Targeting colon cancer cell NF-κB promotes an anti-tumour M1-like macrophage phenotype and inhibits peritoneal metastasis.

In a model of peritoneal metastasis in immune-competent mice, we show that nuclear factor (NF)-κB inhibition in CT26 colon cancer cells prevents metastasis. NF-κB inhibition, by stable overexpression of IκB-α super-repressor, induced differential polarization of co-cultured macrophages to an M1-like anti-tumour phenotype in vitro. NF-κB-deficient cancer cell-conditioned media (CT26/IκB-α SR) induced interleukin (IL)-12 and nitric oxide (NO) synthase (inducible NO synthase (iNOS)) expression in macrophages. Control cell (CT26/EV) conditioned media induced high levels of IL-10 and arginase in macrophages. In vivo, this effect translated to reduction in metastasis in mice injected with CT26/ IκB-α SR cells and was positively associated with increased CD8(+)CD44(+)CD62L(-) and CD4(+)CD44(+)CD62L(-) effector T cells. Furthermore, inhibition of NF-κB activity induced high levels of NO in infiltrating immune cells and decreases in matrix metalloproteinase-9 expression, simultaneous with increases in tissue inhibitor of metalloproteinases 1 and 2 within tumours. CT26/IκB-α SR tumours displayed increased pro-inflammatory gene expression, low levels of angiogenesis and extensive intratumoral apoptosis, consistent with the presence of an anti-tumour macrophage phenotype. Macrophage depletion reduced tumour size in CT26/EV-injected animals and increased tumour size in CT26/IκB-α SR cells compared with untreated tumours. Our data demonstrate, for the first time, that an important implication of targeting tumour cell NF-κB is skewing of macrophage polarization to an anti-tumour phenotype. This knowledge offers novel therapeutic opportunities for anticancer treatment.

Systemic and intestinal pharmacokinetics of methotrexate in patients with inflammatory bowel disease.

BACKGROUND: The pharmacokinetics of low-dose subcutaneous methotrexate have not been determined throughout the standard weekly dosing interval. It is not known whether methotrexate concentrations in the gastrointestinal tract are sufficient for pharmacologic activity in inflammatory bowel disease. METHODS: Ten patients with inflammatory bowel disease participated in the study. After the patients started taking 15 or 25 mg subcutaneous methotrexate once a week, erythrocyte methotrexate concentration was measured every 2 weeks. The absorption, rectal distribution, metabolism, and elimination of methotrexate were measured. The effect of methotrexate on proliferation of an intestinal epithelial cell line was determined. RESULTS: After weekly subcutaneous administration of methotrexate was begun, trough erythrocyte concentration rose to reach a plateau after 6 to 8 weeks, ranging from 150 to 300 nmol/L. More than 90% of subcutaneously administered methotrexate was rapidly excreted in the urine. The methotrexate plasma time course after subcutaneous administration fit a 2-compartment first-order model with biphasic elimination and trough concentration of about 1 nmol/L. Trough and peak methotrexate concentrations (mean value +/- SD) were 64 +/- 33 and 206 +/- 64 fmol/mg in the rectal mucosa and 4 +/- 3 and 51 +/- 26 nmol/L in the rectal lumen. These methotrexate concentrations were in the range found to be pharmacologically active against Caco-2 cell growth, that is, a 50% inhibitory concentration from 10 to 46 nmol/L. CONCLUSION: Subcutaneous methotrexate was well absorbed and distributed to the site of the lesions in patients with inflammatory bowel disease. Methotrexate was concentrated intracellularly in blood and in the rectum. The methotrexate concentration in the rectal mucosa remained within a pharmacologically active range throughout the dosing interval. The findings represent a pharmacologic explanation for the sustained efficacy of weekly methotrexate therapy.

Clinical outcome and pharmacokinetics after addition of low-dose cyclosporine to methotrexate: a case study of five patients with treatment-resistant inflammatory bowel disease.

INTRODUCTION: This study reports the clinical outcome, toxicity, and methotrexate pharmacokinetics after the addition of low-dose cyclosporine to methotrexate in patients with ulcerative colitis or Crohn's disease. METHODS: Three patients with steroid-refractory ulcerative colitis and two patients with steroid refractory Crohn's disease who failed monotherapy with subcutaneous methotrexate 25 mg/week for 16 weeks were treated with the combination of methotrexate and low-dose oral cyclosporine (3 mg/kg/day) for an additional 16 weeks. Clinical response was measured with the Inflammatory Bowel Disease Questionnaire (IBDQ) score. Concentrations of erythrocyte methotrexate, plasma methotrexate, and plasma 7-hydroxymethotrexate were also determined. RESULTS: Both patients with Crohn's disease withdrew from the study for toxicity (headaches, seizure). The three patients with ulcerative colitis experienced clinical improvement with a mean increase in the IBDQ score from 164 to 190 points, p = 0.01. The mean serum creatinine in the three patients who completed the study increased from 0.9 mg/dL at baseline to 1.2 mg/dL at week 16. p = 0.04. One patient developed hypertension. There was no significant change from baseline in the concentrations of erythrocyte methotrexate, plasma methotrexate, and plasma 7-hydroxymethotrexate. CONCLUSIONS: Combination therapy with methotrexate and low-dose oral cyclosporine did not alter methotrexate pharmacokinetics and resulted in high rates of cyclosporine-associated toxicity.

Plasma and rectal adenosine in inflammatory bowel disease: effect of methotrexate.

In animal models, the antiinflammatory mechanism of action of methotrexate has been attributed to elevation of the extracellular concentration of the endogenous autocoid, adenosine. Our goal was to determine if methotrexate elevates adenosine concentrations in plasma and at the site of disease in patients with inflammatory bowel disease. In 10 patients with Crohn's disease or ulcerative colitis, rectal adenosine and plasma adenosine concentrations were measured before and immediately after a subcutaneous injection of methotrexate, 15 or 25 mg. The mean predose rectal adenosine concentration of 2.4 mumol/l was not significantly different from the postdose concentration of 2.1 mumol/l, p = 0.17, (paired two-tailed t test). Rectal adenosine concentration tended to correlate with rectal endoscopic disease activity, r = 0.59, p = 0.067 (Spearman rank order correlation). After methotrexate injection, neither the mean daily plasma adenosine concentration, nor the plasma adenosine at any individual time point, were significantly different from preinjection values. In patients with inflammatory bowel disease, an injection of methotrexate in the clinically effective dose range does not raise rectal or plasma adenosine concentrations. A role for adenosine as the mediator of the antiinflammatory action of methotrexate is not supported.

Methotrexate for inflammatory bowel disease: pharmacology and preliminary results.

OBJECTIVE: To review the limited published experience with methotrexate treatment for inflammatory bowel disease, to examine the proposed anti-inflammatory and immune-modifying mechanism of action and pharmacologic properties of methotrexate, and to detail its limiting toxicities. DESIGN: A comprehensive synopsis of methotrexate is presented to aid physicians who treat patients with inflammatory bowel disease. RESULTS: Methotrexate and its polyglutamate metabolites are folic acid analogues with inhibitory activity against many enzymes in the metabolic pathway of folic acid. Long-term low-dose methotrexate therapy (25 mg or less once a week) inhibits production of thymidylate, purines, and methionine and leads to accumulation of adenosine, a potent anti-inflammatory substance. These actions inhibit cellular proliferation, decrease formation of antibodies, and decrease production of mediators of inflammation such as interleukins and eicosanoids. Three uncontrolled trials and two controlled trials have demonstrated efficacy of low-dose methotrexate therapy for induction of remission in Crohn's disease and have also suggested possible benefit for ulcerative colitis and for remission maintenance indications in both diseases. Although methotrexate is generally well tolerated for long-term use at a low dose, several serious toxicities potentially limit its use. CONCLUSION: Methotrexate is a promising new agent for the treatment of inflammatory bowel disease.

Dialysis of the rectum for sampling drug concentrations in the luminal extracellular fluid of the gut: technique and precision.

BACKGROUND: It is useful to measure the luminal concentration of drugs which act in the gut. Dialysis of the rectum has not previously been used or validated for this purpose. AIM: To determine the precision of rectal dialysis for measuring rectal drug concentrations. METHODS: To establish the duration of dialysis required to approach equilibrium, the rate of methotrexate diffusion into dialysis bags was first determined in vitro. The precision of rectal dialysis for sampling the methotrexate concentration of colonic lumen extracellular fluid was determined in seven subjects who underwent two consecutive dialysis procedures. Subjects treated with subcutaneous methotrexate for refractory inflammatory bowel disease were studied. RESULTS: Methotrexate crossed the dialysis membrane by a first-order process, and after a 2 h in vitro dialysis, equilibration was 74 +/- 2% (mean +/- s.d.) complete. Rectal dialysis was well tolerated by all subjects. The mean +/- s.e. methotrexate concentration of 3.6 +/- 1.1 nmol/L in the first dialysate was not significantly different from 3.6 +/- 0.9 nmol/L in the second dialysate. P = 0.99 (paired two-tailed t-test). Similar precision was obtained for an endogenous molecule, potassium, secreted by the rectal mucosa. CONCLUSIONS: Dialysis of the rectum is a well tolerated and precise technique for sampling the colonic lumen extracellular fluid for quantitative analyses of exogenous and endogenous substances.

A randomized dose-response and pharmacokinetic study of methotrexate for refractory inflammatory Crohn's disease and ulcerative colitis.

BACKGROUND AND AIMS: The optimum initial dose of methotrexate for steroid-requiring inflammatory bowel disease is not known. AIM: To compare directly the efficacy and toxicity of methotrexate 15 and 25 mg/week, and to explore the value of methotrexate blood levels as predictors of outcome. METHODS: A 16-week randomized single-blind comparison of subcutaneous methotrexate 15 or 25 mg/week was performed in 32 patients with steroid-requiring Crohn's disease or ulcerative colitis. Patients who did not respond to methotrexate 15 mg/week were further studied for an additional 16 weeks on methotrexate 25 mg/week. Blood was drawn every 2 weeks for methotrexate levels. RESULTS: After 16 weeks, 17% of patients in each group achieved remission; 39% of patients randomized to 15 mg/week and 33% of patients randomized to 25 mg/week improved (P=N.S. ). Clinical status improved in four out of 11 patients after methotrexate dose escalation from 15 to 25 mg/week. Toxicity was not different between the treatment groups. Methotrexate blood levels did not predict efficacy or toxicity. CONCLUSIONS: For induction of remission in steroid-requiring inflammatory bowel disease, subcutaneous methotrexate at initial doses of 15 and 25 mg/week are equally efficacious. At these doses, response is not associated with blood methotrexate concentrations.

CYP2C19 pharmacogenetics in the clinical use of proton-pump inhibitors for gastro-oesophageal reflux disease: variant alleles predict gastric acid suppression, but not oesophageal acid exposure or reflux symptoms.

BACKGROUND: The rate of metabolic inactivation of proton-pump inhibitors is determined by polymorphisms of CYP2C19. It is not known if CYP2C19 variant alleles affect responses to proton-pump inhibitor therapy in gastro-oesophageal reflux disease (GERD). AIM: To determine if the CYP2C19 genotype is associated with clinical effectiveness of proton-pump inhibitors during GERD therapy. METHODS: GERD patients undergoing ambulatory gastric and oesophageal pH monitoring were genotyped for CYP2C19 polymorphisms. RESULTS: Sixty subjects were enrolled. Forty-four subjects had two wild-type alleles, 15 had one variant, and one had two variant CYP2C19 alleles. The presence of a variant allele was significantly associated with a lower odds of gastric acid breakthrough during proton-pump inhibitor therapy [odds ratio 5.14, 95% confidence interval (CI) 1.17-22.61]. The presence of a variant allele was not associated with a lower odds of significant oesophageal acid exposure (odds ratio 2.50, 95% CI 0.60-10.52), or the occurrence of symptoms (incidence rate ratio 1.06, 95% CI 0.54-2.06). CONCLUSIONS: These results indicate that factors other than gastric acid secretion are important determinants of reflux in GERD patients. This suggests that CYP2C19 genotype testing will not be useful in proton-pump inhibitor therapy of GERD, except perhaps in identifying patients at risk for hypochlorhydria and consequent hypergastrinemia.

Suppression of radiation-induced DNA double-strand break repair by MyD88 is accompanied by apoptosis and crypt loss in mouse colon.

Intestinal microbes promote the injurious effects of radiation on those tissues. However, the molecular factors mediating this effect are largely unknown. In this work, we explored the effects of orally administered antibiotics and MyD88, a key adapter molecule in toll-like receptor signaling, on molecular and cellular responses of mouse colon to radiation. Results show that oral antibiotics lowered radiation-induced colonic damage by protecting epithelial cells against radiation-induced apoptosis, leading to increased survival of crypts. MyD88 deficiency partially phenocopied the effects of oral antibiotics on apoptosis and crypt survival, suggesting that colonic microbes exert their injurious effects in part via that molecule. Analysis of DNA double-strand breaks, the primary genotoxic lesions induced by radiation, showed that their early induction in mouse colon was unaffected by MyD88. However, MyD88 deficiency resulted in the later disappearance of DNA double-strand breaks. Loss of DNA double-strand breaks was accompanied by the evidence of increased activation of both the non-homologous end-joining and homologous recombination pathways of DNA repair in MyD88-deficient mice. These results show that colonic microbes and MyD88 regulate DNA double-strand break repair in irradiated mouse colon, effects which exert significant control over radiation-induced apoptosis and crypt survival.

The monoacylglycerol lipase inhibitor JZL184 attenuates LPS-induced increases in cytokine expression in the rat frontal cortex and plasma: differential mechanisms of action.

BACKGROUND AND PURPOSE: JZL184 is a selective inhibitor of monoacylglycerol lipase (MAGL), the enzyme that preferentially catabolizes the endocannabinoid 2-arachidonoyl glycerol (2-AG). Here, we have studied the effects of JZL184 on inflammatory cytokines in the brain and plasma following an acute immune challenge and the underlying receptor and molecular mechanisms involved. EXPERIMENTAL APPROACH: JZL184 and/or the CB1 receptor antagonist, AM251 or the CB2 receptor antagonist, AM630 were administered to rats 30 min before lipopolysaccharide (LPS). 2 h later cytokine expression and levels, MAGL activity, 2-AG, arachidonic acid and prostaglandin levels were measured in the frontal cortex, plasma and spleen. KEY RESULTS: JZL184 attenuated LPS-induced increases in IL-1ß, IL-6, TNF-α and IL-10 but not the expression of the inhibitor of NFkB (IκBα) in rat frontal cortex. AM251 attenuated JZL184-induced decreases in frontal cortical IL-1ß expression. Although arachidonic acid levels in the frontal cortex were reduced in JZL184-treated rats, MAGL activity, 2-AG, PGE2 and PGD2 were unchanged. In comparison, MAGL activity was inhibited and 2-AG levels enhanced in the spleen following JZL184. In plasma, LPS-induced increases in TNF-α and IL-10 levels were attenuated by JZL184, an effect partially blocked by AM251. In addition, AM630 blocked LPS-induced increases in plasma IL-1ß in the presence, but not absence, of JZL184. CONCLUSION AND IMPLICATIONS: Inhibition of peripheral MAGL in rats by JZL184 suppressed LPS-induced circulating cytokines that in turn may modulate central cytokine expression. The data provide further evidence for the endocannabinoid system as a therapeutic target in treatment of central and peripheral inflammatory disorders.

Pharmacological inhibition of endocannabinoid degradation modulates the expression of inflammatory mediators in the hypothalamus following an immunological stressor.

The endocannabinoid system is an important regulator of the nervous, neuroendocrine, and immune systems, thus representing a novel therapeutic target for stress-related neuroinflammatory and psychiatric disorders. However, there is a paucity of data relating to the effects of endocannabinoids on neuroinflammatory mediators following an immune stress/challenge in vivo. This study investigated the effects of URB597, a selective inhibitor of fatty acid amide hydrolyase (FAAH), the enzyme that preferentially metabolizes anandamide, on lipopolysaccharide (LPS)-induced increases in the expression of immune mediators in the hypothalamus. Systemic administration of URB597 increased the levels of anandamide and the related N-acylethanolamines, N-palmitoylethanolamide, and N-oleoylethanolamide, but not 2-arachidonoyl glycerol, in the hypothalamus and spleen. URB597 attenuated the LPS-induced increase in interleukin (IL)-1ß expression while concurrently augmenting the LPS-induced increase in suppressor of cytokine signalling (SOCS)-3 expression. In addition, URB597 tended to enhance and reduce the LPS-induced increase in IL-6 and IL-10 mRNA expression, respectively. LPS-induced increases in peripheral cytokine levels or plasma corticosterone were not altered by URB597. The present study provides evidence for a role for FAAH in the regulation of LPS-induced expression of inflammatory mediators in the hypothalamus. Improved understanding of endocannabinoid-mediated regulation of neuroimmune function has fundamental physiological and potential therapeutic significance in the context of stress-related disorders.

Endoscopic removal of an embedded biliary Wallstent by piecemeal extraction.

Expandable metal biliary stents are reserved for patients with unresectable malignant biliary obstruction. Occasionally, these stents may cause complications necessitating removal. We describe successful endoscopic removal of a biliary Wallstent one year after insertion in a patient who initially underwent placement of an expandable metal biliary stent for presumed biliary malignancy. The stent was removed after a stent related bleeding duodenal ulcer formed.

New perspectives in gastric acid suppression: genetic polymorphisms predict the efficacy of proton pump inhibitors.

Proton pump inhibitors are highly effective in the management of acid-peptic diseases. These drugs potently inhibit acid secretion from gastric parietal cells by irreversibly inhibiting activity of the H(+), K(+) ATPase (proton pump). Early studies of the pharmacokinetics of proton pump inhibitors demonstrated considerable variation in drug clearance rates among patients and healthy volunteers. This variation was also reflected in a wide range of the efficacy of acid suppression by standard doses of proton pump inhibitors among study subjects; those with slower clearance and higher drug concentrations experienced superior acid suppression. Proton pump inhibitors are predominantly inactivated by the 2C19 isoform of the hepatic cytochrome P450 mixed function oxidase system. The cytochrome P450 2C19 gene is polymorphic, with three known inactivating mutations. Individuals with one or two mutant cytochrome P450 2C19 alleles metabolize proton pump inhibitors more slowly than those with two wild-type alleles and experience higher drug levels. An individual's cytochrome P450 2C19 genotype predicts the degree of acid suppression in response to a standard dose of a proton pump inhibitor. Emerging data suggests that the clinical effectiveness of proton pump inhibitors in the treatment of acid-peptic diseases may also be dependent on cytochrome P450 2C19 genotype.

Drug therapy of inflammatory bowel disease.

The drugs that are effective in inflammatory bowel disease (IBD) act by inhibiting the chronic unregulated intestinal inflammation in these patients. The mainstays of the drug therapy of IBD are a variety of formulations of 5-aminosalicylic acid (5-ASA), the conventional and newer low bioavailability glucocorticoids, the nitroimidazole antibiotic metronidazole, and certain immunomodulating agents. Increased understanding of the mechanisms of inflammation in IBD has permitted the development of effective designer drugs. These agents are products of the biotechnology industry and include antibodies to tumor necrosis factor (TNF)-alpha, antisense oligonucleotides and recombinant human interleukin (IL)-10. In addition, a number of other agents such as nicotine and n-3 fatty acids are useful in certain patients. This review first focuses on the pharmacology and mechanism of action of these drugs in IBD, followed by an approach to the treatment of patients with ulcerative colitis (UC) and Crohns disease (CD). The recommendations consider type and activity of IBD and are based largely on data from controlled trials and systematic reviews in the IBD literature.

Clinical outcome following treatment of refractory inflammatory and fistulizing Crohn's disease with intravenous cyclosporine.

OBJECTIVE: To determine outcome following treatment of refractory Crohn's disease with intravenous (i.v.) cyclosporine (CYA). METHODS: The medical records of 18 patients with refractory Crohn's disease treated with i.v. CYA were reviewed. Nine patients had refractory inflammatory Crohn's disease and nine patients had complex fistulizing Crohn's disease. All patients were initially treated with i.v. CYA (4 mg/kg/day). Patients who responded were converted to standard oral CYA. Patient outcomes were classified as complete response, partial response, or nonresponse. RESULTS: Four of nine patients with severe inflammatory Crohn's disease and seven of nine patients with fistulizing Crohn's disease had a partial response to i.v. CYA. Four of four responding patients in the inflammatory group and four of six responding patients in the fistulizing group (plus one initial nonresponder) maintained or improved their response during oral CYA therapy. After discontinuing oral CYA, all four patients in the inflammatory group and five of seven patients in the fistulizing group relapsed despite 1-17 wk of concomitant treatment with azathioprine or 6-mercaptopurine (AZA/6MP). Two patients who received overlapping CYA and AZA/6MP for 17 and 23 wk maintained long-term responses. CYA toxicity was minimal: reversible nephrotoxicity (n = 2), headache (n = 2), oral candidiasis (n = 1), paresthesia (n = 2). CONCLUSIONS: I.v. CYA appears to benefit both refractory inflammatory and fistulizing Crohn's disease. Most patients who respond to i.v. CYA will maintain their response during oral CYA therapy. However, the majority of these patients relapse when oral CYA is discontinued, probably because of inadequate duration of overlap with the slow acting maintenance drugs, AZA/6MP.

Hereditary deficiency of the seventh component of complement and recurrent meningococcal infection: investigations of an Irish family using a novel haemolytic screening assay for complement activity and C7 M/N allotyping.

Terminal complement component deficiency predisposes to meningococcal infection and is inherited in an autosomal co-dominant manner. An Irish family is described, in which 2 of 3 brothers had recurrent meningococcal infection. A novel screening assay was used to investigate for terminal complement deficiency and the 2 affected brothers were found to be completely deficient in the seventh component of complement (C7). Enzyme-linked immunosorbent assay for C7 revealed lower than normal levels in the remaining brother and parents. C7 M/N protein polymorphism allotyping, used to investigate the segregation of the C7 deficiency genes, showed that the apparently complement sufficient brother was heterozygous C7 deficient and a carrier of one of the deficiency genes. Complement screening should be carried out in any individual suffering recurrent meningococcal infection or infection with an uncommon meningococcal serogroup. Identification of complement deficient patients allows the implementation of strategies to prevent recurrent infection.

Celiac-associated lymphoma. A single institution experience of 30 cases in the combination chemotherapy era.

Celiac sprue (CS) is frequently complicated by malignancy, most commonly small intestinal lymphoma. Our study was performed in an area with a high prevalence of CS to define the clinical features, response to treatment, and outcome of this tumor. Of a total of 31 lymphomas complicating CS identified, 30 case records and 24 tumor specimens were reviewed. Overall 1-year survival was 9 of 29 (31%) and 5-year survival 3 of 27 (11%). Seven previously diagnosed celiac patients developed lymphoma; length on gluten-free diet ranged from 12 to 252 months (median 44 months). In this group, presentation was nonspecific, diagnosis difficult, and survival poor (lymphoma diagnosed in life in four of seven, mean survival 2.25 months). Twenty-three patients had CS and lymphoma diagnosed during the same illness. In this group, 14 of 23 presented with a surgical emergency and were treated with tumor resection and chemotherapy. Nine are disease-free and alive or died of another cause after 10-196 months (mean 74 follow-up). Celiac-associated lymphoma is a frequent, difficult to diagnose, and commonly fatal complication of CS. An aggressive diagnostic approach, including laparoscopy, is recommended. Long-term survival can be expected in a significant number of these patients and in our series was almost exclusively confined to those treated with chemotherapy.

Enteropathy-associated T-cell lymphoma in the West of Ireland: low-frequency of Epstein-Barr virus in these tumors.

The Epstein-Barr virus has been implicated in the etiology of endemic Burkitt's lymphoma, post-transplant lymphoma, large-cell anaplastic CD30 (Ki-1)-positive lymphoma, and in many T-cell lymphomas. A recent report has found Epstein-Barr virus genome in association with 4 of 11 cases (36%) of enteropathy-associated T-cell lymphoma. In a retrospective study, we have characterized 22 consecutive cases of enteropathy-associated T-cell lymphoma from the West of Ireland where celiac disease is endemic. All cases were immunophenotyped with T- and B-cell markers including the anaplastic large-cell lymphoma marker CD30 or Ki-1. Nineteen cases were studied for latent membrane protein expression and 16 for Epstein-Barr virus small RNAs by in situ hybridization using EBER oligonucleotides on routinely processed sections. Only 1 of 16 cases (6%) showed Epstein-Barr virus in tumor cells and no cases stained with latent membrane protein. Eight of 22 cases (36%) including the EBER-positive case were positive for CD30. These results suggest that the Epstein-Barr virus does not commonly play a role in the pathogenesis of enteropathy-associated T-cell lymphoma from this area.