| OBJECTIVE:S037 is an antibody-conjugated drug(Antibody-Drug Conjugate,ADC).The antibody targeting HER2 is covalently linked to the microtubule inhibitor DM1 by stabilizing the thioether linker SMCC,and is clinically used for the treatment of tumors.Firstly,the analytical methods for determination of S037 and KadcylaTM total antibodies and conjugated antibodies in cynomolgus monkey serum and the analytical methods for determination of anti-drug antibodies in cynomolgus monkey serum were verified to ensure the accuracy,reliability and sensitivity of the method.This method was applied to the analysis of serum samples of cynomolgus monkeys.Explore its toxicity and its characteristics of toxicokinetics,to determine the targetAnn organs or target tissues,and to obtain preclinical safety information,when administered S037 via intravenous to cynomolgus monkeys for 8 consecutive cycles and after a 6-weeks recovery phase.Clinical trial design provides a reference.On the other hand,by comparing the toxicity with the marketed drug KadcylaTM,bridging the existing toxicity data of the drug provides a basis for guiding clinical trials.METHODS:The analytical method for determination of S037 and KadcylaTM total antibody and conjugated antibody in cynomolgus monkey serum is ELASA method;methodological verification content,including standard curve reproducibility,selectivity,intra-and inter-assay accuracy and precision,specificity,dilution linearity,Hook effect,parallelism,stability,and reanalysis by sample;S037 and Kadcyla's response to methodological comparability.The analytical method for determination of anti-drug antibodies in cynomolgus monkey serum is a bridging ELISA method;the methodological contents include:screening test Cut point value and confirmatory Cut point value;system adaptability(QC sample acceptance standard);sensitivity;precision;Matrix effect/selectivity;drug tolerance;Hook effect;quality control sample stability.50 Cynomolgus Monkeys(25 males and 25 females)were randomly assigned into 5 groups according to bodyweight.The monkeys were intravenously dose with vehicle control or S037 0.6 mg/kg,2 mg/kg,10 mg/kg or Kadcyla 10 mg/kg(5/sex/group).The drug was continuous intravenous infusion for 8 cycles at a constant rate of 5 mL/kg and 2 mL/min(once dosing every 3 weeks),following with a 6-weeks rsecovery period.Evaluation indicators include clinical observation,body weight,food intake,body temperature,ophthalmology,ECG,hematology,serum biochemistry,serum electrolytes,coagulation,urine,and gross anatomy,organ weight and its coefficient(organ to terminal bodyweight,organ to brain),histopathology and bone marrow smear examination,accompanied by toxicokinetic tests,immunogenic and immunotoxicity tests,safety pharmacology tests and local stimulation tests.RESULTS:The analytical method for determining the total antibody and conjugated antibody content of S037 and KadcylaTM in cynomolgus monkey serum ranged from 1.25 to 80.0 ng/mL with a minimum dilution of 1/100 and a quantitative range of 125 to 8000 ng/mL.The relative standard deviations of the absorbance values and main parameters of the six analytical batch standard curve samples did not exceed 12.9%;the conjugated antibodies did not exceed 16.1%.Dilution of 50 times or more of the substrate did not affect the measurement of S037,and the minimum dilution was set to 1/100.Cynomolgus monkey blank serum meets the selectivity requirements.The intra-assay and inter-assay accuracy of S037 total antibody in cynomolgus monkey serum ranged from 86.6%to 111%.The intra-assay and inter-assay precision was ?13.1%,the total error was ?13.9%.The intra-and inter-assay accuracy of the conjugated antibody ranged from 87.8%to 106%,the intra-assay and inter-assay precision is ?11.8%,and the total error is ?12.7%.The response of the test drug and the marketed drug to the method satisfies the analytical methodological comparability requirements.The total antibody and conjugated antibody of blank samples containing different concentrations of IgG in cynomolgus monkey serum were lower than the lower limit of quantitation.The average antibody accuracy(AR%)of LLOQ and ULOQ samples containing different concentrations of human IgG was 109%,110%,106%?108%;the average accuracy of coupled antibody(AR%)was 82.5%?89.7%,103%?104%.The average recoveries of total antibodies diluted in 1000,2000,4000,8000,16000,and 32000 samples of 1%cynomolgus monkey serum were 104%,103%,98.2%,104%,102%,and 107%,respectively;The average recovery rates were 92.0%,100%,98.8%,100%,100%,and 101%,respectively.The absorbance values of the total antibody and the conjugated antibody Hook effect sample in the serum of 1%cynomolgus monkey were both greater than the absorbance of ULOQ and there was no downward trend.The serum concentration of the four cynomolgus monkeys after individual administration was%CV ?3.62%of the total antibody concentration,and the%CV of the antibody concentration was ?4.29%.The total antibody concentration of the stable samples prepared by cynomolgus monkey serum was 89.3%?115%of the prepared concentration;the measured concentration of the coupled antibody was 80.9?112%of the prepared concentration.The total antibody concentration of the stability samples prepared by 1%cynomolgus monkey serum was 91.3%?110%of the prepared concentration;the measured concentration of the coupled antibody was 96.7%?111%of the prepared concentration.The retest value of 2/3 or more samples is within ±30%of the original measured value.Analytical Methodology for Determination of Anti-drug Antibodies in Cynomolgus Monkey Serum:Eighteen unadministered cynomolgus monkey serum were used for screening test establishment and precision verification of Cutpoint and validation test Cutpoint values.The correction factors for the floating Cutpoints of S037 and Kadcyla obtained through the screening test were 1.074 and 1.104,respectively,for calculating the Cutpoint value of each analysis batch;confirming that the Cutpoint values(CCP,%)of S037 and Kadcyla were 18.4 and 21.5,respectively.In the precision test,the CV(%)within the batch and the batch were less than 20%;the sensitivity of S037 was 39.1 ng/mL,the sensitivity of Kadcyla was 44.7 ng/mL;and 10 normal negative cynomolgus monkeys were selected by the selective test.The serum was tested.The results showed that HPC,LPC and NC met HPC>LPC>NC,and 80%of the individual NC samples were lower than or equal to CP,and the positive control samples were positive.In the drug tolerance test,at the LPC level.At least tolerate the interference of 30 ?g/mL S037 or Kadcyla in serum samples;the Hook effect test of S037 and Kadcyla has no Hook effect;the stability of the quality control sample(room temperature and 2?8 ? for 24 h,room temperature Repeated freezing and thawing 5 times,-20 0 C for 1 month and-80? for 6 months)meet the requirements of immunogenicity studies.The 8-cycle toxicokinetic results of S037 cynomolgus monkeys showed that the concentrations of the three tested substances(total antibody,conjugated antibody,DM1)in the vehicle control group were BLQ.There was no significant gender difference in the exposure levels of the three tested substances in the S037 dose group and the Kadcyla control group.After the first cycle of administration,the AUC0-t ratios of total antibody,conjugated antibody and DM1 in each dose group of S037 were 1:6.88:29.5,1:5.87:21.5 and 1:13.2:79.5,respectively.The ratio of dose increase(dose ratio 1:3:10)showed nonlinear toxicokine dynamics;the other cycles showed similar characteristics;the toxicokinetic characteristics and exposure level of S037 at the same dose of 10 mg/kg.It is comparable to the Kadcyla control group.After repeated administration for 8 cycles,the exposure levels of the three tested substances in S037 group increased with the increase of the number of doses,and no obvious accumulation was observed.Under the condition of 10 mg/kg,S037 and Kadcyla were in the cynomolgus monkey.The level of exposure in the body is comparable.In this trial,S037 0.6 mg/kg,S037 2 mg/kg,and Kadcyla 10 mg/kg groups produced different levels of ADA after administration(c4 to end of recovery)(5/10,1/10,and 1/10);combined with TK data analysis,some animals with higher titer ADA(S037 0.6 mg/kg group 2102#,2104#)after the 5th to 8th cycle of drug concentration is lower than other animals in the group,and the blood concentration at multiple time points was BLQ,suggesting that some ADA-positive animals had an effect on their toxicokinetics,but the other ADA-positive animals did not have a significant effect on toxicokinetics.In addition,the ADA-positive samples were tested for neutralizing activity using a validated Cell-based method(HER2 high expression human breast cancer BT-474).The results showed that some animals produced ADA after S037(0.6 mg/kg)administration.At higher titers,there were different degrees of neutralization activity.Among them,2104#animals detected strong neutralizing activity in ADA samples at 4 time points;ADA produced after Kadcyla(10 mg/kg)administration also had a certain degree of neutralization activity.Under the basic conditions of this material,S037 cynomolgus monkeys were infused intravenously for 8 cycles,and S037 low-dose group 0.6 mg/kg males(1104#)cynomolgus monkeys c2,c3,c4 showed loose stools and red stools.C4-d11,c4-d12 can see symptoms such as weak or paused breathing,mouth breathing and unstable standing,so euthanasia is performed on c4-d12;gross anatomy shows histopathological changes such as pleural effusion and acute pericarditis,combined with Clinical Observation,clinical pathology,toxic exposure,ADA test results,and toxicity and tolerance of animals in the middle and high dose groups were considered as non-drug-related deaths.The remaining animals showed no death,and the main toxicity reactions were summarized as follows:S037 high dose group 10 mg/kg,Kadcyla control group 10 mg/kg visible loose stools(3/10,3/10),skin erythema(7/10,5/10),skin flushing(1/10,1/10),skin damage(2/10,1/10),skin ulceration(0/10,1/10).S037 10 mg/kg group showed poor feeding conditions in w7,w10,w11,w13,w16,w19,w20,but no significant abnormalities in body weight and body weight;in addition,1302#vomiting,hair loss,activity reduction,contracture Into a group,1304#bleeding gums,red gums,dandruff,rash.The hematology indicators(mainly PLT,PCT,MONO#)related to the administration hematology f 10 mg/kg in the S037 high-dose group and 10 mg/kg in the Kadcyla control group occurred mainly on the 5th day of each dosing cycle,showing periodicity.Decrease(PLT,PCT)or increase(MONO#),and the two groups changed to the same extent and returned to normal levels on the 21st day of each dosing cycle.Changes in serum biochemical indicators were mainly observed on the 5th day of each dosing cycle.ALT,AST,ALP,and CK were periodically increased,and returned to normal levels on the 21st day of each dosing cycle.The KadcylaTM control group increased slightly more than the S037 high dose group 10 mg/kg.In addition,TP ?,GLO t,and A/G ? were observed in both groups of c4-d5 and c8-d5,and the results of immunotoxicity test showed IgG?.In the two groups,the coagulation routine indicators showed APTT ?,TT ?,FIB concentration ?,FIB ? in c4-d5 and c8-d5,to the same extent.Histopathology showed increased mitosis in individual Kupffer cells(6/6,6/6),single Kupffer cell hypertrophy(6/6,6/6),subcapsular hepatic sinus congestion(4/6,1/6),spleen red pulp tissue cells mitotic like increased necrosis(6/6,5/6),thymus lymphocytes decreased(6/6,5/6),breast acinar epithelial cells increased mitosis,increased apoptosis(4/5,4/5),mitotic figures of seminal vesicle epithelial cells increased,apoptosis increased(2/3,3/3),erythrocyte hematopoietic cells decreased(6/6,5/6),sciatic axis Mutagenicity(3/6,2/6).Bone marrow smear examination showed that the bone marrow cells counted elevated granules and decreased lymphocytes.S037 medium dose group 2 mg/kg clinical observation showed loose stool 2/10,skin erythema 6/10,rash 1/10,scar 1/10,trauma 1/10,skin ulceration 1/10;Hematology and Serum biochemical indicators showed PCT ?,ALT ?,AST ? at each dosing cycle d5,and the trend of each cycle was the same as that of S037 high dose group 10 mg/kg.Histopathology showed a single Kupffer cell hypertrophy(1/6)in the liver,a decrease in thymic lymphocytes(1/6),an increase in mitosis in breast acinar epithelial cells,increased apoptosis(1/5),and acinar glandular epithelium.The number of mitotic cells increased and apoptosis increased(1/3).There was no drug-related toxicity change in the S037 low-dose group at 0.6 mg/kg compared with the vehicle control group.After a 6-weeks recovery period,the above-mentioned toxic reactions such as blood system,spleen,thymus,liver,breast,seminal vesicle,bone marrow and sciatic nerve all recovered or showed a clear recovery trend(thymus).CONCLUSION:The method for determining S037 in cynomolgus monkey serum and the immunogenicity detection method have been verified and can be used in the study.Under the conditions of this study,S037 cynomolgus monkeys were intravenously infused for 8 cycles of repeated dose toxicity test,NOAEL(no observed adverse effect level)was 0.6 mg/kg(the 8th cycle:the exposure of total antibody,conjugated antibody and DM1 was 1135±342 h·?g/mL,854±168 h·?g/mL and 11.3±10.6 h ng/mL,respectively;HNSTD(highest non-severe toxicity dose)was 10 mg/kg(AUC0-t of total antibody,conjugated antibody and DM1 in cycle 8 was 36220±15200 h ?g/mL,16740±2225 h·?g/mL and 601±149 h ng/mL,respectively).It is mainly manifested as the toxicity of the blood system,spleen,thymus,liver,breast,seminal vesicle,bone marrow,sciatic nerve.At the same dose of 10 mg/kg for S037 and KadcylaTM,both the levels of exposure and the toxicity was similar in cynomolgus monkeys(the total antibody,conjugated antibody and DM1 exposure AUC0-t of the 8th cycle were 41550±19620 h·?g/mL,20460±3817 h·?g/mL and 622±265 h·ng/mL,respectively.). |
PDF Full Text Request