Non-clinical Pharmacokinetcs And Pharmacodynamics Study Of A Long Acting Hematopoietic Growth Factor HHPG-19K

HHPG-19K, a kind of hemopoietic growth factor, is a safe and long acting praeparatum of recombinant human granulocyte colony-stimulating factor (rhG-CSF) modified by PEG 20000. HHPG-19K can specifically stimulate and regulate the proliferation, differentiation of granulocyte series progenitor cell, promote maturity and release of neutrophil, through influencing hematopoietic stem/progenitor cell, microenvironment of bone marrow and apoptosis. It will be of great benefit to anti-infection of acute radiation sickness and treatment of syndrome related to hematopoiesis exhaustion. It aslo can be used in treatment of Neutropenia caused by various disease. As parts of the non-clinical pharmacological study of HHPG-19K, the present study evaluates in vivo pharmacokinetics, pharmacodynamics and tissue distribution of HHPG-19K. It will provide experiment support for further clinical study.Part I. Methodology for pharmacokinetics1. The development and validation of analytical methods for quantitative determination of HHPG-19K in biological matrixA quantitative method was established which was used for determination of HHPG-19K and its reference substance in biological matrix (monkey's plasma). The standard in human G-CSF Duoset kit, HHPG-19K, rhG-CSF and Neulasta all showed typical concentration dependent curve, as well the calibration curves of these four were parallel in linear range. This method with good specificity was no cross interference with the recombinant human G-CSF Receptor and recombinant mice G-CSF. The calibration curves for HHPG-19K, rhG-CSF and Neulasta were linear within the concentration range of 31.25 pg·mL^-1 2000 pg·mL^-1. The limit of quantitation was 31.25 pg·mL^-1. The interday precision of this assay was less than 12.5 %, the intraday precision was less than 6.5 %, and the accuracy of the assay was 93.4 % 105.6 %.The validity studies demonstrated that the method was reliable for the determination of HHPG-19K, rhG-CSF and Neulasta in plasma.2. The labelling, identification of ¹²⁵I-HHPG-19K and validation of TCA precipitation methods for determination of ¹²⁵I-HHPG-19K¹²⁵I-HHPG-19K was labelled successfully, and TCA precipitation method was established used for determination of ¹²⁵I-HHPG-19K. The radioactive purity of ¹²⁵I-HHPG-19K was 99.4 %±0.5 % after triple determination. The specific activity of ¹²⁵I-HHPG-19K was 119.58 kBq·μg^-1. Within the equivalent range of 0.02 27.02 ng, the quantity of ¹²⁵I-HHPG-19K shows good linear relationship with total radioactivity and TCA-precipitable radioactivity, and the correlation coefficient larger than 0.999, the precision was less than 20 %. The radioactivity recovery of different tissues'TCA-precipitable part added in ¹²⁵I-HHPG-19K was 84.0 % ? 7.3 %. The primary form of ¹²⁵I-HHPG-19K mainly resided in TCA precipitation. The validity studies demonstrated that the method was reliable for the determination of¹²⁵I-HHPG-19K in tissues and body fluid.Part II. Study on pharmacokinetics, tissue distribution and excretion of HHPG-19K in normal animals1. Pharmacokinetics of HHPG-19K after administration in normal rhesus monkeysAfter sc administration HHPG-19K at single doses of 30, 100, 300μg·kg^-1 in normal rhesus monkeys, the peak time of plasma concentration reached maximum (Tmax) were 6.67 12.00 h. The terminal half-life (t1/2β) were 16.70±5.14, 12.58±3.23, 23.60±3.71 h, respectively; the clearance (CL) were 5.93±1.92, 2.28±0.50, 1.22±0.18 mL·h^-1·kg^-1. AUC(0-∞) were 5430.64±1762.98, 45155.92±9407.03, 249392.34±34290.96 ng·h·mL^-1, statistically significant difference were found among the groups (P<0.01). Dose ratio was 1 : 3.3 : 10 and the increase of AUC(0-∞) was 1 : 8.3 : 45.9. The increase of dosage was disproportionate with that of AUC. When dosage raised, the clearance growed down by degrees. Among the dosage of 30 300μg·kg^-1, HHPG-19K appeared as nonlinear pharmacokinetics in normal rhesus monkeys. After iv administration HHPG-19K at single doses of 100μg·kg^-1, the plasma concentration reached maximum immediately and decreased quickly. The plasma concentration dropped in background level at 72 h after administration. t1/2βwas 6.13±0.62 h, the plasma clearance was 1.05±0.07 mL·h^-1·kg^-1. Compared to the sc isodose HHPG-19K group, pharmacokinetic parameters were statistically different.After sc administration rhG-CSF at single doses of 10μg·kg^-1, Tmax were 2.83±1.26 h, the plasma concentration peak (Cmax) was 116.23±6.06 ng·mL^-1, t1/2βwas 1.52±0.08 h, AUC(0-12h) was 511.64±59.54 ng·h·mL^-1, AUC(0-∞) was 516.40±59.75 ng·h·mL^-1 and CL was 19.55±2.40 mL·h^-1·kg^-1. Pharmacokinetic parameters were statistically and significantly different with the 100μg·kg^-1 HHPG-19K group. Absolute bioavailability of HHPG-19K (100μg·kg^-1) was 47.5 %2. Tissue distribution and and excretion of ¹²⁵I-HHPG-19K after administration in ratsTissue distribution of ¹²⁵I-HHPG-19K was examined in rats following sc administration. Distribution of total radioactivity was similar to that of TCA-precipitable radioactivity. The organs or body fluid that exhibited the highest radioactive levels of AUC were kidney, serum, lung, urine, urinary bladder, intestinal content. The radioactive exposure level of serum was high, which imply that drug mainly distributed in vascular bed; and the radioactive exposure level of urinary system were much higher than that of other tissues, which imply that drug excreted mostly by urinary system. Organs with lowest radioactive exposure level were bone marrow, brain and fat, which imply that drug was hard to permeate through the blood-brain barrier, also it has a poor lipophilicify.The results of SHPLC showed that the original form of ¹²⁵I-HHPG-19K was mostly existed in serum, and the conjugation of drug with plasma protein was not observed. The original form of ¹²⁵I-HHPG-19K in urine were less, most of which were degraded into metabolite with small molecular weight. The excretion results showed ¹²⁵I-HHPG-19K excreted mostly via urine, little via feces and bile routes. 216 h after administration of ¹²⁵I-HHPG-19K, 82.13 %±4.73 % and 1.47 %±0.16 % of the radioactivity in the urine and feces. 8 h after administration, accumulated excretion of the radioactivity via bile was 0.83 % ? 0.25 %.Part III. Study on pharmacokinetics and pharmacodynamics of HHPG-19K in model animals with 60Coγ-ray irradiation-induced neutropenia1. Pharmacokinetics of HHPG-19K after administration in model rhesus monkeys with 60Coγ-ray irradiation-induced neutropeniaAfter sc administration HHPG-19K at single doses of 30, 100, 300μg·kg^-1 in model rhesus monkeys, Tmax were about 7 h. t1/2βwere 22.94±1.08, 23.48±2.63, 20.62±4.63 h, respectively; CL were 12.22±9.22, 6.46±3.44, 3.10±1.78 mL·h^-1·kg^-1. AUC(0-∞) were 2883.64±1704.86, 15638.09±9354.68, 93541.23±46384.46 ng·h·mL^-1, statistically significant difference were found among the groups (P<0.05). Dose ratio was 1 : 3.3 : 10 and the increase of AUC(0-∞) was 1 : 5.4 : 32.4. The increase of dosage was disproportionate with that of AUC. When dosage raised, the clearance growed down by degrees. Among the dosage of 30 300μg·kg^-1, HHPG-19K appeared as nonlinear pharmacokinetics in model rhesus monkeys with hematopoietic system injuries by 60Coγ-ray. This phenomenon was accorded with pharmacokinetics behavior in normal monkeys.After sc administration Neulasta at single doses of 100μg·kg^-1 in model rhesus monkeys, parameters as Tmax, Cmax, t1/2β, AUC(0-∞), CL were no statistically significant difference with HHPG-19K group. After sc administration rhG-CSF at doses of 10μg·kg^-1 for first time in model rhesus monkeys, pharmacokinetic parameters were statistically and significantly different with the 100μg·kg^-1 HHPG-19K group (P < 0.001 P < 0.01). Especially, t1/2βincreased from 1 2 h to above 20 h (P < 0.001); CL decreased from 47 117 mL·h^-1·kg^-1 to 6.46±3.44 mL·h^-1·kg^-1 (P < 0.01). The plasma concentration exposure levels of HHPG-19K were far more than that of rhG-CSF at the same dose of total protein.2. Pharmacodynamics of HHPG-19K after administration in model rhesus monkeys with 60Coγ-ray irradiation-induced neutropenia24 hours after the irradiation, rhesus monkeys were subcutaneously injected with HHPG-19K, Neulasta or rhG-CSF. After sc administration HHPG-19K at single doses of 30, 100, 300μg·kg^-1 in model rhesus monkeys, the neutropenia duration were 27.8±6.7, 19.2±13.7, 7.0±6.1 d, respectively. HHPG-19K also significantly shortened the neutropenia duration in the medium and high dose groups compared with the control (28.5±7.4 d) (P < 0.001).The time of ANC recovery of low, medium, high dose groups were 39.2±6.4, 34.0±4.9, 24.4±7.7 d, and the time speeded up with the increase of dosage. HHPG-19K groups postponed the time to ANC nadir and elevated the nadir level compared with negative control groups. In HHPG-19K groups, the antibiotic requirements period of low, medium, high dose groups were 3.2±2.9 d, 1.4±1.9 d, 0.2±0.4 d, respectively, which were obviously shorter than negative control group (7.2 d) (P < 0.05, P < 0.01, P < 0.001, respectively). At end stage of observation, the ANC recovery of HHPG-19K groups were higher than that of negative control group. All pharmacodynamics parameters of Neulasta group were no statistically significant difference with HHPG-19K group. Statistically significant difference of the time reached maximum were found between HHPG-19K groups and rhG-CSF group with equivalent dose. Other pharmacodynamics parameters were closed together, with no statistically significant difference.After dosing, the tendency of WBC and ANC in separate groups were with one accord. All medication administration groups had little effect on RBC. Compared to negative control group, HHPG-19K groups,Neulasta group and rhG-CSF group elevated the PLT nadir level, hastened ANC recovery, and behaved a dose-dependent manner.