Ex vivo explant model of adenoma and colorectal cancer to explore mechanisms of action and patient response to cancer prevention therapies.

Colorectal cancer (CRC) is the second leading cause of cancer death in the UK. Novel therapeutic prevention strategies to inhibit the development and progression of CRC would be invaluable. Potential contenders include low toxicity agents such as dietary-derived agents or repurposed drugs. However, in vitro and in vivo models used in drug development often do not take into account the heterogeneity of tumours or the tumour microenvironment. This limits translation to a clinical setting. Our objectives were to develop an ex vivo method utilizing CRC and adenoma patient-derived explants (PDEs) which facilitates screening of drugs, assessment of toxicity, and efficacy. Our aims were to use a multiplexed immunofluorescence approach to demonstrate the viability of colorectal tissue PDEs, and the ability to assess immune cell composition and interactions. Using clinically achievable concentrations of curcumin, we show a correlation between curcumin-induced tumour and stromal apoptosis (P < .001) in adenomas and cancers; higher stromal content is associated with poorer outcomes. B cell (CD20+ve) and T cell (CD3+ve) density of immune cells within tumour regions in control samples correlated with curcumin-induced tumour apoptosis (P < .001 and P < .05, respectively), suggesting curcumin-induced apoptosis is potentially predicted by baseline measures of immune cells. A decrease in distance between T cells (CD3+ve) and cytokeratin+ve cells was observed, indicating movement of T cells (CD3+ve) towards the tumour margin (P < .001); this change is consistent with an immune environment associated with improved outcomes. Concurrently, an increase in distance between T cells (CD3+ve) and B cells (CD20+ve) was detected following curcumin treatment (P < .001), which may result in a less immunosuppressive tumour milieu. The colorectal tissue PDE model offers significant potential for simultaneously assessing multiple biomarkers in response to drug exposure allowing a greater understanding of mechanisms of action and efficacy in relevant target tissues, that maintain both their structural integrity and immune cell compartments.

Renal effects of microwave ablation compared with radiofrequency, cryotherapy and surgical resection at different volumes of the liver treated.

BACKGROUND: Renal changes after microwave tissue ablation (MTA) were compared with those following hepatic resection, cryotherapy (CRYO), and radiofrequency ablation (RFA). Structural damage producing renal impairment has been assessed directly by examining tissue specimens and by serum analysis for two sensitive biomarkers, retinol binding protein (RBP) and the heat shock protein 70 (HSP-70) for each modality at different ablation volumes. METHODS: Live rats underwent MTA, surgical resection, CRYO or RFA of 15, 33 or 66% of total hepatic volume. Urine and tissue samples were collected at the time of death. Percentage of tubules with casts and glomerular damage, tissue expression of HSP-70 and urine RBP were evaluated and compared. Behaviour of the animals was also assessed by means of five different parameters and combined to produce a response score. RESULTS: All RFA and CRYO rats undergoing 66% died and these animals had >60% of damaged tubuli and 8% of altered glomeruli. No animals treated by MTA or surgical resection died. Cut-off values (those predicting fatal treatments) could be identified for levels of HSP-70 and RBP. CONCLUSIONS: Large volume MTA is associated with a significant reduced renal damage and is well tolerated compared with RFA and CRYO.

Pilot study of oral anthocyanins for colorectal cancer chemoprevention.

Naturally occurring anthocyanins possess colorectal cancer chemopreventive properties in rodent models. We investigated whether mirtocyan, an anthocyanin-rich standardized bilberry extract, causes pharmacodynamic changes consistent with chemopreventive efficacy and generates measurable levels of anthocyanins in blood, urine, and target tissue. Twenty-five colorectal cancer patients scheduled to undergo resection of primary tumor or liver metastases received mirtocyan 1.4, 2.8, or 5.6 grams (containing 0.5-2.0 grams anthocyanins) daily for 7 days before surgery. Bilberry anthocyanins were analyzed by high performance liquid chromatography (HPLC) with visible or mass spectrometric detection. Proliferation was determined by immunohistochemistry of Ki-67 in colorectal tumor. Concentrations of insulin-like growth factor (IGF)-I were measured in plasma. Mirtocyan anthocyanins and methyl and glucuronide metabolites were identified in plasma, colorectal tissue, and urine, but not in liver. Anthocyanin concentrations in plasma and urine were roughly dose-dependent, reaching approximately 179 ng/gram in tumor tissue at the highest dose. In tumor tissue from all patients on mirtocyan, proliferation was decreased by 7% compared with preintervention values. The low dose caused a small but nonsignificant reduction in circulating IGF-I concentrations. In conclusion, repeated administration of bilberry anthocyanins exerts pharmacodynamic effects and generates concentrations of anthocyanins in humans resembling those seen in Apc(Min) mice, a model of FAP adenomas sensitive to the chemopreventive properties of anthocyanins. Studies of doses containing <0.5 gram bilberry anthocyanins are necessary to adjudge whether they may be appropriate for development as colorectal cancer chemopreventive agents.

CP-690550, a JAK3 inhibitor as an immunosuppressant for the treatment of rheumatoid arthritis, transplant rejection, psoriasis and other immune-mediated disorders.

Pfizer Inc is developing the novel JAK3 inhibitor CP-690550 for the potential prevention of transplant rejection and treatment of autoimmune diseases, such as rheumatoid arthritis (RA) and psoriasis. The benefits of currently available immunosuppressive drugs are countered by numerous side effects, caused mainly by the ubiquitous distribution of the target molecules of these treatments. CP-690550 is expected to overcome these limitations by selectively targeting JAK3, which is expressed generally only in immune cells and is only bound by gamma-chain-bearing cytokine receptors involved in the JAK/STAT signaling pathway. CP-690550 exhibited potent immunosuppressive activity in preclinical models of RA and organ transplant rejection. Phase I and II clinical trials demonstrated the efficacy and safety of CP-690550 in preventing transplant rejection and alleviating the symptoms of RA and psoriasis. At the time of publication, CP-690550 was in phase II/III trials in patients with RA, phase II trials in patients with psoriasis, ulcerative colitis and Crohn's disease, and transplant recipients, and a phase I/II trial for dry eye disease (xerophthalmia). Thus, the preclinical and clinical data strongly support the use of CP-690550 to produce sufficient immunosuppression to prevent organ transplant rejection and to treat autoimmune diseases; CP-690550 could herald the beginning of a new generation of safe and effective immunosuppressive therapies.

Pilot study of oral silibinin, a putative chemopreventive agent, in colorectal cancer patients: silibinin levels in plasma, colorectum, and liver and their pharmacodynamic consequences.

Silibinin, a flavonolignan from milk thistle, has intestinal cancer chemopreventive efficacy in rodents. It is a strong antioxidant and modulates the insulin-like growth factor (IGF) system by increasing circulating levels of IGF-binding protein 3 (IGFBP-3) and decreasing levels of IGF-I. Here, the hypothesis was tested that administration of oral silibinin generates agent levels in human blood and colorectal and hepatic tissues consistent with pharmacologic activity. Patients with confirmed colorectal adenocarcinoma received silibinin formulated with phosphatidylcholine (silipide) at dosages of 360, 720, or 1,440 mg silibinin daily for 7 days. Blood and biopsy samples of normal and malignant colorectum or liver were obtained before dosing, and blood and colorectal or hepatic tissues were collected at resection surgery after the final silipide dose. Levels of silibinin were quantified by high-pressure liquid chromatography-UV, and plasma metabolites were identified by liquid chromatography-mass spectrometry. Blood levels of IGFBP-3, IGF-I, and the oxidative DNA damage pyrimidopurinone adduct of deoxyguanosine (M1dG) were determined. Repeated administration of silipide was safe and achieved levels of silibinin of 0.3 to 4 micromol/L in the plasma, 0.3 to 2.5 nmol/g tissue in the liver, and 20 to 141 nmol/g tissue in colorectal tissue. Silibinin monoglucuronide, silibinin diglucuronide, silibinin monosulfate, and silibinin glucuronide sulfate were identified in the plasma. Intervention with silipide did not affect circulating levels of IGFBP-3, IGF-I, or M1dG. The high silibinin levels achieved in the human colorectal mucosa after consumption of safe silibinin doses support its further exploration as a potential human colorectal cancer chemopreventive agent.

Epidermal growth factor enemas with oral mesalamine for mild-to-moderate left-sided ulcerative colitis or proctitis.

BACKGROUND: Epidermal growth factor (EGF) is a potent mitogenic peptide produced by salivary glands. We examined whether EGF enemas are an effective treatment for active left-sided ulcerative colitis and ulceration limited to the rectum (proctitis). METHODS: In a randomized, double-blind clinical trial conducted at Leicester Royal Infirmary, 12 patients with mild-to-moderate left-sided ulcerative colitis received daily enemas of 5 microg of EGF in 100 ml of an inert carrier and 12 received daily enemas with carrier alone for 14 days. All also began to receive 1.2 g of oral mesalamine per day or had their dose increased by 1.2 g per day. Patients were assessed clinically at 0, 2, 4, and 12 weeks and by sigmoidoscopy and biopsy at 0, 2, and 4 weeks. The primary end point was disease remission (defined by a St. Marks score of 4 or less without sigmoidoscopic evidence of inflammation) at two weeks. Secondary end points were clinically significant improvements in disease activity (defined by a decrease of more than 3 points in the St. Marks score or the ulcerative colitis disease-activity index) at two and four weeks. Analyses were performed according to the intention-to-treat principle. RESULTS: After two weeks, 10 of the 12 patients given EGF enemas were in remission, as compared with 1 of 12 in the control group (83 percent vs. 8 percent, P<0.001). At the 2-week assessment, disease-activity scores, sigmoidoscopic score, and histologic scores were all significantly better in the EGF group than in the placebo group (P<0.01 for all comparisons), and this benefit was maintained at 4 weeks and at 12 weeks. CONCLUSIONS: This study provides preliminary data suggesting that EGF enemas are an effective treatment for active left-sided ulcerative colitis.