| EGFR is a member of the ErbB family of receptor tyrosine kinases.EGFR signalling in tumor cells is responsible for regulating a diverse network of cellular function that influence neoplastic growth.The expression of EGFR in human cancer has provided a scientific rationale for the development of EGFR antagonists as potentially useful therapeutic agents.Monoclonal antibodies that inhibit EGFR function offer a specific class of EGFR antagonists.Cetuximab(also referred to as C225-03,IMC-C225,C225,ch225) is a chimeric monoclonal antibody that binds with high specificity to the extracellular domain of the human EGFR.The antibody is intended to function as a competitive antagonist that inhibits ligand binding to the EGFR,and may lead to degradation of EGFR.This study includes in vivo and in vitro antitumor activity experiments and pharmacokinetic experiments in healthy monkey,aimed to compare domestic C225 with marketed product Erbitux.Part 1 Antitumor Activity study of C225We have evaluated the growth-inhibitory effect of C225 in tumor cells(human gastric cancer cell line BGC-283,human esophageal cancer cell line Eca109 and human lingual cancer cell line KBV-200) which not express EGFR,and three tumor cells(human breast cancer cell line MCF-7,human colon cancer cell line HT-29 and human non-small lung cancer cell line A549) which highly express EGFR in vitro by the cell-colony method.A concentration-dependent growth inhibition in vitro was observed when EGFR-expressing tumor cells were treated with MAb C225,but it was not inhibiting the growth of no-expressing tumor cells(Eca109,BGC-283 and KBV-200).The study firstly demonstrated that independently use the domestic C225 or combine with carboplatin in A549 xenograft model of high level EGFR could inhibit the xenograft growth.For NSCLC A549 xenograft model,either independently use domestic C225 or carboplatin showed some anti-tumor effect,but without an ideal tumor proliferation rate T/C%.However,when domestic C225 was combined with carboplatin,tumor growth was significantly inhibited,even completely inhibited during drug injection period.The two relative tumor proliferation rates T/C%were 10.7%(P＜0.01) and 8.9%(P＜0.01) in two separate experiments.The growth depressive effect of recombination C225 to HT-29 transplanted nude mice.Histological examination of the tumours showed large areas of necrosis and fibrosis.A marked decrease in tumour cell proliferation was observed after combined treatment,in comparison to control or single agent-treated tumours.A marked decrease in tumor cell proliferation was also observed in C225/irinotecan treated tumors,as measured by anti-Ki-67 IHC,in comparison to control or single agent-treated tumors.In addition,a decrease in microvessel density was observed using anti-CD31 IHC.The precise mechanisms were responsible for the combined effect of C225 and CPT-11 in colorectal tumor model are still unclear and will require additional studies,Nevertheless,the ability of combination therapy to inhibit growth of chemo resistant colorectal carcinoma tumors suggests that EGFR was blockade by C225 combined with CPT-11 refractory colorectal cancer.Part 2 Pharmacokinetics study of recombination C225The KA of domestic C225 and soluble recombined human EGFR was 4.00×108 1/M,KD was 2.50×10-9 M.The KA of reference product Erbitux and soluble recombined human EGFR was 4.25×108 1/M,KD was 2.35×10-9 M.This is similar to the affinitive-kinetic properties of EGFR and Erbitux,which is a product of Merk KGaA Company.Serum concentrations of C225 were determined via surface plasmon resonance (SPR) using a Biacore instrument.In this assay soluble human recombinant EGFR is immobilised to the sensor surface.Test solutions containing C225 flow continuously over the sensor surface.As C225 binds to the immobilised EGFR a response is registered.This biological quantitative analysis method has good specificity,the data are not interfered by matrix.The range of concentration standard curve of measuring was 0.500-50μg.mL-1,LLOQ was 0.500μg.mL-1.The precision and accuracy of intra/inter day of LLOQ and analyzing batch are all consonant with the requirements of the methodology of pharmacokinetic biological products analyzing.This method could be used to measure the concentration of C225 in Cynomolgus monkey serum.a.The results of a pharmacokinetics of C225 after a single intravenous infusion to Cynomolgus monkeys were submitted,Groups of animals received 7.5,24 and 75 mg/kg for 60 minutes,the drug concentration in serum reached peak after 0.5h in most bodys,and the concentration was parallel to the dosage of injected drug. Some time points had statistical variances among the low-medium,medium-high and high-medium dosage groups.The terminal phase half lives significantly extended with the increase of dosages.The drug concentration in serum of low dosage group decreased to 1/20 of the peak concentration 312h after drug injection,while the medium and high dosage groups needed 456h to reach the concentration.b.Comparing the pharmacokinetic parameters of the different groups,the average residence time significantly extended with the increase of dosage,the MRT was 98±6h,142±1h and 161±2h(P＜0.01 vs.intravenous infusion 7.5 and 24 mg·kg-1 group) separately.The dosage rate was 1:3:10,the growth of AUC was 1:7.2:18.7,not increase parallel to the dosage.The dose of of CLs were 0.48±0.03 L·h-1·kg-1,0.21±0.02 L·h-1·kg-1 and 0.26±0.04 mL·h-1·kg-1(P＞0.05 vs. intravenous infusion 7.5 mg.kg-1 group) separately.The clearance decreased and terminal half-life increased with increasing dose,it indicated a saturated clearance at higher doses,showing typical non-linear kinetic characters,This result showed that between the dose range,domestic C225 presented non-linear kinetic character in Cynomolgus monkey.Distribution volume indicated that C225 mainly existed in serum.c.Domestic C225 were intravenous administration to Cynomolgus monkey weekly after four times,the drug concentrations in serum and the pharmacokinetic parameters were similar between the first time administration and the last time.The rates of AUC0-(AUC0-t(4th time)/AUC0-t(1st time) of three Cynomolgus monkeys were 1.1,1.1 and 0.95 after the first time administration and the last time,1.01±0.08 in average,indicating that there was no accumulation after repeat dosing beyond administration weekly 4 times was observed.d.The experiment result of intravenous infusion of comparable product Erbitux and domestic C225 to Cynomolgus monkeys indicated that,generally,the shapes of C-T curves of Erbitux and domestic C225 in every monkey were similar,and the parameters as well.The Student's coupled t-examination of the concentrations of Erbitux and C225 in three Cynomolgus monkeys showed that,no statistical variances were found(P＞0.05) according to the concentrations at all time points after treatment of Erbitux or domestic C225.On the other hand,the results of pharmacokinetics indicated that,all the pharmacokinetic parameters(AUC0-408h, MRT,t1/2,CLS,VSS,Cmax) of the two drugs did not have statistical meanings (P＞0.05),indicating that the pharmacokinetic manners of domestic C225 and the Merk KGaA Company Erbitux were similar. |
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