Jeffrey S. Small

4SC-101, a novel immunosuppressive drug, inhibits IL-17 and attenuates colitis in two murine models of inflammatory bowel disease†

Background: Dihydroorotate dehydrogenase (DHODH) is a key enzyme involved in pyrimidine biosynthesis. DHODH is a known target for the treatment of autoimmune diseases. 4SC‐101 is a novel immunosuppressive drug that inhibits DHODH. A goal of our study was to examine the in vitro effects of 4SC‐101 on IL‐17 production by mononuclear cells. In addition, we evaluated the efficacy of 4SC‐101 against acute TNBS (2,4,6‐tritrobenzene sulfonic acid) and chronic dextran sodium sulfate (DSS)‐induced colitis in mice. Methods: Peripheral blood mononuclear cells (PBMCs) from healthy human donors were used to evaluate cellular proliferation and cytokine (IL‐17, TNF‐&agr;) production. The oral effects of 4SC‐101 (100 or 200 mg/kg) were examined following induction of chronic colitis by the administration of 3% DSS (4 cycles) to Balb/c mice. Morphometric and histological indices of colitis were evaluated as indicators of drug efficacy. 4SC‐101 was also administered for 6 days after the intracolonic administration of TNBS (20 mg in 50% ethanol) to female Balb/c mice. The colons were analyzed for overall macroscopic damage, ulceration, total length, distal segment weight, MPO activity, and histological pathology as indicators for the effectiveness of 4SC‐101. Results: In vitro, 4SC‐101 is a potent inhibitor of human DHODH, inhibits lymphocyte proliferation, and uniquely blocks phytohemagglutinin‐stimulated IL‐17 production by lymphocytes. In vivo, oral administration of 4SC‐101 effectively improved both chronic DSS and acute TNBS colitis in mice. In these colitis models the overall efficacy profile of 4SC‐101 was similar to that of dexamethasone. Conclusions: 4SC‐101 is a novel immunosuppressive drug with excellent potential for the treatment of intestinal inflammation. (Inflamm Bowel Dis 2010)

In vitro and in vivo effects of the probiotic Escherichia coli strain M-17: immunomodulation and attenuation of murine colitis

We examined the in vitro and in vivo effects of a probiotic, Escherichia coli strain M-17 (EC-M17), on NF-kappaB signalling, cytokine secretion and efficacy in dextran sulfate sodium (DSS)-induced murine colitis. NF-kappaB signalling was assessed using an NF-kappaB luciferase reporter cell line that was stimulated with TNF-alpha (100 ng/ml). p65 Nuclear binding and cytokine secretion (TNF-alpha, IL-1beta and IL-6) were evaluated using a RAW 264.7 macrophage cell line that was exposed to lipopolysaccharide (LPS; 5 microg/ml). Mice were administered vehicle, EC-M17, metronidazole, or EC-M17 plus metronidazole for 13 d. During the final 6 d, mice also received 2 % DSS. Parameters evaluated included disease activity index (DAI), histology, myeloperoxidase and NF-kappaB p65. EC-M17 dose dependently inhibited TNF-alpha-induced NF-kappaB signalling. At 5 x 109 colony-forming units/ml, EC-M17 inhibited NF-kappaB by >95 %. LPS-induced nuclear p65 binding was significantly inhibited (78 %; P 90 %) the LPS-induced secretion of TNF-alpha, IL-1beta and IL-6. In mice with DSS-induced colitis, EC-M17, metronidazole, and EC-M17 plus metronidazole significantly reduced DAI and colonic histology scores. Both EC-M17 and metronidazole reduced colonic IL-12, IL-6, IL-1beta and interferon-gamma. The combination of EC-M17 plus metronidazole resulted in more substantial cytokine reductions than were found with either treatment alone, and combination therapy significantly (P < 0.05 in both cases) reduced IL-1beta compared with EC-M17 and colonic histology scores compared with metronidazole. Alone, and in combination with metronidazole, EC-M17 improved murine colitis, probably due to an inhibitory effect on NF-kappaB signalling.

Effects of the probiotic formulation VSL#3 on colitis in weanling rats.

Background: Only a few studies have used models of inflammatory bowel disease (IBD) with weanling animals. Previously, the effects of probiotics have not been assessed in such IBD models. The objectives of our study were 2-fold: to establish a suitable model of dextran sulfate sodium (DSS)–induced colitis in weanling rats and to determine the effects of the probiotic formulation VSL#3 on DSS-induced colitis in weanling animals. Materials and Methods: Rats were weaned on postnatal day 21 and administered 2%, 2.5%, or 3% (wt/vol) DSS in drinking water. In subsequent experiments, newly weaned animals were administered vehicle or VSL#3 (0.06, 0.6, or 6 mg) by orogastric gavage. These treatments were given to animals maintained on water (postnatal days 21–28) and then on DSS (postnatal days 28–35). Disease activity indices were determined on a routine basis. On day 35, rats were euthanized. The total colon length was determined. Other parameters of colitis were measured from the distal colon. These parameters included myeloperoxidase (MPO), interleukin (IL)–1β, inhibitory κB-α (IκB-α), and histological assessment of crypt damage and inflammation. Results: DSS 2% was optimal for inducing colitis in weanling rats without significant morbidity. VSL#3 treatments improved various parameters of 2% DSS–induced colitis in weanling rats. The 0.6- and 6-mg doses of VSL#3 were most effective for attenuating this colitis. Conclusions: The probiotic formulation VSL#3 improved DSS-induced colitis in weanling rats. This improvement of colitis involved changes in colonic IκB-α, IL-1β, and MPO, which are suggestive of immune modulation by VSL#3.

Enhanced Intestinal Expression of the Proteasome Subunit Low Molecular Mass Polypeptide 2 in Patients with Inflammatory Bowel Disease

PurposeLow molecular mass polypeptide 2 is an inducible immunoproteasome subunit. The expression of low molecular mass polypeptide 2 has not been examined in the intestine of patients with inflammatory bowel disease. This study was designed to determine whether the intestinal expression of low molecular mass polypeptide 2 was enhanced in a group of patients with inflammatory bowel disease compared with a group of control patients without inflammatory bowel disease. Moreover, we examined the association between low molecular mass polypeptide 2 expression and histologic pathology in these patients.MethodsTwenty-one patients participated in the study. These included six control subjects without inflammatory bowel disease, eight patients with ulcerative colitis, and seven patients with Crohn’s disease. Intestinal low molecular mass polypeptide 2 expression was evaluated by immunohistochemistry, as well as by Western blot. Histology scores (0–40 severity scale) were determined on the same sections of intestine as those used for low molecular mass polypeptide 2 histochemistry.ResultsBy immunohistochemistry, low molecular mass polypeptide 2 expression was significantly enhanced (P < 0.05 vs. control subjects) throughout visibly diseased areas of colon, rectum, and ileum from patients with inflammatory bowel disease. Low molecular mass polypeptide 2 expression also was increased in macroscopically normal intestine from patients with inflammatory bowel disease compared with normal tissue from control subjects. There was a significant correlation (P < 0.0001) between low molecular mass polypeptide 2 expression and histologic pathology in our patients. Western blot results confirmed that low molecular mass polypeptide 2 expression was enhanced in patients with ulcerative colitis (3.1-fold) and in patients with Crohn’s disease (3.5-fold).ConclusionsIntestinal low molecular mass polypeptide 2 expression is significantly increased in inflammatory bowel disease. The association between intestinal low molecular mass polypeptide 2 expression and histologic pathology suggests that this proteasome subunit plays a role in the pathogenesis of inflammatory bowel disease.

Dextran sulfate sodium-induced colitis is associated with enhanced low molecular mass polypeptide 2 (LMP2) expression and is attenuated in LMP2 knockout mice.

Low molecular mass polypeptide 2 (LMP2) is an inducible proteasome subunit. Our goals were to examine LMP2 expression in mice with dextran sulfate sodium (DSS)-induced colitis and to evaluate colitis in LMP2 knockout (LMP2–/–) mice. Mice were given 2.5% DSS in the drinking water. On day 0, 2, 4, or 6 after DSS treatment, LMP2 expression was determined in the distal colon by western blot and immunohistochemistry. Parameters of colitis were measured in LMP2–/– mice or wild-type mice. LMP2 expression was enhanced in the colon of DSS-treated mice at all time points. Symptoms of DSS-induced colitis were always lower in LMP2–/– mice. Normalized histology scores and colonic IL-1ß levels increased over the 6-day study period in wild-type mice. These parameters were significantly reduced in LMP2–/– mice that consumed DSS for 6 days. Enhanced LMP2 expression contributes to the pathogenesis of DSS-induced colitis in mice.

Bacillus Coagulans GBI-30 (BC30) improves indices of Clostridium difficile-Induced colitis in mice

BackgroundProbiotics have beneficial effects in rodent models of Clostridium difficile (C. diffiicle)-induced colitis. The spore forming probiotic strain Bacillus Coagulans GBI-30, 6086 (BC30) has demonstrated anti-inflammatory and immune-modulating effects in vitro. Our goal was to determine if BC30 improved C. difficile-induced colitis in mice. Starting on study day 0, female C57BL/6 mice were dosed by oro-gastric gavage for 15 days with vehicle (saline) or BC30 (2 × 109 CFU per day). Mice in the C. difficile groups received an antibiotic mixture (study days 5 to 8 in the drinking water), and clindamycin (10 mg/kg, i.p., on study day 10). The C. difficile strain VPI 10463 was given by gavage at 104 CFU to induce colitis on day 11. On day 16, stools and colons were collected for further analyses.ResultsAll mice treated with BC30 survived on study day 13, while two mice treated with vehicle did not survive. On day 12, a significant difference (p = 0.0002) in the percentage of mice with normal stools (66.7%) was found in the BC30/C. difficile group, as compared to the vehicle/C. diffcile group (13.0%). On study day 16, 23.8% of mice treated with BC30 had normal stools, while this value was 0% with vehicle treatment (p value = 0.0187). On this day, the stool consistency score for the BC30/C. difficile group (1.1 ± 0.2) was significantly lower (p < 0.05) than for the vehicle/C. difficile cohort (1.9 ± 0.2). BC30 modestly attenuated the colonic pathology (crypt damage, edema, leukocyte influx) that was present following C. difficile infection. Colonic MIP-2 chemokine contents (pg/2 cm colon) were: 10.2 ± 0.5 (vehicle/no C. difficile), 24.6 ± 9.5 (vehicle/C. difficile) and 16.3 ± 4.3 (BC30/C. difficle).ConclusionThe probiotic BC30 improved some parameters of C. difficile-induced colitis in mice. BC30 prolonged the survival of C. diffiicle infected mice. Particularly, this probiotic improved the stool consistency of mice, in this infectious colitis model.

Bacillus coagulans GBI-30, 6086 limits the recurrence of Clostridium difficile-Induced colitis following vancomycin withdrawal in mice

BackgroundRecently, we found that the probiotic strain Bacillus coagulans GBI-30, 6086 (GanedenBC30) improved indices of Clostridium difficile (C. difficile)-induced colitis in mice (Fitzpatrick et al., Gut Pathogens, 2011). Our goal was to determine if BC30 could also prevent the recurrence of C. difficile-induced colitis in mice, following initial treatment with vancomycin. During study days 0 through 5, mice were treated with antibiotics. On day 6, the C. difficile strain VPI 10463 was given by oro-gastric gavage at ≈ 5x104 CFU to induce colitis. Mice were treated on study days 6 to 10 with vancomycin (50 mg/kg) (vanco) or vehicle (saline) by gavage. On days 10 to16, mice were dosed by gavage with saline vehicle or BC30 (2 x 109 CFU per day). Mice were monitored for mortality, weight loss and diarrhea. On study days 14, 16 and 17, stools and colons were collected for analyzing other parameters of colitis.ResultsThe mean stool consistency score in Vehicle/C.difficile/Vanco mice increased from 0.4 (day 10) to a range of 1.1 to 1.4 (days 14 to 17), indicating the recurrence of colitis. On days 13 through 17, the stool consistency scores for the vancomycin/BC30 mice were significantly lower (p< 0.05) than for the vancomycin/vehicle cohort of animals. On day 17, 88.9% of mice treated with BC30 had normal stools, while this value was 0% with vehicle treatment (p value = 0.0004). Colonic myeloperoxidase (Units/2 cm colon) was significantly (p < 0.05) reduced from 4.3 ± 0.7 (Vehicle/C.difficile/Vanco) to 2.6 ± 0.2 (BC30/C. Difficle/Vanco). The colonic histology score and Keratinocyte derived-chemokine level in the colon were also lower in BC30 treated mice.SummaryIn BC30-treated mice, there was evidence of better stool consistency, as well as improved biochemical and histological indices of colitis, following initial treatment of animals with vancomycin.ConclusionBC30 limited the recurrence of CD-induced colitis following vancomycin withdrawal in mice.

Vidofludimus Inhibits Colonic Interleukin-17 and Improves Hapten-Induced Colitis in Rats by a Unique Dual Mode of Action

Vidofludimus (Vido) is a novel oral immunomodulatory drug that inhibits dihydro-orotate dehydrogenase and lymphocyte proliferation in vitro. Vido inhibits interleukin (IL)-17 secretion in vitro independently of effects on lymphocyte proliferation. Our primary goal was to evaluate the in vivo effects of Vido on IL-17 secretion and the parameters of trinitrobenzene sulfonic acid (TNBS)-induced colitis in rats. To further delineate the mechanism of action for Vido, rats were dosed concomitantly with uridine (Uri). Young Wistar rats received a 150-μl enema of either phosphate-buffered saline (PBS) or TNBS on study day 1. The ex vivo effects of Vido on 24-h colonic IL-17 secretion were determined by using colonic strips from PBS- or TNBS-treated rats. Some rats were dosed with vehicle, Vido, or Vido + Uri for 6 days. On day 6, the parameters of colitis were determined from colonic tissue. These parameters included macroscopic, histological, and transcription factor measurements, IL-17 production, and numbers of CD3+ T cells. Ex vivo Vido completely blocked IL-23 + IL-1β-stimulated secretion of IL-17 by colonic strips. In vivo Vido treatment alone most effectively reduced macroscopic and histological pathology and the numbers of CD3+ T cells. In contrast, similarly reduced nuclear signal transducer and activator of transcription 3 (STAT3) binding and IL-17 levels were observed from animals treated with Vido alone and Vido + Uri. Vido improves TNBS-induced colonic inflammation by a unique dual mode of action: 1) inhibiting expansion of colonic T lymphocytes, and 2) suppressing colonic IL-17 production, which is independent from the control of T-lymphocyte proliferation, by inhibition of STAT3 and nuclear factor-κB activation.

Inhibition of IL-17 Release by the Novel Anti-Inflammatory Drug Vidofludimus Involves Attenuation of STAT3 and NF-kappa B Signaling Pathways in Murine Splenocytes and Hapten-Induced Colitis

IL-17 Secretion: Murine Splenocytes Vidofludimus Inhibits the Nuclear Binding of STAT3 in a Dose Dependent Fashion: Murine Splenocytes Vidofludimus Does Not Affect the Expression of IκB-α: Murine Splenocytes Vidofludimus Attenuates Phospho-p65 Staining: Murine TNBS Colitis The STAT3 and NF-kappa B (NF-κB) pathways play a prominent role in IL-17 production and intestinal inflammation. We have shown that a novel drug named Vidofludimus (Vido, 4SC-101, SC12267) inhibited colonic IL-17 and murine colitis [Inflammatory Bowel Diseases, 2010]. Aims: The aims of our study were: 1) to determine if Vido inhibited IL-17 production by murine splenocytes, 2) to evaluate if Vido inhibited key components of the STAT3 and NF-κB signaling pathways in splenocytes and 3) to determine if Vido inhibited phosphorylated (phospho) STAT3 and NF-κB in the colons of mice with hapten (TNBS)-induced colitis. Methods: Splenocytes were collected from BALB/c mice. These cells were stimulated with IL-23 or IL-1β (10 ng/ml) alone, or in combination. Vido (25 to 100 μM), or vehicle, were added along with the cytokines. IL-17 secretion was determined by ELISA at Vidofludimus (4SC-101, SC12267) is a proprietary compound of 4SC AG Vidofludimus Inhibits Cytokine-Induced IL-17 Secretion: Murine Splenocytes Synergistic activation by IL-23 + IL-1β Vidofludimus Attenuates Phospho-AKT1 Expression: Murine Splenocytes Effects of Vidofludimus on Signaling Pathways: Murine Splenocytes 48 hours. Cytosolic extracts from splenocytes were used for western blots of phospho JAK2, STAT3 and AKT1 (STAT3 pathway proteins). Phospho I-κB and I-κB degradation were evaluated as indices of NF-κB pathway activation. Extracts were also collected from stimulated splenocytes for the evaluation of STAT3 and NF-κB nuclear binding by ELISA. Immunohistochemistry for phospho STAT3 and p65 was done on samples from BALB/c mice that underwent colitis induction with TNBS ± oral treatment for six days with Vido (50-200 mg/kg). Using coded slides, the staining for these proteins in the mucosa/submucosa was graded on a six point intensity scale. Results: IL-23 and IL-1β alone did not induce significant IL-17 production (< 30 pg/ml), but combined cytokine treatment induced a synergistic stimulation of IL-17 secretion (630 pg/ml). IL-17 production was blocked by Vido in a dose dependent fashion (IC50 = 58.1 μM). At 10 to 60 minutes after cytokine stimulation, Vido reduced the expression of phospho proteins (JAK2, STAT3, and AKT1) involved in STAT3 signaling, and also inhibited the nuclear binding of STAT3 (IC50 = 53.9 μM). Vido did not reduce the phosphorylation or degradation of I-κB, but at 100 μM inhibited the nuclear binding of p65 by 47%. Evidence of increased phospho STAT3 and p65 immunostaining was found in leukocytes within the colons of TNBS-treated mice. The staining was attenuated in mice treated with Vido. Phospho STAT3 staining scores (2-3 mice per group) were: 1.3 ± 0.8 (no TNBS), 4.3 ± 0.9 (Vehicle/TNBS), 1.1 ± 0.3 (Vido-50 mg/kg), 1.2 ± 0.7 (Vido-100 mg/kg) and 0.3 ± 0 (Vido-200 mg/kg/TNBS). Similar METHODS immuno-staining scores were found for phospho p65. Summary: Vidofludimus inhibited STAT3 and NF-κB signaling pathways in murine splenocytes. It also normalized the enhanced phospho STAT3 and p65 immunostaining associated with murine colitis. Conclusion: The inhibition of STAT3 and NF-κB pathways by Vidofludmus reduces IL-17 production and contributes to the established anti-colitis activity of this drug. Splenocyte Cultures: Spleens were rapidly harvested from BALB/c mice. Splenocytes were collected by standard techniques, and erythrocytes were removed by using RBC lysis buffer. For the IL-17 secretion studies, we plated 2 x 106 cells/ml in 4 ml of RPMI 1640 medium containing 10% FBS and 5 x 105 β-mercaptoethanol. Therefore, there were 8 x 106 cells per well in the standard six-well plates. We utilized two wells (1.6 x 107 cells) per treatment condition in these studies. Splenocytes were cultured for 48 hours at 37 degrees C. Then, the culture media was collected for the measurement of IL-17 by ELISA (R&D systems, Minneapolis, MN). In subsequent studies (western blots, transcription factor binding assays), splenocytes were plated at a density of 1 x 107 cells/ml in 4 ml of RPMI 1640 medium. Therefore,

M1651 Melanin-Concentrating Hormone Receptor 1 Antagonists Attenuate TNBS-Induced Colitis in Mice

Melanin-concentrating hormone (MCH) is localized mainly in the brain, but it is also found in the intestine. MCH plays a clear role in feeding behavior. Recent data suggests that MCH and the MCH receptor 1 mediate intestinal inflammation in mice. Two diverse MCH receptor 1 antagonists (DABA-821 and DABA-822) have been developed for pharmacological testing. DABA-821 penetrates the blood-brain barrier, while DABA-822 does not. Study Aim: Our goal was to test DABA-821 and DABA-822 in a murine model of TNBS colitis. The efficacy of these compounds was compared to Dexamethasone (Dex).Methods: Female BALB/c mice (n=37) were randomized to receive an intracolonic enema of PBS, 50% ethanol/PBS, or 20 mg/kg TNBS in 50% ethanol. On day 1 (five hours after the enemas) mice were given Vehicle, 30 mg/kg of DABA-821, 30 mg/kg of DABA-822, or 1 mg/kg of Dex by orogastric gavage. Test drugs were also administered bid on days 2 through 6. On day 7, mice were euthanized. The colon was analyzed for overall macroscopic damage, ulceration, total length, distal segment weight, MPO activity and histological pathology. Body and spleen weights were also determined. Results:Macroscopic colonic scores (combination of lesions, diarrhea, adhesions, thickness) were significantly increased in TNBS treated mice, as compared to PBS or 50% ethanol treated animals. These scores were attenuated by 53 to 75% in mice treated with the MCH-1 receptor antagonists. Similar efficacy was found with Dex. Colonic ulceration was clearly apparent in the Vehicle/TNBS treatment group, but ulcer severity was significantly reduced in mice treated with the MCH receptor 1 antagonists. Mean colonic ulcer scores were: 4.7 (Vehicle/TNBS), 2.4 (DABA-821), 2.9 (DABA-822) and 2.8 (Dex). As compared to their vehicle treated cohorts, colons were significantly longer, but weighed less, in drug treated mice. Colonic MPO activity was nearly normalized in mice treated with Dex, but only partially attenuated by treatment with the MCH receptor 1 antagonists. Colonic histology scores were significantly reduced (p < 0.05 vs. Vehicle) in all drug treatment groups. Vehicle/TNBS treated mice lost significant weight throughout the study, as compared to PBS treated animals. None of the treatments normalized this weight loss. Reduced spleen weights were observed in Dex treated mice, as well as in mice treated with DABA-821 and 822. Summary: Two diverse MCH receptor 1 antagonists attenuated various parameters of TNBS-induced colitis in mice. The anti-colitis profile of these antagonists was similar to Dex. Conclusion:MCH receptor 1 antagonismmay represent an effective therapeutic approach for intestinal inflammation.