Pharmacokinetics Investigation Of CpG ODN107, A Novel Radiosensitizer Agent

Malignant gliomas are the most aggressive and common brain tumor with a highmortality. Despite combined postoperative therapeutic efforts such as radiotherapy haveprovided significant survival advantage compared with surgery alone, but the result remainspoor in patients with glioblastoma multiform because the tumor cells are resistant toradiotherapy. Although increased radiation dosage can improve local control of tumordevelopment, it also leads to serious side effects. Therefore, it is significant to search forradiosensitizer to increase sensitivity of malignant glioma to normal dosage of radiotherapy.CpG ODN107(CpG ODN107), a TLR9agonist, is a newly identified stimulatory CpG inour lab. It is consisted of only twelve oligodeoxynucleotides. In the present experiments, therole of CpG107and its possible molecular mechanisms of radiosensitization wereinvestigated to get radiosensitizer with high efficacy and low toxicity. Therefore, it has beeninvestigated as a potential radiosensitizer for glioma. Therefore, the pharmacokinetic (PK)data of CpG ODN107should be investigated in preclinical study, even in clinical research.This work is one of the important reaserch content of major scientific and technologicalspecial project for "Significant New Drugs Creation" of China (2009ZX09103-051), whichconsisted of the absorption, distribution, metabolism and excretion of CpG ODN107in themice or rats.The main research contents:1. Development and validation of LC-MS/MS method for the detection andquantification of CpG oligonucleotides107(CpG ODN107) and it’s metabolites in miceplasma or other biological samplesThe establishment of LC-MS/MS method for determination of CpG ODN107and itsmetabolite in biological samples.Validation of LC-MS/MS method was investigated includespecificity, sensitivity, accuracy, precision, linear and recovery. Based on the revision, theLC-MS/MS method for different biological sample was optimizated by drug extraction, and was applied to analyze a variety of other biological samples, include urine, feces, bile andimportant tissues and organs of homogenate.2. The concentration-time curves of CpG ODN107and its metabolites following asingle intravenous administration in mice.Four doses (2.5,5,10and15mg/kg) were chose as the intravenous injection dose. Theblood collection time points are0h and after administration2min,5min,15min,30min,60min,120min,180min,240min,360min,540min and720min. The plasmaconcentration-time curve (C-T curve) of CpG ODN107with four doses and it’s metabolismwith15mg/kg were traced. Compartment model and non-compartmental model (matrixstatistics) are applied to calculate the PK parameters, and AUC0-tand Cmaxof CpG ODN107are analysis by the linear regression analysis method.3. The distribution of CpG ODN107in mice(1) Three doses were chose to investigate plasma protein binding of CpG ODN107. Invitro binding of CpG ODN107human, mouse plasma protein and HAS were determined byultrafiltration methods.(2) One dose (5mg/kg) was chose to investigate the distribution of CpG ODN107inmice, the tissues and organs collection time points are0h and after administration0.5h,1h,2h,4h,10h and24h. Six time points (5mice/group) were selected to extract liver, heart,brain, spleen, lung, kidney, intestine, muscle and gonads.And then, determinate theconcentrations of CpG ODN107in these various tissues and organs by LC-MS/MS.(3) The distrubtion of CpG ODN107in brain are investigated by optical molecular imagingfor in vivo analysis.4. The excretion of CpG ODN107in miceOne dose was chose for the intravenous injection dose on to feces, urine and bileexcretion studies in mice or rat.The main results:1. Development and validation of LC-MS/MS method for the detection andquantification of CpG oligonucleotides107(CpG ODN107) and it’s metabolites in miceplasma or other biological samplesa novel and sensitive reversed phase HPLC coupled with electrospray triplequadrupole mass spectrometry method followed one-step C18solid-phase extraction (SPE) for biological matrix removal, had been developed and fully validated for the determinationof CpG ODN107and its metabolites such as5’N-1,3’N-1,3’N-2and3’N-3in mice plasma.The analytes were separated on an Extend-C18analytical column (3.5μm,150mm×2.1mm) using an eluent of acetonitrile-0.05%NH3aqueous (20:80, v/v) and detected byelectrospray ionization (ESI) mass spectrometry in the negative multiple reactionmonitoring mode (MRM). The assay was specific, and its linearity was superb (r2≥0.998)for CpG ODN107and its metabolites in the biological matrices. The precision, accuracyand relative recovery values were found to be <15%,±15%, and95-105%, respectively.It was meet for requirement to investigate the PK of CpG ODN107.2. The concentration-time curves of CpG ODN107and its metabolites following asingle intravenous administration in mice.After four dose intravenous administration of CpG ODN107, the results from softwareof DAS showed the mean plasma concentration-time curve of CpG ODN107in mice was anattenuation curve. Plasma concentration-time data was fitted to the three-compartmentmodel. In the non-compartmental model, mean residence time (MRT0-t) were48.37、58.94、54.19and53.74min, respectively.The mean total body clearance (CL) were0.013、0.012、0.013and0.005L/min/kg, respectively. The area under the plasma concentration-timecurve from time zero to the time of the last measurable concentration (AUC0-t) were185974.779，412136.286and732853.511ng/mL*min, respectively. The AUC0-tand CmaxofCpG ODN107suggestted it fitted to the non-linear dynamics in mice, which calculated bythe linear trapezoidal method.After intravenous injection of15mg/kgCpG ODN107, CpG ODN107was rapidlymetabolized into3’N-1,3’N-2,3’N-3and5’N-1. After the Tmax, all of the metabolites levelsdeclined, which was similar to that of the parent drug. At6h post dose, there was nometabolite to be tested. AUCm/AUCp(metabolite AUC0tdivided by CpG ODN107AUC0t)indicated that the relative amounts of the metabolites. The metabolite level of3’N-1,3’N-2,3’N-3and5’N-1was6.93%,3.75%,2.07%and3.39%of the parent drug in blood,respectively.3’N-1was the major metabolite in blood.3. The distribution of CpG ODN107in mice.(1). CpG ODN107was shown to be highly binding with plasma protein of human andmouse (>96%) and HAS (>90%) by in vitro plasma protein binding investigated.The results indicated that CpG ODN107was primarily bound to HAS in human plasma.(2) After intravenous injection of5mg/kg, the CpG ODN107of AUC0-24in descendingwas liver, kidney, spleen, muscle, heart, lung, testis, bowel, brain. There was no detected inbrain, which may infer that CpG ODN107is not easy to get through the blood-brain barrier.The concentration in liver tissue was highly, in addition, it was rapidly distribution andelimination. At1h post dose, the concentration in kidney tissue was highly, may infer kidneyexcretion. The concentration was lower in the muscle, lung, testis and bowel tissue.(3) CpG ODN107distributed into brain tissue of needling after intracalvarium injectionof0.25mg/kg CpG ODN107by marked fluorchrome CY5. At4h post dose, it diffused thewhole brain tissue, even no complete elimination at24h.4. The excretion of CpG ODN107in miceCpG ODN107was excreted in the form of whole sequence and a series of shortenedoligonucleotides after intravenous administration of a single dose to mice and rat. In mice,the accumulated amount of excretion of CpG ODN107contributes2.7%of the dosage inthe first24hours after the injection of the drug, include in the urine of the whole sequencecontributes1.79%of the dosage and feces contributes0.91%of the dosage. The totalamount of the whole sequence of the drug in the two parts, and the amount in urine was amajor part. In rat, the accumulated amount of excretion of CpG ODN107contributes0.84%of the dosage in the first24hours after the injection of the drug, include in the urine of thewhole sequence contributes0.63%of the dosage and feces contributes0.21%of thedosage.CpG ODN107is not detected in the bile of rats.Conclusion：1. A novel and sensitive LC-MS/MS method had been developed and fully validatedfor the determination of CpG ODN107and its metabolites in biological specimen.2. The plasma concentration-time data of CpG ODN107were fitted to athree-compartment model, and fitted to the non-linear dynamics in mice. The majormetabolite of CpG ODN107was3’N-1. All of the metabolites levels declined similar to thatof the parent drug.3. CpG ODN107was highly binding with plasma protein of human and mouse(>96%).CpG ODN107majorly distrubuted in liver, kidey and spleen after intravascularadministration with rapidly distribute and eliminate. But CpG ODN107was not detected in brain. CpG ODN107distributed into brain tissue of needling after intracalvarium injection.4. CpG ODN107was excreted in the form of whole sequence and a series of shortenedoligonucleotides after intravenous administration of a single dose to mice and rat. The majorexcretive path was homaluria. CpG ODN107was not detected in bile.