A multiple-dose pharmacokinetics of polyethylene glycol recombinant human interleukin-6 (PEG-rhIL-6) in rats

A multiple-dose pharmacokinetics of polyethylene glycol recombinant human interleukin-6 (PEG-rhIL-6) in rats 1 2 first-author †E-mail:hxlscu@163.comxmzhang@tom.com †E-mail:hxlscu@163.comxmzhang@tom.com hxlscu@163.com 2 1 3 2 3 3 1 2 3 corresp ‡Corresponding Author ‡Corresponding Author †E-mail:hxlscu@163.comxmzhang@tom.com †E-mail:hxlscu@163.comxmzhang@tom.com hxlscu@163.com Institute of Biomedical Engineering, West China Center of Medical Sciences, Sichuan University, Chengdu 610041, China Institute of Biomedical Engineering, West China Center of Medical Sciences, Sichuan University, Chengdu 610041, China Laboratory Animal Center of Sichuan University, Chengdu 610041, China Laboratory Animal Center of Sichuan University, Chengdu 610041, China Chengdu Institute of Biological Products, China National Biotic Group (CNBG), Chengdu 610023, China Chengdu Institute of Biological Products, China National Biotic Group (CNBG), Chengdu 610023, China Radiation therapy has been widely applied in cancer treatment. However, it often causes thrombocytopenia (deficiency of white blood cells) as an adverse effect. Recombinant human interleukin-6 (rhIL-6) has been found to be a very effective way against this thrombocytopenia, but IL-6 has low stability in blood, which reduces its efficacy. To increases the stability and half-life of rhIL-6, it was modified by polyethylene glycol (PEG). The pharmacokinetics and the tissue distribution of PEG-rhIL-6 labeled with 125 I were examined after subcutaneous injection in rats. The pharmacokinetic pattern of PEG-rhIL-6 was defined with linear-kinetics, and we fitted a one-compartment model with half-lives of 10.44–11.37 h (absorption, t 1/2 K a ) and 19.77–21.53 h (elimination, t 1/2 K e ), and peak concentrations at 20.51–21.96 h ( t peak ) in rats. Half-lives and t peak of PEG-rhIL-6 were longer than those of rhIL-6 previously reported. In the present study, for deposition of PEG-rhIL-6 in rats, the tissue distribution examination showed that blood was the major organ involved, rather than liver. However, as to the elimination of PEG-rhIL-6, the major organ was the kidney. The excretion fraction of the injection dose recovered from urine was 23.32% at 192 h after subcutaneous administration. Less than 6% of PEG-rhIL-6 was eliminated via the feces at 192 h. These results indicate that PEG-rhIL-6 is a good candidate drug formulation for patients with cancer. Polyethylene glycol Recombinant human interleukin-6 Pharmacokinetics Rat Introduction 1 Recombinant human interleukin-6 (rhIL-6) is a novel cytokine produced by monocytes, fibroblasts, and other cell lines, and exhibits a pleiotropic action (Nakanishi et al., 2004 2004 ). Some studies have shown that rhIL-6 is a candidate drug that can prevent thrombocytopenia caused by chemotherapy and irradiation therapy (Drayer et al., 2000 2000 ; Yin et al., 2005 2005 ). The targets of rhIL-6 are the megakaryocytes which are used to promote the formation of platelets: there are high-affinity rhIL-6 receptors in megakaryocytes (rhIL-6 receptor-gp130 complex) (Bracho et al., 2001 2001 ; Kashiwakura et al., 2005 2005 ; Kishimoto, 2005 2005 ). However, the stability of rhIL-6 in vivo is limited because of proteolysis. Also, the half-life of rhIL-6 is short due to rapid renal excretion and broad systemic distribution (Banks et al., 2000 2000 ; Tsunoda et al., 2001 2001 ). Therefore, it is necessary to achieve a sufficient increase in peripheral platelet numbers, and very high dosages and frequent administration of rhIL-6 are required. However, the high dosages and frequent administration give rise to adverse effects. One of the most important adverse effects is that rhIL-6 will induce various acute-phase proteins including C-reactive protein (CRP), β2-fibrinogen, amyloid protein, haptoglobin, hemopexin, and so on. In addition, pyrexia and anorexia are also the common adverse effects of rhIL-6 (Kishimoto, 2010 2010 ). As we know, rhIL-6 is a long-term maintenance therapy, and adverse effects are partly caused by the rhIL-6 accumulation in and transfer to the liver and spleen (Shibata et al., 2005 2005 ). Therefore, modified rhIL-6 was used in this study to improve rhIL-6 retention in blood. Some studies indicated that the properties of biologically active compounds can be improved by covalent attachment to polymers. Polyethylene glycol (PEG) is one of the most widely used polymeric materials for this purpose (Dosio et al., 2001 2001 ; Parveen and Sahoo, 2006 2006 ; Rawat et al., 2010 2010 ), and has been used to modify various enzymes, proteins, and drug delivery systems, including interferon-R (Bailon et al., 2001 2001 ; Wang et al., 2002 2002 ), asparaginase (Kodera et al., 1992 1992 ), granulocyte colony-stimulating factor (G-CSF) (Cindric et al., 2007 2007 ), lactoferrin (Nojima et al., 2008 2008 ), adenosine deaminase (Levy et al., 1988 1988 ), and growth hormone receptor antagonist (Pradhananga et al., 2002 2002 ). Obtaining long half-lives of these proteins depends on the high molecular weight ( M W ) of PEG. Attachment of PEG to small proteins leads to a larger PEG-protein conjugate with a reduced rate of renal clearance. In addition, the attachment leads to the formation of a ‘shell’ around the protein, which reduces or eliminates the immunogenicity and sensitivity of the PEG-protein proteolysis (Mehvar, 2000 2000 ; Kozlowski et al., 2001 2001 ). Chemical modification of rhIL-6 with PEG effectively prolongs its half-life by increasing its molecular size, improving its resistance to proteases, decreasing its renal excretion rate, and reducing the production of antibodies. This could enhance the desirable thrombopoietic effect of rhIL-6 and reduce undesirable effects in vivo. The purpose of this study was to investigate the pharmacokinetics, distribution, and excretion of rhIL-6 chemically modified with PEG (PEG-rhIL-6) in rats using a radiotracer technique. Materials and methods 1 Chemicals 2 PEG-rhIL-6 was provided by Chengdu Rong-sheng Pharmaceuticals Co., Ltd., China. The M W of PEG is about 20 kDa. The mixture ratio of rhIL-6 and PEG for modification is 1:3. The number of mono-PEG-rhIL-6 in all PEG-rhIL-6 was greater than 85%; less than 15% of PEG-rhIL-6 was di-PEG-rhIL-6. The M W of PEG-rhIL-6 was about 46 kDa. All PEG-rhIL-6 was stored at −20 °C. Carrier-free protein iodination Na 125 I ( > 500 MBq/pg) was purchased from the Radiochemical Centre, Amersham, and iodogen (1,3,4,6-tetrachloro-3α,6α-diphenylglycoluril) from Rockford, IL, USA. The other reagents and solvents used in this study were of analytical grade. Iodination of PEG-rhIL-6 2 PEG-rhIL-6 (5 µg) was iodinated according to previous studies (Markwell, 1982 1982 ; Castell et al., 1990 1990 ). Briefly, iodobeads were used as an oxidant, and the reaction was stopped after 20 min by removing the catalyst. A final concentration of 5 g/L bovine serum albumin was added, and then the mixture was gel-filtered by a Sephadex G-25 column, which was equilibrated with 10 g/L bovine serum albumin. The specific activity of the iodinated PEG-rhIL-6 was similar to that reported by Markwell (approximately 150 kBq/pg). 125 I-PEG-rhIL-6 was found to contain only about 3% [ 125 I]iodide by paper chromatography analyzing. Only a single band with an apparent molecular mass of 20 kDa was found by sodium dodecyl sulfate/polyacrylamide gel electrophoresis (SDS-PAGE) and autoradiography. There were no significant differences in biological activities between 125 I-PEG-rhIL-6 and PEG-rhIL-6. Determination of PEG-rhIL-6 activity 2 The capability of PEG-rhIL-6 for inducing rat hepatoma cell line Fao p -fibrinogen mRNA was used to assess its activity. The protocol for the cytoblot and the mRNA hybridization has been described in previous reports (Kishimoto, 2003 2003 ; 2005 2005 ). Animals and treatment 2 Male and female healthy Wistar rats, with weight ranging from 200 to 220 g, were purchased from the Laboratory Animal Center of Sichuan University, China. The animals were acclimated for one week in the Laboratory Animal Center of Sichuan University before any experimental procedures. Then, all rats were housed in a room with controlled temperature of (24±1) °C, a relative humidity of (55±5)%, and a 12-h light/dark cycle (7:00 am to 7:00 pm). All animals had free access to water and a standard rat diet. The animal experimental protocols were approved by the Ethics Committee of Sichuan University and in accordance with the Principles of Laboratory Animal Care of the National Institutes of Health, China. Validation for radioactivity determination in plasma, tissues, and excreta by trichloroacetic acid (TCA) precipitation assay 2 Validation for all samples’ radioactivity was determined according to the method previously reported by Hu et al. ( 2006 2006 ). Briefly, the determination of validation for the PEG-rhIL-6 concentrations was conducted using the precipitation of the iodinated protein by ice-cold TCA (100 g/L). To examine the reliability of TCA precipitation assay in the determination of radioactivity in rats, all blank rat samples (including plasma, tissue homogenate, and excreta) were used in the preliminary experiments. The ratios of radioactivity recovered from TCA precipitable pellet (in vitro) and from supernatant of the excreta (in vivo) were greater than 98% of the added radioactivity. At the same time, the ratios of radioactivity recovered from TCA precipitable to total radioactivities in the rats’ plasma, tissue, and excreta samples decreased in a time-dependent manner after intravenous (i.v.) administration. Moreover, there was only intact protein present in the TCA precipitation using SDS-PAGE assay for plasma, tissue, and excreta samples. To calculate the concentration of 125 I-PEG-rhIL-6 in plasma and tissue homogenate samples of rats, TCA precipitable radioactivity was used instead of total radioactivity. In the excretion studies, however, total radioactivity, rather than TCA precipitable radioactivity, recovered from the excreta, was used to obtain the mass balance information. The plasma, tissue, and excreta homogenate samples of examined blank rats were used to prepare a series of calibration standards by adding seven concentrations (0–50 ng/ml) of radiolabeled PEG-rhIL-6. The relationship between the added concentrations and counted radioactivity of the standards showed a strong correlation ( R 2 > 0.98) and recovery ( > 95%) for the entire matrix. To determine the background counts for each matrix, all the plasma, tissue, and excreta homogenate samples of blank rats were counted. Pharmacokinetics study 2 The rats were grouped randomly (three groups, n =5 per group) based on their genders and body weights (BWs). The three groups of Wistar rats were injected with 125 I-PEG-rhIL-6 at a single dose of 3, 20, or 40 µg/kg BW by subcutaneous (s.c.) administration. After the injection, serial blood samples (approximately 100 µl each) were obtained from the tail vein of the rats and stored in heparinized Eppendorf tubes. All blood samples were centrifuged at 2 000× g for 10 min before 10 µl of plasma was pipetted into plastic tubes, and then stored at −20 °C until assay. Plasma samples were detected in a γ-counter (FT-2008γ, Xi’an, China). Plasma samples were diluted by adding 190 µl 9 g/L saline, and then equal amounts of cold TCA solution (200 g/L) were added to mix. The centrifugation was used to obtain the precipitation. The γ-counter was used to detect the radioactivities of all samples. The concentration of PEG-rhIL-6 at each time point was used to determine its blood clearance rates (Zhang et al., 2007 2007 ). Tissue distribution study 2 Wistar rats were injected with 125 I-PEG-rhIL-6 at a single dose of 2 µg/kg BW s.c. ( n =15). Five rats for each duration, 8, 24, and 48 h post-dose, were sacrificed by decapitation after administration of 125 I-PEG-rhIL-6, respectively. The whole brain, womb, testis, heart, spleen, stomach, kidney, intestine, liver, lung, bladder, muscle (at injection site and skeletal), and plasma were harvested, and all tissues and organs were weighed on an analytical balance (FA1004, Shanghai, China). A γ-counter was used to detect the radioactivities of all weighed tissues. Some of these samples were homogenized in 9 g/L saline. Centrifugation (2 000× g , 10 min) was used to clarify the suspensions. A total of 10 µl supernatants were pipetted into plastic tubes, and then mixed with equal amounts of human plasma. A total of 180 µl 9 g/L saline and 200 µl 200 g/L TCA solution were added and centrifuged (Zhang et al., 2007 2007 ). The radioactivities of the pre-precipitation and post-precipitation were counted. Excretion study 2 Wistar rats were each administered 125 I-PEG-rhIL-6 at a single dose of 2 µg/kg BW s.c. ( n =5). The urine and feces of these rats were collected and weighted in pre-designed intervals. A total of 10 µl urine was pipetted into each plastic tube, and the radioactivity was analyzed by the TCA precipitation method. The radioactive counts of pre-precipitation and post-precipitation were detected. The radioactivity of aliquot feces was detected by the TCA precipitation method. Data analysis 2 Pharmacokinetic analysis was performed using 3P97 computer software (Chinese Pharmacological Society). Results were expressed as the mean±standard derivation (SD). Data were subjected to a one-way analysis of variance (ANOVA) followed by the least significant difference (LSD) post-hoc test, and differences were considered statistically significant at P < 0.05. Statistical analyses were performed with the SPSS software package (SPSS Inc., Chicago, IL, USA). Results 1 Pharmacokinetics study 2 Blood samples of rats were collected at the expected duration after s.c. administration of 125 I-PEG-rhIL-6. The plasma disappearance curves of three groups are displayed in Fig. 1 1 . PEG-rhIL-6 showed a one-compartment model in rats after single s.c. injection (3, 20, and 40 µg/kg BW, respectively), with half-lives of 10.44–11.37 h (absorption, t 1/2 K a ) and 19.77–21.53 h (elimination, t 1/2 K e ), time of peak concentration ( t peak ) 20.51–21.97 h, clearance (CL/F) 20.44–25.59 ml/(kg·h), area under curve up to 72 h (AUC 0–72 ) 120.49–1651.94 ng·h/ml, maximum concentration ( c max ) 2.05–27.40 ng/ml, and mean residence time (MRT) 43.28–46.66 h. The main pharmacokinetics parameters in rats are displayed in Table 1 1 . Except for AUC 0–72 and c max , no significant differences ( P > 0.05) were observed in the pharmacokinetic parameters among the three different doses. Data are expressed as mean±SD ( n =5) c ave : average concentration; K e : elimination rate constant; K a : absorption rate constant; t 1/2 K a : half-life of absorption; t 1/2 K e : half-life of elimination; t peak : time of peak concentration; AUC 0–72 : area under the curve up to 72 h; c max : maximum concentration; CL/F: clearance; MRT: mean residence time. Data are expressed as mean±SD P < 0.05 among the three different doses of 3, 20, and 40 µg/kg Dose (µg/kg BW) c ave (ng/ml) K e (h −1 ) K a (h −1 ) t 1/2 K a (h) t 1/2 K e (h) t peak (h) AUC 0–72 (ng∙h/ml) c max (ng/ml) CL/F (ml/(kg∙h)) MRT (h) 3 9.12±3.57 0.0340±0.0020 0.0690±0.0160 10.44±2.29 20.47±1.33 20.51±2.34 120.49±23.50 2.05±0.46 25.59±4.46 43.28±1.80 20 69.15±14.66 0.0320±0.0010 0.0620±0.0980 11.25±1.65 21.53±1.20 21.30±1.61 988.22±113.26 * * 15.72±2.39 * * 20.44±2.19 46.66±2.55 40 143.39±31.73 0.0354±0.0037 0.0610±0.0036 11.37±0.66 19.77±2.05 21.97±0.83 1651.94±166.33 * * 27.40±2.27 * * 24.41±2.41 43.58±6.70 Tissue distribution study 2 The tissue distribution of 125 I-PEG-rhIL-6 was investigated in Wistar rats. The radioactivities for 125 I-PEG-rhIL-6 in brain, womb, testis, heart, spleen, stomach, kidney, intestine, liver, lung, bladder, muscle (at injection site and skeletal), and plasma of Wistar rats were determined by a γ-counter. Fig. 2 2 illustrates the main tissue distribution of 125 I-PEG-rhIL-6 in rats. Except the plasma and muscle at the injection site, the highest deposition was found in the bladder, followed by the stomach and kidney. Less deposition of 125 I-PEG-rhIL-6 was found in the brain. These results show that the major organs for deposition of PEG-rhIL-6 were the plasma and bladder (Zhang et al., 2007 2007 ). Tissue: (a) 1, blood; 2, heart; 3, liver; 4, spleen; 5, lung; (b) 6, brain; 7, stomach; 8, large intestine; 9, small intestine; 10, uterus; (c) 11, didymus; 12, muscle at the injection site; 13, skeletal muscle; 14, kidney; 15, bladder. Data are expressed as mean±SD ( n =5) Excretion study 2 The urine and feces of Wistar rats were collected, weighed, and counted at the expected duration. The cumulated excretion fractions of rats are illustrated in Fig. 3 3 . The major organ of PEG-rhIL-6 elimination was the kidney. The integral excretion fractions of the injection dose from urine were 9.95% within 24 h, 20.74% within 96 h, and 23.32% within 192 h after injection, respectively. Only about 6% of PEG-rhIL-6 was eliminated via feces during 192 h. Data are expressed as mean±SD ( n =5) Discussion 1 To our knowledge, this is the first report to evaluate the pharmacokinetics, tissue distribution, and excretion of PEG-rhIL-6 in rats. Dose linearity of the pharmacokinetics over the s.c. dosage range examination (3–40 µg/kg BW) was demonstrated. The c max and AUC 0–72 values of the three doses showed a similar dose proportionality. There was an approximately 14-fold increase in c max (2.05 vs. 27.40 ng/ml) and AUC 0–72 (120.49 vs. 1651.94 ng·h/ml) caused by the 14-fold increase in dosage (Table 1 1 ). Although t peak and t 1/2 K a seemed to vary in a dosage-dependent manner ( P > 0.05), there were no significant differences of other parameters including CL/F and t 1/2 K e among the three dosages analyzed by ANOVA ( P > 0.05). Therefore, our results show that PEG-rhIL-6 across the investigated dosage range in rats (3–40 µg/kg BW) displayed a linear plasma pharmacokinetics and fitted a one-compartment model (Castell et al., 1988 1988 ). In some previous studies (Ryffel et al., 1994 1994 ; Zha et al., 1995 1995 ; Banks et al., 2000 2000 ), pharmacokinetic studies showed different results in humans and rats. They have reported that rhIL-6 displayed a very short plasma half-life. In this study, we investigated the plasma half-life of PEG-rhIL-6 in the rat in detail. We found that the half-life of PEG-rhIL-6 in the rat appeared to be 200 times and 20 times, respectively, longer than that in the rat ( t 1/2 K a =3 min, t 1/2 K e =55 min) (Castell et al., 1988 1988 ), and 2 to 10 times longer than that in humans ( t 1/2 =5 h) (Banks et al., 2000 2000 ). The plasma half-life of PEG-rhIL-6 in this study is also longer than that of rhIL-6 in non-human primates (Ryffel et al., 1994 1994 ). The time to reach the maximum concentration of PEG-rhIL-6 appeared to be longer than results reported in some previous studies after s.c. administration (Zha et al., 1995 1995 ; Banks et al., 2000 2000 ). For example, the mean t peak of 4.1 h at doses of 2.5–5.0 µg/(kg·d) was seen in the patients with cancer (Banks et al., 2000 2000 ) and t peak of 0.73 h at the dose of 10 µg/kg BW has been found in mice (Zha et al., 1995 1995 ), in contrast with 20.51, 21.30, and 21.97 h in this study for the rats receiving 3, 20, and 40 µg/kg BW, respectively. These differences may be due to the different administration routes and assay methods utilized. The modification of rhIL-6 with PEG may also contribute to our findings. PEG had been used to modify various enzymes and proteins to improve their properties and increase the size of proteins and enzymes, excluding them from immediate uptake by organs and prolonging blood circulation (Parveen and Sahoo, 2006 2006 ; Rawat et al., 2010 2010 ). In the previous studies, it was reported that the t 1/2 of interferon-alpha-2a (IFNα-2a) was only 2.3 h; however, its t 1/2 reached to 50 h after PEG modification (Zeuzem et al., 2003 2003 ). PEG can obviously prolong the t peak (Hinds and Kim, 2002 2002 ). As a consequence, the mean residence time was increased. In order to ensure the effectiveness and safety of the dosages used in the present study, the results of our pharmacodynamic and toxicological studies in dogs and rats on the tested PEG-rhIL-6 were referenced (Yin et al., 2005 2005 ). PEG-rhIL-6 was found to impede thrombocytopenia caused by cyclophosphamide at the dosages of 1–3 µg/kg BW by once-daily s.c. injection in dogs for one week, and at the dosages of 66 and 40 µg/kg BW by once-daily s.c. injection in rats for 5 d. These results are illustrated in Fig. 4 4 (Yin et al., 2005 2005 ), indicating that PEG-rhIL-6 can alleviate thrombocytopenia significantly and recover the platelet level more rapidly. On the other hand, the toxicological studies on PEG-rhIL-6 demonstrated the increasing risk for pharmacodynamics in dogs at the dosages of 6.0, 12.0, and 30 µg/kg BW by once-daily s.c. injection for 30 d (data not shown). Therefore, it was reasonable for us to choose the 3, 20, and 40 µg/kg BW in our experiments. At the same time, the dosages that were used in the present study keep the results consistent and comparable. (a) Injection of PEG-rhIL-6 (1, 2, and 3 µg/kg BW) to dogs; (b) Injection of PEG-rhIL-6 (40 and 66 µg/kg BW) to rats. Data are from Yin et al. ( 2005 2005 ) Tissue distribution of 125 I-PEG-rhIL-6 was investigated following a single s.c. administration to rats at 2 µg/kg BW dosage. The total radioactivity was determined in various tissues at 4, 24, and 48 h after s.c. injection. The results show that there was a slow and wide distribution in the tissues (or organs) throughout the whole body for the TCA-precipitable PEG-rhIL-6 within the time course examined. The amount of radioactivity in the plasma, muscle at the injection site, and bladder was found to be extremely high. The amount of TCA-precipitable radioactivity sharply increased at 24 h in the bladder. However, there was no marked increase of radioactivity in the liver with time. Considerable TCA-precipitable radioactivity was also found in reproductive tissues. The amount of TCA-precipitable radioactivity in the brain was extremely low, only 0.3 ng/g of brain at 24 and 48 h post-dosing, respectively. Considerable amount of the radioactivity was found in the kidney at 48 h post-dosing. There was an extremely high amount (2.130±1.3 ng/g) of radioactivity in the muscle at the injection site at 48 h post-dosing, indicating that PEG-rhIL-6 was being slowly absorbed. It has been previously reported that, after 20 min i.v. injection, about 80% of the rhIL-6 disappeared from the circulation in the liver. RhIL-6 was exclusively localized on the surface of parenchymal cells, suggesting the existence of an IL-6 receptor on the hepatocytes (Castell et al., 1988 1988 ). However, the radioactivity was relatively low in the liver, but relatively high in the blood at various time points after s.c. administration of PEG-rhIL-6 in this study. These may be accounted for by the modification of rhIL-6 with PEG (Caliceti and Veronese, 2003 2003 ). These findings were thought to be consistent with the literature of a relatively high radioactivity in plasma of human serum albumin-paclitaxel (Dosio et al., 2001 2001 ). There was a relatively low radioactivity of PEG-rhIL-6 in the brain, which means that PEG-rhIL-6 may not pass through the blood-brain barrier (BBB). These findings imply that 125 I-PEG-rhIL-6 is unlikely to accumulate in the tissues (or organs) examined following a single s.c. administration. The data of the excretion after a single s.c. administration of 2 µg/kg BW 125 I-PEG-rhIL-6 are illustrated in Fig. 3 3 . The results suggest that the dominant route of elimination for 125 I-PEG-rhIL-6 is urinary excretion following s.c. administration, as 23% administered radioactivity was recovered in urine, and recovery in the feces was only 6% over 192 h. The elimination for most of PEG-rhIL-6 was through the urine after metabolism. Therefore, it is not surprising that there was a relatively high radioactivity in the bladder. The kidney plays a more important role in the elimination for TCA-precipitable protein than the liver does. Therefore, one should monitor renal function and adjust the dose when PEG-rhIL-6 is used in patients with renal impairment. Conclusions 1 The findings in this study indicate a linear disposition of PEG-rhIL-6 at the used s.c. dosage range, and the c max and AUC showed an similar dose proportionality in rats. The PEG-rhIL-6 has favourable biokinetics. Because of its longer half-life, PEG-rhIL-6 is able to accumulate in blood and effectively increase peripheral platelet numbers. It appears to be a good candidate drug for patients with cancer. References P Bailon , A Palleroni , CA Schaffer , CL Spence , WJ Fung , JE Porter , GK Ehrlich , W Pan , ZX Xu , MW Modi , 等 . Rational design of a potent, long-lasting form of interferon: a 40 kDa branched polyethylene glycol-conjugated interferon alpha-2a for the treatment of hepatitis C . Bioconjug Chem , 2001 . 12 ( 2 ): 195 - 202 . 11312680 DOI: 10.1021/bc000082g http://doi.org/10.1021/bc000082g . Rational design of a potent, long-lasting form of interferon: a 40 kDa branched polyethylene glycol-conjugated interferon alpha-2a for the treatment of hepatitis C RE Banks , MA Forbes , PM Patel , M Storr , S Hallam , D Clarke , D Novick , E Ingham , C Bowmer , J Southgate , 等 . Subcutaneous administration of recombinant glycosylated interleukin 6 in patients with cancer: pharmacokinetics, pharmacodynamics and immunomodulatory effects . Cytokine , 2000 . 12 ( 4 ): 388 - 396 . 10805221 DOI: 10.1006/cyto.1999.0556 http://doi.org/10.1006/cyto.1999.0556 . Subcutaneous administration of recombinant glycosylated interleukin 6 in patients with cancer: pharmacokinetics, pharmacodynamics and immunomodulatory effects F Bracho , MD Krailo , V Shen , S Bergeron , V Davenport , W Liu-Mares , BR Blazar , A Panoskaltsis-Mortari , C van de Ven , R Secola , 等 . A phase I clinical, pharmacological, and biological trial of interleukin 6 plus granulocyte-colony stimulating factor after ifosfamide, carboplatin, and etoposide in children with recurrent/refractory solid tumors: enhanced hematological responses but a high incidence of grade III/IV constitutional toxicities . Clin Cancer Res , 2001 . 7 ( 1 ): 58 - 67 . 11205919 . A phase I clinical, pharmacological, and biological trial of interleukin 6 plus granulocyte-colony stimulating factor after ifosfamide, carboplatin, and etoposide in children with recurrent/refractory solid tumors: enhanced hematological responses but a high incidence of grade III/IV constitutional toxicities P Caliceti , FM Veronese . Pharmacokinetic and biodistribution properties of poly(ethylene glycol)-protein conjugates . Adv Drug Deliv Rev , 2003 . 55 ( 10 ): 1261 - 1277 . 14499706 DOI: 10.1016/S0169-409X(03)00108-X http://doi.org/10.1016/S0169-409X(03)00108-X . Pharmacokinetic and biodistribution properties of poly(ethylene glycol)-protein conjugates JV Castell , T Geiger , V Gross , T Andus , E Walter , T Hirano , T Kishimoto , PC Heinrich . Plasma clearance, organ distribution and target cells of interleukin-6/hepatocyte-stimulating factor in the rat . Eur J Biochem , 1988 . 177 ( 2 ): 357 - 361 . 3263918 DOI: 10.1111/j.1432-1033.1988.tb14384.x http://doi.org/10.1111/j.1432-1033.1988.tb14384.x . Plasma clearance, organ distribution and target cells of interleukin-6/hepatocyte-stimulating factor in the rat JV Castell , J Klapprot , V Gross , E Walter , T Andus , L Snyers , J Content , PC Heinrich . Fate of interleukin-6 in the rat . Eur J Biochem , 1990 . 189 ( 1 ): 113 - 118 . 2331992 DOI: 10.1111/j.1432-1033.1990.tb15466.x http://doi.org/10.1111/j.1432-1033.1990.tb15466.x . Fate of interleukin-6 in the rat M Cindric , T Cepo , N Galic , M Bukvic-Krajacic , N Tomczyk , JP Vissers , L Bindila , J Peter-Katalinic . Structural characterization of PEGylated rHuG-CSF and location of PEG attachment sites . J Pharm Biomed Anal , 2007 . 44 ( 2 ): 388 - 395 . 17448619 DOI: 10.1016/j.jpba.2007.02.036 http://doi.org/10.1016/j.jpba.2007.02.036 . Structural characterization of PEGylated rHuG-CSF and location of PEG attachment sites F Dosio , S Arpicco , P Brusa , B Stella , L Cattel . Poly(ethylene glycol)-human serum albumin-paclitaxel conjugates: preparation, characterization and pharmacokinetics . J Control Release , 2001 . 76 ( 1-2 ): 107 - 117 . 11532317 DOI: 10.1016/S0168-3659(01)00420-5 http://doi.org/10.1016/S0168-3659(01)00420-5 . Poly(ethylene glycol)-human serum albumin-paclitaxel conjugates: preparation, characterization and pharmacokinetics AL Drayer , CT Sibinga , NR Blom , JT de Wolf , E Vellenga . The in vitro effects of cytokines on expansion and migration of megakaryocyte progenitors . Br J Haematol , 2000 . 109 ( 4 ): 776 - 784 . 10929029 DOI: 10.1046/j.1365-2141.2000.02079.x http://doi.org/10.1046/j.1365-2141.2000.02079.x . The in vitro effects of cytokines on expansion and migration of megakaryocyte progenitors KD Hinds , SW Kim . Effects of PEG conjugation on insulin properties . Adv Drug Deliv Rev , 2002 . 54 ( 4 ): 505 - 530 . 12052712 DOI: 10.1016/S0169-409X(02)00025-X http://doi.org/10.1016/S0169-409X(02)00025-X . Effects of PEG conjugation on insulin properties ZP Hu , HS Niu , XX Yang , HF Li , GW Sang , B Li . Recombinant human parathyroid hormone 1–34: pharmacokinetics, tissue distribution and excretion in rats . Int J Pharm , 2006 . 317 144 - 154 . 16621369 DOI: 10.1016/j.ijpharm.2006.03.005 http://doi.org/10.1016/j.ijpharm.2006.03.005 . Recombinant human parathyroid hormone 1–34: pharmacokinetics, tissue distribution and excretion in rats I Kashiwakura , O Inanami , Y Abe , TA Takahashi , M Kuwabara . Different radiosensitive megakaryocytic progenitor cells exist in steady-state human peripheral blood . Radiat Res , 2005 . 164 ( 1 ): 10 - 16 . 15966760 DOI: 10.1667/RR3396 http://doi.org/10.1667/RR3396 . Different radiosensitive megakaryocytic progenitor cells exist in steady-state human peripheral blood T Kishimoto . AW Thomson , MT Lotze . Interleukin-6 (IL-6) . The Cytokine Handbook , 2003 . 1 4th Ed : New York Academic Press , 281 - 304 . . Interleukin-6 (IL-6) T Kishimoto . Interleukin-6: from basic science to medicine-40 years in immunology . Annu Rev Immunol , 2005 . 23 ( 1 ): 1 - 21 . 15771564 DOI: 10.1146/annurev.immunol.23.021704.115806 http://doi.org/10.1146/annurev.immunol.23.021704.115806 . Interleukin-6: from basic science to medicine-40 years in immunology T Kishimoto . IL-6: from its discovery to clinical applications . Int Immunol , 2010 . 22 ( 5 ): 347 - 352 . 20410258 DOI: 10.1093/intimm/dxq030 http://doi.org/10.1093/intimm/dxq030 . IL-6: from its discovery to clinical applications Y Kodera , H Tanaka , A Matsushima , Y Inada . Chemical modification of l-asparaginase with a comb-shaped copolymer of polyethylene glycol derivative and maleic anhydride . Biochem Biophys Res Commun , 1992 . 184 ( 1 ): 144 - 148 . 1567419 DOI: 10.1016/0006-291X(92)91170-U http://doi.org/10.1016/0006-291X(92)91170-U . Chemical modification of l-asparaginase with a comb-shaped copolymer of polyethylene glycol derivative and maleic anhydride A Kozlowski , SA Charles , JM Harris . Development of pegylated interferons for the treatment of chronic hepatitis C . BioDrugs , 2001 . 15 ( 7 ): 419 - 429 . 11520253 DOI: 10.2165/00063030-200115070-00001 http://doi.org/10.2165/00063030-200115070-00001 . Development of pegylated interferons for the treatment of chronic hepatitis C Y Levy , MS Hershfield , C Fernandez-Mejia , SH Polmar , D Scudiery , M Berger , RU Sorensen . Adenosine deaminase deficiency with late onset of recurrent infections: response to treatment with polyethylene glycol-modified adenosine deaminase . J Pediatr , 1988 . 113 ( 2 ): 312 - 317 . 3260944 DOI: 10.1016/S0022-3476(88)80271-3 http://doi.org/10.1016/S0022-3476(88)80271-3 . Adenosine deaminase deficiency with late onset of recurrent infections: response to treatment with polyethylene glycol-modified adenosine deaminase MA Markwell . A new solid-state reagent to iodinate proteins . Anal Biochem , 1982 . 125 ( 2 ): 427 - 432 . 6758629 DOI: 10.1016/0003-2697(82)90025-2 http://doi.org/10.1016/0003-2697(82)90025-2 . A new solid-state reagent to iodinate proteins R Mehvar . Modulation of the pharmacokinetics and pharmacodynamics of proteins by polyethylene glycol conjugation . Pharm Pharmaceut Sci , 2000 . 3 ( 1 ): 125 - 136 . . Modulation of the pharmacokinetics and pharmacodynamics of proteins by polyethylene glycol conjugation H Nakanishi , K Yoshioka , S Joyama , N Araki , A Myoui , S Ishiguro , T Ueda , H Yoshikawa , K Itoh . Interleukin-6/soluble interleukin-6 receptor signaling attenuates proliferation and invasion, and induces morphological changes of a newly established pleomorphic malignant fibrous histiocytoma cell line . Am J Pathol , 2004 . 165 ( 2 ): 471 - 480 . 15277221 . Interleukin-6/soluble interleukin-6 receptor signaling attenuates proliferation and invasion, and induces morphological changes of a newly established pleomorphic malignant fibrous histiocytoma cell line Y Nojima , Y Suzuki , K Iguchi , T Shiga , A Iwata , T Fujimoto , K Yoshida , H Shimizu , T Takeuchi , A Sato . Development of poly(ethylene glycol) conjugated lactoferrin for oral administration . Bioconjug Chem , 2008 . 19 ( 11 ): 2253 - 2259 . 18834167 DOI: 10.1021/bc800258v http://doi.org/10.1021/bc800258v . Development of poly(ethylene glycol) conjugated lactoferrin for oral administration S Parveen , SK Sahoo . Nanomedicine: clinical applications of polyethylene glycol conjugated proteins and drugs . Clin Pharmacokinet , 2006 . 45 ( 10 ): 965 - 988 . 16984211 DOI: 10.2165/00003088-200645100-00002 http://doi.org/10.2165/00003088-200645100-00002 . Nanomedicine: clinical applications of polyethylene glycol conjugated proteins and drugs S Pradhananga , I Wilkinson , RJ Ross . Pegvisomant: structure and function . J Mol Endocrinol , 2002 . 29 ( 1 ): 11 - 14 . 12200225 DOI: 10.1677/jme.0.0290011 http://doi.org/10.1677/jme.0.0290011 . Pegvisomant: structure and function S Rawat , CR Suri , DK Sahoo . Molecular mechanism of polyethylene glycol mediated stabilization of protein . Biochem Biophys Res Commun , 2010 . 392 ( 4 ): 561 - 566 . 20097167 DOI: 10.1016/j.bbrc.2010.01.067 http://doi.org/10.1016/j.bbrc.2010.01.067 . Molecular mechanism of polyethylene glycol mediated stabilization of protein B Ryffel , BD Car , G Woerly , M Weber , F DiPadova , M Kammuller , S Klug , R Neubert , D Neubert . Long-term interleukin-6 administration stimulates sustained thrombopoiesis and acute-phase protein synthesis in a small primate—the marmoset . Blood , 1994 . 83 ( 8 ): 2093 - 2102 . 7512839 . Long-term interleukin-6 administration stimulates sustained thrombopoiesis and acute-phase protein synthesis in a small primate—the marmoset H Shibata , S Nakagawa , Y Tsutsumi . Optimization of protein therapies by polymer-conjugation as an effective DDS . Molecules , 2005 . 10 ( 1 ): 162 - 180 . 18007284 DOI: 10.3390/10010162 http://doi.org/10.3390/10010162 . Optimization of protein therapies by polymer-conjugation as an effective DDS S Tsunoda , T Ishikawa , M Watanabe , H Kamada , Y Yamamoto , Y Tsutsumi , T Hirano , T Mayumi . Selective enhancement of thrombopoietic activity of PEGylated interleukin 6 by a simple procedure using a reversible amino-protective reagent . Br J Haematol , 2001 . 112 ( 1 ): 181 - 188 . 11167800 DOI: 10.1046/j.1365-2141.2001.02508.x http://doi.org/10.1046/j.1365-2141.2001.02508.x . Selective enhancement of thrombopoietic activity of PEGylated interleukin 6 by a simple procedure using a reversible amino-protective reagent YS Wang , S Youngster , M Grace , J Bausch , R Bordens , DF Wyss . Structural and biological characterization of pegylated recombinant interferon alpha-2b and its therapeutic implications . Adv Drug Deliv Rev , 2002 . 54 ( 4 ): 547 - 570 . 12052714 DOI: 10.1016/S0169-409X(02)00027-3 http://doi.org/10.1016/S0169-409X(02)00027-3 . Structural and biological characterization of pegylated recombinant interferon alpha-2b and its therapeutic implications HL Yin , XL He , B Xu , X Liu , G Yang . The impedance effects of the interleukin-6 on hematopoietic damnification of cyclophosphamide-induced dogs . J Biomed Eng , 2005 . 22 ( 4 ): 798 - 801 . (in Chinese) . The impedance effects of the interleukin-6 on hematopoietic damnification of cyclophosphamide-induced dogs S Zeuzem , C Welsch , E Herrmann . Pharmacokinetics of peginterferons . Semin Liver Dis , 2003 . 23 ( Supp1. 1 ): 23 - 28 . 12934165 DOI: 10.1055/s-2003-41631 http://doi.org/10.1055/s-2003-41631 . Pharmacokinetics of peginterferons JQ Zha , QX Zheng , YL Li . Pharmacokinetics of interleukin-6 in mouse . Guangdong Pharm , 1995 . 5 ( 3 ): 53 - 54 . (in Chinese) . Pharmacokinetics of interleukin-6 in mouse YJ Zhang , YJ Xu , R Zhu , MJ Hu , JX Li , YJ Chen , DJ Wang , W Fan . Pharmacokinetics, tissue distribution and excretion of a recombinant fusion protein 125 I-rhTNT-IL2 . J Radioanalyt Nucl Chem , 2007 . 273 ( 1 ): 3 - 8 . DOI: 10.1007/s10967-007-0701-4 http://doi.org/10.1007/s10967-007-0701-4 . Pharmacokinetics, tissue distribution and excretion of a recombinant fusion protein 125I-rhTNT-IL2 PEG-rhIL-6 was kindly provided by Rongsheng Co., Chengdu, China. We wish to thank Prof. Xian-yin ZEN (Sichuan Agricultural University, China) for expert technical assistance. We also would like to thank Ms. Xian-kun XU and Ms. Shu-fang WANG (Sichuan Agricultural University, China) for their valuable assistance in this research. 10.1631/jzus.B1000085 Project (Nos. 10802054 and 30700149) supported by the National Natural Science Foundation of China 2010-03-10 2010-12-12 2010-08-29 2022-01-05 Copyright © Zhejiang University and Springer-Verlag Berlin Heidelberg 2011 2011 Journal of Zhejiang University-SCIENCE B (Biomedicine and Biotechnology) 01 12