Receptor-mediated delivery of drugs to hepatocytes

Advanced Drug Deliver)' Reviews, 4(1989)49-63

49

Elsevier ADR 00035

Receptor-mediated delivery of drugs to hepatocytes R o b e r t J. F a l l o n a and A l a n L. S c h w a r t z a'b The Edward Mallinckrodt Departments of a Pediatrics and bPharmacology, Washington University School of Medicine, Division of Hematology-Oncology, St. Louis Children's Hospital, St. Louis, MO, U.S.A. (Received December 1, 1987) (Accepted June 12, 1988)

Key words: Antineoplastic agent; Asialoglycoprotein receptor; Endosome; Neoligand

Contents Summary .................................................................................................................

50

I. Introduction ...................................................................................................

50

II. Receptor-mediated endocytosis in liver cells: pathways and receptors ........................

51

III. The asialoglycoprotein receptor: a model system for targeting drugs to liver ...............

56

IV. Limitations and future challenges to the use of ligand--drug conjugates ......................

57

V. Conclusions ....................................................................................................

60

Acknowledgements ....................................................................................................

60

References ...............................................................................................................

60

Abbreviations: ASGP, asialoglycoprotein; ASOR, asialoorosomucoid; CURL, compartment of uncoupling receptor and ligand; CT, CURL tubule; LDL, low-density lipoprotein; EGF, epidermal growth factor; HDL, high-density lipoprotein. Correspondence: R.J. Fallon, The Edwardt Mallinckrodt Department of Pediatrics, Washington University School of Medicine, Division of Hematology-Oncology, St. Louis Children's Hospital, 400 South Kingshighway Boulevard, St. Louis, MO 63110, U.S.A. 0169-409X/89/$03.50 © 1989 Elsevier Science Publishers B.V. (Biomedical Division)

50

R.J. F A L L O N A N D A.L. S C H W A R T Z

Summary Standard pharmacologic approaches to liver diseases have been frustrated by ir~adequate delivery of active agents into liver cells as well as nonspecific toxicity ~owards other organs. Drug targeting to hepatocytes via the binding of a drug-ligand or macromolecule-ligand complex to a cell surface receptor with subsequent endocytosis and release within the cell may improve the drug's therapeutic ratio. Several receptor molecules specific for liver parenchymal cells exist and could serve such a drug-delivery function. One such receptor, the asialoglycoprotein (ASGP)receptor has been studied in hepatocytes as well as in a well-differentiated hepatoma cell line, HepG2. This cell line resembles mature hepatocytes in many metabolic, synthetic, and structural features. In this model system, ligand bound to receptor is internalized via coated pits and enters an acidic compartment where ligand-receptor dissociation occurs followed by receptor recycling to plasma membrane and eventual transfer of ligand to lysosome for degradation. Other intracellular routes not resulting in degradation of ligand have also been elucidated in hepatocytes and the HepG2 cell line. This has been demonstrated for the ASGP receptor, the transferrin receptor and the polymeric Ig-IgA receptor. It is apparent that macromolecular pharmacologic agents complexed to ligands for any of these receptors (such as monoclonal antibodies, enzymes, or nucleic acids) must dissociate from the ligand and permeate the endosomal membrane to reach their intracellular sites of action. Recent studies of viral pathogenesis and intracellular protein targeting have revealed key features of protein domain structure that are necessary for membrane permeation. Further understanding of the mechanisms responsible for these pathways of intracellular sorting will enable drug-carrier design that will fully exploit the potential of hepatocyte receptor-mediated endocytosis for drug delivery. I.

Introduction

The liver occupies a central role in the synthesis and secretion of proteins as well as the metabolism of drugs and macromolecules. Many pathologic states unique to this organ such as chronic hepatitis, inborn metabolic errors, cirrhosis, toxicologic states and metal storage diseases are not satisfactorily managed by current pharmacologic approaches. This results from several factors: incomplete understanding of the pathologic mechanisms responsible for the disorder, lack of active drugs, antitoxins, proteins or other molecules that could potentially reverse the pathologic condition, or an inability to achieve sufficient intracellular quantities of these drugs to reverse the disease process. This review will deal with the last of these obstacles to effective drug therapy of the hepatocyte and will address the utility of receptor-mediated processes for specific access of drugs to this cell. The discussion will emphasize the diseases of liver possibly amenable to such an approach. Optimization of receptor-mediated delivery of drugs to the hepatocyte requires that the candidate receptor protein meet the following criteria: (1) abundant

Rt~(F(t,I ()I(-MtDIAFED DELIVERY OF DRUGS TO HEPATOCYTES

51

exprcs~io~ tm hepatocyte sinusoidal (i.e., blood front) surface; (2) selective cxpr~'ssicm ~f receptor protein on the hepatocyte; (3) lack of adverse metabolic ct)nsequences (e.g., mitogenesis) of prolonged ligand-receptor binding; (4) precise undcrst~mding of trafficking pathways of ligand and receptor following internalization including intracompartmental conditions encountered; and (5) lack of interference with normal functioning of receptor protein .during therapy (minimal down-regulation). One receptor potentially suitable for specific drug targeting due to its exclusive locz~tion on the hepatocyte surface is the asialoglycoprotein (ASGP) rc~cpt~r. Background information on the cell biology of this receptor protein and its recycling pathways during endocytosis has been obtained in intact liver, isolated heputocyte and a continuous hepatoma cell line. This receptor has been utilized for delivery into cells of antitoxins, antineoplastic drugs and antiviral compounds. These data will be reviewed below and compared to other receptors putatively specific to liver cells. A drug conjugated to a ligand (neoligand) must obviously be constructed to preserve functional integrity of the active species following release from the ligand, whether the ligand is a small molecule or a macromolecule such as protein. The biochemical synthesis of these conjugates is beyond the scope of this review. The discussion below will focus on the intracellular compartments and environments encountered by a ligand-drug conjugate internalized by a receptor-mediated process. The hydrolytic enzymes and acidic conditions in endosomes and lysosomes of liver cells clearly present obstacles to current employment of many hypothetical ligund-enzyme or ligand-macromolecule conjugates. It is also true, however, that a more complete understanding of these endosomal conditions will lead to advances in conjugate design permitting their exploitation for the selective intracellular generation of active pharmacologic agents. II. Receptor-mediated endocytosis in liver cells: pathways and receptors

Receptor-mediated endocytosis is a well-described system for cellular uptake of many substances critical for cell metabolism and growth [1,2]. Specialized receptor protcins have beera identified for nutrients (LDL-cholesterol, transferrin-iron), growth factors (epidermal growth factor, insulin), viruses (influenza), toxins (diphtheria) and glycoproteins. These receptor molecules are heterogeneous in structure but contain the following common features: a hydrophilic extracellular (ligand-binding) domain which contains glycosylation sites, a hydrophobic membrane-spanning region and an intracytoplasmic tail. This intracellular domain may possess intrinsic protein kinase activity (in the case of the epidermal growth factor and insulin receptors) and varies widely in molecular mass from 1(I3 to 105 daltons. Receptors lacking intrinsic kinase activity (for example, beta-adrenergic receptor) may also become associated with an active kinase following binding of ligand [3]. This ability of selected receptors to initiate a cascade of protein phosphorylation as well as other second messenger signals upon ligand association suggests that caution must be exercised in the design of ligand-drug chimeras. Maximal binding to rcceptors and mimicry of mitogenic signals may lead to significant cellular toxicity.

52

R.J. FALLON AND A.L. SCHWARTZ

cv

\,k, "~)-~

CURL

Internalization

L-R Uncoupling R Recycling

LYSOSOME @

L Degradation

Fig. 1. Pathways of receptor-mediated endocytosis. The routing pathways for receptor (R) and ligand (L) are shown in a schematized hepatocyte. See text for discussion. CV, coated vesicle; CURL, compartment of uncoupling receptor and ligand.

The pathways common to many receptors and ligands found in hepatocytes are presented in Fig. 1. After binding of ligand to receptor at the cell surface and clustering in coated pits, internalization of the receptor-ligand complex occurs via a clathrin-coated vesicular intermediate [4]. For many receptors (e.g., LDL, ASGP, transferrin receptors) this internalization process appears to occur constitutively (without iigand binding) while for others (e.g., insulin, EGF receptors) formation of a ligand-receptor complex is required. The ligand-receptor complex then becomes located in an acidic compartment through a maturation or fusion mechanism. This pre-lysosomal sorting compartment has been referred to as CURL (compartment of uncoupling of receptor and ligand) endosome, or receptosome [5-7]. Electron microscopy has shown this to be a tubulovesicular structure located in the cell periphery. Double-label immunogold electron microscopy has further demonstrated the vesicular part of this structure to contain both receptor and ligand, while the tubular arms bear receptors alone [5]. Fig. 2 shows an example of such a study. ASGP-receptor has been detected in ultrathin cryosections of fixed liver cells by means of anti-receptor antibody incubation followed by localization of antibody with staph protein A linked to colloidal gold particles. This figure shows ASGP-receptor predominantly within CURL tubules (CT) at the periphery of the cell. From this location receptor recycles to plasma membrane in vesicles. The endosomal structure (end) is the site of ligand-receptor dissociation and can be demonstrated by serial sections to be continuous with tubules. Numerous functions relevant to endocytosis and drug delivery occur in the endosomal compartment. These include acidification, ligand-receptor dissociation, and sorting of receptor-ligand to lysosome, plasma membrane, or Golgi. The acidic nature of the endosome was demonstrated by Tycko and Maxfield [8], and appears to be responsible for the dissociation of most ligands and receptors. They employed pH-sensitive fluorescent ind~ators coupled to ligand molecules to show that an intravesicular p H of 5 to 5.6 is rapidly achieved following internalization. A proton-translocating ATPase responsible for this acidification is associated with

Rf:CI!PFOR-MEDIATED DELIVERY OF DRUGS TO HEPATOCYTES

53

Fig. 2. Immunogold labeling of asialoglycoprotein (ASGP) receptor in hepatocyte. Cryosection of hepatocyte cell periphery near sinusoidal surface (top). Gold particles indicate subcellular location of .kSGP-R within CURL tubules (CT) and on membrane. Note absence of particles within endosome (end) or smooth ER (ser) x 65 000 (by courtesy of H. Geuze).

vesicle m e m b r a n e s [9,10]. F o l l o w i n g l i g a n d - r e c e p t o r dissociation, recycling o f rec e p t o r s to t h e p l a s m a m e m b r a n e can o c c u r , e n a b l i n g t h e cell to e n d o c y t o s e e x t r a cellular l i g a n d s at l i n e a r r a t e s for h o u r s , e v e n in t h e a b s e n c e o f p r o t e i n synthesis. T h e t i m e r e q u i r e d for this p a t h w a y f r o m p l a s m a m e m b r a n e to e n d o s o m e a n d re-

54

R.J. F A L L O N A N D A.L. S C H W A R T Z

turn to plasma membrane is between 10 and 15 min for each round-trip cycle. Thus for receptors such as the ASGP, transferrin, and LDL receptor, several hundred rounds of endocytosis are possible during a single receptor lifetime [11]. Some receptors of hepatocytes are recycled in a less efficient manner. Epidermal growth factor and insulin receptors are to a large extent retained within the cell following ligand-receptor complex internalization [12,13]. This phenomenon of 'down-regulation' or desensitization has been observed for many receptor systems [14,15]. It also occurs following anti-receptor antibody binding to its cell surface receptor [16,17]. The chemical mechanisms responsible for this process are incompletely understood. This effect could potentially limit the utility of a given receptor for a potential drug-delivery application. Subsequent to ligand-receptor dissociation the ligand is transported to lysosome for degradation (Fig. 1). Proteolytic degradation in this organelle as well as ligandreceptor uncoupling in endosomes requires an acidic environment. Weak bases such as chloroquine or ammonium chloride or ionophores capable of collapsing H ÷ gradients (e.g., monensin) will disrupt these functions [18-20]. These studies imply that drug-ligand complexes released from receptors into the endosomal compartment will eventually be located within lysosomes. It follows that pharmacologic agents to be used in receptor-mediated drug delivery must be stable at pH 5, resistent to proteolytic degradation, or capable of crossing the membrane of the endosomal compartment prior to transport to lysosomes. A prototypic example of the latter would be influenza virus which enters the cytoplasm following a low pHdependent fusion of its coat proteins with the endosomal membrane [21]. It has also been recognized that several receptors follow different pathways in the same cell type. The ASGP receptor and the polymeric Ig-IgA receptor are examples of this phenomenon. As studied in rat liver, these receptors colocalize in liver cells to the same CURL vesicles but differ in their subsequent fate in several important respects [22]. First, the ligand-receptor interactions of the IgA-receptor is stable to the pH of 5.0-5.5 found in endosomal compartments. In addition, the ligand-IgA receptor complex has a final destination of bile canalicular membrane, where the ligand and a bound portion of the receptor molecule (secretory component) are endoproteolytically released [23]. In the future, this system could potentially be used to target drugs to the biliary system. As will be further discussed below, the ASGP-receptor, in contrast, dissociates from ligand in the acidic milieu of CURL, the receptor is recycled to the plasma membrane while the ligand is transported to lysosome. Table I lists receptor molecules found in greater numbers or exclusively on hepatocytes. The biochemical characterization of these receptors as well as their intracellular routing are given where available from the literature. The chylomicron remnant receptor is found on hepatocytes and the HepG2 hepatoma cell line. It binds apolipoprotein E and serves physiologically to remove chylomicrons traversing liver from the portal vein after absorption in the gut. The hemopexin receptor resides on hepatocytes, several transformed cell lines, and recently has been purified from the human placenta. This preparation yielded a single molecular species of molecular weight of 91000. Its apparent physiological role is to recycle

RECEFFC,RL~t

)

\ 1 [ 1) I)[.ii IV[ RY O F D R U G S T O H E P A T O C Y T E S

55

TABLE 1 SPECIFICt~._:{ i i> ~ R ' , t)F t ~ E P A I ( ) ( Y I [ ~ n.a., not a'~aikt ,] Fate of ligand

Refs.

lysosome

24, 25

Receptor

F,~ochcmiczd data

Chylomicr~,n r~'r~: :,

binds apo [7.: 3,1,

Hemopexi~

3', ~ 01 l)l~0 (! um~m placenta)

Haptoglobia-h~: ~ T , t i n

n .L

c y t o p l a s d (heme); ex- 26--28 tracellular m e d i u m (hemopexin) lysosome 29, 30

Albumin ~

rl .~

cytoplasm

31

Asialoglyc(,i)io~.~ T .ct,l,~r

gtycoprotein: 3 L = 460)0; no kinase activit3

lysosome

32, 33

n.a.

q ' h e existenc,." , t tl)'(IH',lll ~cccp~ors is controversial.

free heine t,, ~h: hcp~tocytc. A similar function is ascribed to the h a p t o g l o b i n h e m o g l o l , i n ~,',c.,,t,,r which is liver-specific and whose binding has been characterized in vcholc p.:z-tuscd liver and hepatocytes. No biochemical information concerning the ~c~cptor molecule nor its endocytosis is available. The presence of an a l b u m b , re.z, f,lo, ,,n hcpatocytes which facilitates the unloading from albumin of small moie,-'t:lv~s ,r drugs has been hypothesized though its existence remains controversial. T h e aaiat~ ,,,, v', ( ,!,r(,,'i,~ rA S G P) receptor is the most completely characterized hepatocyte-s~x.citic rcccptor. Its physiologic function as originally outlined by Ashwell ar, d c,)llaborators is the removal from the sinusoidal circulation of galactose-terrnim~t I~sialo-) glycoproteins. Consequently, abundant physiologic ligands (asiai~.c
56

R.J. FALLON AND A.L. S C H W A R T Z

surface to proteolytic digestion. There is considerable evidence that the ASGP receptor molecule, in contrast, is recycled to plasma membrane where it participates in additional cycles of endocytosis. For example, over a 6 hour period of ligand uptake, Steer and Ashwell [38] showed that 34 times more ligand was internalized and degraded than could be accounted for by surface binding. Furthermore, from pulse-chase labeling studies with radioisotopes, it is known that accelerated endocytosis in the presence of excess ligand does not shorten the receptor lifetime (@2 = 22 h in HepG2 cell line). The regulation of the pathway of receptor recycling to plasma membrane is not well understood at present. Receptors recycling back to membrane can be detected in a coated vesicle population that also contains newly synthesized secretory proteins, suggesting a common site of vesicle formation for these processes [39]. This may involve the trans Golgi reticulum, C U R L tubules, or another structure in continuity with both. In addition, data from this laboratory suggest there is a protein kinase-C-sensitive step subsequent to ligand dissociation which is necessary for ASGP-receptor recycling to plasma membrane [36]. Dissection of the regulation of this pathway clearly has relevance to alteration of receptor number on the cell surface and the eventual utility of a receptor system for drug delivery. Evidence has been presented in recent years demonstrating the existence of bidirectional ligand movement. A significant fraction (20-30%) of ligand internalized by the A S G P receptor in hepatocytes or the mannose receptor in macrophages is released into the extracellular medium in undegraded form [40--42]. This phenomenon, termed ligand recycling or diacytosis, occurs via a preacidic sorting compartment. Free ligand or ligand-receptor complexes may be recycled, and the latter can further be observed to reenter the cell through the coated pit/coated vesicle pathway and be transported to lysosome. The physiological significance of this pathway is unclear; it may represent an overflow pathway, an abortive C U R L structure which then becomes recycled to the cell surface, or a physiologic mechanism for membrane flow in a polarized epithelium. In either case, the regulation of the proportion of ligand-receptor complexes entering this pathway may be critical to drug delivery applications. Though this ligand recycling compartment is known not to be acidic, little is known about other internal constituents. Hence its ability to serve as an internal site for drug-ligand association may vary with each drug-ligand species constructed. In conclusion, the cell biology and recycling characteristics of one receptor specific for hepatocytes, the ASGP receptor, is known in some detail. Information on other hepatic receptors is emerging though as yet few biochemical studies are available. Reported studies using this group of receptors for neoligand delivery to hepatocytes will be discussed below. III.

The asialoglycoprotein receptor: a model system for targeting drugs to liver

Receptor sites on cell lines and intact liver have been used as targets to facilitate endocytosis and selective delivery to cytoplasm of pharmacologic agents. The ASGP-receptor serves as a convenient delivery system, since both abundant nat-

RECEP'I'OR-M|~!,I'~ F [, [~[ [ I\ ! I¢'~ ()t: I)R[i(;S TO HEPATOCYTES

57

ural ligands ~t~ h a.~ ~i:~l~qc~ lii1 z~ ~cll a~ .~ynthetic galactose conjugates are readily availab e [i"~ I '1 h~ ~.cti~ u ~ill pt-cscnt examples from the literature of this approach. Two a:2~lt, t,,~i< t~ hc&~t~c3tcs or hepatic malignant cells, methotrexate and acetarninc>~d~er~, h ~ c b,:cra rendered nontoxic by the subsequent addition of specific antido~, +c~,t~plcd tt~ .~izd~ffctuin. a ligand which binds to the ASGP-receptor. Wu arid ~+',:,ii~b,,t ~t~,r-, [u+41 h:tve reported that these ligand-antidote conjugates (folinic acid ,~r '~ +<~.t~ lc~ ~tciuc. respectively) are able to 'rescue' ASGP-receptorbearing ceils (i..~l ~,~I vcccpl,~r-ncgative ceils). For example, after 7 days of culture, methotve:,:~:c-t~.zttcd c'~.ll cultures had 5%, of the cell number of control (untreated) ct~ltur~.~. ] I,~ u~ct. ~ucthotrcxz~tc and asialofetuin-folinic acid addition together to rec~.'t?l,~r-t~>~iti~c c~.ll~ (t tcpG2) led to preservation of 90% of the control cell number in tho,c c~ltutc~. :\ receptor-negative cell line (PLC/PRF/5) was not protected by tills c~m/u~:~tc 44.45]. A similar ~tpl)r,3:~ch h:t~ bcc~) utilized to deliver specific antimicrobial (here antiviral) thera]~.~ ~, h¢?zttt)c3'tc~ infected with Ectromelia virus. The antiviral agents arabinoside A :~nd tri/]u~t~)tta~midinc were complexed to asialofetuin [46,47] and selectively deh~ cred t~) hc?:~t~c p~trenchymal cells where they inhibited viral D N A replication. In order to tz~g.e~ w~,z~k b~t~c.s t~) hcpc~tocytes to neutralize potentially acidic sorting compartme~tr~. ~c~ ct:d i~wc~tig:~t~rs have developed conjugates of primaquine with asial ~ receptor-positive cell line [51]. The constructed neoligand consisled ~>~ ~i~dt~w~omucoid cowflently linked to poly-L-lysine to which was electrosta~calt3 cc~t~?lcd z~ ph~.~mid containing the chloramphenicol acetyl transferase gent,. Ttaesc c×pcrimcnt~ resulted in gene transformation only in receptor-posati~e ccil~. Ihim ~ccurted t~t low ligand concentrations near the affinity constant t"or t:hc AS(iP tccc?tor and could be blocked by 10 nM free ASOR. In addition to dcmon~trztti~g ~cnctic transformation in these cells, the findings of Wu and Wu indicate, their mactomoleculcs may be internalized, escape immediate lysosomal degradztti~m. :tnd permeate cellular internal membranes in a functional form. The further ina?lic:~ti<)n of these results for therapy of hepatocyte disorders is discussed itl the ~_:xt ~cctit>n. IV. Limitations and future challenges to the use of ligand-drug conjugates InformatioE1 presented :d~oxc indicates the feasibility of selective drug entry into cells bearing oil t:hcir .~utf~ce a receptor of interest. In this section, the findings on the cell biolo~D of qv.~tcuas of receptor-mediated endocytosis discussed above will be presented as they ~cl~ttc to potential difficulties in the pre-clinical or clinical use of these neoligai~ds, as well a~ potential areas for exploitation in future drug-ligand design. While most stadics of hep~tocyte targeting have been performed with isolated cells, several importzmt studies have been performed in whole isolated organs (i.e., perfused liver) :~s ~cll as intact animals [52-54]. O f note, Van Berkel et al. (1985) found that galacN',sc-modified lipoproteins were differentially targeted to different

58

R.J. FALL()N A N D A . L S([{x.~:\I ~.I/

T A B L E II H E P A T I C D I S E A S E S P O T E N T I A L L Y A P P R O A C H A B I . F . B'~ P, E C E I ' T O R - M E D I A IF!~ DRUG D E L I V E R Y

Disease Toxic drug toxin ethanol Storage metal glycogen Infectious

Neoplastic Metabolic

Example

Agent delivered

acetaminophen, h,alothane, isoniazid Amanita

phylloides,

CCI~

antidote: antitoxin: fr
Fe, Cu (Wilson's diseasc)

chelator:

Pompe's disease Hepatitis B, CMV, Delta agent chronic active hepatitis, malaria, leishmaniasis hepatoceilular carcinoma hepatoblastoma enzyme and receptor delicicncies

cllzyrllc

antimicrobial

c,;totoxic drug enz} filc~ gone

(e.g., Lesch-Nyhan, porphyria.

Defective plasma protein synthesis

familial hypercholesterole mia, galactosemia) coagulation factors alpha-l-antitrypsin deficiency

gcnc

liver cells (hepatocytes versus Kupffer cells) dependent upon the t y p e and ,izc ,~t the lipoprotein particle (i.e., HDL vs. L D L ) . It is important to emphasize that t!~' targeted drug must survive the intracellular environment of the endocytotK pati> way in order to be useful therapeutically. Furthermore. there is potential f,~r designing novel conjugates which may release their products at different points witl:in the endocytotic pathway. Table II indicates the clinical states of liver, both acquired and congenital, which are potential targets of this type of therapy. The chronic viral infections, re~.ulting from infection with hepatitis B virus, cytomegalovirus, Epstein-Barr virus, ~,r the Delta agent are candidates for this approach because they are limited prima~-ily ~o liver, are all severe illnesses often carrying a poor prognosis, and are condition> for which current antiviral agents are either ineffective or excessively toxic. As demonstrated above in studies on hepatocytes, antiviral medications can be selectively introduced. Other infectious diseases of liver, e.g., leishmania or malaria. may have organisms living intracellularly protected in vact, oles. By recept<,r-mcdiated entry of drug-ligand complexes into liver cells, and consequently into endosomes and vacuoles, the drug should be brought directly into contact wi~h the parasite. Such an approach has been employed to access enzymes to the parasitophorous vacuole of infected macrophages [55]. The treatment of malignancy by immunotoxins (neoligands where monoclonal antibodies are substituted ior ligand) directly against tumor-specific antigens or receptors has been addressed in several recent reviews [56,57] and will be discussed ihere only as it relates !o he-

RECEPTOR-MEDIATED DELIVERY OF DRUG,'-."I',3 HI PA'}~C'CIYI"E-S

5~)

patio malignancy. In this case the F,roblem~ encountered even ;tt this c~rly stage are several and fundamental. First, the most ,lbundant liver-specilic r e c e p t o r that might be used as a target molecule, ASGP-reccpt~,r, i~ almost alw~y~ absent from the cell surface of hepatocellular carcinoma and hep~ttot)last~ma. Secondly, a problem which is not exclusively one of live:r, is that ~antigcnic modt~l;~tion o c c u r s leading to decreased number of immunotoxin binding sites at the cell surface and consequently lower effectiveness with time. Third, the t:oxir~,molecule moiety often appears to be less effective in this setting than when occarring natuv~ally as the intact molecule [58]. Nevertheless, an asialofetuin-diphtheria toxin coTaiugate has been shown to kill a receptor-bearing cell population [59]. It c:~n be c~)ncluded, however, that at the present time a promising combinatiol~ ,:)f sensitive tumor-specific receptor system, and cytotoxic drug does not ~tt)p~'ar to exist. The tc,~
60

R.J. FALLON AND A.L. SCHWARTZ

cell (e.g., I g A receptor vs. ASGP-receptor) of newly internalized molecules; (4) regulation of receptor m o v e m e n t within the cell by processes such as down-regulation or antigenic modulation. This will permit a maximal continued efficiency in delivery of neoligands to hepatocytes. V.

Conclusions

Studies presented here have demonstrated the feasibility of delivery of pharmacologic agents to hepatocytes by utilization of the pathways of receptor-mediated endocytosis, both for toxic (immunotoxins) and nontoxic molecules. Basic approaches to the cell biology of receptor-mediated endocytosis have highlighted potential obstacles to this area of pharmacotherapy, as well as potential areas for exploitation. Though progress in understanding of intracellular pathways and c o m p a r t m e n t s in hepatocyte and h e p a t o m a cells has been made in the last five years, additional advances will be required to translate these gains into clinically useful programs. Orthotopic liver transplantation, for example, is one currently available approach to overwhelming liver involvement by neoplastic, congenital, or toxic processes. The end result of drug-ligand development as discussed in this review would be to produce options to this surgical approach which would combine lower cost and morbidity with hepatocyte-specific therapy. Acknowledgements

We t h a n k the National Institutes of Health, the American H e a r t Association, the National Foundation, Monsanto, and the American Cancer Society for support, and Ms Millicent Schainker for secretarial assistance. References

1 Brown; M.S., Anderson, R.G.W. and Goldstein, J.L. (1983) Recycling receptors: the round trip itinerary of migrant membrane proteins, Cell 32,663-667. 2 Stahl, P. and Schwartz, A.L. (1986) Receptor-mediated endocytosis, J. Clin. Invest. 77, 657-662. 3 Benovic, J.L., Strasser, R.H., Caron, J.G. and Lefkowitz, R.J. (1986) B-Adrenergic receptor kinase: identification of a novel protein kinase that phosphorylates the agonist-occupiedform of the receptor, Proc. Natl. Acad. Sci. USA 83, 2797-2801. 4 Fine, R.E. and Oekleford, C.D. (1984) Supramolecular cytology of coated vesicles, Int. Rev. Cytol. 91, 1--43. 5 Geuze, H.J., Slot, J.W., Strous, G.J.A.M., Lodish, H.F. and Schwartz, A.L. (1983) IntraceUular site of asialoglycoproteinreceptor-liganduncoupling: double-label immunoelectronmicroscopyduring receptor-mediated endocytosis, Cell 32, 277-287. 6 Helenius~ A., Mellman, I., Wall,D. and Hubbard, A. (1983) Endosomes, Trends Biochem. Sci. 8, 245--250. 7 Pastan, I.H. and Willingham, M.C. (1981) Journey to the center of the cell. Role of receptosome, Science 214, 504-509. 8 Tycko, B. and Maxfield, F.R. (1982) Rapid acidification of endocytic vesicles containing alpha2macroglobulin, Cell 28, 643-651. 9 Stone, D.K, Xie, X.-S. and Racker, E. (1983) An ATP-driven proton pump in clathrin-coated vesicles, J. Biol. Chem. 259, 4059--4062.

RECEPTOR-MEDIATED DELIVERY O[: DRLiGS T() t[~!PA I'(1( Y IES

61

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