| IL-12 is a complex heterodimer composed of two subunits of P40 and P35 linked by pairs of disulfide bonds and is a bridge connecting innate immunity and acquired immunity and also is regarded as a key anti-virus regulator in immune system. rhIL-12 as a therapical drugs for infectious disease, cancer, autoimmune diseases and asthma have entered clinical trials abroad, while domestic rhIL-12 study is still at pre-clinical stage. Our preliminary studies have shown that rhIL-12 combined with rHBsAg coordinately regulates the immune response through the third and the first signal in chronic hepatitis B (CHB) patients and it may be an innovative and promising therapeutic strategy against HBV. For this purpose, we conducted a systematic pharmacological research on rhIL-12 alone or in combination with rHBsAg for the treatment of CHB to develop a kind of new biological agents against HBV.The transcripts of one P35 subunit and one P40 subunit of IL-12 were constructed in the same carrier, and the CHO engineering cell line with stable and high-level expression of rhIL-12 protein was successfully obtained by introduction of regulatory elements Intron and SV40 Enhancer. The rhIL-12 with more than 95% purity was acquired after continuous culture in fermentor and supernatant purification by HPLC. The comprehensive quality tests such as contents of components, physical and chemical properties, biological activity, immunological properties, purity and impurity, as well as the identification of pollutants were all finished, and the prepared rhIL-12 meets the quality standards of genetic engineering drug.This thesis includes the pre-clinical primary pharmacology and its mechanism and pharmacokinetics of rhIL-12 alone or combined with rHBsAg for CHB treatment.1 Primary pharmacology1.1 Pharmacodynamics in vitroIFN-γwas served as the key indicators for evaluating the biological activity of rhIL-12. IFN-γsecretion was increased in peripheral blood mononuclear cells (PBMCs) in healthy stimulated by rhIL-12 with a concentration of 0.005,0.01,0.1, 0.5,1.0,2.0,10,80 ng/ml. The dose-effect curve of IFN-γshowed a typical S-figure and ED50 was 0.0863±0.0032 ng/ml, R2=0.999. IFN-γsecretion was dose-dependently increased by rhIL-12 at the range of 0.01～1.0 ng/ml and therefore the preferred doses of rhIL-12 were determined as 0.01,0.1,1.0 ng/ml.PBMCs in healthy were stimulated by 1.0 ng/ml rhIL-12, the levels of IFN-γwere measured at five points:24h,48h,72h,96h and 120h. The results showed that there was IFN-γproduction 24 hours after rhIL-12 stimulation and the content of IFN-γwas increased over time and reached the peak value at 72h or 96h. The time point of 72h was preferred as the IFN-γdetection point based on analysis and references.The difference of biological activity between rhIL-12-GZ (Guangzhou Kai-tai Bio-Technology Co., Ltd) and rhIL-12-IS (international standard, purchased from the UK NIBSC) was compared by the method of PBMCs inducing IFN-γin vitro. The both dose-effect curves of rhIL-12-GZ and rhIL-12-IS showed a typical S-figure in the range of 0.005,0.02,0.078,0.313,1.25,5.00,20.00,80.00 ng/ml, and the two curves were fitted well (correlation coefficient R=0.991). The dose-effect curves drew from the 1g concentration of 0.005～1.25 ng/ml rhIL-12 and the content of IFN-γpresented linear and the regression equations were rhIL-12-GZ:Y= 3.016+1.147 X; rhIL-12-IS:Y=3.030+1.048X, and the two regression lines were also fitted well (P=0.217). The results indicate that the biological activity of this product is substantially equal to the international standard.To investigate the effect of rhIL-12 on IFN-γand IL-4 induced by PBMCs in the healthy and CHB patients, peripheral blood was collected from 20 healthy volunteer and 68 CHB patients (21 in immune tolerant phase,22 in immune clearance phase,19 in non-active HBsAg carrying phase), and PBMCs were prepared and then incubated with rhIL-12 for 72h. There were three groups of rhIL-12:low, medium and high concentration (0.01,0.1, and 1.0 ng/ml), and saline control group. The results showed that rhIL-12 dose-dependently increased IFN-γlevels produced by PBMCs in all people and more obviously in CHB patients, and there were no significant difference of IFN-γlevels among CHB patients with the different sub-types, but the IL-4 could not be detected in all groups. This indicates that rhIL-12 can enhance the Th1 cellular immune function of CHB patients with different clinical sub-type, but has no effect on Th2 cell-mediated immunity. The result provided an evidence for rhIL-12 as an immune modulator for the treatment of CHB.The coordinative effect of rhIL-12 combined with rHBsAg:PBMCs were prepared from 9 healthy subjects and 15 CHB patients and incubated with rhIL-12 (0.01,0.1,1.0 ng/ml), rHBsAg (0.5μg/ml) or rhIL-12 (0.01,0.1,1.0 ng/ml) combined rHBsAg (0.5μg/ml) for 72 h, respectively. The results showed that the IFN-γinduced by rhIL-12 in combination group was significantly higher than those in groups of rhIL-12 or rHBsAg alone (P<0.01), suggesting that rhIL-12 combined with rHBsAg had a role of increasing specific cell-mediated immune response.1.2 Pharmacodynamics in vivoThe researches were carried out on four kinds of animal models:BALB/c, C57BL/6J, HBV transgenic mice and cynomolgus.BALB/c mice were injected subcutaneously low, medium and high dose rmIL-12 (0.05,0.15,0.45μg/10g), once every three days, totally 2 times, and a saline group as control. Blood was orbitally collected predose and at 24h,72h after the first postdose and at 24h after the second postdose to detect serum concentrations of IFN-γ. The results showed that after the first administration of rmIL-12, the serum concentration of IFN-γpeaked at 24h in mice and presented an obvious dose-dependence. The content of IFN-γalmost returned to the pre-dose level at 72h. For the second administration, although there was still a clear dose-response relationship, IFN-γproduced by rmIL-12 at 24h was significantly less than that of the first administration. The results of this study show that rmIL-12 can dose-dependently induce IFN-γand enhance the role of cell-mediated immunity in BALB/c mice, and the role of rmIL-12 on IFN-γinduction presents the pre-conditioning effect.C57BL/6J mice were injected subcutaneously low, medium high and dose rmIL-12 (0.05,0.15,0.45μg/10g) and a normal saline group as control, once every three days, totally 3 times. Blood was orbitally collected predose, at 24h after the first postdose and at 24h after the third postdose, respectively. The results showed that the orderlinesses of serum IFN-γsecretion were similar between C57BL/6J mice and BALB/c mice, both of peak time were at 24h, the content of IFN-γsignificantly and dose-dependently increased in all the three groups, P<0.05, compared with the saline group. This proved that rmIL-12 also enhanced the cellular immune function in C57BL/6J mice.HBV transgenic BALB/c mice were intraperitoneally administrated low, medium and high dose rmIL-12 (0.05,0.25,0.75μg/10g) and a normal saline group as control, twice weekly, totally 8 times. The serum HBV DNA was measured predose and the eighth postdose. The results showed that rmIL-12 can decrease the serum HBV DNA load in HBV transgenic mice.The contents of IFN-y, IL-2, IL-4 and TNF-α, neutralizing antibody response to rhIL-12, and the distribution of sub-type of T cells in peripheral blood were detected after SC administration-of 0.5,5.0,40.0μg/kg rhIL-12 and 0.5,40.0μg/kg rhIL-12 combined with rHBsAg in cynomolgus. rhIL-12 was administered three times a week, totally 40 times and rHBsAg vaccine was administered once every 4 weeks, totally 4 times by IM injection of 20μg/kg.After the first administration, the results showed that IFN-γwas detected only in some of animals in groups of three doses rhIL-12 and rhIL-12 combined with rHBsAg at 6h,24h, but the number increased in all groups at 48h, and IFN-γcould be detected in all animals of high-dose group, and rhIL-12 combined with rHBsAg group increase more than rhIL-12 group. After the second and forth administration, IFN-γcould be detected in all animals treated with rhIL-12, and peaked at 48h. The content of IFN-y presented a certain dose-effect relationship, and rhIL-12 combined with rHBsAg group increase more than rhIL-12 group. After the seventh (3rd week) and thirteenth (5th week) administration, IFN-γcould be detected in some of animals and dropped significantly at 24h. After the administration of rhIL-12 for 3 weeks and rHBsAg vaccine for 4 weeks, all the animals produced neutralizing antibodies of anti-IL-12 and antibodies against rHBsAg. IFN-γalmost could not be detected at 24h after the thirty-eighth administration. rhIL-12 could significantly increase the percentages of CD3 and CD8 cells, and decrease the percentages of CD4 and the ratio of CD4/CD8. rHBsAg alone had no effect on the above item and rhIL-12 combined with rHBsAg had coordinationThere was no significant difference among IL-2, IL-4 and TNF-αlevels of predose and postdose measured at the same time point in all experimental animals. This indicated that rhIL-12 could induce IFN-γand had no effect on IL-2, IL-4 and TNF-α, that is, Thl-type cellular immune response is dominant.2. Studies on mechanismThe effect of rhIL-12 against HBV is due to its ability to induce endogenous IFN-γand inhibit HBV DNA replication. Therefore, we explored the cell source of IFN-γand the signal transduction pathways of rhIL-12.(1) PBMCs from 8 healthy volunteers were respectively incubated with rhIL-12 (1ng/ml), rHBsAg (0.5μg/ml) and lng/ml IL-12+0.5μg/ml rHBsAg in vitro for 24h:. The percentages of CD4+ IFN-γ+ T cells, CD8+IFN-γ+ T cells and CD56+ IFN-γ+ NK cells were measured by flow cytometry. The results showed that the percentages of CD8+ IFN-γ+ T cells and CD56+ IFN-γ+ NK cells in rhIL-12 group increased significantly compared with the control group, but the percentage of rHBsAg group showed no significant difference compared with the control group (P>0.05), the percentage of IL-12 combined with rHBsAg group had very significant difference compared with the control group (P<0.01). The results suggest that the IFN-y induced by rhIL-12 derives mainly from CD56+ NK cells and CD8+ T cells and secondly from CD4+ T cells, and the combination of IL-12 and rHBsAg has synergy.(2) The specificity of IL-12 biological activity must be mediated by the receptor with high affinity to IL-12. PBMCs were prepared and respectively incubated with rhIL-12 (1ng/ml), rHBsAg (0.5μg/ml) and lng/ml IL-12+0.5μg/ml rHBsAg for 72h for the further study of the effect of rhIL-12 on the IL-12 receptor (IL-12R) expression in seven healthy volunteers, IL-12Rβ1 and IL-12Rβ2 on the surface of CD4+, CD8+ and CD56+ cells were detected by flow cytometry.The results showed that all the five kinds of cellular receptors of CD4+ IL-12Rβ1+, CD8+ IL-12Rβ1+, CD56+ IL-12Rβ1+, CD8+ IL-12Rβ2+ and CD56+ IL-12Rβ2+ were elevated to various extents in rhIL-12 group, particularly, CD8+ IL-12Rβ2+ and CD56+ IL-12Rβ1+ increased significantly (P<0.01) compared with the control group.In rHBsAg group, The receptors increased more than in rhIL-12 group and there were very significant differences in CD8+ IL-12Rβ1+ and CD56+ IL-12Rβ1+ (P<0.01) compared with the control group. CD8+ IL-12Rβ1+ and CD56+ IL-12Rβ1+ in IL-12 combined with rHBsAg group increased more than those in rhIL-12 alone and combined with rHBsAg groups, and there were very significant differences (P<0.01) compared with the control group. This indicates that IL-12 can upregulate the expression of IL-12 receptor on the surface of PBMCs in healthy human and has synergistic effect in combination with HBsAg.3 The pharmacokinetics of rhIL-12 in cynomolgus3.1 Distribution and Pharmacokinetics of 125I-rhIL-12The distribution, kinetics, excretion and plasma protein binding of rhIL-12 in vivo were investigated by the method of Na125I label and proteins precipitation by trichloroacetic acid (TCA) in cynomolgus. The biological activity of 125I-rhIL-12, with the radiochemical purity of 95.6±1.9 (%) and average specific activity of 197.72 Bq/ng, had no significant difference compared with the potency of unlabeled reference substances, and the lowest limit of quantification (LLOQ) of 125I-rhIL-12 detection was 0.02 ng-equivalent/g(ml). The identification by TCA precipitation showed that it could basically meet the requirements for distribution study.After SC injection of 125I-rhIL-12 in cynomolgus, both of the AUCs of TCA precipitated radioactivity and the total radioactivity were ordered as the following sequence:intestinal contents>blood>kidney>muscle>the brain, which indicates that the drug mainly distributes in intestines, blood and kidney and it is not easy to pass the blood-brain barrier. The AUCs of the total radioactivity of urine and bile were more than intestinal contents because of lots of destructive small molecular radioactivity in urine and bile.The AUC (0-t)s of the serum TCA precipitation radioactivity and the total radioactivity after administration were 158.77±16.00 and 216.07±28.18 ng-equivalent h/ml; Cmax were 6.62±0.57 and 7.91±0.39 ng-equivalent/ml, Tmax were 5.33±1.15 h; MRT were 21.82±1.21 and 22.96±2.02 h; CL(S) were 11.30±1.25 and 8.36±1.36 ml/kg/h; Vd were 0.35±0.02 and 0.26±0.01 L/kg; elimination phase half-life were 21.82±1.02 and 22.19±3.47 h, respectively.The intake of radioactivities were totally discharged 84.4±6.0(%) and 0.96±0.17(%)respectively by urine and feces during 168 h, the total radioactivities discharged by urine and feces were 85.33±5.82(%). This shows that rhIL-12 is mainly excreted through urine and minor through feces and bile. There was metabolites in urine and there were both of metabolites and antetype in bile.Another study on plasma protein binding in vitro showed 125I-rhIL-12 did not bind to plasma protein.3.2 Pharmacokinetics and pharmacodynamics rhIL-12 in cynomolgusTo study the PK and PD of rhIL-12 in cynomolgus, experimental animals were assigned to 5 groups:low, middle and high dose groups for SC injection of rhIL-12 0.15,0.30,1.50μg/kg; a group forⅣinfusion of 0.60μg/kg rhIL-12; and a group for rhIL-12+HBsAg vaccine (SC rhIL-12 0.30μg/kg, IM HBsAg 20μg/kg) combination; the animals in the above groups were all administrated single dose. The low-dose group also served as the SC injection continuous dosing group (once for 4 days, totally 5 times).Blood Sample Collection for PK StudyFor animals in the single dose SC injection groups (0.15,0.30,1.50μg/kg rhIL-12, and the vaccine combined group), 1mL of blood was collected pre-dosing and at 2,4,6,8,10,24,48,72,120, and 168 h post-dosing. For animals in the constant-dosing SC injection group, 1mL of blood was collected at each pre-dosing and at 2,4,6,8,10,24,48, and 72 h after first dosing, at 8 h after the second, third, and fourth dosing, and at 2,4,6,8,10,24,48,72,120, and 168 h after the fifth dosing. For animals in the IV infusion group, 1mL of blood was collected pre-dosing and at 1,15,30 min and 1,2,4,6,8,24,48,72,120, and 168 h post-dosing.Blood Sample Collection for PD StudyFor the constant-dosing SC injection group, 1mL of blood was collected every pre-dosing and at 8,24,48, and 72 h after the first dosing, at 8,24, and 72 h after the second, third and fourth dosing, and at 8,24,48, and 72 h after the fifth dosing.The serum rhIL-12 concentrations were measured by ELISA to calculate PK parameters, and its LLOQ for this assay was 0.05ng/ml. The serum levels of IFN-y were also measured by ELISA to research the PD characteristics of rhIL-12 in the constant-dosing SC injection group.The AUC(0-t) of this protein after SC injection of 0.15,0.30 and 1.50μg/kg were 9.03±2.85,25.34±7.20 and 186.65±67.32 h×ng/mL, respectively; AUC(0-∞) were 10.52±3.26,26.43±8.25 and 190.66±70.55 h×ng/mL, respectively; Cmax were 0.50±0.14,0.95±0.33 and 8.14±3.21 ng/mL, respectively; Tmaxs were 6.50±1.91, 7.00±1.15 and 6.00±2.31 h; MRTs were 11.69±2.73,27.67±14.50 and 23.74±7.21 h; T1/2s were 10.04±4.82,22.91±15.52 and 19.75±10.06 h; CLs were 15.88±7.00, 12.25±3.85 and 8.89±3.85 mL/h/kg; Vds were 213.92±85.72,341.88±147.37 and 287.53±273.27mL/kg; absolute bioavailability(F) were 65.16%,54.57%,78.72%, respectively, and the mean F was 66.15±12.11 (%).The ratio of low-, medium-, and high-dose was 1:2:10, the ratio of Cmax was 1:1.91:16.31, and the ratio of AUC(0-t) was 1:2.81:20.68. This indicates the metabolism of rhIL-12 presents linear kinetic characteristics.There was no statistical significance among all parameters of medium dose rhIL-12 combined with the vaccine group compared with the group of medium dose rhIL-12 alone, suggesting that rHBsAg had no effect on the metabolism behavior of rhIL-12. The serum rhIL-12 was undetectable after the fifth SC injection times because of producing neutralizing antibody against rhIL-12, so the pharmacokinetic parameters could not be calculated.The IFN-gamma was very low after SC continuous dosing of low dose rhIL-12, and not detectable or less than LLOQ in samples at most time points. IFN-γcan be detected only in No.3 animal in the first measurement and Tmax was 72 h, later than the Tmax of rhIL-12(4-8h).IFN-γwas detectable in all animals of SC single injection of high dose rhIL-12 and the value of serum IFN-y was obviously higher than that of SC continuous dosing group at the same time points. Tmax was 24-72 h, later than the Tmax of rhIL-12(4-8 h). |
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