Pharmacokinetics Study of Recombinant Hirudin in the Plasma of Rats Using Chromogenic Substrate, ELISA, and Radioisotope Assays

Aim To compare the analytical methods used to study the pharmacokinetics of recombinant hirudin in the plasma of rats that had been injected with 125I-recombinant hirudin. Methods 2.0 mg/kg 125I-recombinant hirudin were injected intravenously into rats. The recombinant hirudins in the plasma was analyzed by chromogenic substrate assay, enzyme-linked immunosorbent assay (ELISA), total radioisotope assay (RA) and trichloroacetic acid pre-treated total radioisotope assay (TCA-RA). Results The chromogenic substrate assay standard curve was linear over the concentration range from 3.12 to 40.00 ng/ml for the recombinant hirudin in plasma. The relative standard deviations (RSD) for the intra- and inter-day variation were 5.0 to 6.3% and 11.9 to 12.6%, respectively. The recoveries of recombinant hirudin was 89.8% to 100.7%. The limit of quantification (LOQ) was 3.12 ng/ml. The concentration-time curve of the recombinant hirudin in the plasma could be explained as a two-compartment model. Pharmacokinetic parameters, including the half-life of distribution phase (t1/2 α), the half-life of elimination phase (t1/2 β), volume of apparent distribution (Vd), and area under the concentration-time curve from zero to infinite time (AUC0–t) were 7.59 min, 46.99 min, 0.17 L/kg, and 204.5 mg/L/min, respectively, as determined by chromogenic substrate assay; 6.41 min, 47.28 min, 1.24 L/kg, and 575.18 mg/L/min, respectively, as determined by ELISA; 3.69 min, 701.90 min, 0.04 L/kg, and 4189 mg/L/min, respectively as determined by RA; and 4.57 min, 724.9 min, 0.09 L/kg, and 2329 mg/L/min, respectively, as determined by TCA-RA. Conclusions The chromogenic substrate assay on the concentration dynamics of the recombinant hirudin in the plasma is a specific, sensitive, and accurate analytical method for pharmacokinetic studies. Moreover, the pharmacokinetic parameters determined by the chromogenic substrate assay and ELISA are congruent except for AUC.


Introduction
Hirudin is a small anti-coagulant protein originally isolated from the saliva of medicinal leeches [1]. Hirudin consists of 65 amino acid residues, with a molecular weight of about 7000 Da, and possesses anti-thrombin activity [2]. In contrast to heparin, hirudin does not require cofactors for thrombin inhibition [3]. Hirudin is the most powerful thrombin inhibitor with both anticoagulant and antithrombotic effects and is superior to the traditional anticoagulant heparin for treating thrombotic diseases [4]. A recombinant form of hirudin, in which the residue tyrosine 63 was replaced with a SO 3 -base form of the residue, exhibits similar pharmacological activity as that of the wildtype [5,6].
The bioassay, immunoassay and widely used isotope tracer techniques [7,8,9] have been reported to be used in rH pharmacokinetic studies. The total radioactivity detected in the isotope tracer techniques does not represent the real hirudin concentration in blood samples and therefore other methods should be adopted for the pharmacokinetic study [9]. We have used enzyme-linked immunosorbent assay (ELISA) to study the pharmacokinetic parameters of recombinant hirudin [10]. Most recently, chromogenic substrate assay method was employed to study the metabolites of recombinant hirudin in rats [11]. However, the pharmacokinetic parameters determined from these assays vary, depending on the assay employed [9]. In Markwardt's study [12], the thrombin time detection and chromogenic substrate method were used to for rH pharmacokinetic research in dog and human plasma, and the elimination half-life (t 1/2b ) of the pharmacokinetic parameters calculated was 73 min for dog and 60-100 min for human. Richter et al [9] reported the t 1/2b was 65 min for rats and 56.6 min for dog by using the 125 I-rH combined with thrombin titration assay. The t 1/2b of rH in baboons was determined as 24 min and 23 min by using the 131 Ilabeled rH combined the ecarin Clotting Time (ECT) method by Meiring et al [13]. Recently, Huang et al [8] reported that in rats, the t 1/2b was 172.8 min by the total plasma 125 I-labeled rH radioactivity (RA) method and 107 min by the trichloroacetic acid precipitate plasma 125 I-labeled rH radioactive activity (TCA-RA) method. Importantly, the pharmacokinetic parameter t 1/2b of rH varies not only between different experimental animals (73 min for dog; 60-100 min for human; 65 min for rat; 56.6 min for dog; 24 min for baboons; and 107-172 min for rats), but also in the same animals when different methods are employed (172 min by the RA vs. 102 min by the TCA-RA). When different methods are used for rH analysis in same animals, the pharmacokinetic parameters differences may be found by analyzing and comparing data determined by these methods. In the present study, we analyze the 125 I-recombinant hirudin in the plasma of the rats that had been injected with the radiolabeled recombinant hirudinuse, using chromogenic substrate assay, ELISA, total radioisotope assay (RA), and trichloroacetic acid pre-treated total radioisotope assay (TCA-RA). By comparing the pharmacokinetics parameters obtained from these assays, we determine the optimal assay to study the pharmacokinetics of recombinant hirudin.

Ethics Statement
Animals were maintained and experiments were conducted in accordance with the Institutional Animal Care and Use Committee, Dalian University of Technology, and with the 1996 Guide for the Care and Use of Laboratory Animals (Institute of Laboratory Animal Resources on Life Sciences, National Research Council, National Academy of Sciences, Washington DC). The study was approved by the Education and Research Committee and the Ethics Committee of Dalian University of Technology (approval # DUT2010553A).

Drugs and reagents
The recombinant hirudin used in this study (Lot Number 99110) was provided by the Dalian Gaoxin Biopharmaceutical Company (Dalian, China). The recombinant hirudin was dissolved in saline to 1.0 mg/ml. This standard stock solution was stored at 230uC. 125 I-recombinant hirudin was radiolabeled and prepared using the modified chloraine-T method with a specific activity of 45.361.48 MBq/ml. The purity of the 125 I-recombinant hirudin was determined to be 99.460.47% by HPLC (Chinese Academy of Military Medical Sciences; Beijing, China). The biological activity of the 125 I-recombinant hirudin remained unchanged during the labeling procedure. Thrombin (1000 U/vial) was purchased from Sigma (St Louis, MO).

Chromogenic Substrate Assay Method
Basic principle. This method is based on the principle that thrombin hydrolyzes the chromogenic substrate TGPApNA and releases pNA, which has an absorption at 405 nm. The thrombin is present in excess relative to the recombinant hirudin in the reaction. The quantity of recombinant hirudin is calculated from the difference between the total thrombin and the residual thrombin.
Preparation of solutions. Thrombin was diluted to 10 U/ ml with 50 mM Tris-buffer pH 8.3 (containing 6.05 g Tris, 1.00 g sodium azide, 13.30 g NaCl, 1.00 g bovine serum albumen in 1000 ml distilled water). The chromozym chromogenic substrate (TGPApNA) was diluted to 2 mM with distilled water.
Preparation of the blank plasma. Blank plasma was obtained from the orbital sinus of rats that had not been injected with recombinant hirudin, using procedures described by Van Herk et al (1998). 3 ml of the blank plasma sample was treated with 0.3 ml of 1 M HCl at 65uC for 5 min. The blank plasma samples were rapidly cooled to room temperature. After an addition of 0.3 ml of 1 M NaOH, the blank samples were centrifuged at 5000 rpm for 15 minutes. The supernatant was collected and diluted 20-fold with 50 mM Tris-buffer pH 8.3 to serve as the blank samples.   Preparation of the sample from rat plasma. The rat plasma sample preparation followed the same procedure as that for the blank, described above, except 0.5 ml of the rat plasma was treated with 0.05 ml of 1 M HCl, and 0.05 ml of 1 M NaOH accordingly.
The rH concentration in plasma was determined as previously described [11].

ELISA method
Solution preparation. Wash buffer was prepared by dissolving the content of the PBS/Tween 20 package according to the manufacturer's instructions. The sample buffer (T-buffer) was prepared by dissolving 3 g of bovine serum albumin in 100 ml of wash buffer.
Preparation of the rat plasma blank. Plasma was obtained from rats that have not been injected with recombinant hirudin from the orbital sinus. The plasma blank was diluted 100-fold with saline, followed by an additional 10-fold dilution with T-buffer.
The rH concentration in plasma was determined as previously described [10].
3. Total radioisotope assay and trichloroacetic acid pretreated total radioisotope assay method Standard curve of total radioisotope assay and the trichloroacetic acid pre-treated total radioisotope assay. The 125 I-recombinant hirudin solutions prepared above were diluted with saline to obtain a solution of specific radioactivity of 725 Bq/ml and recombinant hirudin was added to obtain a standard stock solution concentration of 0.4 g/L. The standard stock solution was diluted to concentrations of 0.026, 0.128, 0.64, 3.2, 16, and 80 mg/ml with diluted rat plasma blank. 100 ml of the standard solution was added to each sample tube. The radioactivity of the samples was measured by a gamma counter. After an addition of 100 ml of trichloroacetic acid solution, the samples were centrifuged at 10,000 rpm for 10 minutes. The supernatant was discarded and the radioactivity of the precipitate was determined.
The plasma samples were analyzed using the total radioisotope assay and the trichloroacetic acid pre-treated total radioisotope assay method. The radioactivity was plotted against the corresponding concentrations of recombinant hirudin standard to generate the standard curve. The recombinant hirudin concentration was calculated using this standard curve.

Statistical methods
The statistical comparison between two groups was analyzed by the ANOVA variance analysis and the multiple comparisons between groups were analyzed by the LSD method [14].
The difference between the two groups is p,0.05. 1. The Chromogenic substrate assay; 2. ELISA; 3. the total radioisotope assay; 4. the trichloroacetic acid pre-treated total radioisotope assay. The statistical comparison between two groups was analyzed by the ANOVA variance analysis and the multiple comparisons between groups were analyzed by the LSD method. doi:10.1371/journal.pone.0064336.t002
The pharmacokinetics of recombinant hirudin in plasma. The concentration-time curve (Figure 1) showed that after intravenous administration, the recombinant hirudin in plasma decayed bi-exponentially, with an extremely rapid initial phase followed by a relatively slow elimination phase. A twocompartment model for recombinant hirudin was further confirmed. The main pharmacokinetic parameters are presented in Table 1. The distribution half-life (t 1/2 a) was 7.5964.56 minutes. The elimination half-life (t 1/2 b) was 47.0622.2 minutes. These results suggest that the recombinant hirudin was distributed from the central to the peripheral compartment and eliminated rapidly from the body. These results are similar to those of the chromogenic substrate assay below except that the area under the curve was 204.5619.9 mg/L/min by the chromogenic substrate assay and 575.2639.4 mg/L/min by ELISA.
The recombinant hirudin pharmacokinetic in plasma. The plasma concentration-time curve (Figure 2) from the ELISA assay showed that, similar to the concentration-time curve derived from the chromogenic substrate assay, the 125 Irecombinant hirudin decayed bi-exponentially with an extremely rapid initial phase followed by a relatively slow elimination phase.
The two-compartment model for recombinant hirudin was further confirmed. The main pharmacokinetic parameters are presented in Table 1. The distribution half-life (t 1/2 a) was 6.4262.60 minutes. The elimination half-life (t 1/2 b) was 47.3613.1 minutes. In agreement with the results obtained from chromogenic assay, these results also suggest that the recombinant hirudin was distributed from central to peripheral compartment, and eliminated rapidly from the body.
3. Total radioisotope assay and the trichloroacetic acid pre-treated total radioisotope assay Radioactivity (Y) versus concentration (X, mg/ml) displayed a linear relationship, as described by the equation of Y = 141.64X 263.93 (r = 1.00, n = 5) from the total radioisotope assay); and Y = 24.15X 26.95 (r = 1.00, n = 5) from the trichloroacetic acid pre-treated total radioisotope assay). The limit of detection (LOD) was 0.026 mg/ml. The intra-day variation RSD were 1.7%, 4.5% and 4.9%, and inter-day variation RSD were all less than 10%. The recombinant hirudin recovery was 98.560.5%, as determined by the total radioisotope assay method.
The recombinant hirudin concentration-time curve in rat plasma, determined by total radioisotope assay and the trichloroacetic acid pre-treated total radioisotope assay method are shown in Figure 3. The main pharmacokinetic parameters are shown in Table 1. The distribution half-life (t 1/2 a) was 3.6961.71 minutes by the total radioisotope assay and 4.5861.73 minutes by the trichloroacetic acid pre-treated total radioisotope assay. The elimination half-life (t 1/2 b) was 701.96198.8 minutes and 724.9681.2 minutes respectively.

Discussion
The principle of the chromogenic substrate assay method is that thrombin can hydrolyze the chromogenic substrate TGPApNA to release pNA, which has absorption at 405 nm. The thrombin concentration is in excess relative to the recombinant hirudin in the reaction. The quantity of recombinant hirudin is calculated by the difference between the total thrombin and the residual thrombin. The acid treatment of samples at 65uC deactivates the intrinsic thrombin in plasma prior determining the recombinant hirudin concentration, as described previously [15]. The limit of detection of the chromogenic substrate assay method was 3.12 ng/ml, of the ELISA assay method is 0.25 ng/ml; hence these assays could also be used in the pharmacokinetic study of recombinant hirudin in rats.
The isotope tracer method is the classic method for protein (including peptide) drug pharmacokinetic studies. In this paper, the total radioactivity of 125 I-recombinant hirudin (total radioisotope assay) and the total radioactivity of 125 I-recombinant hirudin after precipitating the samples with trichloroacetic acid (the trichloroacetic acid pre-treated total radioisotope assay) were employed to study the pharmacokinetic behavior of recombinant hirudin. The pharmacokinetic parameters (except for AUC) showed little difference, indicating the recombinant hirudin sedimentation rate in vivo in this study is consistent to that reported in the literature [9], although the trichloroacetic acid concentration was low (less than 20%). The recombinant hirudin could not be completely precipitated because of its low molecular weight (7000 Da) and higher solubility.
The three main pharmacokinetic parameters calculated by the three methods are listed in Table 1 and the multiple comparisons of the pharmacokinetic parameters within each group are listed in Table 2. The AUC (0-t) values, are significantly different from each other, as measured by chromogenic substrate assay, ELISA, the total radioisotope assay and the trichloroacetic acid pre-treated total radioisotope assay. The half-life of the recombinant hirudin is less than 1 hour, as analyzed by the chromogenic substrate assay and ELISA, but the half-life is more than 10 hours, as analyzed by the total radioisotope assay and the trichloroacetic acid pre-treated total radioisotope assay. This discrepancy may be due to long halflife of radioisotope 125 I. These results suggest that the total radioisotope assay is not a suitable assay to study for pharmacokinetics of recombinant hirudin.