Kinetic Analysis of In Vivo Receptor-Dependent Binding of Human Epidermal Growth Factor by Rat Tissues

Kinetic Analysis of In Vivo Receptor-Dependent Binding of Human Epidermal Growth Factor by Rat Tissues DONGCHOOL KIM*, YUlCHl MANABU HANANO*

SUGIYAMA*,

HIROAKISATOH*, TOHRUFUWA*, TATSUJI lGA*'X,

AND

Received May 5, 1987, from the *Departmentof Pharmaceutics, Faculty of Pharmaceutical Sciences, University of Tokyo, Hongo, Bunkyo-ku, Tokyo 1 13, Japan, and the *Central Research Laboratories, Wakunaga Pharm. Co., Lfd., Shimokotachi, Koda-cho, Takada-gun, Accepted for publication October 8, 1987. Hiroshima 729-64, Japan. Abstract 0 Kinetic analysis of the tissue distribution of human epidermal growth factor (hEGF) in rats was performed in vivo. The plasma disappearance half-life of [lz5I]hEGF was prolonged by coadministration of unlabeled hEGF, indicating saturation of the mechanism for hEGF removal from the systemic circulation. To analyze the contribution of each tissue to the uptake of hEGF, the amount of [lZ51]hEGFtaken up by each tissue was determined after coadministration of various amounts of unlabeled hEGF. Kinetic analysis of the data yielded the following results. (1) Among the tissues examined, the distribution of ['251]hEGF to the liver, kidney, small intestine, stomach, and spleen was much greater than that accounted for by the distribution to the extracellular space of each tissue. (2) The binding (or uptake) of hEGF by these tissues showed remarkable saturation, which may represent the receptor-dependent binding (or uptake) mechanism. (3)The apparent binding (or uptake) clearance per gram of tissue at the low dose (in the range of first-order kinetics),defined with regard to the arterial plasma concentration, was greatest in the kidney, followed by the liver and small intestine. The larger binding (or uptake) clearance of the kidney compared with that of the liver can be attributed to the higher plasma flow rate (per gram of tissue) in the kidney. However, the intrinsic ability to take up hEGF was much greater in the liver than that in the kidney. The hepatic binding (or uptake) of hEGF at the low dose was almost limited by the hepatic plasma flow rate. (4) In the whole animal, the bulk of the removal of hEGF from the systemic circulation was accounted for mainly by hepatic clearance, both at the low and high doses.

binding (or uptake) by all tissues in in vivo conditions. We therefore undertook in vivo studies to define the kinetic characteristics of EGF binding (or uptake) by all major tissues in the rat. These data yielded the basis for defining the apparent maximum binding (or uptake) rates (Vmm), Michaelis constants (K,) for the receptor-dependent process, and proportionality constants (P& for the receptor-independent distribution in the tissues.

Experimental Section

Materials-Biosynthetic human epidermal growth factor (hEGF), obtained from Escherichia coli via the synthesized codin sequence described previously,1z,13was used in all experiments. 51-labeled sodium (100 mCi1mL) was purchased from the Radio Chemical Center (Amersham Corp., Arington Heights, IL). The hEGF was radiolabeled with [12511Naby the chloramine-T method. l4 Unreacted [1251]Na was removed by a Sephadex G-25 column, and the [1261]hEGFwas eluted in the void volume. The ['2511hEGF had a specific activity of 0.5-1.0 mCi/nmol and was >95% precipitable in 15% trichloroacetic acid (TCA). Furthermore, >98% of ['2511hEGF binding to a specific antiserum was displaced by excess amount of unlabeled hEGF (6 nM). All other reagents were commercially available and of analytical grade. Trichloroacetic Acid Precipitation Method-Plasma (50KL) was added to 1mL of 15%(wh) TCA containing 0.1%(w/v) bovine serum albumin (BSA; designated as TCA solution) and mixed well. After standing at 4 "C for 30 min, the mixture was centrifuged at 3000 rpm Epidermal growth factor (EGF), which has been isolated for 20 min and the supernatant was transferred to a separate tube by from the submandibular gland of the mouse and from human aspiration. The percent of radioactivity in the precipitate (designat~ r i n e , ' . stimulates ~,~ cell proliferation in the epidermal and ed as TCA-precipitable percent) was calculated as (cpm in precipitaepithelial tissues of animals and in various cell types in te)/[(cpm in precipitate) + (cpm in supernatant)] x 100, where cpm is culture.1,2.3It is also well known that EGF induces various the radioactivity of 1251,To determine the tissue concentrations of immediate and delayed biological effects, such as early stimthe TCA-precipitable [1261]hEGF,each tissue was homogenized in a ulation of nutrient t r a n ~ p o r t , activation ~.~ of a membrane 0.9% NaCl solution in a motor-driven Potter homogenizer, and 0.9 protein kinase;,? inhibition of gastric acid ~ e c r e t i o n de, ~ ~ ~ mL of the homogenate was added to 0.4 mL of 50%(w/v) TCA. The subsequent procedure was the same as that described above for layed activation of cytoplasmic enzymes, and stimulation of plasma. DNA synthesis. Animal Experiments-Adult male Wistar rats (Nihon Seibutsu Thus, although EGF has been shown to have various Zairyo, Tokyo, Japan), weighing 240-280 g, were used throughout biological effects in a variety of in vivo and in vitro cell the experiments. Food and water were available ad libitum. Under systems, its physiological role and the mechanisms for mainlight ether anesthesia, the femoral vein and the femoral artery were taining its plasma concentration are poorly understood. In cannulated with polyethylene tubing (PE-50) that was filled with any physiological situation, the steady-state plasma EGF isotonic saline, for drug administration and blood sampling, respecconcentration is determined by the rate of EGF production tively. Blood samples were obtained from the femoral artery after iv and/or release into the systemic circulation relative to the administration of [1261]hEGF. The plasma was quickly separated from the blood by centrifugation. rate of EGF removal by the tissues from the systemic Binding of I2'I-Labeled Human Epidermal Growth Factor by circulation. Epidermal growth factor is known to be cleared the Tissues of Intact Rats-Tracer amounts of ['251]hEGF or from the plasma by a receptor-dependent hepatic uptake unlabeled hEGF dissolved in 0.3 mL of rat plasma were injected into process.lo.ll However, considering that EGF has biological the femoral vein and blood samples were obtained from the femoral effects on several tissues other than the liver, it is possible artery at designated times (20, 40, 60, 80, 120, and 150 s) after iv that the receptor-dependent binding (or uptake) mechanism administration. Three minutes after administration, the rats were may also exist in other tissues. sacrificed by cutting the carotid artery. The liver, kidney, stomach, From these considerations, it is obvious that the mechaspleen, duodenum, jejunum, lung, heart, muscle, skin, and brain nisms of regulation of the plasma EGF level can only be were quickly excised, rinsed with ice-cold saline, and blotted dry, and a portion of each tissue was weighed and counted for the total understood if detailed kinetic data are available on EGF

2001Journal of Pharmaceutical Sciences Vol. 77, No. 3, March 1988

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0022-3549l8810300-200$01,0010 1988, American Pharmaceutical Association

radioactivity in a gamma counter (model ARC-300, Aloka Co., Ltd., Tokyo, Japan) with a counting efficiency of -80%. The remaining portion was quickly stored a t -40 "C until the time of assay. The amount of [12611hEGFtaken up by each tissue during a period of 3 min after administration was determined by the total radioactivity per gram of tissue. In the experiments where the tracer amount of ['2511hEGF was injected, the TCA-precipitable radioactivities in the tissues were also determined as described above. Distribution of '"SI-Labeled Human Epidermal Growth Factor to Blood Cells-Blood was sampled via the carotid artery into a heparinized tube and pooled from three rats. An aliquot (1mL) of the blood was transferred to the test tube and incubated for 5 min at 37°C prior to the addition of the tracer amount of [12'I]hEGF (-50,000 cpdmL) alone or the tracer with unlabeled hEGF (30 nM). One hundred microliters of blood was withdrawn from the incubation medium 10 and 60 min after the addition of ['2611hEGF alone or with unlabeled hEGF (30 nM). The blood cells were separated from the blood by centrifugation. The radioactivities of TCA-precipitable [12'I]hEGF in the plasma and in the blood cells were determined. The percentage of the ratio of TCA-precipitable cpm in blood cells to that in the blood was calculated by: 100 x [(TCA-precipitable cpm i n the blood cells)/(TCA-precipitablecpm in the blood cells + TCAprecipitable cpm in the plasma)]. Kinetic Analysis of the Tissue Binding (or Uptake) of Human Epidermal Growth Factor-Method I-The following assumptions were made in the kinetic analysis of the receptor-dependent hEGF binding (or uptake) by the tissues of the rat in vivo. (1) The tissue compartment is well stirred. (2) Little or no degradation of [1251]hEGFoccurred during the 3-min period after iv administration. (3)The venous concentration of ['261]hEGF in the tissues, except for that in the liver and spleen, is almost the same as the arterial concentration. ( 4 ) There is rapid equilibrium of [lz6I1hEGFbetween the capillary bed and the interstitial fluid. When a tracer dose of [l2'I1hEGF was intravenously administered, the tissue binding of [l2'I1hEGF can be described by linear kinetics as:

where AUC(o_t,represents the area under the plasma concentrationtime curve from time 0 to t. Consequently, the plot of V&#) versus AUCtoA,yields a straight line within a short period of time when the efflux (or dissociation) is much smaller than the influx. The elimination of hEGF from the plasma is mainly due to the endocytosis process which is mediated by a cell surface receptor." After the binding of hEGF to the receptor occurred, the hEGF-receptor complex is carried out into the intracellular compartment where the hEGF-receptor complexes or hEGF are degraded by the lysosomal enzymes.ll The time which is needed for the cell surface-bound hEGF to he carried out into the intracellular lysosomal compartment is -10-20 min.11 Therefore, it could be considered that at 3 min after hEGF administration, the distribution process to the cell surface receptor is the main mechanism for the disappearance of hEGF from the plasma. As shown in Figure 1, the linear relation between V K T ( t )and AUC,,,, passing through the origin (where t is from 1to 5 rnin), in the liver, kidney, small intestine, and spleen, indicates that the efflux of ['261]hEGF from these tissues is much smaller than the influx. If one assumes that both the receptor-dependent and the receptor-independent processes are concerned with hEGF binding (or uptake) by the tissue and that the tissue binding (or uptake) is measured within a short time when the efflux (or dissociation) is much smaller than the influx, the binding (or uptake) can be expressed by? VT-dCT =

dt

vmxcp

Km + C,

+ pd&,

where K, and V , represent the Michaelis constant and the maximum binding (or uptake) rate for the receptor-dependent binding (or uptake) process, respectively, and P d i f represents the proportionality constant for the receptor-independent binding (or uptake) process. Within a short period of time (<3 rnin), during which the change in the plasma concentration of hEGF is not so large, eq 3 may be approximated as:

(4)

(1)

where J,, is the average binding (or uptake) rate of hEGF (for 3 min), Km,app,Vma,,,, , and pdif,app are the apparent paramEters corresponding to K,,, , % , and P d i f in eq 3, respectively, and C, is the average plasma concentration of hEGF which can be obtained by:

where CT and VT are the tissue concentration ( c p d g of tissue) and the tissue distribution volume ( m u g of tissue) of [1261]hEGFa t time t after administration, respectively, C, is the plasma concentration of ['2SI]hEGF (cpdmL), kl represents the clearance for the binding process (or the uptake process into the cell), and k2 represents the clearance for the efflux process from the tissue (or the dissociation process from the cell surface binding sites). Integration of eq 1gives:

V&(t)

=

(5)

Jav/cp

A plot of versus Jay(EadkHofstee-type plot) will give us an idea whether or not the second term in eq 4 is necessary. That is, the second term is not necessary if the plot shows a straight line with a negative slope, while the second term is essential if the plot shows curvature and the plot is parallel to the abscissa in the range of large JaVvalues. To get the kinetic parameters, we fitted the experimental

kl{ C,dt - kzlfC& = 0

r*

0

(2) I

(3)

8 1

0 1 2 3 4 5 6

0 1

AUC(O-t)

(%

2 of

3 4 5 6

0 1 2 3 4

doselmin/ml plasma)

Figure 1-Relationship between the amount taken up by each tissue at time t and the area under the plasma concentration-time curve after iv administration of a tracer dose of ['25flh€GFfrom time 0 to t, where t is from 0 to 5 min. Key: A, liver; 6,kidney; C, duodenum (A)and jejunum (A). Each point represents one animal. Journal of Pharmaceutical SciencesllQl Vol. 77, No. 3, March 1988

data (listed in Table I) to eq 4 by use of the nonlinear iterative leastsquares method.16 Method ZZ-Equation 4,which is used in Method I, was obtained by assuming the approximation that the averaged plasma concentration (C,)can be used. Therefore, in Method 11, we also attempted to get kinetic parameters by fitting the experimental data directly to the differential equation (eq 3). The fitting was performed by use of the Multi-Runge program.'' In the fitting procedure, 14 simultaneous differential equations corresponding to each r at (shown in Table I) were used, since the plasma concentration-time profiles (C,) are different among the rats, depending on the dose. In advance, the time courses of the plasma concentration of hEGF were analyzed by the monoexponential equation (C, = Ae-"7, to obtain the A and a values of each rat. The binding (or uptake) of hEGF in the liver and spleen was also analyzed by a perfusion model, since the extraction ratios of these tissues were so high that the extracellular concentration of hEGF cannot be approximated by the arterial plasma concentration as discussed later. The mass balance equations of hEGF in the extracellular space and the cell (or the cell surface) compartment of the liver are described as follows, based on an assumption that both the extracellular and the cell compartments are well stirredlA

(7) where V H , E and CHVE are the extracellular volume and the hEGF concentration in the extracellular space of the liver, respectively, Qa is the hepatic plasma flow, and X, is the amount of hEGF associated with the hepatocytes. The liver receives venous blood flow from the GI tract and spleen. For the spleen, whose binding (or uptake) clearance is -64% of the plasma flow, the venous concentration is not necessarily close to the arterial concentration of [lZ6I]hEGF.Therefore, in a strict sense, the term of the inflow from the spleen should be included in eq 6.But the plasma flow in the spleen (0.2 m u m i d r a t ) is almost 1/50of the hepatic plasma flow (10mu mi d r at ) . Therefore, the contribution of the inflow of ['2611hEGF from the spleen to the liver can be ignored in eq 6,even though the extraction of hEGF in the spleen is 0.64.In the kinetic analysis, the values of 0.29m u g of liver and 1.0 rnUmin/g of liver were used as VHeEand QH, respectively.19.20The fitting was performed as described above by use of the Multi-Runge program." The mass balance equations of hEGF in the extracellular space and the cell (or the cell surface) compartment in the spleen are as follows:

(9) where V,,, and CSp,E are the extracellular volume and the hEGF concentration in the extracellular space of the spleen, respectively, QSpis the plasma flow in the spleen, and Xsp is the amount of hEGF associated with the spleen. In the kinetic analysis, the values of 0.30 m u g of spleen and 0.22 mLJmin/g of spleen were used as V,,, and QSP,respectively. The fitting was performed as described above by use of the Multi-Runge program.17

Results Plasma Disappearance of I2'I-Labeied Human Epidermal Growth Factor-Plasma disappearance of [lZ5I]hEGF was determined for a short period of time (<3 min) after iv administration of [lZ5I]hEGFwith varied amounts of unlabeled hEGF. As shown in Figure 2, the plasma disappearance curve of ['2511hEGF can be expressed by a monoexponential equation for such a short period of time, and the plasma disappearance was more delayed as the dose of unlabeled hEGF was increased. A dose-de endent change in the plasma disappearance half-life (t1J of Lg51]hEGF is shown in Table 1. The half-life increased, particularly at doses above -14 nmol/kg of body weight, indicating that the hEGF binding (or uptake) by the tissues was saturated as the dose increased. All the experimental results for the plasma and tissue concentrations of hEGF are listed in Table I. Tissue Distribution of '"1-Labeled Human Epidermal Growth Factor-Table I1 lists the apparent tissue distribution volume (Vd,,,) of [lZ5I]hEGFmeasured 3 min after the iv administration of the tracer amount of [lz5I1hEGF.The Vd,, values of ['251]hEGF in the kidney, liver, small intestine, stomach, and spleen are much larger than those accounted for by the distribution to the extracellular space. The most feasible mechanism for such large Vdap, values in these tissues is the specific binding of [lZ5I]hEGFto the cell surface receptor of these tissues, as has already been found for the liver.*OJ1Concerning the lung, heart, muscle, and brain, the

Table I-Concentratlons of Human Epidermal Growth Factor in Various Rat Tissues' Rat No.

Dose, nmov kg

1 0.035 2 0.053 3 0.058 4 0.083 5 0.118 6 0.531 7 1.21 8 2.14 9 3.01 10 5.00 5.04 11 12 14.0 13 19.7 14 22.7

igc

G,pmol/g of tissue'

AUC, AUCIt, nM.mind M ,,e Liver

Kidney

num "Ode-

0.301 0.77 0.10 0.596 0.088 0.29 0.76 0.12 0.337 0.37 0.733 0.090 0.346 0.66 0.39 0.13 0.697 0.108 1.18 0.73 0.22 0.499 0.66 0.140 1.01 0.77 0.25 0.781 0.76 0.167 0.74 4.27 3.79 3.23 1.08 0.759 7.46 3.09 0.76 7.26 9.28 1.53 19.5 7.80 22.9 0.88 23.4 3.62 21 .o 8.07 25.3 0.77 24.2 4.00 12.4 0.95 37.3 32.8 35.1 4.32 32.1 14.2 0.92 42.6 35.5 4.56 77.5 2.11 189 63 185 6.60 65.5 105 2.41 31 6 262 15.6 2.17 385 100 128 19.3 237

Jeiunum

Stomach Spleen

0.088 0.039 0.087 0.097 0.101 0.098 0.156 0.080 0.140 0.088 0.692 0.523 1.21 1.23 1.82 3.07 3.05 3.84 3.72 4.57 4.21 3.27 7.57 7.24 10.2 12.0 16.3 14.0

Lung

0.053 0.024 0.054 0.023 0.062 0.020 0.088 0.042 0.112 0.036 0.342 0.186 0.625 0.469 1.75 1.66 1.63 0.979 1.81 2.45 2.27 2.50 9.38 12.2 15.6 17.6 20.1 24.0

Heart

Muscle

0.012 0.004 0.007 0.010 0.006 0.014 0.012 0.023 0.012 0.027 0.054 0.103 0.153 0.226 0.318 0.759 0.357 0.571 1.14 0.715 0.750 1.20 2.53 6.08 4.15 10.5 4.76 9.23

0.022 0.022 0.023 0.048 0.046 0.202 0.555 1.36 1.39 1.93 2.10 13.3 17.1 21.1 ~

a Determined

Skin

Brain

0.002 0.001 0.002 0.003 0.003 0.014 0.039 0.092 0.065 0.141 0.167 1 .oo 1.90 2.21

~

3 min after iv administration of [1251]hEGFwith varied doses of unlabeled hEGF. Doses are normalized by the rat body weight. "Biological half-life (min). dArea under the plasma concentration-time curve after iv injection of hEGF from 0 to 3 min; calculated by the trapezoidal rule. eMean plasma concentration; t is 3 min in these experiments. 'Tissue concentration of hEGF in each tissue 3 min after iv injection. 202IJournal of Pharmaceutical Sciences Vol. 77, No. 3, March 1988

apparent distribution volume is almost the same as the extracellular volume in these tissues. This means that hEGF distribution in these tissues is mainly due to the extracellular space. On the other hand, for the skin, the value of Vd,, is smaller than that of VE in Table 11. It may be considerei that the capillary membrane permeability in the skin is very low, so the distribution equilibrium of hEGF between the capillary bed and interstitial fluid in the skin is not reached during 3 min after administration of [lZ5I]hEGF. Not only the total radioactivity, but also the TCA-precipitable radioactivity was determined in all the tissues tested 3 min after iv administration of the tracer dose of ['2511hEGF. The percentages of the TCA-precipitable [ lZ5I]hEGFwere 84, 90,88,82,78,68,67,65, and 62% in the plasma, liver, spleen, small intestine, kidney, lung, heart, muscle, and brain, respectively. Thus, the radioactivity associated with the tissues (kidney, liver, small intestine, stomach, spleen) which show large Vdtissuevalues was found to come mostly (-- >80%) from the TCA-precipitable [lZ5I]hEGF,suggesting

2 31

that metabolism (degradation) cannot account for the large V&issuevalues in these tissues. The time courses of Vdappvalues of these tissues (Figure 3) show that the Vdappvalues continue increasing at least up to 5 min after the iv injection of 11251]hEGF. This suggests that the distribution (or the binding to the cell surface) equilibrium cannot be obtained within 5 min. In Figure 1, the tissue concentrations of [lz5I1hEGFa t time t (C,) were plotted against corresponding AUCm,, values according to eq 2. Almost straight lines passing through the origin were obtained for the liver, kidney, and small intestine (up to -5 min), suggesting that the efflux of [lZ5I]hEGFfrom these tissues (or the dissociation from the cell surface binding sites) may be much smaller than the influx within such a short period of time. Dose-Dependent Tissue Uptake of Human Epidermal Growth Factor-The results of the tissue uptake study of [1251]hEGFafter iv administration are summarized in Table I. In order to know whether or not the binding (or uptake) of hEGF by the tissues shows saturation, J J C , versus JaV was plotted according to eq 4 (Figure 4). For all the tissues that exhibited large Vd,,, values, the plots had negative slopes at smaller Cp values, indicating the existence of a saturable uptake (or binding) mechanism. Kinetic parameters calculated by the two methods (see details in the Experimental Section) are listed in Tables I11 and IV. The two methods gave comparable results for the tissues tested, except for the liver and spleen. Both the K, and V, values showed large

0.05 0

20

40

60

80

100

120

140

TIME b e d Figure 2-Plasma concentration-time curves of ['251]hEGF after iv administration. The doses of tracer only (closed symbol) or the coadministered unlabeled hEGF (open symbol) in each case are 0.035 (O), 0.053 (A),0.058 (a),22.7 (0),19.7 (A), and 14.0 (0)nmol per kg of body weight. Table Il-Apparent Tlssue Distrlbutlon Volume (Vd,,) of '251-LabeledHuman Epidermal Growth Factor for Various Rat Tissues' Tissue

Vdappg mug of tissue

V,, mug of tissueb

Kidney Liver Duodenum Jejunum Stomach Spleen Lung Heart Muscle Skin Brain

9.53 f 0.21 4.86f 0.26 1.35f 0.08 1.31 f 0.07 0.86f 0.19 0.63f 0.03 0.32f 0.03 0.31 ? 0.01 0.12f 0.03 0.13 f 0.04 0.025'

0.40' 0.29 0.094d 0.094d 0.094d 0.30 0.38 0.30 0.13 0.31 0.015-0.035'

aDetermined 3 min after iv administration of a tracer dose of ['25i]hEGF; Vd,, was calculated by dividing the total cpm of 1251 per gram of tissue by the total cpm of lZ5lper milliliter of plasma at 3 min after administration; each value represents the mean f SD derived from three rats. bObtainedfrom ref 19."Obtained from ref 32.dExtraceIIular volume of GI tract was obtained from ref 19.eThe mean of two animals. 'Capillary volume was obtained from Rapoport, S. K.; Klee, W. A.; Pettigrew, K. D.; Ohno, K. Science 1980, 207, 84-86.

0

1

2 3 Time (min)

4

5

Figure &Time profiles of the apparent tissue distribution volume (Vd,,) of tracer ['251]hEGFafter iv administration. The tracer dose of ['251JhEGFin 0.3 mL of rat plasma was injected into the femoral vein. At the designated times (1, 2, 3, 4, and 5 min), the blood was withdrawn from the femoral aflery and the rats were sacrificed soon after the blood sampling. The h e r , kidney, stomach, spleen, duodenum, and jejunum were quickly excised and the total cpm per gram of tissue was determined. The apparent tissue distribution volume (Vd-) was calculated by dividing the total cpm/g of tissue by the total cpm/mL of plasma. In panel A, liver (0)and kidney (0)results are shown, and in panel B, duodenum (A), jejunum (A),stomach (O), and spleen (m) results are shown. Journal of Pharmaceutical Sciencesl203 Vol. 77, No. 3, March 7988

L-

0.31

B

1

C

0

0 0

10

30

20

0

1550

5

2

0

4

6

0.2 0.1

-

- ,

0 0

2

4

6

0 ,J,

2

4

(Pmol/rnin/g

6

0

I

I

I

2

4

6

tissue)

Flgure IbEadieHofstee-type plot showing the tissue binding (or uptake) of hEGF. Key: A, liver; B, kidney;C, duodenum; D, jejunum; E, stomach; F, spleen. Fitted curves, caiculated by method I, are shown in each panel. The abscissa is the tissue binding (or uptake) velocity (Jay,pmol/min/g of tissue) and the ordinate is the tissue binding (or uptake) clearance (Ja&, mUmin/g of tissue). Equation 4 was used for data fitting, except for the liver and spleen (see details in the text).

Table llCKinetlc Parameters for the Tissue Binding (or Uptake) of Human Epidermal Growth Factor (Method 1)'

Kidney Liver Stomach Duodenum Jejunum Spleen

0.4422 0.330 37.4 5.7 1.83 1.25 1.76 f 0.65 1.38 f 0.35 0.0975 0.031

0.306 2 0.364 38.0 2 7.3 15.0 f 10.3 8.06 2 3.26 7.11 f 2.06 0.798f 0.327

* *

1.444 0.986 0.122 0.218 0.194 0.122

0.769 f 0.055 N.D.

0.019 2 0.012 0.0242 0.009 0.027 f 0.005 0.049 f 0.003

aSee details in the Experimental Section; eqs 4 and 5 were used for the fitting; the parameter values are shown as mean f computer calculated SD. Not determined.

Table IV-Kinetic Tissue Kidney Liver Stomach Duodenum Jejunum Spleen

Parameters for the Tissue Binding (or Uptake) of Human Epidermal Growth Factor (Method 11)' Vmax v pmollminlg of tissue

Krnv nM

VrnaJKm, mUmin/g of tissue

0.306 5 0.484 27.9 2 3.1 39.4 2 6.6 2.40 f 1.95 2.06 2 0.91 1.67 -t 0.49 0.076 2 0.042 0.105 2 0.040

0.426 2 0.372 7.25 2 1.40 50.6 2 10.8 24.4 2 19.3 11.8 2 5.7 10.7 2 3.5 0.208f 0.196 1.03 2 0.52

0.718 3.85 0.779 0.098 0.175 0.156 0.365 0.102

Pdil,appg

mUmin/g of tissue

0.609 5 0.045 0.004 f 0.004 N.D.

0.013 f 0.013 0.018 2 0.009 0.020f 0.005 0.071 ? 0.008 0.0392 0.002

VmJKrn + Pdh mUmin/g of tissue

1.33 3.85 0.779 0.111 0.193 0.176 0.436 0.141

VmaxIKm, mUmin/ratc

'

1.44 38.5 7.79 0.13: 1.66 0.365 0.102 (11,l)e

a See details in the Experimental Section; eq 3 was used for the fitting; the parameter values are shown as mean ? computer calculated SD. For the liver and spleen, the perfusion model was also used for the fitting (using eqs 6,7,8,and 9)since the extraction ratios of the liver and spleen were so high that the extracellular concentrationof hEGF cannot be approximated by the arterial plasma concentration (see details in the Discussion);the values in the upper row were calculated accordingto the perfusion model, while those in the lower row were calculated by the conventionalMethod II. 'The parameter values expressed as those per whole rat (250g) are shown; the tissue mass used in these calculations was obtained from refs 19, 20,23,24,and 32.dValue for the GI tract is shown; the mass of the GI tract is assumed to be 10 g per rat (ref 19).eSum of the clearances corresponding to the saturable uptake process; the values of 7.79 and 0.102 were used for the liver and spleen, respectively.

204lJournal of Pharmaceutical Sciences Vol. 77, No. 3, March 1988

intertissue differences. For example, the liver has a relatively low affinity and high capacity system, while the kidney has a high affinity and low capacity system. The activity of a unit weight of the kidney (V,,IK, + F'di~) to bind (or take up) hEGF is 1.5 times that of the liver, and at least seven times those of the other tissues. On the other hand, as shown in Figure 5 and Table IV, if one calculates the activity of each tissue to take up hEGF on the basis of the whole tissue weight (not per gram of tissue), the liver is found to play a dominant role both at a tracer dose and at a large dose (23 nmol/kg of body weight).

-

Discussion The present studies provide the first quantitative description of the kinetics of the saturable and unsaturable processes that remove hEGF from the systemic circulation under in vivo conditions. The saturable process may be concerned with the receptor-dependent binding (or uptake) mechanism which has been identified previously in the liver." We used the assumption of a rapid equilibium of hEGF between the capillary bed and the interstitial fluid in the tissues examined in the kinetic analysis of hEGF tissue binding. Although Sat0 et a1.21reported the application of the capillary membrane-limited model of pendorphine, there is little information concerning the capillary membrane permeability of peptide hormones, except that across the blood-brain barrier.22But, if the transport process of hEGF between the capillary bed and the interstitial fluid is the rate-limiting step, the saturation of [lZ51]hEGFbinding (or uptake) to the tissues shown in Figure 4 would not be observed. Therefore, the assumption of rapid equilibrium between the capillary bed and the interstitial fluid could be applicable in the tissues shown in Figure 4.However, there still remains the possibility that the saturable transport system across the capillary membrane in these tissues may be observed instead of cell surface receptor binding. As shown in Table IV, this receptor-dependent process clears -11 mL of plasma containing hEGF per minute per rat. And, -70% of this receptor-dependent binding (or uptake) activity (Vmax/Km) is found in the liver (Table IV). The receptor-dependent hepatic binding (or uptake) clearance (Vmm/Km)of hEGF in the first-order kinetic range is 0.78 mL/min/g of liver, and is comparable with the hepatic plasma

-

0 0 c

0

n .-

2

.c_

o

m

Liver

Kidney

G.I. T r a c i

Muscle

Figure 5-Tissue distribution of ['251]hEGF in each tissue 3 min after administration of tracer only or tracer with excess unlabeled h€GE The open bars represent the mean f SD of three animals corresponding to the administration of a tracer dose of ['251]hEGF (Rat Nos. 1, 2, and 3 shown in Table I); the dotted bars represent the mean of two animals corresponding to the coadministration of tracer with unlabeled hEGF (Rat Nos. 13 and 14 shown in Table I).

flow.2334 The percentage of [lZ51]hEGFdistributed into the blood cells was negligible either in the presence or absence of unlabeled hEGF (30 nM; see details in the Distribution of 1125ZJhEGFto the Blood Cells). This finding, together with our present result that the distribution of [lz511hEGFinto the blood cells can be ignored, suggests that the hepatic uptake of hEGF is plasma flow limited. In other words, the single-pass hepatic extraction of [lZ51]hEGFa t the low dose is almost complete, which supports the previous findings obtained by a liver perfusion experiment.11 Despite this predominant role of the liver in removing hEGF from the systemic circulation, saturable transport has also been identified in vivo in other organs, including the kidney, small intestine, stomach, and spleen. Specific binding of hEGF was previously identified in the small intestineZ6 and in the stomach,zg as well as in the liver,11 in an in vitro experimental system using isolated cells or tissue homogenates. The K, values of EGF binding in the perfused liver and liver homogenate were 1-2 nM and 8-15 nM, respectively, while the K, value obtained from our in vivo experiments in the liver was 7.3 nM, a value which is comparable with those reported previously. As for the reason for the various K, values between tissues (Table IV), we can consider the following possibilities: the heterogeneity of the hEGF receptor between tissues, the release of endogeneous binding inhibitors of hEGF from tissues, and the different distribution between the capillary bed and the extracellular space of hEGF among the tissues. Therefore, it could be considered that the several factors mentioned above were the reason for the discrepancy of K , values of the small intestine between our present results (10.7-11.8 nM), determined in in vivo, and the previously reported ones (1-2 nM), determined in in vitro.26 Matrisian et aL27reported that iodinated EGF was heterogeneous and that [1251]EGFpooled into five groups revealed markedly different binding behaviors to the EGF receptor. Therefore, it is possible that the heterogeneity of 1251-labeled hEGF induced some effects on the binding behavior of hEGF in our in vivo experiment. There exist many factors influencing the binding of hEGF with its receptor in in vivo conditions, so it is very difficult to determine the exact binding parameters of K , and V,,, in vivo. In our in vivo experiment, we demonstrated the contribution of each tissue clearance (VmJKm) to the total body distribution clearance, rather than the K , values of the tissues. The in vitro experiment using isolated rat intestinal epithelial cells28 also indicated the presence of specific binding, internalization, and processing of [1251]hEGF.However, Rao et a1.Z8 did not clarify on which side (luminal or antiluminal side) hEGF may act at first. The present in vivo study identified the saturable binding of hEGF to the small intestine shortly after iv administration of hEGF (<3 min). This may suggest that the specific binding site exists at least on the antiluminal membrane, although the possibility of the presence of a specific binding site on the luminal membrane still exists. These hEGF receptors are compatible with the physiological involvements of hEGF in the regulation of the function of the intestinal epithelium with regard to the mitogenic action of hEGF.28 In this paper, we have presented for the first time the presence of specific binding of hEGF in the kidney and spleen in vivo. Yeh et a1.29.30previously found that EGF generally stimulated DNA synthesis in the kidney, liver, lung, cornea, aorta, testis, and parotid, and inhibited it in the spleen, bone marrow, and thymus of the male mouse, although the growth response of each tissue to EGF depended in part on the EGF injection and kill times. However, at present, we cannot tell whether the specific binding sites of hEGF on the kidney and spleen are related to the growth Journal of Pharmaceutical ScienceslSO5 Vol. 77, No. 3, March 7988

response. The hEGF detected in the kidney after iv administration may be partly accounted for by hEGF which undergoes glomerular filtration and then remains in the lumen of the kidney. An estimate of the maximum contribution of this mechanism can be attempted as follows. The amount of hEGF which undergoes glomerular filtration (AGFR)during 3 min after iv administration of a tracer dose of [1261]hEGFcan be calculated by AGFR= GFRf AUC(03,, where fp is the plasma unbound fraction of hEGf, and GFR is the glomerular filtration rate. The AGFR/AUC(~-~) value was calculated to be 0.5 mllmin/g of kidney using the GFR value of 0.5 mL/min/g of kidney1*and the maximum fp value of 1.0. Comparing this value with the value (1.9 mL/min/g of kidney) obtained by dividing the experimentally determined amount of hEGF in the kidney by AUC(o-3),the maximum contribution of the glomerular filtration of hEGF to the apparent amount of hEGF detected in the kidney is <30%. Thus, the bulk of [ ‘251]hEGF associated with the kidney may be explained by specific binding to the cellular components (probably on the cell surface) of the kidney. In fact, our preliminary in vitro binding experiments using rat kidney homogenates revealed specific binding of hEGF with the dissociation constant of 6.0 nM and the binding capacity of 0.05 pmol/mg of tissue rotei in.^^ In the present kinetic analysis, we used two methods. Method I is a simpler method, although it is based on an assumption that the plasma concentration of hEGF is constant for a relatively short period of time (<3 min). This assumption, however, does not hold in a strict sense. On the other hand, Method I1 can be used without the approximation of mean plasma concentration, although the inevitable use of the numerical solution of simultaneous differential equations made it complicated and time consuming to use this method. We found that these two methods yielded comparable parameter values (Tables I11 and IV),suggesting that the assumption made in the analysis by Method 1may be allowed, except in the liver. In the present analysis, the basic mass balance equation (eq 2) was written by assuming that the tissue binding (or uptake) of hEGF occurs from the circulating plasma. However, it may be more reasonable to consider that the compartment from which the tissue uptake occurs is capillary plasma. In this sense, the use of the perfusion model considering the plasma flow rate into and from each tissue is more rational, particularly for the tissues which show high hEGF binding (or uptake) clearance (V,,/K, + Pdif) compared with the tissue plasma flow rate. As shown in Table IV, the hEGF binding (or uptake) clearances in the liver, kidney, small intestine, stomach, and spleen are 0.78, 1.33, 0.18, 0.11, and 0.14 mL/min/g of tissue, respectively. On the other hand, the plasma flow rates in the liver, kidney, GI tract, and spleen have been reported to be 0.6-1.2, 3.1, 0.6, and 0.22 mL/min/g of tiss~e,’9.20,23,~~,~2 respectively. The hEGF binding (or uptake) clearances are thus smaller than the corresponding plasma flow rates in all tissues except the liver and spleen. This means that the drop in plasma concentrations of hEGF from the artery to the tissue vein after a single pass through the tissues is minimal, and that the use of eq 4 is appropriate for these tissues. However, in the case of the liver and spleen, the extraction of hEGF is so large that the use of a flow model is necessary. Consequently, the kinetic analysis of the binding (or uptake) of hEGF in the liver and spleen was carried out using eqs 6 , 7 , 8 , and 9. The K, value calculated based on the perfusion model was much lower than that obtained based on the conventional model. This result is reasonable if we consider that the conventional model and the perfusion model assume that the tissue binding (or uptake) occurs from the arterial plasma and the hepatic venous plasma, respectively. The apparent tissue POBIJournal of Pharmaceutical Sciences Vol. 77, No. 3, March 1988

binding (or uptake) per gram of tissue is higher in the kidney than in the liver (Tables 11, 111, and IV, Figure l),although the intrinsic ability (Vmax/Km)to take up hEGF is much higher in the liver. This is due to the much greater plasma flow rate in the kidney (3.1 mL/min/g of kidney) compared with that in the liver (0.6-1.2 mL/min/g of liver). The hepatic extraction of hEGF a t the low dose is almost complete, hence the hepatic binding (or uptake) clearance is close to the hepatic plasma flow. On the other hand, the renal extraction of hEGF is relatively small (-0.4), but the renal binding (or uptake) clearance calculated by multiplying the extraction ratio by the renal plasma flow is larger than the hepatic uptake clearance, due to the large plasma flow in the kidney. In conclusion, we present the first quantitative analysis of the kinetics of the saturable removal process of hEGF from the systemic circulation in vivo. The analysis shows that the saturable binding (or uptake) mechanism exists in the liver, kidney, small intestine, stomach, and spleen, and the contribution of each tissue to the removal of hEGF from the systemic circulation is greatest in the liver followed by the kidney.

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finfi

18. FiLg, K. S.; Rowland, M. J . Pharmacokinet. Biopharm. 1977,5, 625-653. 19. Tsuji, A.; Yoshikawa, T.; Nishide, K.; Minami, H.; Kimura, M.; Nakashima, E.; Terasaki, ,T.; Miyamoto, E.; Nightingale, C. H.; Yamana, T. J . Pharm. Sci. 1983, 72, 1239-1251. 20. Machida, M.; Morita, Y.; Hayashi, M.; Awazu, S. Biochem. Phurmacol. 1982,31,787-791. 21. Sato, H.; Sugiyama, Y.; Sawada, Y.; Iga, T.; Hanano, M. Drug Metab. Dispos. 1987. in press.

22. Sharma, R.R.; Vimal, L. P. Bruin Res. 1984,308,201-214. 23. Sasaki, Y.; Wa er, N. J. A p l . Physiol. 1971,30,879-884. 24. Lutz, R. J.; D e g c k , R. L.; &thews, H. B.; Eling, T. E.; Anderson, M. W. Drug Metub. Dispos. 1977,5, 386396. 25. Forgue-Lafitte, M.E.;Laburthe, M.; Chamblier, M. C.; Moody, J. A.; Rosselin, G. FEBS Lett. 1980,114,243-346. 26. Nomura, H.; Iwakawa, S.; Okumura, K.; Hori, R. Abstract, 106th Annual Meeting of the Pharmaceutical Society of Japan, Chiba, 1986. 27. Matrisian, L. M.; Planck, S. R.; Finch, J. S.; Magun, B. E. Biochim. Bio hys. Actu 1985,839,139-146. 28. Rao, R. Thornbur , W.; Korc, M.; Matrisian, L. M.; Magun, B. E.; Koldovsky, 0. i m . J. Physwl. 1986,250,G8504855. 29. Scheving, L. A.;Yeh, Y. C.; Tsai, T. S.; Scheving, L. E. Endocrinology 1980,106,1498-1503.

<

30. Yeh, Y. C.; Scheving, L. A,; Tsai, T. H.; Schering, L. E. Endocrinology 1981,109,644-651. 31. Yanai, S.;Su 'yama, Y.; Kim, D. C.; Sato, H.; Fuwa, T.; Iga, T.; Hanano, M. &em. Phurm. Bull. 1987,35,4891-4897. 32. Itoh, N.; Sawada, Y.; Sugi ama, Y.; Iga, T.; Hanano, M. Am. J. Physiol. 1986,251,F103-$114.

Acknowledgments This study was supported b a ant-in-aid for Scientific Research rovided by the Ministry oPEgcation, Sciences and Culture of japan.

Journal of Pharmaceutical Sciencesl207 Vol. 77, No. 3, March 1988