The Sphingosine Kinase 2 Inhibitor Opaganib Protects Against Acute Kidney Injury in Mice.

Introduction: Acute kidney injury (AKI) is a common multifactorial adverse effect of surgery, circulatory obstruction, sepsis or drug/toxin exposure that often results in morbidity and mortality. Sphingolipid metabolism is a critical regulator of cell survival and pathologic inflammation processes involved in AKI. Opaganib (also known as ABC294640) is a first-in-class experimental drug targeting sphingolipid metabolism that reduces the production and activity of inflammatory cytokines and, therefore, may be effective to prevent and treat AKI. Methods: Murine models of AKI were used to assess the in vivo efficacy of opaganib including ischemia-reperfusion (IR) injury induced by either transient bilateral occlusion of renal blood flow (a moderate model) or nephrectomy followed immediately by occlusion of the contralateral kidney (a severe model) and lipopolysaccharide (LPS)-induced sepsis. Biochemical and histologic assays were used to quantify the effects of oral opaganib treatment on renal damage in these models. Results: Opaganib suppressed the elevations of creatinine and blood urea nitrogen (BUN), as well as granulocyte infiltration into the kidneys, of mice that experienced moderate IR from transient bilateral ligation. Opaganib also markedly decreased these parameters and completely prevented mortality in the severe renal IR model. Additionally, opaganib blunted the elevations of BUN, creatinine and inflammatory cytokines following exposure to LPS. Conclusion: The data support the hypotheses that sphingolipid metabolism is a key mediator of renal inflammatory damage following IR injury and sepsis, and that this can be suppressed by opaganib. Because opaganib has already undergone clinical testing in other diseases (cancer and Covid-19), the present studies support conducting clinical trials with this drug with surgical or septic patients at risk for AKI.

IBD and Bile Acid Absorption: Focus on Pre-clinical and Clinical Observations.

Inflammatory bowel disease (IBD) causes chronic inflammation affecting the GI tract. It is classified as consisting of Crohn's Disease (CD) and Ulcerative Colitis (UC). Bile Acid absorption is altered in both pre-clinical models of Inflammatory Bowel Disease (IB) and in human IBD. The bile acid transporter apical sodium dependent bile acid transporter (ASBT) showed decreased expression in rats with TNBS colitis. Decreased ASBT expression has also been described in murine, canine and rabbit models of intestinal inflammation. Human IBD studies have shown that an inflamed ileum can interrupt enterohepatic recirculation of bile acid, which could be due to inflammatory cytokine induced repression of the ASBT promoter. There are different hypotheses as to why ASBT is downregulated during CD. In addition, one study has demonstrated the beneficial effect of a glucocorticoid on ASBT expression, when treating IBD. Our aim in this paper was to systematically review various aspects of bile acid malabsorption in animal models of intestinal inflammation, as well as in IBD.

Role of the δ-Opioid Receptor in 2 Murine Models of Colitis.

Crohn disease and ulcerative colitis, collectively referred to as inflammatory bowel disease (IBD), are chronic inflammatory disorders of the gastrointestinal tract. Currently, the etiology of IBD is unknown, and immunosuppressive therapies have become the standard of care to reduce the inflammation; however, these agents only induce remission 50% of the time in patients and can have serious side effects. Recently, endogenous opioids and opioid receptors have been shown to play a role in the mediation of inflammation. In addition, opioid receptor blockade with a nonselective antagonist, naltrexone, has been shown to reduce colitis in both murine models and human subjects. The goal of the current study was to determine if the antiinflammatory effects of naltrexone are mediated through the delta (δ) opioid receptor. Male C57BL/6NCrl (6 to 8 wk.; n = 110) and female BALB/cAnNCrl (6-8 wk.; n = 91) mice were studied using 2 animal models of chemically induced colitis: dextran sodium sulfate (DSS) and 2, 4, 6-trinitrobenzenesulfonic acid (TNBS). The selective δ-receptor antagonists naltrindole and 7-benzylidenenaltrexone were administered to examine the role of the δ-opioid receptor in colonic inflammation. The quantitative measurement of colitis activity, colon weight and length, Hct, WBC count, and gross and microscopic aberrations were analyzed. Administration of naltrexone in the DSS colitis model significantly improved overall disease activity indices on day 5 of therapy. The use of δ-antagonists and naltrexone had limited to no effect on TNBS colitis. Similar findings were obtained by using the DSS colitis model. Based on the current findings, the authors conclude that naltrexone therapy has limited effect on the improvement of colitis in 2 murine models; however, the δ-opioid receptor was not responsible for mediating the effects.

VPR-254: an inhibitor of ROR-gamma T with potential utility for the treatment of inflammatory bowel disease.

INTRODUCTION: Retinoic Acid Related Orphan Nuclear Receptor gamma T (RORγT) is a lineage specifying transcription factor for IL-17 expressing cells, which may contribute to the pathogenesis of Inflammatory Bowel Disease (IBD). VPR-254 is a selective in vitro inhibitor of RORγT. AIMS: The main goals of our study were twofold: (1) To determine if ex vivo treatment with VPR-254 reduced relevant cytokine (IL-17 and IL-21) secretion from colonic strips of mice with colitis; (2) To determine if treatment of mice with VPR-254 attenuated parameters of colitis, using three murine IBD models. METHODS: VPR-254 was evaluated ex vivo in a colonic strip assay, using tissue from mice with Dextran sulfate sodium (DSS)-induced colitis. In vivo, VPR-254 was evaluated for efficacy in DSS, Trintirobenzenesulfonic acid (TNBS) and Anti-CD40 antibody-induced murine models of colitis. RESULTS: VPR-254 reduced the production of key pro-inflammatory cytokines (e.g., IL-17) in ex vivo and in vivo models of colitis. This small molecule inhibitor of RORγT also improved various morphometric and histological parameters associated with three diverse murine models of IBD. CONCLUSION: Our results support the concept that an inhibitor of ROR-gamma T may have potential utility for the treatment of IBD.

Therapeutic potential of flavonoids in inflammatory bowel disease: A comprehensive review.

The inflammatory process plays a central role in the development and progression of numerous pathological situations, such as inflammatory bowel disease (IBD), autoimmune and neurodegenerative diseases, metabolic syndrome, and cardiovascular disorders. IBDs involve inflammation of the gastrointestinal area and mainly comprise Crohn's disease (CD) and ulcerative colitis (UC). Both pathological situations usually involve recurring or bloody diarrhea, pain, fatigue and weight loss. There is at present no pharmacological cure for CD or UC. However, surgery may be curative for UC patients. The prescribed treatment aims to ameliorate the symptoms and prevent and/or delay new painful episodes. Flavonoid compounds are a large family of hydroxylated polyphenolic molecules abundant in plants, including vegetables and fruits which are the major dietary sources of these compounds for humans, together with wine and tea. Flavonoids are becoming very popular because they have many health-promoting and disease-preventive effects. Most interest has been directed towards the antioxidant activity of flavonoids, evidencing a remarkable free-radical scavenging capacity. However, accumulating evidence suggests that flavonoids have many other biological properties, including anti-inflammatory, antiviral, anticancer, and neuroprotective activities through different mechanisms of action. The present review analyzes the available data about the different types of flavonoids and their potential effectiveness as adjuvant therapy of IBDs.

A Novel Mathematical Model for Determining Faculty Workload.

Objective. To develop a mathematical model for determining faculty workload at a college of pharmacy with a team-based learning curriculum. Methods. Using faculty provided data, our model calculated activity and weighted means in teaching, scholarship and service. Subsequently, these data were used to develop departmental and institutional workload models. Results. For the pharmaceutical and biomedical sciences department, percent faculty activity mean values were greatest for service followed by teaching and scholarship. These values in the clinical sciences department were greatest for teaching followed by service and scholarship. Overall, the institutional workload model had the largest maximum faculty activity value for teaching, followed by service and then scholarship. Conclusions. A novel faculty workload model proved to be effective in optimizing faculty workload within a college of pharmacy. Since the workload analysis, the faculty service commitment has been substantially changed, by reducing the number of committees at our institution. This type of workload analysis may particularly benefit colleges of pharmacy that employ a team based learning curriculum, with a large time commitment to teaching.

The synthetic triterpenoid (CDDO-Im) inhibits STAT3, as well as IL-17, and improves DSS-induced colitis in mice.

INTRODUCTION: Synthetic triterpenoids inhibit IL-17 and improve autoimmune disease in mice. A prototype triterpenoid, 1-[2-cyano-3-,12-dioxooleana-1,9(11)-dien-28-oyl] imidazole (CDDO-Im), also inhibits signal transducer and activator of transcription 3 (STAT3) activation. AIMS: The goals of our study were twofold: (1) To determine if ex vivo treatment with CDDO-Im attenuated colonic IL-17 secretion from isolated splenocytes and colonic strips; (2) To determine if oral treatment with CDDO-Im improved DSS-induced colitis in mice. METHODS: Splenocytes were isolated from male Balb/c mice. Colitis was induced in rodents, with either trinitrobenzene sulfonic acid or dextran sulfate sodium (DSS). Colonic strips were collected 5 or 6 days after colitis induction. Splenocytes or colonic strips were exposed to CDDO-Im (0.5-2 µM) concomitantly with IL-23 + IL-1ß. Supernatants were collected after 48 or 24 h, and IL-17 was measured by ELISA. Using a DSS colitis model, mice were dosed orally with vehicle or CDDO-Im (20 mg/kg) over a 5-day period. Subsequently, various parameters of colitis were determined on study day 6. RESULTS: Ex vivo treatment with CDDO-Im inhibited IL-17 secretion from splenocytes and colonic strips. The IC50 values were ≤0.62 µM. In vivo, CDDO-Im improved the altered colonic histology, and cytokine (IL-6, and IL-17) contents. Colonic STAT3 activation was also significantly reduced by CDDO-Im treatment. CDDO-Im attenuated IL-17 secretion in ex vivo models of inflammation. In vivo, histological and biochemical parameters of colitis were improved in CDDO-Im treated mice. CONCLUSION: CDDO-Im has a unique pharmacological profile, which supports further testing in animal models of IBD.

Inflammation-induced functional connectivity of melanin-concentrating hormone and IL-10.

Melanin-concentrating hormone (MCH) was identified in mammals as a hypothalamic neuropeptide regulating appetite and energy balance. However, similarly to most of the brain peptides, MCH is also produced in the gastrointestinal system and can act locally as an immunomodulator. We have previously reported high expression of MCH and its receptor MCHR1 in the affected mucosa of patients with inflammatory bowel disease. Furthermore, MCH deficiency in mice attenuated experimental colitis, pointing to MCH as a mediator of intestinal inflammation. In the present study, in order to gain further insights into the underlying mechanisms of such effects of MCH, we treated mice with established experimental colitis due to IL-10 deficiency with a MCHR1 antagonist (DABA-822). While treatment with the same drug was successful in attenuating TNBS-induced colitis in previous studies, it offered no benefit to the IL-10 knockout mouse model, suggesting that perhaps IL-10 is a downstream target of MCH. Indeed, in experiments focusing on monocytes, we found that treatment with MCH inhibited LPS-mediated IL-10 upregulation. Conversely, in the same cells, exogenous IL-10 prevented LPS-induced MCHR1 expression. Taken together, these findings indicate a functional cross-talk between MCH and IL-10 which prevents resolution of inflammation.

Probiotics for the treatment of Clostridium difficile associated disease.

The purpose of this review paper is to update the current and potential future role of probiotics for Clostridium difficile-associated disease (CDAD). Included in this review, is an update on the testing of newer probiotics (e.g., Bacillus coagulans GBI-30, 6086) in animal models of CDAD. There is a focus on the modulation of signal transduction pathways (i.e., transcription factors like cAMP response element-binding, activator protein 1, and nuclear factor kappa B), as well as the inhibition of certain kinases (e.g., p38 mitogen activated protein kinases) by probiotics. Inhibition of signal transduction by probiotics, such as Saccharomyces boulardii, result in multiple effects on intestinal fluid secretion, neutrophil influx into the colon, inflammation, and colonocyte apoptosis that may positively impact CDAD. Recent clinical approaches with probiotics, for the prevention of primary and recurrent CDAD, are also summarized in this review paper. Future directions for the treatment of CDAD by probiotics are also mentioned in this review. In particular, the use of multi-strain probiotic formulations such as Ecologic(®) AAD and VSL #3(®) may represent a rationale pharmacological approach, particularly as adjunctive therapies for CDAD. Understanding the mechanistic basis of CDAD, and how probiotics interfere at ceratin steps in the pathogenic process, may also present the opportunity to design other multi-strain probiotics that could have a future impact on CDAD.

Inhibition of IL-17 as a pharmacological approach for IBD.

Several experimental approaches have been utilized, in order to critically examine the roles of IL-17 family members in intestinal inflammation. These approaches have included: (1) the use of IL-17A and IL-17F-deficient mice, (2) specific antibodies directed against IL-17, (3) an IL-17 vaccine, (4) methods to block the IL-17 receptor and (5) small-molecule inhibitors of IL-17. Previous studies found somewhat conflicting results in preclinical models of Inflammatory Bowel Disease (IBD), using specific strains of IL-17-deficient mice. This paper will review the preclinical results using various pharmacological approaches [specific IL-17 antibodies, an IL-17 receptor fusion protein, IL-12/IL-23 p40 subunit and IL-17 vaccine approaches, as well as a small molecule inhibitor (Vidofludimus)] to inhibit IL-17 in animal models of IBD. Recent clinical results in patients with IBD will also be discussed for Secukinumab (an IL-17A antibody), Brodalumab (an IL-17 receptor antibody) and two small-molecule drugs (Vidofludimus and Tofacitinib), which inhibit IL-17 as part of their overall pharmacological profiles. This review paper will also discuss some pharmacological lessons learned from the preclinical and clinical studies with anti-IL-17 drugs, as related to drug pharmacodynamics, IL-17 receptor subtypes and other pertinent factors. Finally, future pharmacological approaches of interest will be discussed, such as: (1) Retinoic acid receptor-related orphan nuclear receptor gamma t (Rorγt) antagonists, (2) Retinoic acid receptor alpha (RARα) antagonists, (3) Pim-1 kinase inhibitors and (4) Dual small-molecule inhibitors of NF-κB and STAT3, like synthetic triterpenoids.

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

BACKGROUND: Recently, 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. RESULTS: The 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. SUMMARY: In 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. CONCLUSION: BC30 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-1ß-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.

Novel Pharmacological Approaches for Inflammatory Bowel Disease: Targeting Key Intracellular Pathways and the IL-23/IL-17 Axis.

This review identifies possible pharmacological targets for inflammatory bowel disease (IBD) within the IL-23/IL-17 axis. Specifically, there are several targets within the IL-23/IL-17 pathways for potential pharmacological intervention with antibodies or small molecule inhibitors. These targets include TL1A (tumor necrosis factor-like molecule), DR3 (death receptor 3), IL-23, IL-17 and the receptors for IL-23 and IL-17. As related to IBD, there are also other novel pharmacological targets. These targets include inhibiting specific immunoproteasome subunits, blocking a key enzyme in sphingolipid metabolism (sphingosine kinase), and modulating NF-κB/STAT3 interactions. Several good approaches exist for pharmacological inhibition of key components in the IL-23 and IL-17 pathways. These approaches include specific monoclonal antibodies to TL1A, IL-17 receptor, Fc fusion proteins, specific antibodies to IL-17F, and small molecule inhibitors of IL-17 like Vidofludimus. Also, other potential approaches for targeted drug development in IBD include specific chemical inhibitors of SK, specific small molecule inhibitors directed against catalytic subunits of the immunoproteasome, and dual inhibitors of the STAT3 and NF-κB signal transduction systems. In the future, well-designed preclinical studies are still needed to determine which of these pharmacological approaches will provide drugs with the best efficacy and safety profiles for entrance into clinical trials.

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

BACKGROUND: Probiotics 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. RESULTS: All 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). CONCLUSION: The 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.

Experimental osteoarthritis in rats is attenuated by ABC294640, a selective inhibitor of sphingosine kinase-2.

BACKGROUND/AIMS: Osteoarthritis (OA) is a progressive degenerative disease characterized by cartilage degradation and chondrocyte apoptosis, which may involve aberrant sphingolipid metabolism. ABC294640 is a compound that selectively inhibits sphingosine kinase-2, a key enzyme in the sphingolipid pathway. Our goal was to assess the pharmacological effects of ABC294640 in the monosodium iodoacetate (MIA) model of OA. METHODS: MIA (3 mg) was injected into the right knee joint to induce osteoarthritis in rats. Subsequently, the rats were treated with vehicle, ABC294640 or tramadol over a 28-day period. To assess pain, incapacitance readings were obtained weekly. MIA-injected knee joints were evaluated for histological damage, cartilage degradation and chondrocyte apoptosis (terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling histochemistry). RESULTS: The percent weight bearing in vehicle/MIA rats significantly (p < 0.01) decreased from 48.8 ±0.8 (day 0) to 41.9 ±2.9 (day 28). In contrast, these values in ABC294640-treated rats were virtually the same on days 0 and 28. Knee joint histology scores were less severe in ABC294640-treated rats. Cartilage proteoglycan staining was more prominent in ABC294640/MIA animals than in vehicle/MIA rats. The percentage of apoptotic chondrocytes was decreased from 39.5% (vehicle treatment) to 25.8% (ABC294640 treatment). CONCLUSION: ABC294640 attenuated the knee joint histological damage and pain associated with MIA-induced OA in rats.

Attenuation of arthritis in rodents by a novel orally-available inhibitor of sphingosine kinase.

UNLABELLED: Pro-inflammatory cytokines like TNF-α activate sphingosine kinase (SK). Therefore, inhibition of SK is a potential molecular target for the treatment of rheumatoid arthritis. AIMS: The primary goal of this study was to assess the efficacy of ABC249640 (a selective SK-2 inhibitor) in two models of rodent arthritis. A secondary goal was to evaluate the pharmacological profile of ABC294640, when given in combination with methotrexate. METHODS: The efficacy of ABC294640 was determined by paw diameter/volume measurements, histological evaluations, and micro-CT analyses. RESULTS: ABC294640 attenuated both collagen-induced arthritis in mice, as well as adjuvant-induced arthritis in rats. With the adjuvant arthritis model, the prophylactic efficacy profile of ABC294640 was similar to indomethacin. Of note, ABC294640 reduced the bone and cartilage degradation, associated with adjuvant-induced arthritis. Rats treated with a suboptimal dose of MTX (50 µg/kg/day) in combination with ABC249640 (100 mg/kg/day) had better anti-arthritis effects in the adjuvant model, than treatment with either agent alone. CONCLUSION: Our results suggest that ABC249640 is an orally available drug candidate with a good pre-clinical efficacy profile for the prevention and/or treatment of RA.

Skeletal muscle catabolism in trinitrobenzene sulfonic acid-induced murine colitis.

The present study determined whether the muscle atrophy produced by colitis is associated with altered rates of muscle protein synthesis or degradation, as well as the potential role of the local (eg, muscle) insulin-like growth factor (IGF) system and muscle-specific ubiquitin E3 ligases atrogin-1 and MuRF1 in mediating altered muscle protein balance. Colitis was induced in C57BL/6 mice by intrarectal administration of trinitrobenzene sulfonic acid (TNBS), and blood and tissues were collected on day 10. Mice with inflammatory bowel disease demonstrated reduced skeletal muscle mass and protein content, whereas colonic segment weight and gross damage score were both increased in mice with colitis, compared with time-matched control values. There was no change in muscle protein synthesis in mice with inflammatory bowel disease; but there was an increased protein breakdown (45%), proteasome activity (85%), and messenger RNA (mRNA) expression for atrogin-1 and MuRF1 (200%-300%) in muscle. These changes were associated with a reduction in liver (but not muscle) IGF-I mRNA as well as a reduction in both total and free IGF-I in the blood. Colitis decreased the hepatic content of IGF binding protein (IGFBP)-3 mRNA by 40% and increased IGFBP-1 mRNA by 100%. In contrast, colitis did alter IGFBP mRNAs in muscle. The tumor necrosis factor-α, interleukin-6, and nitric oxide synthase 2 mRNA content of both liver and skeletal muscle was increased in TNBS-treated mice; and plasma tumor necrosis factor-α and interleukin-6 concentrations were also elevated. These data suggest that TNBS-induced colitis is independent of a change in muscle protein synthesis but dependent on stimulation of protein degradation via increased expression of muscle-specific atrogenes, which may be mediated in part by the reduction in circulating concentration of IGF-I and the concomitant increase in inflammatory mediators observed in the blood and muscle per se.

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-α) 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.

A new model of chronic hapten-induced colitis in young rats.

AIM AND OBJECTIVE: : Chronic models of inflammatory bowel disease are lacking in preadult rodents. The primary goal of our study was to develop a chronic model of hapten-induced intestinal inflammation and fibrosis in young rats. Second, we aimed to determine the profiles of key Th-1, Th-2, and Th-17 proinflammatory and profibrotic cytokines, during the progression of colitis in young rats. MATERIALS AND METHODS: Chronic hapten-induced colitis was induced by the administration of intracolonic 2,4,6-trinitrobenzene sulfonic acid (TNBS) in young Wistar rats (postnatal days 23, 35, 48, and 59). After 1, 3, or 4 cycles of TNBS, rats were euthanized and the colons were removed for the measurement of macroscopic, histologic, and biochemical parameters of colitis. RESULTS: Young rats developed moderate to severe colitis in the distal colon, without significant morbidity or mortality. Macroscopic severity, histologic pathology, and colonic weights increased progressively with repeated TNBS administration. Cobblestone-like ulceration and fibrosis was evident in the colon, particularly after 4 cycles of TNBS. There was a unique cytokine pattern associated with colitis in young rats. Interleukin (IL)-12 and tumor necrosis factor (TNF)-alpha peaked during the earlier postnatal time points (days 28 and 54) and then declined after repetitive administration of the hapten (day 67). In contrast, IL-13 and IL-17 were consistently elevated after administration of TNBS to the colon of young rats. CONCLUSIONS: A new model of colitis was established in young rats, which has a unique pattern of Th-1, Th-2, and Th-17 cytokine induction. This chronic TNBS model may be useful for studying the development of inflammation and fibrosis in preadult animals.

Efficacy of a novel sphingosine kinase inhibitor in experimental Crohn's disease.

AIM: Activation of sphingosine kinase (SK) is a key response to many inflammatory processes. The present studies test the hypothesis that an orally available SK inhibitor, ABC294640, would be effective in rodent models of Crohn's disease. METHODS: Trinitrobenzene sulfonic acid (TNBS) was administered rectally to mice and rats. Rats were treated with ABC294640 orally alone or in combination with olsalazine and disease progression was monitored. RESULTS: For both rodent species, treatment with ABC294640 attenuated disease progression. Colon samples from the ABC294640-treated animals had improved histology and cytokine parameters when compared with vehicle-treated animals. The expression of SK was similarly increased in TNBS-treated animals and in human colon tissue specimens from inflammatory bowel disease patients relative to normal, control patients. CONCLUSIONS: Sphingosine kinase may be a critical mediator of colonic damage during intestinal inflammation, and pharmacologic inhibitors of this enzyme may prove useful in the treatment of Crohn's disease.