WO2005077389A1 - An anti-inflammatory, cytoprotective factor derivable from a probiotic organism - Google Patents
An anti-inflammatory, cytoprotective factor derivable from a probiotic organism Download PDFInfo
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- WO2005077389A1 WO2005077389A1 PCT/US2005/003765 US2005003765W WO2005077389A1 WO 2005077389 A1 WO2005077389 A1 WO 2005077389A1 US 2005003765 W US2005003765 W US 2005003765W WO 2005077389 A1 WO2005077389 A1 WO 2005077389A1
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- 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
- A61K35/741—Probiotics
- A61K35/744—Lactic acid bacteria, e.g. enterococci, pediococci, lactococci, streptococci or leuconostocs
- A61K35/745—Bifidobacteria
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- 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
- A61K35/741—Probiotics
- A61K35/744—Lactic acid bacteria, e.g. enterococci, pediococci, lactococci, streptococci or leuconostocs
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- 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
- A61K35/741—Probiotics
- A61K35/744—Lactic acid bacteria, e.g. enterococci, pediococci, lactococci, streptococci or leuconostocs
- A61K35/747—Lactobacilli, e.g. L. acidophilus or L. brevis
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
- G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
- G01N33/5044—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving specific cell types
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/52—Assays involving cytokines
- G01N2333/521—Chemokines
- G01N2333/523—Beta-chemokines, e.g. RANTES, I-309/TCA-3, MIP-1alpha, MIP-1beta/ACT-2/LD78/SCIF, MCP-1/MCAF, MCP-2, MCP-3, LDCF-1or LDCF-2
Definitions
- the invention relates generally to the field of inflammatory disorders. More particularly, it concerns inflammatory bowel diseases, such as ulcerative colitis and Crohn's disease.
- inflammatory bowel diseases such as ulcerative colitis and Crohn's disease.
- BACKGROUND OF THE INVENTION Inflammatory bowel disease (HBD) is a group of chronic disorders, such as ulcerative colitis and Crohn's disease, that cause inflammation or ulceration of the digestive tract.
- HBD Inflammatory bowel disease
- Ulcerative colitis causes inflammation and ulceration of the inner lining of the colon and rectum. It rarely affects the small intestine except for the end that connects to the colon, called the terminal ileum. Ulcerative colitis may also be called colitis or proctitis. Ulcerative colitis may occur in people of any age, but most often it starts between ages 15 and 30. Ulcerative colitis affects men and women equally and appears to run in some families.
- ulcerative colitis The extent and severity of mucosal injury in inflammatory bowel diseases are determined by the disequilibrium between two opposing processes, reparative and cytoprotective mechanisms versus infl-immation-induced injury. Treatment for ulcerative colitis depends on the seriousness of the disease. Most people are treated with medication. In severe cases, a patient may need surgery to remove the diseased colon. Some people whose symptoms are triggered by certain foods are able to control the symptoms by avoiding foods that upset their intestines, like highly seasoned foods, raw fruits and vegetables, or milk sugar (lactose). Some people have remissions that last for months or even years. However, most patients' symptoms eventually return.
- the goal of therapy is to induce and maintain remission, and to improve the quality of life for people with ulcerative colitis.
- Aminosalicylate drugs such as those that contain 5-aminosalicylic acid (5- ASA)
- Sulfasalazine is a combination of sulfapyridine and 5-ASA and is used to induce and maintain remission.
- the sulfapyridine component carries the anti-inflammatory 5-ASA to the intestine.
- sulfapyridine may lead to side effects such as nausea, vomiting, heartburn, diarrhea, and headache.
- 5-ASA agents such as olsalazine, mesalamine, and balsalazide, have a different carrier, offer fewer side effects, and may be used by people who cannot take sulfasalazine.
- 5-ASAs are given orally, through an enema, or in a suppository, depending on the location of the inflammation in the colon. Most people with mild or moderate ulcerative colitis are treated with this group of drugs first.
- Corticosteroids such as prednisone and hydrocortisone, also reduce inflammation. They may be used by people who have moderate to severe ulcerative colitis or who do not respond to 5-ASA drugs.
- Corticosteroids can be given orally, intravenously, through an enema, or in a suppository. These drugs can cause side effects such as weight gain, acne, facial hair, hypertension, mood swings, and an increased risk of infection. For this reason, they are not recommended for long-term use.
- Immunomodulators such as azathioprine and 6-mercapto-purine (6-MP), reduce inflammation by affecting the immune system. They are used for patients who have not responded to 5-ASAs or corticosteroids or who are dependent on corticosteroids. However, immunomodulators are slow-acting and it may take up to 6 months before the full benefit is seen.
- Cyclosporine A may be used with 6-MP or azathioprine to treat active, severe ulcerative colitis in people who do not respond to intravenous corticosteroids.
- other drugs may be given to relax the patient or to relieve pain, diarrhea, or infection.
- About 25-40% of ulcerative colitis patients must eventually have their colons removed because of massive bleeding, severe illness, rupture of the colon, or risk of cancer.
- the doctor will recommend removing the colon if medical treatment fails or if the side effects of corticosteroids or other drugs threaten the patient's health.
- Crohn's disease differs from ulcerative colitis in that it may affect any part of the digestive tract. It causes inflammation and ulcers that may affect the deepest layers of lining of the digestive tract.
- Anti-inflammatory drugs such as 5- aminosalicylates (e.g., mesalamine) or corticosteroids, are typically prescribed, but are not always effective. Immunosuppression with cyclosporine is sometimes beneficial for patients resistant to or intolerant of corticosteroids. Nevertheless, surgical correction is eventually required in 90% of patients with Crohn's disease; 50% undergo colonic resection. (Leiper et al., 1998; Makowiec et al., 1998). The recurrence rate after surgery is high, with 50% requiring further surgery within 5 years.
- Hsp Inducible heat shock proteins
- Induction of heat shock proteins by a mild "stress” confers protection against subsequent insult or injury, which would otherwise lead to cell death. This well-described phenomenon is known as “stress tolerance” (Parsell and Lindquist, 1993).
- stress tolerance In intestinal epithelial cells, inducible heat shock proteins convey a degree of cytoprotection against stressors such as inflammatory cell-derived oxidants and preserve the integrity of intestinal epithelial cell barrier function under hostile conditions (Chang, 1999; Musch et al, 1996; Musch et al, 1999).
- the induction of heat shock proteins in intestinal epithelial cells prolongs viability under conditions of stress (Musch et al, 1996) and preserves tight junctions as measured by transepithelial resistance (Musch et al, 1999).
- Activation of the pro-inflammatory NF- ⁇ B pathway is thought to be a key molecular event involved in the pathogenesis of IBD (Neurath et al unfamiliar 1998; Jobin and Sartor, 2000; Sch id and Adler, 2000; Boone et al. , 2002).
- Administration of antisense oligonucleotides targeting the NF- ⁇ B subunit p65 was more effective than steroid treatment in reducing inflammation in two different murine models of colitis (Neurath et al, 1996).
- Immunohistochemical studies have shown that colonic biopsies from Crohn's patients display increased levels of expression of the NF- ⁇ B subunit p65 in areas of active inflammation (Neurath et al, 1998).
- NF- ⁇ B In the noninflammatory state, NF- ⁇ B is held in its inactive, cytosolic form complexed to the inhibitory protein I ⁇ B. Once a signal is received to activate NF- ⁇ B, its inhibitor I ⁇ B is phosphorylated and targeted for degradation by the ubiquitin proteasome pathway. The release of NF- ⁇ B from inhibition and its translocation to the nucleus, results in the transcriptional activation of a broad spectrum of cytokine and chemokine genes, cell adhesion molecules, and immunoreceptors, all important mediators of the inflammatory response (Neurath et aladmi 1998; Jobin and Sartor, 2000; Schmid and Adler, 2000; Boone et al. , 2002).
- probiotics which are defined as ingestible microorganisms having health benefit beyond their intrinsic nutritive value, in the treatment of a variety of gastrointestinal ailments including inflammatory bowel diseases (Gionchetti et al, 2000a), irritable bowel syndrome (Niedzielin et al, 2001), pouchitis (Gionchetti et al, 2000b; Gionchetti et al, 2003), as well as rotavirus and antibiotic-associated diarrhea (Isolauri et al, 1991; Majamaa et al, 1995; Arvola et al, 1999).
- probiotics Although little is known about, their mechanisms of action, probiotics appear to have protective, trophic, and anti-inflammatory effects on bowel mucosa. Proposed mechanisms by which probiotics may act include the production of ammonia, hydrogen peroxide (Kullisaar et al, 2002; Annuk et al, 2003; Ocana et al, 1999), and bacteriocins (Cleveland et al, 2001; Paraje et al, 2000; Braude and Siemienski, 1968), which inhibit the growth of pathogenic bacteria, the competition for adhesion sites on intestinal epithelia (Lee et al, 2000; Lee et al, 2003), and an adjuvant-like stimulation of the immune system against pathogenic organisms (Maassen et al, 2000).
- probiotic VSL#3 (comprised of Streptococcus thermophilus, and several species of Lactobacillus and Bifidobacteria) attenuates intestinal inflammation in the IL-10 knockout mouse model of enterocolitis (Madsen et al, 2001) and has been shown to improve the clinical outcome of chronic intestinal inflammation in clinical trials (Gionchetti et al, 2000b).
- probiotics are highly dependent on the ability to establish and maintain bacterial colonization, and is limited by unregulated composition of formulations and homeopathic delivery of active agents.
- composition of formulations and homeopathic delivery of active agents there is a need to elucidate the mechanisms of probiotic activity and develop more effective therapies for inflammatory bowel diseases.
- the invention disclosed herein satisfies at least one of the aforementioned needs in the art by providing at least one soluble factor from the probiotic NSL#3, wherein the soluble factor(s) is useful in treating or preventing inflammatory disorders, such as inflammatory bowel disease.
- the soluble factor(s) inhibits the chymotrypsin-like activity of the proteasome in, e.g., intestinal epithelial cells. Proteasome inhibition occurs relatively soon after exposure of the epithelial cells to the probiotic-conditioned medium containing the soluble factor(s).
- the conditioned medium has shown a capacity to inhibit the pro-inflammatory ⁇ F- ⁇ B pathway, and does it through a mechanism different from type-Ill secretory mechanisms that have been described.
- the soluble factor(s) also induces expression of cytoprotective heat shock proteins (Hsp, e.g., Hsp25 and Hsp72) in intestinal epithelial cells. Without wishing to be bound by theory, these effects appear to be mediated through the common unifying mechanism of proteasome inhibition.
- the resulting inhibition of NF- ⁇ B and increased expression of one or both of Hsp25 and Hsp72 are consistent with the anti-inflammatory and cytoprotective effects of the soluble factor(s) and reveal a new mechanism underlying microbial-epithelial interaction. .
- the invention provides bioactive compounds or agents secreted by probiotic bacteria that attenuate the TNF- ⁇ -mediated induction of NF- ⁇ B activation in intestinal epithelial cells and induce the expression of cytoprotective heat shock proteins, thus affecting at least one, and perhaps two, "arms" of current inflammatory bowel disease models. These beneficial effects on the gut mucosa appear to stem from a common mechanism mediated by proteasome inhibition.
- the compounds of the invention provide the basis for therapies for the treatment of IBD that are superior to those currently available in the art.
- a composition is provided that comprises an isolated, anti-inflammatory, cytoprotective compound.
- the compound is present in a probiotic-conditioned medium.
- One suitable probiotic- conditioned medium is medium conditioned by the probiotic, NSL#3.
- the compound is present in an ether-extracted fraction of the probiotic-conditioned medium.
- the compound is an organic acid.
- the invention provides an isolated, anti-inflammatory, cytoprotective compound comprised in medium conditioned with one or more of Streptococcus salivarius subsp. thermophilus, Lactobacillus casei, Lactobacillus plantarum, Lactobacillus acidophilus, Lactobacillus delbrueckii subsp. bulgaricus, Bifidobacteria longum, Bifidobacteria infantis, and Bifidobacteria breve.
- the compound is present in a medium conditioned with Lactobacillus plantarum and in other embodiments, the conditioned medium is NSL#3-conditioned medium.
- the compound induces the expression of at least one heat shock protein.
- the heat shock protein is at least one of Hsp25 and Hsp 72.
- the compound is an inhibitor of ⁇ F- ⁇ B activation, such as by inhibiting the NF- ⁇ B pathway.
- the compound inhibits the NF- ⁇ B pathway by stabilizing I ⁇ B, such as by stabilizing unphosphorylated I ⁇ B, phosphorylated I ⁇ B, or both forms of I ⁇ B.
- the compound is both an inducer of heat shock protein expression and an inhibitor of the NF- ⁇ B pathway.
- the compound is a proteasome inhibitor.
- the compound may be a selective inhibitor of the proteasome.
- the selectivity of the proteasome inhibitor may be with regard to the protease activity of the proteasome, the type of cells in which it inhibits the proteasome, or both.
- the compound selectively inhibits the chymotrypsin-like activity of the proteasome. In other embodiments, the compound does not significantly inhibit the trypsin-like activity of the proteasome.
- the compound weakly inhibits the caspase-like activity of the proteasome, wherein "weak inhibition” refers to a level of inhibition equivalent to that caused by 10 ⁇ M lactacystin.
- the compound selectively inhibits the proteasome in epithelial cells.
- the compound selectively inhibits the proteasome in intestinal, or gut, epithelial cells.
- Another aspect of the invention is drawn to a pharmaceutical composition comprising an isolated, anti-inflammatory, cytoprotective compound derived from a probiotic-conditioned medium and at least one pharmaceutically acceptable excipient.
- An exemplary pharmaceutical composition comprises an isolated, anti-inflammatory, cytoprotective compound derived from an ether-extracted fraction of a conditioned medium, such as a NSL#3-conditioned medium.
- the compound is an organic acid.
- the compound induces expression of at least one heat shock protein, e.g., Hsp25 and/or Hsp 72.
- the compound is an inhibitor of ⁇ F- ⁇ B activation, such as by stabilizing I ⁇ B, whether phosphorylated I ⁇ B or not.
- the compound is a proteasome inhibitor, such as a selective inhibitor of the chymotrypsin- like activity of a proteasome.
- An exemplary proteasome is an epithelial cell proteasome, such as an intestinal epithelial cell proteasome.
- Yet another aspect of the invention is a method for treating a patient with an inflammatory disorder comprising administering to the patient an effective amount of an isolated, anti-inflammatory, cytoprotective compound derived from a probiotic- conditioned medium.
- the compound is administered in an amount effective to slow, halt or reverse the progress of an inflammatory disorder, such as an inflammatory disease or condition; however, also contemplated is the administration of a compound as described herein in an amount effective to ameliorate a symptom associated with an inflammatory disorder.
- Symptoms associated with inflammatory disorders are known in the art, as are methods for measuring or assessing such a symptom to determine whether that symptom has been ameliorated.
- the inflammatory disorder may be an autoimmune disorder.
- autoimmune disorders that may be treated according to the invention include rheumatoid arthritis, juvenile rheumatoid arthritis, osteoarthritis, psoriatic arthritis, atopic dermatitis, eczematous dermatitis, psoriasis, Sjogren's Syndrome, Crohn's disease, aphthous ulcer, ulcerative colitis, asthma, allergic asthma, cutaneous lupus erythematosus, scleroderma, vaginitis, leprosy reversal reactions, erythema nodosum leprosum, autoimmune uveitis, polychondritis, Stevens- Johnson
- the inflammatory disorder is an inflammatory bowel disease.
- the inflammatory bowel disease is Crohn's disease.
- the inflammatory bowel disease is ulcerative colitis.
- a preferred probiotic-conditioned medium for use in this aspect of the invention is a NSL#3-conditioned medium.
- the compound is derived from an ether-extracted fraction of the medium (i.e., the compound is extracted from the medium using ether). It is contemplated that compounds useful in the practice of the method will include organic acids and acid-stable proteins or peptides.
- the compound induces the expression of at least one heat shock protein, such as Hsp25 and/or Hsp72.
- the compound may inhibit ⁇ F- ⁇ B activation (e.g., by stabilizing I ⁇ B in a phosphorylated or unphosphorylated form) without or, preferably, with the induction of at least one heat shock protein.
- the compound used in the method is an inhibitor of a protease activity, such as a protease activity of a proteasome.
- a compound used in the method may selectively inhibit the chymotrypsin-like activity of a proteasome, such as an epithelial cell proteasome (e.g., an intestinal epithelial cell proteasome).
- Embodiments according to this aspect of the invention include the method of treating a patient with an inflammatory disorder wherein the anti-inflammatory, cytoprotective compound does not alter the ubiquitination level of at least one protein amenable to ubiquitination in an epithelial cell exposed to the compound.
- a related aspect of the invention is directed to a method of preventing an inflammatory disorder comprising administering an effective amount of an anti- inflammatory, cytoprotective compound derived from a probiotic-conditioned medium.
- This aspect of the invention includes embodiments analogous to the above- described embodiments of treatment methods, with apparent modification of those embodiments to suit the prophylactic use of a compound according to the invention to prevent, rather than to treat, a patient with an inflammatory disorder.
- Yet another aspect of the invention is drawn to a kit for treating (including ameliorating a symptom thereof) or preventing an inflammatory disorder comprising a pharmaceutical composition as described above and instructions for administration of the composition to treat or prevent the disorder.
- Another aspect of the invention provides a method of producing an isolated, anti-inflammatory cytoprotective compound comprising obtaining a NSL#3- conditioned medium; and isolating an anti-inflammatory, cytoprotective compound from the NSL#3-conditioned medium, thereby producing an isolated, anti- inflammatory, cytoprotective compound.
- the method further comprises characterizing the anti-inflammatory, cytoprotective compound. More preferably, the method further comprises identifying the anti-inflammatory, cytoprotective compound.
- the method further comprises obtaining more anti-inflammatory, cytoprotective compound.
- the more anti-inflammatory, cytoprotective compound is obtained by isolation from NSL#3.
- the more anti-inflammatory, cytoprotective compound is obtained by chemical synthesis.
- the method further comprises placing the more anti-inflammatory, cytoprotective compound in a pharmaceutical composition.
- the method further comprises administering the pharmaceutical composition to a subject.
- the subject is a human.
- the subject has an inflammatory disorder. More preferably, the inflammatory disorder is an inflammatory bowel disease. In some embodiments the inflammatory bowel disease is Crohn's disease. In other embodiments the inflammatory bowel disease is ulcerative colitis.
- Another aspect of the invention is drawn to a method of screening for a modulator of monocyte chemoattractant protein - 1 (MCP-1) release, comprising: (a) combining a candidate modulator, a probiotic-conditioned medium, and an epithelial cell; (b) measuring MCP-1 release by the cell; and (c) comparing the MCP-1 release in the presence, and absence, of the candidate modulator, wherein a difference in the MCP-1 release identifies the candidate modulator as a modulator of MCP-1 release.
- MCP-1 monocyte chemoattractant protein - 1
- the invention provides a method of screening for a modulator of heat shock protein expression, comprising (a) combining a candidate modulator, a probiotic-conditioned medium, and an epithelial cell; (b) measuring heat shock protein expression in said cell; and (c) comparing the heat shock protein expression in the presence, and absence, of said candidate modulator, wherein a difference in said heat shock protein expression identifies the candidate modulator as a modulator of heat shock protein expression.
- the method will identify a modulator of expression of Hsp25 and/or Hsp 72. Also contemplated are screening methods wherein the modulator alters the activity of Heat Shock Transcription Factor- 1 (HSF-1).
- HSF-1 Heat Shock Transcription Factor-1
- FIG. 1 Probiotic-conditioned medium inhibits TNF-alpha stimulation of
- NF- ⁇ B NF- ⁇ B.
- YAMC young adult mouse colon
- VSL#3-CM NSL#3 -conditioned medium
- T ⁇ F- ⁇ 50 ng/ml 6 hours prior to harvest.
- Experimental conditions are as indicated below each column, "ctrl" column is untreated control, i.e., baseline level of ⁇ F- KB activity in YAMC cells prior to T ⁇ F- ⁇ stimulation.
- FIG. 2 Probiotic-conditioned medium stabilizes and prevents degradation of I ⁇ B ⁇ . Immunoblot of I ⁇ B ⁇ and the phosphorylated form of I ⁇ B ⁇ , 20 ⁇ g protein lane. YAMC cells were treated with NSL#3-conditioned medium for 16 hours, then stimulated with T ⁇ F- ⁇ (50 ng/ml) and harvested at the times indicated. Shown in the upper two panels, T ⁇ F- ⁇ stimulates a transient phosphorylation of I ⁇ B ⁇ (5 minutes), is associated with decreased total I ⁇ B ⁇ (5-30 minutes) as I ⁇ B ⁇ is targeted for degradation.
- FIG. 3 Global Ubiquitination is not inhibited by VSL#3-conditioned medium. Immunoblot analysis of ubiquitinated proteins from YAMC cells following treatment with NSL#3-CM for 16 hours, demonstrating that global blockade of ubiquitination does not occur when cells are treated with NSL#3-CM.
- MG132 a compound known to inhibit proteasome function and increase accumulation of ubiquitinated proteins, is also shown, as is thermal stress (HS) and untreated control cells (C).
- HS thermal stress
- C untreated control cells
- the pattern of ubiquitinated proteins observed after NSL#3-CM treatment most closely resembles the pattern seen with thermal stress.
- Molecular weight markers (kDa) are indicated to the right.
- FIG. 4 Probiotic-conditioned medium inhibits proteasome activity.
- FIG. 5 shows immunoblot analysis of levels of Hsp25 and Hsp72 in YAMC cells following exposure to NSL#3 bacteria for the times indicated, demonstrating a time-dependent increase in inducible Hsp expression.
- Hsc73 heat shock cognate 73, serves as a loading control.
- FIG. 5B shows immunoblot analysis of levels of Hsp25 and Hsp72 in YAMC cells following exposure to NSL#3 -conditioned medium or sonicated organisms at the concentrations of bacteria indicated (cfu/ml).
- Bacteria cultures were separated into either conditioned medium fraction (CM) or sonicated pellet (Pellet).
- CM conditioned medium fraction
- Pellet sonicated pellet
- a concentration-dependent increase in Hsp expression can be seen upon exposure to NSL#3 -conditioned medium, which is not seen with sonicated pellet, indicating that the active factors produced by the bacteria are secreted into conditioned medium and are not cell wall components.
- Hsc 73 serves as a loading control.
- FIG. 5C shows immunoblot analysis of Hsp72, comparing different cell lines following exposure to NSL#3-conditioned medium for 16 hours, 20 ⁇ g protein lane.
- NSL#3 -conditioned medium induces a robust Hsp72 response in both colonic (YAMC) and small intestinal (MSIE) epithelial cells which is not seen in 3T3 fibroblast cells, suggesting that the probiotic effect is specific to epithelial cells.
- Untreated cells are indicated (-), thermal stress (HS) serves as a positive control.
- FIG. 6 Probiotic compounds induce intestinal epithelial heat shock proteins through an apical (luminal) membrane specific process.
- YAMC intestinal epithelial cells exposed to NSL#3 -conditioned medium from the apical (luminal) side demonstrate robust Hsp25 and Hsp72 protein expression, hi contrast, cells exposed to NSL#3 -conditioned medium from the basolateral side are not stimulated to express Hsp25 and Hsp72 proteins.
- NSL#3- CM has a similar effect to what is seen when it is added only to the apical side.
- the constitutive heat shock cognate Hsc73 was used as a control.
- FIG. 7 Time course of Hsp induction by the proteasome inhibitor MG132 is similar to that produced by VSL#3-conditioned medium.
- Hsp25 and Hsp72 protein expression is stimulated by components of NSL#3.-CM that reside in fractions that were prepared through Centricon filters with a molecular weight cut-off of 10 kDa.
- the constitutive heat shock cognate Hsc73 was used as a control.
- Control (C) NSL#3-CM (CM), NSL#3-CM passed through 10 kDa filter ( ⁇ 10 kDa), heat shock (HS).
- FIG. 10: VSL#3 bioactivity is pH-dependent.
- the induction of Hsp25 and Hsp72 by NSL#3-CM (CM) is influenced by the pH of the medium prior to its addition to the luminal fluid of YAMC monolayers.
- the pH values shown in the figure indicate the pH of the CM prior to its addition to the YAMC cells.
- the pH of the medium is 4.0 after being conditioned by the bacteria.
- the final pH after the 1:10 dilution in the luminal buffer is between 6.5 and 7.0, which is the approximate pH of the acid microclimate of intestinal epithelial cells in situ.
- FIG. 11 Ether-extracted compounds of VSL#3-CM inhibit T ⁇ F- ⁇ - stimulated ⁇ F- ⁇ B activity.
- the effects of ether-extracted compounds (EEC) and MG132 on NF-KB activity were determined using an NF- ⁇ B ELISA assay (Active Motif).
- FIG. 12 Ether-extracted compounds of VSL#3-CM directly inhibit proteasomal function.
- FIG. 13 Probiotic-conditioned medium displays differential inhibition of proteasome activity. YAMC cells were treated with NSL#3-conditioned medium for 16 hours and then harvested for proteasome assay using the fluorogenic substrate Bz- val-gly-arg-AMC (FIG. 13 A) or Z-leu-leu-glu- AMC (FIG. 13B). Fluorescence is expressed in arbitrary units over time.
- FIG. 14 Figure 1. Probiotic-conditioned media inhibits T ⁇ F-alpha stimulation of ⁇ F- ⁇ B. YAMC cells were transfected with a NF- ⁇ B luciferase reporter gene and treated with VSL#3 -conditioned media for 16 hours, then stimulated with TNF- ⁇ (50 ng/ml 6 hours prior to harvest). Experimental conditions are as indicated below each column.
- YAMC cells were treated with NSL#3-conditioned media (NSL-CM) for 16 hours, then stimulated with T ⁇ F- ⁇ (50 ng/ml) 6 hours prior to harvest and compared to untreated control (No Tx), TNF- ⁇ treatment alone (TNF- ⁇ only ), or cells pretreated with conditioned media from the E.coli strain DH5 ⁇ with and without TNF- ⁇ .
- Supernatants were assayed for release of the chemokine MCP-1 by ELISA (as described herein). Experimental conditions are as indicated below each column.
- YAMC cells pretreated with NSL-CM show a reduction in the amount of MCP-1 released in response to T ⁇ F- ⁇ stimulation compared to controls (mean ⁇ SE for three separate experiments, in each experiment each group was performed in triplicate, * p ⁇ 0.05 compared to controls).
- Figure 16 Probiotic-conditioned media stabilizes and prevents degradation of I ⁇ B ⁇ . YAMC cells were treated with NSL#3-conditioned media for 16 hours, then stimulated with T ⁇ F- ⁇ (50 ng/ml) and harvested at the times indicated. Shown in the upper panel, T ⁇ F- ⁇ stimulates a transient phosphorylation of I ⁇ B ⁇ (5 minutes) and is associated with decreased total I B ⁇ (5-15 minutes) as I ⁇ B ⁇ is targeted for degradation.
- VSL has a dramatic inhibitory effect on the chymotrypsin-like activity, no inhibitory effect on the trypsin-like activity, and a partial inhibitory effect on the caspase-like activity of the proteasome.
- YAMC cells were treated with NSL#3 -conditioned media for 16 hours and then analyzed as described but instead of MG132, the proteasome inhibitor lactacystin was used at a concentration of 10 ⁇ M (use of higher concentrations of lactacystin was limited due to cell toxicity). Data is expressed as means for three separate experiments, with error bars expressed as standard errors of the mean.
- Figure 18 Proteasome inhibition by probiotic-conditioned media is an early event.
- Hsp25 and Hsp72 expression is induced by probiotics, does not involve cell wall components, and is specific to epithelial cell types.
- Panel B Immunoblot analysis of levels of Hsp25 and
- Hsp72 in YAMC cells following exposure to NSL#3-conditioned media or sonicated organisms at the concentrations of bacteria indicated (cfu/ml). Bacteria were grown as described herein, then separated into either a conditioned media fraction (CM) or a sonicated pellet fraction(Pellet). A concentration-dependent increase in Hsp expression can be seen upon exposure to NSL#3 -conditioned media which is not seen with the sonicated pellet, indicating that the active factors or agents produced by the bacteria are secreted into conditioned medium and are not cell wall components. Hsc 73 serves as a loading control.
- Panel C Immunoblot analysis of Hsp72, comparing different cell lines following exposure to NSL#3 -conditioned media (CM) for 16 hours, 20 ⁇ g protein lane.
- NSL#3-conditioned media induces a robust Hsp72 response in both colonic (YAMC) and small intestinal (MSIE) epithelial cells which is not seen in 3T3 fibroblast cells, suggesting that the probiotic effect is specific to epithelial cells.
- Thermal stress (HS) serves as a positive control.
- Figure 20 Hsp induction by probiotics is at least partly transcription al and involves HSF-1.
- Panel A Electrophoretic mobility shift assays (EMS A) show that the induction of Hsp expression by NSL#3-CM was transcriptional in nature.
- NSL#3-CM induces binding of the heat shock transcription factor HSF, reaching a maximal signal around 4 or 5 hours after exposure and then tapering off after 6 hours, indicating that.
- Hsp induction by NSL#3- CM is at least partly transcriptional in nature.
- Panel B EMS A showing specificity of this binding by using antibodies against the transcription factors HSF-1 and HSF-2 (panel B).
- the major transcription factor involved in Hsp induction by NSL#3-CM is HSF-1; HSF-2 does not appear to play a role in this Hsp induction.
- Figure 21 Probiotic-conditioned media protects epithelial cells against oxidant stress.
- Panel A Chromium release assay demonstrating that NSL#3-CM protects YAMC cells from oxidant injury. YAMC cells were treated with NSL#3- CM for 16 hours. Cells were labeled with 51 Cr for 60 minutes and stimulated with monochloramine ( ⁇ H C1, 0.6 mM) for 60 minutes and the ratio of released 51 Cr to intracellular 51 Cr was determined (mean ⁇ SE for three separate experiments, in each experiment each group was performed in triplicate, * p ⁇ 0.05 compared to controls).
- Panel B NSL#3-CM prevents oxidant-induced actin depolymerization from the F- actin to the G-actin form.
- YAMC cells were treated with NSL#3-CM for 16 hours, when appropriate, and then treated with the oxidant monochloramine (0.6 mM, 60 minutes) along with untreated control (Con) cells.
- Cells were processed for globular (G) and filamentous (F) actin as described herein. Images shown are representative of three separate experiments.
- IBDs Inflammatory bowel diseases
- IBDs are a group of chronic disorders that affect the digestive tract of susceptible individuals.
- the extent and severity of mucosal injury in IBD is determined by the disequilibrium between inflammation- induced injury versus reparative and cytoprotective mechanisms.
- various probiotics have been shown to be effective in either preventing or mitigating intestinal mucosal inflammation associated with experimental colitis (Madsen et al, 2001; Gionchetti et al, 2000b; Campierei et al, 2000).
- probiotics appear to reduce the rate of malignant transformation of colonic mucosa in the setting of chronic inflammation (Wollowski et al, 2001).
- probiotics are effective in the treatment of pouchitis and IBD.
- Several multicenter clinical trials are also under way to determine the effectiveness of these agents and to optimize dosage in IBD patients.
- the mechanism(s) of probiotic action remains unclear. It follows that there is no appreciation in the art that the beneficial effects of crude probiotic materials, such as unrefined probiotic-conditioned media, can be ascribed to, and hence achieved, with one or more discrete compounds. Moreover, the therapeutic use of crude conditioned media of uncharacterized content presents significant health concerns.
- the probiotic NSL#3 is disclosed herein as producing soluble factor(s) with anti-inflammatory and cytoprotective properties.
- Isolated in the context of describing the invention disclosed herein means that a given substance is separated from at least one other substance with which it is typically found in nature.
- a bioactive agent "isolated" from a conditioned medium is separated from at least one other component of the relevant crude conditioned medium.
- “Selective inhibition,” in the context of the selective inhibition of protease functions of the proteasome, means that less than all, and preferably one, protease function of a proteasome is reduced to a level comparable to the level of that protease measured in the presence of up to 10 ⁇ M lactocystin.
- reduction of a chymotrypsin-like activity of a proteasome to a level found in the presence of no more than 10 ⁇ M lactocystin, without the concomitant reduction in the activity of at least one of the trypsin-like or the caspase-like proteasome activities is illustrative of selective inhibition.
- Anti-inflammatory has a plain meaning well known in the art as a substance or process that reduces inflammation, a physiological process generally characterized by heat, redness, swelling and pain. "Anti-inflammatory” is given its plain meaning herein.
- Inflammatory disorder means any disease, malady, or condition known in the art to be characterized by involvement of inflammation. The term includes diseases, maladies and conditions of epithelial cells and, by way of particular example, of intestinal (i.e., gut) epithelial cells.
- Cytoprotective has a plain meaning well known in the art as a substance or process that protects at least one cell or cell type, and it is this plain meaning that is given the term throughout this application.
- Probiotic-conditioned media means a cell culture medium that has been exposed to viable cells. Suitable culture media include all media known in the art to be suitable for the growth, and/or maintenance, of a cell amenable to maintenance or growth in vitro and includes numerous media useful for maintaining or growing a variety of prokaryotic or eukaryotic cells. "Media,” and “medium,” are given their plain meanings of compositions containing compounds required for the maintenance and/or growth of at least one cell type. For example, a medium may contain an energy source, nutrients, growth factors, and the like, as would be known in the art. These terms are used throughout this application without strict adherence to number and, accordingly, may be used as synonyms, as would be apparent to one of skill from the context of a particular recitation.
- NSL#3 is given the meaning it has acquired in the art of a group of gram- positive bacterial species collectively known and marketed as a probiotic.
- Heat shock protein refers to any one of a group of proteins known in the art to exhibit a detectable increase in activity, typically reflective of an increase in expression, upon exposure to a thermal stress in at least one cell type.
- Stabilizing IkB means the act of preserving an IkB protein for a physiologically significant period of time, without regard to whether the protein being stabilized is unmodified or modified, for example by phosphorylation.
- ⁇ F- ⁇ B activation means that intracellular NF- ⁇ B exhibits an increased level of at least one activity characteristic of this protein, as would be known in the art. Such activation may result from a decreased rate of destruction of NF- ⁇ B, an increased rate of production (e.g., expression) of NF- ⁇ B, or a combination thereof.
- "Chymotrypsin-like” proteasome activity means a protease activity exhibiting at least one characteristic in common with chymotrypsin, such as the common recognition of a cleavage site or structurally related cleavage sites, as would be known in the art.
- “Pharmaceutically acceptable excipient” is a phrase given its plain meaning of a substantially inert substance admixable with a pharmaceutical or bioactive agent as a vehicle to provide a consistency or form suitable for pharmaceutical administration. Such vehicles typically do not produce an allergic or similar untoward reaction when administered to a human.
- “MCP-1 release” refers to the separation of Monocyte Chemoattractant
- Protein- 1 from a cell that had produced or harbored it, such as by secretion, as would be known in the art.
- Module means a substance that affects a detectable activity (e.g., of a protein) or process (e.g., a physiological process such as MCP-1 release), regardless of whether the effect is one of promotion (e.g., enhancement) or inhibition.
- a detectable activity e.g., of a protein
- process e.g., a physiological process such as MCP-1 release
- the compounds of the invention provide therapies for the treatment of inflammatory disorders, such as IBD, that are superior to those currently available in the art.
- the invention provides a composition comprising an isolated, anti-inflammatory, cytoprotective compound derivable from a probiotic-conditioned medium.
- the invention provides methods for treating a patient with an inflammatory disorder comprising administering to the patient an isolated, anti-inflammatory, cytoprotective compound derivable from a probiotic-conditioned medium
- the invention provides methods for isolating and characterizing at least one compound from a probiotic-conditioned medium that has anti-inflammatory and/or cytoprotective properties, and preferably both types of properties.
- the invention provides methods of identifying and characterizing compounds derivable from cell cultures, such as bacterial cultures, that have anti-inflammatory and cytoprotective properties.
- the invention provides isolated, anti-inflammatory, cytoprotective compounds derivable from probiotic organisms.
- the invention also provides compositions and methods useful in treating, and/or preventing, inflammatory diseases, particularly inflammatory disease of an epithelium.
- any bacterial strain or probiotic formulation may be screened for anti- inflammatory and cytoprotective compounds.
- the bacteria are non- pathogenic, enteric bacteria.
- the probiotic formulation NSL#3 (NSL Pharmaceuticals, Gaithersburg, MD), was used. This formulation contains Streptococcus salivarius subsp. thermophilus, Lactobacillus casei, Lactobacillus plantarum, Lactobacillus acidophilus, Lactobacillus delbrueckii subsp. bulgaricus, Bifidobacteria longum, Bifidobacteria infantis, and Bifidobacteria breve.
- NSL#3 is cultured in mammalian tissue culture medium.
- NSL#3 grows readily in mammalian tissue culture medium (e.g., RPMI 1640 or DMEM) under aerobic conditions. Growth in tissue culture medium makes the isolation of secreted factors much more straightforward than if a complex broth is used.
- the anti-inflammatory cytoprotective compounds of the invention are soluble factors derivable from a cell-conditioned medium such as a NSL#3 -conditioned, medium. To facilitate the identification and characterization of these compounds it is preferable to remove the bacterial cells from the medium.
- One of skill in the art would be familiar with methods of separating cells from the soluble factors in the medium.
- the cells may be removed by centrifugation, filtration or a combination of both, hi a preferred embodiment, overnight NSL#3 cultures grown at 37°C in tissue culture medium (e.g., RPMI 1640) are prepared and then centrifuged at 10,000g for 5 min at 4°C. The medium is then removed and filtered through a 0.2 ⁇ m cellulose acetate filter to exclude all live and intact bacteria. This "conditioned medium" is then used as the source from which anti-inflammatory and cytoprotective compounds are identified.
- tissue culture medium e.g., RPMI 1640
- This "conditioned medium" is then used as the source from which anti-inflammatory and cytoprotective compounds are identified.
- A. Organic Extraction The anti-inflammatory and cytoprotective compounds can be further isolated from the conditioned medium by extraction with organic solvents. Organic extraction separates organic from aqueous compounds.
- the organic extraction is performed with ether.
- the ether extraction process generally removes organic acids and their derivatives, as well as lipid and phospholipid molecules, whereas inorganic salts, hydrophilic peptides, hydrophilic proteins, carbohydrates and polysaccharides tend to remain in the aqueous phase.
- the anti-inflammatory and cytoprotective compounds of the invention are present in the ether-extracted fraction and many, if not all, have a molecular weight of less than 10 kDa. It is expected that an anti-inflammatory and cytoprotective compound of the invention is an organic acid.
- the compounds of the invention may be purified from the ether-extracted fraction of the conditioned medium using thin layer chromatography (TLC), which is a chromatographic technique that is useful for separating organic compounds such as organic acids and their derivatives.
- TLC thin layer chromatography
- ether extracts of NSL#3-conditioned medium will be subjected to thin layer chromatography (TLC) on a silica gel G TLC plate that has been activated at 150°C for 6 hours.
- the plate will be developed using ethanol/ammonia/water in a ratio of 50:8:6 by volume for the first dimension, and benzene/methanol/acetic acid in a ratio of 45:8:4 for the second dimension. Because of differences in their partitioning behaviors between the mobile liquid phase and the stationary phase, the different components in the ether-extracted mixture will migrate at different rates, allowing for their separation. The chromatogram will then be developed reversibly under iodine vapor, which binds to carbon double bonds and allows visualization of the individual components of the ether-extracted mixture. The separated components will be individually isolated by scraping the visualized spots off with a spatula, allowing the iodine vapor to evaporate, and then back extracting again with ether.
- conditioned medium from the DH5 ⁇ laboratory strain of E. coli will be treated in the same manner as above and used as a negative control for the ether extraction process.
- C. Other Separation Techniques Other separation techniques known to those of skill in the art may also be employed in the invention to fractionate the conditioned medium.
- High Performance Liquid Chromatography HPLC is characterized by a very rapid separation with extraordinary resolution of peaks. This is achieved by the use of very fine particles and high pressure to maintain an adequate flow rate. Separation can be accomplished in a matter of minutes, or at most an hour.
- Gel chromatography or molecular sieve chromatography, is a special type of partition chromatography that is based on molecular size. The theory behind gel chromatography is that the column, which is prepared with tiny particles of an inert substance that contain small pores, separates larger molecules from smaller molecules as they pass through or around the pores, depending on their size. As long as the material of which the particles are made does not adsorb the molecules, the sole factor detennining rate of flow is the size.
- the conditioned medium may be passed through filters with specific molecular weight cutoffs. For example, some fractions of the invention were parsed by passage through Centricon filters with a 10 kDa molecular weight cutoff.
- the medium or fraction may be screened for the ability to induce cytoprotective heat shock proteins, inhibit NF- ⁇ B activity, and inhibit proteasomal function of intestinal epithelial cultured cells.
- HPLC high performance liquid chromatography
- a RP-HPLC fractionation of VSL#3 -conditioned medium was performed using a C18 reverse-phase (RP) analytical column (3.9 mm x 300 mm).
- the mobile phase contained buffer A (0.1% trifluoroacetic acid, i.e., 10 mM TFA) and buffer B (60% acetonitrile in 0.1% TFA), with filtering and degassing of buffers before use.
- the injection volume was 200 ⁇ l of conditioned-medium supernatant, the flow rate was 1.0 ml/minute and the chromatography was performed at room temperature.
- the elution profile was: 5% Buffer B for 5 minutes, 5% Buffer B to 100% Buffer B for 60 minutes, 100% Buffer B for 10 minutes, and 100% Buffer B to 5% Buffer B for 5 minutes.
- Detection was at 214 ran and 280 ran (UN) and fractions were collected, frozen at -80°C and lyophilized. Lyophilized fractions were subsequently dissolved in a suitable solvent (e.g., a buffer compatible with cell viability), as would be known in the art. Fractions showing biological activity were used to identify the bioactive agent(s).
- a suitable solvent e.g., a buffer compatible with cell viability
- the compounds of the invention may be identified using mass spectrometry.
- Mass spectrometry provides a means of "weighing" individual molecules by ionizing the molecules in vacuo and making them “fly” by volatilization. Under the influence of combinations of electric and magnetic fields, the ions follow trajectories depending on their individual mass (m) and charge (z). Mass spectrometric methods are well-known to those of skill in the art. and are routinely used for the analysis and characterization of a variety of molecules.
- Heat shock proteins are a family of proteins that protect a cell against environmental stressors. NSL#3-conditioned medium induces the expression of heat shock proteins, specifically Hsp72 and Hsp25. Hsp 72 binds and stabilizes critical cellular proteins, preventing their denaturation.
- Hsp25/27 is an actin-stabilizing agent and preserves cytoskeletal and tight junction functions.
- Methods of analyzing the induction of heat shock proteins are known to those of skill in the art. For example, the induction of Hsp72 and Hsp25 can be performed by standard Western blot analysis using monoclonal antibodies specifically recognizing and binding specific Hsp isoforms (Stressgen).
- hnmunoblots for the constitutive heat shock cognates, Hsp60 and Hsc73 can be performed to check the specificity of response and to ensure equal loading of lanes (the expression of these proteins usually remains constant).
- antibodies can be used to detect the expression of heat shock proteins by immunofluorescence and ELISA.
- Other methods of analyzing the induction of heat shock proteins include assaying Hsp mRNA levels using, for example, RT-PCR, genomic microarrays, and real-time PCR.
- Another approach for analyzing the induction of heat shock proteins is the use of electrophoretic mobility shift assays, for example to look at binding of the transcription factor HSF-1.
- HSE-luciferase reporter assays can be employed to measure activity of the transcription factor HSF- 1.
- B. The NF- ⁇ B Pathway A number of approaches are known to those of skill in the art to assess the inhibition of NF- ⁇ B activation, such as inhibition of the NF- ⁇ B pathway. For example, electrophoretic mobility shift assays (EMSA or gel shifts) using an oligonucleotide labeled with 32 P can be performed to determine activation of NF- ⁇ B. Activation of NF- ⁇ B and release from the inhibitor IKB results in binding to this mimic, which can be easily detected on polyacrylamide gels. At least two additional measures may be used to corroborate NF- ⁇ B activation.
- ESA electrophoretic mobility shift assays
- NF- ⁇ B-sensitive reporter construct such as a construct having five copies of the NF- ⁇ B responsive promoter element cloned in front of a firefly luciferase reporter
- data from the three assays may help identify unique steps at which the compounds of the invention modulate, e.g., inhibit, NF- ⁇ B activity.
- ELISA-based assays for the detection of NF- ⁇ B activation are also known in the art.
- an NF- ⁇ B ELISA-based assay kit is commercially available from Ninci-Biochem (Ninci, Italy).
- ⁇ F- ⁇ B regulates a wide variety of genes encoding, for example, cytokines, cytokine receptors, cell adhesion molecules, proteins involved in coagulation, and proteins involved in cell growth.
- Another approach to the study of the NF- ⁇ B pathway is through the analysis of the expression of genes known to be regulated by NF- ⁇ B.
- Those of skill in the art will be familiar with a variety of techniques for the analysis of gene expression. For example, changes in mRNA and/or protein levels may be measured.
- Changes in mRNA levels can be detected by numerous methods including, but not limited to, real-time PCR and genomic microarrays. Changes in protein levels may be analyzed by a variety of immuno-detection methods known in the art. It is also worthwhile to monitor changes in the NF- ⁇ B regulator, I ⁇ B. As the compounds of the invention are expected to affect the activity of I ⁇ B in more than one form, antibodies to both the native as well as the phosphorylated form of I ⁇ B are useful and may be used for Western blotting and immunohistochemical localization. C. The Proteasome Finally, the compounds of the invention may be screened by assessing their effects on cellular proteasomal function.
- the proteasome is a large complex, which contains several protease activities with different specificities. It exists in two forms, a 20S complex and a 26S complex.
- Cellular proteasomes play an important role in degrading cellular proteins as well as in providing viral and endogenous peptide fragments for loading of MHC I molecules for antigen presentation. Inhibitors of the proteasome block the degradation of many cellular proteins.
- Proteasome inhibitors are broadly categorized into two groups: synthetic analogs and natural products. Synthetic inhibitors are peptide-based compounds with diverse pharmacophores. These include peptide benzamides, peptide ⁇ -ketoamides, peptide aldehydes, peptide ⁇ -ketoaldehydes, peptide vinyl sulfones, and peptide boronic acids. Known natural product proteasome inhibitors include linear peptide epoxyketones, peptide macrocycles, ⁇ -lactam thiol ester, and epipolythiodioxopiperazine toxin.
- proteasome inhibitors include MG132, ALLN, E64d, LLM, quinacrine, chloroquine, clioquinol, (R)-(-)-3- hydroxybutyrate, dopamine 5 L-DOPA, PR39, gliotoxin, and green tea (EGCG). Additional examples of proteasome inhibitors are disclosed in Kisselev and Goldberg (2001) and Myung et al. (2001), both of which are incorporated herein by reference in their entireties. Inhibition of proteasomal function by NSL#3 -conditioned medium provides a potential unifying mechanism for the inhibition of ⁇ F-KB and induction of cytoprotective heat shock proteins.
- proteasome assays are performed using a fluorometric assay by preparing crude cell lysates from YAMC cells treated with NSL#3 -conditioned medium, then adding the proteasome substrate SLLNY-AMC and measuring hydrolysis of this product over time (see FIG. 4).
- the substrate is a five amino acid peptide attached to a fluor (4-amino-7-methylcoumarin) which, upon cleavage by the chymotrypsin-like activity of the proteasome, results in a fluorescent signal that can be measured and plotted over time.
- the activity of the proteasome is reflected by the rate, or slope of the line.
- the inhibition of proteasome activity by a candidate molecule may be compared to that of a known proteasome inhibitor, such as MG 132.
- Another method for assaying proteasome function is immunofluorescence using antibodies that recognize active proteasomes. For example, LMP2 antibodies specifically recognize the proteasome beta subunit.
- proteasome assay kits are commercially available from Biom ⁇ l International LP.
- the characterization of the compounds of the invention may involve the use of various animal models, including transgenic animals that have been engineered to have specific defects, or to carry markers that can be used to measure the ability of a candidate substance to reach and affect different cells within the organism. Due to their size, ease of handling, and information on their physiology and genetic make-up, mice are a preferred animal model, especially for transgenics.
- mice are suitable as well, including rats, rabbits, hamsters, guinea pigs, gerbils, woodchucks, cats, dogs, sheep, goats, pigs, cows, horses, and monkeys (including chimps, gibbons and baboons).
- Assays may be conducted using an animal model derived from any of these species:
- Some examples of mouse models for colitis include the DSS-induced colitis model, IL- 10 knockout mouse, A20 knockout mouse, TNBS-induced colitis model, IL--2 knockout mouse, TCRalpha receptor knockout mouse, and the E-cadherin knockout mouse.
- Treatment of animals with test compounds will involve the administration of the compound, in an appropriate form, to the animal.
- any animal model of inflammatory disease known to those of skill in the art can be used in the practice of a method according to the invention.
- Administration will be by any route that could be utilized for clinical or non-clinical purposes.
- the compound may be delivered by gavage or by rectal administration.
- the protective effects of a compound may be assayed by administering a compound before inducing colitis in the animal model.
- inflammation can be measured by histological assessment and grading of the severity of colitis.
- Other methods for assaying inflammation in a subject include, for example, measuring myeloperoxidase (MPO) activity, transport activity, villin expression, and transcutaneous electrical resistance (TER) or transepithelial electrical resistance (TEER).
- MPO myeloperoxidase
- TER transcutaneous electrical resistance
- TEER transepithelial electrical resistance
- the effectiveness of a compound can also be assayed using tests that assess cell proliferation. For example, cell proliferation may be assayed by measuring 5- bromo-2'-deoxyuridine (BrdU) uptake.
- compositions of the invention comprise an effective amount of an anti- inflammatory, cytoprotective compound, which may be dissolved and/or dispersed in a pharmaceutically acceptable excipient, such as a carrier and/or aqueous medium.
- a pharmaceutically acceptable excipient such as a carrier and/or aqueous medium.
- the anti- inflammatory, cytoprotective compounds of the invention maybe delivered by any method known to those of skill in the art (see for example, "Remington's Pharmaceutical Sciences” 15th Edition).
- the pharmaceutical compositions may be delivered orally, rectally, parenterally, or topically.
- Solutions comprising the compounds of the invention may be prepared in water suitably mixed with a surfactant, such as polyethylene glycol (PEG) of low (less than 8 kDa) or high (greater than 8, and preferably greater than 15, kDa) average molecular weight, or hydroxypropylcellulose.
- PEG polyethylene glycol
- these preparations contain a preservative to prevent the growth of microorganisms.
- the pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions.
- the form should usually be sterile and must be fluid to the extent that effective syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi.
- parenteral administration in an aqueous solution for example, the solution may be suitably buffered if necessary and the liquid diluent first rendered isotonic with sufficient saline or glucose.
- aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous, intratumoral, and intraperitoneal administration.
- sterile aqueous media that can be employed will be known to those of skill in the art in light of the present disclosure.
- a suppository may also be used.
- Suppositories are solid dosage forms of ⁇ various weights and/or shapes for insertion into the rectum, vagina and/or the urethra. After insertion, suppositories soften, melt and/or dissolve in the cavity fluids.
- traditional binders and/or carriers may include, for example, polyalkylene glycols and/or triglycerides; such suppositories may be formed from mixtures containing the active ingredient in the range of 0.5% to 10%, preferably l%-2%.
- the pharmaceutical compositions of the invention. may also be delivered by enema.
- Oral formulations include such normally employed excipients as, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, and/or the like. These compositions take the form of solutions, suspensions, tablets, pills, capsules, sustained-release formulations and/or powders.
- oral pharmaceutical compositions will comprise an inert diluent and/or assimilable edible carrier, and/or they may be enclosed in hard- and/or soft-shell gelatin capsule, and/or they may be compressed into tablets, and/or they may be incorporated directly with the food of the . diet.
- the active compound(s) may be incorporated with excipients and/or used in the form of ingestible tablets, buccal tables, troches, capsules, elixirs, suspensions, syrups, wafers,, and/or the like.
- Such compositions and/or preparations should contain at least 0.1% of active compound.
- the percentage of the compositions and/or preparations may, of course, be varied and/or may conveniently be between about 2 to about 75% of the weight of the unit, and/or preferably between 25-60%.
- the amount of active compounds in such therapeutically useful compositions is such that a suitable dosage will be obtained.
- the tablets, troches, pills, capsules and/or the like may also contain the following: a binder, such as gum tragacanth, acacia, cornstarch, and/or gelatin; excipients, such as dicalcium phosphate; a disintegrating agent, such as corn starch, potato starch, alginic acid and/or the like; a lubricant, such as magnesium stearate; and/or a sweetening agent, such as sucrose, lactose and/or saccharin may be added and/or a flavoring agent, such as peppermint, oil of wintergreen, and/or cherry flavoring.
- a binder such as gum tragacanth, acacia, cornstarch, and/or gelatin
- excipients such as dicalcium phosphate
- a disintegrating agent such as corn starch, potato starch, alginic acid and/or the like
- a lubricant such as magnesium stearate
- a sweetening agent
- compositions include, creams, ointments, jellies, gels, epidermal solutions or suspensions, and the like, containing the active compound.
- preparations should meet sterility, pyrogenicity, general safety and purity standards as required by the FDA Office of Biologies standards.
- the dosage of the anti-inflammatory, cytoprotective compounds and dosage schedule may be varied on a subject-by-subject basis, taking ' into account, for example, factors such as the weight and age of the subject, the type of disease being treated, the severity of the disease condition ⁇ previous or concurrent therapeutic interventions, the manner of administration, and the like, which can be readily determined by one of ordinary skill in the art. Administration is in any manner compatible with the dosage formulation, and in such amount as will be therapeutically effective.
- the quantity to be administered depends on the subject to be treated. Precise amounts of an active ingredient required to be administered depend on the judgment of the practitioner and such judgments may involve routine procedures to determine an effective amount on a case-by-case basis. The following examples are included to demonstrate preferred embodiments of the invention.
- Example 1 describes basic techniques used in the work disclosed herein, including tissue culture and cell lysate preparation, NF- ⁇ B, 51 Cr, G/F actin and proteasome activity assays, electrophoretic mobility shift assays, Western blot analysis of proteins, MCP-1 assays, and statistical analyses of the observed data;
- Example 2 discloses the inhibition of NF- ⁇ B activity by probiotic-conditioned medium;
- Example 3 describes the inhibition of Monocyte Chemoattractant Protein- 1 (MCP-1) release by probiotic-conditioned medium;
- Example 4 describes the inhibition of I ⁇ B degradation (including phosphorylated IKB) by probiotic-conditioned medium;
- Example 5 describes the failure of probiotic- conditioned medium to universally inhibit protein ubiquitination;
- Example 6 shows that probiotic-conditioned medium inhibits proteasome activity, and does so more rapidly than protein expression is induced;
- Example 7 addresses the induction of heat shock protein expression by probiotic-conditioned medium, including the time course of such induction, and provides evidence that the induction is
- the probiotic formulation contains Streptococcus salivarius subsp. thermophilus, Lactobacillus casei, Lactobacillus plantarum, Lactobacillus acidophilus, Lactobacillus delbrueckii subsp. bulgaricus, Bifidobacteria longum, Bifidobacteria infantis, and Bifidobacteria breve at a concentration of 5 x 10 ⁇ lyophilized bacteria/gram.
- NSL#3 (batch number 2034- A2, NSL Pharmaceuticals, Gaithersburg, MD) was grown to a concentration of approximately 2 x 10 14 (as determined by colony counts) in phenol red-free RPMI 1640 medium for 16 hours, then centrifuged at low speed in a tabletop Sorvall centrifuge (3000 x g, 4°C, 10 minutes). The supernatant (conditioned medium) was then passed through a 0.22 micron low protein-binding filter (Millipore, Bedford, MA) to remove all bacterial cells and debris. Aliquots of conditioned medium were stored in sterile microcentrifuge tubes at -80°C until further use.
- Tissue Culture YAMC young adult mouse colon
- Tissue Culture YAMC young adult mouse colon
- tsA58 temperature-sensitive SN40 large T antigen
- YAMC cells were maintained under permissive conditions (33°C) in RPMI 1640 medium with 5% (vol/vol) fetal bovine serum, 5 U/ml murine IF ⁇ - ⁇ (GibcoBRL, Grand Island, ⁇ Y), 50 ⁇ g/ml streptomycin, 50 U/ml penicillin, supplemented with ITS+ Premix (BD Biosciences, Bedford, MA).
- RPMI 1640 medium 5% (vol/vol) fetal bovine serum
- 5 U/ml murine IF ⁇ - ⁇ GibcoBRL, Grand Island, ⁇ Y
- 50 ⁇ g/ml streptomycin 50 U/ml penicillin
- ITS+ Premix BD Biosciences, Bedford, MA
- Cells were plated at a density of 2 x 10 5 per 60 mm tissue culture dish (for Western blot analysis and proteasome assays), or at 1 x 10 5 per well in 6-well plates (for ⁇ F- B luciferase transfection experiments). After 24 hours of growth at 33°C to allow for cell attachment, the medium was replaced with IFN-free medium and cells were moved to 37°C (non-permissive conditions) for 24 hours to allow the development of the differentiated colonocyte phenotype for all experiments. Cells were treated with NSL#3 -conditioned medium (1:10 dilution) overnight, and then used the following day in various experiments.
- ⁇ F- ⁇ B luciferase reporter assays murine TNF- ⁇ (Peprotech, Rocky Hill, NJ) at a concentration of 50 ng/ml was added directly to the cells at this time and left for 6 hours before harvest. Heat shock controls were exposed to 42°C for 23 minutes and allowed to recover at 37°C for 2 hours before harvest. MG132-treated control cells were treated for 2 hours with 25 ⁇ M MG132 (Biomol, Plymouth Mtg, PA) at 37°C prior to harvest unless otherwise specified. Two other cell lines, MSIE (a small intestine YAMC counterpart cell line) and 3T3 fibroblasts, were used in this study and maintained as previously described (Kojima, 2003), incorporated herein by reference.
- MSIE small intestine YAMC counterpart cell line
- 3T3 fibroblasts were used in this study and maintained as previously described (Kojima, 2003), incorporated herein by reference.
- Membranes were blocked in 5% (wt/vol) non-fat milk in TBS-Tween (Tris-buffered saline (150 mM ⁇ aCl, 5 mM KCl, 10 mM Tris, pH 7.4) with 0.05% (vol/vol) Tween 20) for one hour at room temperature.
- TBS-Tween Tris-buffered saline (150 mM ⁇ aCl, 5 mM KCl, 10 mM Tris, pH 7.4) with 0.05% (vol/vol) Tween 20) for one hour at room temperature.
- TBS-Tween Tris-buffered saline (150 mM ⁇ aCl, 5 mM KCl, 10 mM Tris, pH 7.4) with 0.05% (vol/vol) Tween 20) for one hour at room temperature.
- TBS-Tween Tris-buffered saline (150 mM ⁇ aCl, 5 m
- VSL#3-CM VSL#3-conditioned media
- NF- ⁇ B response element-driven firefly luciferase reporter plasmid (Clontech, Palo Alto, CA)
- TK Thymidine Kinase
- VSL#3 -conditioned medium VSL#3-CM was added to each well at a dilution of 1:10 and left overnight, unless otherwise specified.
- Murine TNF- ⁇ was added at 50 ng/ml the next morning and cells were harvested 6 hours later.
- NF- B luciferase assays were performed as per the manufacturer's instructions and luminescence measured in a Berthold Luminometer (Oakridge, TN).
- Co-transfection with TK-Renilla which displays constitutive low levels of activity, is used as an internal control against which to normalize the NF- ⁇ B luciferase data. Experiments were performed in triplicate. Young adult mouse colon cells transiently transfected with the reporter gene expressed a low level of baseline NF- ⁇ B activity, which increased upon stimulation with TNF- ⁇ , as reflected by an increase in luciferase activity (FIGS. 1 and 14).
- VSL#3-CM Pretreatment with VSL#3-CM for 16 hours resulted in the attenuation of TNF- ⁇ - induced NF- ⁇ B activity in epithelial cells by 45% compared to TNF- ⁇ treatment alone (FIG. 14, 1.80 +/- 0.41 for VSL#3-CM-treated cells vs. 3.25 +/- 0.41 with TNF- ⁇ alone, p ⁇ 0.05).
- This effect was specific to VSL#3-CM, as pretreatment of epithelial cells with conditioned medium from the E. coli strain DH5 ⁇ did not attenuate TNF- ⁇ - induced NF- ⁇ B activation (FIG. 1, column 4; FIG. 14, column 5).
- MCP-1 Monocyte Chemoattractant Protein 1
- MCP-1 Monocyte Chemoattractant Protein 1
- IL-8 studies have shown that MCP-1 is highly expressed in areas of active inflammation in Crohn's disease and its expression depends on NF- ⁇ B activation.
- YAMC cells were grown and treated with VSL#3-CM and subsequently treated with TNF- ⁇ , as described herein, to stimulate NF- ⁇ B activation.
- VSL#3-CM inhibits release of MCP-1 establishes a role for an isolated, anti-inflammatory compound derivable from VSL#3-CM in the prevention and/or treatment of inflammatory disorders, such as IBD (e.g., Crohn's disease, ulcerative colitis).
- IBD e.g., Crohn's disease, ulcerative colitis
- EXAMPLE 4 Probiotics Inhibit Degradation of the NF- ⁇ B Inhibitor I ⁇ B in Intestinal Epithelial Cells
- the regulation of the NF- ⁇ B inhibitory molecule, I ⁇ B ⁇ was investigated.
- the effects of VSL#3-C on total I ⁇ B ⁇ and phosphorylated I ⁇ B ⁇ protein in YAMC cells treated with TNF- ⁇ were examined.
- Pretreatment with VSL#3-CM inhibits degradation, of the phosphorylated form of I B ⁇ in TNF- ⁇ -treated cells (FIG. 16, bottom two panels or rows).
- TNF- ⁇ stimulates phosphorylation of I ⁇ B ⁇ within 5 minutes, followed by rapid degradation of I ⁇ B ⁇ at 15 and 30 minutes (FIG. 16, top two panels or rows). Subsequently, NF- ⁇ B stimulates I B ⁇ expression, shutting down further NF- KB activation (FIG. 16, see lane on top panel or row at 60 minutes).
- phosphorylated I ⁇ B ⁇ is stabilized and resists degradation for over 2 hours (FIG. 16, lower bottom panel or row).
- VSL#3-CM immunoblot analyses using anti-ubiquitin ⁇ antibodies were performed on total cell protein from intestinal epithelial cells treated ' with VSL#3-CM (FIG. 3).
- VSL#3-CM treatment of epithelial cells with VSL#3-CM does not result in a decrease of ubiquitinated proteins compared to untreated cells.
- VSL#3-CM treatment actually results in accumulation of certain ubiquitinated proteins.
- Proteasome inhibitors which block NF- ⁇ B activation through inhibition of I ⁇ B ⁇ degradation have already been described (Gao, 2000). Since probiotic treatment results in both attenuation of NF- ⁇ B activity and inhibition of I ⁇ B ⁇ degradation, the effect of VSL#3-CM on proteasome activity, as measured by cleavage of the SLLVY- AMC substrate, was investigated (FIG. 17). Proteasome activity from cell lysates was determined using a 20S Proteasome assay kit (Calbiochem, San Diego, CA). Briefly, ice-cold cell lysate containing 20 ⁇ g protein was added to proteasome assay reaction buffer (25 mM HEPES, 0.5 mM
- EDTA pH 7.6 activated with 0.03% (wt/vol) SDS.
- the sample was allowed to come to room temperature, then placed in a quartz cuvette and lO ⁇ M of the substrate suc- leu-leu-val-tyr-AMC (SLLVY-AMC), Bz-val-gly-arg-AMC, or Z-leu-leu-glu-AMC was added.
- the SLLNY-AMC substrate is cleaved by the chymotrypsin-like activity of the proteasome
- the Bz-val-gly-arg-AMC substrate is cleaved by the trypsin-like activity of the proteasome
- the Z-leu-leu-glu-AMC substrate is cleaved by the PGPH, or caspase-like, activity of the proteasome.
- Proteasome activity was determined by measuring the fluorogenic signal generated by cleavage of AMC (7- amino-4-methylcoumarin) from the peptide moiety of the proteasome substrate.
- Fluorescence (excitation at 380 nm, emission at 460 nm) was measured every minute for the first 10 minutes, then every 15 minutes thereafter in a Hitachi F-2000 fluorometer (Hitachi, Japan).
- Cells were treated with either MG132 (25 ⁇ M) or lactacystin (lO ⁇ M) as a positive inhibitor control.
- Untreated cells were treated with DMSO as a vehicle control for MG132 and lactacystin. Experiments were perfonned in triplicate. For each experiment, all time points were performed in duplicate. Extracts from cells treated with NSL#3-CM were compared with untreated controls, cells treated with MG132 (a potent proteasome inhibitor), and DH5 ⁇ (E.
- an isolated, anti-inflammatory compound derivable from VSL#3-CM is expected to be more therapeutically acceptable than such known proteasome inhibitors as MG132.
- a. Time Course of Proteasome Activity Inhibition A time course of VSL#3-CM treatment was performed in order to determine the speed with which VSL#3-CM is able to elicit the proteasome inhibition described above. Cells were treated for 30 minutes, 60 minutes, and 6 hours, then harvested and assayed for their ability to inhibit the CTL-like activity of the proteasome (FIG. 18). It was found that the most pronounced proteasome inhibition occurs early after probiotic treatment, with over 50% of the inhibition occurring within the first 30 minutes, consistent with what is reported with other proteasome inhibitors.
- VSL#3-CM proteasome inhibition by VSL#3-CM is an early event, occurring almost immediately after exposure of the epithelial cells to the probiotic-conditioned medium.
- This finding considered in view of the relative lack of toxicity of VSL#3- CM, indicates that an isolated, anti-inflammatory compound derivable from VSL#3- CM would be a safe and quick-acting, i.e., an effective, prophylactic and/or therapeutic for inflammatory disorders such as IBD.
- VSL#3-CM possesses some weak inhibitory activity against the caspase-like proteolytic function of the proteasome and has no inhibitory effect on its . trypsin-like activity.
- YAMC cells were treated with VSL#3-conditioned medium for 16 hours and then harvested for proteasome assay. Proteasome activity was measured in cell lysates using either the fluorogenic substrate Bz-Val-Gly-Arg-AMC, which measures the trypsin-like protease activity (FIG.
- VSL#3-CM does not globally inhibit all proteolytic functions of the proteasome, but rather displays some specificity or affinity for some functions.
- Treatment with VSL#3-CM is well tolerated by the epithelial cells, which is not the case with most of the synthetic proteasome inhibitors.
- the lower toxicity of VSL#3-CM may be due to its differential affinity, which likely allows some normal functioning of the proteasome to continue while the degradation of certain proteins such as IKB is blocked. Without wishing to be bound by theory, this may in part explain why the pattern of accumulated ubiquitinated proteins is different in cells treated with VSL#3 from what is observed with the more powerful and toxic synthetic inhibitors such as MG132.
- EXAMPLE 8 Probiotics Induce Heat Shock Proteins in Intestinal Epithelial Cells It has been shown that enteric flora (luminal bacteria of the colon) in the gut influence expression of epithelial heat shock proteins (Kojima- 2003; Beck, 1995). Proteasome inhibitors are potent inducers of heat shock protein expression through activation of the heat shock transcription factor, HSF-1 (Pirkkala, 2000). Accordingly, experiments were conducted to determine whether heat shock protein expression occurred concomitantly with proteasome inhibition. YAMC cells were co-cultured with the probiotic VSL#3 to test its ability to induce heat shock protein expression.
- VSL#3 was shown to induce Hsp25 and Hsp72 expression in YAMC cells beginning at 6 to 12 hours (FIG. 19 panel A).
- Hsc73 constitutively expressed heat shock protein Hsc73, serving as a loading control, was not affected by VSL#3.
- Untreated cells left in culture for 48 hours do not mount an Hsp response, demonstrating that the effect is specific to the probiotic treatment and not a time-dependent characteristic of the cells grown in culture.
- YAMC cells were either treated with VSL#3-CM (for times varying from 15 minutes to 6 hours), or heat shocked as described herein.
- Cell extract containing ten micrograms protein was mixed with 32 P-labeled HSE oligonucleotide (containing four tandem inverted repeats of the heat shock element (nGAAn): CTAGAAGCTTCTAGAAGCTTCTAG (SEQ ID NO: 1)) and 0.5 ⁇ g poly (dl-dC) in binding reaction buffer (final concentrations 20 mM Tris, pH 7.4, 100 mM NaCl, 1 mM EDTA, 10% vol/vol glycerol). The binding reaction was allowed to incubate for 25 minutes at 25°C and then analyzed on a 4% non-denaturing polyacrylamide gel run in 0.5X TBE buffer. Gels were dried and autoradiographed to detect DNA-protein complexes.
- nGAAn heat shock element
- YAMC cells were incubated with VSL#3-CM for 6 hours before harvest and l ⁇ g of specific antibody to either HSF-1 (SPA-950, Stressgen, Victoria, BC, Canada) or HSF-2 (sc-8062X, Santa Cruz Biotechnology, Santa Cruz, CA) were pre-incubated with cell extracts at 25°C for 30 minutes prior to the HSE-binding reaction. After this preincubation, the binding reaction and analysis were performed as described above.
- HSF-1 SPA-950, Stressgen, Victoria, BC, Canada
- HSF-2 sc-8062X, Santa Cruz Biotechnology, Santa Cruz, CA
- VSL#3 was added to YAMC cells, as filter- sterilized conditioned medium or sonicated bacterial pellets, in a concentration- dependent manner (FIG. 19 panel B).
- live gram-negative bacteria and lipopolysaccharide (LPS) a cell wall component found only in gram-negative bacteria, can induce Hsp expression in epithelial cells (Kojima, 2003)
- the bacteria which comprise VSL#3 are all gram-positive organisms and thus none of them contain LPS.
- MSIE is a murine small intestine epithelial cell line; these cells respond in a similar fashion as YAMC cells.
- 3T3 fibroblasts are able to mount a heat shock response to thermal stress, they do not respond to treatment with VSL#3-CM, indicating that the effects of VSL#3-CM are specific to epithelial cell types.
- Attempts to induce heat shock protein expression in all of these cell lines using E. coli DH5 ⁇ - CM were unsuccessful.
- the probiotic compounds induce intestinal epithelial heat shock proteins through apical (luminal) membrane-specific processes (see FIG. 6).
- YAMC intestinal epithelial cells When YAMC intestinal epithelial cells are grown on a permeable support, they form tight junctions and exhibit a high degree of polarity. Cells exposed to conditioned medium from the luminal side demonstrate robust Hsp25 and Hsp72 protein expression. Basolateral addition fails to stimulate a response. When added to both sides, VSL#3-CM has a similar effect to what is seen when it is added only to the apical side. These data suggest the presence of specific receptors or entry pathways for the probiotic-derived bioactive factors or agents. The proteasome inhibitor M.G132 was then used to determine whether the time course of Hsp induction following proteasomal inhibition in epithelial cells would parallel the induction attributable to VSL#3-CM treatment.
- FIG. 7 Treatment with MG132 results in a very strong induction of both Hsp25 and Hsp72 that is mote robust than thennal stress (FIG. 7).
- FIG. 7 A comparison of FIG. 7 with the time course of FIG. 19A shows that the appearance of a signal by 7 to 14 hours more closely parallels what is seen with VSL#3-CM than the time course normally observed with thennal stress, which induces Hsp expression by two hours in this cell line (Kojima, 2003).
- VSL#3- CM acting through a mechanism of proteasome inhibition would be expected to display a time course comparable to a known proteasome inhibitor such as MG132 and not as comparable to a mere physical stress such as heat shock.
- VSL#3 was added to YAMC cells, as filter-sterilized conditioned medium or sonicated bacterial pellets, in a concentration- dependent .manner (FIG. 5 panel B).
- live gram-negative bacteria and lipopolysaccharide (LPS) a cell wall component found only in gram-negative . bacteria, can induce Hsp expression in epithelial cells, the bacteria which comprise VSL#3 are all gram-positive organisms and, thus, lack LPS.
- MSIE is a murine small intestine epithelial cell line; these cells respond in a fashion similar to YAMC cells.
- 3T3 fibroblasts are able mount a heat shock response to thermal stress, they do not respond to treatment with VSL#3-CM.
- VSL#3-CM the effects of VSL#3-CM are specific to epithelial cell types. Attempts to induce heat shock protein expression in all of these cell lines using E. coli DH5 ⁇ -CM were unsuccessful.
- the data establish that a compound derivable from VSL#3-CM exhibits a cytoprotective function by inducing the expression of heat shock proteins, in addition to exhibiting an anti-inflammatory function.
- the invention contemplates an isolated, anti-inflammatory, cytoprotective compound derivable from VSL#3-CM, related compositions such as pharmaceutical compositions and kits comprising the compound(s), as well as methods of producing the compound(s) and methods of using the compound(s) to prevent or treat an inflammatory disorder such as IBD or to ameliorate a symptom of such a disorder.
- Hsp Expression Induction is Mediated by HSF-1 Activation Electrophoretic mobility shift assays were performed to determine whether or not the induction of Hsp expression by VSL#3-CM was transcriptional in nature. From FIG. 20 it can be seen that VSL#3-CM induces binding of the heat shock.
- transcription factor HSF reaching a maximal binding signal around 4 or 5 hours after exposure and then tapering off after 6 hours (panel A). Specificity of this binding was confirmed using antibodies against the transcription factors HSF-1 and HSF-2 (panel B), which demonstrates that the major transcription factor involved in Hsp induction by VSL#3-CM is HSF-1 ; HSF-2 does not appear to play a role.
- panel B The time course of Hsp induction by the proteasome inhibitor MG132 is similar to that produced by probiotics, but MG132 is more toxic. As the proteasome time course data indicated that VSL#3-CM acted quickly like other proteasome inhibitors such as MG132 (see FIG.
- VSL#3-CM were acting through a mechanism of proteasome inhibition, it would display a time course more comparable to a known proteasome inhibitor such as MG132 and less similar to a mere physical stress such as heat shock. Unlike VSL#3, which did not result in any change in viability compared to untreated control cells even after 24 hours of treatment, prolonged exposure of YAMC cells to MG132 resulted in markedly increased cell death, suggesting that MG132 is significantly more toxic than VSL#3-CM.
- EXAMPLE 9 Probiotics Protect Intestinal Epithelial Cells Against Oxidant Stress
- the oxidant monochloramine (NH 2 C1) was used.
- Monochloramine is a pathophysiologically relevant oxidant produced in large quantities when hypochlorous acid, released from innate immune cells within inflamed tissues, reacts with ammonia in vivo. Once formed, monochloramine causes loss of tight junction barrier function, mitochondrial injury, cytoskeletal disruption, impaired membrane transport functions, and eventual cell death. Cells were treated with VSL#3-CM overnight and, after exposure to monochloramine, cell viability was assessed using 51 Cr release (FIG. 21 , panel A).
- YAMC cells were grown in 24-well plates and either left untreated (control), or treated with VSL#3-CM overnight.
- Cells were loaded with 51 Cr (50 ⁇ Ci/ml; Sigma Chemical Co.; 250 ⁇ l/well in a 24-well plate format, or 12.5 ⁇ Ci per well) for 60 minutes, washed, and incubated in medium with 0.6 mM of the oxidant monochloramine to induce cell injury.
- Medium was harvested after 60 minutes and the 51 Cr remaining in the cells extracted with IN HNO 3 for 4 hours. Cr m the released and cellular fractions was counted by liquid scintillation spectroscopy. 51 Cr released was calculated as amount released divided by released plus cellular remainder.
- VSL#3-CM pretreatment results in a mild but statistically significant protective effect, decreasing the NH 2 Cl-stimulated 51 Cr release and improving epithelial cell viability in the face of oxidant injury by about one-third compared to control cells treated with monochloramine alone (P ⁇ 0.05).
- the ability of VSL#3-CM treatment to protect epithelial cells against cytoskeletal damage from oxidant stress was determined using F/G actin assays.
- Filamentous actin (F-actin) carries out important functions involved in the maintenance of cellular scaffolding and shape, as well as acting as an anchoring point for numerous integral membrane proteins.
- actin cytoskeleton is particularly vulnerable to injury which can result in cellular compromise. Exposure to monochloramine causes rapid dissociation of filamentous actin. Hence, we determined whether VSL#3-CM treatment would protect the integrity of cytoskeletal filamentous actin in the face of oxidant stress. F/G actin assays were conducted by initially shifting confluent YAMC cell monolayers to 37°C in IFN- ⁇ -free medium and treating with VSL#3-CM overnight. Prior to assessment, cells were treated with phalloidin (3 O ⁇ g/ml for 2 hours; Molecular probes, Eugene, OR), cytochalasin D (1 O ⁇ g/ml for 15 min), or the oxidant monochloramine (0.6 mM for 30 minutes).
- Cells were rinsed in PBS, harvested, centrifuged (14,000 x g for 20 seconds at room temperature) and the pellets resuspended in 200 ⁇ l of 30°C lysis buffer (1 mM ATP, 50 mM PIPES, pH 6.9, 50 mM NaCl, 5 mM MgCl 2 , 5 mM EGTA, 5% (vol/vol) glycerol, 0.1% (vol/vol) Nonidet P-40, Tween 20, and Triton X-100, containing complete protease inhibitor cocktail). Cells were homogenized by gently pipetting up and down ten times and incubated at 30°C for 10 minutes.
- 30°C lysis buffer (1 mM ATP, 50 mM PIPES, pH 6.9, 50 mM NaCl, 5 mM MgCl 2 , 5 mM EGTA, 5% (vol/vol) glycerol, 0.1% (vol/vol) Nonidet P-40, Twe
- phalloidin which binds and stabilizes the barbed ends of F-actin filaments, thus increasing the amount of F-actin
- cytochalasin D an F-actin disrupting agent which greatly increases the amount of globular (G) actin
- panel B The results of the experiment disclosed in this example are consistent with NSL#3-CM exhibiting reduced toxicity relative to known proteasome inhibitors such as MG132, and also are consistent with the disclosure herein that NSL#3-CM induces the expression of at least one heat shock protein in epithelial cells, in establishing a cytoprotective role for at least one compound derivable from NSL#3-CM.
- the bioactive agent(s) were not peptides or proteins susceptible to pepsin digestion.
- the bioactive agent(s) are non-proteinaceous compounds, such as non-polar compounds like organic acids; of course, the bioactive agent(s) could be peptides, such as relatively small peptides, that are refractory to pepsin digestion or that retain an active peptide fragment following exposure to pepsin.
- the proteasome inhibitors in NSL#3 maybe small organic molecules. Some proteasome inhibitors found in nature are small molecular weight organic esters or organic acid derivatives, such as those in green tea.
- compositions and methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of prefened embodiments, it will be apparent to those of skill in the art that variations may be applied to the compositions and methods and in the steps or in the sequence of steps of the methods described herein without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain agents which are both chemically and physiologically related may be substituted for the agents described herein with the same or similar results being achieved.
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AU2005212311A AU2005212311A1 (en) | 2004-02-06 | 2005-02-04 | An anti-inflammatory, cytoprotective factor derivable from a probiotic organism |
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FR2912917A1 (en) * | 2007-02-26 | 2008-08-29 | Oreal | CONDITIONED MEDIUM AND USES THEREOF |
FR2912916A1 (en) * | 2007-02-26 | 2008-08-29 | Oreal | COSMETIC OR DERMATOLOGICAL COMPOSITION COMPRISING A CELL CULTURE MEDIUM |
US20130209411A1 (en) * | 2010-02-11 | 2013-08-15 | Technische Universitat Munchen | Lactocepins For Use in the Treatment of IP-10-Mediated Inflammatory Diseases |
CN116115582A (en) * | 2022-11-10 | 2023-05-16 | 重庆大学 | Engineering probiotics packaged by prodrug as well as preparation method and application thereof |
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MADSEN K.L. ET AL: "inflammatory Bowel Disease: Lessons from the IL-10 Gene Deficient Mouse.", CLIN.INVEST.MED, vol. 24, no. 5, October 2001 (2001-10-01), pages 250 - 257, XP009116945 * |
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FR2912917A1 (en) * | 2007-02-26 | 2008-08-29 | Oreal | CONDITIONED MEDIUM AND USES THEREOF |
FR2912916A1 (en) * | 2007-02-26 | 2008-08-29 | Oreal | COSMETIC OR DERMATOLOGICAL COMPOSITION COMPRISING A CELL CULTURE MEDIUM |
EP1974718A1 (en) * | 2007-02-26 | 2008-10-01 | L'Oreal | Cosmetic or dermatological composition comprising a cell culture medium |
EP1974719A1 (en) * | 2007-02-26 | 2008-10-01 | L'Oreal | Conditioned medium and its uses |
US8101167B2 (en) | 2007-02-26 | 2012-01-24 | L'oreal | Conditioned medium and uses thereof |
CN101254213B (en) * | 2007-02-26 | 2012-10-10 | 莱雅公司 | Conditioned medium and its uses |
CN101254164B (en) * | 2007-02-26 | 2013-10-23 | 莱雅公司 | Cosmetic or dermatological composition comprising cell culture medium |
CN103462876A (en) * | 2007-02-26 | 2013-12-25 | 莱雅公司 | Cosmetic or dermatological composition comprising a cell culture medium |
US20130209411A1 (en) * | 2010-02-11 | 2013-08-15 | Technische Universitat Munchen | Lactocepins For Use in the Treatment of IP-10-Mediated Inflammatory Diseases |
CN116115582A (en) * | 2022-11-10 | 2023-05-16 | 重庆大学 | Engineering probiotics packaged by prodrug as well as preparation method and application thereof |
CN116115582B (en) * | 2022-11-10 | 2024-04-16 | 重庆大学 | Engineering probiotics packaged by prodrug as well as preparation method and application thereof |
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