Introduction

Abstract Introduction Materials & Methods Results Discussion References

EPO¹ is one of the hematopoietic growth factors, such as granulocyte colony-stimulating factor and granulocyte macrophage colony stimulating factor; it is a 34 kDa glycoprotein mainly produced by the kidney, and it stimulates the proliferation and differentiation of colony-forming unit erythroid (1). rh-EPO is currently used as a treatment for anemia in patients with endstage renal diseases. Such patients with renal failure have anemia because of a low production of EPO. Treatment of rh-EPO improves this type of anemia in such patients (2).

It has been shown that the pharmacokinetics of many biologically active polypeptides exhibits nonlinearity due to saturation of their disposition mainly governed by RME (3, 4). In our previous study, we showed the extent of the contribution of RME to the nonlinear elimination of rh-EPO from the circulation in rats (5). Our study also indicated that repeated administration of rh-EPO caused up- and downregulation of receptor-mediated uptake by target tissues (5). Not only the saturation of receptor binding and/or receptor-mediated uptake, but also such up- and downregulation of the receptor may affect the pharmacokinetics of rh-EPO.

Since our previous study (5) was performed over a short period (<1 week), specific antibody against the injected rh-EPO might have not been produced. However, in the preclinical studies performed for a longer period in experimental animals, it may be possible that specific antibody is produced against the injected biologically active polypeptides because recombinant human polypeptides are used in the experimental animals. It is possible that the production of antibody against them causes a reduction in their pharmacological effect. In fact, a significant reduction in pharmacological effect was observed after multiple administration of rh-EPO (6). To evaluate the pharmacological and toxicological effects of such an antibody in vivo, it is important to understand the pharmacokinetics of these peptides in the presence of antibody. The present study showed that the production of antibody against rh-EPO exhibits interindividual differences in rats, producing a biphasic effect on the pharmacokinetics of rh-EPO.

	Materials and Methods

Abstract Introduction Materials & Methods Results Discussion References

rh-EPO was produced using Chinese hamster ovarian cells transfected with expression vector harboring the human erythropoietin cDNA at Production Technology Laboratories, Chugai Pharmaceutical Co., Ltd. (Tokyo, Japan). ¹²⁵I-sodium iodine (17.4 Ci/mg) was obtained from Amersham plc (Amersham, UK). Iodo-Gen (1,3,6-tetrachloro-3,6-diphenylglycouril) was obtained from Pierce Chemical Company (Rockford, IL). Protein A was obtained from Behring Diagnostics (La Jolla, CA). All other reagents were obtained as the purest grade available.

Radiolabeling. ¹²⁵I-rh-EPO was prepared by the Iodo-Gen method described previously (7). The specific radioactivity was 6.87 µCi/µg as determined by gel filtration assay. The radiochemical purity was 95.2% as determined by gel filtration.

Animals. Male Sprague-Dawley rats (JCL:SD, Clea Japan, Inc., Tokyo, Japan) were allowed to acclimatize to the laboratory environment for 1 week, and then the experiment was started at 7 weeks of age when the animals had a body weight of 240-290 g. Animal rooms were maintained at constant ambient temperature and relative humidity of 24°C and 55%, respectively, throughout the experimental period. A standard rodent feed in pellet form (CE-2, Clea Japan, Inc.) and tap water ad libitum were available throughout the study.

Multiple Administration Study. ¹²⁵I-rh-EPO was administered subcutaneously to the dorsal back region of 10 animals at dose of 1 µg/kg polypeptide equivalent to rh-EPO once a week for 4 weeks. The control rats received the vehicle solution 3 times instead of the first three administrations (from first to third) of ¹²⁵I-rh-EPO. Blood was withdrawn from each rat through the tail vein into a heparinized tube at 4, 8, 10, 12, 18, 24, 34, and 48 hr after injection of ¹²⁵I-rh-EPO. Blood was centrifuged at 15,000 rpm for 3 min. Anti-rh-EPO antiserum was obtained 4 days after the fourth administration.

Determination of Antibody. Antibody detection was conducted according to the method reported by Shimane et al. (8). Binding properties were determined by following method. One hundred microliters of ¹²⁵I-rh-EPO solution (0.25, 1, 4, 8, 16, and 32 ng/ml) and 400 µl of phosphate buffer (0.8% NaCl, 2.7 mM KCl, 8.1 mM Na₂HPO₄, 1.47 mM KH₂PO₄, 1% BSA, 0.05% NaN₃; pH 7.2) was added to 100 µl of diluted (1,000-fold) anti-rh-EPO antiserum. The mixtures were allowed to stand for 4 hr at room temperature. They were then incubated for 1 hr with 400 µl of Bio Mag Goat Anti-Rat IgG (Advanced Magnetics, Inc., Cambridge, MA) and were then centrifuged for 10 min at 3,000 rpm. Five hundred microliters of supernatant and the precipitated fraction were counted in a gamma counter. Nonspecific binding was measured by the aforementioned procedure without addition of antiserum.

Gel Filtration of Plasma Samples. Gel filtration of plasma samples was performed on a column of TSK gel G3000SW_XL at a flow rate of 0.5 ml/min with 0.1 M phosphate buffer (pH 7.2) containing 0.2 M NaCl solution. The elution volume of the molecular weight marker (cytochrome c, 12.4 kDa; BSA, 67 kDa; catalase, 232 kDa; thyroglobulin, 669 kDa) was determined by measuring the absorbance at 280 nm. Eluates were collected using a fraction collector, and the radioactivity in each fraction was measured.

Effect of Antiserum on Pharmacokinetics of rh-EPO. Anti-rh-EPO antiserum was obtained from rat 5 and administered to normal rats in volumes of 5, 50, and 500 µl. ¹²⁵I-rh-EPO was administered intravenously in doses of 0.1 µg/kg of polypeptide equivalent to rh-EPO via a femoral vein, 1 hr after antiserum administration. Blood was withdrawn from each rat through an arterial cannula into a heparinized tube at 2, 5, 10, 20, and 30 min, and at 1, 2, 4, 6, 8, and 24 hr after injection of ¹²⁵I-rh-EPO. Blood was centrifuged at 15,000 rpm for 10 min.

TCA Precipitation Assay and Immunoprecipitation Assay. For a TCA precipitation assay, 200 µl of 25% TCA solution and 150 µl of 1 M NaF were added to 50 µl of plasma. The reaction mixture was allowed to stand for 10 min at room temperature and then centrifuged for 5 min at 3,000 rpm, followed by measurement of the radioactivity in the TCA-precipitated fractions. For immunoprecipitation assay, 200 µl of phosphate buffer and 200 µl of diluted (100-fold) anti-rabbit serum were added to 50 µl of plasma. The mixture was allowed to stand for 16 hr at room temperature and then incubated for 1 hr with 100 µl of a suspension containing 5% protein A. Then 200 µl of 0.9% NaCl was added after centrifugation at 15,000 rpm using a microcentrifuge for 3 min. The supernatant was removed, and the precipitated fraction was counted in a gamma counter.

Data Analysis. The plasma concentration data after intravenous administration were fitted to eq. 1 using a nonlinear regression program MULTI (9). The plasma concentration declined biexponentially and could be described by a two-compartment open model according to eq. 1:

(1)

where C is the plasma concentration with time; and A, B, , and  correspond to the coefficients and exponents of the biexponential equation. Akaike's information criteria was used to judge the appropriateness of the models (9). The calculated SD (percentage of coefficient of variation) for A, B, , and  was 12.0 ± 4.3%, 11.7 ± 9.6%, 27.2 ± 9.7%, and 14.6 ± 9.2% (mean ± SD), respectively. Half-lives in - and -phases were calculated as ln2/ and ln2/, respectively. The V_c was calculated as dose/(A + B). The AUC was calculated by the trapezoidal rule with extrapolation to infinity. CL_total was calculated as dose/AUC. AUC_0-48h after subcutaneous administration was estimated by trapezoidal rule. The plasma concentration data at 18, 24, 34, and 48 hr after subcutaneous administration were fitted to C = A exp(t). Half-life was calculated as ln2/.

(2)

Statistical Method. Comparison of B_max was performed using Mann-Whitney test. Comparisons of pharmacokinetic parameters were performed using a one-way analysis of variance followed by Scheffé's test. Statistical significance was taken as p < 0.05.

	Results

Abstract Introduction Materials & Methods Results Discussion References

The pharmacokinetics of ¹²⁵I-rh-EPO was examined after the first and fourth subcutaneous administrations of ¹²⁵I-rh-EPO in rats (fig. 1). In all rats, the plasma levels of immunoreactive radioactivity reached a maximum 10-12 hr after the first dose and declined with a half-life of ~10 hr (fig. 1). On the other hand, the plasma levels of TCA-precipitable radioactivity after the fourth administration of ¹²⁵I-rh-EPO was minimal and increased little in any of the rats, except nos. 6 and 9, which exhibited an AUC after the administration comparable with both that in control rats and after the first administration (fig. 1). The plasma levels of TCA-precipitable radioactivity after administration of ¹²⁵I-rh-EPO in the control rats, which received the vehicle solution 3 times instead of three sequential administrations of ¹²⁵I-rh-EPO, were almost identical to those of immunoreactive radioactivity after the first administration of ¹²⁵I-rh-EPO in the 10 rats (fig. 1).

View larger version (27K): [in this window] [in a new window]   Fig. 1.   Plasma concentration of radioactivity after repeated subcutaneous administration of 125I-rh-EPO. 125I-rh-EPO was administered to rats (nos. 1-10) at a dose of 1 µg/kg once a week for 4 weeks. The plasma concentration () after first administration represents immunoreactive radioactivity. The plasma concentration () after fourth administration represents TCA-precipitable radioactivity. The control rats (nos. 1-4) received the vehicle solution 3 times instead of three sequential administrations (from first to third) of 125I-rh-EPO.

The antibody against rh-EPO in serum was detected in all rats receiving multiple administration of ¹²⁵I-rh-EPO, whereas in control rats no antibody was observed. The gel filtration chromatogram of plasma obtained from rat 9, in which the plasma concentration of TCA-precipitable radioactivity rose even after the fourth administration, showed that the most of the radioactivity was eluted in the higher molecular weight fraction (232-669 kDa) than free ¹²⁵I-rh-EPO (34 kDa) (fig. 2B). The gel filtration chromatogram of plasma obtained from rat 5, in which plasma concentration of TCA-precipitable radioactivity did not increase after the fourth administration, showed only one peak corresponding to free iodide ion and no peak corresponding to higher molecular weight material (fig. 2A). The gel filtration chromatogram of plasma obtained from control rats showed two peaks: the major one corresponding to ¹²⁵I-rh-EPO and the other, minor, one corresponding to iodide ion, respectively (fig. 2). Binding of ¹²⁵I-rh-EPO to immunoglobulin in individual rat serum was determined in the in vitro serum binding study using goat anti-rat IgG antibody (fig. 3). The obtained B_max values in rats in which plasma levels of radioactivity did not increase after the fourth administration (rats 1-5, 7, 8, and 10) were higher (490 ng/ml) than those in the other two rats, nos. 6 and 9 (254 and 156 ng/ml, respectively) (table 1). In these two rats (nos. 6 and 9), the specific binding (B_max/K_d) was also much less (0.1 and 0.4, respectively) than in the other rats (0.9-6.8) (table 1). AUC_0-48h after the fourth administration was plotted against B_max (fig. 4). Only two rats (nos. 6 and 9) showed an AUC_0-48h comparable with the control level. The B_max values in these two rats were lower than those in the other eight rats showing smaller AUC_0-48h values (p < 0.05) (fig. 4). Thus, there seems to exist a threshold for the B_max beyond which the AUC_0-48h suddenly fell as the B_max slightly increased (fig. 4).

View larger version (21K): [in this window] [in a new window]   Fig. 2.   Gel filtration chromatogram of plasma samples obtained from rat 5 (A), rat 9 (B), and control (C) 10 hr after fourth administration of 125I-rh-EPO. 125I-rh-EPO was administered to rats at the dose of 1 µg/kg once a week for 4 weeks. The control rats received vehicle solution 3 times instead of three sequential administrations (from first to third administrations) of 125I-rh-EPO.

View larger version (19K): [in this window] [in a new window]   Fig. 3.   Binding of 125I-rh-EPO to immunoglobulin in serum obtained from rats given repeated subcutaneous administration of 125I-rh-EPO. Individual rat serum was obtained 96 hr after the fourth administration. 125I-rh-EPO was added to serum diluted 10,000-fold. The mixture was allowed to stand for 4 hr, then incubated for 1 hr with Bio Mag Goat Anti-Rat IgG and centrifuged at 3,000 rpm for 10 min. Five hundred microliters of supernatant and the precipitated fraction were counted in a gamma counter. Binding parameters are shown in table 1. Solid lines were obtained by fitting using eq. 2.

View larger version (18K): [in this window] [in a new window]   Fig. 4.   Relationship between Bmax of serum immunoglobulin and AUC0-48h. AUC0-48h was calculated by the trapezoidal rule from the individual rat data shown in fig. 1. Bmax values are taken from table 1.

Figure 5 shows plasma concentrations of TCA-precipitable radioactivity after intravenous administration of 0.1 µg/kg ¹²⁵I-rh-EPO to rats pretreated with anti-rh-EPO serum from rat 5 (0, 5, 50, and 500 µl). The t_1/2 was prolonged from 3.15 to 4.66 hr, and 7.30 hr as the volume of anti-rh-EPO antiserum increased from 0 to 5 and 50 µl, respectively. However, t_1/2 was reduced to 3.81 hr in rats receiving 500 µl of anti-rh-EPO antiserum (table 2). CL_total decreased significantly from 16.6 ml/hr/kg to 10.5 ml/hr/kg, and 6.70 ml/hr/kg as the volume of anti-rh-EPO antiserum increased from 0 to 5 and 50 µl, respectively. However, CL_total rather increased to 21.4 ml/hr/kg in rats receiving 500 µl of anti-rh-EPO antiserum (table 2). The V_c did not change after administration of anti-rh-EPO anti-serum (table 2).

View larger version (19K): [in this window] [in a new window]   Fig. 5.   Effect of pretreatment with anti-rh-EPO serum on pharmacokinetics of 125I-rh-EPO. 125I-rh-EPO was administered at the dose of 0.1 µg/kg to rats treated with rat anti-rh-EPO serum, the volume used being 0 (), 5 (), 50 (), and 500 () µl. Plasma concentrations of TCA-precipitable radioactivity were measured. Pharmacokinetic parameters are shown in table 2.

	Discussion

Abstract Introduction Materials & Methods Results Discussion References

Recently, rapid advances in recombinant DNA technology have made it possible to use human biologically active polypeptides for therapeutic purposes. However, human polypeptide is administered to experimental animals in preclinical studies, and this may cause the production of antibody against it. In this study, we have shown that such production of antibody shows interindividual differences, and the effect of antibody on the pharmacokinetics of rh-EPO is biphasic, depending on the level of antibody in plasma.

We studied the pharmacokinetics of ¹²⁵I-rh-EPO after repeated administration once a week for 4 weeks (fig. 1). Although antibody against ¹²⁵I-rh-EPO was detected in all rats (from nos. 1 to 10) repeatedly given doses of ¹²⁵I-rh-EPO, there were two cases in whichafter the fourth administrationthe plasma levels of TCA-precipitable radioactivity were much lower than those of immunoreactive radioactivity after the first administration of ¹²⁵I-rh-EPO in rats 1-5, 7, 8, and 10 (case 1) (fig. 1). On the other hand, the plasma concentration profiles were similar between the first and fourth administration in rats 6 and 9 (case 2) (fig. 1). Thus, it has been shown that the effect of the antibody produced on pharmacokinetics of rh-EPO takes two forms: plasma clearance of ¹²⁵I-rh-EPO is increased (case 1) or not increased (case 2). For proteins other than rh-EPO, several studies have been examined concerning the antibody produc-tion and its effect on the pharmacokinetics of the proteins. The half-life of TCA-precipitable radioactivity after administration of ¹²⁵I-BSA in rats that had been immunized with BSA to produce anti-BSA antibody (IgG) was 4 min and significantly shorter than that in control rats where the half-life was 24 hr (10). In addition, neutralizing antibodies were produced in most of the rats receiving multiple intramuscular administrations of rIFN-2A for 28 days (see ref. 12), and the plasma clearance of rIFN-2A after its administration was much larger in rats with a high antibody titer, compared with that in the rats with a low antibody titer (12). However, Tagliaro et al. (11) reported that the plasma concentrations of eel calcitonin in patients with the antibody against it after repeated treatment with eel calcitonin were significantly greater than those in the patients without this antibody. These findings also support the hypothesis that the effect of antibody on the pharmacokinetics of polypeptides takes two forms: case 1an antigen is rapidly eliminated from the circulation; and case 2an antigen becomes more stable in circulation.

Such an interindividual difference in the effect of antibody on the pharmacokinetics of rh-EPO may come from different binding characteristics between rh-EPO and antibody. Therefore, the plasma protein binding of ¹²⁵I-rh-EPO was examined by gel filtration (fig. 2). The radioactivity in plasma from rat 9 was found in the high molecular fraction (~232-669 kDa), corresponding to the dimer or trimer of the immune complex, and the free form of ¹²⁵I-rh-EPO (34 kDa) was not observed (fig. 2). This result suggests that the ¹²⁵I-rh-EPO is stable in blood when ¹²⁵I-rh-EPO exists as a dimer or trimer immune complex (fig. 2). In addition, B_max and specific binding (B_max/K_d) for serum from rats 6 and 9 was much lower than that for other rats (p < 0.05) (table 1). Thus, rh-EPO is stable when the plasma level of antibody is relatively lower. On the other hand, most of the radioactivity was recovered as a degradation product in rat 5 (fig. 2), implying that the stability of ¹²⁵I-rh-EPO is markedly lower in the circulating blood of this rat. Antibody against rh-EPO was also detected in the blood of rat 5, and the B_max was much higher than that in rats 6 and 9 (table 1). Thus, although the immune complex could not be detected in the gel filtration chromatogram (fig. 2), there is a high level of antibody against rh-EPO in the blood of rat 5. These results indicate that the plasma clearance of ¹²⁵I-rh-EPO is large when the antibody concentration is high, and the clearance is smaller when the antibody concentration is low.

This hypothesis was also supported by the following finding. Namely, t_1/2 was prolonged significantly as the pretreatment volume of anti-rh-EPO antiserum increased from 0 to 50 µl, whereas it became shorter in rats receiving 500 µl of anti-rh-EPO antiserum (fig. 4, table 2). Additionally, CL_total decreased significantly as the volume of anti-rh-EPO antiserum increased from 0 to 50 µl and increased to 21.4 ml/hr/kg in rats receiving 500 µl of anti-rh-EPO antiserum (fig. 4, table 2). In our preliminary study, CL_total also increased to 47.2 ml/hr/kg in the rat receiving antiserum (2 ml) (data not shown). These results suggest that elimination of rh-EPO is slower when there is only a small amount of antibody in plasma and more rapid when there is a large amount.

It has been reported that the disappearance from the circulating plasma of the antigen-antibody complex of large size (more than a trimer) is fast, compared with that of a small one (monomer or dimer) because a large antigen-antibody complex will be eliminated by the liver in a few minutes (13). Therefore, our present finding that the pharmacokinetics depends on the amount of antibody may be explained as follows: when the amount of antibody in plasma is relatively small, ¹²⁵I-rh-EPO bound to the antibody in plasma is in the monomeric or dimeric form. In this case, the clearance of ¹²⁵I-rh-EPO is small because such an immune complex can escape from the clearance mechanism of EPO, such as RME in target organs and glomerular filtration in kidneys. On the other hand, when the amount of antibody is relatively large, the immune complex is taken up rapidly by the liver because ¹²⁵I-rh-EPO and the antibody form an immune complex with a molecule weight greater than a trimer. It has also been reported that the disappearance of plasma rIFN was delayed and the AUC increased ~15 times when the monoclonal antibody against rIFN was administered before administration of rIFN (14). Sato et al. (15) reported that the CL_total of rIL-2 after administration of rIL-2 mixed with its monoclonal antibody is only one-sixth that after administration of rIL-2 alone. This may be reasonable inasmuch as monoclonal antibody can never form a large immune complex, but can only be in the monomer form with antigen.

In the development of biologically active polypeptides as therapeutic drugs, antibodies against them might be produced in clinical and preclinical studies. Generally, it is thought that their pharmacological effect should be reduced when the antibody that inhibits polypeptide binding to the receptorthe so-called neutralizing antibodyis produced. However, even when the antibody produced cannot inhibit ligand binding to the receptor, it is possible that no pharmacological effect is observed, because the immune complexes may be eliminated rapidly from the circulation if a lot of antibodies are produced and the resulting immune complexes have high molecular weights. On the other hand, when the amount of antibody is small, resulting in formation of small immune complexes and an increase in the stability of rh-EPO in blood, there are two possibilities: in one case, no pharmacological effect can be observed because the antibody inhibits the receptor binding of the polypeptide; in the other, antibody cannot inhibit receptor binding and, therefore, a pharmacological effect is still observed. The plasma concentrations and pharmacological effect of eel calcitonin in patients with an antibody against eel calcitonin after treatment with eel calcitonin were greater than those in patients without antibody (11). This finding suggests that the immune complex can exert a pharmacological effect when the immune complex is stable in the circulating blood and can still bind to receptor. Therefore, to evaluate the pharmacological effect of biologically active polypeptides, it is by all means important to monitor the production of the antibody and its effect on both receptor binding and pharmacokinetics.