Therapeutic treatment with a novel hypoxia-inducible factor hydroxylase inhibitor (trc160334) ameliorates murine colitis brain anoxia

Introduction

Inflammatory bowel disease (IBD) is comprised of crohn’s disease (CD) and ulcerative colitis (UC) and is a chronic gastrointestinal inflammation disorder with considerable morbidity and health care burden. 1 although the pathophysiological understanding of IBD has enhanced over time, its exact etiology is yet to be established and the disease remains incurable. IBD remains a complex multifactorial disease involving dysfunctional intestinal epithelial barrier, aberrant innate immune system responses, contribution by intestinal flora along with genetic predisposition 2 and stress. 3 however, recent research indicates that breach in intestinal barrier function is one of the early and crucial factors in the pathogenesis of IBD. 4 breach of intestinal integrity allows nonselective influx of luminal antigens which acts as a trigger for immune cells in the lamina propria, resulting in prolonged, chronic inflammation ultimately leading to clinical presentation of disease in the form of abdominal pain, intestinal bleeding, weight loss, fever, and diarrhea of different severity. 5

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Current therapeutic strategies for IBD involve multidisciplinary management targeting inflammation and immune components associated with the disease. However, several limitations exist with current therapeutic options such as inadequate response or nonresponsiveness, loss of response, and immunosupression. 5 more recently, approval of anti-tumor necrosis factor (TNF) α antibodies for IBD have revolutionized the treatment approach. However, in addition to high cost, adverse effects such as immunogenicity, infusion site reaction, and the loss of efficacy have been observed for anti-tnfα antibodies. 6 recently, mucosal healing has emerged as a key treatment goal in IBD and as a predictor for sustained clinical remission and-resection free survival of patients.Brain anoxia while current therapeutic strategies for IBD reduce disease activity and improve disease symptoms, these agents are inadequate in achieving mucosal healing. 7 indeed, despite therapeutic developments, the number of IBD patients ultimately requiring surgical intervention remain high. 8 this highlights the significant unmet need which can be addressed by new therapeutics aimed at effective mucosal healing for IBD. Effective blockade of breach in intestinal barrier function is expected to bring about such mucosal healing in IBD.

Under physiological conditions, intestinal tissue presents a unique steep oxygen gradient with an oxygen rich vascular bed and almost anoxic luminal epithelial interface. 9 however, when inflamed, a high degree of intestinal mucosal hypoxia is observed in the animal model for colitis, an observation that has been further confirmed in human IBD patients. 10 hypoxia-inducible factor (HIF) activation is one of the prominent adaptive mechanisms associated with hypoxia/ischemia.Brain anoxia HIF activation induces transcription of genes in a concerted manner that performs multiple functions to adapt and to recover from hypoxic/ischemic conditions. 11 recently, we demonstrated that TRC160334, a novel HIF hydroxylase inhibitor, ameliorates ischemic acute kidney injury in rats. 11 the potential of inhibiting HIF hydroxylase to bring about subsequent HIF activation has been identified for the treatment of diseases like anemia 12 and IBD. 13

In the context of IBD, HIF activation results in upregulation of a set of target genes linked with maintenance of intestinal barrier function (heatshock protein 70 [HSP70], multidrug resistant gene 1, intestinal trefoil factor). 14 karhausen et al 14 have shown that transgenic mice with targeted mutation for HIF-1α in intestinal epithelial cells (inability to form HIF-1) demonstrate a more severe colitic phenotype.Brain anoxia in addition, animals mutant for von hippel-lindau gene (forming constitutively active HIF-1) were protected from developing colitis. Pharmacological inhibition of HIF hydroxylase and subsequent HIF activation has resulted in protection of colitis in mice. 13 however, this study employed only the prophylactic treatment of HIF hydroxylase inhibition and did not explore the therapeutic treatment regimen. 13 in the present study we have studied the potential of TRC160334 for treatment of colitis in clinically relevant animal models simulating human CD and UC disease conditions using prophylactic as well as therapeutic treatment regimens.

DAI for colitis

For assessment of clinical severity of colitis, DAI was calculated as reported earlier. 16, 17 DAI is a composite score derived on the basis of loss in body weight, stool consistency, and appearance of blood in feces.Brain anoxia briefly, body weights were monitored daily, whereas stool consistency and rectal bleeding were examined every alternate day. The loss in body weight was scored as: 0, no weight loss; 1, weight loss of 1% to 5%; 2, weight loss of 6% to 10%; 3, weight loss of 11% to 20%; and 4, weight loss 20%. The assessment of diarrhea (stool consistency) was scored as 0 for normally formed pellets; 2 for pasty and semiformed pellets; and 4 for liquid stools. Bleeding was scored using the standard guaiac paper test (hemospot; crest biosystems, goa, india) as follows: 0, negative hemoccult; 2, positive hemoccult; and 4, visible blood/gross bleeding from the rectum. These scoring parameters were added together resulting in a total clinical score ranging from 0 (healthy) to 12 (maximal ill/activity of colitis).Brain anoxia at the end of the experiment, the colons were harvested for further macroscopic/microscopic damage scoring and expression study.

Histological evaluation of colonic damage

The colonic tissues were fixed in 10% neutral buffered formalin, processed, sectioned at 4 μm thickness, and stained with a routine hematoxylin and eosin method. 19 stained sections were evaluated in a blinded manner by a pathologist.

Sections from TNBS treated mice were semiquantified for severity of lesion using a score of 0 to 6: 0, normal crypt architecture; 1, focal to multifocal inflammatory cell infiltration or focal necrosis; 2, diffuse inflammatory cell infiltration or focal to multifocal erosions with necrosis extending up to muscularis mucosa; 3, multifocal erosions or ulceration with and without partial loss of crypts along with inflammatory cell infiltration; 4, diffuse erosions or focal to multifocal ulceration along with partial loss of crypts and inflammatory cell infiltration; 5, multifocal to diffuse ulceration with necrosis extending up to serosa, complete loss of crypts, inflammatory cell infiltration, and mineralization in mucosa and submucosa; 6, multifocal to diffuse ulceration with necrosis perforating serosa, complete loss of crypts, inflammatory cell infiltration, and mineralization in mucosa and submucosa.Brain anoxia

Sections from DSS treated mice were semiquantified for severity of lesion using a scoring of 0 to 4: 0, normal crypt architecture; 1, crypt shortening, loss of basal one third crypt, lamina propria prominent with minimal inflammatory changes; 2, crypt shortening, loss of basal two third crypt, thinning of epithelium with mild inflammatory changes; 3, loss of entire crypt with intact epithelial layer and moderate inflammatory changes; 4, loss of entire crypt along with the epithelial layer (erosion) with severe inflammatory changes. 20, 21

Discussion

Recent advances in the pathophysiology of IBD have revealed that HIF activation may be beneficial in IBD. 13, 14 in the present study, we first demonstrated that prophylactic treatment with TRC160334, a novel HIF hydroxylase inhibitor, is protective in TNBS-induced colitis.Brain anoxia further, we demonstrated efficacy of TRC160334 in a clinically relevant therapeutic setting with the DSS-induced colitis model. The disease attenuating potential of TRC160334 in animal models of CD (TNBS-induced colitis) 14, 22 and UC (DSS-induced colitis) 23, 24 at remarkably low doses via an oral route makes TRC160334 an attractive prospective therapeutic for the treatment of IBD.

TNBS-induced colitis is a clinically relevant model of CD as it displays similar functional and anatomical microvascular abnormalities as observed in human CD. 14, 22 treatment with TRC160334 results in amelioration of body weight loss, significant improvement in DAI, reduction in colon injury severity, and better survival rate in TNBS-induced colitis.Brain anoxia however, prophylactic treatment has limited clinical applicability as in most of the cases of IBD, treatment is initiated upon clinical disease presentation and in such cases therapeutic intervention rather than a preventive treatment strategy is warranted. The therapeutic efficacy of HIF hydroxylase inhibitors in clinically relevant therapeutic settings for colitis has not been reported. Therefore, we explored the therapeutic intervention of TRC160334 in a DSS-induced colitis model.

In the present study, we demonstrated that therapeutic treatment with TRC160334 is protective in DSS-induced colitis. The DSS-induced colitis model is a clinically relevant model of UC. DSS induces intestinal barrier dysfunction, stimulating local and systemic inflammation and thus reproduces many of the histopathologic and clinical features of human colitis. 23, 24

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Treatment with TRC160334 was initiated on day 5 of DSS induction after ascertaining that DAI had reached a value of around 50% of its maximum attainable value. We continued DSS treatment for a duration of 11 days and then kept three DSS free days in our protocol. This ensured that the trigger which leads to an acute episode of colitis is still present while the treatment is initiated and continued, thus enabling us to explore the therapeutic potential of TRC160334 in the disease model. TRC160334 treatment resulted in improvement in body weight loss and DAI, along with improvement in colon histopathology in DSS-induced colitis. This finding is the first to demonstrate that therapeutic intervention with a HIF hydroxylase inhibitor is efficacious in a colitis model.Brain anoxia

Therapeutic treatment with TRC160334 favorably impacted inflammation in the colitis model as trends of reduction in expression of the proinflammatory cytokines, tnfα and infγ, and elevation in the expression of the anti-inflammatory cytokine, IL-10, were observed. Treatment with TRC160334 also resulted in a significant increase in HSP70 expression which is one HIF target protein. Interventions leading to enhanced HSP70 response have been suggested as a potential approach to improve outcomes of IBD. 25 HSP70 transgenic mice demonstrated significantly milder colitis in response to DSS than wild type. 26 HSP70 expression is found to be significantly reduced in actively inflamed mucosa from both human IBD biopsies and IL-10 −/− mice with spontaneous colitis. 27 intestinal epithelial HSP70 plays an important role in protecting mucosal integrity and function by stabilizing the tight junctions between intestinal epithelial cells.Brain anoxia such intestinal epithelial protection is associated with restricted bacterial translocation and a reduction in inflammation. 28 conversely, compromised expression of HSP70 renders the mucosa highly susceptible to inflammatory and immune processes, and to potentially otherwise harmless enteric flora. 27

An earlier report with another HIF hydroxylase inhibitor, FG-4497, has shown a beneficial effect of HIF activation by attenuating TNBS-induced colitis. 13 however, benefits upon therapeutic treatment with FG-4497 are not known. Secondly, the prophylactic efficacy of FG-4497 was shown at higher doses (in a dose range of 20 to 40 mg/kg/day) and by an intraperitoneal route of administration, 13 whereas TRC160334, as reported in current study, has shown both prophylactic and therapeutic benefits in relevant animal models of CD and UC, respectively.Brain anoxia additionally, comparatively lower doses and ease of administration by oral route is likely to improve compliance and convenience in clinical settings.

Our findings with TRC160334, a novel HIF hydroxylase inhibitor, confirms a prophylactic effect of such inhibitors for IBD. In addition, our findings are the first to demonstrate that therapeutic intervention with a HIF hydroxylase inhibitor can also ameliorate IBD. These findings highlight the potential of TRC160334 for its clinical application in the treatment of IBD.