| Objective:This thesis was designed to study the Preclinical Pharmacokinetic of Fusion proteinE2HSA.Methods:A chemiuminescent enzyme immumoassay(CLEIA) for analyzing E2HSA in bloodserum samples of rat and rhesus was established and validated. CLEIA was employed tostudy pharmacokinetics of E2HSA in Wistar rat and rhesus monkeys. Radioactive isotope125I marked E2HSA was given to rat to study characteristics of tissues distribution andelimination.Results:1. Develop and validate a chemiluminescent enzyme immunoassay toDetermination of drug concentration in biological samplesTo develop a chemiluminescent enzyme immunoassay of speciality,sensitivity,precision and accuracy for E2HSA. This method can specifically recognize the epitope ofE2HSA. A good linearity was obtained over the serum concentration range2~200ng·mL-1.Methodology validation shows that the standard E2HSA in the range of2~200ng·mL-1,the concentration and absorbance was Interdependent. Fitted by four parameters, Logistic:Y (OD)=A2+(A1-A2)/(1+(X/X0)^P), correlation coefficient (r) is larger than0.99, thelimit of quantification was2ng·mL-1. The mean inter-and intra-variation coefficients ofdetermination results for E2HSA was1.3~8.1%, The accuracy was-3.6~2.3%. Thesamples were stable after3months placed in-20℃, stock solution were stable in7daysUnder the same conditions, meanwhile, there was no dilution effect when iniluted no more than1000times.2. The pharmacokinetic evaluation in Wistar rat and rhesus monkey after singlesubcutaneouly administration of E2HSAFollowing a single subcutaneouly administration with1.98mg·kg-1of E2HSA toWistar rat, the blood samples were collected at different time points after dosing. Allcollected blood samples were centrifuged to obtain serum and the concentration of E2HSAin serum were determined by ELISA method described as above. Pharmacokineticparameter calculations and pharmacokinetic modeling were carried out using theWinNonlin5.2Statistical software. The elimination half-live (t1/2) of E2HSA was12.2±1.2h, the MRTlastof E2HSA was34.9±1.9h, the CL was19.1±5.6mL·h-1·kg-1, the Vd was381.4±50.7mL·kg-1, the AUClastwas91652.8±8119.1ng·h·mL-1. Following asubcutaneouly administration with0.3、0.9、2.7mg·kg-1of E2HSA to rhesus monkeys, Theelimination half-lives (t1/2) of E2HSA were53.4±8.0h、50.9±9.1h、56.8±8.1h respectively,the differences between the three group were not statistically significant by student’s t-test.The MRTlast of E2HSA were78.3±6.2h、89.0±7.6h、83.3±7.7h respectively. The kewere0.013±0.003、0.014±0.003、0.015±0.006;The CL were1.66±0.27mL·h-1·kg-1、1.39±0.15mL·h-1·kg-1、1.36±0.12mL·h-1·kg-1respectively， there were statisticallysignifican differences between the low-dose and mid-dose group, also the low-dose andhigh-dose group, but not between the mid-dose and high-dose group.The Vd were125.5±8.8mL·kg-1、103.8±28.4mL·kg-1、110.4±12.2mL·kg-1respectively, there were nostatistically significant differences between the three group. The AUClastwere179182±27148ng·h·mL-1，630956±68878ng·h·mL-1，1928196±168615ng·h·mL-1respectively (the proportion was1:3.5:10.8), the proportion of the three groups was1：3：9，The AUClast proportional increased with dose.A single subcutaneouly administration with0.9mg·kg-1of E2HSA to rhesus monkeysevery week, After a5-times, the accumulation factors (R) of5animals in this groupwere0.91、1.07、0.80、0.62、0.63respectively, the average was0.81±0.19,Comparation between the single subcutaneouly administration with2.7mg·kg-1ofE2HSA and intravenous administration with0.9mg·kg-1indicate that the bioavailability ofsubcutaneouly administration was81.9%. 3. Tissue distribution and excretion of Wistar rats by subcutaneous injection of125I-E2HSAResults of tissue distribution indicate that after given drug to Wistar rat, tissuedistribution of2h:serum> lung> stomach contents> kidney> spinal> intestinalcontents>adrenal> stomach> ovary> heart> bladder> vas deferens> liver> spleen>pancreas> skin> intestine> bones> uterus> fallopian> colon> thymus> testes>brain>muscle> colon contents> fat.8h:serum> lung> adrenal gland> kidney> stomachcontents> ovarian> stomach> small intestine contents> tubal> heart> vas deferens>liver> intestine> spleen> spinal> bladder> testes> uterine> skin> pancreas> colon>bones> fat> thymus> brain> muscle> colon contents.24h:serum> stomach contents>skin> lung> bladder> stomach> tubal> kidney> ovary> vas deferens> intestine> largeintestine contents> adrenal> heart> uterus> liver> testes> spleen> intestinalcontents>colon> bones> pancreas> fat> spinal> thymus> muscle> brain.96h:stomach contents> bladder> serum> colon contents> brain> skin> lung> kidney>adrenal> vas deferens> liver> stomach> ovarian> intestinal contents> tubal> uterus>spinal> heart> intestine> spleen> colon> pancreas> testes> bones> thymus> musclefat.Following a single subcutaneouly administration with1.98mg·kg-1of125I-E2HSA toWistar rats (♂), all the animals were fed in independent metabolic cages, collecte thebile、urine and feces respectively. The account of E2HSA collected from bile in0~144htake over3.97±1.29%of the total account was given to animals, it was62.86±10.47%and14.47±4.37%in urine and feces respectively, in this period of time, washing themetabolic cages also recalled2.83%±0.63%of the total account. The total recovery ratewas80.16%.Conclusions:1. Develop and validate a chemiluminescent enzyme immunoassay toDetermination of drug concentration in biological samplesWe have developed a chemiluminescent enzyme immunoassay of speciality, sensitivity, precision and accuracy for E2HSA and it was in the line with guidelines for bioanalytical method requirements.2. The pharmacokinetic evaluation in Wistar rat and rhesus monkey after singlesubcutaneouly administration of E2HSAThese results of t1/2of monkeys indicate that the differences between the three group were not statistically significant by student’s t-test. The proportion of AUClastwas1:3.5:10.8, the proportion of the three groups was1:3:9，The AUClast proportionalincreased with dose. the result point out that E2HSA appeared as a linear kinetic changesbetween0.3~2.7mg·kg-1in rhesus monkeys.The result point out that it will not accumulate in serum after a5-times subcutaneoulyadministration. But the concentration of E2HSA falled in someone, this result maybecome from anti-drug antibodies.Following a single subcutaneouly administration with2.7mg·kg-1of E2HSA tomonkeys,we found out than the absolute bioavailablity of subcutaneouly administrationwas81.9%,which hint that subcutaneous injection may be a more rapid, convenient andeffective route of administration.3. Tissue distribution and excretion of Wistar rats by subcutaneous injection of125I-E2HSAAfter a single subcutaneouly administration with1.98mg·kg-1of125I-E2HSA toWistar rats, the animals were slaughtered after2h、8h、24h、96h respectively and28kindsof tissues was collected. The data indicated that the concentration of E2HSA in kidneywas high, this result may illustrate the main way of elimination was urine. The low levelsof E2HSA in fat、muscle and brain indicated that it is difficult to reach tissues with lowlipid soluble, meanwhile, it cannot get through the blood-brain barrier (BBB). Content inthe gastrointestinal tract was high, probably because some basic metabolism of theproteolytic fragments secrete into the contents of gastrointestinal.The data of excretion of Wistar rats proved that the main way of elimination was renalexcretion. |
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