Importance of receptor-mediated endocytosis in peptide delivery and targeting: kinetic aspects

ELSEVIER Advanced Drug Delivery Reviews 19 (1996) 445-467

advanced drugdeliiry reviews

Importance of receptor-mediated endocytosis in peptide delivery and targeting: kinetic aspects

Hitoshi Sate”‘” , Yuichi Sugiyamab, Akira Tsuji”, Isamu Horikoshi” A Department of Hospital Pharmacy, Toyama Medical and Pharmaceutical University, 2630 Sugitani. Toyama 9.30-01. Japan

hDepartment of Pharmaceutics, Faculty of Pharmaceutical Sciences, University of Tokyo, Tokyo, Japan ‘Department of Pharmaceutics, Faculty of Pharmaceutical Sciences. Kanazawa University. Kanazawa, Japan

Abstract

Receptor-mediated endocytosis (RME) is a general mechanism by which eukaryotic cells internalize peptide hormones, growth factors, cytokines, plasma glycoproteins, lysosomal enzymes, toxins and viruses concomitantly with their receptors on the cell surface. Receptor-mediated drug targeting is considered to show promise as a drug delivery system (DDS). The success of RME-utilizing drug targeting is dependent not only on the in vitro efficacy but on the in vivo behavior of the drug-polypeptide complex. In fact, there has not been much progress from the “natural” targeting of glycoproteins and LDL to hepatocytes or macrophage lineages, due to unfavorable effects on the pharmacokinetics and pharmacodynamics (PK/PD) of polypeptides. In this review, we considered the kinetic modelling of polypeptide RME in isolated or cultured cells in vitro, in perfused organs and in the whole body. Typical RME analyses are described to estimate the rate constants for polypeptide-receptor internalization, the degradation of internalized polypeptide and receptor externalization, based upon the “receptor recycling” model. The kinetic RME analysis enables not only the non-linear clearance and distribution in the liver to be predicted, but also the surface-bound and internalized polypeptide in the target cell, which is important for the intracellular efficacy of drug-polypeptide complex. In particular, the kinetics of down-regulation and subsequent recovery of surface receptors were precisely elucidated, because if the receptors are once down-regulated, the next dosage should be given only after the surface receptors are restored. The therapeutic efficacy/toxicity of a drug-carrier conjugate should be assessed from its microscopic pharmacology based on the RME mechanisms, in conjunction with macroscopic pharmacokinetic modelling.

Keywords: Peptide delivery and targeting; Receptor-mediated endocytosis (RME); Receptor recycling

Contents

1.

2. 3.

4. 5 . 6.

Introduction .......................................................................................................................................................................... 446

Factors influencing polypeptide pharmacokinetics/pharmacodynamics.. .............................................................................. 447

Kinetic analysis of RME in vitro and in perfused organs.. .................................................................................................... 449

3.1. Estimation of RME parameters from ligand movement.. .............................................................................................. 450

3.2. Estimation of RME parameters from receptor movement ............................................................................................ 452

Kinetic analysis of RME in vivo.. .......................................................................................................................................... 456

Implications of RME kinetics for clinical application of polypeptides .................................................................................. 459

Perspectives .......................................................................................................................................................................... 459

Acknowledgments ..................................................................................................................................................................... 460 References.. ............................................................................................................................................................................... 460

*Corresponding author. Fax: +81 764 345089; e-mail: [email protected]

0169-409X/96/$32.00 @ 1996 Elsevier Science B.V. All rights reserved

PII SO169-409X(96)00013-0

1. Introduction

Receptor-mediated endocytosis (RME) is a general mechanism by which animal cells internalize a wide variety of selected extracellular materials concomitantly with their specific receptors (for review, see Refs [l-S]). Such materials include peptide hormones, growth factors, cytokines, plasma glycoproteins, lysosomal enzymes, toxins [9,10] and viruses [11,12]. First, a ligand binds to its specific receptor on the cell surface membrane: the ligand-bound receptors cluster in discrete regions called coated pits, which invaginate into the cell to form endocytotic vesicles or endosomes. The ligand and receptor then dissociate at an acidic pH (5-5.5) within the endosomes. The internalized receptor usually recycles back to the cell surface for further binding, while the internalized ligand is sorted and delivered for the most part to lysosomes for degradation.

According to current understanding of RME, the receptor-mediated targeting of drugs has been considered promising as a drug delivery system (DDS) (for review, see Refs [13-201). In this DDS, drug or peptide is covalently or noncovalently coupled to a polypeptide carrier; the prepared drug-carrier complex should still bind to receptor, without significantly reducing the internalization capability. Moreover, gene fusion technology has produced cytokine-toxin complexes [21,22]. Fig. 1 indicates how a drug-polypeptide complex may undergo cellular processing via the RME. The nature of receptor recycling enables the continuous delivery of drug-polypeptide complexes into the target cells. Lysosom- al degradation of the drug-carrier complex (with an acid-sensitive linkage) may result in the release of a drug into the cytosol, where it can exert its effect. Sometimes, endosomes merely cross polarized endothelial or epithelial cells in such a manner that the ligand remains intact (transcytosis). For example, transferrin remains bound to its receptor in the endosome because of its high affinity at acid pH, and the transferrin- receptor complex recycles back to the plasma membrane without degradation [23-261. where- upon the ligand dissociates from the receptor. Transcytosis of other polypeptides has also been

(outside)

WCeptClr binding

r* w+

.*

.* ‘r’r Cdl surface y y y y y y

membrane

(Inside) t recycling

internalization

Fig. I. A schematic diagram for receptor-mediated endo-

cytosis (RME) of drug-polypeptide complex. Intracellular

routings of polypeptide and receptor are described in the

text.

reported [27-301. Thus, drug-polypeptide complexes have been designed to cross the blood- brain barrier (BBB) by means of transcytosis. These include insulin [31] and transferrin [32,33], although the brain selectivity of these complexes in vivo remains unclear. The findings that ligand translocates from the endosome to the nucleus [34-371 has opened an avenue with which to develop a carrier for nuclear drug targeting. For example, albumin conjugated with the peptide RRRGL, corresponding to residues 31-35 of human angiogenin, has been specifically im- ported into the nucleus [38]. Endosome-disruptive peptides [39] may help drug-carrier complexes leave the endosome for the cytosol before sorting to lysosomes. This strategy is important for drugs sensitive to inactivation in lysosomes. In the RME-utilizing DDS, therefore. a polypeptide ligand can be recognized as a transport carrier or a “vector” to efficiently deliver other- wise impermeable substances into, or across, cells. Gene transfer into endothelial cells and lymphocytes from patients ex vivo and grafting them to the same individual (i.e., gene therapy)

H. Sato et al. I Advanced Drug Delivery Reviews 19 (1996) 445-467 447

has allowed several kinds of human genetic diseases, especially those caused by single gene enzyme deficiencies, to be treated [40-421. Ac- cordingly, there is increasing usage of polypeptides for DNAioligonucleotide delivery due to their capability to undergo RME in vitro. As an example, Fig. 2 illustrates an insulin-[N-acylurea albumin] conjugate bound electrostatically to NE0 gene, which was used for the DNA delivery into HepGz cells via endocytosis with insulin receptors [43]. Table 1 summarizes applications of receptor-mediated drug delivery between 1990-1995 (prior to 1990, see [15] and [16]).

The success of the RME-utilizing drug targeting is dependent not only on the in vitro efficacy of targeting but on the in vivo behavior of the drug-carrier complex. Therefore, it is important to kinetically analyze the RME processes of polypeptides in order to estimate both their targeting efficiency and pharmacokinetics in a comprehensive manner. In this review, therefore, we will focus upon the kinetic modeling analysis of polypeptide RME using isolated or cultured cells in vitro, perfused organs and the whole body in vivo, in order to provide kinetic insights

CELL MEMBRANE r 1 DNA

-

INSULIN RECEPTOR COMPLEX

, J lNsuLlN - [N-ACYLUREA

ALBUMIN] CONJUGATE

Fig. 2. Binding interactions of a conjugate consisting of a

polypeptide ligand such as insulin (designated as I) covalently

cross-linked to N-acylurea albumin. The N-acylurea albumin

(A-CDI). produced by carbodiimide modification of al-

bumin, bears numerous positive charges and is electrostatical-

ly bound with negatively charged DNA.

into receptor-mediated drug-polypeptide targeting.

2. Factors influencing polypeptide phannacokinetics/pharmacodynamics

General considerations about the various factors influencing peptide pharmacokinetics have been provided by McMartin and Bennett (44,451, and some of these factors have been incorpo- rated into physiologically based models [46-491 to predict the pharmacokinetics of peptides and proteins [50-521. If the target site (receptor-rich cells) resides within the vascular compartment where exogenously administered polypeptide can easily gain access, targeting efficiency may be relatively high. On the other hand, if the target site is located outside the vascular compartment, the polypeptide will encounter several limiting factors before reaching the target cells. These are enzymatic degradation either in blood or on the endothelial cell surface, non-specific elimination by the liver and kidney, binding with plasma proteins [53], especially a,-macroglobulin [54- 581, binding with heparin [59-611, blood flow to the target site, transcapillary permeability [62- 65] and interstitial diffusion [66,67]. Thus, the in vivo efficacy of polypeptides may be offset by some of these physiological barriers. Moreover, macromolecules with high receptor binding affinity may not diffuse into the kernel of the target tissue due to efficient binding and uptake at the perivascular site (so-called “binding site barrier”), as demonstrated for antibody percola- tion into cancer tumors [68-701.

When a polypeptide is mainly cleared by the RME system, the RME should be considered as a predominant factor in its PK/PD. For example, the remarkable dose-dependence and analogue

( or antibody) blockade of peptide pharmacokinetics may result from saturable, receptor-mediated clearance [71-781. A clearance- mediating receptor (C-receptor) distinct from that (B-receptor) mediating biological actions has been reported for atria1 natriuretic factor (ANF) [79] and its pharmacokinetic role has been demonstrated [72,73,80]. Reversible receptor binding serves as a substantial depot of

448 H. Sato et al. I Advanced Drug Delivery Reviews 19 (1996) 445%467

Table 1 Recent applications of receptor-mediated drug delivery (1990-1YYS)

Drug/gene Carrier Experimental method Ref No.

(i) Genes and antisense oligonucleotides

PEPCK-hFIX gene Galactosylated polylysine

CAT DNA Galactosylated histone

Luciferase gene Lactosylated polylysine

c-myb antisense Transferrin-polylysine

Luciferase gene

CAT DNA

CAT antisense

DTA”

Oligonucleotides

Neo gene

Factor IX gene

DTA”

Interleukin-6

Transferrin-polylysine

Asialoglycoprotein-polylysine

Asialoorosomucoid-polylysine

Asialoorosomucoid

Asialofetuin-polylysine

Insulin

Adenovirus

Virosome

Gene engineered diphteria toxin

Rat in vivo

HepGz cells

HepGz cells

Leukemia cells

Eukaryotic cells

Rat in vivo

NIH 3T3 cells

Hepatocytes

HepGl cells

HepGz cells

Hepatocytes

BHK-21 cells

Eukaryotic cells

11641

[I651 [166]-

[I671 [168,169]

11701 [I711 u721 [I731 I431 I1741 I1751 [I761

(ii) Antineoplastic agemr

Doxorubicin

Methotrexate

Eiliptinium-oleate

ILEh

Neocarzinostatin

(iii) Antiviral agents

ara-C

AZT

PMEA’

(iv) Peptides and proteins

Horseradish peroxidase

Superoxide dismutase

HIV-1 derived

synthetic peptide

(v) Miscellaneous agents

Chlorin e6

Naproxen

Maleylated BSA

Maleylated BSA

LDL

LDL

Transferrin

Glycosylated dextran

Mannosylated HSA

Mannosyl polymer

Insulin fragment

Mannosylated derivative

a,-Macroglobulin

Insulin

Lysozyme

Lymphoma cell line

Macrophages

916 melanoma

A549 cells

Leukemia K562 cells

[I771 [I781 [I791

[W [loll

Mouse in vivo

MT-4 cells

Macrophages

[1SIl

[I821

(1831

Mouse in vivo

Macrophages 1311 [I841

Mouse in vivo [I851

HepGL cells

Rat in vivo 1341

N61

” Diphtheria toxin fragment A.

h 2-morpholinomethyl-2’,3’,4’-trimethoxy acrylophenone hydrochloride.

’ 9-(2-phosphonylmethoxyethyl)adenine

polypeptide in its in vivo distribution, at the level of the whole body [81] and organs [82-861. Moreover, polypeptide pharmacokinetics may vary depending on alterations in receptor num- bers (up- and down-regulation) [87-891, endogenous ligands [90] and soluble receptors [91-961. The importance of RME in the overall pharmacokinetics of polypeptide hormones has been emphasized from a kinetic viewpoint [97-991.

At the cellular level, the pharmacological

effect of a drug-polypeptide complex may be dependent on the targeting efficiency and cellular processing via the RME. Potential problems of receptor-mediated drug targeting have been described comprehensively in previous reviews [15,16,100]. Ultimately, post-endocytotic intracellular routing of a drug-polypeptide complex should affect RME-utilizing drug delivery. The use of transferrin as a carrier would be useful for helping cell-impermeable drugs traverse endo-

H. Sato et al. I Advanced Drug Delivery Reviews 19 (19%) 445-467 449

thelial cells without degradation, but not for launching drugs within target cells. The complex- ity associated with RME, however, is that the kinetic properties of RME may vary depending on the ligands and cell types. For example, 25% of the internalized neocarzinostatin-transferrin conjugate was degraded in the lysosomes of leukemia cells, while transferrin alone was not degraded at all [loll. Moreover, fibroblasts degrade complexes of internalized epidermal growth factor (EGF) and its receptor 11021, whereas pancreatic carcinoma cells recycle them back to the cell surface [103].

For any kinetic analysis of polypeptide, an adequate assay system has to be established with which to measure intact polypeptide. There are several means of doing so, including bioassays, trichloroacetic acid (TCA)-precipitation [104], gel chromatography, radioimmunoassays (RIA), radioreceptor assays (RRA), enzyme-linked im- munosorbent assays (ELISA) and high-performance liquid chromatography (HPLC). A comparison of these methods is not within the scope of this review. However, it must be pointed out that assay results can differ depending on the method used, due to differences in the resolution of metabolite separation [105,106] or in the recognizable locus of the polypeptide between the receptor and an antibody [107]. Moreover, when a radiolabeled peptide is used, the labeled material has to have sufficient receptor binding activity. Occasionally, labeled polypeptide loses its receptor binding activity due to a conforma- tional change, or iodination at a biologically important tyrosine residue. It is not surprising that receptor-mediated clearance and distribution cannot be demonstrated using such a radiolabeled ligand in vivo. This problem has been experienced with some peptide hormones including insulin [108,109] and /3-endorphin [85]. Thus, experimental conditions (receptor-binding affinity, administered dose and the assay method) should also be included as variables influencing peptide pharmacokinetics. These factors should be considered especially when comparing peptide kinetic studies from different laboratories. Table 2 summarizes the factors which may influence polypeptide PK/PD as mentioned above.

Table 2 Various factors which may influence the pharmacokinetics and pharmacodynamics of drug-polypeptide complex

(i) Physiological factors’ * Enzymatic degradation in the blood or on endothelial cells * Non-specific elimination by the liver and the kidney . Receptor-mediated clearance by the liver and the kidney - Binding to plasma proteins (such as cu,-macroglobulin) * Binding to heparin * Binding to soluble receptor . Blood flow to target site . Transcapillary permeability . Interstitial diffusion * Prevascular distribution (“binding-site barrier”)

(ii) RME activity of target cell * Competitive endogenous Iigand . Receptor number on cell surface . Rate of receptor-mediated endocytosis * Rate of receptor synthesis and degradation * Rate of receptor recycling * Up- and down-regulation of surface receptor

(iii) Intracellular fate in target cell Trafficking and translocation from endosome Disruption of drug-polypeptide linkage in lysosome Release of drug into cytosol Stability of drug in lysosome

iv) Experimental conditions Dose administered

. Route of administration * Molecular ratio of drug/carrier - Effect of labeling on receptor binding activity * Analytical methods

a Pathophysiological changes of these factors should be also considered.

These factors have to be considered to better incorporate PK/PD principles in the develop- ment of polypeptides and proteins and their drug delivery systems for therapeutic use.

3. Kinetic analysis of RME in vitro and in perfused organs

So far, a large number of kinetic studies have been performed with regard to the RME of polypeptides. Using epidermal growth factor (EGF) as an example, its in vitro RME kinetics

450 H. Suto et al. I Advanced Drug &livery Reviews 19 (1996) 44.f-467

were first elucidated by Wiley and Cunningham using human fibroblasts [llO,ll l] and later ex- tended to a state-of-the-art kinetic model (112,113]. Later, the hepatic handling of EGF via the RME mechanism was described kinetically using isolated and cultured hepatocytes [97,114- 1181 and perfused livers [83,89,97-99,114, 119,120]. As such, the RME processes of EGF in the liver have been extensively studied from various aspects. It is difficult to cover all of them here. Therefore, only typical RME analyses are described in this review.

Among the several processes of RME (Fig. l), binding of ligand to its receptor is the first step that triggers the internalization of ligand-receptor complex. Hence, the binding kinetic parameters of k,,, (association) and k,,rr (dissociation), as well as surface receptor number ([R,]), have to be measured in order to quantify the dynamic ligand-receptor interaction. However, intracellular degradation of ligand and subsequent ac- cumulation of degradation products in the extracellular medium may significantly hamper the accurate assay of receptor binding, based on unchanged ligand concentrations. Moreover, receptor binding equilibrium should be altered by the internalization process. Even if the ligand concentration bound to receptors is appro- priately measured as opposed to that which was internalized, the apparent dissociation constant (K,,app) obtained by Scatchard analysis is not necessarily a valid measurement of ligand-receptor affinity (i.e.. K, = k,,,,lk,,,) when RME is taking place. According to the formulation by Wiley and Cunningham [ill], K,,app can be expressed as follows:

K+pp = koff + kint ’ kt

k k Cl” Ill,

where k,,, and k, represent the endocytotic rate constants of occupied and unoccupied receptors, respectively. If the endocytosis of unoccupied receptors is neglected from the formulation process, then the following equation holds:

K,,app = k,” + km,

k 011

Therefore, it is a prerequisite to suppress RME

activity during the receptor binding assay. The temperature can be easily decreased to 4°C for this purpose. However, when receptor-binding kinetics are temperature-dependent, the use of phenylarsine oxide (PhAsO), NaN,, or KCN to inhibit RME may be an appropriate means of measuring true receptor-binding parameters in intact cells at 37°C (110,121,122].

The multiple indicator dilution (MID) tech- nique developed by Goresky et al. [123,124] has been employed to measure kinetic parameters representing the relatively rapid interactions between polypeptides and the liver cell surface in the perfused liver [83,89,98,11.5,125]. In a MID experiment, a test polypeptide and an extracellular reference compound ( 14C-labeled inulin) are administered as a bolus into the portal vein, and the outflow in the hepatic vein is sampled in about 0.5-l s aliquots. The concentration-time profiles (“dilution” curves) of both compounds in the outflow are simultaneously analyzed by a mathematical model [126]. Thus, some receptor- related parameters, including R,, k,, and k,,(,, can be determined. The key features of the MID method are, 1) the structural architecture of the liver as an in vivo system is maintained, 2) the effect of endogenous substances can be ignored, 3) multiple injectates can be consecutively tested, and 4) the concentration of drug, inhibitors, or plasma proteins, plasma flow-rate and temperature can be controlled.

-3.1. Estimation of RME parameters ,from l&and movement

A mathematical model for the movement of extracellular and intracellular ligands, as well as of intracellular degradation products, is presented in Fig. 3. This model is a topology of the RME scheme shown in Fig. 1 with respect to the movement of ligand, and is essentially the same as those previously developed for EGF [127] and insulin [128]. Since lysosomal delivery of polypeptide is supposed to take place before peptide degradation (or digestion), the fraction (f) of internalized ligand that is sorted to lysosomes is parameterized. When the intracellular drug concentration ( [Di]) is to be simulated, considerations should include the generation of the drug

H. Sato et al. I Advanced Drug Delivery Reviews 19 (1996) 445-467 4.51

ligand

cell surface degradation

products

kint A

extracellular extracellular space space

Fig. 3. A generic model for the internalization and degradation of drug-ligand (polypeptide) complex via the RME mechanism. 12,. L,, and Ldcg represent the amounts of surface-bound ligand, internalized ligand, and intracellular degradation products, respectively; k,“, represents the rate constant for internalization of drug-ligand-receptor complex; k_, is the externalization rate constant of intracellular receptor; k,,, and krV, define the degradation of L, and subsequent release of L,,, into the extracellular fluid, respectively: and f denotes the fraction of internalized ligand that is sorted to lysosomes; on the other hand, (1 -f) is assumed to be the fraction of internalized ligand that is not degraded but transcytosed (e.g., f=l for most polypeptides but f=O for transferrin).

from an intralysosomal drug-polypeptide complex (f.Li), with the rate constant, kgen, as well as the sequestration (degradation and extracellular release) of the generated drug with the rate constant, kXeq. Thus, the equation d[D,]ldt= k,,;f[L,] -k,,,[D,] may hold for an intracellularly generated drug which is pharmacologically active. However, the feasibility of this equation remains to be confirmed due to the lack of corresponding experimental data. In fact, polypeptide sorting in endosomes is a complex process and has been elaborately modelled [129,130].

Our task is to estimate the endocytotic rate constant (k,,,) and the intracellular degradation rate constant (kdcg) of the labeled polypeptide. The experimental procedure for this analysis is to incubate a labeled polypeptide (conjugated with drug) with isolated cells or perfused tissue at 37°C for a short period and then to measure the surface-bound and internalized ligands by an acid-wash [110,111,122,131]. The logic of the

acid-wash is that the radioactivity removed from the cells at low pH at O-PC represents “surface- bound” ligand, while the acid-resistant radioactivity associated with the cells represents “internalized” ligand. In a perfused organ, the acid- wash should be performed after removing the extracellular polypeptide ligand that is not bound with surface receptors [119,128,132]. The internalized ligand is further analyzed for the unchanged (L,) and degraded (Ldeg) ligands. For simplicity, let us assume that no transcytosis of internalized ligand (Li) with its receptor (i.e., f=O) takes place. Thus, the mass-balance equations for the internalized ligand (L,) and its intracellular degradation products (Ldeg) can be written as:

g&l dt = kint[L,l - kdcg[Lil

Wdegl ~ = k&,1 - kelLegl dt

The summation of Eq. (3) and Eq. (4) and assuming the condition of kre,[Ldeg] <<k,,,[L,] (i.e., when the amount of intracellular degradation products is relatively low compared with surface-bound ligand) yields:

Wil + dL_J dt = kint[I. ] 5 (5)

The integration of Eq. (5) from time 0 to t yields:

[LB1 + [Ldegl= kint _/ Psldt 0

(6)

Thus, kint can be estimated from the initial slope of an “integration plot”, that is, a plot of internalized radioactivity ([L,] + [Ldeg]) versus the integrated amount of surface-bound ligand

WJ j- 1 L, Id t can be calculated numerically by the trap%zoidal rule. As far as the acid-wash is used to measure the surface-bound and internalized ligands, the same equations can be applied to a perfused organ system. This is feasible when the intracellular degradation products cannot be neglected, but the release of degradation products into the medium is not yet significantly increased [128,132]. Moreover, when intracellular degradation of the ligand is negligible during the

452 H. Saio et al. I Advanced Drug Delivery Reviews 1Y (1996) 44-T-467

experiment, Eq. (6) is approximated to [L,], so that:

This equation has been readily applied to a relatively stable polypeptide, EGF [ 114,119]. Moreover, if we assume that [L,] is almost constant, Eq. (7) can be solved as:

[L,I kl

= k,,, . t

This equation corresponds to the simple plot of the “In/Sur ratio” as a function of time. which was first derived by Wiley and Cunningham for EGF [llO,lll] and that has been applied to other polypeptides [122,133-1361.

Assuming in Eq. (4) that [L,,,] is small enough for the condition of k,,,[L,,,]<k,,,[L,] and subsequent integration from time 0 to t yields:

lLdegl = kdeg 1 CLildt 0

(9)

Thus, kdrg can be estimated from the initial slope of a plot of the intracellular degradation products versus the integrated amount of internalized ligand, as demonstrated for EGF [128,137].

These methods of estimating k,,, and kdeg are feasible only for a short time interval (e.g., 5-20 min), where a significant amount of degradation products may not be generated and released from the cell. Therefore, the calculated values may serve as “initial” estimates for the overall fitting of the data to Eq. (3) and Eq. (4). Moreover, these methods cannot be used to monitor the intracellular routing of a ligand for a relatively long interval. Alternatively, “pulse- chase” can be applied to investigate the intracellular itinerary of ligand more precisely [26,138- 1421. Briefly, labeled polypeptide is incubated with cells at 0°C for 60 min, and the extracellular polypeptide is removed by washing. Endocytosis of the surface-bound ligand is initiated by warm- ing the cells to 37°C. At various times after the start of incubation, the cells are harvested and the surface-bound (L,) and internalized (L,)

ligands are separated by an acid-wash at 4°C. Thus, the pulse-chase method allows the “single- wave” internalization of surface receptors to be observed, whereas the conventional steady-state method [ 1 LO,11 l] allows the continuous internalization (or multiple rounds) of receptors.

3.2. Estimation of RME parameters from receptor movement

Generally, internalized receptors behave differently from their ligand. A mathematical model for the surface and intracellular receptors is presented in Fig. 4. This model is a topology of the RME scheme shown in Fig. 1 with respect to the movement of the receptor. Our aim is to estimate the endocytotic rate constants (kinr and k,) and the externalization rate constant of the internalized receptor (k,,,). The experimental procedure for this analysis is to incubate isolated cells or perfused tissue with a large excess of unlabeled polypeptide to cause rapid internalization of the surface receptors, then to measure the ligand binding activity on the cell surface at 4°C. For simplicity, both the degradation rate constant of internalized receptor (k,,,,,) and the

unoccupied occupied

1 II\

k ext

+, } 1 $ kinl

intracellular space

Fig. 4. A generic model for internalization and externaliza-

tion of receptor movement via the RME mechanism. R, and

R, define the number of surface and internalized receptors,

respectively; v represents the fraction of receptor occupancy

on the cell surface; k,,, and k, represent the endocytotic rate

constants of occupied and unoccupied receptors on the cell

surface, respectively; v,~” denotes the de novo synthesis rate

of receptor; and k,,,,, is the degradation rate constant of

internalized receptor.


new receptor synthesis rate (v,,,) are assumed to be zero, so that the total number of mobile receptors ( [RT]) is constant (= [R,] + [Ri]), for a short time. The mass-balance equations for the surface receptor number ([R,]) can then be written as:

dR1 dt = kext[‘il - {kt(l - v) + kint . ~>[kl

(10)

where Y represents the fraction of receptor occupancy (= [L,]/[R,]). When a high concentration of ligand is exposed to cells, v falls to unity and Eq. (10) reduces to:

4R51 dt = kextlIR~l - kint[Rsl (11)

The integration of Eq. (11) from time 0 to t and rearrangement yields:

ln ( Rlll - Psleq = (k + k

RI - RL, ) tnt ext

)t

(12)

where subscripts 0 and eq represent the initial condition (t =0) and the equilibrium condition where [R,] reaches the minimum, respectively. According to Eq. (11) [R,]” and [R,],, are given by:

No = k, :;Iex, ’ rRTl (13)

[R~leq = k,,, + k,,, ’ LRTI

The rearrangement of Eq. (12) yields:

In [R,]:[R,;1J, - (Y = (kin, + k,,,t)

= k down-regulation *t

where (Y and kdown_regu,at,on are defined

RI,, k + Lt a=[R,lo= kint + kcxt

(14)

(15)

as:

(16)

(17)

The kdown-rcgulatmn value can be determined from the slope of the plot of ln{(l-(Y)/([R,]/ [R,],,-a)} versus the time curve when the sur-

face receptors are being “down-regulated”. Di- viding Eq. (14) by [RT] gives:

kxt [RJe, _ RT km, + Lt

(18)

Thus, the RME parameters can be estimated as follows:

down-regulation

=al/3.k down-regulatmn

k,,, = k down-rrgulatm - kext

k,=a.k down-regulation - L,

where p is defined as:

(19)

(20)

(21)

LRTI

p = ~W,, (22)

Standaert and Pollet [143] derived some equations equivalent to Eqs. (10-22) and determined the values of CX, p and kdown_regu,at,on as 0.55, 1.11 and 0.22 h ’ , respectively, according to the rapid “down-regulation” of surface receptors in vitro (Fig. 5). The values of k,,,, kint and k, were estimated as 0.11, 0.11 and 0.011 h- ’ , respectively, using Eqs. (19-21) for the movement of insulin receptors in myocytes (Fig. 5) [143].

The next objective is to examine whether the down-regulation of surface receptors is reversible. The extracellular ligand should be removed and the cells washed by ligand-free medium after the maximum down-regulation of the surface receptors. The surface receptor number may then increase with time depending on the rate of externalization of internalized ligand. However, it should be confirmed by using cyclohexamide that the increase in the receptors is attributed not to newly synthesized receptor but receptor reap- pearance. Now the extracellular ligand is removed and there should be no surface-bound ligand, so the receptor occupancy (v) can be set at zero and Eq. (10) reduces to:

(23)

44 H. Sate et al. I Advanced Drug Delivery Reviews 19 (1996) 44.7-467

I. I. I. 10 20 30 40

TIME (hours)

Fig. 5. Kinetics of insulin-induced down-regulation and suh-

sequent recovery of cell surface insulin receptors in the

myocytes. The cells were incubated at 37°C for the indicated

times in the presence or absence (control) of 20 nM insulin.

After washing extensively. surface “rf-labeled insulin binding

was determined. Following maximum down-regulation of

surface receptors (0.45 at 20 h), the cells were washed. and

insulin-free conditioned media was added. The inset shows

these data plotted as first-order kinetic processes as described

in the text. Reproduced from Ref [143] with permission.

The integration of Eq. (23) from time 0 to t and rearrangement yields:

,n (24)

where subscript ss represents the steady-state condition where [R,] is maximum. According to Eq. (23) [R,],, is given by:

k, [Rslss = k, ;;,,, ’ lRTl (25)

receptor recovery reflects not only k,,, but also k,. However, krecovery can be approximated as k,,,, since k, is usually smaller than k,,,. The value of krCCOVCrV can be determined from the slope of the plot’of ln{(r- l)l(y- [R,]/[R,],,)} versus time. This parameter, as well as k down-rcgulallon’ is very important to optimize the interval of drug-polypeptide administration, because if the receptors are once down-regulated, the next dosage should be given only after the surface receptors are restored. Standaert and Pollet [143], determined krecovery as 0.12 h-’ from the kinetics of surface receptor recovery (Fig. 5). This value is entirely consistent with the sum of k, and k,,, estimated from the kinetics of receptor down-regulation (Fig. 5) indicating the feasibility of the “receptor-recycling” model (Fig.

4). Rearrangement of Eq. (1.5) gives:

z = (1 - LY). exp{ - (k,,, + k,,,)t} + a (29) \ 0

Essentially the same approach was employed to estimate k,,, (0.22 min-‘) and k,,, (0.11 mini’) [128] to accommodate the ligand-induced “rapid down-regulation” of surface receptors in vivo (Fig. 6) [144]. Insulin was injected into rats at a high dose and the time course of ligand- binding activity on the cell surface was measured. In a perfused organ system. the receptor recovery can be chased with the single-pass ex- traction measurement [83] after the surface receptor is down-regulated. Analysis of the recovery phase of the curve in Fig. 6 with Eq. (24)

Rearrangement of Eq. (24) yields:

In Y-l

y - [R,] / [R,],, = (k’ + kCx’)r

= krec<,oerr . t

where y and k I

rL‘o\‘CT\I are defined as: 8 16 24 40 80 120 180

k.. 1 =k,+k,,, (27) Time of sacrifice after insulin injection (min) r“OL’LI”

VU,

’ = RI,, (28)

The apparent slope (krCCOVelV) of the surface

Fig. 6. Liver insulin binding activity at various times following

insulin injection in rats. The binding assay was performed

both on intact (filled circles) and Triton X-lOO-solubilized

(untilled circles) liver particles. Reproduced from Ref [I441

with permission.

H. Sato et al. I Advanced Drug Delivery Reviews 19 (1996j 445-467 45s

is not adequate, because the receptor recovery process is inevitably affected by high insulin concentrations in plasma (delayed recovery of surface receptors due to continuous internalization). For the same reason, the discrepancy between the k,,, of the insulin receptor (0.035 min-‘) estimated by Frank et al. [145] and that (0.11 mini’) we estimated as above might be attributed to the high plasma concentration (45 nM) of insulin in their study.

Moreover, a dynamic RME model of polypeptide in an intact, perfused organ (Fig. 7) can be developed by combining a recirculatory system with generic RME models (Fig. 3 and Fig. 4) under some of the assumptions described above (i.e., f=O; kdeg, R=O; vSyn=O). The nonlinear clearance and surface binding of insulin in the perfused livers were analyzed [128] based on this model. The mass-balance differential equations for Fig. 7 are as follows:

d[R,l dt = ‘ext[R,l- ‘in,&,1 - ‘,([R,l- &,I)

(30)

Reservoir (V,)

Fig. 7. The “receptor-recycling” model of hepatic insulin

clearance and distribution. Solid and broken arrows indicate

the movement of insulin and its receptor, respectively. The

liver is subdivided into three compartments. i.e., extracellular

fluid. cell surface and intracellular (vesicular) space, which

are assumed to be well-mixed. C, and C, denote the poly-

peptide concentrations in the perfusate and extracellular

fluid, respectively. The other nomenclature is presented in

the legends to Fig. 3 and Fig. 4. The total number of mobile

receptors R., (= R, + R,) and receptor affinity K, (=k,,,, lk,,,)

are assumed to be constant.

WI 2 = k,nt[L,l + k,([R,l - Psi) - kcxt[Ril dt

(31)

v W,l - = Q,W,l - [C,l> p dt (32)

v d[C,l - = Q,W,l - [Ccl> e dt

- k,“~cJRI - [U + k&s1 (33)

$$ = k,,[C,IRI - [Ll) - (k,,, + k,Ll

(34)

d[Lil dt = k,nt[Lsl - kdeg[Lil

4Lde,l ~ = kcieg[Lil - krel[Ldrgl dt

(35)

(36)

The nomenclature is presented in the legends to Fig. 3 and Fig. 4. These equations were simultaneously fitted to the observed time courses of

C,, L L, and Ldel! by a nonlinear least-squares regression analysis, to optimize R,, k,,, and k_. For this fitting procedure, the initial estimates of

k,“, and k&g were used as obtained by the “ratio plot” analysis (i.e., Eq. (6) and (9)). The initial estimation of R, is described in Ref [128]. The other parameters (VP, V,, Q,, k,,, kc,,.,, k, and k,,,) were experimentally measured, cited, or calculated from published references. The theo- retical curves for C,, L,, L, and Ldeg were in good agreement with the observed data (Fig. 8) [128]. The optimized values of R,, k,,, and kdcg were 8.66 pmol per g liver, 0.62 min -’ and 0.91 min -‘, respectively. Thus, the k,,, of insulin in different types of cell is in the order of hepatocytesbkidney (0.022 mini ‘) [132]> adipocytes (0.0027 min-‘) [146] >myocytes (0.0018 mini’) [143], in consistent with the ability of these tissues to degrade insulin. The subsequent computer-simulation (Fig. 9) clearly showed that the recovery half-life of the surface receptor is not constant, but varies with the initial dose added to the perfusate, reflecting the insulin disappearance from the circulation. The dynamic “receptor-recycling” model (Fig. 7) is

4% H. Sate et al. I Advanced Drug Delivery Reviews 19 (19%) 44-f-467

Time (min) Time (min)

Fig. 8. The disappearance of “‘I-labeled insulin by cyclically

perfused mouse livers at low (0.018 nM) and high (2 nM)

initial concentrations (in panel A). In panel B, the timc-

courses of surface-bound (unfilled circles) and internalized

(filled circles). as well as of intracellular degradation products

(tilled triangles), are presented. Solid lines show computer-

generated simulation curves using the model shown in Fig. 6.

Reproduced from Ref 1128) with permission.

useful when predicting the changes in surface receptor number and surface/intracellular ligand concentrations in the target tissue, which should be important for the pharmacodynamics (onset, offset and duration of effect) of a drug-polypeptide complex in general. An important advan-

0.01 I 0 40 80 120

Time (min)

tage of physiologically based models relative to the traditional compartmental types is the possi- bility of predicting the changes in drug levels and physiological factors in practically inaccessible or specific tissue sites, thus making it possible to predict data by changing the selected parameters involved in the model.

4. Kinetic analysis of RME in vivo

Among several means of evaluating the in vivo effect of tissue uptake via the RME [97-99,147], in vivo tissue uptake [84] is a rapid and simple procedure applicable to various polypeptides and tissues. The experimental protocol is as follows. Animals receive an intravenous (i.v.) injection of labeled polypeptide with several doses of unlabeled polypeptide, and arterial blood is col- lected at designated times (4-5 points over 20- 150 s). The amount of labeled ligand taken up by

40 80

Time (min)

120

Fig. 9. Computer simulation of the time-dependent changes in the perfusate concentration of insulin (A) and surface receptor

number (B) at varying initial concentrations of insulin in the reservoir. using the “receptor-recycling” model of hepatic insulin

clearance and distribution.

H. Sato et al. I Advanced Drug Delivery Reviews 19 (1996) 44.5-467 451

each tissue is determined at 3 min postdose. The apparent tissue uptake rate is described by:

dL1, - = ~Lptake~qJ, dt (37)

where [C,],, [L,], and CLuprakt: represent the plasma concentration, the amount of polypeptide associated with tissue at time t and the tissue uptake clearance, respectively. Integration of Eq. (37) from time 0 to t yields:

Ll, = CLptake . AUC”-, + MO (38)

where AUC,_, and [L,], represent the area under the plasma concentration (AUC)-time profile from time 0 to t and the amount of polypeptide in tissue at time 0, respectively.

Thus, CLuptakc can be estimated from the initial slope of an “integration plot” of [L,], versus

AUC,,_,. In each organ, the integration plot gives

0

-2

-4 t

Liver (22 min) \

%

\

+,

Jejunum (2.4 hr)

2 4 6 2 4 6

Ileum (2.2 hr) Spleen 14.5 hrl

0 2 4 6 0 2 4 6 0 2 4 6 0

Stomach (3.5 hr)

0 2 4 6

Or

a straight line passing through the y-axis inter- cept, [L,],. Therefore, dividing the [L,],, value by the initial plasma concentration of the polypeptide ([C,],) yields the initial distribution volume of the tissue ([K,],,), which represents the volume to which polypeptide can be distributed in the tissue of interest within a short time. The tissue uptake of EGF [84] and hepatocyte growth factor (HGF) [148] have been analyzed by this procedure. From the CLuptak, values in various tissues, it was found that the hepatic and renal uptake clearances were approximately 50% and 15% of the early-phase clearance of EGF, respectively [88]. A useful application of this tech- nique is to analyze the down-regulation and subsequent recovery of surface receptors (Fig. 10) [Ml. To do so, an i.v. injection of a high dose of polypeptide is followed either by a binding assay of the tissue surface receptors or by a second injection of tracer polypeptide to measure

I Kidney (4.6 hrl I Duodenum (2.0 hr)

The (h)

Fig. 10. Sigma-minus plot for the determination of recovery half-lives of tissue uptake clearance of [‘ZSI]-labeled epidermal growth

factor (EGF). In normal rat, tissue uptake clearance [CL,,,,,_(O)] was determined after intravenous injection of [“‘I]-labeled

EGF. At time t after the administration of excess (300 pglkg) unlabeled EGF, tissue uptake clearance [CL,,,,,,(t)] of

[“SI]-labeled EGF was determined. Adopted from Ref [88] with permission.

Drliverv Rrview.c 1Y (lYY6) 44.5-467

CLupltik, during the recovery of surface receptors. The “sigma-minus” plot of ln[CL,,,,,,(O) - CL uptake(t)] (difference of CLuprakc between time 0 and t) versus time will give a straight descend- ing line, and the ratio of 0.693/t,,, represents

krccWcry which is the sum of k,,, and k,, as mentioned above. It is assumed that the plasma concentration of exogenously administered polypeptide becomes low enough to neglect the continuous endocytosis of surface receptors at time t. As shown in Fig. 10, the rate constant for the surface receptor recovery in the liver is much higher than those in other tissues, suggesting that the kinetic properties of EGF receptors differ among tissues,

If we further consider the capillary permeability (PS) and intrinsic uptake clearance (CL,,,) of a ligand under a pseudo steady-state condition, CL,,,,,,, can be also expressed as follows [149]:

PS . CL,“, CLupNX = ps + CL

I I, I

This equation assumes that the blood flow is not rate-limiting the ligand uptake into the tissue. If PS is relatively large compared with CL,,,,. CL uptnkc is approximately equal to CL,,,,, and

CL,,,, reflects the endocytotic uptake of the ligand as:

CLJC,l = k,,,,F-,I (40)

Since [L,] is the concentration of occupied surface receptors. Y [R,] (v=[C,], Eq. (40) can be formulated as:

CLI”JC,J = k”, . [WqJ

K d’ app + [C P ]

Substituting K,,app using Eq. (2:

~(&app + [C,l b

(41)

1 (a simpler form of K,,app) and solving for CL,,, yields:

k ,n, kJR,l cLm’ = k,, + k ,,,, + k,,,[C,,l (42)

If [C,] is low (i.e.. at a tracer concentration) enough for the condition k,,,, + k,,, >> k ,,,, [C,] to hold, then Eq. (42) simplifies to:

k . km l&l cL1nl = i:,,, + k,,,, (43)

Eq. (43) was already utilized in a previous paper [87] without an exact formulation. On the other hand, if [C,] is high enough for the condition k,,f, +k ,,,, <<k,,,[C,,] to hold, then Eq. (42) reduces to:

cL _ k,Rl I”[ --Kg- (44)

This equation indicates that the uptake rate, CL,,,,[C,], is almost constant at high ligand concentrations. According to Eq. (18), [R,] at high ligand concentrations under a steady-state condition ([R,],,) is expressed as:

L, L&l IR& = k,,, + k,,,

Therefore, Eq. (44) can be formulated as:

cL~~~~[c,~l = km, . LPGI

k,,, + k,,,

(45)

(46)

Since k,,, is usually smaller than k,,,, the above equation can be approximated to:

CL,,[C,l = k,,[%l (47)

This equation suggests that ligand uptake via RME is limited for the most part by receptor externalization when the ligand concentration is very high and the surface receptor is significantly down-regulated. Therefore, receptor recycling (or reutilization) may be important for the polypeptide clearance at its higher concentrations. but not at lower concentrations, as suggested in a previous paper which compared “receptor-recycling” and “non-receptor recycling” models for insulin clearance in the perfused liver [128]. Eqs. (42-44) and Eqs. (46,47) are useful when predicting explicitly the in vivo clearance of polypeptides in an organ using the in vitro parameters of RME, as follows:

Q . fs . CL,,,, cLr,an = Q + fB . CL,,, (48)

where Q is the blood flow rate and fB is the fraction of a polypeptide unbound in blood.

H. Sate et al. I Advanced Drug Delivery Reviews 19 (1996) 44.5-467 459

5. Implications of RME kinetics for clinical application of polypeptides

From the RME kinetics of insulin in the perfused liver [128], the mean time of receptor recycling (T,,,) was calculated to be 11 min (= I lki”t + l lk,,,), suggesting that the recycling of insulin receptors will harmonize with plasma insulin concentrations, if insulin is administered in pulses at about ll-min intervals. In fact, reports [150-1.541 have shown that the pulsatile administration of insulin with 11-13 min intervals reduces hypoglycemia (or hyperinsulinemia) compared with continuous administration. More- over, the pulsatile administration of insulin may reduce insulin resistance by reversing the down- regulation of insulin receptors. The general no- tion that harmonization of drug administration with receptor recycling could provide more efficient and safer therapy, will be useful for the clinical application of other polypeptides and proteins.

In practice, the clinical application of polypeptides will be targeted to patients for a particular disease. Therefore, the PKlPD analysis of polypeptides in diseased model animals is important [87,155]. For example, a significant decrease and subsequent recovery of CLuptak, in the liver was observed after Ccl, treatment, whereas no change was seen in kidney, spleen, or lung [155].

6. Perspectives

In fact, there has not been much progress from the “natural” targeting of glycoproteins and LDL to hepatocytes or macrophage lineages, for the most part due to the effects of unfavorable factors on the PK/PD of polypeptides (see Table 2). Drug delivery using neoglycoproteins [156], such as mannosylated and lactosylated albumin, and nonprotein macromolecules such as galactosylated polylysine, has also been limited to the same types of cells (Table 1). Nevertheless, the ability to manipulate even these two cell types would have a major impact in the therapy of a large number of disease states related to these cells (such as hepatocellular carcinoma). To fur-

ther accomplish extrahepatic drug targeting via RME, we need to identify or engineer other types of polypeptide carriers that are little taken up by the liver and macrophages, but efficiently distributed into selected target sites such as tumors. Polypeptides such as cytokines and growth factors have been identified and cloned, and their structural and functional characteristics have been classified and stored in databases [157,158]. Some of the available polypeptides, including soluble receptors for cytokines [91-961, might be good carriers for receptor-mediated drug delivery.

Although there are some physiological barriers for polypeptides as listed in Table 2, it may be possible to overcome these factors by miniaturiz- ing polypeptides to increase their tissue/capillary diffusibility, and by modification of polypeptides to protect against non-receptor-mediated sequestration mechanisms. For instance, the systemic administration of HGF as a heparin-bound form was proposed (Fig. 11) [13]. The heparin-HGF complex was two-three times more stable than HGF itself, with the hepatocyte proliferative activity retained. The same strategy could be applied to other heparin binding polypeptides, such as tumor necrosis factor (TNF) [59] and fibroblast growth factor (FGF) [60,61]. In general, the polypeptides classified as “paracrines” and “autocrines” are not appropriate for systemic administration unless they are stabilized some- how.

Intracellular trafficking of receptors is directly influenced by the pH sensitivity of the receptor/ ligand dissociation [159,160]. The gene-engineered LDL receptor without a property of ligand release at acid pH was no longer recycled back, but was degraded intracellularly [160]. Moreover, molecular mechanisms of lysosomal sorting have been clarified [161-1631. Therefore, neo polypeptides that can regulate their own destination in the cell may be engineered in future. Perhaps the ideal complex for receptor- mediated drug delivery is a multi-functional protein, which consists of a homing device (“navigator”), receptor targeting device (“recog- nize?), intracellular targeting device (“door- opener”) and a drug.

Finally, the therapeutic efficacy/toxicity of a

460 H. Sato et al. I Advanced Drug Delivery Reviews 19 (1996) 44-f-467

l HGF

0 ep Heperin

$J HCF recepror

y tleperin-Ike substance

l P L$Aa eP

eP l P ,BQv v

/

00 a. I

out I

I VV

In HGF receptor Heparin-like substance

receptor-mediated endocytosle

\/ 1

StitWfation of DNA Synthesis non-specific uptake

Fig. 11. Schematic diagram of a drug delivery system of HGF based on the mechanism of its hepatic handling. HGF molecules

carried in the blood flow can bind to both HGF receptor and a heparin-like substance [ 1871, followed by internalization via each

binding site. On the other hand, the binding of the heparin-HGF complex to a heparin-like substance is reduced by pre-masking

of the heparin-binding domain of HGF with heparin. Adopted from Ref [13] with permission.

drug-carrier conjugate should be assessed from its microscopic pharmacology based on the RME mechanisms, in conjunction with macroscopic pharmacokinetic modelling.

Acknowledgments

This work was supported by a grant from the Research Foundation for Pharmaceutical Sci- ences and Uehara Memorial Foundation, awarded to H.S.

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Importance of receptor-mediated endocytosis in peptide delivery and targeting: kinetic aspects