The Study On Preclinical Pharmacokinetics And Major Pharmacodynamics Of Recombinant Humanized Antibody SM09

Recombinant humanized antibody SM09（short for SM 09）is directed against the CD20 antigen found on the normal and malignant B lymphocytes.SM09 is presently under the development as the first class of national biotechnology new drug.The work of this paper was designed to evaluate the pharmacokinetic feature and to support for clinical pharmacokinetics study.The determination of affinity constant of SM09The protein quantification study on SM09 and Rituximab（a chimeric CD20 monoclonal antibody is used for clinical therapy of CD20 positive B-cell non-Hodgkin’s lymphoma）shows that SM09 and Rituximab have unified protein level.The determination of the dissociation constant（Kd）and affinity constant（Ka）of SM09 by Surface Plasmon Resonance（SPR）technique is 3.65×10^-9M and 0.27×10⁹ M^-1respectively.It shows that SM09 has a better binding affinity for the CD20 antigen,and similar with Rituximab Kd 9.55×10^-9M.The study on pharmacodynamics of SM09 in vitroSM09-FITC is applied for direct immunofluorescence staining Raji,Ramos and Daudi cell lines （Burkitt’s lymphoma）for flow cytometric analysis.CD20 antigen presents on more than 98%Raji, Ramos and Daudi cells.Raji present the highest level of CD20 antigen,Ramos present the lowest level.In viro cell experiment study indicated possible mechanisms of cell lysis include direct lethal effect,complement-dependent cytotoxicity（CDC）,antibody-dependent cellular cytotoxicity（ADCC） and induce apoptosis. The study on pharmacodynamics of SM09 in vivoI.V.infusion of 30mg·kg^-1SM09 in 3 rhesus monkeys,CD20⁺ cell was rapid deplete after 1 hour of the adminstration and stay in low level for a long term.T cell and mononuclear cell/ macrophage were not significant changed.Red blood cell count,white blood cell count and mean corpuscular hemoglobin were not significant changed.The establishment of analytical method of SM09 in biological matrixSM 09 was labeled ¹²⁵I by Chloramines T method,the radioactivity purity of ¹²⁵I- SM09 was 96.0±0.3%.¹²⁵I- SM09 can compete with non-radiolabeled SM09 in binding to CD20 antigen present on Raji cell.SHPLC and TCA precipitation methods in possession of speciality,sensitivity, precision,accuracy and recovery was consistent with demand of preclinical pharmacokinetics study.Pharmacokinetics study of SM09 in rhesus monkeyFollowing a single administration with different dose of SM09 to rhesus monkey(100 mg·kg^-1、30 mg·kg^-1、10 mg·kg^-1).It was shown that the pharmacokinetic parameters of rhesus monkey were fit to tow-compartment model and linear kinetics characteristic in the range of administration dosage.Analytical method of SM09 by gross radioactivity,TCA precipitation and SHPLC method were comparability.Multiple iv infusion with 30mg.kg^-1/week for 4weeks of ¹²⁵I -SM09 to rhesus monkey,the pharmacokinetic parameters of rhesus monkey were fit to tow-compartment model.There was no significant increase of AUC_0-tand t_1/2βafter the fourth administration.Fluctuation（FI）and degree of fluctuation（DF）of the fist and fourth administration had no statistical changes.It suggested that SM09 did not accumulate in vivo.Tissue distribution and excretion study of SM09 in rhesus monkeyAfter i.v.administration(10 mg·kg^-1)of SM09,it showed a higher distribution in blood,gastric contents,liver,spleen and lymph node of neck.Urinary system also had a higher radioactivity.The result suggested that SM9 is excreted mainly by kidney and urine.The analysis of serum sample after administration by SHPLC method showed that 8.0min retention time of radiochromatogram is origin 125I-SM09 peak.There is no obviously ¹²⁵I labeled degradation or Macromolecular binding peak. SM9 is excreted mainly by kidney and urine,a small amount is excreted from feces.After 72 hour of administration,70%radioactivity was excreted.The excretion was slow from 72 hour to 31 day.¹²⁵I-SM09 was added in blank rhesus monkey blood plasma,incubated 6 hours at room temperature before SHPLC analysis.There is no plasma protein binding or degradation.