the non-lipidated form of PSA).
• the invention provides, inter alia, a more potent form of PSA (i.e., PSA- LT) having enhanced IL-10 inducing activity and Treg maturation activity.
• PSA-LT is significantly more protective and therapeutic in animal models of inflammatory conditions such as but not limited to multiple sclerosis (i.e., the EAE animal model) and inflammatory bowel disease than the originally described non-lipidated form of PSA.
• the invention therefore provides, inter alia, compositions comprising the lipidated form of PSA, methods for its isolation and purification, and in vitro and in vivo methods of use thereof.
• the invention provides a method comprising extracting, into an aqueous phase, a capsular complex from B. fragilis using a mixture of phenol and water at high temperature, precipitating polysaccharide A (PSA) from the aqueous phase using ethanol, acid-treating the precipitate using 2% acetic acid at high temperature, wherein the method is performed at a pH equal to or less than about 9.
• the pH ranges from about 4 to about 9, or from about 4 to less than about 9.
• the invention provides a method comprising extracting, into an aqueous phase, a capsular complex from B. fragilis using a mixture of phenol and water at high temperature, precipitating polysaccharide A (PSA) from the aqueous phase using ethanol, acid-treating the precipitate using 2% acetic acid at high temperature, wherein the method is performed in the absence of sodium deoxycholate.
• PSA polysaccharide A
• each method may further comprise, after acid-treatment, a step of purifying PSA by size exclusion in a detergent-free buffer. In some embodiments, each method may further comprise dialysis of size excluded PSA.
• extraction occurs at 60-75°C. In some embodiments, extraction occurs at about 68°C.
• acid-treatment occurs at about 90°C for about 3 hours.
• each method is performed in the absence of sodium deoxycholate. In some embodiments, each method is performed in the absence of detergent. In some embodiments, B. fragilis is a PSA-overexpressing strain of B. fragilis such as but not limited to strain 9343.
• the invention provides a composition comprising isolated lipid- containing polysaccharide A (PSA-LT) produced by any of the foregoing methods.
• PSD-LT polysaccharide A
• the isolated lipid-conjugated polysaccharide A comprises one or more non-LPS lipids.
• the composition is formulated for oral administration to a subject.
• the invention provides a composition comprising isolated B. fragilis non-LPS lipid-conjugated polysaccharide A (PSA).
• PSA polysaccharide A
• the non-LPS lipid may be covalently conjugated to PSA.
• the isolated B. fragilis non-LPS lipid-conjugated PSA comprises less than 1%, less than 0.5%, less than 0.1%, less than 0.05%, or less than 0.01% (w/w) of non-LPS lipid conjugated to PSA.
• the isolated B. fragilis non-LPS lipid-conjugated PSA comprises less than 1%, less than 0.5%, less than 0.1%, less than 0.05%, or less than 0.01% (w/w) of non-LPS lipid conjugated to PSA.
• fragilis non-LPS lipid-conjugated PSA comprises non-LPS lipid in the range of about 0.1% to 2% (w/w) or about 0.1% to 1% (w/w), including about 0.5% (w/w).
• the isolated lipid- conjugated polysaccharide A comprises one or more non-LPS lipids.
• the composition is formulated for oral administration to a subject.
• the invention provides a composition comprising isolated B. fragilis lipid-conjugated polysaccharide A (PSA) and less than or about 0.5% (w/w) LPS, wherein the composition comprising the isolated B. fragilis lipid-conjugated polysaccharide A, when treated with 2% acetic acid and run on a 16.5% Tris-Tricine SDS-PAGE gel and reverse- stained using zinc sulphate and imidazole, demonstrates a band at above 60 kD (PSA-LT) and a band at about 5 kD.
• the isolated B. fragilis lipid-conjugated polysaccharide A comprises one or more non-LPS lipids.
• the lipid(s) may be covalently conjugated to the carbohydrate portion of PSA-LT. Under some circumstances, the bond between the lipid(s) and the carbohydrate portion of PSA-LT is not susceptible to cleavage by snake venom phosphodiesterase.
• the composition demonstrates LPS bands at 6 and 8 kD of less intensity than the band at about 5 kD. In some embodiments, the composition is formulated for oral administration to a subject.
• the invention provides a composition comprising isolated B. fragilis lipid-conjugated polysaccharide A wherein the composition comprises about 99% PSA, about 0.5% non-LPS lipid, and about 0.5% LPS.
• the invention provides a composition comprising isolated B. fragilis lipid-conjugated polysaccharide A wherein the composition comprises about 97% - 99% PSA, about 0.5% - 2% non-LPS lipid, and about 0.5% LPS.
• the invention provides a composition comprising isolated PSA-LT wherein the isolated PSA-LT comprises about 99.5% (w/w) of the composition and LPS represents about 0.5% (w/w) of the composition.
• compositions are essentially free of nucleic acids, proteins and/or other bacterial contaminants.
• the invention provides a method comprising administering to a subject having a condition associated with inflammation an effective amount of any of the foregoing PSA-LT-comprising compositions. In another aspect, the invention provides a method comprising administering to a subject at risk of a recurrence of a condition associated with inflammation an effective amount of any of the foregoing PSA-LT-comprising compositions.
• the method is a method of reducing the likelihood of a recurrence of the condition or reducing the frequency of future recurrences. In some embodiments, the method is a method of reducing the severity of symptoms associated with the condition, whether such symptoms are present in the first manifestation, in a recurrence, or chronically.
• the condition is an autoimmune disease.
• the autoimmune disease may be multiple sclerosis, Crohn's disease, ulcerative colitis, rheumatoid arthritis, or type I diabetes.
• the condition is asthma. In some embodiments, the condition is obesity.
• the composition may be administered to the subject by inhalation (e.g. nebulization), by oral administration, or by injection. In some embodiments, the composition is orally administered to the subject.
• the invention provides a method comprising administering to a subject at risk of developing a post-surgical adhesion an effective amount of any of the foregoing PSA-LT comprising compositions.
• the PSA-LT comprising composition is administered prior to surgery, during surgery, after surgery, or any combination thereof.
• the invention provides a method comprising administering to a subject having or at risk of developing an abscess an effective amount of any of the foregoing PSA-LT comprising compositions.
• the subject is also administered an anti-bacterial agent such as an antibiotic.
• the PSA-LT comprising composition is administered prior to development of an abscess and/or prior to the
• the PSA-LT comprising composition is administered after an abscess has been detected or diagnosed and/or after symptoms associated with an abscess are manifested.
• the invention provides a method comprising analyzing a composition suspected of comprising PSA-LT using a Tris-Tricine SDS-PAGE gel that is reversed stained with zinc sulphate and imidazole.
• the gel is a 16.5% Tris-Tricine SDS-PAGE gel.
• the composition is a bacterial fraction, such as a B. fragilis fraction.
• the bacterial fraction has been subjected to a hot phenol/water extraction, an ethanol precipitation, a mild acid-treatment (e.g., at an acidic pH at or above 4, typically in the range of 4-5), and/or size exclusion.
• the composition, including the bacterial fraction has not been contacted with sodium deoxycholate. In some embodiments, the composition, including the bacterial fraction, has not been contacted with a detergent. In some embodiments, the method is a method of detecting presence of a non-LPS lipid in the composition. In some embodiments, the method is a method of detecting PSA-LT in the composition. The presence of PSA, LPS and/or non-LPS lipid may be detected using the method. The amount of PSA, LPS and/or non-LPS lipid may be measured using the method.
• FIG. 1 is an elution profile of an S400 column.
• FIG. 2 is a proton nuclear magnetic resonance spectroscopy profile of purified PSA-
• FIG. 3 is a histogram of IL-10 production in a splenic dendritic cell (DC) T cell co- culture system using lipidated (PSA24) and non-lipidated (PSA23) forms of PSA.
• DC splenic dendritic cell
• FIG. 4 is a histogram of IL-10 production in a splenic dendritic cell (DC) T cell co- culture system using lipidated (PSA24) and non-lipidated (PSA23) forms of PSA, and non- lipidated (PSA23) form of PSA with added LPS (PSA23 + LPS).
• DC splenic dendritic cell
• FIG. 5 is a photograph of a gel showing the results of an acid treatment time course of PSA-LT containing composition.
• FIG. 6 is a photograph of a gel showing the effect of acid treatment on B. fragilis LPS compared to lipidated-PS A (PS A24) .
• FIG. 7 is a photograph of a gel showing the presence of lipid (arrow) in the PSA-LT preparation (PSA26).
• the higher molecular weight PSA shown in the box
• the gel-extracted PSA shows the lipid band (labeled with the arrow).
• FIG. 8 is a histogram showing IL-10 production as a function of PSA-LT preparation protocol, acid hydrolysis and/or organic solvent dissolution.
• FIG. 9 is a histogram showing the effect of sodium deoxycholate during PSA-LT preparation on IL-10 production.
• FIG. 10 is a photograph of a gel showing loss of the lipid band (Band 3) after sodium deoxycholate based extraction.
• the gel in this Figure, as in other Figures, is a 16.5% Tris- Tricine-SDS-PAGE gel that is reversed- stained with zinc sulphate and imidazole.
• FIG. 11 is a graph showing the effect of non-lipidated (PSA) and lipidated (PSA-LT) forms of PSA on outcome in an experimental autoimmune encephalitis (EAE) animal model system.
• FIG. 12 is a schematic illustrating an embodiment of PSA-LT attachment to the outer membrane of B. fragilis.
• FIG. 13 is a photograph of a gel showing the results of an acid treatment time course of a PSA-containing composition (PSA23) produced using a prior art isolation method and a PSA-LT containing composition (PSA24). Notably, there is no Band 3 following acid treatment of PSA23.
• the invention is premised on the surprising and unexpected discovery of a lipidated form of polysaccharide A (PSA), referred to herein as PSA-LT, from B. fragilis. It has been found, according to the invention, that the prior art methods for isolating and purifying PSA from B. fragilis inadvertently and unknowingly removed a lipid component that is conjugated to native PSA.
• PSA-LT polysaccharide A
• the lipidated form of PSA is better able to induce IL-10 production (and therefore better able to interact with Treg cells) and better able to prevent an experimentally induced murine form of multiple sclerosis (i.e., experimental autoimmune encephalitis, EAE).
• the invention provides compositions comprising the isolated lipidated form of PSA (PSA-LT), methods of isolating and purifying PSA-LT from B. fragilis, and methods of using isolated PSA-LT in vitro and in vivo.
• PSA-LT isolated lipidated form of PSA
• the invention contemplates that the isolated PSA-LT may be completely (or fully) lipidated or it may be partially lipidated.
• the degree of lipidation will depend upon the isolation methods, including for example the degree of acid hydrolysis.
• the compositions of the invention typically comprise a plurality of PSA-LT molecules, and that the plurality may exhibit variation in the degree of lipidation. Accordingly, in some instances the characteristics provided herein relate to the composition comprising a plurality of PSA-LT rather than a single PSA-LT molecule.
• the invention relates in part to a newly discovered lipidated form of PSA from B. fragilis.
• the lipidated form is referred to as PSA-LT.
• This lipidated form is comprised of a polysaccharide and one or more lipid chains (or tails).
• the carbohydrate portion of the newly discovered molecule is referred to as PSA.
• PSA This is also the form of the polysaccharide that has been previously isolated and analyzed. It was not known prior to the invention that this polysaccharide existed in nature in a lipid-conjugated form.
• Polysaccharide A comprises a tetrasaccharide repeating unit that is -3) a-D- AAT Galp-(1 -> 4)-[P-D-Galf-(l -> 3) -D-GalpNAc-(l -> 3) - [4,6-pyruvate] - ⁇ -D-Galp- (1 It possesses zwitterionic behavior as conferred by a positive charge on its free amine group and a negative charge on its free carboxyl group (per repeating tetrasaccharide unit).
• Its naturally occurring state is composed of over 60 tetrasaccharide repeating units (e.g., up to and including in some instances about 100, or about 200, or about 300 repeated units on average), and it has an average molecular size of about 150 kD (with a range of about 60 kD to 2000 kD).
• the repeating tetrasaccharide unit of PSA has a structure as follows:
• the invention is based in part on the isolation of a lipidated form of PSA from B. fragilis using isolation and purification methods that maintain the lipid conjugated to the PSA.
• the lipidated form of PSA referred to herein as PSA-LT, comprises tetrasaccharide repeating unit structure of PSA covalently conjugated to one or more lipid chains (or tails or moieties, as used interchangeably herein).
• the bond between the lipid and PSA is not susceptible to cleavage using phosphodiesterases extracted from snake venom.
• the lipid moieties may be conjugated to PSA via ester or amide linkages. There may be one or more lipid chains (or tails) per PSA molecule.
• compositions comprising isolated PSA-LT As used herein, with respect to PSA-LT, the term "isolated” intends that the PSA-LT is physically separated from its natural environment (i.e., a B. fragilis cell).
• Compositions comprising isolated PSA-LT may comprise other components including LPS and/or non-lipidated PSA. In some embodiments, the amount of LPS present in such compositions is about 0.5% (w/w) or less.
• compositions of the invention comprise about 99% PSA, 0.5% non-LPS lipid, and about 0.5% LPS. It will be understood that in some instances the compositions comprise PSA conjugated to its lipid(s) (i.e., PSA-LT), even though
• compositions may require cleavage of the lipid from PSA (e.g., in the gel systems described herein).
• PSA e.g., in the gel systems described herein.
• LPS refers to
• a non-LPS lipid is a lipid that is not LPS.
• the amounts of PSA, LPS and non-LPS lipid can be determined using a gel system such as that described herein.
• the compositions are essentially free of contaminants such as nucleic acids such as DNA and RNA and proteins. Essentially free, as used herein, intends that these contaminants represent about or less than 0.1% (w/w) of the composition. In some instances, such contaminants may be undetectable.
• compositions for use in vitro and in vivo may be used as analytical tools or assay standards.
• compositions may be used or in experimental models, such as animal models, of human disease or in humans or other subjects in need of immune regulation.
• experimental models such as animal models, of human disease or in humans or other subjects in need of immune regulation.
• compositions are pharmaceutically acceptable, intending that they are suitable for administration into a subject. They may or may not be used prophylactically or
• the method provides general and specific methods for isolating and purifying PSA- LT from B. fragilis. It is to be understood that these methods may be performed on any strain of B. fragilis provided it produces PSA. Such strains include naturally occurring strains or mutant strains such as the overexpressing strain 9343.
• the isolation methods of the invention differ from the prior art isolation methods in a number of ways.
• the instant methods perform hydrolysis with mild acid (e.g., at a pH of about 4, or in the range of 4-5) at an early step in the purification process rather than a later step as was done in the prior art.
• the instant methods perform molecular sieve chromatography in a neutral buffer solution (Tris) in the absence of sodium deoxycholate (DOC) which was used in the prior art methods.
• Tris neutral buffer solution
• DOC sodium deoxycholate
• the pH throughout the isolation is 9 or less (e.g., about 4 to about 9 or less), and in most steps is maintained in a neutral range.
• isolation and purification of PSA-LT comprises (1) growth of B. fragilis (wild type or mutant strain that produces PSA, including strains that overexpress PSA) under anaerobic conditions, (2) isolation of the capsular complex from B. fragilis, and (3) ethanol precipitation of PSA-LT, (4) mild acid-treatment.
• the capsular complex may be isolated using for example a hot phenol/water extraction.
• Ethanol precipitation of PSA-LT may be preceded by treatment with DNase, RNase and/or pronase.
• the acid-treatment may be performed using dilute acid (e.g., 2% acetic acid) at elevated temperature.
• the elevated temperature may range from 80-100°C, 85-95°C, and in some instances is about 90°C.
• the treatment may last for 1 hour, 2 hours, 3 hours or longer. In some instances, the acid treatment is performed using 2% acetic acid at 90°C for 3 hours.
• PSA-LT may be further purified from the precipitate by size exclusion.
• the precipitate may be dissolved in a neutral buffer such as but not limited to PBS, and then applied on a S-400 size exclusion column. This size exclusion step is performed in the absence of sodium deoxycholate, and in some instances in the absence of any detergent.
• PSA-LT containing fractions are then pooled, optionally analyzed, and dialyzed. The final dialysate may be lyophilized.
• FIG. 1 shows an elution profile of an S400 column in Tris buffered saline monitored by absorption at 280 nm. As stated above, fractions were tested by double diffusion in agar with an antibody to PSA-LT.
• FIG. 2 is a proton nuclear magnetic resonance spectroscopy profile of purified PSA- LT done on a 600mHz NMR.
• the carbohydrate structure of this molecule precisely fits the published spectrum for PSA. This indicates that the carbohydrate portion of PSA-LT is identical to PSA.
• the very small percentage (m/m) of PSA-LT that is attributable to lipid is not observed by this high-resolution technique.
• FIG. 5 shows the results of an acid treatment time course of PSA-LT using a 16.5% Tris-Tricine SDS-PAGE gel reverse stained with zinc sulphate/imidazole staining.
• This staining protocol allows one to observe both the carbohydrate (i.e., PSA) and lipid portions of PSA-LT in the same gel system.
• PSA-LT carbohydrate
• lipid portions of PSA-LT in the same gel system.
• Four mg of a 5 mg/ml PSA-LT (Lot 26) was treated with 2% acetic acid at 90°C for various periods of time followed by neutralization with NaOH and dialysis.
• One hundred micrograms of the resultant product was run on a 16.5% Tris-Tricine SDS-PAGE gel and reverse- stained as described above.
• the Figure clearly shows the disappearance of the LPS bands (at about 6 and 8 kD) and the emergence of a lipid band (labeled Band 3) at about 5 kD with increasing
• Band 3 was a hydrolytic product of PSA-LT
• the large molecular weight PSA-LT as observed in the gel on the left in FIG. 7, was further analyzed.
• the box indicates the band cut out of the gel for further analysis.
• the product eluted from the gel was then hydrolyzed and studied again by running it in the same gel system, as seen in the right hand column of FIG. 7.
• Band 3 can be observed, as indicated by the arrow, after hydrolysis of the large molecular weight PSA-LT.
• PSA-LT (Lot 24) was hydrolyzed very early in the purification process at a time immediately after separation of the aqueous phase from the initial phenol/water extraction step.
• PSA-LT was tested for its ability to induce IL-10 production in a splenic dendritic cell and T cell co-culture.
• This assay was performed as follows: (1) splenic DCs are isolated using mouse anti-CD 11c microbeads (Miltenyi Biotec cat#130-052-001); (2) CD4 + T cells are isolated using Mouse T cell CD4 Subset Column Kit (R&D systems cat#MCD4C-1000); (3) 2xl0 4 CD1 lc + DCs and 10 5 CD4 + T cells are mixed and 1 ⁇ anti-CD3 (BD
• Pharmingen cat#553057) is added; (4) the culture is then stimulated with 100 ⁇ g/ml PSA and the cells are incubated for 5 days; and (5) supernatants are harvested and analyzed by ELISA for the presence of IL-10.
• FIG. 3 demonstrates that, in terms of IL-10 production in this co-culture system, PSA-
• LT (shown as PSA24) is approximately 3 fold more potent than PSA made by the prior art method (shown as PSA 23).
• PSA24 had small amounts of contaminating LPS (approximately 0.5%, w/w) we added purified B. fragilis LPS to PSA Lot 23 (PSA23) and determined that the addition of LPS to a PSA lot containing no LPS did not enhance (and thus did not reconstitute) its ability to induce IL-10 in the co-culture system. (FIG. 4)
• PSA-LT 24 was quite active in inducing IL-10 production in the DC T cell co-culture system.
• PSA-LT (Lot 26, and shown as PSA26-PD in this Figure) was similarly active prior to any hydrolysis step. Following further purification of unhydrolyzed PSA-LT (Lot 26) by S400 chromatography, this material was hydrolyzed under the same conditions as the earlier phase hydrolysis of PSA-LT (Lot 24). In contrast to PSA-LT hydrolyzed early in the purification process, the PSA-LT hydrolyzed later in the process had lost much of its capacity to induce IL-10 in the co-culture system.
• the invention provides methods for detecting the presence of PSA-LT and in some instances quantitating the amount of PSA-LT in a sample or a composition. These methods employ the Tris-Tricine SDS-PAGE gel system described herein. Samples are run on such 16.5% Tris-Tricine SDS-PAGE gels and then reverse stained with zinc sulphate and imidazole. The presence of PSA-LT is indicated by the presence of a band at about 5 kD. The major band above 60 kD represents PSA (lipidated or non-lipidated versions).
• compositions analyzed using this system may be obtained as described herein, including for example following a phenol/water extraction, ethanol precipitation, mild acid hydrolysis, and size exclusion, or they may be obtained and/or prepared in other ways.
• the invention provides various methods of in vitro and in vivo use of the compositions provided herein.
• In vitro uses include use as an analytical tool (e.g., as a marker of the presence of B. fragilis) and as an assay standard or control (e.g., as a positive marker of PSA-LT).
• In vivo uses include but are not limited to those involving human subjects.
• in vivo uses include administration of the compositions of the invention to a non- human subject in order to modulate an immune response.
• the invention generally provides methods of modulating immune responses in a subject having or likely to develop an aberrant immune response.
• the aberrant immune response is an enhanced immune response and the composition acts to down- regulate the immune response.
• Enhanced immune responses are typically associated with inflammatory conditions, such as but not limited to autoimmune diseases.
• compositions of the invention may be used to modulate (and typically down-regulate) immune responses in subjects having or at risk of developing autoimmune diseases.
• subjects having autoimmune diseases typically experience one or more "events" or recurrences associated with the autoimmune disease.
• a subject having inflammatory bowel disease may experience temporally isolated attacks of the disease, characterized by the presence of symptoms or increased severity of symptoms.
• the invention contemplates that the compositions may be used in such subjects to reduce the likelihood of such future recurrences of the disease or to reduce the severity of symptoms associated with the disease (e.g., pain, fever, discomfort, fatigue, etc.).
• compositions may be administered prior to such recurrence, and in this manner may be chronically administered, optionally at a regular frequency. Examples include once a day, once every 2, 3, 4, 5 or 6 days, or once a week, etc.
• the invention also contemplates that the compositions may be administered to the subject during a recurrence in order to reduce the severity of symptoms or shorten the time of the recurrence.
• autoimmune diseases include but are not limited to multiple sclerosis, inflammatory bowel disease including
• the autoimmune disease is multiple sclerosis. In other important embodiments, the autoimmune disease is an inflammatory bowel disease including but not limited to ulcerative colitis and Crohn's disease.
• compositions of the invention may be administered to a subject who has yet to manifest an autoimmune disease (including symptoms thereof) yet is at risk of developing such as disease based on a known genetic or familial predisposition.
• a subject may have one or more family members that are afflicted with the disease.
• compositions of the invention are administered to subject having or at risk of developing graft-versus-host disease. Administration may occur prior to, during and/or after transplantation of an organ or tissue (including blood or a blood product) into the subject.
• compositions may be administered to subjects having or at risk of developing a conditions associated with inflammation.
• the composition may be administered to a subject having asthma.
• a subject having asthma typically experience asthmatic attacks or events characterized by impaired breathing.
• the invention contemplates that the
• compositions described herein may be administered acutely (e.g., a single large dose) or chronically (e.g., repeated, smaller doses) to asthmatic subjects. Accordingly, in some instances, the compositions may be administered prior to an asthmatic attack in order to prevent the occurrence of the attack, reduce the frequency of attacks, and/or to lessen the severity of the attack. In some instances, the compositions may be administered during an attack in order to reduce its severity and/or reduce its duration.
• compositions described herein are administered prior to, during, and/or immediately following surgery in order to prevent the occurrence of such adhesions and/or reduce their severity.
• the compositions may be administered repeatedly following surgery, including for example every day, every two days, every three days, etc. for a week, two weeks, three weeks, a month, or several months post- surgery.
• Another condition associated with inflammation is an abscess, including but not limited to an abdominal abscess as may occur upon leakage of intestinal contents into the peritoneum.
• the subjects being treated may also be administered antibacterial agents such as antibiotics.
• compositions described herein are administered to subjects that are obese.
• Such subjects are typically defined as having a body mass index (BMI) of 30 or more.
• BMI body mass index
• the compositions may be administered to a subject having a BMI greater than 20 or greater than 25.
• the compositions are intended to prevent further weight gain and/or induce weight loss in such subjects.
• a subject intends any subject that would benefit from administration of a composition of the invention or that could be administered the composition of the invention.
• the subject is a human subject.
• the subject may also be a companion animal such as a dog or cat, agricultural livestock such as horses, cattle, pigs, sheep, etc., laboratory animals such as mice, rats, rabbits, monkeys, etc., or animals such as those maintained in zoos or otherwise in captivity.
• a variety of administration routes are available. The particular mode selected will depend, of course, upon the particular condition being treated, the severity of the condition being treated, and the dosage required for therapeutic efficacy.
• the methods of the invention may be practiced using any mode of administration that is medically acceptable, meaning any mode that produces effective levels of the active compounds without causing clinically unacceptable adverse effects.
• modes of administration include oral, rectal, topical, nasal, inhalation, or parenteral routes.
• parenteral includes subcutaneous, intravenous, intramuscular, intraperitoneal, or infusion.
• the active agents of the invention are formulated as
• compositions or preparations Such compositions or preparations. Such compositions or preparations.
• compositions may routinely contain pharmaceutically acceptable carriers, concentrations of salt, buffering agents, preservatives, other immune modulators, and optionally other therapeutic ingredients.
• pharmaceutically-acceptable carrier as used herein, and described more fully below, means one or more compatible solid or liquid filler, dilutants or encapsulating substances which are suitable for administration to a human or other animal.
• carrier denotes an organic or inorganic ingredient, natural or synthetic, with which the active ingredient is combined to facilitate the application.
• the components of the pharmaceutical compositions also are capable of being comingled with active agents of the present invention, in a manner such that there is no interaction which would substantially impair the desired pharmaceutical efficacy.
• compositions may conveniently be presented in unit dosage form and may be prepared by any of the methods well-known in the art of pharmacy. All methods include the step of bringing PSA-LT (and/or compositions comprising PSA-LT) into association with a carrier which constitutes one or more accessory ingredients. In general, the compositions are prepared by uniformly and intimately bringing the PSA-LT composition into association with a liquid carrier, a finely divided solid carrier, or both, and then, if necessary, shaping the product.
• Compositions suitable for oral administration may be presented as discrete units, such as capsules, tablets, lozenges, each containing a
• compositions include suspensions in aqueous liquids or non-aqueous liquids such as a syrup, elixir or an emulsion.
• the active agent may be administered per se (neat) or in the form of a
• pharmaceutically acceptable, but non-pharmaceutically acceptable salts may conveniently be used to prepare pharmaceutically acceptable salts thereof and are not excluded from the scope of the invention.
• Such pharmacologically and pharmaceutically acceptable salts include, but are not limited to, those prepared from the following acids: hydrochloric, hydrobromic, sulphuric, nitric, phosphoric, maleic, acetic, salicyclic, p-toluene sulphonic, tartaric, citric, methane sulphonic, formic, malonic, succinic, naphthalene-2-sulphonic, and benzene sulphonic.
• pharmaceutically acceptable salts can be prepared as alkyline metal or alkyline earth salts, such as sodium, potassium or calcium salts of the carboxylic acid group.
• Suitable buffering agents include: acetic acid and a salt (1-2% w/v); citric acid and a salt (1-3% w/v); boric acid and a salt (0.5-2.5% w/v); and phosphoric acid and a salt (0.8-2% w/v).
• Suitable preservatives include benzalkonium chloride (0.003-0.03% w/v); chlorobutanol (0.3-0.9% w/v); parabens (0.01-0.25% w/v) and thimerosal (0.004-0.02% w/v).
• compositions suitable for parenteral administration conveniently comprise a sterile aqueous preparation of the polysaccharide, which can be isotonic with the blood of the recipient.
• acceptable vehicles and solvents that may be employed are water, Ringer's solution, and isotonic sodium chloride solution.
• sterile, fixed oils are conventionally employed as a solvent or suspending medium.
• any bland fixed oil may be employed including synthetic mono- or di-glycerides.
• fatty acids such as oleic acid find use in the preparation of injectables.
• Carrier formulations suitable for subcutaneous, intramuscular, intraperitoneal intravenous, etc. administrations may be found in Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pa.
• a therapeutically effective amount is that amount necessary to delay the onset of, inhibit the progression of, or halt altogether the particular condition being treated, including reducing the likelihood, frequency and/or severity of a recurrence of the condition.
• the effective amount may be that amount which serves to reduce, alleviate, or delay the onset of the symptoms (e.g., pain, fever, etc.) of the disorder being treated or prevented. The effective amount will depend upon the mode of administration, the particular condition being treated and the desired outcome.
• doses of active compounds of the present invention may be from about 0.01 mg/kg per day to 1000 mg/kg per day, preferably from about 0.1 mg/kg to 200 mg/kg and most preferably from about 0.2 mg/kg to about 20 mg/kg, in one or more dose administrations daily, for one or more days. It is expected that doses ranging from 1-500 mg/kg, and preferably doses ranging from 1-100 mg/kg, and even more preferably doses ranging from 1-50 mg/kg, will be suitable. The preferred amount can be determined by one of ordinary skill in the art in accordance with standard practice for determining optimum dosage levels of the agent. It is generally preferred that a maximum dose is the highest safe dose according to sound medical judgment be used.
• the total daily dose for a human subject may range from about 50-
• the pharmaceutical preparation may be administered alone or in conjunction with other compounds.
• the pharmaceutical preparation is given in conjunction with one or more anti-bacterial agents including antibiotics selected from the group consisting of penicillin G, penicillin V, ampicillin, amoxicillin, bacampicillin, cyclacillin, epicillin, hetacillin, pivampicillin, methicillin, nafcillin, oxacillin, cloxacillin, dicloxacillin, flucloxacillin, carbenicillin, ticarcillin, avlocillin, mezlocillin, piperacillin, amdinocillin, cephalexin, cephradine, cefadoxil, cefaclor, cefazolin, cefuroxime axetil, cefamandole, cefonicid, cefoxitin, cefotaxime, ceftizoxime, cefmnenoxine, ceftriaxone, moxalactam, cefotet
• B. fragilis was grown in anaerobic conditions.
• the capsular complex from B. fragilis was isolated with hot phenol/water extraction.
• PSA-LT was precipitated with ethanol after DNAse, RNAse and pronase treatments. The precipitate was then acid treated with 2% acetic acid at 90°C for 3 hours.
• PSA-LT was further purified on S-400 size exclusion column in PBS. The fractions were analyzed and pooled, then dialyzed and lyophilized. The purity of PSA was assessed by nuclear magnetic resonance spectroscopy and SDS PAGE gel, as described herein.
• B. fragilis strain 9343 which overexpresses polysaccharide A (PSA) was plated onto a blood agar plate and grown overnight at 37°C.
• a swab from a heavily colonized plate was sub-cultured into a 500 ml starter culture of peptone yeast broth.
• the starter culture was inoculated into 16 liter culture of the same media and pH was titrated to neutrality with 5M NaOH. An anaerobic gas mix was bubbled into the sealed culture.
• bacteria were checked by Gram stain and subculture. Organisms were collected by centrifugation at 8,000x g for 20 minutes. Bacterial pellets were washed two times with saline yielding approximately one liter of bacterial pellet.
• the bacterial pellet was suspended in 68°C melted crystalline phenol to a final concentration of phenol of about 37% v/v (yielding a phenol/water preparation) and mixed for 30 minutes at 68°C followed by stirring at 4°C for 48 hours.
• the phenol/water preparation was aliquoted into glass bottles which were then centrifuged at 1500 rpm. The upper water layer was harvested. Any residual phenol contained in the harvested aqueous phase was extracted with an equal volume of ethyl ether. The ether phase was then removed using a separatory funnel and any residual ether in the aqueous phase was evaporated, yielding the final aqueous phase from the phenol/water preparation.
• the aqueous phase was dialyzed versus water with multiple changes over 5 days at
• the PSA-LT was precipitated by adding 5 volumes of ethanol at 4°C to the mixture.
• Acid treatment was then carried out by adding 8 mis acetic acid to the solution, and heating it for three hours at 90°C. The solution was then cooled slightly, neutralized with NaOH, and cooled to 4°C.
• PSA-LT was then lyophilized. Recovery of total PSA was 739 mg.
• the degree of contaminating LPS was determined by Pro Q LPS stain on an SDS PAGE gel system and determined to be approximately 0.5%. Polysaccharide purity and structure was determined by proton nuclear magnetic resonance spectroscopy on a 600 mHz spectrometer. EQUIVALENTS
• inventive embodiments are presented by way of example only and that, within the scope of the appended claims and equivalents thereto, inventive embodiments may be practiced otherwise than as specifically described and claimed.
• inventive embodiments of the present disclosure are directed to each individual feature, system, article, material, kit, and/or method described herein.
• a reference to "A and/or B", when used in conjunction with open-ended language such as “comprising” can refer, in one embodiment, to A only (optionally including elements other than B); in another embodiment, to B only (optionally including elements other than A); in yet another embodiment, to both A and B (optionally including other elements); etc.
• the phrase "at least one,” in reference to a list of one or more elements, should be understood to mean at least one element selected from any one or more of the elements in the list of elements, but not necessarily including at least one of each and every element specifically listed within the list of elements and not excluding any combinations of elements in the list of elements.
• This definition also allows that elements may optionally be present other than the elements specifically identified within the list of elements to which the phrase "at least one" refers, whether related or unrelated to those elements specifically identified.
• At least one of A and B can refer, in one embodiment, to at least one, optionally including more than one, A, with no B present (and optionally including elements other than B); in another

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