USOO9248120B2
(12) United States Patent (10) Patent No.: US 9.248,120 B2 Penny et al. (45) Date of Patent: Feb. 2, 2016
(54) REVERSING INTESTINAL INFLAMMATION (56) References Cited BY INHIBITING RETNOIC ACID METABOLISM U.S. PATENT DOCUMENTS 6,486,187 11, 2002 Venet et al. (75) Inventors: Hweixian Leong Penny, Singapore 7,265,143 9, 2007 Njar et al. (SG); Edgar G. Engleman, Palo Alto, 7,807,650 10, 2010 Jimenez et al...... 514.44 R CA (US) 2002.0193578 12, 2002 Vasudevan et al. 534,753 2004/0228871 11, 2004 Hasan et al...... 424,178.1 2005/O187298 8, 2005 Vasudevan et al. (73) Assignee: THE BOARD OF TRUSTEES OF 2006, OOO9645 1, 2006 Smith et al...... 548,265.8 THE LELAND STANFORD JUNOR 2008.0031984 2, 2008 Quartet al. UNIVERSITY, Stanford, CA (US) 2010.0048415 A1 2/2010 Croner et al. 2010, O292284 A1 11/2010 Barrett et al. (*) Notice: Subject to any disclaimer, the term of this OTHER PUBLICATIONS patent is extended or adjusted under 35 Terzic et al.(Gastroenterology .2010, 138, pp. 2101-2114).* U.S.C. 154(b) by 531 days. Collins; etal. “Retinoic acid attenuates ileitis by restoring the balance between T-helper 17 and Tregulatory cells'. Gastroenterology (Nov. (21) Appl. No.: 13/592,180 2011), 141(5):1821-1831. Duester; etal. "Cytosolic retinoid dehydrogenases govern ubiquitous (22) Filed: Aug. 22, 2012 metabolism of retinol to retinaldehyde followed by tissue-specific metabolism to retinoic acid”. Chem Biol Interact (Feb. 2003), 143 Prior Publication Data 144:201-210. (65) Eisinger; et al. “Retinoic acid inhibits beta-catenin through Suppres US 2013/O131129 A1 May 23, 2013 sion of Cox-2: a role for truncated adenomatous polyposis coli'. J Biol Chem (Oct. 2007), 282(40):29394-294.00. (Continued) Related U.S. Application Data Primary Examiner — Savitha Rao (60) Provisional application No. 61/526,574, filed on Aug. (74) Attorney, Agent, or Firm — Bozicevic, Field & Francis 23, 2011. LLP. Kyle A. Gurley (51) Int. C. (57) ABSTRACT A6 IK3I/428 (2006.01) An agent that increases local concentration of retinoic acid A6 IK3I/484 (2006.01) (RA) in the intestine through modifying enzymatic pathways (52) U.S. C. involved in RA metabolism is administered in a dose effective CPC ...... A61 K3I/428 (2013.01); A61 K3I/4184 to inhibit or reverse production of inflammatory mediators by (2013.01) intestinal dendritic cells and thereby reduce intestinal inflam (58) Field of Classification Search mation and tumor growth associated with intestinal inflam CPC ...... A61K 31/4184: A61 K31/428 mation. USPC ...... 514/367 8 Claims, 16 Drawing Sheets See application file for complete search history. (12 of 16 Drawing Sheet(s) Filed in Color)
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(56) References Cited Shelton; et al. "Up-regulation of CYP26A1 in adenomatous polyposis coli-deficient vertebrates via a WNT-dependent mecha OTHER PUBLICATIONS nism: implications for intestinal cell differentiation and colon tumor development”, Cancer Res (Aug. 2006), 66(15): 7571-7577. Goss; et al. “Effects of liarozole fumarate (R85246) in combination Verfaille; etal. “Oral R115866 in the treatment of moderate to severe facial acne vulgaris: an exploratory study', Br J Dermatol (Jul. 2007), with tamoxifen on N-methyl-N-nitrosourea (MNU)—induced mam 157(1): 122-126. mary carcinoma and uterus in the rat model. BMC Cancer (Jan. Verfaille; etal. “Oral R115866 in the treatment of moderate to severe 2007), 7:26. plaque-type psoriasis”. J. Eur Acad Dermatol Venereol (Sep. 2007), Huynh; etal. "Inhibitory effects of retinoic acid metabolism blocking 21(8):1038-1046. agents (RAMBAs) on the growth of human prostate cancer cells and Xavier; et al. "Unravelling the pathogenesis of inflammatory bowel LNCaP prostate tumour xenografts in SCID mice', Br JCancer (Feb. disease”. Nature (Jul 2007), 448(7152):427-434. 2006), 94(4): 513-523. Bai, et al., “All-trans retinoic acid down-regulates inflammatory Nadauld; et al. "Adenomatous polyposis coli control of C-terminal responses by shifting the Treg Th17 profile in human ulcerative and binding protein-1 stability regulates expression of intestinal retinol murine colitis”, Journal of Leukocyte Biology (Oct. 2009), dehydrogenases”, J Biol Chem (Dec. 2006), 281 (49):37828-37835. 86(4):959-969. Patel; et al. “Novel retinoic acid metabolism blocking agents Klopcic; et al., “Indomethacin and Retinoic Acid Modify Mouse endowed with multiple biological activities are efficient growth Intestinal Inflammation and Fibrosis: A Role for SPARC. Dig. Dis inhibitors of human breast and prostate cancer cells in vitro and a Sci (Jun. 2008), 53(6): 1553-1563. human breast tumor xenograft in nude mice'. J Med Chem (Dec. Osanai; et al., "Cellular Retinoic Acid Bioavailability Determines 2004), 47(27):6716-6729. Epithelial Integrity: Role of Retinoic Acid Receptor (alpha) Agonists Rai; etal. “DNA demethylase activity maintains intestinal cells in an in Colitis'. Molecular Pharmacology (Jan. 2007), 71(1):250-258. undifferentiated state following loss of APC". Cell (Sep. 2010), 142(6):930-942. * cited by examiner U.S. Patent Feb. 2, 2016 Sheet 1 of 16 US 9.248,120 B2
U.S. Patent Feb. 2, 2016 Sheet 3 of 16 US 9.248,120 B2
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US 9.248,120 B2 1. 2 REVERSING INTESTINAL INFLAMMATION individual, where the agent increases local concentration of BY INHIBITING RETNOIC ACID retinoic acid (RA) in the intestine through modifying enzy METABOLISM matic pathways involved in RA metabolism. In some embodiments, an inhibitor of CYP26A1 is admin GOVERNMENT RIGHTS istered in a dose effective form to neutralize a pre-existing inflammatory environment, prevent the development of This invention was made with Government support under inflammation, or maintain the tolerogenic functions of intes contract CA141468 awarded by the National Institutes of tinal dendritic cells that maintain intestinal tolerance by Health. The Government has certain rights in this invention. inducing Treg formation. Alternatively, an agent that 10 increases activity of retinaldehyde dehydrogenase or retinol BACKGROUND OF THE INVENTION dehydrogenase may be administered in a dose effective to Pathological inflammation is emerging as an underlying neutralize or prevent inflammation in the intestine. The mechanism for numerous diseases. For example, the major appropriate dose may be determined by evaluating the effect forms of idiopathic IBD, ulcerative colitis and Crohn's dis 15 of the agent on functions of intestinal dendritic cells, for ease are chronic inflammatory disorders of the gastrointesti example by monitoring synthesis of cytokines such as IL-23 nal tract. Animal studies have shown that chronic intestinal and IL-17, or by overall analysis of intestinal inflammation. inflammation precipitates as well as propagates tumor The methods of the invention exclude administration of ret growth. inoic acid directly. A number of sentinel cell populations in the intestinal Dendritic cells are shown to be reprogrammed due to local mucosa continuously monitor luminal microbes and other loss of the vitamin A metabolite, retinoic acid (RA), which antigens. Among the Subsets of antigen-presenting cells, occurs as a result of insufficient retinaldehyde dehydrogenase myeloid-derived dendritic cells are a dominant subtype in the and an overabundance of the transcriptional co-repressor, intestinal lamina propria and show considerable functional Ctbp 1. The reprogrammed dendritic cells secrete mainly pro plasticity depending on the location, state of maturation, and 25 inflammatory cytokines and induce Th17 formation. Treat stage of inflammation. Dendritic cells form an extensive net ment by the methods of the invention replenish RA and neu work beneath the intestinal epithelium and project long pro tralize the inflammatory phenotype of the dendritic cells. cesses through the interstices of epithelial cells to sample Other aspects and features will be readily apparent to the luminal antigens. In response to TLR ligands, immature den ordinarily skilled artisan upon reading the present disclosure. 30 dritic cells produce IL-23, which contributes to development BRIEF DESCRIPTION OF THE DRAWINGS of intestinal inflammation in murine models of colitis and intestinal inflammation. The remarkable capacity of dendritic The patent or application file contains at least one drawing cells to orchestrate distinct immune responses is aided by a executed in color. Copies of this patent or patent application panoply of environmental cues, which condition the cells to publication with color drawing(s) will be provided by the adopt specific phenotypes in different settings. DCs in gut 35 Office upon request and payment of the necessary fee. associated lymphoid tissue are of particular interest because The invention is best understood from the following they maintain tolerance to commensal flora as well as mount detailed description of exemplary embodiments when read in protective inflammatory responses in the face of pathogen conjunction with the accompanying drawings. It is empha incursion. sized that, according to common practice, the various features Migration of innate immune cells such as neutrophils, mac 40 of the drawings are not necessarily to-scale. On the contrary, rophages, and dendritic cells into target mucosal tissues the dimensions of the various features are arbitrarily depends on the expression of cytokines, chemokines and expanded or reduced for clarity. Included in the drawings are adhesion molecules. Recruitment of activated neutrophils, the following figures: dendritic cells and macrophages into the lamina propria in FIG. 1. APC' SI-LPDCs accumulate over time and general amplifies the local immune response, whereas acti 45 vated natural killer cell recruitment seems to enhance antimi secrete more pro-inflammatory cytokines compared to WT crobial factors, leading to attenuation of inflammation. SI-LPDCs upon TLR stimulation. DCs were defined as PI The CD4 T-cell lineage (TH17) is characterized by the EpCAM-CD45+ DX5-CD3e-CD19- CD11c MHCII+ in production of IL-17. Its development is promoted by IL-23, in all the tissues. TWT: APC" (a) A representative addition to IL-6 and TGFbeta. Evidence indicates that RORct 50 contour plot depicting how DCs are gated by CD11c and (retinoic acid-related orphan nuclear hormone receptor MHC II expression. (b) A bar graph of the mean frequency gamma-t) is necessary for TH17 commitment and differentia (with SEM) of CD11c-- MHCII+ DCs as a percentage of tion. In addition to its ability to support the development of PI-Lineage-cells. (c) Representative contour plots of SPL, TH17 cells, IL-23 induces the secretion of IL-17 by non-T- mLN/PP and SI-LP DCs divided by CD11b and CD103 cells in an inflammatory environment, and both T cells and 55 expression. Below each FACS plot are bar graphs showing the monocytes serve as Sources of increased expression in the mean frequency (with SEM) of each subset in that tissue as a mucosa of IBD patients. percentage of total CD11c-- MHCII+ DCs. (d) Mean fre An understanding and manipulation of inflammatory cells quency (with SEM) of cells expressing the co-stimulatory in the gut is of great interest. The present invention addresses molecules CD40, CD80, CD83, CD86 and PDL1 as a per this issue. 60 centage of the different CD11b CD103 subsets. Data for (a-d) were obtained at intermediate stage disease. Inset values in SUMMARY OF THE INVENTION the contour plots are the mean frequencies of each population pooled using at least 4 mice per strain (WT and APC") per Methods are provided for reducing intestinal inflamma experiment, from 5 independent experiments. (e) 5x10' tion, particularly chronic inflammation, and tumor growth 65 FACS-purified WT and APC' SI-LPDCs were stimulated precipitated by intestinal inflammation. In the methods of the with a panel of 6 different TLR agonists—Pam3Csk4 (1 invention, an effective dose of an agent is provided to the ug/ml), Poly I:C (10 ug/ml), LPS (10 ug/ml), flagellin (1 US 9.248,120 B2 3 4 ug/ml), R848 (10 ug/ml), CpG 2336 (10 ug/ml). Supernatants luorescence with DC-SIGN and 8-catenin (b). Ctbp 1 (c), were collected after 48 hours. Representative bargraphs show Raldh1a1 (d), Raldhla2 (e), CYP26A1 (f). Shown are mean production (with SEM) of the cytokines IL-6, TNFC. samples from one representative normal colon, two represen and IL-2p40 as measured by standard ELISA. Data in (e) was tative FAP adenoma and one representative SSP patient. obtained at late stage of disease. Data are representative of 4 Images for FAP P1 show serial sections of the same polyp independent experiments, with DCs pooled from 8 mice per with underlying adjacent normal grossly uninvolved tissue. strain (WT and APC") per experiment. An unpaired stu Overall, samples from a total of 11 normal colon, 8 FAP dent's t test with 95% confidence interval was performed. adenoma, 2 FAP adenocarcinoma and 4 SSP patients were P<0.05=*; p<0.001=**; p<0.0001=***. analyzed. All images were captured using the same exposure FIG. 2. APC' SI-LPDCs have a reduced capacity to 10 time and the same brightfieldorfluorescence settings for each induce Foxp3+T and IL-10 producing CD4+ T cells, but protein of interest. White magnification bars in (a-f) are 100 instead generate Th17 cells, in an RA-dependent manner. uM. 2x10" of sorted CD11c MHCII DCs were co-cultured for 5 FIG. 5. A vitamin A-deficient diet exacerbates disease in days with 1x10 MACS-enriched CD4+CD62L+Foxp3 APC" mice. (a-d) 10 week old WT (open circles) and naive T cells from the spleen and lymph nodes of OT-II 15 APC' (filled triangles) mice were placed on 1500 ppm TCR-transgenic mice, along with OVA, so peptide and 10 ng/ml recombinant human TGF-B. 5 ng/ml of recombinant Celecoxib (* , s ); 100 ppm Rosiglitazone (*, ); a human IL-2 was added to the cultures every other day begin vitamin A deficient diet (0 IU/g of vitamin A) (* , s ); or a ning on day 2. m. WT: APC" (a) DCs used in these base diet (4 IU/g of vitamin A) ( , " : ) for 10 consecutive cultures were purified from SPL, mLN/PP and SI-LP, and weeks. Mice were monitored for disease progression on the stimulated with 5 nM OVAs. Representative bar graph basis of hematocrit and body weight change. Line graphs shows the mean frequency (with SEM) of Foxp3 induction show Kaplan-Meier Survival curves (a), mean change in per from 9 independent experiments, with DCs pooled from at centage body weight over original body weight (b) and hema least 5 WT and at least 3 APC" mice per experiment. (b) 25 tocrits (c), with SEMs. 5 mice were used per strain for each SI-LPDCs were used in these co-cultures, and stimulated diet. Data are representative of 2 independent experiments. with 200 nM, 1 uM and 5uMOVAs. Representative bar Liarozole, a CYP26A1 inhibitor, ameliorates disease in graph shows the meanfrequency (with SEM) of Foxp3 induc APC" mice by increasing local RA which reverses the tion from 4 independent experiments, with DCs pooled from SI-LPDC pro-inflammatory phenotype. 8 week old WT and at least 5 WT and at least3 APC" mice perexperiment.(c) 30 APC" mice were placed on 40 ppm of Liarozole for 6 CD11c-- MHCII+ SI-LP DCs were further sorted into the CD103+ and CD103- Subsets and cultured with naive CD4 T consecutive weeks. APC'-Base diet :: APC"-Liaro cells as before. Bar graph shows the mean frequency (with zole : ; WT-Liarozole 8. Disease was monitored as SEM) of Foxp3 induction in a representative experiment of 2 described in (a-d). In these experiments, Small intestinal pol independent experiments, with DCs pooled from at least 5 yps were enumerated at 14 weeks, the point of euthanasia. A WT and at least 3 APC' mice per experiment. (d) In these 35 scatter plot shows the mean number of polyps (with SEM) (h) experiments, 10 nM all-trans RA or 1 of the RAR antagonist between APC" mice on Liarozole compared to base diet. LE540 was added to whole SI-LPDC-T cell co-culture wells, 5 mice were used per strain in each diet. Data are represen in addition to TGF-31. Cells were re-stimulated on day 4 with tative of 2 independent experiments. Significance in disease plate-bound anti-CD3 and anti-CD28 (1 lug/ml each) for 18 indicators of the different diets was calculated by comparing hours. Supernatants were collected on day 5 to assay for 40 values to those obtained on the base diet. For calculating cytokine production by standard ELISA. Representative bar significance in DAI, a Wilcoxon-rank-Sum test was used. (i) graphs show the mean production (with SEM) of IL 10 (e), All-trans retinoic acid (RA) was extracted from the duode IL17A(f) and IL6 (g). Data in this figure were obtained at late num, jejunum, ileum, colon and eye and quantified by tandem stage disease, and are representative of 4 independent experi 45 mass spectrometry. WT-base diet m; APC"-Base diet ments, with DCs pooled from at least 5 WT and at least 3 : APC'-Liarozole . Bar graphs show mean APC" mice per experiment. An unpaired student's t test amounts of retinoic acid per gram tissue (with SEM) among with 95% confidence interval was performed. P<0.05=*: mice of the same strain. 5 WT, 5 APC' and 4 Liarozole p-0.001=**; p<0.0001=***. treated APC" mice were used in this experiment. For data FIG.3. APC' SI-LP DCs and IECs have lower expres 50 in this figure, an unpaired student's t test with 95% confidence sion of Raldhs than WT, resulting in loss of RA in the tumor interval was performed unless otherwise stated. PK0.05=*: milieu. WT: APC'Total RNA from FACS-puri p<0.001=**; p<0.0001=***. 8 week-old WT and APC''“ fied SI-LPDCs, splenic DCs (A) and epithelial cells (IECs) mice were placed on 40 ppm of Liarozole for 6 consecutive (B) was extracted and assayed in triplicate by qPCR for the genes Raldh1a1, Raldh1a2, Raldhla3 and Ctbp1 (C). Each 55 weeks as described in FIG. 5. WT-base diet M: APC'- sample was normalized to ubiquitin b expression. Represen Base diet : APC'-Liarozole . (a) At the 14 week tative bar graphs show mean relative expression (with SEM) time point of euthanasia, 2x10 FACS-purified WT and of triplicate samples. Data are representative of 3 independent APC' SI-LPDCs were stimulated with Pam3Csk4 (1 qPCR experiments, with DCs and IECs pooled from 3 sorts ug/ml), LPS (10 ug/ml) and R848 (10 g/ml). Supernatants per timepoint, using at least 5 WT and at least 3 APC'" 60 were collected after 48 hours. Representative bargraphs show mice per sort. An unpaired student's t test with 95% confi mean production (with SEM) of the cytokines IL-6, TNFC. dence interval was performed. P-0.05=*; p<0.001=**: and IL-2p40 as measured by standard ELISA. Data are rep p-0.0001=***. resentative of 2 independent experiments, with at least 2 mice FIG. 4. FAPadenoma tissue exhibits IL-17-driven inflam per strain. (b) A T induction assay was performed as mation and loss of RA. Normal colon, FAP adenoma and 65 described in FIG. 2 on SI-LPDCs sorted from these mice. sessile serrated polyp (SSP) sections were stained by immu Representative bar graph shows the mean frequency (with nohistochemistry for IL-17A (a) or co-stained by immunof SEM) of Foxp3 induction from 2 independent experiments, US 9.248,120 B2 5 6 with DCs pooled from at least 2 mice per strain. (c) T cells in diet (4 IU/g of vitamin A), for 10 consecutive weeks. Line the SI-LPDC-T cell co-culture was re-stimulated as described graph show mean change in percentage body weight over in FIG. 2 and assayed for IL 10, IL17A and IL6 by standard original body weight. Data are representative of 2 indepen ELISA as before. For data in this figure, an unpaired students dent experiments. t test with 95% confidence interval was performed. 5 FIG. 13. APC' SI-LPDCs display higher expression of P<0.05=*; p<0.001=**; p<0.0001=***. Cox2 compared to WTSI-LPDCs. RT-qPCR on FACS-puri FIG. 6. Proposed Model: In the APC' mouse, the fied WT and APC' SI-LP DCs was performed as immune state of the intestine, departs from homeostatic tol erance to chronic inflammation. Truncated APC protein leads described in FIG. 3." WT: APC" Representative 10 bar graph shows mean relative expression (with SEM) of to an accumulation of the transcription factor Ctbp1, which in triplicate samples. Data is from 1 independent qPCR experi turns suppresses retinol dehydrogenases in the epithelium ment, with DCs and pooled from 3, using at least 5 WT and at and may lead to a reduction in local concentrations of RA. Upregulation of Cox2 in the inflamed tissue leads to greater least 3 APC" mice per sort. production of PGE, which has been reported to suppress FIG. 14. Direct supplementation of RA does not affect Raldh1a2 in DCs and likely does so to the surrounding epi 15 tumor frequency or intestinal RA levels in APC" mice, but thelium as well. Lack of RA in the intestinal milieu conditions Liarozole, a CYP26A1 inhibitor, improves all tested disease or reprograms. SI-LPDCs to a pro-inflammatory phenotype, parameters. Groups of 8 week-old APC' mice were in which IL6 production combined with TGFB found locally, injected i.p. with or without 200 g/mouse RA in sunflower contributes to a deleterious Th17 response that promotes oil injected twice weekly while on a base diet, or placed on 40 tumor growth. ppm of Liarozole for 6 consecutive weeks. APC" on Base FIG. 7 Morphology of sorted SI-LPDCs from WT and diet?: APC'" on Liarozole?: APC' given RA i.p./; WT APC' mice. Sorted PI-EpCAM-CD45+ DX5-CD3e on Liarozole. (a) Small intestine tumors were enumerated at CD19->CD11c' MHCII+ LP DCs were stained with May 14 weeks. Scatter plot shows the mean number of tumors Grumwald-Giemsa. (with SEM) in APC" mice on base, Liarozole or RA i.p. FIG. 8. APC" SI-LPDCs have a reduced capacity to 25 (a). Line graphs show mean change in percentage body induce Foxp3+Trina tissue-specific, dose-dependent and weight (b) and hematocrit (c). For (a-c), data for mice on base time-dependent manner. A T induction assay was per diet and LiaroZole are aggregated from 4 independent experi formed as described in FIG. 2. (a-c) Representative dot plots ments, with at least 4 mice per group, while data for RA of Foxp3 versus CFSE in Thy 1.2+ CD4+ cells. The inset 30 i.p.-injected mice are aggregated from 2 independent experi values of the bordered population show the mean frequency ments, with at least 3 mice per group. (d) Concomitant to of Foxp3+ cells as a percentage of Thy 1.2+ CD4+ cells. (a) experiments performed in FIG. 4d. RA was quantified by DCs used in these cultures were purified from SPL, mLN/PP LC/MS in 5 Liarozole treated and 5 RA i.p.-injected APC and SI-LP, (b) SI-LPDCs were incubated with 200 nM, 1 uM '"" mice. Shown are mean RA levels (with SEM) in each and 5uMOVA. (c) SI-LPDCs were isolated from early, 35 tissue isolated at the 14 week time point. (e) Dot plots show intermediate and late stage disease. the mean frequency of IL-17A- and IFNY-producing CD4"T FIG. 9. ADH class I and II expression in APC'". IECs cells in freshly-isolated SI-LP from WTAPC" and Liaro and SI-LPDCs increased at intermediate stage and decreased zole-treated APC" mice. Also shown are bar graphs at Subsequent late stage compared to WT counterparts. RT depicting mean frequency (with SEM) of IL-17A- and IFNY qPCR on FACS-purified WT and APC' SI-LP DCs, 40 producing CD4+ T cells from these mice. (f) CD103- SI splenic DCs (a) and epithelial cells (IECs) (b) was performed LPDCs from WT, APC' and Liarozole-treated APC' as described in FIG.3. T WT: APC" Representative mice were co-cultured in the T cell differentiation assays bar graphs show mean relative expression (with SEM) of performed as in FIG.1. Inset values on the representative dot triplicate samples. Data are representative of 3 independent plots show the mean frequency of IL-10- and IL-17A-pro qPCR experiments, with DCs and IECs pooled from 3 sorts 45 ducing CD4 T cells induced. In (ef) IL-17A, IFNY", or per time point, using at least 5 WT and at least 3 APC'" IL-10" cells are shown as a percentage of CD4 T cells. Data mice per sort. Shown is the ADH class I and II expression in in (e-f) are from 2 independent experiments, with at least 4 DCs (a) and in IECs (b). mice per group. DCs obtained in (f) were pooled from all mice FIG. 10. Quantification of retinyl esters and retinol in vivo. in the same group in each experiment. P-0.05=*: Retinyl esters (RE) and all-trans retinol (ROL) were extracted 50 p-0.001=**; p<0.0001=***. from the duodenum, jejunum, ileum, colon and eye and quan FIG. 15. Talarozole, a highly specific inhibitor of tified by HPLC. Bar graphs show mean amounts of retinoid CYP26A1, ameliorates disease in APC' mice.8 week-old per gram tissue (with SEM) among mice of the same strain. 5 APC" mice were placed on 8 ppm of Talarozole for 6 WT, 5 APC' and 4 Liarozole-treated APC' mice were consecutive weeks. APC'-Base diet: APC'-Talaro used in this experiment. An unpaired student's t test with 95% 55 Zole. At 14 weeks, tumors in the Small and large intestine were confidence interval was performed. P<0.05=*; p<0.001=**: enumerated. Results shown are aggregated from 2 indepen p-0.0001=***. dent experiments, with 5 mice per group. FIG. 11. Isotype control for immunohistochemistry and FIG. 16. Paraffin embedded normal, inflamed, dysplastic immunofluorescence. Normal colon sections were stained or carcinoma colon sections were stained by immunofluores with an isotype control (Rabbit IgG) to the rabbit primaries 60 cence for CYP26A1 (in red) and DC-SIGN (in green). Shown used against B-catenin, Ctbp 1, Raldh1a1. Raldh1a2 and are samples from 2 different patients. Patient 1: Normal, CYP26A1. Dysplastic and Carcinoma sections from a collectomy sample FIG. 12. Wildtype mice on base, rosiglitazone, celecoxib and Patient 2 Inflamed and dysplastic regions from a collec and vitamin A-deficient diets gain in weight comparably. tomy sample. All images were captured using the same expo Wildtype mice on the various diets 10 week-old WT mice 65 Sure time and the same brightfield or fluorescence settings for were placed on 1500 ppm Celecoxib, 100 ppm Rosiglitazone, each protein of interest. Similar results were obtained from a vitamin A deficient diet (0 IU/g of vitamin A), or on a base staining with Raldh1a1 and Raldh1a2. US 9.248,120 B2 7 8 DETAILED DESCRIPTION OF THE "polypeptide' is recited herein to refer to an amino acid EMBODIMENTS sequence of a naturally-occurring protein molecule, "polypeptide' and like terms are not necessarily limited to the It is to be understood that the invention is not limited to amino acid sequence to the complete, native amino acid particular embodiments described, as such may, of course, sequence associated with the recited protein molecule, but vary. It is also to be understood that the terminology used instead can encompass biologically active variants or frag herein is for the purpose of describing particular embodi ments, including polypeptides having Substantial sequence ments only, and is not intended to be limiting, since the scope similarity or sequence identify relative to the amino acid of the present invention will be limited only by the appended sequences provided herein. In general, fragments or variants claims. 10 retain a biological activity of the parent polypeptide from Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower which their sequence is derived. limit unless the context clearly dictates otherwise, between As used herein, "polypeptide' refers to an amino acid the upper and lower limits of that range is also specifically sequence of a recombinant or non-recombinant polypeptide disclosed. Each Smaller range between any stated value or 15 having an amino acid sequence of i) a native polypeptide, ii) intervening value in a stated range and any other stated or a biologically active fragment of an polypeptide, or iii) a intervening value in that Stated range is encompassed within biologically active variant of an polypeptide. Polypeptides the invention. The upper and lower limits of these smaller Suitable for use can be obtained from any species, e.g., mam ranges may independently be included or excluded in the malian or non-mammalian (e.g., reptiles, amphibians, avian range, and each range where either, neither or both limits are (e.g., chicken)), particularly mammalian, including human, included in the Smaller ranges is also encompassed within the rodenti (e.g., murine or rat), bovine, Ovine, porcine, murine, invention, Subject to any specifically excluded limit in the or equine, particularly rat or human, from any source whether stated range. Where the stated range includes one or both of natural, synthetic, semi-synthetic or recombinant. In general, the limits, ranges excluding either or both of those included polypeptides comprising a sequence of a human polypeptide limits are also included in the invention. 25 are of particular interest. Unless defined otherwise, all technical and scientific terms The term "derived from indicates molecule that is used herein have the same meaning as commonly understood obtained directly from the indicated Source (e.g., when a by one of ordinary skill in the art to which this invention protein directly purified from a cell, the protein is "derived belongs. Although any methods and materials similar or from the cell) or information is obtained from the source, equivalent to those described herein can be used in the prac 30 tice or testing of the present invention, exemplary methods e.g. nucleotide or amino acid sequence, from which the mol and materials are now described. All publications mentioned ecule can be synthesized from materials other than the source herein are incorporated herein by reference to disclose and of information. describe the methods and/or materials in connection with The term "isolated indicates that the recited material (e.g. which the publications are cited. It is understood that the 35 polypeptide, nucleic acid, etc.) is substantially separated present disclosure Supercedes any disclosure of an incorpo from, or enriched relative to, other materials with which it rated publication to the extent there is a contradiction. occurs in nature (e.g., in a cell). A material (e.g., polypeptide, It must be noted that as used herein and in the appended nucleic acid, etc.) that is isolated constitutes at least about claims, the singular forms “a”, “an', and “the include plural 0.1%, at least about 0.5%, at least about 1% or at least about referents unless the context clearly dictates otherwise. Thus, 40 5% by weight of the total material of the same type (e.g., total for example, reference to “a cell' includes a plurality of such protein, total nucleic acid) in a given sample. cells and reference to “the polypeptide' includes reference to The terms “subject' and “patient” are used interchange one or more polypeptides and equivalents thereof known to ably hereinto mean a member or members of any mammalian those skilled in the art, and so forth. or non-mammalian species that may have a need for the It is further noted that the claims may be drafted to exclude 45 pharmaceutical methods, compositions and treatments any element which may be optional. As such, this statement is described herein. Subjects and patients thus include, without intended to serve as antecedent basis for use of such exclusive limitation, primate (including humans), canine, feline, ungu terminology as “solely”, “only' and the like in connection late (e.g., equine, bovine, Swine (e.g., pig)), avian, and other with the recitation of claim elements, or the use of a “nega Subjects. Humans and non-human animals having commer tive' limitation. 50 cial importance (e.g., livestock and domesticated animals) are The publications discussed herein are provided solely for of particular interest. As will be evidence from the context in their disclosure prior to the filing date of the present applica which the term is used, subject and patient refer to a subject or tion. Nothing herein is to be construed as an admission that patient susceptible to infection by a Flaviviridae virus, par the present invention is not entitled to antedate Such publica ticularly HCV. tion by virtue of prior invention. Further, the dates of publi 55 "Mammal’ means a member or members of any mamma cation provided may be different from the actual publication lian species, and includes, by way of example, canines; dates which may need to be independently confirmed. felines; equines: bovines; Ovines; rodentia, etc. and primates, The term “effective dose” or “effective dosage” is defined particularly humans. Non-human animal models, particularly as an amount Sufficient to achieve or at least partially achieve mammals, e.g. primate, murine, lagomorpha, etc. may be the desired effect. The term “therapeutically effective dose' is 60 used for experimental investigations. defined as an amount Sufficient to cure or at least partially The term “unit dosage form,” as used herein, refers to arrest the disease and its complications in a patient already physically discrete units Suitable as unitary dosages for suffering from the disease. Amounts effective for this use will human and animal Subjects, each unit containing a predeter depend upon the severity of the disorder being treated and the mined quantity of compounds calculated in an amount Suffi general state of the patients own immune system. 65 cient to produce the desired effect in association with a phar “Polypeptide' and “protein’ as used interchangeably maceutically acceptable diluent, carrier or vehicle. The herein, can encompass peptides and oligopeptides. Where specifications for the novel unit dosage forms depend on the US 9.248,120 B2 9 10 particular compound employed and the effect to be achieved, of interferon-gamma (IFNG). In contrast to IL12, which acts and the pharmacodynamics associated with each compound mainly on naive CD4(+) T cells, IL23 preferentially acts on in the host. memory CD4 (+) T cells. A“pharmaceutically acceptable excipient,” “pharmaceuti Dendritic cell. As used herein, the term refers to any mem cally acceptable diluent,” “pharmaceutically acceptable car ber of a diverse population of morphologically similar cell rier and pharmaceutically acceptable adjuvant’ means an types found in lymphoid or non-lymphoid tissues. Dendritic excipient, diluent, carrier, and adjuvant that are useful in cells are a class of “professional antigen presenting cells, preparing a pharmaceutical composition that are generally and have a high capacity for sensitizing MHC-restricted T safe, non-toxic and neither biologically nor otherwise unde cells. Dendritic cells may be recognized by function, or by sirable, and include an excipient, diluent, carrier, and adju 10 vant that are acceptable for veterinary use as well as human phenotype, particularly by cell Surface phenotype. These pharmaceutical use. A pharmaceutically acceptable excipi cells are characterized by their distinctive morphology, inter ent, diluent, carrier and adjuvant” as used in the specification mediate to high levels of surface MHC-class II expression and and claims includes both one and more than one such excipi ability to present antigen to T cells, particularly to naive T ent, diluent, carrier, and adjuvant. 15 cells (Steinman et al. (1991) Ann. Rev. Immunol. 9:271; As used herein, a “pharmaceutical composition' is meant incorporated herein by reference for its description of such to encompass a composition Suitable for administration to a cells). Subject, Such as a mammal, especially a human. In general a The vitamin A metabolite all-trans-retinoic acid (RA) is an “pharmaceutical composition' is sterile, and is usually free of essential signaling molecule in embryonic development and contaminants that are capable of eliciting an undesirable throughout life; a potent regulator of cell differentiation, pro response within the Subject (e.g., the compound(s) in the liferation, and apoptosis in various cell types. RA acts pharmaceutical composition is pharmaceutical grade). Phar through specific RA nuclear receptors (RARC, B, and Y) and maceutical compositions can be designed for administration their heterodimeric counterparts, the retinoid-X-receptors (C. to subjects or patients in need thereof via a number of differ B, and Y) to positively or negatively regulate expression of RA ent routes of administration including oral, buccal, rectal, 25 target genes by binding to their respective response elements. parenteral, intraperitoneal, intradermal, intracheal and the Vitamin A deficiency has been linked to increased suscepti like. bility to carcinogenesis in animal models. Although essen T helper 17 cells (Th17) are a subset of T helper cells, tially all cell types express nuclear retinoic acid receptors, characterized by their production of interleukin 17 (IL-17). cellular responsiveness is determined by RA bioavailability They are considered developmentally distinct from Th1 and 30 regulated by the coordinated balance between vitamin A Th2 cells and excessive amounts of the cell are thought to play nutritional status and RA biosynthesis and catabolism. The a key role in autoimmune disease. In humans, a combination RA-metabolizing cytochrome P450s CYP26A1. B1, and C1 of TGF-B, IL-1B and IL-23 induces Th17 differentiation from convert RA into rapidly excreted oxoderivatives (4-OHRA, naive T cells. Both interferon gamma (IFNY) and IL-4, the 4-oxo RA, 18-OHRA), while retinaldehyde dehydrogenase main stimulators of Th1 and Th2 differentiation respectively, 35 generates RA. Inhibitors of the CYP26 gene family are of negatively regulate Th17 differentiation. interest for use in the methods of the invention, including T helper 1 cells (Th1). Proliferating helper T cells that without limitation CYP26A1. develop into effector T cells differentiate into two major CYP26A1 is a member of the cytochrome P450 superfam subtypes of cells known as Th1 and Th2 cells. Th1 cells ily of enzymes. The cytochrome P450 proteins are monooxy primarily produce IFN-Y and TNF-B cytokines. IFN-y 40 genases. This endoplasmic reticulum protein acts on retin increases the production of interleukin-12 by dendritic cells oids, including all-trans-retinoic acid (RA), with both and macrophages, and via positive feedback, IL-12 stimulates 4-hydroxylation and 18-hydroxylation activities. Two alter the production of IFN-Y in helper T cells, thereby promoting natively spliced transcript variants of this gene, which encode the Th1 profile. IFN-Y also inhibits the production of cytok the distinct isoforms, have been reported. ines such as IL-4. Conditions that polarize to the TH1 type 45 Inhibitors of CYP26A1 can increase local concentrations include antigen presenting cells and IL-12. of RA. For example, an orally administered inhibitor, particu Interleukin-17 (IL-17) refers to a group of cytokines called larly a formulation that provides for enteric delivery, can raise the IL-17 family. IL-17 shows high homology to viral IL-17 the RA concentration in intestinal tissues. Inhibitors of encoded by an open reading frame of the T lymphotropic CYP26A1 are known in the art, and include, without limita rhadinovirus Herpesvirus saimiri. To elicit its functions, 50 tion, talarozole, ketoconazole; liarozole; IS (R*.R*) N IL-17 binds to a type I cell surface receptor called IL-17R of 4-2-(dimethylamino)-1-(1H-imidazole-1-yl)propyl-phe which there are at least three variants IL17RA, IL17RB, and nyl 2-benzothiazolamine (R116010); (R) N-4-2-ethyl-1- IL17RC. Members of the IL-17 family include IL-17B, (1H-1,2,4-triazol-1-yl)butylphenyl-2-benzothiazolamine IL-17C, IL-17D, IL-17E (also called IL-25), and IL-17F. All (R115866); the retinoic acid receptor (RAR)Y agonist members of the IL-17 family have a similar proteinstructure, 55 CD1530; the pan-RAR agonist 4-(E)-2-(5,6,7,8-tetrahydro with four highly conserved cysteine residues critical to their 5,5,8,8-tetramethyl-2-naphthalenyl)-1-propenylbenzoic 3-dimensional shape, although with no sequence similarity to acid; peroxisome proliferator-activated receptor ligands any other known cytokines. Numerous immune regulatory rosiglitaZone and pioglitaZone; etc. functions have been reported for the IL-17 family. Aldehyde dehydrogenase 1 (retinal dehydrogenase, IL-23 alpha subunit is a protein encoded by the IL23A gene 60 RALDH1) is a liver cytosolic isoform of acetaldehyde dehy (see Oppmann et al. (2001) Immunity 13 (5): 715-25). This drogenase. Retinaldehyde is generated by ADH1 from ret gene encodes the p19 subunit of the heterodimeric cytokine inol, and its concentration is determined in large part by its interleukin 23 (IL23). IL23 is composed of this protein and subsequent catabolism by RALDH1 to retinoic acid, see the p40 subunit of interleukin 12. The receptor of IL23 is Hempel et al. (1984) Europ. J. Biochem. 141: 21-35; Hsu et formed by the beta 1 subunit of IL12 (IL12RB1) and an IL23 65 al. (1985) Proc. Nat. Acad. Sci. 82: 3771-3775. Agonists of specific subunit, IL23R. Both IL23 and IL12 can activate the RALDH1, or other agents that increase activity of RALDH1 transcription activator STAT4, and stimulate the production are of interest for the methods of the invention. US 9.248,120 B2 11 12 Retinol dehydrogenase 10 (RDH10) generates all-trans (especially fissures and fistulas), which is sometimes the most retinal from all-trans retinol and may plan an important role in prominent or even initial complaint. In children, extraintesti the photic visual cycle. The first oxidative step of vitamin A nal manifestations frequently predominate over GI symp metabolism is catalyzed in large part by RDH10 and is critical toms; arthritis, fever of unknown origin, anemia, or growth for spatiotemporal synthesis of retinoic acid, Sandell et al. retardation may be a presenting symptom, whereas abdomi (2007) Genes Dev. 21: 1113-1124; Wu et al. (2002) Invest. nal pain or diarrhea may be absent. Ophthal. Vis. Sci. 43: 3365-3372. Agonists of RDH10, or Established Crohn's disease is rarely cured but is charac other agents that increase activity of RDH10 are of interest for terized by intermittent exacerbations and remissions. Some the methods of the invention. patients Suffer severe disease with frequent, debilitating peri The term colorectal cancer includes all cancers of the colon 10 ods of pain. However, with judicious medical therapy and, and/or rectum, but particularly adenocarcinoma of the colon where appropriate, Surgical therapy, most patients function (e.g., mucinous (colloid) adenocarcinoma or signet ring well and adapt successfully. Disease-related mortality is very adenocarcinoma). Other types of colorectal cancer included low. GI cancer, including cancer of the colon and Small bowel, by the term include the following varieties of colon cancer: is the leading cause of excess Crohn's disease-related mor neuroendocrine, lymphoma, melanoma, squamous cell, sar 15 tality. coma and carcinoid. The term colorectal cancer also includes Irritable bowel syndrome consists of recurring upper and all stages of colorectal cancer, for example, under the Modi lower GI symptoms, including variable degrees of abdominal fied Duke Staging System or TNM system (Tumor, Node, pain, constipation or diarrhea, and abdominal bloating. Diag Metastasis). The stages associated with these systems are nosis is clinical. Treatment is generally symptomatic, consist well known by practitioners of ordinary skill in the art. ing of dietary management and drugs, including anticholin In the methods of the invention, the agent(s) may be admin ergics and agents active at Serotonin receptors. There are no istered to a Subject to treat an inflammatory condition, which consistent motility abnormalities. Some patients have an inflammation may predispose to colorectal cancer, and may abnormal gastro-colonic reflex, with delayed, prolonged include individuals having familial adenomatous polyposis colonic activity. There may be reduced gastric emptying or (FAP), hereditary nonpolyposis colon cancer (HNPCC) (i.e., 25 disordered jejunal motility. Some patients have no demon Lynch I Syndrome or Lynch II Syndrome), inflammatory strable abnormalities, and in those that do, the abnormalities bowel disease, such as chronic ulcerative colitis (UC) or may not correlate with symptoms. Small-bowel transit varies: Crohn's disease, other family cancer syndromes (e.g., Peutz Sometimes the proximal Small bowel appears to be hyperre Jegher Syndromem and Familial Juvenile Polyposis), active to food or parasympathomimetic drugs. Intraluminal adenomatous polyps (e.g., sessile (flat with a broad base and 30 pressure studies of the sigmoid show that functional consti no stalk); tubular (composed of tubular glands extending pation can occur with hyperreactive haustral segmentation downward from the outer surface of the polyp); villous (com (ie, increased frequency and amplitude of contractions). In posed of fingerlike epithelial projections extending outward contrast, diarrhea is associated with diminished motor func from the surface of the bowel mucosa); pedunculated (at tion. Thus strong contractions can, at times, accelerate or tached by a narrow base and a long stalk), and sporadic forms 35 delay transit. of colon cancer. Diagnosis is based on characteristic bowel patterns, time Familial adenomatous polyposis (FAP) is an inherited con and character of pain, and exclusion of other disease pro dition in which numerous polyps form mainly in the epithe cesses through physical examination and routine diagnostic lium of the large intestine. In general, while these polyps start tests. Diagnostic testing should be more intensive when “red out benign, malignant transformation into colon cancer 40 flags' are present: older age, weight loss, rectal bleeding, occurs when not treated. Familial juvenile polyposis (FJP) is vomiting. Proctosigmoidoscopy with a flexible fiberoptic an autosomal dominant condition characterized by multiple instrument should be performed. Introduction of the sigmoi juvenile polyps of the gastrointestinal (GI) tract. Kindreds doscope and air insufflation frequently trigger bowel spasm have been described in which there is involvement of the and pain. The mucosal and vascular patterns in IBS usually colon only, the upper GI tract or both upper and lower GI 45 appear normal. Colonoscopy is preferred for patients >40 tracts. FJP is a hamartomatous polyposis syndrome. with a change in bowel habits, particularly those with no Adenomatous polyps (adenomas) of the colon and rectum previous IBS symptoms, to exclude colonic polyps and may be benign (noncancerous) growths that may be precursor tumors. In patients with chronic diarrhea, particularly older lesions to colorectal cancer. In general, polyps greater than women, mucosal biopsy can rule out possible microscopic one centimeter in diameter are associated with a greater risk 50 colitis. of cancer. If polyps are not removed, they typically continue to grow and can become cancerous. METHODS OF THE INVENTION Crohn's Disease (Regional Enteritis; Granulomatous Ile itis or Ileocolitis) is a chronic transmural inflammatory dis Methods are provided for reducing intestinal inflamma ease that usually affects the distal ileum and colon but may 55 tion, particularly chronic inflammation, and tumor growth occur in any part of the GI tract. Symptoms include diarrhea precipitated by intestinal inflammation. In the methods of the and abdominal pain. Abscesses, internal and external fistulas, invention, an effective dose of an agent is provided to the and bowel obstruction may arise. Extraintestinal symptoms, individual, where the agent increases local concentration of particularly arthritis, may occur. Diagnosis is by colonoscopy retinoic acid (RA) in the intestine through modifying enzy and barium contrast studies. The most common initial pre 60 matic pathways involved in RA metabolism. In particular, an sentation is chronic diarrhea with abdominal pain, fever, inhibitor of a CYP26, enzyme, for example CYP26A1, may anorexia, and weight loss. The abdomen is tender, and a mass be administered in a dose effective to neutralize an inflam or fullness may be palpable. Gross rectal bleeding is unusual matory environment and/or maintain the tolerogenic func except in isolated colonic disease, which may manifest simi tions of intestinal dendritic cells that maintain intestinal tol larly to ulcerative colitis. Some patients present with an acute 65 erance by inducing Treg formation. Alternatively, an agent abdomen that simulates acute appendicitis or intestinal that increases activity of retinaldehyde dehydrogenase or ret obstruction. About 33% of patients have perianal disease inoldehydrogenase may be administered in a dose effective to US 9.248,120 B2 13 14 maintain the tolerogenic functions of dendritic cells. The Parenteral Medications Dekker, N.Y.; Lieberman, et al., appropriate dose may be determined by evaluating the effect (eds.) (1990) Pharmaceutical Dosage Forms: Tablets Dekker, of the agent on functions of dendritic cells, or by overall N.Y.; and Lieberman, et al., (eds.) (1990), Pharmaceutical analysis of intestinal inflammation. The methods of the inven Dosage Forms: Disperse Systems Dekker, N.Y. tion exclude administration of retinoic acid directly. The A pharmaceutical composition can be prepared using con administration of inhibitors of CYP26A1, including without ventional pharmaceutically acceptable excipients and addi limitation liarozole and talarozole, are of particular interest. tives and conventional techniques. Such pharmaceutically The term “therapeutically effective amount’ or “therapeu acceptable excipients and additives include non-toxic com tically effective dosage' may mean that amount or dosage of patible fillers, binders, disintegrants, buffers, preservatives, an agent or combination thereof of the invention or compo 10 anti-oxidants, lubricants, flavorings, thickeners, coloring sition thereofthat will elicit a biological or medical response agents, emulsifiers and the like. All routes of administration ofa tissue, system, patient, Subject or host that is being sought are contemplated including, but not limited to, parenteral by the administrator (such as a researcher, doctor or veteri (e.g., Subcutaneous, intravenous, intraperitoneal, intramuscu narian) which includes any measurable alleviation of the lar, topical, intraperitoneal, inhalation, intra-cranial) and non signs, symptoms and/or clinical indicia of intestinal inflam 15 parenteral (e.g., oral, transdermal, intranasal, intraocular, mation, including colorectal cancer (e.g., tumor growth and/ Sublingual, rectal and topical). or metastasis) including the prevention, slowing or halting of Oral administration is of interest, including enteric formu progression of the inflammation to any degree whatsoever. lations, which may include acid stable agents that maintain Dosage regimens may be adjusted to provide the optimum activity undergastrointestinal conditions, enteric coatings of desired response (e.g., a therapeutic response). For example, pills, and the like, where there is a significant activity of the a single dose may be administered or several divided doses agent in intestinal tissues. may be administered over time or the dose may be propor Injectables can be prepared in conventional forms, eitheras tionally reduced or increased as indicated by the exigencies or liquid solutions or Suspensions, solid forms suitable for solu the particular circumstances or requirements of the therapeu tion or Suspension in liquid prior to injection, or as emulsions. tic situation. For example, dosage may be determined or 25 The injectables, Solutions and emulsions can also contain one adjusted, by a practitioner of ordinary skill in the art (e.g., or more excipients. Excipients include, for example, water, physician or veterinarian) according to the patient’s age, saline, dextrose, glycerol or ethanol. In addition, if desired, weight, height, past medical history, present medications and the pharmaceutical compositions to be administered may also the potential for cross-reaction, allergies, sensitivities and contain minor amounts of non-toxic auxiliary Substances adverse side-effects. For example, the physician or veterinar 30 Such as wetting or emulsifying agents, pH buffering agents, ian could start doses of the agent at levels lower than that stabilizers, solubility enhancers, and other Such agents, such required in order to achieve the desired therapeutic effect and as for example, sodium acetate, sorbitan monolaurate, trietha gradually increase the dosage until the desired effect is nolamine oleate and cyclodextrins. achieved. The effectiveness of a given dose or treatment regi In an embodiment of the invention, pharmaceutically men of an agent of the invention can be determined, for 35 acceptable carriers used in parenteral preparations include example, by determining the concentration of pro-inflamma aqueous vehicles, nonaqueous vehicles, antimicrobial agents, tory cytokines such as IL-23; whether a tumor being treated in isotonic agents, buffers, antioxidants, local anesthetics, Sus the Subject shrinks or ceases to grow. The size and progress of pending and dispersing agents, emulsifying agents, seques a tumor can be easily determined, for example, by X-ray, tering or chelating agents and other pharmaceutically accept magnetic resonance imaging (MRI) or visually in a Surgical 40 able Substances. procedure. In general, tumor size and proliferation can be Examples of aqueous vehicles include Sodium chloride measured by use of a thymidine PET scan (see e.g., Wells et injection, Ringers Injection, isotonic dextrose Injection, Ster al., Clin. Oncol. 8: 7-14 (1996)). Generally, the thymidine ile water injection, dextrose and lactated Ringers Injection. PET scan includes the injection of a radioactive tracer, such as Nonacqueous parenteral vehicles include fixed oils of veg 2-'C-thymidine, followed by a PET scan of the patient’s 45 etable origin, cottonseed oil, corn oil, Sesame oil and peanut body (Vander Borght et al., Gastroenterology 101: 794-799, oil. Antimicrobial agents in bacteriostatic or fungistatic con 1991; Vander Borght et al., J. Radiat. Appl. Instrum. Part A, centrations may be added to parenteral preparations packaged 42: 103-104 (1991)). Other tracers that can be used include in multiple-dose containers which include phenols or cresols, F-FDG (18-fluorodeoxyglucose), ''IIIUdR (5-II mercurials, benzyl alcohol, chlorobutanol, methyl and propyl iodo-2'-deoxyuridine), ''BrBrdUrd (Bromodeoxyuridine), 50 p-hydroxybenzoic acid esters, thimerosal, benzalkonium IFFLT (3'-deoxy-3' fluorothymidine) or 'CIFMAU (2'- chloride and benzethonium chloride. Isotonic agents include fluoro-5-methyl-1-?3-D-arabinofuranosyluracil). sodium chloride and dextrose. Buffers include phosphate and Methods for treating or preventing intestinal inflammation, citrate. Antioxidants include sodium bisulfate. Local anes including inflammation leading to colorectal cancer by thetics include procaine hydrochloride. Suspending and dis administering a pharmaceutical composition comprising an 55 persing agents include Sodium carboxymethylcellulose, agent that increases RA levels by altering enzymatic activity hydroxypropyl methylcellulose and polyvinylpyrrolidone. involved in RA metabolism, in association with a pharmaceu Emulsifying agents include Polysorbate 80 (TWEEN-80). A tically acceptable carrier are also within the scope of the sequestering or chelating agent of metal ions includes EDTA present invention (e.g., in a single composition or separately (ethylenediaminetetraacetic acid) or EGTA (ethylene glycol in a kit) as are combinations and compositions including Such 60 tetraacetic acid). Pharmaceutical carriers may also include pharmaceutical compositions. The pharmaceutical composi ethyl alcohol, polyethylene glycol and propylene glycol for tions may be prepared by any methods well known in the art water miscible vehicles; and sodium hydroxide, hydrochloric of pharmacy; see, e.g., Gilman, et al., (eds.) (1990), The acid, citric acid or lactic acid for pH adjustment. Pharmacological Bases of Therapeutics, 8th Ed., Pergamon In an embodiment of the invention, preparations for Press; A. Gennaro (ed.), Remington’s Pharmaceutical Sci 65 parenteral administration can include sterile Solutions ready ences, 18th Edition, (1990), Mack Publishing Co., Easton, for injection, sterile dry soluble products, such as lyophilized Pa.; Avis, et al., (eds.) (1993) Pharmaceutical Dosage Forms: powders, ready to be combined with a solventjust prior to use, US 9.248,120 B2 15 16 including hypodermic tablets, sterile Suspensions ready for yet to be examined in models such as APC". The remark injection, sterile dry insoluble products ready to be combined able capacity of these cells to orchestrate distinct immune with a vehicle just prior to use and sterile emulsions. The responses is aided by a panoply of environmental cues, which Solutions may be either aqueous or nonaqueous. condition the cells to adopt specific phenotypes in different The concentration of the agent of the invention, which is settings. DCs in gut-associated lymphoid tissue are of par optionally in association with a further chemotherapeutic ticular interest because they maintain tolerance to commensal agent, can be adjusted so that a dose provides an effective flora as well as mount protective inflammatory responses in amount to produce the desired pharmacological effect. As the face of pathogen incursion. discussed herein, the exact dose depends, in part, on the age, The experiments described in this report indicate that small weight and condition of the patient or animal as is known in 10 intestine LPDCs (SI-LPDCs) play a critical role in the inflam the art. matory process that underlies tumor progression in the Implantation of a slow-release or Sustained-release system, APC' mouse. Whereas SI-LPDCs in healthy control mice Such that a constant level of dosage is maintained, is also induce the formation of T. APC" SI-LPDCs are repro contemplated herein. Briefly, an active agent is dispersed in a grammed to induce Th17 formation. A marked reduction in Solid inner matrix, e.g., polymethylmethacrylate, polybutyl 15 the intestine of the vitamin A metabolite, retinoic acid (RA), methacrylate, plasticized or unplasticized polyvinylchloride, which under homeostatic conditions maintains the tolero plasticized nylon, plasticized polyethyleneterephthalate, genic functions of LPDCs, explains the reprogramming of natural rubber, polyisoprene, polyisobutylene, polybutadi these cells. Since we find identical defects in RA metabolism ene, polyethylene, ethylene-vinylacetate copolymers, sili in FAP tissue, the same mechanism that drives inflammation cone rubbers, polydimethylsiloxanes, silicone carbonate in APC" mice contributes to tumor formation in FAP. copolymers, hydrophilic polymers such as hydrogels of Inflammatory DCs accumulate in the SI-LP of APC'" esters of acrylic and methacrylic acid, collagen, cross-linked mice. We initially compared the frequency of DCs in various polyvinylalcohol and cross-linked partially hydrolyzed poly tissues in APC' mice and their WT littermate controls. vinyl acetate, that is Surrounded by an outer polymeric mem DCs were identified as EpCAMCD45"Lin MHCII" brane, e.g., polyethylene, polypropylene, ethylene/propylene 25 CD11c and analyzed by flow cytometry at 10, 14 and 18 copolymers, ethylene/ethyl acrylate copolymers, ethylene? weeks of age, time points that correspond to minimal (<30 vinylacetate copolymers, silicone rubbers, polydimethyl polyps, <2 mm diameter), intermediate (30-60 polyps, 0.5-4 siloxanes, neoprene rubber, chlorinated polyethylene, poly mm diameter) and late stage (>60 polyps, 1-6 mm diameter) vinylchloride, vinylchloride copolymers with vinyl acetate, disease, respectively. As shown in FIG. 1a-b, DCs accumu vinylidene chloride, ethylene and propylene, ionomer poly 30 lated in the SI-LP as disease progressed. In the steady state, ethylene terephthalate, butyl rubber epichlorohydrin rubbers, DCs in the gut environment are comprised of three pheno ethylene/vinyl alcohol copolymer, ethylene/vinyl acetate?vi typically distinct populations CD103"CD11b, CD103" nyl alcohol terpolymer, or ethylene/vinyloxyethanol copoly CD11b and CD103CD11b DCs. CD103*CD11b DCs mer, that is insoluble in body fluids. The antibody or fragment are enriched in the Peyer's patches, whereas CD103"CD11b" diffuses through the outer polymeric membrane in a release 35 and CD103CD11b DCs are found mainly in the LP'. rate controlling step. The percentage of active agent con Although there were some differences in the frequency of tained in Such parenteral compositions is highly dependent on splenic DC subsets between APC" and WT control mice, the specific nature thereof, as well as the activity of the anti no significant differences in the percentages of the three key body or antigen-binding fragment, which is optionally in subsets were observed in the mesenteric lymph nodes (mLN), association with a further chemotherapeutic agent, and the 40 Peyer's Patches (PP) and most importantly, the SI-LP (FIG. needs of the subject. 1c). These analyses were performed at intermediate stage, as Agents set forth herein can be formulated into a Sustained the APC' mice lose their Peyer's Patches by late stage release formulation including liposomal formulations such as disease, accompanying other developmental abnormalities unilamellar vesicular (ULV) and multilamellar vesicular Such as early thymic involution, splenomegaly and lym (MLV) liposomes. 45 phodepletion. We considered the possibility that the DCs accumulating in Example 1 the SI-LP of APC' mice may be responsible for generat ing the altered CD4 T cell response in the gut demonstrated The present invention is intended to exemplify the present in previous studies. No significant changes in the expression invention and not to be a limitation thereof. Methods and 50 of costimulatory molecules (CD40, CD80, CD83, CD86 or compositions disclosed below fall within the scope of the PDL1) were seen on these cells (FIG. 1d). Nonetheless, when present invention. stimulated with a panel of TLR agonists, purified SI-LPDCs In the majority of sporadic colorectal cancers and in FAP, from APC" mice, especially those with late-stage disease, tumorigenesis is initiated from mutations that arise in the secreted much larger amounts of the pro-inflammatory cytok APC tumor suppressor gene. In the APC" murine model, 55 ines TNFO, IL-6 and IL-12p40 compared to SI-LPDCs from numerous adenomas develop in the intestines, closely resem WT mice (FIG. 1e). bling FAP APC'" homozygotes die in utero, and APC" SI-LPDCs are impaired in their ability to induce APC" heterozygotes spontaneously lose their wildtype Tr and instead promote Th17 formation. To address allele during puberty and start developing polyps by week 10. whether APC'* SI-LPDCs are able to induce Foxp3"T Reg's Although these mice do not develop invasive metastases, they 60 de novo, we cultured CFSE-labeled naive CD4-CD63f. eventually Succumb to a fatal anemia through excessive intes Foxp3 T cells from OT-IITCR transgenic mice with purified tinal bleeding. DCs in the presence of TGFB and the ovalbumino pep Prior studies in APC' mice show that chronic intestinal tide. After 5 days of co-culture, the frequency of Foxp3"CD4" inflammation precipitates as well as propagates tumor growth T cells was determined. The results show that splenic DCs and that Th17 cells are the responsible immune effector cells. 65 from both genotypes induced Foxp3 cells weakly, while Since chronic inflammation in the intestine predisposes to mLN/PP DCs were more potent inducers of Foxp3" cells, colorectal cancer, it is Surprising that the role of local DCS has consistent with previous studies. Interestingly, APC" SI US 9.248,120 B2 17 18 LPDCs were impaired in their capacity to induce Foxp3 cells levels of Raldh1a1 and Raldh1a2 compared to WTSI-LPDCs compared to WT controls (FIG. 2a, FIG. 7a). This impair (FIG. 3a). However, as the disease progressed in APC'" ment was observed beginning at intermediate stage and mice, expression of these enzymes, especially Raldhlal, became more apparent at late stage (FIG. 7c). Similar results declined markedly. were obtained across a 25 fold range of peptide concentration 5 Recent studies have shown that RA in the gut induces (FIG.2b, FIG.7b). Raldh expression. Since IECs constitutively express these When we measured the levels of key immunomodulatory retinol-metabolizing enzymes and can efficiently generate cytokines in the supernatants of these DC-T cell co-cultures, RA, we postulated that defective RA production in the IECs the results revealed a striking six-fold reduction in IL-10 might help explain the loss of Raldh expression observed in generated in APC" LPDC co-cultures compared to the 10 WT control cultures (FIG. 2e). Moreover, there was a con the APC' LPDCs. As shown in FIG. 3b, Raldh1a1 and comitant and similarly dramatic increase in IL-17A (FIG. 2.f), Raldh1a2 expression in sorted CD45EpCAM" IECs did not consistent with the documented role of Th17 cells inadenoma change significantly through early and intermediate stage development. Intracellular staining of the T cells in the disease. At late stage, however, APC" IECs exhibited APC" co-cultures confirmed the presence of IL-17A pro- 15 approximately five- and two-fold reduction in the expression ducing cells and a reduction of IL-10 producing cells. of Raldh1a1 and Raldh1a2, respectively, compared to WT As CD103" SI-LPDCs are the main cells responsible for IECs. Of note, compared to WT IEC, ADH class I and II generating immune tolerance in the intestinal environment, expression in APC" IEC increased at intermediate stage we sorted SI-LPDCs into CD103 and CD103 Subsets to and decreased at subsequent late stage (FIG. 9). Taken determine which subset accounted for the observed impair together, these data show that both APC'". SI-LPDCs and ment in Foxp3 induction. APC'* CD103* SI-LPDCs were IECs lose expression of the critical Raldh1a1 and Raldh1a2 four-fold less able to induce Foxp3T cells compared to their enzymes by late stage disease, and have a reduced RA-pro WT counterparts (FIG.2c). In contrast, CD103. SI-LPDCs duction capacity compared to their WT counterparts. from both genotypes were equally poor in inducing Foxp3 To confirm that the changes observed at the transcript level expression (FIG. 2c), consistent with previous studies. In 25 of these enzymes correlated with a loss of RA in the local addition, co-cultures of APC'* CD103* SI-LPDCs and T tumor milieu, we utilized tandem mass spectrometry to quan cells, but not CD103 LPDC-T cell co-cultures, contained titatively profile endogenous retinoids in late stage APC'" large amounts of IL-17A. These results show that the CD103" duodenum, jejunum, ileum, colon and eye. As shown in FIG. subset of SI-LPDCs in APC" mice is not only responsible 5i, RA levels in APC" tissues were diminished compared for defective induction of T, but also for the generation of 30 to WT controls, with the difference reaching statistical sig Th17 cells. nificance in the ileum, where there is the highest frequency of Retinoic acid (RA) reverses the inflammatory phenotype of polyps, and in the colon. Although retinol was reduced in the APC'* LPDCs. The unique capacity of the CD103* SI APC" ileum, tissues from both genotypes had comparable LPDC subset to store and metabolize vitamin A into RA amounts retinyl esters, indicating that the deficit in RA is not explains the specialized role of these cells in generating and 35 due to a lack of substrate (FIG. 10). maintaining intestinal tolerance. We hypothesized that the Previous studies have demonstrated that the APC protein cytokine profile of the pro-inflammatory APC'". SI-LPDCs can directly bind to Ctbp 1 in both Drosophila melanogaster could be modulated by changes in the local RA concentration. and human cell lines. APC-mutant Zebrafish were shown to To address this possibility we added RA or the RA receptor express abnormally high levels of Ctbp 1, which correlated antagonist, LE540, to the LPDC-T cell co-cultures and 40 with low levels of the intestinal retinol dehydrogenase Rdh1 I assessed the induction of Foxp3' T cells. LE540 abrogated and intestinal differentiation defects. Moreover, reintroduc Foxp3 induction in both WT and APC'* SI-LPDC co tion of the APC protein into human colon carcinoma cell lines cultures (FIG. 2d), whereas RA enhanced Foxp3 induction in led to a proteasome-dependent destruction of Ctbp1, con the APC'". SI-LPDC co-cultures to the levels seen in WT comitant with increased expression of the retinol dehydroge co-cultures (FIG. 2d). Addition of RA also strongly inhibited 45 nase, DHRS9. To determine if the loss of RA we observed in IL17A production in the APC" co-cultures. Since Th17 the APC" gut may be due to upregulation of Ctbp1, we differentiation requires IL-6 in conjunction with TGFB, and assessed Ctbp1 transcript levels in SI-LPDCs and IECs. A since no exogenous IL-6 was added to our cultures, we pos significant elevation of Ctbp1 was seen in APC'". IECs tulated that the APC' LPDC co-culture supernatants may compared to WT IECS beginning at intermediate stage dis contain IL-6. This was confirmed (FIG.2g). RA also potently 50 ease (FIG. 3c). Surprisingly, there was a parallel accumula inhibited IL-10 production in both WT and APC" co tion of Ctbp1 in the APC' SI-LPDCs as well. These cultures, consistent with a previous study. findings Suggest that along with loss of Raldh expression, Loss of RA is linked to both reduced expression of retinal elevated levels in Ctbp1 in both cell types may suppress dehyde dehydrogenases (Raldh) and defective regulation of intestinal retinol dehydrogenase expression, thereby contrib the transcriptional co-repressor C-terminal binding protein 1 55 uting to the loss of RA observed in the tumor milieu. (Ctbp1). VitaminA, or retinol, when absorbed in the intestine, FAPadenomas exhibit the same inflammatory changes and is either converted to a storage form or metabolized to RA. In abnormalities in RA metabolism as seen in APC" mice. the latter, retinol is catalyzed to retinal by two families of To assess whether our findings in the APC" model are enzymes, the alcohol dehydrogenases (ADHS) and the short predictive of the human condition, we used immunohis chain dehydrogenase reductases (RDH), and Subsequently 60 tochemistry and immunofluorescence to analyze the colonic oxidized to bioactive RA by the Raldh enzymes. To ascertain polyps of FAP patients. For comparison, we analyzed biop whether APC" LPDCs are able to produce RA, we used sies of normal colon as well as sessile serrated polyps (SSP), RT-qPCR to measure the expression of several key Raldh the latter serving as a source of non-APC mutated adenomas. enzymes in these cells. In line with previous studies, Raldh IL17A expression was strikingly elevated in FAPadenomas expression in the SI-LP of both WT and APC" mice was 65 compared to adjacent uninvolved colon, normal colon or SSP consistently higher than control splenic DCs (FIG. 3b). At (FIG. 4a). Staining of the same tissues with an isotype control early stage, APC" SI-LPDCs expressed equal or higher antibody is shown in FIG. 11. These findings indicate that the US 9.248,120 B2 19 20 inflammatory environment in FAP polyps is similar to that deteriorated to the same extent as untreated controls (FIG. seen in the APC' mouse model. 6a-d). To confirm that Liarozole mediated its effects by We next examined the tissues for expression of key vitamin increasing local RA concentration in the intestine, we mea A metabolizing enzymes and the proteins that modulate them. sured retinoid levels in the treated mice. As shown in FIG. 6e, Consistent with published reports, cytoplasmic B-catenin was RA levels returned to normal in Liarozole treated mice, espe expressed at higher levels in FAP adenomas compared to cially in the ilium. adjacent uninvolved tissue and to SSP. Interestingly, however, To assess whether the Liarozole mediated increase in intes B-catenin was not expressed by cells expressing dendritic tinal RA influenced immune outcome by re-reprogramming cell-specific Intercellular Adhesion Molecule-3-Grabbing the pro-inflammatory APC' SI-LPDCs, SI-LPDCs from Non-integrin (DC-SIGN) in the tissues tested (FIG. 4b). 10 Liarozole-treated mice were isolated and tested functionally. Ctbp1, the transcription factor that negatively regulates intes The results show that APC" SI-LPDCs from Liarozole tinal RDHs, was present at much higher concentration in FAP treated miceno longer produced Substantial amounts of pro IECs than in the IECs of normal or SSP (FIG.5c), consistent inflammatory cytokines when stimulated with TLRagonists with previous reports, Remarkably, Raldh1a1 and Raldhla2 (FIG. 6f), and instead of inducing Th17 formation, promoted expression was almost completely abolished in IECs of FAP 15 the formation of IL-10 secreting Foxp3+Tregs (FIG. 6g-h) at adenoma tissue and not SSP (FIG. 5d-e), validating our find levels similar to wildtype mice. Taken together, these findings ings in the APC" mouse. Finally, the RA catabolizing show that whereas exacerbating RA deficiency in the intes enzyme CYP26A1 was markedly upregulated in the epithelia tine accelerates disease, restoring RA reverses the reprogram (FIG. 5f) as has been reported. Since FAPadenomas contain ming of SI-LPDCs, thereby preventing deleterious Th17 little RALDH activity (possibly due to excess Ctbp 1), but responses and ameliorating disease. excessive CYP26A1, the net result is likely a dearth of RA in The role of intestinal inflammation in the development of the local tumor milieu. adenomas in the APC" model of spontaneous neoplasia Polyposis is exacerbated by a vitamin A deficient (VAD) has been well documented. However, the few immunological diet and ameliorated by Liarozole, a CYP26A1 inhibitor. studies performed on APC" mice and related APC muta Mice fed a vitamin A-deficient diet have dramatically reduced 25 tion models have focused almost exclusively on altered T cell DC Raldh expression. Given the likelihood that diminished responses, with no attention directed at the underlying cause RA explains the pro-inflammatory profile of SI-LPDCs in of these changes. Our findings indicate that as disease APC" mice, we postulated that prolonged vitamin A-de progresses, there is an accumulation of pro-inflammatory ficiency would intensify Raldh loss, drive inflammation and DCs in the SI-LP that induce the formation of Th17 cells. This accelerate polyp development. To test this hypothesis, groups 30 stands in dramatic contrast to the SI-LPDCs of healthy mice, of WT and APC" mice were placedon a diet containing no which do not promote inflammation but instead maintain vitaminA for 10 weeks, beginning at 8-10 weeks of age, after immune tolerance through the generation of T. CD103+ which they were returned to a normal rodent chow diet. LPDCs, believed to be responsible for tolerance induction in Groups of mice treated with Celecoxib (a cyclooxygenase-2 the intestine, were present in greater numbers in APC'" inhibitor) and RosiglitaZone (a peroxisome proliferator-acti 35 than healthy control mice, but secreted pro-inflammatory vated receptor-Yagonist), were included as positive and nega cytokines and induced the formation of Th17 cells rather than tive controls, respectively. These compounds were added to T. The induction of Th17 cells by APC1/* SI-LPDCs “base' mouse chow which contained 4 IU vitamin Afg. Dis likely represents a critical control point in shaping the inflam ease development and severity were monitored on the basis of matory milieu that drives tumor growth, and explains the Survival, changes in body weight, hematocrit and polyp count 40 predominance of IL-17 in the inflamed APC' intestine. at the point of euthanasia. Since SI-LPDCs play such an important role in tumor WT mice on each diet gained weight comparably (FIG.12) promoting inflammation, identifying the mechanism that and did not develop polyps. APC" mice on the base diet induces their unusual phenotype in APC" mice is key to survived throughout the 10-week period of disease monitor understanding how the inflammatory cascade is triggered. We ing, but all succumbed by week 24 (FIG. 5a). Compared to 45 hypothesized that these cells could have undergone repro mice on the base diet APC" mice treated with Celecoxib gramming in response to one or more factors in the tumor had markedly prolonged survival, while mice that received microenvironment. Our efforts focused on RA because of the RosiglitaZone had reduced Survival consistent with previous well documented role of this molecule in maintaining a tol reports (FIG.5a). Interestingly, VAD diet-fed APC' mice erant state in the intestine. Addition of RA to the T induc had particularly aggressive disease and Succumbed even 50 tion cultures not only enhanced the capacity of APC'" more rapidly than mice on Rosiglitazone (FIG. 5c-d). The SI-LPDCs to induce Foxp3+ T cells but also prevented the body weights of APC' mice placed on base or VAD diets, induction of Th17 cells. Conversely, addition of the RAR or treated with RosiglitaZone, decreased comparably, while antagonist, LE540, resulted in further diminution of Foxp3 those treated with Celecoxib gained weight steadily (FIG. induction and augmented the production of IL17A by more 5b). 55 than five-fold. Since decreasing RA in the intestinal environment exacer The absolute amount of RA in the intestines of mice with bates disease in APC" mice, we wanted to test the hypoth APC mutations has not been reported previously, likely due to esis that increasing intestinal RA would ameliorate disease. the technical difficulty of carrying out such measurements. Oral administration of RA was not feasible, since it is known By preventing exposure of tissues to white light, which rap to stimulate further polyp growth in APC" mice due to the 60 idly degrades retinoids, and utilizing mass spectroscopy to induction of CYP26A1 and consequent loss of RA. Instead, measure RA, we could achieve accurate RA quantitation. Our we sought to increase intestinal RA by preventing its break results revealed highly significant reductions of RA in the down with a CYP26A1 inhibitor, Liarozole, which was added ileum and colon of APC' mice, but not in other sites. to the base diet of 8 week old mice for a period of 10 weeks, Although some RA remained in the intestines, the reductions as above. Remarkably, the Liarozole-treated mice exhibited a 65 seen are nonetheless noteworthy, as retinol bound to its spe striking reduction in the number of polyps as well as a sig cific retinol-binding protein (RBP) is strictly regulated and nificant increase in body weight, although their hematocrits maintained in plasma at about 2 LM despite daily fluctuations US 9.248,120 B2 21 22 in dietary intake of vitamin A. Only in situations of severe, Materials and Methods prolonged vitaminA-deficiency, where stores of retinyl esters Mice. Breeding pairs of APC't male and WT C57BL/6 in the liver are depleted, is there a drop in plasma concentra female mice were purchased from The Jackson Laboratory tion of retinol-RBP, and by association, RA. and bred on-site. OT-II TCR transgenic Rag mice were 5 purchased from Taconic. TNF^' mice were kindly pro The loss of RA in the tumor milieu appears to be due to both vided by Dr. Frank Jirik of the University of Calgary, Canada. diminished synthesis and excessive breakdown of this mol All mice were housed in an American Association for the ecule. We found that the critical Raldh enzymes that control Accreditation of Laboratory Animal Care-accredited animal RA production declineas disease progresses, and this is likely facility, maintained in pathogen-free conditions on a standard explained, in part, by the overexpression of Ctbp1, which rodent chow ad libitum unless otherwise stated. suppresses Rdh. Ctbp1 is normally degraded by APC, but 10 Isolation of DCs. Epithelial cells from the small intestine accumulates in the absence of functional APC. Our immun were washed in PBS with vigorous stirring and the remaining ofluorescence data confirmed a marked accumulation of intestinal pieces digested twice with Type VIII Collagenase Ctbp 1 in FAPIECs. Inactivation of the APC gene also results (Sigma-C2139) and DNase I (Sigma). Spleen and mesenteric in constitutive expression off-catenin, which upregulates the lymph nodes were digested with collagenase Type IV (Wor 15 thington, 200 U/ml). For purification of DCs, cells were major RA catabolic enzyme, CYP26A1. Indeed, the stained with monoclonal antibodies (Biolegend) against CYP26A1 transcript has been found to be upregulated in CD45.2, CD49b, CD3e, CD19, CD11c, MHC II, EpCAM whole tissue isolated from both APC' and FAPadenomas, and propidium iodide (PI), and sorted using a FACS Aria (BD sporadic colorectal carcinomas, and the intestine of APC Biosciences). In some cases, DCs were additionally sorted mutant Zebrafish embryos. Here we validate these observa using anti-CD103. tions, specifically identifying epithelial cells as one of per Flow Cytometric analysis. Isolated small intestine lamina haps several cell types in FAP colon displaying CYP26A1 propria (SI-LP) cells, splenocytes and mesenteric lymph node upregulation. Finally, prostaglandin E2 (PGE2), which has cells were resuspended in 1% BSA in PBS (FACS buffer). been reported to inhibit Raldh1a2 expression in DCs, may After Fc blockade with anti-FcyRIII/II (BD Biosciences), contribute to the loss of RA. It is well established that FAP 25 cells were stained with Live/Dead Blue (Invitrogen) or PI, and APC' epithelium exhibit constitutively high Cox2 and monoclonal antibodies (all Biolegend) against CD40, expression. In addition, we found that the Cox2 transcript is CD80, CD83, CD86, PDL1, Thy 1.2 and CD4. Intracellular overexpressed in late stage APC'". SI-LPDCs compared to Foxp3 and cytokines were stained using antibodies against their WT counterparts (FIG. 13). This finding is consistent Foxp3, IL 10, IL6, IL17A (all eBioscience) per manufactur with the possibility that Cox2-overexpression and abundance 30 er's instructions. Flow cytometric analysis was performed on a LSRII flow cytometer (BD Biosciences). of PGE2 in the FAP colon may lead to suppression of T cell differentiation assay. 2x10 sorted CD11e's Raldh1a2 in both DCs and epithelia. Taken together, our MHCII+ DCs were co-cultured with 1x10 MACS-enriched results point to at least 3 mechanisms that cooperate to Sup CD4+CD62L+Foxp3- naive T cells from the spleen and press the RA levels in the tumor milieu of APC" mice. 35 lymph nodes of OT-II TCR-transgenic mice, along with Moreover, these data indicate that the APC mutation under OVA so peptide (New England Peptide) and 10 ng/ml lying malignant transformation of intestinal epithelia is recombinant human TGF-31 (Peprotech). On day 5, cells directly linked to the immune defect driving tumor growth. were harvested and analyzed for intracellular Foxp3 or intra We Summarize our key findings and others in an illustration cellular cytokines. In some experiments, cells were restimu depicted in FIG. 6d. 40 lated on day 4 with 1 g/ml each of plate-bound anti-CD3 and Importantly, human FAPadenoma tissue exhibited several anti-CD28 (BDBiosciences), with or without Brefeldin A for of the same defects that we found in the APC' mouse 18 hr. Where indicated, 10 nM all-trans RA (Sigma), or 1 uM intestine. Not only was there a marked elevation in IL17 LE540 (Wako Chemicals) was added to culture wells. expression, signifying Th17 driven inflammation, but in addi ELISA. Purified SI-LPDCs were stimulated for 48 hr with tion we observed concomitant Raldh downregulation and 45 Toll-like receptor agonists—1 ug/ml Pam3Csk4, 10 ug/ml CYP26A1 upregulation in colonic epithelial cells. Defective Poly I:C, 10ug/ml LPS, 1 lug/ml flagellin, 10ug/ml R848, 10 RA production combined with enhanced RA breakdown ug/ml CpG 2336 (Invivogen). Supernatants from these would produce a deficit of similar magnitude to that seen in experiments were assayed for IL-6, TNFO, IL12-p40 and APC mice. those from were DC-T cell co-cultures were assayed for IL-6, To investigate the role of RA in disease development and 50 IL-10 and IL17A, performed according to manufacturers progression, we sought to alter RA concentration in the intes instructions (eBioscience). tine in Vivo, by removing or administering molecules in the Quantitation of gene expression using real-time PCR. Total diet that are known to affect RA production or breakdown. RNA from purified LPDCs, splenic DCs and epithelial cells Our results revealed that a diet deficient in Vitamin A exac was extracted using RNAeasy kits (Qiagen), and the DNase erbates disease, while the CYP26A1 inhibitor, Liarozole, 55 treated total RNA was reverse-transcribed using High-Capac ameliorates disease as indicated by a dramatic reduction in ity Reverse Transcription Kit (Applied Biosystems) accord the number of intestinal as well as steady weight gain. ing to manufacturers instructions. Gene expression of ADH Since increasing intestinal RA proved highly efficacious in class I-III, Raldh1a1-3, Ctbp1, and Cox2 was determined by APC" mice, doing so inpatients with APC-mutation asso quantitative PCR with Power SYBR Green PCR Master Mix ciated bowel disease may be initiated. Small molecule ago 60 (Applied Biosystems) per manufacturers instructions using a nists of Rdhs and Raldhs, or inhibitors of CYP26 such as 7900HT real-time PCR instrument (Applied Biosystems). Liarozole, are logical candidates. Indeed, Liarozole has been Ubiquitin levels were measured in a separate reaction and evaluated in clinical trials for diseases unrelated to colorectal used to normalize the data. cancer and is apparently well-tolerated. Maintaining Suffi Quantitation of tissue retinoids. Retinyl esters (RE), all cient RA in the intestinal environment could therefore, 65 trans retinol, and all-trans retinoic acid (RA) were extracted reverse inflammation and reduce tumor burden, as we from the duodenum, jejunum, ileum, colon and eye using observed in APC' mice. procedures to those described previously. RA was quantified US 9.248,120 B2 23 24 by LC/MS/MS with atmospheric pressure chemical ioniza AOM/DSS mice, corresponding to the very locations where tion. RE and retinol were quantified by HPLC. Tissues were inflammation and carcinoma was occurring respectively. harvested and retinoids handled under yellow light using only Taken together, an RA deficiency can occur in generalized glass laboratory equipment. Results were normalized to per settings of chronic intestinal inflammation and in colorectal gram tissue weight, or to control groups as fold-change. cancer, Suggesting that the beneficial effects of modulation of Histology. local intestinal RA levels extends to generalized chronic Immunohistochemistry. inflammation in the gut. Formalin-fixed, paraffin-embedded, 5 uM-thick, human As with many other hormones and vitamins that can be tissue sections were stained with the primary antibody rabbit dysregulated in disease, reinstating RA to the appropriate IL17A (Protein Tech Group (PTG) 13082-1-AP), and the 10 dynamic range may be key. RA has a short elimination half secondary rabbit-HRP polymer (Dako Envision). Antigen life in vivo and in cultured cells just 6-7 hours. Retinol bound retrieval was performed using Human Diva Decloaker citrate to its specific retinol-binding protein in plasma is strictly buffer (Biocare Medical). regulated and maintained at about 2 uM despite daily fluc Immunofluorescence. tuations from dietary intake of vitamin A. Only in situations Primary antibodies, all from PTG and rabbit anti-human 15 of severe vitamin A-deficiency where stores of retinyl esters unless otherwise noted, were mouse DC-SIGN (Dendritics in the liver are depleted, is there a drop in plasma concentra DDX0202), B-catenin (51067-2-AP), Ctbp 1 (Human Protein tion of retinol-RBP. Corresponding RA levels in plasma are in Atlas (HPA) HPAO18987), Raldh1a1 (15910-1-AP), the range of 5-10 nM, more than two orders of magnitude Raldh1a2 (HPAO10022), CYP26A1 (HPA C6498). Chicken lower than that of its substrate retinol, reinforcing the notion secondary antibodies include anti-rabbit Alexa647 and anti that retinol metabolism is tightly regulated and often mouse Alexa488 (Invitrogen). 11 normal colon, 8 FAP restricted to certain settings. Local RA concentrations in the adenoma, 2 FAPadenocarcinoma and 4 sessile serrated polyp APC" ileum and colon may be reduced to a point where patients were analyzed. Images were collected using a Leica tolerance is broken, but not to the point where compensatory DM2500 confocal laser Scanning microscope, and analyzed mechanisms that maintain tolerance in severe RA deficiency using the LASAF software. 25 starts to kick in; or to the point that Th17 cells cannot induce Drug Treatment. inflammation anymore. 1500 ppm of Celecoxib (Celebrex, Pfizer), 100 ppm of We measured RA levels in Liarozole-treated APC'" Rosiglitazone (Avandia, Glaxo Smith Kline), or 40 ppm of mice and observed a return to wildtype levels of RA in the LiaroZole (Tocris Pharmaceuticals) was incorporated into a illeum, so we validated that Liarozole did indeed increase base diet (4 IU/g of vitamin A) by Research Diets. A vitamin 30 local concentrations of RA to homeostatic levels, and not to A deficient (OIU/g) diet (VAD) was also studied. Hematocrits Supraphysiological levels. were measured every 2 weeks, while weights and DAI were Since decreasing RA in the intestinal environment exacer measured every week. At the point of euthanasia, polyps were bates disease in APC' mice, we tested the reciprocal enumerated and measured using a stereomicroscope at 10x hypothesis that increasing intestinal RA would ameliorate magnification, from the gastro-duodenual junction to the 35 disease. RA was administered intraperitoneally (i.p.) twice ceco-colic junction. weekly for 6 weeks to APCMin/+ mice, using the same pro Statistics. tocol that has been reported to attenuate ileitis in a mouse An unpaired student's t test (2-tailed) with a 95% confi model of Crohn's disease. Surprisingly, compared to con dence interval was performed in Prism (Graphpad) in all trols, this regimen did not improve disease outcome in terms experiments unless otherwise stated. Kaplan-Meier survival 40 of tumor frequency, body weight or hematocrit. Consistent curves were analyzed with the log-rank test. Differences of with these findings, SI-LPDCs isolated from APC" mice DAI in the drug treatment studies were analyzed using the that received RA i.p. retained their proinflammatory pheno Wilcoxon Rank-Sum test in R. Where needed, meantSEM type and ability to induce the formation of Th17 cells. A likely was represented on graphs. P<0.05=*; p<0.001=**: explanation for this apparent paradox is that RA i.p. did not p-0.0001=***. 45 increase RA levels in the ileum, due to persistent breakdown of RA from upregulated CYP26A1 in APC" tissue. Example 2 Indeed, direct RA supplementation via the diet has been shown to stimulate tumor formation in the APC" model. Drug Treatment of Inflammatory Bowel Disease Given these results, we decided to try an alternative strat 50 egy to increase intestinal RA by targeting the upregulated Inflammatory DCs that infiltrate the GALT during autoim CYP26A1 with Liarozole, an inhibitor of this enzyme. Com mune colitis appear to amplify Th17 responses that exacer pared to untreated and RA i.p.-treated mice, Liarozole treated bate intestinal pathology, similar to our observations in the mice exhibited a striking reduction in tumor number in the APC' model of intestinal cancer. To address whether the jejunum and ileum (FIG. 14a), as well as a Substantial loss of local RA as observed in our studies extended beyond 55 increase in body weight (FIG. 14b), and hematocrit (FIG. APC-associated GI cancers, we quantified endogenous tissue 14c). Importantly, Th17 cells were reduced to WT levels retinoids as before in TNF^' and azoxymethane/dextran (FIG. 14e), and SI-LPDCs from these mice failed to induce sodium-sulphate (AOM/DSS)-treated mice. A model of Th17 cells, instead promoting the formation of IL-10-secret Crohn's disease that exhibit chronic ileitis, TNF^' mice ing CD4+ T cells (FIG. 14f). Moreover, RA in the ileum of provide a model to examine whether there was a similar RA 60 Liarozole-treated mice was restored to WT levels (FIG. deficiency in cases of generalized chronic intestinal inflam 145d), likely explaining the therapeutic effect. Consistent mation. The AOM/DSS carcinogen and mucosal injury-in with this interpretation, treatment of APC" mice with duced model of colorectal carcinogenesis provided another Talarozole, a more potent and more specific CYP26A1 model of colorectal cancer, which is chemically-induced inhibitor than Liarozole 38, also ameliorated disease (FIG. instead of spontaneously-arising, like the APC'". Intrigu 65 15). ingly, compared to WT tissue, we observed a marked reduc Taken together, these findings indicate that whereas tion in RA in both the ileum and colon of TNF^' and increasing the RA deficit in the intestine exacerbates disease, US 9.248,120 B2 25 26 restoring RA to normal levels reverses the pro-inflammatory in APC" mice is responsible for the observed defect in phenotype of SI-LPDCs, attenuates Th17-driven inflamma TReg induction, and that both CD103 and CD103* SI-LP tion and ameliorates disease. DCs likely contribute to the Th17-skewed inflammation observed in these mice. Example 3 5 To directly assess the role of DCs in tumor progression, we generated bone marrow (BM) chimeras to selectively and Inflammatory DCs. Accumulate in the SI-LP of constitutively ablate DCs using BM donor cells in which APC" Mice and Promote Th17 Formation and Diphtheria Toxin A (DTA) is activated in CD11c expressing Tumor Growth cells. Chimeras generated with WT and APC' BM were 10 used for comparison. Depletion of DCs resulted in a signifi The frequency of DCs in various tissues was compared in cant decrease in total tumor number, due mainly to a marked APC' mice and their WT littermate controls. DCs (PI tumor reduction in the ileum where the incidence of tumors in EpCAMCD45"LinCD11'MHCII) were analyzed at 10, APC" mice is highest, providing strong evidence that DCs 14 and 18 weeks of age, which correspond to early—(<30 are a key driver of tumor development. In contrast, APC'" adenomas, <2 mm diameter), intermediate—(30-60 15 mice reconstituted with WT BM had similar numbers of adenomas, 0.5-4 mm diameter) and late-stage (>60 tumors compared to control APC' BM-reconstituted adenomas, 1-6 mm diameter) disease, respectively. DCs APC" mice, suggesting that the intestinal environment of accumulated in the SI-LP as disease progressed and by inter the APC' host overrides any potential benefit afforded by mediate stage the frequency of SI-LPDCs was more than 3 a reconstituted WT immune system. times greater in APC" than WT mice. 2O In the steady state. DCs in the gut consist of three pheno Example 4 typically distinct populations: CD103"CD11b, CD103" CD11b" and CD103CD11b DCs. Although there were Tissue Histology some differences in the frequencies of splenic DC subsets between APC" and WT control mice, no significant dif- 25 Immunohistochemical and immunofluorescence staining ferences in the percentages of the three main DC subsets were of intestinal biopsies from FAP and APC" mice shows the observed in the mesenteric lymph nodes (mLN)/Peyer's presence of identical markers of inflammation as well as Patches (PP) or SI-LP. These analyses were performed at abnormal RA metabolism, including Raldh1a1, Raldh1a2 intermediate-stage, as APC" mice lose their Peyer's and CYP26A1. These findings support the view that the Patches by late-stage disease. Further studies of SI-LPDCs 30 underlying pathology in APC" disease is similar to that of from APC" mice revealed that, although their expression FAP. Tumors in the APC" model are primarily in the small of costimulatory molecules was similar to that of WT SI intestine, whereas the tumors in FAP are primarily in the LPDCs, they secreted much higher levels of the pro-inflam colon. One reason for this difference is that the mouse colon matory cytokines TNFO, IL-6 and IL-12p40 under basal con has very few DCs, while DCs are abundant in the human ditions and in response to a panel of Toll-like receptor 35 colon. Drugs that work in APC" mice also work in FAP, agonists. Moreover, APC'". SI-LPDCs induced fewer providing additional evidence that the pathogenesis of dis Foxp3 TRegs in a conventional TReg induction assay. This ease is the same in the two species. impairment was observed at intermediate-stage and became Patients with ulcerative colitis, a type of inflammatory even more apparent at late-stage disease. Similar results were bowel disease, are at greatly increased risk for developing obtained across a 25-fold range of peptide concentrations. 40 colon cancer due to the presence of chronic inflammation. Splenic DCs from both genotypes weakly induced Foxp3"T Once regions of microscopic dysplasia are identified in the cells, while mLN/PP DCs were more potent inducers of colons of Such patients, the colons are typically removed Foxp3' T cells, consistent with previous studies. because dysplasia almost always becomes cancer. Supernatants obtained from APC" SI-LPDC-T cell co Based on staining data, the same abnormalities in retinoic cultures contained a striking six-fold reduction in IL-10 com- 45 acid metabolism that were found in APC" mice, patients pared to WT SI-LPDC co-cultures. Moreover, there was a with FAP and mice with experimentally induced colitis and concomitant and similarly dramatic increase in IL-17A, a key Crohn's disease were present in the dysplastic areas of cytokine involved in adenoma development. Since Th17 dif patients with ulcerative colitis. ferentiation requires IL-6 in addition to TGFB and no exog enous IL-6 was added to our cultures, we measured IL-6 in 50 What is claimed is: co-culture Supernatants and, as expected, there were larger 1. A method of reducing chronic intestinal inflammation in amounts in APC' SI-LPDC co-cultures. Consistent with an individual with familial adenomatous polyposis (FAP), the these findings, IL-23, known to be essential for the mainte method comprising: nance of Th17 cells, though not detectable by ELISA, was administering to the individual an effective dose of an expressed at higher levels in APC'". SI-LPDCs compared 55 agent that increases local concentration of retinoic acid to WT cells in situ. (RA) through modifying enzymatic pathways involved As CD103" SI-LPDCs are the main cells responsible for in RA metabolism, wherein the agent is not retinoic acid, generating immune tolerance in the intestinal environment, and wherein inflammation is reduced. we sorted SI-LPDCs into CD103 and CD103 Subsets to 2. The method of claim 1, wherein the agent inhibits activ assess the role of each subset in the observed impairment in 60 ity of a retinoic acid catabolizing enzyme. Foxp3 induction. APC'* CD103* SI-LPDCs were four 3. The method of claim 1, wherein the agent increases fold less efficient at inducing Foxp3' T cells compared to activity of retinaldehyde dehydrogenase or retinol dehydro their WT counterparts. In contrast, CD103. SI-LPDCs from genase in intestinal tissues. both genotypes were equally poor at inducing Foxp3 expres 4. The method of claim 2, wherein the retinoic acid catabo sion. Both CD103 and CD103* APC'* SI-LPDCs 65 lizing enzyme is a protein of the CYP26 family. induced more Th17 cells compared to the WT SILPDC sub 5. The method of claim 1, wherein the agent is orally sets. These results show that the CD103" Subset of SILPDCs administered. US 9.248,120 B2 27 28 6. The method of claim 4, wherein the retinoic acid catabo lizing enzyme is CYP26A1. 7. The method of claim 6, wherein the agent comprises one or more of liarozole, talarozole, ketoconazole, S (R*, R*) N-4-2-(dimethylamino)-1-(1H-imidazole-1-yl)pro pyl-phenyl]2-benzothiazolamine (R116010), and (R) N 4-2-ethyl-1-(1H-1,2,4-triazol-1-yl)butylphenyl-2- benzothiazolamine (R115866). 8. The method of claim 7, wherein the agent comprises one or more of liarozole and talarozole. 10
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