inflammatory bowel diseases are chronic inflammatory diseases of the gastrointestinal tract resulting from a complex interaction of genetic , immunologic , and environmental factors . two major forms of ibd are described : crohn 's disease and ulcerative colitis . increased numbers of activated immune cells are present in the intestinal mucosa of ibd patients , resulting in enhanced cytokine levels of tumor necrosis factor- and interleukins ( il ) , such as il-6 , il-17 , il-10 , and il-8 . these cytokines are important mediators for interaction between immune , epithelial , and mesenchymal cells . biologic agents , antagonizing tnf- , are effective in ibd patients to induce and maintain disease remission . however , not all patients benefit from this strategy [ 4 , 5 ] . other current approaches , such as biologic agents antagonizing proinflammatory cytokines ( e.g. , il-6 ) or the ones that favour increased levels of the suppressive cytokine il-10 , did not result in a substantial reduction of ibd disease activity in clinical trials . therefore , new therapeutic strategies are urgently required that may be based on small - molecular compounds , rather than biologic agents . here we present a new class of small - molecular compounds , the uca derivatives that originated from uv - exposure of the skin . the incidence of inflamed bowel diseases ( ibd ) varies according to geographical location , that is , near the equator there is a smaller incidence than in northern countries in europe and north america [ 711 ] . exposure to solar uv near the equator is more intense and is known to cause immunosuppression or anti - inflammatory effects . an inverse correlation may exist between daily uv - doses and the severity of ibd . one proposed mechanism is related to the occurrence of relatively high concentrations of trans - uca in the stratum corneum . the absorption of uv - radiation by this compound leads to a partial isomerisation to cis - uca , or to ( photo ) oxidation yielding uca oxidation products . both have shown to have anti - inflammatory effects both in vivo and in vitro [ 1416 ] . in the case of cis - uca , attenuation of experimental colonic epithelial damage , induced by dextran sodium sulfate ( dss ) , has recently been obtained in vivo . urocanic acid ( uca ) derivatives , studied here , are natural and synthetic derivatives of uca . several uca derivatives are potential anti - inflammatory agents , primarily developed for topical applications on inflamed skin [ 15 , 16 ] . two natural uca derivatives , imidazole-4-carboxylic acid ( imcooh ) and imidazole-4-acetic acid ( imac ) , were found to have an excellent safety profile after the preclinical test phase , including partial systemic exposure through skin penetration ( unpublished report ) . the natural derivatives of uca are photo ( oxidation ) products of uca and can occur in low concentrations in uvb - exposed skin , for example , skin exposed to bright sun . synthetic uca derivatives are derived from their natural precursors and were alkylated to obtain enhanced lipophilicity . a smoother passage through biological membranes and together , six uca derivatives were tested for anti - inflammatory effects on inflamed human colon tissue , derived from patients with ulcerative colitis in an ex vivo model of ulcerative colitis . two amide - like uca - derivatives were included in the ex vivo experiments next to two ethyl esters and the natural compounds imcooh and imac . an anti - tnf - antibody was included in the ex vivo experiments as well , to compare effects of the uca derivatives with a well - established anti - inflammatory agent . in addition , an experimental dss mouse model was used to study the anti - inflammatory effects of the uca derivatives on the reduction of dss - induced epithelial damage and the relation between both experimental approaches is evaluated . test compounds imidazole-4-carboxylic acid ( imcooh ) and imidazole-4-acetic acid ( imac ) were synthesized by chemshop bv . imidazole-4-acetamide ( im - acetamide ) and ethyl imidazole-4-acetate ( et - imac ) were synthesized in our laboratory , starting with esterification of imidazole-4-acetic acid and ammonialysis to obtain the acetamide . the anti - tnf antibody , known as infliximab , was obtained from schering - plough / msd . the immunohistological staining of neutrophils was performed on snap frozen sections with a primary rat anti - mouse ly-6b.2 antibody ( clone 7/4 , number mca771gt , abd serotec , followed by a secondary antibody donkey anti - rat horse radish peroxidase ( jackson immunoresearch laboratories ) ) . color development proceeded with a staining kit , based on 3,3-diaminobenzidine tetrahydrochloride ( dako north america inc . ) . the uca derivatives imcooh , imac , im - carboxamide , im - acetamide , ethyl imidazole-4-carboxylate ( imcooet ) , and ethyl imidazole-4-acetate ( et - imac ) were dissolved in a concentration of 100 mmol / l in phosphate - buffered saline ( pbs ) . in the case of imcooh and imac , the ph value was readjusted to 7 with a sodium hydroxide ( naoh ) solution . all solutions were argon saturated and kept under an argon atmosphere until use , because the stability of uca derivatives towards oxygen has not been fully defined . each uca derivative was added at a final concentration of 2  mmol / l . argon - saturated pbs was added to the medium to obtain a positive control sample , reflecting nonmodified cytokine patterns , and set to 100% in figure 1 . intraperitoneal ( i.p . ) injections of 0.2  ml of combined imcooh ( 85  mm ) and imac ( 85  mm ) , referred to as imidazoles  , each at a dose of 0.85  mmol / kg , were given to one group and 0.2  ml of combined imcooet ( 42.5  mm ) and et - imac ( 42.5  mm ) , referred to as  ethyl esters  , each at a dose of 0.425  mmol / kg , to a separate group . as a positive control , 0.2  ml pbs i.p . stock amounts of uca derivative solutions were freshly prepared on day 1 and day 4 of the mouse experiment . stability checks on uca derivative solutions were performed with hplc , in particular for monitoring eventual hydrolysis of the ethyl esters . the analyses showed sufficient stability across the period of use ( 3 - 4 days ) , with minor hydrolysis of et - imac into imac ( approx . female c57bl/6 mice of 7 weeks were purchased from charles river ( l'arbresle , france ) . three weeks prior to the start of the experiment , three groups of 10 mice were divided across separate boxes , kept in light- , humidity- , and temperature - controlled rooms in the in - house animal facility . they were given water , containing 1.5% dss , ad libitum , and crm - e food of special diets services ( sds , witham , essex , uk ) . approval of the animal welfare committee was obtained for all dss experiments performed ( approbation number dix102231 ) . cytokines il-6 , il-8 , and il-10 were quantified in the medium obtained from the ex vivo culture experiments by means of elisa ( quantikine , r&d systems ) . inflamed colon tissue from ibd patients was obtained as residual material from clinical procedures according to the ethical guidelines of the academic medical center ( amc ) , amsterdam , the netherlands . tissue was subdivided into small pieces of 1020 milligrams with the aid of a skin biopsy puncher ( 4  mm  ) , followed by submersion in 200  l rpmi culture medium , containing fetal calf serum ( 10% ) , l - glutamine ( 1% ) , and penicillin ( 1  u / ml)/streptomycin ( 1  g / ml ) . test compound solution ( 100  mmol / l ) was added to 200  l medium to obtain a final concentration of 2  mmol / l , followed by a 24-hour incubation period at 37c in an atmosphere containing 5% co2 . subsequently , the supernatant was collected ( approx . 180  l ) , centrifuged to remove debris , and frozen at 20c until further use for cytokine quantification . the weight of each drinking bottle , as well as body weight of the c57bl/6 mice , was noted each day . intraperitoneal ( i.p . ) injections were given once a day on days 1 to 3 , followed by twice daily injections on days 4 to 6 . one half was rolled up on a plastic waver for paraffin embedding and the other half was snap frozen and stored at 80c . disease activity score ( dai ) was based on the cumulative scores of three different parameters : stool consistency ( 04 ) , presence of blood in stool ( 04 ) , and categorized weight loss ( 04 ) . histological examination was performed on paraffin - embedded , rolled - up colons , stained with hematoxylin and eosin ( he ) and on snap - frozen tissue , immunohistologically stained for ly-6b.2 . a pathologist , blinded for this study , performed the scores for aspects as inflammation involved areas , edema , fibrosis , erosion / ulceration , crypt loss , granulocytes , and mononuclear cell infiltration . the absence of inflammation aspects was assigned as  0  , and the most severe inflammation aspects were assigned as  3  . scores were summed per slide and per test group of 10 mice , providing an overall score and a score per histological aspect . statistical evaluation on all parameters was performed using a kruskal - wallis test , eventually followed by dunn 's posttest . the focus of the experiment was on the following cytokines ( i.e. , il-6 , il-8 , and il-10 ) which are representative of an innate response which is known to be elevated during dss - induced epithelial damage [ 1 , 3 , 6 , 12 , 14 , 17 ] . the uca derivative - induced effects on il-6 , il-8 , and il-10 all uca derivatives , except imac , showed suppressive tendencies on il-6 and il-8 levels . an opposite , agonistic effect of imac might be apparent on il-6 and il-8 levels . the anti - tnf antibody , im - carboxamide , and im - acetamide did not seem to affect il-6 and il-8 levels much under this test condition . interestingly , the anti - inflammatory effect of the uca derivatives seemed to be confirmed by the effect on il-10 levels . here , imcooh , both amides , imcooet , and the anti - tnf antibody increased il-10 levels ; however , significance is not reached ( p > 0.05 , kruskal - wallis ) . the ex vivo experiments were found to be susceptible for large variations in equally treated samples . to enhance reliability , inflamed ulcerative colitis tissue of 3 to 4 donors body weight of the female c57bl/6 mice was measured every day at the same time in the morning . the consumption of drinking water was monitored each day of the experiment , and a steep reduction in drinking water consumption was observed in the positive control group during days 6 - 7 , as compared to the two groups receiving the uca derivatives , who maintained their consumption of drinking water ( figure 2(b ) ) . the ratio of colonic weight ( mg ) per length ( cm ) was calculated as a parameter for disease severity . decreased ratios indicate anti - inflammatory effects , as can be seen with the administration of the uca derivatives ( figure 2(c ) ) . the mean weight per cm colon length was reduced in the group of mice receiving both ethyl esters , as compared to the positive control group . considering a limited number of samples in this explorative study , a mann - whitney t - test did reveal a significant difference ( p < 0.05 ) , between positive control and ethyl esters , in contrast to a kruskal - wallis multiple comparison test ( p > 0.05 ) . the disease activity index ( dai ) was determined as outlined in section 3 . a tendency for a lower dai , induced by the imidazoles ( imcooh / imac ) , is shown in figure 2(d ) , but conclusive effects were not observed . blinded histological examination of he - stained paraffin slides were performed according to the histological aspects explained in section 2 . the administration of the ethyl ester combination showed a significant reduction in the areas of inflammation ( figure 3(a ) ) . however , when observing the other aspects , only the administration of imidazoles ( i m ) reduced fibrosis in a significant manner ( figure 3(c ) ) . the other parameters tend to be reduced , predominantly in case of the imidazoles ( figures 3(e ) , 3(f ) , and 3(g ) ) , together resulting in a significant reduction of the total histological score for the administration of the imidazoles ( figure 3(h ) ) . the degree of edema ( figure 3(b ) ) and erosion / ulceration ( figure 3(d ) ) were not affected by the test compounds . a separate investigation was performed on neutrophil infiltration with immunohistological staining of the ly-6b.2 epitope on neutrophils . typical extensive infiltrations are shown in colonic tissue slides of the positive control mice ( figure 4(a ) ) and a substantially reduced number of neutrophils in colonic tissue of the mice treated with both test compound combinations ( figures 4(b ) and 4(c ) ) . semiquantitative analysis revealed the following degrees of inflammation : + + for the positive control group ( figure 4(a ) ) , + for the group that administered the imidazoles ( figure 4(b ) ) , and + for the group that administered the ethyl esters ( figure 4(c ) ) on a scale ranging from + + + ( most severe inflammation ) to  ( no inflammation ) . the decrease of inflammatory cytokines ( i.e. , il-6 and il-8 ) in combination with the increase in il-10 by the uca derivatives in the ex vivo experiments is encouraging for further development of the uca derivatives as potential , low - risk anti - inflammatory drug candidate compounds . the in vivo results confirm findings in a recent dss study that also revealed anti - inflammatory effects on ibd by a uv - induced compound of the skin . their target compound is cis - uca , the uv - induced stereoisomer of trans - uca , and is closely related to the uca derivatives of this study . compared to this study , the way of administration and the dosage of cis - uca was different , as well as the mouse strain . in contrast to our study , albert et al . obtained anti - inflammatory effects with daily subcutaneous injections of 0.36  mol ( 50  g ) cis - uca , whereas we injected intraperitoneally 17  mol of each imidazole and 8.5  mol of ethyl ester . several anti - inflammatory effects were found , indicating that effects of uv - exposure of the skin may attenuate ibd . remarkably , albert et al . found a decrease in neutrophil chemoattractant cxcl1 , a finding that is in accordance to the reduction of infiltrating neutrophils , shown in this study . the ethyl esters imcooet and et - imac are expected to have enhanced membrane passage , which property may have enhanced the anti - inflammatory trends , seen in the ex vivo experiments . the anti - inflammatory effects of the ethyl esters in the dss experiment were however not superior possibly due to yet unknown pharmacological events . routes of administration and delivery of the uca derivatives at body compartments in general and especially delivery at the mucosal side of the colon should be studied . in that particular layer , permeability and subsequent invasion of antigens are expected to be increased due to the effect of orally ingested dss . we chose for an intraperitoneal administration route to exclude an unknown intervention with dss by the oral route . the anti - inflammatory agent , infliximab , was introduced in the ex vivo tests as a reference . however , the overall effects of the anti - tnf agent are less convincing for anti - inflammatory behaviour under our ex vivo test conditions . the documented use for this agent in ibd patients indicates that a longer interaction time is needed for the anti - inflammatory effects to appear . imac showed agonistic effects in the ex vivo experiments and may have counteracted on imcooh , while in the dss experiments during their combined administration . this late conclusion can be drawn from the ex vivo results , where imac seems to increase il-8 levels ( figure 1 ) . consequently , the anti - inflammatory effect of imcooh could have been dampened by imac . if indeed true , the ex vivo results correlate with the results obtained in vivo . the ex vivo model could add value to the question of the usefulness of anti - inflammatory candidate compounds for drug development . if the variation in the results could be decreased , an improved ex vivo model may reduce the use of experimental animals in the future . in this study , many other uca derivatives with potential anti - inflammatory properties can be synthesized within the context of a submitted patent ( international patent number pct / nl2008/050367 ) . this family of uca derivatives may form a novel class of anti - inflammatory agents for ibd .
urocanic acid ( uca ) derivatives were tested for their anti - inflammatory activity in inflammatory bowel disease ( ibd ) in two models : ex vivo and an experimental mouse model . 
 ex vivo : inflamed colonic tissue was incubated in culture medium with or without the uca derivatives . 
 biopsies , incubated with uca derivatives , produced lower levels of proinflammatory cytokines il-6 and il-8 as compared to control biopsies . 
 the same compounds also showed increased levels of il-10 , providing an additional indication for anti - inflammatory properties . in vivo : a combination of two imidazoles and a combination of two of their ethyl esters were administered to mice while colitis was induced by oral administration of dextran sodium sulfate ( dss ) . 
 some parameters did not show conclusive effects , but the imidazoles and their ethyl esters reduced the area of inflammation and the number of infiltrating neutrophils . 
 fibrosis and the sum of all histological aspects were reduced by the imidazoles , whereas the ethyl esters reduced the colon weight to length ratio . 
 these results suggest that the uca derivatives have anti - inflammatory effect on ibd . 
 in addition , fine tuning of the ex vivo model may provide an elegant way to predict anti - inflammatory effects of potential drugs in humans , which may decrease the need for animal experiments .