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Characterization of the Hepatic Disposition of Lanoteplase, a Rationally Designed Variant of Tissue Plasminogen Activator in Rodents

Department of Business Planning and Development, Daiichi Asubio Pharma Co. Ltd., Tokyo, Japan (K.K.); Department of Molecular Pharmacokinetics, Graduate School of Pharmaceutical Sciences, University of Tokyo, Tokyo, Japan (Y.K., Y.S.); Institute for Medicinal Research and Development, Daiichi Asubio Pharma Co. Ltd., Gumma, Japan (Y.H., K.O.); and Department of Pharmacokinetics and Biopharmatics, Faculty of Pharmaceutical Sciences, Toho University, Chiba, Japan (R.N.)

(Received September 3, 2006; accepted December 15, 2006)

	Abstract

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Lanoteplase is a recombinant mutant of tissue-type plasminogen activator (t-PA) that was developed with an aim to overcome the drawback of rapid systemic elimination of t-PA. In this study, we examined the disposition profile of lanoteplase in vivo and the kinetics of receptor-mediated endocytosis (RME) of this recombinant t-PA in vitro to kinetically characterize the mechanism(s) underlying its tissue distribution and elimination. Integration plot analysis of the initial-phase tissue distribution in rats revealed a much lower uptake clearance (CL_uptake) of lanoteplase in the liver than that of t-PA. Rate constants for cell surface binding, internalization, and degradation of lanoteplase were also lower than those for t-PA in primary cultured rat hepatocytes. These results suggest that the improved stability of lanoteplase in vivo could be accounted for by the delay in the RME of this recombinant protein. The CL_uptake in the liver decreased with coadministration of lactoferrin, a ligand for the low-density lipoprotein receptor-related protein (LRP) and the asialoglycoprotein (ASGP) receptors in normal mice, and in lrpap1^(–/–) mice, which have a hereditary deficiency of LRP; In contrast, CL_uptake was not affected by mannose, whereas that of t-PA decreased with both ligands and in the lrpap1^(–/–) mice. Thus, the hepatic disposition of lanoteplase seems to be mediated by common specific receptors for t-PA, including LRP and the ASGP receptors, whereas the mannose receptor seems to be only minimally involved in the disposition of lanoteplase.

Lanoteplase is one of the mutant forms of t-PA, which was developed with the recombinant DNA technology to achieve such an aim. Lanoteplase was produced by removing the epidermal growth factor domain, a part of the finger domain, and by substituting Gln for the Asn residue at position 117, resulting in a removal of the sugar chain. Despite such a major structural modification, lanoteplase still exhibited a potent thrombolytic activity at much lower dose than t-PA both in vivo and in a rabbit model of jugular vein thrombosis (Furuya et al., 1999). In addition, the efficiency of uptake by the liver of lanoteplase was much lower than that of t-PA, resulting in its longer plasma half-life and lower systemic clearance than those of t-PA (Larsen et al., 1989; Hata et al., 1997a,b). The lower plasma clearance of lanoteplase, compared with that of t-PA, has also been confirmed in patients with acute myocardial infarction (Kostis et al., 2002). Thus, lanoteplase is expected to become a therapeutic thrombolytic agent, although its pharmacokinetic properties still remain uncertain.

Meanwhile, the clearance via receptor-mediated endocytosis (RME), in which protein ligands are internalized via receptors on the cell surface, and subsequently lysosomal degradation is induced, has been recognized as an important mechanism for the systemic elimination of many bioactive proteins, including certain types of cytokines (Sugiyama and Hanano, 1989; Liu et al., 1992; Kuwabara et al., 1996; Tang et al., 2004). Moreover, numerous studies have suggested that the low-density lipoprotein receptor-related protein (LRP) receptor, the asialoglycoprotein (ASGP) receptor, and the mannose receptor presented on the liver cell surface might play important roles as clearance mechanisms for t-PA (Camani and Kruithof, 1994). Furthermore, Bu et al. have suggested that the plasma proteins such as the plasminogen activator inhibitor (PAI)-1 are also associated with such receptor binding (Bu et al., 1992; Otter et al., 1992; Camani and Kruithof, 1994). Although pharmacokinetic analyses have been performed for various types of recombinant mutant t-PAs, only the plasma elimination profiles have been investigated in most of these studies (Kuiper et al., 1995, 1996; Aoki et al., 2000, 2001). It is noteworthy that Oikawa et al. (2000a,b) attempted to characterize the kinetics of RME in the hepatocytes for pamiteplase, one of the mutant t-PAs, but the elimination mechanism of pamiteplase still remains unclear. The plasma level of t-PA mutants would be expected to be governed by the balance between the administration rate and the elimination efficiency. Therefore, it would be important to elucidate the elimination mechanism of these mutant proteins to clearly understand their pharmacology in vivo.

In the present study, we attempted to kinetically characterize the hepatic elimination mechanism of lanoteplase compared with that of t-PA to elucidate the differences in the elimination pathways between the two proteins. First, we performed integration plot analyses to assess the hepatic influx process of each protein in rats in vivo. Second, we characterized the RMEs of the two proteins in primary cultures of rat hepatocytes. Finally, we conducted inhibition studies on hepatic uptake and pharmacokinetic analysis in gene knockout mice to analyze the hepatic uptake mechanism of these proteins.

	Materials and Methods

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Materials. Lanoteplase was produced by gene-recombination technology at the production technology laboratories at Suntory Co. Ltd. (now known as Daiichi Asubio Pharma Co. Ltd., Gumma, Japan). Wild-type t-PA was purchased from Boehringer Ingelheim Pharma GmbH and Co. KG (Biberach, Germany). ¹²⁵I-Sodium iodine (644 GBq/mg) was obtained from GE Healthcare (Chalfont, St. Giles, UK). All the other materials and reagents used were of analytical grade.

Radiolabeling. Lanoteplase and t-PA were radiolabeled with ¹²⁵I-sodium iodide by the peroxidase method (Miyachi et al., 1972). In brief, lanoteplase or t-PA dissolved in a buffer containing 50 mM citric acid, 50 mM creatinine, 50 mM histidine, and 0.1% Tween 80, pH 4.6, was first loaded on to a Sephadex G50 column (GE Healthcare), followed by the addition of 24 MBq of ¹²⁵I-sodium iodide dissolved in a buffer containing 0.5 M arginine, 0.4 M sodium acetate, pH 5.6, and 0.3 µg/ml lactoperoxidase (Calbiochem, Darmstadt, Germany). Then, after the addition of 0.02% H₂O₂, the column was incubated at 30°C for 5 min, and the ¹²⁵I-lanoteplase or ¹²⁵I-t-PA fraction was eluted. The specific radioactivity of ¹²⁵I-lanoteplase and ¹²⁵I-t-PA was 0.2 to 0.5 and 0.1 to 0.5 Bq/g, respectively.

Animals. Six-week-old male Sprague-Dawley rats (190–240 g) and 6-week-old male C57BL/6N Crj mice (16–20 g) were purchased from Charles River Japan Inc. (Tsukuba, Japan). In addition, 6- to 8-week-old male lrpap1^(–/–) mice (13–14 g) were purchased from The Jackson Laboratory (Bar Harbor, ME).

Tissue Distribution Studies in Vivo. ¹²⁵I-Lanoteplase and ¹²⁵I-t-PA (1 µg/kg; 0.37 MBq/kg) with or without unlabeled ligand (100 µg/kg) were dissolved in a solution for injection (50 mM citric acid, 50 mM creatinine, 50 mM histidine, and 0.1% Tween 80, pH 7.6) and injected via the femoral vein into rats under light pentobarbital (Dainippon Pharmaceuticals Co. Ltd., Osaka, Japan) anesthesia (40 mg/kg i.p.). Blood samples were then collected from the femoral artery at designated times for 10 min. Meanwhile, biopsy samples of the liver (approximately 100 mg) were obtained at 1.5, 3, 5, and 7 min after the injection. At 10 min after the i.v. administration, the rats were sacrificed, and the liver, kidney, heart, spleen, and brain were immediately removed, weighed, and the radioactivity counts were conducted with a gamma counter (COBRAII; PerkinElmer Life and Analytical Sciences, Boston, MA).

For the next experiment in mice, ¹²⁵I-lanoteplase and ¹²⁵I-t-PA (1 µg/kg; 0.37 MBq/kg) were administered via the femoral vein into male C57BL/6N Crj mice or male lrpap1^(–/–) mice under light anesthesia. Blood samples were then collected from the jugular veins of the mice with disposable syringes at designated intervals for 5 min. The mice were sacrificed, and the liver, kidney, heart, lung, spleen, and brain were immediately removed and weighed. Radioactivity counts were performed.

Plasma samples were obtained by the addition of 3.8% sodium citrate to the blood specimens from the animals (1:9, v/v) and subsequent centrifugation at 8000g for 10 min at 4°C. Ice-cold 4% trichloroacetic acid (TCA; 200 µl) was added to 200 µl of the plasma, followed by incubation for 30 min at 4°C and centrifugation at 8000g for 30 min at 4°C. Then, the TCA-precipitable radioactivity in the plasma was counted.

Kinetic Analysis for Calculation of the Total Body Clearance and Tissue Uptake Clearance. The plasma concentration (Cp)-time profiles were fitted to a biexponential equation with a nonlinear least-squares method using the MULTI program (Yamaoka et al., 1981). Total body clearance (CL_tot) was calculated as the dose divided by the area under the plasma concentration-time curve (AUC) for an infinite time.

In regard to the hepatic distribution, the initial slope in a plot (designated as an integration plot) of the tissue/plasma concentration ratio (Kp) versus the AUC/Cp yielded the tissue uptake clearance (CL_uptake), where AUC was calculated by the use of the trapezoidal rule with extrapolation to the final sampling (Liu et al., 1992). For tissues other than the liver, the Kp at time 0 [Kp₍₀₎] was assumed to be the same as that in the extracellular space, which was obtained as the Kp of ¹²⁵I-human serum albumin (Liu et al., 1992), and the CL_uptake was calculated as [Kp – Kp₍₀₎]/(AUC/Cp).

Primary Cultures of Rat Hepatocytes. Rat parenchymal cells were isolated by in situ perfusion of the liver with collagenase (Tomita, et al., 1981). Cells were disseminated on a collagen-coated 24-well plate (Sumitomo Bakelite Co. Ltd., Tokyo, Japan) containing the culture medium (William's medium E containing 5% calf serum, 1 nM insulin, 1 nM dexamethasone, and 30 mg/l kanamycin monosulfate) at the density of 1.5 x 10⁵ cells/well (0.32 mm^–1) and incubated for 3 h in an atmosphere containing 5% CO₂ at 37°C. The cells were washed twice and consecutively cultured for 21 h under the same conditions. The monolayer formed was then washed with standard medium (Hanks' buffer with 20 mM HEPES, pH 7.4, containing 0.2% bovine serum albumin) and preincubated for 30 min before starting the following experiments.

Binding Assay. After setting the cell plate on the ice, the cells were incubated in the ice-cold standard medium containing ¹²⁵I-lanoteplase or ¹²⁵I-t-PA (0.1 pmol/0.45 kBq/ml) and various concentrations (0.1–100 nM) of unlabeled ligands for 240 min at 4°C. The cells were washed, solubilized in 1 N NaOH, and their radioactivity count was performed. The binding was calculated as the ratio of the surface-bound amount thus obtained to the ligand concentration in the medium.

Determination of Surface Binding, Internalization, and Degradation. The cell monolayer was incubated in standard medium containing ¹²⁵I-lanoteplase or ¹²⁵I-t-PA (0.1 pmol/0.45 kBq/ml) in an atmosphere containing 5% CO₂ at 37°C, and the reaction was terminated by aspiration of the medium at designated times. The monolayer was then washed twice with the ice-cold standard medium, and the washed medium was mixed with the reaction medium aspirated as mentioned above. An aliquot (1 ml) of the mixture was then subjected to the TCA precipitation technique, and TCA precipitable (intact) and soluble (degraded) radioactivities were counted (Hata et al., 1997b). In our previous analysis comparing the TCA precipitation method with gel filtration, polypeptides with molecular masses of higher than 15 kDa were regarded as TCA-precipitable radioactivities (Hata et al., 1997b). In our preliminary study, both ¹²⁵I-lanoteplase and ¹²⁵I-t-PA were incubated with rat liver homogenate, which contains endogenous type I deiodinase, and time-dependent increases in the TCA soluble fraction were similar between the two proteins, suggesting that the rate of deiodination of the two proteins is the same. Accordingly, the monolayer was incubated for 20 min at 4°C with ice-cold acid buffer (Hanks' buffer with 20 mM MES and 0.2% bovine serum albumin. pH 3.0) and washed twice with the same acid buffer (Haigler et al., 1980). The ligand released in this series of acid washings was designated as the surface-bound ligand. The monolayer was solubilized by incubation with 0.5 N NaOH for 30 min at 37°C and washed twice with 0.5 N NaOH. The NaOH extract thus obtained was regarded as the internalized ligand. Cellular protein was measured by the Bio-Rad protein assay kit (Bio-Rad, Hercules, CA) using bovine serum albumin as the standard. All the radioactivities measured were normalized by both the cellular protein and the initial ligand concentration in the medium, and they are expressed as distribution volume (microliter per milligram of protein).

View larger version (14K): [in this window] [in a new window]   FIG. 1. Plasma concentration-time profiles of 125I-lanoteplase (filled circles) or 125I-t-PA (open circles) after intravenous administration in rats. TCA-precipitable radioactivity in the plasma was determined after i.v. administration of 125I-lanoteplase and 125I-t-PA at the dose of 1 (A) and 100 µg/kg (B). Each point and vertical bar represent the mean ± S.D. from three to eight animals.

(1)

(2)

(3)

where X_s, X_int, and X_deg are the amounts of surface-bound, internalized, and degraded ligand, respectively, at time t, and k_on, k_off, k_int, and k_deg indicate the rate constants for association, dissociation, internalization, and degradation, respectively. The L and R_free are ligand concentration in medium and free (unoccupied) receptor on cell surface. At the earlier time period, eq. 2 can be simplified to the following:

(4)

The integration of eq. 4 and eq. 3 yields the following:

(5)

(6)

where AUC_s and AUC_int are area under the time course of X_s and X_int, respectively. Thus, initial slope in the plot of X_int versus AUC_s yields k_int and that in the plot of X_deg versus AUC_int yields k_deg. Since we used a tracer concentration of the ligand, the R_free was assumed to be constant. In addition, L was also assumed to be constant, since the decrease in medium ligand concentration was found to be minimal in the present study. Thus, the eq. 1 can be simply integrated to be the following:

(7)

Therefore, the slope in -minus plot for X_s yields sum of k_off and k_int, and subtraction of k_int from the slope yields k_off.

Statistical Analysis. Statistical comparisons between the treatment group were performed using one-way analysis of variance. When analysis of variance showed significant differences among the groups, multiple pairwise comparisons of each experimental group versus the control group were performed using Dunnett's test to identify which group differences accounted for the significant p values. The p value of less than 0.05 with two-tailed was considered statistically significant.

	Results

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Comparison of the Disposition Profile between ¹²⁵I-Lanoteplase and ¹²⁵I-t-PA in Rats. The plasma concentration-time profiles after i.v. administration of 1 and 100 µg/kg ¹²⁵I-lanoteplase and ¹²⁵I-t-PA are shown in Fig. 1. The disappearance of lanoteplase from the plasma was significantly slower than that of t-PA, with the plasma concentration of lanoteplase at 10 min after its administration being approximately 5-fold higher than that of the corresponding concentration of ¹²⁵I-t-PA (Fig. 1).

Since the liver has been considered to be the major clearance organ for t-PA (Camani and Kruithof, 1994), the hepatic uptake profile of both lanoteplase and t-PA were examined by the integration plot analysis. The integration plot for the liver was almost linear up to 10 min for both proteins, although the slope for t-PA was much steeper than that for lanoteplase, after administration of either 1 or 100 µg/kg (Fig. 2). The CL_uptake values in the liver and other organs are shown in Fig. 3. The CL_uptake values of lanoteplase in the liver, spleen, and heart were significantly lower than the corresponding values of t-PA, in particular, that of lanoteplase in the liver was just approximately 7% of that of t-PA (Fig. 3A). Meanwhile, the CL_uptake of lanoteplase in the kidney was approximately 1.6 times higher than that of t-PA (Fig. 3A). In addition, the CL_uptake of t-PA per kilogram of body weight in the liver was the highest among all organs examined, whereas such values for lanoteplase in the liver and kidney were comparable (Fig. 3B). Furthermore, those of lanoteplase and t-PA in the liver were around 60 and 89% of the CL_tot, respectively, showing consistency with the study results reported by Camani and Kruithof (1994) that the major organ of elimination of t-PA from the circulating blood was the liver. The difference in the CL_tot between lanoteplase and t-PA was comparable with the difference in the CL_uptake per kilogram of body weight in the liver between the two proteins (Fig. 3, B and C). Thus, the improved stability in the systemic circulation of lanoteplase compared with that of t-PA can be principally explained by the lower hepatic elimination of this protein, as hypothesized by us in our previous report (Hata et al., 1997b).

View larger version (16K): [in this window] [in a new window]   FIG. 2. Integration plots for estimating CLuptake. After i.v. administration of 125I-lanoteplase (filled circles) and 125I-t-PA (open circles) at 1 (A) and 100 µg/kg (B), the time profiles of TCA-precipitable radioactivity in the plasma and radioactivity in the liver were determined for 10 min. The data are expressed as integration plots, with the initial slope representing the CLuptake.

View larger version (13K): [in this window] [in a new window]   FIG. 3. CLuptake of 125I-lanoteplase (filled bars) and 125I-t-PA (open bars) per gram of tissue (A) or per kilogram of body weight (B) and the CLtot values (C). The CLuptake in the liver was obtained from Fig. 2, whereas that in other tissues was obtained from the time profiles of the plasma concentrations and Kp at 10 min after i.v. administration. Each point and vertical bar represent the mean ± S.D. from three to eight animals.

View larger version (32K): [in this window] [in a new window]   FIG. 4. Time courses of the surface binding, internalization, and degradation of 125I-lanoteplase (filled circles) and 125I-t-PA (open circles) in primary cultures of rat hepatocytes. The cells were incubated with a tracer concentration (0.1 nM) of 125I-lanoteplase or 125I-t-PA at 4°C (A) or 37°C (B–D), and the surface bound (A and B), internalized (C), and degraded (D) amounts were measured. All these values were normalized by both the cellular protein and the initial ligand concentration in the medium, and they are expressed as microliters per milligram of protein. Each point and vertical bar represent the mean ± S.D. of four determinations.

View larger version (26K): [in this window] [in a new window]   FIG. 5. Effect of inhibitors of hepatic receptors and gene knockout of Lrpap1 on the hepatic and renal distribution of lanoteplase and t-PA in mice. After i.v. administration in normal mice of 125I-lanoteplase (A and C) or 125I-t-PA (B and D) at the dose of 1 µg/kg, the time profiles of TCA-precipitable radioactivity in the plasma, and the radioactivity in the liver (A and B) and kidney (C and D) at 1, 2, 3, and 5 min were measured and are shown as integration plots (filled circles). The time profiles of radioactivity in plasma until 3 min, and radioactivities in the liver and kidney at 3 min were also measured after i.v. administration under four different experimental conditions (difference in inhibitors or experimental animals): Those in the lrpap1(–/–) are shown as open circles, and those in the normal mice with coadministration of 120 µg/kg mannose, 180 µg/kg lactoferrin, or both 120 µg/kg mannose and 180 µg/kg lactoferrin are shown as open squares, open triangles, and open diamonds, respectively. The arrows indicate the data at 3 min after i.v. administration of 125I-lanoteplase or 125I-t-PA alone in normal mice. The asterisks represent significant difference in Kp values compared with that in normal mice at 3 min after i.v. administration (*, p < 0.05; **, p < 0.01).

Characterization of the Tissue Uptake Mechanism(s) of Lanoteplase in Mice. To analyze the hepatic uptake mechanism for lanoteplase, a tissue distribution study was performed in mice by coadministration of ligands for receptors that have already been suggested to be involved in the uptake of t-PA. Lactoferrin and mannose were used as the ligands for LRP and the ASGP receptors and for mannose receptors on liver cells, respectively. The dose of each ligand was set so as to be sufficient to saturate each receptor (180 and 120 mg/kg, respectively), based on previous reports (Emeis et al., 1985; Brock, 1997; Crawford and Borensztajn, 1999). The Kp of lanoteplase in the liver at 3 min after its administration was not affected by the coadministration of mannose; however, it decreased significantly by the coadministration of lactoferrin alone and by the coadministration of lactoferrin plus mannose (Fig. 5A). In contrast, mannose and/or lactoferrin coadministration decreased the Kp of t-PA in the liver (Fig. 5B). The trend of Kp of lanoteplase in the kidney was similar to that in the liver (Fig. 5, A and C), whereas that of t-PA in the kidney was not significantly affected by the coadministration of mannose, lactoferrin, or lactoferrin plus mannose (Fig. 5D).

It has been suggested that the hepatic disposition of t-PA was at least partially mediated by the LRP (Bu et al., 1992). The LRP-associated protein (LRPAP in humans, RAP in mice) may act as a folding chaperone for LRP (Willnow et al., 1996), and it has been widely used as a specific inhibitor of LRP. Target mutation in the lrpap1 gene causes functional deficiency of LRP, with reduction in its expression levels in the liver and lrpap1^(–/–) mice are now commercially available. Therefore, in the present study, lrpap1^(–/–) mice were used to investigate the possible involvement of LRP in the hepatic disposition of lanoteplase. Although the Kp of lanoteplase in the liver tended to be decreased in the lrpap1^(–/–) mice (Fig. 5A), an obvious decrease of the Kp of t-PA was observed in the liver of these mice (Fig. 5B).

	Discussion

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It has been obligatory to administer t-PA by intravenous infusion in emergency medical situations due to its short half-life (Collen et al., 1984; Verstraete et al., 1985). Lanoteplase is a new mutant t-PA produced as a result of a major structural modification with a molecular size four fifths that of t-PA. It has similarly potent thrombolytic activity as t-PA (Furuya et al., 1999) and pharmacokinetic properties that should enable bolus intravenous administration for the treatment of acute emergencies. The present study indicated that the major clearance organ for both t-PA and lanoteplase is the liver (Fig. 3). However, the hepatic CL_uptake of lanoteplase was substantially smaller than that of t-PA (Figs. 2 and 3), and such smaller hepatic uptake could be a reason for the prolonged retention of lanoteplase in the circulating blood.

Previous reports have revealed that approximately 60% of hepatic distribution of t-PA was associated with parenchymal cells, and the remaining 40% was associated with nonparenchymal cells of the liver (Kuiper et al., 1988; Rijken et al., 1990). The LRP and ASGP receptors on the parenchymal cells and also the mannose receptors on the nonparenchymal cells of the liver could represent the clearance receptors for t-PA on the liver cell-surface (Bu et al., 1992; Otter et al., 1992; Camani and Kruithof, 1994). We therefore investigated the RME mechanism for lanoteplase and t-PA in primary cultured rat hepatocytes. The amounts of surface-bound, internalized, and degradation products of lanoteplase were smaller than those of t-PA (Fig. 4, B–D), suggesting that lanoteplase also underwent RME in the parenchymal cells just like t-PA, but its clearance via RME was smaller than that of t-PA. Cell-surface binding of lanoteplase assessed at 4°C was lower than that of t-PA (Fig. 4A), suggesting lower receptor binding affinity of lanoteplase. Furthermore, the kinetic analyses suggested that a reduction in internalization and degradation (Table 1) could also be the reason for lower clearance efficiency of lanoteplase via RME.

It is considered important from a perspective of safety in the clinical use to clarify which receptors among the three receptors in the liver cells are responsible for the elimination of lanoteplase. The CL_uptake of lanoteplase in the liver was decreased only when coadministered with lactoferrin or administered to lrpap1^(–/–) mice (Fig. 5A), even though that of t-PA decreased when coadministered under the same experimental conditions, including mannose (Fig. 5B). These results suggested that the ASGP receptor and the specific receptor for LRPAP, presumably the LRP receptor, were responsible for the distribution of lanoteplase into the liver. Meanwhile, the LRP, the ASGP and the mannose receptors were responsible for the distribution of t-PA into the liver. Based on these results, it is considered that the binding of lanoteplase to the mannose receptor may have disappeared because of the removal of the mannose-rich sugar chain from its molecule at Asn 117. This is compatible with the previous report that the CL_tot of another structural mutant of t-PA, in which Asn at 117 was substituted by Gln and a sugar chain was removed, was decreased by 38% of that of t-PA (Aoki et al., 2001). LRP is known to consist of four clusters, and the t-PA·PAI-1 complex binds to cluster II of LRP (Neels et al., 1999). The binding affinity of t-PA·PAI-1 complex to the LRP receptor is 10 times higher than that of nonprotein binding (free) t-PA (Horn et al., 1997). Moreover, it has been reported that lactoferrin binds to clusters II and IV of the LRP (Bennatt et al., 1997; McAbee et al., 1998). Considering that the distribution of lanoteplase into the liver was inhibited by coadministration of lactoferrin (Fig. 5) and that lanoteplase was a mutant of t-PA, it would seem that lanoteplase is also taken up by the liver mainly as a protein-bound complex. Alternatively, the ASGP receptor binds to t-PA, and this receptor is known to recognize the structural carbohydrates of t-PA at Thr 61, Arg 184, and Asn 448 (Camani et al., 1998; Nagaoka et al., 2003). Because the carbohydrate at Asn 448 is also retained in lanoteplase, the drug seems to bind to the ASGP receptor.

It is noteworthy that the CL_uptake per gram of tissue of lanoteplase in the kidney was greater than that in the liver and that the CL_uptake values per kilogram of body weight of this compound in the liver and kidney were comparable (Fig. 3). This result showed that the kidney may also be an important clearance organ for lanoteplase. Hepatic dysfunction may severely affect the pharmacokinetics when the clearance organ is only the liver, and multiple elimination pathways may be beneficial for the clearance of therapeutic agents. Therefore, further clinical studies are necessary to examine whether lanoteplase might be safer than the traditional t-PA when it is administered to the specific target population such as the patients with hepatic dysfunction, those receiving concomitant estradiol administration or older patients. Until now, there have been few reports about the renal elimination mechanism of t-PA even though it was suggested that t-PA might be taken up by one of the members of LDL receptor family in this organ (Camani and Kruithof, 1994). Considering the significance of renal elimination (Fig. 3), it would also be important for lanoteplase to clarify the mechanism of renal elimination in detail. The present study indicated the similar patterns in decrease of the Kp of lanoteplase between kidney and liver (Fig. 5C), implying that RME may also be involved in renal disposition, although the higher CL_uptake of lanoteplase in the kidney (Fig. 3) may presumably be explained by the easier glomerular filtration of lanoteplase because of its smaller molecular size (50 kDa) than that of t-PA (64 kDa).

In our previous study using gel filtration chromatography, the major plasma proteins that bind to t-PA and lanoteplase were different; that is, the binding fractions for t-PA to PAI-1 and 2-macroglobulin were 19 and 8%, whereas the corresponding values for lanoteplase were 6 and 14%, respectively (Hata et al., 1997b). In contrast, the uptake of t-PA into the liver has been reported to be affected by its binding to PAI-1 or 2-macroglobulin, presumably because t-PA was taken up into the liver as a complex with these proteins (Camani and Kruithof, 1994; Camani et al., 2000). Thus, it is possible that the binding of lanoteplase to the plasma proteins or these receptors is changed because of conformational changes following the removal of several structural domains in t-PA. To conduct an in-depth analysis of the elimination mechanisms, further examination using the protein complexes might be needed.

In conclusion, lanoteplase showed long blood retention, mainly because of a decrease in the efficiency of RME in the liver. The primary receptors related to the hepatic uptake for lanoteplase seemed to be the LRP and the ASGP receptors, and the contribution of mannose receptor seemed to be small.

	Acknowledgments

 
We thank Dr. Ke-Xin Liu (presently in College of Pharmacy, Dalian Medical University) for helpful advice concerning in vivo studies. We thank Drs. Takeshi Hanada and Yasushi Kanai (Daiichi Asubio Pharma Co. Ltd.) for support during this study.

	Footnotes

 
Article, publication date, and citation information can be found at http://dmd.aspetjournals.org.

doi:10.1124/dmd.106.012518.

ABBREVIATIONS: t-PA, tissue-type plasminogen activator; RME, receptor-mediated endocytosis; LRP, low-density lipoprotein receptor-related protein; ASGP, asialoglycoprotein; PAI, plasminogen activator inhibitor; TCA, trichloroacetic acid; CL_tot, total body clearance; CL_uptake, tissue uptake clearance; Cp, plasma concentration; AUC, area under the plasma concentration-time curve; Kp, tissue/plasma concentration ratio; MES, 2-(N-morpholino)ethanesulfonic acid; LRPAP, low-density lipoprotein receptor-related protein-associated protein.

Address correspondence to: Prof. Yuichi Sugiyama, Graduate School of Pharmaceutical Sciences, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan. E-mail: sugiyama{at}mol.f.u-tokyo.ac.jp

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