Animal studies
Specific pathogen-free wild type female C57BL/6 mice were obtained from Jackson Laboratories (Bar Harbor, ME, USA) and typically used at an age of 6–8 weeks. Starting weights (mean ± SD) for acute and chronic DSS studies were 18.8 ± 1.0 g and 19.2 ± 0.9 g. RAG-2-/- female mice on the C57BL/6 background were obtained from Taconic Farms (Germantown, NY, USA) (mean weight 22.5 ± 1.2 g). Mice were housed in polycarbonate micro-isolator or individually ventilated cages and were allowed access to food and water ad libitum. All animal studies were approved by the Duke University Institutional Animal Care and Use Committee.
A bacterial lipopolysaccharide (LPS) challenge model was used to test the ability of LMP-420 to block TNF production in vivo. Mice were treated for 16 days with 2 mg (100 mg/kg) LMP-420 given intraperitoneally (i.p.) once daily or with LMP-420 doses of up to 145/mg/kg/day given orally mixed in food. The i.p. dose of LMP-420 used was chosen to represent the highest parenteral dose reasonable given the solubility of the drug (~10 mg/ml in 5% sorbitol). The 16 day treatment period was chosen to assess the toxicity of repeated daily dosing of LMP-420 and because a treatment period of 16 days or longer is typically needed to result in histologically detectable differences in inflammatory activity. Vehicle- or LMP-420 treated mice were challenged with a lethal dose of 0.5 mg LPS (from E. coli strain O111:B4; catalog#L-2630, Sigma-Aldrich, St. Louis, MO, USA) given 4 hrs after the last LMP-420 dose. Mice were euthanized 2 hrs after LPS challenge to measure TNF levels in serum and colon tissues.
Acute colitis was induced by addition of 3% DSS (40 kDa molecular weight; obtained from ICN, Costa Mesa, CA, USA) into the drinking water for 7 days. Chronic colitis was induced by 3 cycles of DSS administration, each consisting of 5 days DSS followed by 16 days of recovery. The clinical severity of colitis was assessed by daily observations for weight loss, stool consistency, and the presence of gross bleeding. Freshly passed stool pellets were tested periodically for occult blood using Hemoccult Sensa II cards (Beckman Coulter, Palo Alto, CA, USA).
Specific pathogen and Helicobacter-free female IL-10-deficient (IL-10-/-) mice on the C57BL/6 background (strain name = B6.129P2-Il10tm1Cgn/J; stock # 002251) were obtained from Jackson Laboratories (Bar Harbor, ME, USA). Powdered food containing 200 ppm piroxicam was administered for 7 days at 6 – 7 wks of age (weights = 18.6 ± 1.6 g, mean ± SD) to accelerate the development of chronic colitis [26]. Piroxicam was discontinued and mice were treated for 16 days with a dose range of LMP-420 (0, 5, 15, or 45 mg/kg/day) given i.p. once daily or food containing LMP-420 that delivered mean LMP-420 doses of 0, 41, 62, or 138 mg/kg/day.
In vitro studies
Thioglycollate-stimulated macrophages were obtained for in vitro studies by peritoneal lavage (ice-cold PBS; 0.1% BSA; 10 u/ml sodium heparin) of euthanized mice injected intraperitoneally 3–4 days earlier with 1 ml of sterile thioglycollate broth (DIFCO; Voight Global Distribution, Kansas City, MO, USA). Peritoneal exudates were centrifuged, washed once with PBS and resuspended in complete RPMI medium containing 10% heat-inactivated (56°C, 45 min) fetal bovine serum (FBS). Macrophages were isolated by subsequent overnight adherence to plastic by incubation at 37°C in humidified 5% CO2, then plated into 96 well plates at 1 × 105 cells/well in RPMI1640 with 5% heat-inactivated FBS, 100 u/ml penicillin, 100 μg/ml streptomycin and the indicated concentration of LMP-420. Cells were incubated for 2 hrs, LPS (1 μg/ml; from E. coli strain O111:B4, Sigma-Aldrich, St. Louis, MO, USA) was added, and after an additional 24 hrs TNF content of the media was measured.
Murine splenocytes were prepared from spleens harvested from normal mice after euthanasia. Briefly, the spleens were removed aseptically and placed in plastic Petri dishes containing ~10 ml of tissue culture medium. The medium was drawn up in a 27 gauge needle on a 10-ml syringe and then repeatedly injected under the splenic capsule, forcing leukocytes from the splenic tissue into the medium. The leukocytes were pelleted by centrifugation (15 min, 200 × g, 4°C), washed once with PBS and contaminating red blood cells removed by a brief (< 20 s) hypotonic lysis in 9 volumes of sterile H2O followed by the addition of 1 volume of 10× PBS. The leukocytes were washed once with complete RPMI medium and resuspended to a concentration of 1 × 106/ml. For studies of LPS stimulation, splenocytes were cultured for 2 hrs in 96 well plates at 2 × 105 cells/well in RPMI1640 with 5% heat-inactivated FBS, 100 u/ml penicillin, 100 μg/ml streptomycin with the indicated concentrations of LMP-420. One μg/ml LPS was added and TNF content of the media was measured after an additional 24 hrs of culture. For studies using CD3 stimulation, splenocytes (1 × 106/ml) were incubated with media (RPMI1640 with 5% heat-inactivated FBS, 100 u/ml penicillin, 100 μg/ml streptomycin) or the indicated concentration of LMP-420 in media for 2 hrs at 37°C in polypropylene tubes and then 200 ml of cell suspension was added to each well of a 96-well BioCoat™ antimurine CD3 plate (BD Biosciences, Franklin Lakes, NJ, USA). The TNF content of culture supernatants was measured at both 24 and 48 hr time points for CD3-stimulated splenocytes.
Treatment with LMP-420
LMP-420 was custom synthesized by Scynexis Inc. (Research Triangle Park, NC, USA) under provisions of a Material Transfer Agreement between LeukoMed (Raleigh, NC, USA) and Duke University. For i.p. injections, a 10 mg/ml stock solution was prepared in 5% sorbitol, pH 9.0 in sterile water and further diluted as necessary to deliver the desired dose in a volume of 0.2 ml. For oral delivery, LMP-420 was mixed with powdered rodent diet containing 20 μg omeprazole per 3.5 g food to minimize gastric degradation of LMP-420. All control mice in oral dosing studies also received food containing omeprazole. The method of serial dilutions was used to ensure uniform mixing of drugs into powdered food. The weight of food consumed daily was recorded and averaged to determine the mean dose received per kg body weight during therapy. For studies involving both i.p. and oral administration of LMP-420, the doses listed are as administered. Serum and tissue levels of LMP-420 were not measured in this study.
Tissue analysis
Mice were euthanized by CO2 asphyxiation in accordance with the American Veterinary Medical Association Panel on Euthanasia. The entire colon (cecum to anus) was removed and colon length was measured from the ileocecal junction to the anus. The colon was then divided into segments representing the cecum, proximal, mid-, distal, and terminal colon/rectum. Colon tissue obtained from the proximal ends of the mid-, distal, and terminal colon segments was harvested for TNF measurement. Five colors of permanent tissue marking dye (Bradley Products, Bloomington, MN, USA) were used to specifically identify each colonic segment. These tissues were fixed in Carnoy's solution for 2 – 4 hrs, then processed and embedded into paraffin.
The severity of inflammation seen in hematoxylin and eosin-stained sections was scored independently by a pathologist blinded to treatment group. Histologic scores were calculated as described, using a scale that takes into account mucosal changes in 5 different bowel segments, including hyperplasia and ulceration, degree of inflammation, and % of each bowel segment affected by these changes [[26]; modified from [27]]. Using this scale, the maximum score is 75 and a score > 12 indicates colitis.
Cytokine/cytokine receptor measurements
Stool for cytokine analysis was collected before DSS exposure began (day 0), at the end of each 5 day cycle of DSS administration (day 5, 26, and 47, and after 16 days of recovery prior to beginning the next DSS cycle (days 21, 42, and 63). Freshly obtained stool was kept on ice until it was homogenized at 100 mg stool/ml buffer in PBS containing 1% bovine serum albumin, 0.1% Kathon (a microbiocide; Supelco, Bellefonte, PA, USA), and Protease Inhibitor Cocktail (Sigma-Aldrich, St. Louis, MO, USA). Insoluble material was removed by centrifugation at 4°C at 15,000 × g in a microfuge for 10 min. Stool extracts were filtered at 0.2 μm then stored at -20°C until assayed. Fresh colon samples were homogenized at 100 mg tissue/ml buffer using a PowerGen 125 tissue grinder (Fisher Scientific, Suwanee, GA, USA) and the BioPlex Cell Lysis Kit (BioRad, Hercules, CA, USA) according to the manufacturer's instructions. Colon tissue extracts were then frozen at -20°C until analysis. TNF and TNF-RII were quantitated in tissue culture supernatants, stool, and colon tissue extracts by enzyme immunoassay using Duo-Set reagents (R&D Systems, Minneapolis, MN, USA). Results were expressed as pg/ml for culture supernatants or as pg/100 mg tissue or stool.
Statistical analysis
Statistical comparison of groups was performed using Student's t test or analysis of variance (ANOVA). A value of p ≤ 0.05 was considered to be significant.