Introduction

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Tissue-type plasminogen activator (t-PA)¹ is an endogenous glycoprotein that plays a central role in fibrinolysis. Recently, recombinant wild-type t-PA (rwt-PA) has been widely used as a thrombolytic agent to treat acute myocardial infarction. Many clinical trials have been conducted to evaluate the pharmacologic efficacy of various rwt-PAs, and the results of these studies revealed lower patient mortality compared with the use of streptokinase or urokinase (Verstraete et al., 1985; Kanemoto et al., 1991).

One major disadvantage of rwt-PA is its short plasma half-life due to rapid uptake by the liver (Camani et al., 1994). Two kinds of hepatocytes, parenchymal and endothelial cells, are responsible for the uptake of rwt-PA. To improve the short plasma half-life of rwt-PA, various modified rwt-PAs have been developed. Pamiteplase is a novel recombinant t-PA bearing a deletion in the Kringle-1 domain and a point mutation (Arg₂₇₅  Glu) in the Kringle-2 domain (Kawauchi et al., 1991). Despite these structural modifications, pamiteplase possesses almost the same in vivo affinity for fibrinous thrombi as rwt-PA (Katoh et al., 1991). Furthermore, plasma concentrations after administration of pamiteplase to rats or dogs are higher than those of rwt-PA (Oikawa et al., 1996a). Pamiteplase at one-fifth the dose of rwt-PA has almost the same thrombolytic activity as rwt-PA in rats or dogs with induced thrombi (Kawasaki et al., 1993a,b,c). Consequently, pamiteplase has the same biological effect as t-PA but much lower clearance compared with rwt-PA.

The clearances of high-molecular weight compounds from plasma are usually due to cellular uptakes by endocytosis, especially in the liver and the kidneys, and/or to irreversible bindings of these compounds to plasma proteins. In the case of pamiteplase and rwt-PA, an unchanged form after administration of either pamiteplase or rwt-PA to rats was not excreted in urine, indicating their eliminations are due to these kinds of metabolic clearance (Iida et al., 1988; Kizen et al., 1988; Komuro et al., 1989; Okumura et al., 1989; Oikawa et al., 1996b). The greatest tissue distribution of both compounds is in the liver, where uptake and degradation occur (Iida et al., 1988; Kizen et al., 1988; Komuro et al., 1989; Okumura et al., 1989; Oikawa et al., 1996b). Therefore, these findings suggest a large difference in hepatic uptake between pamiteplase and rwt-PA.

In this study, the distribution of radioactivity in tissues was examined after single i.v. administration of the same dose of ¹²⁵I-labeled forms of both drugs. A large difference in hepatic distribution between the drugs was observed, so the uptake of pamiteplase and rwt-PA to the liver in rats was examined using the liver uptake index (LUI) measured by a tissue-sampling single-injection technique. Furthermore, because both drugs formed complexes with plasma protein, the rates of formation of these complexes were examined.

Experimental Procedures

All animal experiments complied with the regulations of Yamanouchi Pharmaceutical's Animal Experimentation Ethics Committee.

Materials. The pamiteplase gene was constructed by combining a part of the t-PA gene and a synthesized DNA fragment coding the finger and epidermal growth factor (EGF) domains, and was then cloned into a mammalian expression vector, pVY1, under the control of the SV 40 early promoter (Kawauchi et al., 1991). Plasmid pVY-1-pamiteplase was transfected into Chinese hamster ovary cells (Kawauchi et al., 1991). The pamiteplase molecule contains a finger domain, an EGF domain, a Kringle-2 domain, a serine protease domain, and a site mutation at the Kringle-2-serine protease linkage site (Kawauchi et al., 1991). A lyophilized pharmaceutical preparation of pamiteplase was used in this study. rwt-PA (Alteplase) was purchased from Genentech, Inc. (San Francisco, CA). Carrier-free Na¹²⁵I and ³H₂O were purchased from DuPont NEN (Boston, MA). Other reagents were commercially available and of analytical grade.

Animals. Fischer male rats aged 6 weeks (body weight, 162-201 g) were purchased from Charles River Japan, Inc. (Yokohama, Japan), and were acclimated for more than 1 week before the study. They were housed in an air conditioned room (temperature, 23 ± 2°C; relative humidity, 55 ± 5%) and kept on a light/dark cycle of 12 h/12 h. They had free access to pelleted food (MF; Oriental Yeast Co., Ltd., Tokyo, Japan) and water.

Preparation of ¹²⁵I-Labeled Pamiteplase (¹²⁵I-pamiteplase) and ¹²⁵I-Labeled rwt-PA (¹²⁵I-rwt-PA). Lyophilized formulations containing either 4 mg of pamiteplase or 50 mg of rwt-PA were dissolved in physiologic saline to make stock solutions of both drugs (1 mg/ml), respectively. ¹²⁵I-pamiteplase or ¹²⁵I-rwt-PA was synthesized by the chloramine-T method using carrier-free Na¹²⁵I and stock solutions (Iida et al., 1988; Kizen et al., 1988; Komuro et al., 1989; Okumura et al., 1989; Oikawa et al., 1996b). The specific activity, concentration, and purity of radioactivity of ¹²⁵I-pamiteplase were 214.3 MBq/mg, 11.4 MBq/ml, and >95%, respectively; those of ¹²⁵I-rwt-PA were 177.0 MBq/mg, 9.4 MBq/ml, and >95%, respectively.

Intravenous Administration. Dosing solutions of ¹²⁵I-pamiteplase or ¹²⁵I-rwt-PA were prepared from the stock solution, ¹²⁵I-labeled compound, and vehicle A solutions (0.127 M phosphoric acid solution containing 0.2 M arginine and 0.01% Tween 80) to yield a final concentration of 0.15 mg/ml. These dosing solutions were i.v. administered at 0.3 mg/kg to rats via the tail vein.

Analysis of GFC. Analysis of GFC was: precolumn: TSK guard column SW_XL (6.0-mm × 4-cm; Tosoh Corp., Tosoh, Japan); column: TSK-GEL G-3000 SW_XL (7.5-mm × 30-cm; Tosoh); column temperature: room temperature; eluent: 0.1 M Na₂HPO₄-NaH₂PO₄ buffer (pH 6.0) containing 0.01% Tween 80 and 0.2 M L-arginine; flow: 0.5 ml/min; and detection: UV 280 nm.

Pharmacokinetic Analysis of Plasma Concentrations. Plasma concentrations were analyzed using WinNonlin software (Version 1.5; Pharsight Inc., Mountain View, CA) to calculate the half-life in -phase (t_1/2), half-life in -phase (t_1/2), area under the plasma concentration-time curve from zero to infinity (AUC₀), total clearance (CL_total), distribution volume at the steady state (Vd_ss), and mean residence time (MRT).

Kinetic Analysis of Liver Concentrations. The hepatic uptakes of pamiteplase or rwt-PA after i.v. administration of ¹²⁵I-pamiteplase or ¹²⁵I-rwt-PA can be described by linear kinetics as follows:

(1)

where C_T is the liver concentration, V_T is the liver distribution volume at time t after administration, C_P is the plasma concentration, k₁ is the clearance constant for the binding process (or the uptake process into the cells), and k₂ is the clearance constant for efflux from the liver (or the dissociation process from cell surface binding sites). Integration of eq. 1 gives:

(2)

(3)

where AUC_0t represents the area under the plasma concentration-time curve from time 0 to t. When the efflux (or dissociation) is much smaller than the influx within a short period of time, eq. 3 can be simplified to eq. 4:

(4)

The plot of V_T · C_T(t) versus AUC_0t yields a straight line, and the slope of the line represents k₁.

Intravenous Administration through the Hepatic Portal Vein. The portal vein injection technique to obtain the LUI was performed according to the methods described by Tsuji et al. (1990). Dosing solutions containing ³H₂O (a freely diffusible reference) were prepared from stock solutions, the ¹²⁵I-labeled compounds, ³H₂O, and Ringer HEPES buffer [10 mM HEPES buffer (pH 7.4) containing 141 mM NaCl, 4 mM KCl, 2.8 mM CaCl₂, 0.2 M arginine, and 0.01% Tween 80]. Three rats were used at each point. Rats were anesthetized by ketamine (235 mg/kg) and xylazine (2.3 mg/kg). After laparotopy, the hepatic artery was ligated, and the portal vein was cannulated with a 27-gauge needle. Two hundred microliters of the dosing solution was injected within 0.5 s. The portal vein was transected at 18 s after injection, and a part of the right major lobe of the liver was immediately removed. The removed liver was minced, and approximately 200 mg of minced tissue was dissolved in 2.0 ml of Soluene 350 (Packard) at 50°C for 4 h. After the addition of hydrogen peroxide, acetic acid was added to neutralize the solution. Radioactivity in the liver samples was measured in a gamma counter (Autogamma 5530; Packard) and in a liquid scintillation counter (Model CA2000; Packard).

Determination of LUI and Hepatic Clearance (CL_hepatic). The LUI is defined in eq. 5 and experimentally determined using eq. 6.

(5)

(6)

where E_drug and E_reference are the fractional extraction of test and reference compounds on a single pass. The E_drug was given by:

(7)

The E_reference value of ³H₂O has been previously reported as 84% (Tsuji et al., 1990). The vascular volume of the liver is not negligible. Therefore, the extravascular hepatic extraction (E_x,drug, the extraction is only due to the cellular uptake) is calculated as follows:

(8)

where E_ns represents the extraction of albumin for distribution in hepatic vessel, and a value of 13% has been reported (Tsuji et al., 1990). Furthermore, hepatic clearance in LUI studies [CL_hepatic (LUI)] was calculated with C_in (concentration of a drug in dosage solution) and hepatic plasma flow as follows:

(9)

Effect of rwt-PA on the E_x,drug of Pamiteplase. To determine the effect of rwt-PA on the E_x,drug of pamiteplase, a dosing solution was prepared using stock solutions of the drugs, ¹²⁵I-pamiteplase, ³H₂O, and Ringer-HEPES buffer to yield final concentrations of 10 nM pamiteplase and that of rwt-PA ranging from 5 to 500 nM. The experimental procedure is the same as described above.

Examination of Plasma Clearance. Ten micrograms per milliliter of incubation solutions were prepared using the stock solutions, ¹²⁵I-labeled compounds, and vehicle A solutions. After adding an incubation solution (final concentration 1 µg/ml) to rat plasma preincubated at 37°C for 5 min, these mixtures were incubated at 37°C, and samples were collected at 2, 5, 10, 15, 20, 30, 45, and 60 min after incubation. Unchanged drug concentrations in these samples were analyzed using GFC. As the plasma concentrations of both drugs declined in a monophasic manner, the elimination rate constant (k_el) was calculated using a first-order exponential equation:

(10)

where C_P is unchanged drug concentration in plasma at time t and A is the coefficient, respectively.

	Results

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Tissue Distribution. Figures 1 and 2 show concentrations and percentages of the administered dose distributed in tissues after a single i.v. administration of 0.3 mg/kg of ¹²⁵I-pamiteplase or ¹²⁵I-rwt-PA to rats, respectively. As shown in Fig. 1, the concentration at 5 min after administration of ¹²⁵I-pamiteplase was highest in the plasma, followed by the blood, liver, and kidneys. The concentration of ¹²⁵I-pamiteplase in the liver was 37% of that in the plasma. In contrast to pamiteplase, the concentration of ¹²⁵I-rwt-PA at 5 min after administration was highest in the liver, followed by the plasma, spleen, and kidneys. The liver concentration of ¹²⁵I-rwt-PA was 2.9 times higher than that of the plasma. At 120 min postdosing, no difference was observed in the tissue concentrations of the drugs. As shown in Fig. 2, the percentage distribution of radioactivity in the liver at 5 min after administration of ¹²⁵I-rwt-PA was 2.5-fold higher than that after administration of ¹²⁵I-pamiteplase.

View larger version (28K): [in this window] [in a new window]   Fig. 1.   Concentrations of radioactivity in tissues at 5 and 120 min after i.v. administration of 0.3 mg/kg of 125I-pamiteplase or 125I-rwt-PA to rats. Each value represents mean and standard error of three rats.

View larger version (21K): [in this window] [in a new window]   Fig. 2.   Percentages of the administered dose distributed in tissues at 5 and 120 min after i.v. administration of 0.3 mg/kg of 125I-pamiteplase or 125I-rwt-PA to rats. Each value represents mean and standard error of three rats.

Plasma Concentration. Figure 3 shows a time profile of unchanged drug concentration in plasma after a single i.v. administration of 0.3 mg/kg of ¹²⁵I-pamiteplase or ¹²⁵I-rwt-PA to rats, and Table 1 shows the pharmacokinetic parameters of both drugs. Plasma concentrations of both drugs declined biexponentially. t_1/2 of ¹²⁵I-pamiteplase was 5.5 times longer than that of rwt-PA, t_1/2 was 4.8 times longer, and MRT was 7 times longer, respectively. CL_total of pamiteplase decreased to 22% of that of rwt-PA.

View larger version (17K): [in this window] [in a new window]   Fig. 3.   Concentrations of unchanged drug in plasma after i.v. administration of 0.3 mg/kg of 125I-pamiteplase or 125I-rwt-PA to rats. Each value represents mean and standard error of three rats.

Concentration of Radioactivity in the Liver. Figure 4 shows liver concentrations, Fig. 5 percentages of the administered dose distributed in the liver, and Fig. 6 percentages of concentration in the liver relative to that in the plasma after a single i.v. administration of 0.3 mg/kg of ¹²⁵I-pamiteplase or ¹²⁵I-rwt-PA to rats. As shown in Figs. 4 and 5, the maximum liver concentration after administration of ¹²⁵I-rwt-PA was 2.5 times higher than that after administration of ¹²⁵I-pamiteplase, and the maximum percentage of radioactivity distribution also was 2.6 times higher. As shown in Fig. 6, the percentage of concentration in the liver relative to that in the plasma after administration of ¹²⁵I-pamiteplase leveled out from 10 min after dosing, and ranged from 0.69 to 0.95, whereas that of ¹²⁵I-rwt-PA ranged from 1.4 to 14.2. 

View larger version (14K): [in this window] [in a new window]   Fig. 4.   Concentrations of radioactivity in liver after i.v. administration of 0.3 mg/kg of 125I-pamiteplase or 125I-rwt-PA to rats. Each value represents mean and standard error of three rats.

View larger version (22K): [in this window] [in a new window]   Fig. 5.   Percentages of the administered dose distributed in the liver after i.v. administration of 0.3 mg/kg of 125I-pamiteplase or 125I-rwt-PA to rats. Each value represents mean and standard error of three rats.

View larger version (15K): [in this window] [in a new window]   Fig. 6.   Ratios of radioactivity concentration in the liver relative to plasma after i.v. administration of 0.3 mg/kg of 125I-pamiteplase or 125I-rwt-PA to rats. Each value represents mean and standard error of three rats.

CL_hepatic Calculated by an Integration Plot. Figure 7 shows a plot of liver drug concentration versus AUC_0t of plasma concentration, and the slope reveals k₁ (ml/min/g of tissue) by eq. 4. Table 2 shows CL_hepatic calculated using k₁, liver weight, and body weight. The CL_hepatic of pamiteplase decreased to 19% of that of rwt-PA. The CL_hepatic of both drugs was 75 and 86%, and accounted for most of the CL_total calculated using unchanged drug concentrations in the plasma.

View larger version (15K): [in this window] [in a new window]   Fig. 7.   Plots for estimating CLhepatic according to eq. 4. Each value represents mean and standard error of three rats.

Hepatic Extraction Ratio by a Portal Vein Injection Technique. Figure 8 shows E_x,drug and CL_hepatic after a single administration of ¹²⁵I-pamiteplase or ¹²⁵I-rwt-PA (ranged from 0.057-57 µg/ml) into the portal vein of rats. E_x,drug ranged from 8.94 to 15.36% for ¹²⁵I-pamiteplase and from 30.25 to 33.58% for ¹²⁵I-rwt-PA, and it fluctuated very little from these values. E_x,drug of ¹²⁵I-rwt-PA was 2.2 to 3.7 times higher than that of ¹²⁵I-pamiteplase. CL_hepatic ranged from 5.26 to 9.03 ml/min/kg for pamiteplase and from 17.8 to 20.0 ml/min/kg for rwt-PA.

View larger version (18K): [in this window] [in a new window]   Fig. 8.   Ex,drug and CLhepatic (LUI) after bolus intraportalvenous administration of 125I-pamiteplase or 125I-rwt-PA to rats. Each value represents mean and standard error of three rats.

View larger version (25K): [in this window] [in a new window]   Fig. 9.   Effect of rwt-PA on Ex,drug of pamiteplase after intraportalvenous administration of 10 nM 125I-pamiteplase with rwt-PA to rats. Each value represents mean and standard error of three rats.

Examination of Plasma Clearance In Vitro. Figure 10 shows a time profile of plasma unchanged drug concentration after incubation of ¹²⁵I-pamiteplase or ¹²⁵I-rwt-PA with rat plasma, and Table 3 shows the kinetic parameters. The unchanged drug concentrations of both drugs declined monoexponentially, and the elimination rate constant (k_el) calculated by a first-order exponential equation was 0.0172 l/min for pamiteplase and 0.0167 l/min for rwt-PA. CL_plasma was calculated using k_el, and plasma volume was almost comparable for both drugs. The percentage of CL_plasma relative to CL_total was 10.2% for pamiteplase and 2.19% for rwt-PA.

View larger version (17K): [in this window] [in a new window]   Fig. 10.   Concentrations of unchanged drug after incubation of 125I-pamiteplase or 125I-rwt-PA with rat plasma. Each value represents mean and standard error of three experiments.

	Discussion

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Pamiteplase possesses the same biological activity as rwt-PAs despite its structural modifications (Katoh et al., 1991), although having a longer plasma half-life than that of rwt-PAs (Oikawa et al., 1996a). By prolonging the half-life, a bolus administration of pamiteplase at a lower dose compared with other rwt-PAs exhibited a comparable thrombolytic activity to rwt-PA administered by infusion (Kawasaki et al., 1993a,b,c). In this study, the prolonged half-life of pamiteplase was examined in the terms of the clearance mechanism of both drugs in the body.

¹²⁵I-Labeled materials are usually used in drug metabolism studies of bioactive peptides, such as pamiteplase and rwt-PA, and attention should be paid to the relationship of properties between ¹²⁵I-labeled and nonlabeled materials. Before this study, the relationship between pamiteplase and rwt-PA was examined using a GFC analysis of plasma after administration of ¹²⁵I-labeled materials to rats. The concentrations calculated by radioactivity, ELISA, and bioassay were comparable with each other. In addition, the pharmacokinetic parameters after administration of nonlabeled materials were comparable with those following the administration of ¹²⁵I-labeled materials. Therefore, the iodinated materials are thought to represent nonradiolabeled drug behavior.

Recently, bioactive peptides, such as granulocyte colony-stimulating factor (Kuwabara et al., 1994), EGF (Yanai et al., 1991), erythropoietin (Kato et al., 1997), and t-PA, have been mass-produced for use as therapeutic agents. Clearance of these bioactive peptides from systemic circulation is mainly due to receptor-mediated endocytosis (RME), especially in the liver and kidneys. The clearance mechanism of rwt-PA is reported to be the RME in two hepatocytes and the irreversible binding with plasma protein, and leads to a markedly short plasma half-life (Camani et al., 1994). RME of t-PA involves the mannose receptor on endothelial cells and the low-density lipoprotein receptor-related protein/₂-macroglobulin receptor on parenchymal cells (Camani et al., 1994).

The difference in pharmacokinetics between pamiteplase and rwt-PA under identical conditions (dose, animal, researcher, and laboratory) was examined. The prolongations of t_1/2, t_1/2, and MRT, and the marked decrease in CL_total of pamiteplase in plasma compared with rwt-PA were also confirmed in this study, showing the reason why a bolus pamiteplase administration at a one-fifth dose of rwt-PA exhibits comparable pharmacologic effects. On the other hand, the main site of clearance for both drugs was the liver, from their distributions in tissues. Furthermore, the level of pamiteplase in the liver was markedly lower than that of rwt-PA, suggesting a large difference in hepatic uptake between the drugs. CL_hepatic calculated by an integration plot accounted for most of CL_total for both drugs (75% for pamiteplase and 86% for rwt-PA), and was markedly lower in pamiteplase than in rwt-PA. Consequently, the difference in CL_hepatic was thought to be the direct cause of differences in pharmacokinetics.

Differences in drug uptakes in the liver were directly compared for both drugs by employing the LUI using a tissue-sampling single-injection technique. This technique is widely used to measure transport rates at cell membranes without damaging tissues (Tsuji et al., 1990). This technique has been applied to compare in vivo transport rates of a drug in the blood-brain barrier (BBB), the liver, and kidneys (Tsuji et al., 1990). Examples include studies of the BBB transport of acidic drugs (Kang et al., 1990), the carrier-mediated uptake of -lactam antibiotics in the kidneys (Tsuji et al., 1990), and the hepatic uptake of asialoglycoprotein (Pardridge et al., 1983).

The concentration range (from 0.057-57 µg/ml) examined in this study covered plasma unchanged drug concentrations after administration of the clinical dose (pamiteplase, 0.3 mg/kg; rwt-PA, 1 mg/kg) of pamiteplase or rwt-PA to rats. The E_x,drug of ¹²⁵I-rwt-PA was comparable to previous reports studying the high-capacity hepatic uptake of t-PA (Bakhit et al., 1987; Einarsson et al., 1988; Tanswell et al., 1990). In contrast, the lower E_x,drug of ¹²⁵I-pamiteplase compared with rwt-PA was thought to result in increased bioavailability of pamiteplase. Furthermore, the values of CL_hepatic (LUI) of both drugs were comparable to those calculated by the integration plot, indicating this technique accurately represents in vivo events.

The concentration-dependent inhibition effect of rwt-PA on ¹²⁵I-pamiteplase hepatic uptake suggested that pamiteplase would be eliminated by the same mechanism as rwt-PA but would have a lower affinity than rwt-PA to the specific receptor comprising the RME mechanism. The Asn₁₁₇ binding mannose-rich carbohydrate chain in the Kringle-1 domain and the Tyr₆₇ in the EGF domain are reported to be responsible for the hepatic uptake of rwt-PA (Bassel-Duby et al., 1992). Pamiteplase does not possess the Asn₁₁₇ binding mannose-rich carbohydrate chain, and has both the Asn₃₃₆ binding N-glycoside-type carbohydrate chain and the Thr₆₁ binding O-glycoside-type carbohydrate chain (Kawauchi et al., 1991). Consequently, the affinity of pamiteplase for the mannose receptor on endothelial cells may be lower than that of rwt-PA because of these modifications. Furthermore, possible structural changes in the EGF and finger domains neighboring the deleted Kringle-1 domain may reduce the affinity of pamiteplase for the low-density lipoprotein receptor-related protein/₂-macroglobulin receptor on parenchymal cells.

Various second generation t-PAs improving the short plasma half-life of rwt-PA, such as reteplase (Kuiper et al., 1995) and monteplase (Mizuo et al., 1996), have been developed. The decreases in CL_total and liver distribution of reteplase and monteplase compared with rwt-PA are similar to those of pamiteplase (Kuiper et al., 1995; Mizuo et al., 1996). The distribution of reteplase to parenchymal cells is three times larger than to nonparenchymal cells (Kuiper et al., 1995). The binding of reteplase is inhibited by rwt-PA, but the affinity of reteplase for parenchymal cells is lower than for rwt-PA, suggesting that reteplase has the same mechanism as rwt-PA but lower affinity than rwt-PA to the specific receptors (Kuiper et al., 1995). These results suggest that pamiteplase may have the variation of main distribution hepatocytes and the decrease in affinity to hepatocytes in the same way as reteplase.

The serine residue at the active site of both pamiteplase and rwt-PA is thought to bind ₂-macroglobulin and ₂-plasmin inhibitor irreversibly, resulting in both protease inhibition and plasma clearance (Iida et al., 1988; Kizen et al., 1988; Komuro et al., 1989; Okumura et al., 1989; Oikawa et al., 1996b). In this study, a kinetic analysis of the binding of both drugs with these glycoproteins was conducted in vitro. The unchanged drug k_el was almost the same for both compounds, and the contribution of CL_plasma of both drugs to CL_total was relatively small.

The previously described observations lead to the following conclusions. By the i.v. administration study and a tissue-sampling single-injection technique, the difference in CL_total between pamiteplase and rwt-PA was shown to be caused by the difference in CL_hepatic. The same receptors would be responsible for pamiteplase and rwt-PA uptake in the liver. The affinity of pamiteplase for these receptors, however, appeared to be lower than that of rwt-PA, resulting in the difference in CL_total.