Species Difference in Radioactivity Elimination from LiverParenchymal Cells after Injection of Radiolabeled Proteins

Takahiro Mukai,1 Yasushi Arano,2 Koyo Nishida,1 Hitoshi Sasaki,3

Hiromichi Akizawa,2,* Kazuma Ogawa,2 Masahiro Ono,2 Hideo Saji2

and Junzo Nakamura1

1SCHOOL OF PHARMACEUTICAL SCIENCES, NAGASAKI UNIVERSITY, NAGASAKI, JAPAN; 2GRADUATE SCHOOL OF

PHARMACEUTICAL SCIENCES, KYOTO UNIVERSITY, KYOTO, JAPAN; AND 3DEPARTMENT OF HOSPITAL PHARMACY,

NAGASAKI UNIVERSITY SCHOOL OF MEDICINE, NAGASAKI, JAPAN

ABSTRACT. To elucidate the cause for the different levels of hepatic radioactivity among mammals afterinjection of protein radiopharmaceuticals, the metabolism of radiolabeled proteins and the fate of theirradiometabolites in the parenchymal cells of rat liver were investigated and compared with those of mice. Weused galactosyl-neoglycoalbumin (NGA) as a carrier protein, and NGA was labeled with 111In via1-(4-isothiocyanatobenzyl)ethylenediaminetetraacetic acid (SCN-Bz-EDTA) or 1-[p-(5-maleimidopen-tyl)aminobenzyl]ethylenediaminetetraacetic acid (EMCS-Bz-EDTA) and with 125I via direct iodination. Allradiolabeled NGAs exhibited rapid accumulation in liver parenchymal cells after intravenous injection intorats. Radioactivity was eliminated following NGA-125I injection at similar rates from rat and mouse liver. Incontrast, both 111In-labeled NGAs demonstrated much slower elimination of radioactivity in rat whencompared with mouse liver. Analyses of radioactivity in bile and liver indicated that both SCN-Bz-EDTA andEMCS-Bz-EDTA rendered mono-amino acid adducts as the final radiometabolites, which were generated inrat liver within 1 h postinjection. Subcellular distribution studies suggested that these radiometabolites werecopurified with lysosome in rat liver. Because similar results were observed in mice previously, the differencebetween rats and mice in radioactivity elimination from liver parenchymal cells would be predominantlyattributable to the different efflux rate of the 111In-labeled metabolites from the lysosome between thesespecies. Such differences in the efflux rates of radiometabolites from the lysosome among mammals may alsoaccount for the different hepatic radioactivity levels of radiolabeled proteins between animal and clinicalstudies. NUCL MED BIOL 26;3:281–289, 1999. © 1999 Elsevier Science Inc. All rights reserved.

KEY WORDS. Liver, Lysosome, Metabolism, Protein radiopharmaceuticals, Species difference, Radiometabolite

INTRODUCTION

Radiolabeled monoclonal antibodies (MAbs) against tumor-associ-ated antigens are being evaluated extensively for tumor imaging andtherapy. Indium-111 has been used widely as a diagnostic radionu-clide, because of its relatively short half-life and its suitable g energyfor imaging. And cyclic diethylenetriaminepentaacetic anhydride(cDTPA) has long been used as a bifunctional chelating agent(BCA) to label MAbs with 111In, due to its simple conjugationreaction with proteins and its ready availability from commercialsources. Tumor imaging with the use of 111In-cDTPA-MAbs,however, is hindered by nonspecific sequestration of radioactivity inthe liver.

Numerous BCAs rendering higher in vivo stability of the resulting111In-chelates have been developed to decrease the undesirableradioactivity localization (9–11, 26, 30). Although some of thesenew BCAs demonstrated reduced radioactivity levels in animalliver, high hepatic radioactivity is still visualized in clinical studies(34). These results indicate that factors other than in vivo stability

of the radiometal chelates are responsible for hepatic radioactivitylocalization of 111In-labeled MAbs. These findings also suggest thatbiodistribution studies of radiolabeled MAbs in animal models maynot always coincide with clinical results.

Recently, we have reported that not only in vivo stability of theradiometal chelates but also in vivo behaviors of radiometabolitesgenerated after lysosomal proteolysis play a critical role in thehepatic radioactivity levels of protein radiopharmaceuticals (3–5).Our previous studies indicated that when 1-(4-isothiocyanatoben-zyl)ethylenediaminetetraacetic acid (SCN-Bz-EDTA) is used as aBCA, which forms highly stable 111In-chelates (11), lysine-SCN-Bz-EDTA-111In is generated as a final radiometabolite in bothparenchymal and nonparenchymal cells of mouse liver within 1 hpostinjection (4, 5). Although this metabolite is eliminated fromparenchymal cells via hepatobiliary excretion, it is retained innonparenchymal cells for longer postinjection periods. Althoughthe parenchymal cells have been reported to be a common locationof MAbs in the mouse liver (25), the cell type of the liver in whichMAbs accumulate after injection into humans remains unknown.Thus, the above findings suggest that discrepancies between animaland clinical studies with the use of 111In-labeled MAbs might bedue to: (i) the slower elimination rate of radioactivity from theparenchymal cells of human liver than that of mouse liver, (ii) thelocalization of radiolabeled MAbs in the nonparenchymal cells ofhuman liver, or (iii) a contribution of both (i) and (ii).

The aim of this study was to obtain information about the

Address correspondence to: Dr. Takahiro Mukai, School of PharmaceuticalSciences, Nagasaki University, 1-14 Bunkyo-machi, Nagasaki 852-8521,Japan; e-mail: mukai@net.nagasaki-u.ac.jp.

*Present address: Faculty of Pharmaceutical Sciences, Okayama Univer-sity, 1-1-1 Tsushima-naka, Okayama 700-0082, Japan.

Received 16 May 1998.Accepted 28 November 1998.

Nuclear Medicine & Biology, Vol. 26, pp. 281–289, 1999 ISSN 0969-8051/99/$–see front matterCopyright © 1999 Elsevier Science Inc. All rights reserved. PII S0969-8051(98)00114-0

difference among mammals in the metabolism of radiolabeledproteins and the fate of their radiometabolites in liver parenchymalcells. Thus, we analyzed quantitatively the pharmacokinetics ofradioactivity after injection of radiolabeled galactosyl-neogly-coalbumins (NGAs) into another animal model, rats. By usingNGA as a carrier protein, which is known to be incorporated byliver parenchymal cells via receptor-mediated endocytosis immedi-ately after injection (7, 28), the identification of radiometabolitesproduced after lysosomal proteolysis and the estimation of their invivo behaviors in the parenchymal cells can be achieved withoutconsidering the transchelation and redistribution of radiolabelsgenerated in tissue and fluid outside liver (4, 5, 13, 16). Themetabolism of radiolabeled NGAs and fate of their radiometabolitesin the parenchymal cells of rat liver were investigated and comparedwith those of mice.

MATERIALS AND METHODSReagents111InCl3 (74 MBq/mL in 0.02 N HCl) was supplied by NihonMedi-Physics (Takarazuka, Japan). Na125I was obtained from Daii-chi Kagaku (Tokyo, Japan) and was diluted with 0.1 N NaOH to370 MBq/mL. Both SCN-Bz-EDTA and 1-[p-(5-maleimidopen-tyl)aminobenzyl]ethylenediaminetetraacetic acid (EMCS-Bz-EDTA) were purchased from Dojindo Laboratories (Kumamoto,Japan). Human serum albumin (HSA; A-3782) and Type I colla-genase (C-0130) were obtained from Sigma Chemical Co. (St.Louis, MO). All other reagents were reagent grade products ob-tained commercially.

Animals

For animal studies, male Wistar rats weighing 240–260 g were used.Rats were maintained on standard rat foods and water ad libitum. Allexperiments in the study conformed to the Guidelines for AnimalExperimentation in Nagasaki University.

Synthesis of NGA

NGA was prepared by the reaction of HSA with cyanomethyl-2,3,4,6-tetra-O-acetyl-thio-b-D-galactopyranoside, according to theprocedures of Stowell and Lee (29). The numbers of sugars incor-porated into HSA were determined by the phenol-sulfuric acidmethod (12) to be 44.

Preparation of NGA-SCN-Bz-EDTA

NGA-SCN-Bz-EDTA was prepared according to the proceduredescribed previously (5). Briefly, to a solution of NGA (10 mg/mL)in borate-buffered saline (0.05 M, pH 8.5), a 10-molar excess ofSCN-Bz-EDTA in dimethylformamide (16 mg/mL) was added.After incubating for 20 h at 30°C, the conjugate was purified bySephadex G-50 (Pharmacia Biotech, Tokyo, Japan) column chro-matography (1.8 3 40 cm), equilibrated and eluted with 0.1 Macetate buffer (pH 3.0). The conjugate fractions were collected andsubsequently concentrated to 10 mg/mL with the Diaflow system(8 MC model, Amicon Grace, Tokyo, Japan). The concentration ofNGA conjugates was determined spectrophotometrically at280 nm.

Preparation of NGA-EMCS-Bz-EDTA

EMCS-Bz-EDTA was conjugated with NGA using maleimido-thiolchemistry (3). Briefly, freshly prepared 0.5 mL of dithiothreitol

(DTT; 0.45 mg/mL) in 0.1 M phosphate buffer (pH 6.8) containing0.3 M NaCl and 10 mM EDTA was added to 0.5 mL of NGA(40 mg/mL) in the same buffer. After incubation of the mixture for30 min at room temperature, excess DTT was removed by theDiaflow system (8 MC model) with 20 volumes of well degassed 0.1M phosphate buffer (pH 6.0) containing 10 mM EDTA beforeadjusting the protein concentration to 10 mg/mL. To the freshlythiolated NGA (10 mg/mL), a 5-molar excess of EMCS-Bz-EDTAin dimethylformamide (10 mg/mL) was added, the reaction mixturewas stirred for 2 h. The conjugate was purified and concentrated to10 mg/mL in 0.1 M acetate buffer (pH 3.0) according to theprocedure similar to that of NGA-SCN-Bz-EDTA.

111In-labeling of NGAs

Both NGA-SCN-Bz-EDTA and NGA-EMCS-Bz-EDTA were la-beled with 111In according to the procedure described previously(3). Briefly, 111InCl3 (10–200 mL) was added to each proteinsolution (1 mg, 5–10 mg/mL) in 0.1 M acetate buffer (pH 3.0).After incubation for 1 h at room temperature, a 144-mL aliquot of10 mM EDTA in acetate buffer (0.1 M, pH 3.0) was added, and thereaction mixture was gently agitated for 1 h at room temperature.Then, each 111In-labeled protein was purified by a centrifugedcolumn procedure (22) using Sephadex G-50, equilibrated with 0.1M acetate buffer (pH 3.0). Radiochemical purities of 111In-labeledNGAs were determined by cellulose acetate electrophoresis (CAE)and size-exclusion high performance liquid chromatography (SE-HPLC). CAE was run at an electrostatic field of 0.8 mA/cm for45 min in veronal buffer (I 5 0.06, pH 8.6) and SE-HPLC(Cosmosil 5Diol-300, 7.5 3 600 mm, Nacalai Tesque Inc., Kyoto,Japan) was eluted with 0.1 M phosphate buffer (pH 6.8) at a flowrate of 1 mL/min.

Lysine-SCN-Bz-EDTA-111In and cysteine-EMCS-Bz-EDTA-111In were prepared as reported previously (3, 5).

Radioiodination of NGA

Radioiodination of NGA with Na125I was achieved by the chlora-mine T method (6), followed by purification by the centrifugedcolumn using Sephadex G-50, equilibrated with 0.1 M phosphatebuffer (pH 7.4). In a manner similar to 111In-labeled proteins, theradiochemical purity of NGA-125I was determined.

Biodistribution of Radiolabeled NGAs

The protein concentration of each radiolabeled NGA was adjustedto 750 mg/mL with 0.1 M phosphate-buffered saline (pH 6.0). Eachradiolabeled NGA was injected into the right femoral vein of rats ata dose of 75 mg (12–18 kBq). Rats were housed in metabolic cagesfor urine and feces collection. At 1, 6, 24, 48, and 72 h afterinjection in the case of 111In-labeled NGAs, and after 5, 10, 15, and30 min, and 1, 6, and 24 h in the case of NGA-125I, blood wascollected from the inferior vena cava under ether anesthesia and therats were killed. Organs of interest were removed and weighed, andthe radioactivity in blood, urine, feces, and organs were determinedwith a well counter (ARC 2000, Aloka, Tokyo, Japan). From thesedata and the rat blood volume estimated from the body weight (20),the percent of injected dose of each sample was calculated.

In Vivo Studies by Cannulation into Bile Duct

Rats were anesthetized with sodium pentobarbital (50 mg/kg intra-peritoneally). An incision of approximately 3 cm was made in the

282 T. Mukai et al.

middle abdomen and the common bile duct was cannulated with apolyethylene tube (i.d. 0.28 mm, o.d. 0.61 mm, Becton Dickinson,Parsippany, NJ). The body temperature of the rats was kept at 37°Cby a heat lamp during the experiment. After intravenous injectionof each 111In-labeled NGA (75 mg, 12–18 kBq), bile samples werecollected at appropriate time intervals for 4 h. The radioactivity ofeach sample was determined with a well counter (ARC 2000).

Cellular Localization of 111In-Labeled NGAs in Liver

To assess the cellular localization of radioactivity in rat liver,parenchymal and nonparenchymal cells were fractionated by colla-genase perfusion in situ from the portal vein at 10 min postinjectionof each 111In-labeled NGA (75 mg, 12–18 kBq) with Type Icollagenase dissolved in an EDTA-free solution (27).

Identification of Radiometabolites of 111In-Labeled NGAs

To identify the radiometabolites, each 111In-labeled NGA (75 mg,0.74–1.11 MBq) was injected intravenously into rats. At 1 and 24 hpostinjection, liver of ether-anesthetized rats was treated accordingto the method as described previously (2). Briefly, the rat liver wasperfused in situ with cold 0.1 M Tris-citrate buffer (pH 6.5)containing 0.15 M NaCl, 0.02% (w/v) sodium azide, 1 TrypsinInhibitor Unit (TIU)/mL aprotinin, 2 mM benzamidine-HCl, 2mM iodoacetamide, 2 mM phenylmethylsulfonyl fluoride, and 5mM diisopropyl fluorophosphate. One gram of each liver wasexcised, placed in a tube, and subjected to three cycles of freezing(dry ice-acetone bath) and thawing. After adding the buffer (5 mL)containing an additional 35 mM of b-octyl-glucoside, the liver washomogenized by a Polytron homogenizer (PT 10-35, KinematicaGmbH Littau, Switzerland) set at full speed with three consecutive30-s bursts prior to centrifugation at 48,000 3 g for 20 min at 4°C(Himac CS-120 centrifuge; Hitachi Co Ltd., Tokyo, Japan). Super-natants were separated from the pellets, and the radioactivity wascounted. At 24 h postinjection, bile samples were collected accord-ing to the procedures described above. The liver supernatants andbile were analyzed by SE-HPLC, after filtering through a celluloseacetate membrane with a diameter of 0.20 mm (Dismic, AdvantecToyo, Tokyo, Japan). Each sample was also analyzed by CAE andTLC (Merck Art. 5553) without any pretreatment and by reversed-phase HPLC (RP-HPLC) after filtering through a 10-kDa cut-offultrafiltration membrane (Microcon 10; Amicon Grace). TLCplates were developed with 10% (w/v) ammonium chloride-meth-anol (1:1 by vol.). RP-HPLC analyses (Cosmosil 5C18-MS, 4.6 3150 mm, Nacalai Tesque Inc.) were eluted with a mixture ofmethanol and 20 mM aqueous ammonium acetate (5:95 for NGA-SCN-Bz-EDTA-111In, 17:83 for NGA-EMCS-Bz-EDTA-111In byvol) at a flow rate of 1 mL/min. Each analysis was also carried outby cochromatography with either lysine-SCN-Bz-EDTA-111In orcysteine-EMCS-Bz-EDTA-111In.

Subcellular Distribution of Radiometabolites in LiverParenchymal Cells

Subcellular distribution of radioactivity in rat liver was investigatedby perfusing the organ in situ with a cold solution of 0.25 M sucrosebuffered with 10 mM phosphate (pH 7.4) at 24 and 48 h postin-jection of 111In-labeled NGA (75 mg, 0.74–1.11 MBq). The liverwas then treated according to the procedure of Yamada et al. (32)with slight modifications. In brief, the organ was excised, mincedwith scissors, and suspended in four volumes of the same buffer prior

to homogenization for 20 strokes by hand with a Dounce homog-enizer. The homogenization was complemented by 2 final strokes inan ice-cooled Potter-Elvehjem homogenizer with a Teflon pestlerotated at 800 rpm. The resulting 20% (v/v) homogenate wascentrifuged for 5 min at 340 3 g. The isolated supernatant, whichwas again centrifuged under the same conditions, was then layeredon the top of iso-osmotic (0.25 M sucrose) Percoll (PharmaciaBiotech Co. Ltd.) at a density of 1.08 g/mL. After centrifugation at20,000 3 g in a RP 30 rotor (Hitachi Co. Ltd.) at 4°C for 90 min,the gradients were collected in 14 fractions, and b-galactosidaseactivity (31), density, and radioactivity of respective fraction wereanalyzed.

Compartment Model Analysis

The pharmacokinetic model to estimate the fate of the radiom-etabolites in rat liver parenchymal cells was constructed based onthe mouse model reported previously (23). The model is depictedschematically in Figure 1. The coefficient F is calculated as follows:

F 5

[Total radioactivity in feces at 72 hpostinjection of 111In-labeled NGA]

[Total radioactivity in feces and urine at 72 hpostinjection of 111In-labeled NGA]

(1)

In this model, the amount of radiometabolites in liver (X1), blood(X2), and intestine (X3) are expressed by the following equations:

X1 5 D z e2K1z~t2t! (2)

X2 5~1 2 F! z K1 z D

K1 2 K2z ~e2K2z~t2t! 2 e2K1z~t2t!! (3)

X3 5F z K1 z DK1 2 K3

z ~e2K3z~t2t! 2 e2K1z~t2t!! (4)

FIG. 1. Pharmacokinetic model used for the analysis ofradioactivity profiles following intravenous injection of 111In-labeled galactosyl-neoglycoalbumin (NGA) into rats. X1, ra-diometabolites in liver parenchymal cells compartment; X2,radiometabolites in blood compartment; X3, radiometabolitesin intestine compartment; (1 2 F) z K1, transport rate constantof radiometabolites from liver to blood; F z K1, transport rateconstant of radiometabolites from liver to intestine; K2,transport rate constant of radiometabolites from blood tourine; K3, transport rate constant of radiometabolites fromintestine to feces. When this model is applied to the simula-tion of biliary excretion of radiometabolites, X3 refers to theradiometabolites in the bile compartment and the radio-metabolites do not disappear from this compartment.

Kinetics of Radiometabolites in Rat Liver 283

where D (%) and t (h) represent the percent of injected radioac-tivity accumulated in the liver and the postinjection time to reachthe maximum level of radioactivity in the liver, respectively. K1, K2,and K3 are the first-order rate constants representing an eliminationfrom liver parenchymal cells, urinary excretion from the blood, andan excretion from intestine to feces, respectively. Initial conditions(t 5 t) for each compartment are defined as X1 5 D and X2 5X3 5 0 (23).

First, the radioactivity profiles in the liver, blood, and intestineafter injection of 111In-labeled NGAs were simultaneously fitted toEqs. (2)–(4) by the nonlinear least-squares method (33), and thefirst-order rate constants (K1, K2, and K3) were determined.

Next, the hepatic radioactivity profile of each radiolabeled NGAwas fitted to Eq. (2) by the nonlinear least-squares method, and thefirst-order rate constant (K1) was determined.

The elimination half-life (t1/2) was calculated by dividing 0.693by K1. The multiple correlation coefficient (R) was calculated toestimate the goodness of curve-fitting.

When this model (Fig. 1) is applied to the simulation of biliaryexcretion of radiometabolites, X3 is the radiometabolites in bilecompartment and the radiometabolites do not disappear from thiscompartment. The cumulative radioactivity in bile after injection ofeach 111In-labeled NGA was simulated by the obtained parametersand the following equation:

X3 5 F z D z ~1 2 e2K1z~t2t!! (5)

RESULTS

Radiochemical purities of all the radiolabeled NGAs exceeded97.7%. Collagenase digestion of liver cells at 10 min postinjectionof each 111In-labeled NGA indicated that more than 95.9% of theinjected radioactivity were found in the parenchymal cells of ratliver.

The biodistribution of radioactivity after intravenous injection ofNGA-SCN-Bz-EDTA-111In or NGA-EMCS-Bz-EDTA-111In intorats is shown in Table 1. More than 94% of the injected radioac-tivity accumulated in the liver at 1 h postinjection of NGA-SCN-

Bz-EDTA-111In, followed by biliary excretion. No evidence ofenterohepatic recycling of the radioactivity was observed. At 72 hpostinjection, 68% and 9% of the injected radioactivity of NGA-SCN-Bz-EDTA-111In were recovered in the feces and urine, respec-tively. The percentages of radioactivity in any other organs thanliver and intestine were below 0.4 at any time. In a similartendency, more than 96% of the injected radioactivity was presentin the liver at 1 h postinjection of NGA-EMCS-Bz-EDTA-111In,followed by biliary excretion. As the radioactivity elimination fromthe liver after injection of NGA-EMCS-Bz-EDTA-111In was fasterthan that of NGA-SCN-Bz-EDTA-111In, a lower percentage (11%)of the injected radioactivity level remained in the liver coupledwith 80% and 6% of the injected radioactivity registered in thefeces and urine, respectively, during this 72-h interval. Only lowradioactivity, amounting to less than 0.7% of that injected, weredetected in any other organs than liver and intestine.

The above biodistribution data were analyzed by the compart-ment model shown in Figure 1. The F values for NGA-SCN-Bz-EDTA-111In and NGA-EMCS-Bz-EDTA-111In were calculated tobe 0.886 and 0.931, respectively, from Eq. (1). The radioactivityprofiles in the liver, blood, and intestine after injection of each111In-labeled NGA were simultaneously fitted to Eqs. (2)–(4), andthe first-order rate constants were determined. As shown in Figure2, the fitting curves were in good agreement with the experimentaldata for each 111In-labeled NGA (R . 0.98), suggesting the validityof this model. The obtained pharmacokinetic parameters are shownin Table 2.

To further understand the biliary excretion rate of radioactivity,each 111In-labeled NGA was injected into rats and the radioactivityexcreted in the bile was assessed. The biliary excretion of theradioactivity after injection of NGA-EMCS-Bz-EDTA-111In wasfaster than that of NGA-SCN-Bz-EDTA-111In in accordance withthe biodistribution studies (Fig. 3). When the cumulative radioac-tivity in bile was simulated by the obtained K1 values and Eq. (5),good agreements were obtained between simulation curves andexperimental data (Fig. 3), suggesting that each K1 value wasappropriate.

Supernatants of the liver homogenate at 1 and 24 h postinjection

TABLE 1. Biodistribution of Radioactivity after Intravenous Injection of 111In-Labeled-NGA into Rats

Tissue

Percent of injected dose per tissue

1 h 6 h 24 h 48 h 72 h

NGA-SCN-Bz-EDTA-111InBlood 0.04 6 0.04 0.02 6 0.01 0.03 6 0.02 0.06 6 0.04 0.00 6 0.00Liver 94.76 6 4.21 87.59* 6 2.47 50.51* 6 2.83 30.25 6 1.90 19.63 6 1.31Kidney 0.08 6 0.00 0.16 6 0.01 0.25 6 0.03 0.36 6 0.04 0.36 6 0.09Spleen 0.02 6 0.01 0.02 6 0.00 0.02 6 0.01 0.03 6 0.01 0.01 6 0.01Intestine 2.85 6 0.76 11.40 6 0.94 19.80 6 4.84 11.82 6 1.37 4.17 6 0.63Urine 4.52 6 0.90 7.40 6 0.88 8.68 6 0.84Feces 25.07 6 4.38 57.69 6 3.18 67.78 6 3.19

NGA-EMCS-Bz-EDTA-111InBlood 0.12 6 0.03 0.04 6 0.01 0.03 6 0.02 0.01 6 0.01 0.01 6 0.01Liver 96.16 6 0.86 65.77* 6 6.92 29.90* 6 0.56 16.85 6 1.96 10.64 6 0.95Kidney 0.32 6 0.01 0.62 6 0.05 0.75 6 0.05 0.69 6 0.12 0.68 6 0.02Spleen 0.13 6 0.01 0.13 6 0.04 0.10 6 0.02 0.07 6 0.03 0.04 6 0.02Intestine 5.10 6 1.00 31.89 6 4.01 18.87 6 7.07 5.10 6 1.60 1.08 6 0.36Urine 2.56 6 0.30 4.41 6 0.34 5.95 6 0.32Feces 51.02 6 6.38 73.09 6 3.55 79.92 6 1.94

NGA 5 galactosyl-neoglycoalbumin. Each value represents the mean 6 SD of four animals.* Statistical analysis was performed using unpaired Student’s t test. Significantly higher (p , 0.0005) than the results for mice (3, 5).

284 T. Mukai et al.

of NGA-SCN-Bz-EDTA-111In were extracted with efficiencies ofmore than 91%. The SE-HPLC profiles of the liver and bile samplesdepicted a single radioactivity peak at 25 min, a retention time thatis representative of small molecular weight compounds such aslysine-SCN-Bz-EDTA-111In. Under similar conditions, the parental111In-labeled NGA was eluted at 18 min (Fig. 4A). In RP-HPLC,the radioactivity was eluted as a single peak at the retention time(10 min) similar to that of lysine-SCN-Bz-EDTA-111In (Fig. 4B).CAE analyses of each sample indicated a single radioactivity peak at1.5 cm anode from the origin, which was well correlated tolysine-SCN-Bz-EDTA-111In (Fig. 4C). TLC analyses of each sam-ple again illustrated a radioactivity peak with an Rf value of ca. 0.6,which was equivalent to that of lysine-SCN-Bz-EDTA-111In (Fig.4D). In each analytical system, all samples showed a single radio-activity peak even when cochromatographed with lysine-SCN-Bz-EDTA-111In (data not shown).

Chromatographic analyses of liver supernatants at 1 and 24 h andbile samples at 24 h postinjection of NGA-EMCS-Bz-EDTA-111Inwere also performed (Fig. 5). Extraction of radioactivity from theliver homogenates at 1 and 24 h postinjection of NGA-EMCS-Bz-EDTA-111In was performed with efficiencies of more than 91%. Ineach analytical system, all the samples exhibited a single radioac-tivity peak, which had similar retention time, migration distance,and Rf value to those of cysteine-EMCS-Bz-EDTA-111In, as con-firmed by cochromatographic analyses.

To further pursue the fate of radiometabolites after hepaticincorporation, the subcellular distribution of radioactivity in liverhomogenates at 24 and 48 h postinjection of each 111In-labeledNGA was investigated by Percoll density gradient centrifugation.Each liver homogenate demonstrated a single radioactivity peak ata density of ca. 1.10 g/mL, which correlated well with the b-galac-tosidase activity profile. The Percoll density gradient centrifugationprofile of liver homogenate at 48 h postinjection of NGA-SCN-Bz-EDTA-111In is shown in Figure 6 as a typical example.

The biodistribution study of 125I-labeled NGA was performed asa reference (Table 3). More than 86% of the injected radioactivityaccumulated in the liver at 5 min postinjection of NGA-125I.Following rapid radioactivity elimination from the liver, NGA-125I

FIG. 2. Radioactivity profiles in the liver (A), intestine (B),and blood (C) following intravenous injection of NGA-SCN-Bz-EDTA-111In (F) or NGA-EMCS-Bz-EDTA-111In (E) intorats. Each point was expressed as the percent of injected dose(mean 6 SD of four experiments). Fitting curves were drawnusing the obtained values of first-order rate constants.

TABLE 2. Pharmacokinetic Parameters for RadioactivityElimination from the Liver after Intravenous Injection ofRadiolabeled NGAs

Conjugates Species K1 (h21) t1/2 (h) R

NGA-SCN-Bz-EDTA-111In Rata 0.025 28 0.998Rat 0.024 29 0.996Mouseb 0.45 1.5 0.990

NGA-EMCS-Bz-EDTA-111In Rata 0.056 12 0.982Mouseb 1.6 0.43 0.987

NGA-125I Rat 2.9 0.24 0.985Mousec 3.0 0.23 0.989

NGA 5 galactosyl-neoglycoalbumin.a Calculated from the simultaneous curve-fitting of the radioactivity profiles

in the liver, blood and intestine after intravenous injection of 111In-labeledNGA into rat.

b Results were reported previously (23).c Results were obtained previously (Mukai et al., unpublished data).

FIG. 3. Cumulative radioactivity in bile for 4 h after intrave-nous injection of NGA-SCN-Bz-EDTA-111In (F) or NGA-EMCS-Bz-EDTA-111In (E) into rats. Each point representsthe mean 6 SD of four experiments. Simulation curves weredrawn using the values listed in Table 2.

Kinetics of Radiometabolites in Rat Liver 285

demonstrated high radioactivity accumulation in the stomach andintestine. At 24 h postinjection, 45% of the injected radioactivitywas excreted in the urine. On the other hand, only 2% of theinjected radioactivity was excreted in the feces during this 24-hinterval, although 13% of the injected radioactivity was present inthe intestine at 1 h postinjection. This result indicated that most ofthe radioactivity eliminated from the liver via biliary excretion wasreabsorbed from the gut lumen to blood and eventually excreted inthe urine. Thus, the pharmacokinetics of radioactivity after lysoso-mal proteolysis of NGA-125I in liver parenchymal cells is toocomplicated to be explained by the compartment model shown inFigure 1. Therefore, the only radioactivity profile in the liver afterinjection of NGA-125I was fitted to Eq. (2), and the first-order rateconstant (K1) was determined. To confirm the validity of thisanalysis, the K1 value for NGA-SCN-Bz-EDTA-111In was calcu-lated in the same way. As shown in Figure 7, the fitting curves werein good agreement with the experimental data for each radiolabeledNGA (R . 0.98) and the obtained K1 value for NGA-SCN-Bz-EDTA-111In was similar to that calculated by the simultaneousfitting (Table 2).

DISCUSSION

Specific radioactivity accumulation in the parenchymal cells of ratliver was demonstrated after injection of NGA-SCN-Bz-EDTA-111In or NGA-EMCS-Bz-EDTA-111In, followed by collagenaseperfusion. In addition, radiolabeled NGAs with high radiochemicalpurities were obtained. Therefore, in vivo behaviors of radioactivityafter injection of the three proteins would reflect the fate ofradiolabels after lysosomal proteolysis in liver parenchymal cells.

Both 111In-labeled NGAs demonstrated significantly higher ra-dioactivity levels in rat liver than those of mice (Table 1: p ,0.0005). Comparison of K1 values revealed that the radioactivityelimination from the rat liver after injection of NGA-SCN-Bz-EDTA-111In and NGA-EMCS-Bz-EDTA-111In were 18 and 29times slower than those from the mouse liver, respectively (Table2). Analyses of radiolabeled species following injection of NGA-SCN-Bz-EDTA-111In or NGA-EMCS-Bz-EDTA-111In into ratssuggested strongly that the final radiometabolites, which weregenerated within 1 h postinjection in liver parenchymal cells, weremost likely to be lysine-SCN-Bz-EDTA-111In or cysteine-EMCS-Bz-EDTA-111In, respectively (Figs. 4 and 5). In our previous studiesof NGA-SCN-Bz-EDTA-111In and NGA-EMCS-Bz-EDTA-111In,the same metabolites were generated as the final radiometabolites inthe parenchymal cells of mouse liver within 1 h postinjection (3, 5).Hence, it is most likely that protein metabolism in both rat andmouse liver is fast, and that the different K1 values between rats andmice are not ascribed to the difference in metabolism of radiola-beled NGA. Analyses of bile and liver at 24 h postinjection ofNGA-SCN-Bz-EDTA-111In or NGA-EMCS-Bz-EDTA-111In indi-cated that these radiometabolites were excreted without furthermetabolism (Figs. 4 and 5) in accordance with earlier observationsin mice (3, 5). Therefore, it is clear that both SCN-Bz-EDTA andEMCS-Bz-EDTA render 111In-chelates of high in vivo stability, andthat the different radioactivity levels caused by two animal models

FIG. 4. Chromatographic analyses of liver supernatants at 1(F) and 24 h (E), and bile samples (Œ) at 24 h postinjectionof NGA-SCN-Bz-EDTA-111In using lysine-SCN-Bz-EDTA-111In (‚) as a reference. All samples were analyzed bysize-exclusion high performance liquid chromatography (SE-HPLC) (A), reversed-phase (RP)-HPLC (B), cellulose acetate

electrophoresis (CAE) (C), and thin layer chromatography(TLC) (D). The parental NGA-SCN-Bz-EDTA-111 In (l) waseluted at 18 min on SE-HPLC. The peak of NGA-SCN-Bz-EDTA-111In was observed at 0.5–1.0 cm anode on CAE or theorigin on TLC.

286 T. Mukai et al.

are not due to the differences in stability of the 111In-chelates.Because a large difference in K1values was observed between ratsand mice, the in vivo behaviors of the radiometabolites (lysine-SCN-Bz-EDTA-111In and cysteine-EMCS-Bz-EDTA-111In) in theparenchymal cells of both rodents are apparently responsible for thedifferent elimination rate of radioactivity from the liver.

For further pursuit of the cause for the difference in the K1 values,the subcellular distribution of radioactivity was investigated. At 24and 48 h postinjection of NGA-SCN-Bz-EDTA-111In or NGA-EMCS-Bz-EDTA-111In, all radioactivity counts were detected inthe lysosomal fraction without intracellular transport to otherorganelles and interaction with biomolecules in the cytoplasm (Fig.6). Because similar results were observed in mice (3, 5), the differentK1 values between rats and mice would be due to the differentelimination rate of each radiometabolite from the lysosomal frac-tion between these species, not to the difference of the subcellulardistribution of the radiometabolites.

No systematized results to indicate the species difference in thesolute translocation across the lysosomal membrane are presentlyavailable, although there are several evidences for heterogeneity oflysosomes (15). Furthermore, the mechanisms for the egress of theseradiometabolites from the lysosome are not clear at present. There-fore, the reason for the difference between these species in thedisappearance of the radiometabolites from the lysosome remainsunknown. The past decade has witnessed the discovery and char-acterizations of numerous carrier-mediated transport mechanismssuch as amino acid transporters in the lysosomal membrane (21,24). Thus, one possible explanation is that these radiometabolitesmay cross the membrane by a certain transport system and thedifferent elimination rate from the lysosome might be attributed tothe difference in the amount and substrate specificity of thetransporter between rats and mice. But unfortunately, the speciesdifference in these transporters has not yet been reported in theliterature.

Previous studies in our laboratory have indicated that the

phoresis (CAE) (C), and thin layer chromatography (TLC)(D). The parental NGA-EMCS-Bz-EDTA-111In (l) waseluted at 18 min on SE-HPLC. The peak of NGA-EMCS-Bz-EDTA-111In was observed at 0.5–1.0 cm anode on CAE or theorigin on TLC.

FIG. 5. Chromatographic analyses of liver supernatants at 1 (F)and 24 h (E), and bile samples (Œ) at 24 h postinjection ofNGA-EMCS-Bz-EDTA-111In using cysteine-EMCS-Bz-EDTA-111In (‚) as a reference. All samples were analyzed by size-exclusion high performance liquid chromatography (SE-HPLC)(A), reversed-phase (RP)-HPLC (B), cellulose acetate electro-

FIG. 6. Percoll density gradient profiles of liver homogenateat 48 h postinjection of NGA-SCN-Bz-EDTA-111In. A singleradioactivity peak (F) that coincided with b-galactosidaseactivity (‚) was detected at a density (E) of ca. 1.10 g/mL.

Kinetics of Radiometabolites in Rat Liver 287

radiometabolite efflux from the lysosomal fraction in liver paren-chymal cells would be affected by the bile secretion characteristics(3, 5). Because the bile flow and bile acid excretion per body weightof mice are larger than those of rats (17), these factors might causethe difference between these species in the disappearance of theradiometabolites from the lysosome.

In this study, NGA was used as a carrier protein to investigate thefate of radiolabels derived from SCN-Bz-EDTA-111In and EMCS-Bz-EDTA-111In after lysosomal proteolysis in liver parenchymalcells. Although SCN-Bz-EDTA and EMCS-Bz-EDTA covalentlylink to e-amine groups of lysine residue and to thiol groups ofcysteine residue of proteins, respectively, the major radiometabolite

of each 111In-labeled NGA in the mouse and rat liver manifested asingle component that correlated with the mono-amino acid ad-duct, where all peptide bonds in these proteins were cleaved.Because these BCAs are conjugated to MAbs via similar conjuga-tion chemistries, similar radiometabolites would be generated afterlysosomal proteolysis of 111In-labeled MAbs in the parenchymalcells. Furthermore, the present findings raise the possibility that thedisappearance of the 111In-labelled metabolites from the parenchy-mal cells of human liver may be as slow as, and possibly much slowerthan, that of rat liver. Because the cell type of the liver in whichMAbs accumulate after injection into humans remains unknown, itcannot be denied that the accumulation of 111In-labeled MAbs inliver nonparenchymal cells and the slow radioactivity eliminationfrom the nonparenchymal cells may lead to the high radioactivitylevels in human liver. However, the parenchymal cells have beenreported to be the common site where radiolabeled MAbs in animalliver are located (18, 25) and to be by far the most prominentcomponent of liver cells, in terms of both volume and membranesurface area (8). Thus, the slow disappearance of 111In-labeledmetabolites from the parenchymal cells might contribute to thehigh and prolonged retention of the radioactivity in the liver afterinjection of 111In-labeled MAbs into humans.

The K1 value for NGA-125I in rats was determined to be 2.9 h21,which was similar to that in mice (Table 2). This value would beappropriate because the calculated t1/2 value was equivalent to thehalf-life (10–25 min) of the radioactivity elimination from rat liverafter injection of 125I-labeled asialofetuin (14). These findingsindicated that there was no difference between these species in themetabolism of NGA-125I and fate of the radiometabolite in liverparenchymal cells. It has been reported that radioiodinated proteinsare metabolized to iodotyrosine in the lysosome (19) and thatiodotyrosine is transported across the lysosomal membrane by theamino acid transporter, system h (1). The present result mightsuggest that there is no difference in the amount and substratespecificity of system h between rats and mice. Because no hepaticradioactivity localization was observed in clinical use of radioiodi-nated MAbs, the disappearance of the radiometabolite from humanliver, generated after lysosomal proteolysis of radioiodinated MAbs,would be as fast as those from mouse and rat liver.

CONCLUSION

The present study provides the first demonstration of the speciesdifferences in the disappearance of 111In-labeled metabolites fromthe lysosome in liver parenchymal cells. This finding raises thepossibility that the efflux of 111In-labeled metabolites from the

TABLE 3. Biodistribution of Radioactivity after Intravenous Injection of NGA-125I into Rats

Tissue

Percent of injected dose per tissue

5 min 10 min 15 min 30 min 1 h 6 h 24 h

Blood 0.45 6 0.20 3.14 6 4.81 7.74 6 4.02 11.05 6 0.98 8.19 6 1.04 4.48 6 0.91 0.66 6 0.22Liver 86.48 6 2.20 77.55 6 6.49 62.91 6 11.09 14.65 6 3.10 3.78 6 0.39 1.76 6 0.27 0.45 6 0.13Kidney 0.15 6 0.01 0.24 6 0.10 0.49 6 0.27 0.90 6 0.05 0.86 6 0.08 0.43 6 0.08 0.07 6 0.03Spleen 0.03 6 0.01 0.03 6 0.02 0.09 6 0.06 0.19 6 0.03 0.19 6 0.07 0.11 6 0.03 0.02 6 0.01Intestine 0.24 6 0.03 0.96 6 0.76 2.45 6 1.88 9.80 6 1.78 12.60 6 2.83 7.66 6 0.73 1.17 6 0.23Neck 0.00 6 0.00 0.03 6 0.02 0.08 6 0.07 0.16 6 0.04 0.21 6 0.12 0.11 6 0.03 0.05 6 0.07Stomach 0.05 6 0.01 0.12 6 0.08 0.51 6 0.32 4.63 6 0.79 10.25 6 1.76 12.99 6 3.10 1.42 6 0.12Urine 10.96 6 5.04 44.50 6 6.30Feces 0.22 6 0.15 1.70 6 0.34

NGA 5 galactosyl-neoglycalbumin. Each value represents the mean 6 SD of four animals.

FIG. 7. Hepatic radioactivity profiles following intravenousinjection of NGA-125I (A) or NGA-SCN-Bz-EDTA-111In (B)into rats. Each point was expressed as the percent of injecteddose (mean 6 SD of four experiments). Fitting curves weredrawn using the values listed in Table 2.

288 T. Mukai et al.

lysosome in human liver might be slower than that in animal liver.Such differences may also account for the different hepatic radio-activity levels of 111In-labeled proteins between animal and clinicalstudies.

We are grateful to Nihon Medi-Physics Co. Ltd., Takarazuka, Japan forits kind gift of 111InCl3. This work was supported in part by Grant-in-Aid for Scientific Research from the Ministry of Education, Science,Sports and Culture, Japan.

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