生物活性

Infliximab, a chimeric human-murine anti-TNFαmonoclonal antibody, is widely used for the treatment of inflammatory boweldisease (IBD).

体外研究

Infliximabdirectly affects psoriatic T cells and impairs the antigenpresenting capacityof DCs. Infliximab strongly and dose-dependently impaired in vitro activationof psoriatic as well as antigen (nickel)-specific skinhoming T cells, in termsboth of proliferation and of IFN-γ release.[1]

体内研究

激酶实验

Western blot[2]

Binding of IFX to recombinant (r) TNF-α was studied using standard denaturing Western blot techniques. Briefly, 2-mercaptoethanoland heat-treated rTNF-α was loaded onto a preprepared iBlot sodium dodecylsulphate (SDS) denaturing gel (Novex) in denaturing SDS running buffer (NuPAGETris-Acetate SDS). After transferring to a nitrocellulose membrane, the gel wasblocked (5% fish gelatin for 1 h, shaking), washed [1% phosphate-buffered saline(PBS)-Tween20 x 4] and primary antibodies added [infliximab, 2μg/ml;polyclonal rabbit anti-mouse TNF-α, 10μg/ml]. Membraneswere incubated for 16 h at 4 ^oC, washed and secondary antibodies[rabbit anti-human IgG, 1μg/ml; goat anti-rabbit, 2μg/ml] were added(1 h withshaking). Membranes were then washed and treated with ECL (1 min) beforeimaging using Bio-Rad Chemidoc. Data were processed using Image Lab Software.

细胞实验

TNF-Capture ELISA[3]

The TNF-binding capacity of Infliximab wasquantified by a capture ELISA. The method in brief as follows: a 96-well ELISAplate was coated overnight at 4 °C with mouse-anti-human TNFα capture antibody(1:400; Pelikan TNF kit) 100μL per well. The plate was washed three times withwash buffer (PBS containing 0.05% Tween80) followed by blocking for 2 hourswith the blocking buffer (1% m/v fish skin gelatin). Later, 100μL of solution containing10ng/mL in blocking buffer Human-rTNFα was added and incubated for 1h at room temperature.After washing, 100μL of diluted Infliximab control in blocking buffer(500ng/mL) and reconstituted samples were added to the corresponding wells. Thiswas followed by addition of 100μL of detection antibody–goat-anti-IgG HRP wasadded and incubated for 1h at room temperature. The bound antibody was detectedwith tetramethyl benzidine (TMB) as peroxidase substrate. The enzymaticreaction was allowed to proceed at roomtemperature for 3 min and stopped byadding 50μL of 1M sulfuric acid. The absorbance was measured at a wavelength of450 nm using an Envision plate reader. The data is represented as actualconcentration vs absorbance.

MSCs were obtained from the bone marrow of3‑week‑old SD rats. Following euthanasia, whole bone marrow was flushed withDMEM from the tibia and femur of the SD rat. The marrow was pooled andcollected in fresh tubes. The marrow suspension was then centrifuged at 157 x gfor 10 min. The supernatant was removed and the pellet was resuspended with low‑glucoseDMEM containing 10% FBS. Cells were plated in a 25 cm² flask andincubated in a humidified atmosphere with 5% CO₂ at 37˚C. The medium was changed after 2 days and the nonadherent cellswere removed. The medium was then changed every 3 days. When the cells were at80‑90% conflu­ence, the adherent cells were detached with 0.25% trypsin EDTAand replated at a 1:2 ratio. The cells were further puri­fied with passages.

To evaluate the effects of infliximab onthe MSCs derived from the bone marrow of SD rats, the MSCs at passage 4 wereused. The cells were grown in various concentrations of infliximab‑supplemented(0, 0.10, 0.20, 0.30 and 0.40 mg/ml) DMEM with 10% FBS. The MSCs were incubatedin a humidified atmosphere with 5% CO₂ at 37˚C.

Cellproliferation assay

The MSCs at passage 4 were cultured with infliximab‑supplemented DMEM as described above in 6‑well culture dishes with 5x10⁴ cells/well for threereplicate wells for each treatment and each day. The cells were trypsinized andcounted every 24 h for eight consecutive days, respectively. The growth curvesof the MSCs from each group were obtained from three independent experiments.

动物实验

Rat experiments[5]

Animalsand treatment

Male Wistar rats weighing 180–200 g andaged 12 weeks were housed with a maximum of four animals per cage undertemperature- and light-controlled conditions room (23 ± 1.5 ^oC).

Experimental colitis was induced bytreating animals with 5% DSS (dextran sulfate sodium) in drinking water. Theanimals were divided into two experimental models: i) rats treated with DSS for6 d and sacrificed on the sixth day (acute/induction phase), and ii) rats treatedwith DSS for 6 d and sacrificed on the fifteenth day (recovery phase). In eachexperimental phase, three groups of rats were employed: healthy rats (controlgroup) and rats subjected to experimental colitis, without infliximab (IFX)treatment (DSS group) or with IFX treatment (DSS + IFX group). In the DSS + IFXgroups, IFX (5 mg/kg, i.p.) was administered on days 0 and 5 (acute/inductionphase), or on days 0, 5 and 10 (recovery phase). In acute and recovery phaseexperiments, five or six animals were used per experimental group. Once therats had been sacrificed, serum and tissues from the colon were obtained. Thetreatment of rats with DSS in drinking water is the most widely used UC model.

Generalassessment of colitis

To characterize the extent of the disease,we determined daily body weight evolution and colon length after sacrifice. Colonlength is a good morphologic parameter to indicate the degree of inflammation.Fecal counts, stool consistency and rectal bleeding were monitored daily. Wealso measured daily food and water consumption. The disease activity index(DAI) was determined by scoring body weight, stool consistency and blood in stoolsas described previously, with minor modifications (Table 1A). The diarrhealindex (DI) was calculated by substituting weight loss by the number of fecal countsper day/g of food consumed (frequency) in the DAI (Table 1B).

Biochemicalparameters

Serum samples for biochemical analysis werecollected from the rats at the time of sacrifice by cardiac puncture (Table 2).Glucose levels were analyzed using a hematology analyzer, determination ofserum IFX levels was performed by ELISA, detection of serum cytokines (IL-1a,IL-4, IL-17A, MCP-1, IFN-γ and TNF-α) was performed using Rat Cytokine 5 plex Kit FlowCytomix and serumAIF-1 levels (using a rabbit anti-AIF-1) were studied by immunoblot techniques.

Tissuepreparation

Proximal colon samples were fixed with neutralformalin, washed with PBS, dehydrated with graded ethanol series and embeddedin paraffin. Five-micron thick sections were obtained and mounted on silanizedglass slides.

Colonmucosal integrity evaluation

Periodic acid Schiff (PAS) and alcian blue(AB) stains were used to evaluate colon mucosal integrity, to visualize goblet cellsand above all to differentiate neutral and acidic mucin. Briefly, the sampleswere dehydrated with ethanol and n-butanol for 48 h at 4 ^oC, afterbeing included in paraffin. Subsequently, they were cut with the microtome; somesamples were also stained with AB (15 min) and washed with water. Then, thesamples were treated with periodic acid (1%) daily for 5 min and washed withdistilled water. Finally, treated with the Schiff’s reactive (5 min) and againwashed and stained with Harris hematoxylin (2 min). The tissue expression ofthe neutral mucins was determined by means of the histochemical technique ofPAS while the expression of acid mucins was determined using the AB technique.The neutral mucins stained magenta, while the acid mucins stained blue. Only inmagenta stained cells is recorded.

不同实验动物依据体表面积的等效剂量转换表（数据来源于FDA指南）

例如，已知某工具药用于小鼠的剂量为88 mg/kg , 则用于大鼠的剂量换算方法：将88 mg/kg 乘以小鼠的Km系数（3），再除以大鼠的Km系数（6），得到该药物用于大鼠的等效剂量44 mg/kg。

参考文献

[1] Bedini, C.; Nasorri, F.; Girolomoni, G.; Pita, O.; Cavani, A., Antitumour necrosis factor-alpha chimeric antibody (infliximab) inhibits activation of skin-homing CD4+ and CD8+ T lymphocytes and impairs dendritic cell function. Br J Dermatol 2007, 157 (2), 249-58.
[2] Assas, B. M.; Levison, S. E.; Little, M.; England, H.; Battrick, L.; Bagnall, J.; McLaughlin, J. T.; Paszek, P.; Else, K. J.; Pennock, J. L., Anti-inflammatory effects of infliximab in mice are independent of tumour necrosis factor alpha neutralization. Clin Exp Immunol 2017, 187 (2), 225-233.
[3] Kanojia, G.; Have, R. T.; Bakker, A.; Wagner, K.; Frijlink, H. W.; Kersten, G. F.; Amorij, J. P., The Production of a Stable Infliximab Powder: The Evaluation of Spray and Freeze-Drying for Production. PLoS One 2016, 11 (10), e0163109.
[4] Huang, H. R.; Zan, H.; Lin, Y.; Zhong, Y. Q., Effects of azathioprine and infliximab on mesenchymal stem cells derived from the bone marrow of rats in vitro. Mol Med Rep 2014, 9 (3), 1005-12.
[5] Roman, I. D.; Cano-Martinez, D.; Lobo, M. V.; Fernandez-Moreno, M. D.; Hernandez-Breijo, B.; Sacristan, S.; Sanmartin-Salinas, P.; Monserrat, J.; Gisbert, J. P.; Guijarro, L. G., Infliximab therapy reverses the increase of allograft inflammatory factor-1 in serum and colonic mucosa of rats with inflammatory bowel disease. Biomarkers 2017, 22 (2), 133-144.

体内溶解度

Clinical Trial Information ( data from http://clinicaltrials.gov )

注：以上所有数据均来自公开文献，并不保证对所有实验均有效，数据仅供参考。

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