CA2180348A1 - Hepatocyte-targeted drug conjugates - Google Patents

Abstract

The invention provides conjugates for targeting a therapeutic agent to a cell with asialoglycoprotein receptors. The conjugates comprise a therapeutic agent and ligand for the asialoglycoprotein receptor, wherein the therapeutic agent and the ligand are linked by a bridging agent. The bridging agent can be a crosslinker, a polyfunctional carrier molecule or a crosslinker and a polyfunctional carrier molecule. In a preferred embodiment, the therapeutic agent is a nucleoside analog or colchicine and the ligand is asialoorosomucoid, arabinogalactan or a Tris-(N-acetyl galactosamine aminohexyl glycoside) amide of tyrosyl(glutamyl)-glutamate. Preferred crosslinkers include aminoacyl derivatives, carboxyacyl derivatives, phosphate, peptides and reductively-labile crosslinkers. Preferred polyfunctional carrier molecules include polyamino acids and polysaccharides. The conjugates of the invention can be used to target a therapeutic agent to a cell, for example to inhibit viral DNA replication in a virally-infected hepatocyte.

Description

~ WO 95/18636 , ~"~
HEPATOCYTE-TARGETED lDRUG CONJUGATES
of f~- T ~-~Tu~ ,;rlc toxicity is a major ;................... ,,~.1; " .l to the ;I.,v.,lu~u~ of ,1,. . ". .Ih- A~ agents for the treatment of diseases such as viral infections and cancer.
Since the molecular " ~r - I IA. 1 . _ lC by which both of these disease types propagate are intimately associated with normal cellular mf~tol~r~ m, the discovery of drugs which selectively block propagation of these diseases has been slow. Thus, conventional .. 1,.. ,Ih.. I,y relies on the balanced use of therapeutic agents, many of which have a narrow rangeofactive...." ~ .ibeforetoxicityismanifestedinuninfectedor~ rlll~llr~
cells, thereby prohibiting the use of greater < "".. 1. Al ;1 l. ' - of the therapeutic agent.
Additional i ~ to the efficacy of certain therapeutic agents include inactivation of the agents witbin the body and/or rapid excretion of the agents from the body, thus limiting their therapeutic activity An approach that has been taken to increase the therapeutic activity of a drug has been to conjugate the drug to a Illa~,lvlllOlc~,ldc which acts as a carrier for the drug.
~ r njllg?tinn of a drug to a Il~ u~ lc~ can slow the rate of drug excretion and increase cellular uptake of the drug, I"c~l.~ly by non-specific pinocytosis. Certain drugs have been conjugated to polymeric ~l~a~,~ul~ol~,~,ul~A such as pul~a~,~,lla~ and polyamino acids, resulting in decreased illa~Li~aiiull of the drug and/or decreased excretion of the drug from the body. See for example, Bernstein, A., et al., (1978) ~ Natl Cancer Inst. 60:379-384; Kato, Y., et al., (1984) Cancer Res. ~L:25-30; Kéry, V., et al., (1990) Int. ~ Biochem. ~:1203-Z5 1207; Onishi, H., et al., (1991 ) Drug Design and Delivery _:139-145.
However, CC II; _,~ " of a drug to a Illa.,lulll~ c which does not function as a ligand for a specific receptor does n~t address the problem of non-specific toxicity of therapeutic agents. One general strategy for increasing the specificity of therapeutic agents 30 mvolves attachment of a drug to a cell-s]~ecific ligand to effect t~rgeted delivery of the drug to a desired cell population. Conjugatiol[~ of a drug to a ligand which binds to a structure on the surface of a cell to be targeted for drug delivery (e.g., a virally-infected or malignant cell) can incrcose the specific uptake of the drug by the tArget cell via receptor-mediated ~lldu~ylu ~ H and reduce non-specific ~;ylulu~ y.
One type of cell-specific ligand which has been used for drug conjugation is a ,,..,I~. .l, .,.~ amtibody directed against a surface structure present on a target cell. For example, targeted delivery of therapeutic agents to tumor cells by .... ,;- -~ t;.,~ the agents to 2l 80348 WO 95/18636 P~ IL
' ;~
an anti-tumor cell antibody has been ill~. _ ' extensively (for a review see Pietersz, G.A.
( 1990) ~io. ~ u~ule Chemistry 1(2): 89-95).
Another type of cell-specific ligand which has been used for drug ~.. ., j, .~,.l ;""
5 is a ~]y~,u,ul~ ' which binds to a membrane receptor for the ~Iy~,vul Ut~,ill present on a target cell (for a review see Bodmer, J.L. and Dean, R.T. (1988) Mefhods in Enzymology, 112:298-306). One target cell which is of particular clinical import~mce is the ~ ,llylllal liver cell, or hepatocyte, which is a primary site of infection for hepatitis viruses, such as hepatitis B
virus. The nucleoside analogs adenine ' ~ ' ; ' . ' (QraAMP) amd 10 acyclovir l ' - . ' (ACVMP) have shown promise as therapeutic agents for treatment of hepatitis B virus (HBV), but their use as free drugs has been associated with problems such as toxicity, rapid clearance (for araAMP) and poor cellular uptake (for ACVMP) (see for example Jacyna, M.R. amd Thomas, H.C. (1990) British Medical ~ulletin, 46:368-382;
Sacks,S.L.,etal.(1979)JAMA2~1:28;Whitley,R.,etal.(1980)Drugs~:267;Balfour, H.H. (1984) Ann Rev. Med. ~:279; Weller, I.V.D., et al., (1983) J. An~imicrob. Chemother.
11:223). AraAMP amd ACVMP have been targeted to liver cells by cnnj~g~tin~ them to ' human serum albumin (hereinafter L-HSA) which binds to the ~ o~ IY~,VIVlUL~.;.l receptor present on liver cells (Fiume, L., et al. (1981 ) FEBS Letters L2:261-264; Fiume, L., et al. (1988) Pharm. Acta Helv. ~:137-139; Fiume, L., (~989) Nu~ ,f' , :Zh:74-76; U.S. PatentNo. 4,725,672; U.S. PatentNo. 4,794,170). In these cases, the phosphate moiety was used to crosslink the nucleoside analogs to L-HSA.
ACVMP has also been conjugated to L-HSA via a glutarate crosslinker or a succinate crosslinker (U.S. Patent No. 4,725,672). A conjugate of araAMP coupled to L-HSA was selectively cleared from circulation by the ~ _~u, ,ly~,vlllvt~;ll receptor and inhibited DNA
synthesis in hepatocytes (Fiume, L., et al. (1981) FEBS l etters 129:261-264). A conjugate of ACVMP coupled to L-HSA waS shown to release the free drug in liver cells (Fiume, L., (1989) N..lu, ~v ~ ,A ~:74-76). Both conjugates lowered woodchuck hepatitis virus DNA levels at doses lower than the I ; ., ' drugs (Ponzetto, A., et al. (1991) Hepatology L:16-24) amd the araAMP-L-HSA conjugate ir~hibited HBV replication in30 humans (Fiume, L., et al. (1988) Lancet ~:13-15). However, there can be drawbacks to using - ' albumin as a cell-specific ligand. The specificity of L-HSA for the V,VlVt~;l. receptor results from the galactosyl residues that are attached to the albumin upon l - I~ n If this process is inefficient, the number of galactosyl residues which are coupled to the albumin may not be sufficient to produce a ligand with high 35 affinity for the ' ~ .U~IUL~;II receptor, thereby reducing the targeting ability of the conjugate.
rolyrull~liulldl carrier molecules have also been used to increase the therapeutic activity of drugs. rOIyrul.~,~iul.dl carrier molecule have a ll~ul~iuli~ y of reactive 0 WO 95/18636 r~
side chains to which other molecules c~m be conjugated and thus have the advamtage that many small molecules can be coupled ~o a single molecule of carrier. Drugs have been conjugated to polyf~mctional carrier molecules, such as polyglutamic acid, to increase the non-specific uptake of the drug (see fol example Kato, Y., et al., (1984) Cance~ Res. 44:25-5 30). However, attempts at combining the use of a pvly r ~ carrier molecule and a cell-specific ligand to target a therapeutic agent have shown limitations. In one study (Fiume, L.
et al. (1986) FEBS l,etters 2Q~:203-206), polylysine was used in conjugates for targeting antiviral agents to virus-infected I . ~ ~.r tbrough the ASGR by coupling galactose residues to polylysine. ~IraAMP and A.CVMP were then conjugated to the galætosyl-10 polylysine. Of the two constructr~, only araAMP-galactosyl-polylysine effectively targeted ,Y ~ and inhibited DNA synthesis in Ectromelia virus-infected mice; the ACVMP-galactosyl-polylysine conjugate was in;lctive.
S of " ~
This invention pertains ~o drug conjugates which can target a therapeutic agent to a cell which expresses ' ~ /C,UIJIVt~ receptorr,. The the:rapeutic agent is targeted to the ' ~ ,~y~v~lv~ receptor by ~onjl~o~tin~ it to a ligand for the ~ ~v~ly~ù~JIvt~
receptor. The therapeutic agent is conj~Igated to the ligand by one or more bridging agents 20 which function to couple the therapeutic agent and the ligand. The bridging agent(s) has the property that it allows the therapeutic agent and the ligand to be coupled without destroying the therapeutic ætivity of the agent or the binding ætivity of tbe ligand. Binding of the ligand to the ~ y~,v~ulvt~ receptar facilitates uptake of the conjugate by the cell via receptor-mediated cn~o."~ v,is.
In a preferred ~IIIbOd;lllC,III, the conjugate comprises a therapeutic agent and a ligand for the: ~ ly.,U~JlU~,;.I receptor selected from l ~, _.,~l,;", ,~,"l and the synthetic ligand YEE(GalNAc~H)3. The therapeutic agent is cûnjugated to the ligand via a bridging agent which can be a crosslinker, a pOly r ' carrier molecule or 30 both a crosslinker and a pOly r ~ carrier molecule. When a crosslinker is used alone, the crosslinker covalently binds to both the therapeutic agent and the ligand, thereby coupling the therapeutic agent to the ligamd. For example, C~IU~ ~lillc l~ which react with amino groups, carboxyl groups or sulfhydryl groups oln the ligand can be used. Similarly, when a I.olyru.l~,liul.al carrier molecule is used alone, the pOlyrull..liull~l carrier molecule covalently 35 bmds to both the therapeutic agent and the ligand, thereby coupling the therapeutic agent to the ligand. For example, a polyfunctiollal carrier molecule with reactive aldehyde groups, such as luuly~ lyd~ dextran, can be u~;ed. ~ , when both a crosslinker and a pOIyrl l~,Liul~l carrier molecule are used, the reagents are chosen such that the crosslinker covalently binds to both the therapeutic agent and the polyr~ .l;ullal carrier molecule, and

2 1 803~8 WO 95/18636 ~ . . ' IE
the l~vlyru-l-,Liullàl earrier moleeule eovalently binds to the ligand, thereby eoupling the therapeutie agent to the ligand. The crosslinker can be coupled to the ~ulyrh.l~,Liu~dl earrier molecule by an amide bond, a 1,~ i ir bond or a disulfide bond. For example, an aminoacyl crosslinker can be used with a ~ùlyru~ iullal carrier molecule having multiple 5 reactive earboxyl groups, or dlt~ aii~ y~ a ~,a~vv~.~al,yl crosslinker can be used with a pvl.yru~l~,Liulldl earrier moleeule having multiple reactive amino groups.
Preferred ~,.. " ' for use in the conjugates include phosphate derivatives, Wl vv~,~a~,yl derivatives of sueeinate and glutarate, aminoacyl derivatives of trans-~
10 ~ In~ vù;~yk.~or~ L~aderivativeof(3-(2-pyridyldithio)propionate or a peptide eomprising an arnino aeid sequenee Leu-Ala-Leu.
Preferred ~ùlyrull-,Livll~:l carrier molecules include polyamino acids, such a polylysine, polyu..l;l.il.~, polyglutarnie acid and pvlya~ l Lic aeid and polysaecharides such as polyaldehyde dextran.
The conjugates of the invention can be used to target a therapeutie agent to h~ Lu."~.~., whieh express ~ .v~u.~,;.. reeeptors. Therapeutie agents whieh are effeetive against viral infections of l~ JaLu~ can thus be conjugated to a ligand for the asialoOI~v,v.uL~ reeeptor. For example, antiviral drugs effeetive against a hepatitis virus, 20 sueh as hepatitis B virus, ean be used in the conjugates. A preferred type of antiviral therapeutie agent is a nucleoside analog. The invention ~ - conjugates comprising a nueleoside analog and an ' ~ ,vlJ~vt~ reeeptor ligand, sueh as ' - . 1, ineluding eonjugates wherein the nueleoside analog is 9-~-D-~Ilv;l~vrulal~v ~h, ~ Lv ~ c, 9-(2-I.~d.v~ y~-l.,Ll~yl)guanine~ v~ L;d;--, 9-~3-D-: ~ r ylav~,ll 1~ and 3'-25 azido-3~-d~ yLlly~l~;di~ The eonjugates ofthe invention are useful for inhibiting vira;i DNA replication in viraily-infected cells, such as hepatitis B virus-infected hepatocytes.
The invention further provides methods for targeting a therapeutie agent to a eell expressing d~ ,vlJlvt~ reeeptors in a subject. The method involves forming a 30 conjugate of the therapeutic agent and an asialoOly~vl~lvt~;ll receptor ligand, such as . . . v~i and d' i" ' " ~ the conjugate in a l.I.y ,;vlog;~lly aeceptable vehicle to the subjeet.
Brj-~f 1- . r ~' ~ D' Figure I is a graph depieting the effeet of increasing ~. ...1.,.1;.~..~ of an araC-glutarate-PL-ASOR eonjugate on the intr~ r a~c~ tinn of replieation ' (Rl) and relaxed eireular (RC) forms of HBV DNA in HBV DNA-transfeeted 2.2.15 eells.

=

~ WO95/18636 r~,l" '~ '_ Figure 2 is a graph depicting the effect of increasing ~.. .. 1, ~l ;.. - of free ACV and an ACVMP-PL-ASOR conjugate on the intr ~ Ar ~ ." of relaxed circular HBV DNA in HBV DNA-transfected 2.2.15 cells.
.
Figure 3 is a graph depi~,ting the effect of increasing ~.1 .l l. . . ~".1 ;.~. ,~ of free ACV and am ACVMP-PL-ASOR conjugate on the (~Ytr~r~.ll ' r ' ' ' of HBV DNA
in the culture medium from HBV DNA-transfected 2.2.15 cells.
Figure 4 is a graph depicting the effect of inaeasing c~ of a ddC-PAD-ASOR conjugate on the intrA~r~ rc~n~ tion of relaxed cr~cular HBV DNA in HBV DNA-transfected 2.2.15 cells.
15 I) ' ' Il` of T
The invention relates to conjugates which can target a therapeutic agent to a cell expressing . ' ~ UUI~ ;II rece~)tors. The therapeutic agent is targeted to the a~;~lv~;ly~u~lui~;ll receptor (hereinafter ASGR) by ~__ _ it to a ligamd for ASGR. The 20 ligand serves to target the therapeutic a~ent to a cell with ~ u~lut~ l receptors and to facilitate uptake of the conjugate by the cell via receptor-mediated ~ u., y ~usis. Because the conjugates of the invention achieve the effect of targeting the therapeutic agent to a cell, a lower dosage of a conjugate is needed to achieve a desired therapeutic effect compared to the therapeutic agent. ~' "!o because the conjugates are directed away from 25 cells which do not express the: ~ U~lVt~;ll receptor it is to be expected tb~t the non-specific ~:y ~UlUlU~ y of the therapeutic ~Igent will be decreased in vivo.
The term "conjugate" is intended to include two or more molecular species which are covalently bonded to each ot~ler. The conjugates of the invention are composed of 30 at least a therapeutic agent and a ligand for ASGR, and usually at least one additional molecular species which functions as a bridging agent between the therapeutic agent amd the ligamd. Thus, there are essentially three ~ of the conjugate to be considered: the therapeutic agent, the ligand and the means by which tbe two are conjugated together (i.e., the bridging agent(s)), which will be discussed in more detail in the sections below.

1. Th~r~PIltif', The term '~ a~ tic agent" is intended to include molecules which are r ~ d to a subject with the intenl of changing, in a beneficial way, a ~ .Iulo~icdl WO g~/18636 2 1 8 ~ 3 4 8 P~
function in the subject or with the intent of treating, in a beneficial way, a disease or disorder in the subject. Therapeutic agents irlclude drugs, cu~ ..iv.,al antiviral agents (including nucleoside analogs), Cu~ iullal anti-tumor agents, reverse ~ r irlhibitors, ~u,u~ . I inhibitors, l~ II inhibitors, prokaryotic DNA gyrase inhibitors, S DNA binding agents, hormones, growth factors, vitamins, proteins and peptides and analogs thereof, nucleic acids amd analogs thereof, and other bioactive molecules. For example, the the~apeutic agent can be an antiviral drug which is targeted to virally-infected cells which express ASGR as a means of treating the viral infection.
I û A preferred ASGR-expressing cell type to which therapeutic âgents are targeted is a hepatocyte. Thus, an antiviral drug which is effective against a viral infection of ~ -yt-~ can be corljugated to a ligand for ASGR to target the antiviral drug to virâlly-infected l r ' ,y a,~ A viral infection of h~ ..u~ can be due to infection by any h~ ~l.u~;~, virus. Examples of l,~ .u~;c viruses include hepatitis virus A, hepâtitis virus 15 B, hepatitis virus C and hepatitis virus D. A preferred l~ Ja~ullu~;~ virus against which a therapeutic agent is directed is hepatitis B VilUs.
One therapeutic approach to treating viral infections, such as hepatitis B vircs, is to use a drug which interferes with viral DNA synthesis, such as a nucleoside amalog. For 20 example, two nucleoside analogs, 9-t3-D-~.,..l,: lr~ lal~lf,l,e (araA) and 9-(2-I~yLu7~ yu~ /l)guanine (also known as acyclovir; ACV), have been tested in patients with chronic hepatitis B virus infection (Hoofnagle, J.H. et al. (1984) Gu..hu.,.:~, ùlû,~
~:150^157;Weller,I.V.D.,etal.,(1985)Guf2t~:74S-751;Sacks,S.L.,etal.,(1979)JAM4 2~.:28; Weller, I.V.D., et al., (1983) J. An~imicroh. Chemot~2er. 11:223-231; Alexander, 2S G.J.M., et al., (1986) J. HepatoL :~(Suppl. 2):S 123-S 127). While the free nucleoside analogs displayed a blocking effect on viral growth, dose-related side-effects were observed.
Ill~,OllJula~iull of a nucleoside analog into a conjugate of the invention can increase the therapeutic activity of the dlug, thereby decreasing the dosage of the drug necessary for therapeutic t~ . Accordingly, in one ,1,.~ .1 the therapeutic agent of the conjugate is a nucleoside analog. As used herein, the term "nucleoside analog" is intended to include molecules having the general formula:
RO- ~ B
xh wo 9~/18636 . ~1/ll... 1 1.
wherein Z is oxygen, sulfur o} carbon, I~ is a nucleoside base or analog, X and Y are substituent groups such as OH, H, N3, F etc. and R is a functional group that allows attachment of the nucleoside ana~og to a crosslinker and/or carrier molecule by a coYalent 5 bond. Examples of suitable functional groups include those that provide a free -OH, -NH2, -COOH or -SH moiety.
The term " ' ' analog" is also intended to include acyclic . ,. ,. l~ . .ci,l~
of the general formula:

RO ~ z B
\/ \/
wherein B is a nucleoside base or analog, Z is oxygen, sulfur or carbon, and R is a functional group tnat allows attachment of the nucleoside analog to a crosslinker and/or carrier molecule 15 by a covalent bond. Examples of suitab] e functional groups include those that provide a free -OH, -NH~, -COOH or -SH moiety. Ex.~mples of acyclic nucleotides which are effective antiviral agents which can be used in the conjugates of the invention are described in U.S.
Patent No. 4,199,574 by Schaeffer.
Preferred nucleoside anall~gs for use in conjugates of the invention include 9-~-D ~ .r~ yla.lc;lfillf (araA), 9-3-D-.~ r~ yl~,yLv~ille(araC), 2',3'-dhl~,vAy~y~idillf (ddC) amd 3'-azido-3'~ ,vAya~J~ .u., (AZT) A preferred acyclicnucleoside amalog is 9-(2-llJLuA~.,alyv~.yll~.,llyl)guanine (ACV). Other possible nucleoside analogs include ~ ,y~,luvil~ L~u-~,y~.lvv;ll, p~ ,y-,lvv;l, hlullluvlllyldLvAy~
1 l h r ', the 2',3'-d;d~VAy ' ' of adenosine (ddA), inosine (ddI), guanosine (ddG), thymidine (ddl) and uracil (ddU), 9-~3-D-ol~;llu-ul~lvaylal~,l.h~-crythro-9-(2-lly~uAy-lvllyl)adenine (AraA-EHNA), 2~-fluoro-l-~-D-~Al~ r~ vayl-s-~ ylul~lci (FMAU),2'-fluoro-1-~3-D; ~ r~ uayl-5-etnyluracil(FEAU), 2'-fluoro-1-~-D-Ar~hin( ~ yl-5-iodouracil (FIAU), 2'-fluoro-1-13-D ~, I.;....rl... .~yl-5-iodul,yi ' 30 (FIAC), 3'-fluoro-ddC, 5-chloro-ddC, 3'-.fluoro-5-chloro-ddC, 3'-azido-5-chloro-ddC, 3'-fluoro-ddT, 3'-fluoro-ddU, 3'-fluoro-5-chloro-ddU, 3'-azido-ddU, 3'-azido-5-chloro-ddU, 2'-6'-~' . 2', 3'-~' ' yl;bos;ve (ddDAPR) aQd a carbocylic analog of dCvA~, (2'-CDG).
35 In addition to nucleoside analogs, other types of therapeutic agents can be used to inhibit viral infections, such as hepatitis virus infections. For example, reverse A_~_ inhibitors, lu~..l: ,..., .,~. inhibitors, gyrase inhibitors and DNA binding agents have been shown to inhibit hepatitis B vil us DNA replication (Civitico, G., et al. (1990) ~
, WO95/18636 21 8 0348 r~ 5 ~~ O
Med. riroL ~:90-97). Th- ~ A11y effective compoumds which c~in be used a-a therapeutic agents in the conjugates of the invention include the ~u;. - .:~ .. r` Il inhibitors ellipticine, amsacrine, adriamycin and Ill;LIv~ciilLiu~ the ,uluhyiyuLiC DNA gyrase inhibitor UU~.lll.,llll,r.~;ll Al and the DNA binding agents lf U' -' ~ nd .1~ (which 5 either interc_'iate or nick DNA).
Tl. T. ' f~-~r thP A~ t~ pAp~tt~r A therapeutic agent is targeted to a cell expressing ASGR by uu..; Uod~illo it to 10 a ligamd for the ' ~ ,U,UiUt~,;ll receptor (aliso referred to as the hepatic Gai/Ga'iNAc specific receptor). The term "a ligand for the -a;dliûOly~,uulut~,;ll receptor" is intended to include any molecule which binds to the asia~iuoly~ui~lut~;ll receptor. One type of ligand for the da;dloOly-,uulut~,;ll receptor is an ~ yuui~lvt~;ll with clustered terminal ga'iactose residues. Such an: ' o'~,uj~lut~,;.. cam be prepared from sialic acid terminating 15 Olyl ujulut~,;lla with j~ ' galactosyl residues. The galactose residues are exposed by desialation of the olywjulu~;ll using standard techniques. For example, O~y~,u,uluL~illa can be desialated by treating them with the enzyme ~ Alternatively, Ol~,ujulut~,;lla can be desia'iated by acid hydrolysis as described in Example I . Examples of asialoOly.,u~.ut~ .a include iia;~ uluaulllu~oid~ and 20 desialylated vesicular stomatitis virus. OluaullluuOi~'i, fetuin, c~lu'iu~ alllill and ..gl~.l...l;.. can be obtained from blood plasma and then desialated.
A preferred ASGR ligand for use in the conjugates of the invention is the I o~ ,ujl,.ut~,;ll da;-'iOul~ ~hereinafter ASOR). It should also be appreciated that certain alterations (e.g, amino acid deletions or point mutations) of the u~u~v~u~,oid protein, or derivatives of the protein or attached ~ b~ ' moiety, can be made without destroying theabilityoftheOly.,uju.ut~;lltobindtoASGR. SuchalteredorderivatizedformsofASOR
are intended to be within the scope of the term "Qi;lùu~u~u~u~,ù;~!i as used herein. ASOR
can be prepared from uluaulll i~,Oid (also referred to as ~-I acid joly~U,UlUt~Lil), isolated from humam plasma, by desialation of tiLie isolated uluaulll i~,u;d to expose I~ ~- ' ';, . gaiactose groups (such as described in Example I ). It has been foumd that when severa!i . _~li.-.l_l ,. If plasma-derived ' ~ /.,vu.u~;l.a are coinjected in vivo into the circulation, 1 is cleared most rapidly from the circulation, indicatmg that ASOR is taken up rapidly by the liver ( see J. Biol. Cf'2em. (1970) 245:4397; and PCT Application WO
92/22310). ~ ' ' "y, ASOR is rich in carboxylic acid groups which allow for coupling of therapeutic agents or bridging agents to ASOR through these groups.
Another type of ligand for ASGR is a Il~,uolyuu~u;~ , a protein which has been modified to be a ligand for ASGR For example, terminai galiactosyl residues can be _ _ _ _ WO 9~/18636 r~ s coupled to a protein to convert it to a li~and for ASGR. For example, ~al~Lv~
vvl~.~L ~ such as lactose, can be coupled to a protein by reductive ammation.
Anotber type of ligand vvhich can be used in the conjugates of the invention is S a ~bul~ ' which binds to ASGR. For example, a poly~a.,~ al;le with terminal galactose residues can be used to target a therapel~tic agent to ASGR. A preferred calbOlly~' ligand is . . ,.1,;, I.~S..IA. ;A- ~ A ' ,, ' is a component of the cell walls of many species of trees amd plamts. Structurally, ~ 1 consists of a galactose backbone with branch chains of arabinose and galactose. Generally, the ratio of galactose to arabinose is between 5 :1 and 10:1 (see Glickman, ed. (1982) Food ~lydrocolloids, CRC Press). Derivatives of orto-l can be prepared which provide functional groups that allow att~chment of . ,.1 .;, ~oL~ to a therapeutic agent, a crosslinker or a uvl~rull~,Livllal carrier molecule. For example, an amino derivative or a carboxyl derivative of ~.,.1.:",,~,,1-. 1~.. can be used to prepare a conjugate ofthe invention in which "",I,;.,.~g~l -;,.., serves as the ligand for ASGR.
Another type of ligand v~hich cam be used in the conjugates of tne invention is a synthetic ligand for ASGR. The syntlletic ligand comprises a ~albvllydl~.~C moiety having a binding specificity for ASGR linked to a peptide via an amide bond. A preferred ~,albvll~l' moiet,v is N-àc~;lyl~ 1- lu~ For example, two or more ~,albvllyl' 20 moieties can be linked to a di- or tri-pe~tide to form a cluster ligand specific for ASGR. The synthetic ligand also comprises an organic structure having a functional group available for forming a covalent bond with a therapeutic agent, a crosslinker or a polyru~ iu.~dl carrier molecule. A preferred synthetic ligand is the Tris-(N-acetyl ~ v~ - .. -.~ ~I.;IIVII.Ayl glycoside) amide of tyrvsyl(glutOmyl)glutoOmate~ referred to herein as YEE(GalNAcAH)3.
YEE(GalNAcAH)3 can be synthesized as described in Lee et al. (1987) Cly~,u~u~yu,~ ~.h Journal :~:317, or as described in U.S. I'atent Application Serial No. 08/045,985 by Findeis et al., the contents of which are hereby ill~Vl U~ ' ~ by reference. T~Le structure of YEE(GalNAcAH)3 can be represented by the following formula:

W0 95/18636 . ~ . ~

OH
O AcHN OH OH
HJlNH~ Oæ~
OH
R--HN o HN o Ac~OH
~NH--~O o OH
O=~ Ac~OH
NH ~ ,O o_~
OH
wherein R is H or COCH2CH2CO2H.
5 T~T. C/~ ~slti-.n ofth! Thrr~,n.-lltic A~nt to thr A!~GR r.~nrl Bri~in,~ ent~
A therapeutic agent is conjugated to a ligand for the ' ~ n~uulu~
receptor by means of an ill.~,llll~,di~r which functions as a bridging agent to conmect the therapeutic agent to the ligand. The bridging agent must allow for coupling of the therapeutic 10 agent to the ligand without destroying either the therapeutic activity of the t_erapeutic agent or the binding activity of the ligand and should be stable in the circulation in vivo.
Additionally, the bridging agent preferably should allow for release of the therapeutic agent 'l ' y in an ætive, fimctional form, although this may not be am absolute lcl_ cThe bridging agent and uu..; Ll,_;iUII mechsmism are critical Cu~ ,r of the conjugate t_at 15 can play a major role in ~' v vhether a conjugate is ætive or not (i.e., a conjugate in which the therapeutic agent and/or the ligand is covalently bonded to the bridging agent(s) in am ill~ JlU~ manner may not maintain functional activity). For example, an ACVMP-L-HSA conjugate displayed hepatocyte targeting and antiviral activity (Fiume, L., (1989) N..t..",; "A Z.~i:74-76; U.S. Patent No. 4,725,672) whereas an ACVMP-polylysine-2û galactose conjugate was inactive (Fiume, L. et al. (1986) FI~BS ~etters ~:203-2(~6) ~s used herein, the term "bridging agent" is intended to include molecules which couple a therapeutic agent to am ASGR ligand. The bridging agent used m Lhe conjugates of the invention can be a crosslinker, a polyrul~Liullal carrier molecule or both a crosslinker and a polyrullcLiull~l carrier molecule. Accordingly, in one c.~ o.l;- : of the W095/18636 P~l/l 'I !.-//
invention, the conjugate has the general formula A-B-C-D, wherein A is the therapeutic agent, B is a crosslinker, C is a pOIyr - I carrier molecule and D is: ' ~ , .1 In another . ,.,1 ,o.1;",. ll, the conjugate has the general formula A-C-D, wherein A is the therapeutic agent, C is a polyrull~,liollal carrier molecule and D is I ' ~ u~u~ ,oid. In yet S another culL " t, the conjugate has ihe general fonmula A-B-D, wherein A is the therapeutic agent, B is a crosslirlker and D is a I Ui~UlllU~Ui~
The term "crosslinker" is intended to include molecules which can function as bridging molecules between two other n1olecules by way of having two reactive functional 10 groups, one of which reacts to form a cavalent bond with the first molecule and the other of which reacts to form a covalent bond with the second molecule, thereby effectively connecting the two molecules together. Preferably, the crosslinker has two reactive functional groups of different functional moieties. Examples of suitable functional groups include amino groups, carboxyl groups, sulfhydryl groups and hydroxy groups. When one 15 functional group of the crosslinker is re~lcted with a molecule (e.g., a therapeutic agent), the other functiona~ group can be, if necessary, prevented from reacting with that molecule by means of a protecting group which modifies the second functional group of the crosslinker so that it cannot react with the molecule. After the first reaction is completed, the protecting group can be removed, restoring the sec~nd functional group, and then the second functional 20 group can be reacted with another molel,ule (e.g., an ASGR ligand such as ~ usulll~ iJ).
The term ",uolyr ul~,liu~ carrier molecule" is intended to include molecules which cam function as bridging molecul~ s between two or more other molecules by way of 25 having multiple (i.e., more than two) re~lctive functional groups which can form covalent bonds with the other molecules, thereby effectively comnecting the other molecules together.
In general, a pol~ ' ' carrier has a polymeric structure and, preferably, the multiple functional groups of the poly ' 1 ~carrier are of the same functional moiety. Examples of suitable functional groups include amino groups, carboxyl groups and aldehyde groups.
30 Because of the multiple reactive functional groups present on the polyru~ iu~lal carrier molecule, multiple molecules can be co1Ipled to it (i.e., many molecules of a therapeutic agent and/or ligand can be coupled to a single molecule of carrier). Thus, the molar ~ratio of conjugates containing a polyfunctional carrier molecule generally is increased relative to conjugates which do not contain a polyrull~,liullal carrier molecule. Increasing the molar

3 5 ~ ;. ., . ratio of a conjugate cam pro vide a means by which to increase the therapeutic index (i.e., therapeutic activity) of a conjugate.
According to this invention, several different coupling strategies can be used to conjugate a therapeutic agent to 1. u~l ' or other ligand for ASGR using WO95/18636 2 1 80348 1 "~ "~
/~
different ~,.. ' ' and/or polyfunctional carrier molecules. The strategies and reactions are described in detail in the Examples. Different types of l.,lU~I;IILCID and ,uvlyrLIlllliullàl carrier molecules which can be used are . , ~ briefly in the following ;. .~.,. . I ;. ."~
5 A. Acyl (~ " ' A therapeutic agent can be conjugated to a ligand (or a carrier-ligand complex) by preparing an acyl derivative of the agent, wherein the acyl derivative has a functional group which can react with another functional group on the ligand or on the carrier-ligand 10 complex. The functional group of the acyl derivative can be, for example, a carboxyl group (which can then be reacted with amino groups on the ligand or carrier to form amide bonds), an amino group (which can then be reacted with carboxyl groups on the ligand or carrier to form amide bonds) or a phosphate group (which can then be reæted with amino groups on the ligand or carrier to form ~ ";.l bonds).
~`~rboxyacyl C~
A therapeutic agent can be conjugated to a ligand (or a carrier-ligand complex) by active ester coupling of a ~albvAya~,yl derivative of the agent to a ligand or carrier-ligand 20 having reactive amino groups. Briefly, a therapeutic agent is acylated at an amino or hydroxy group to form an acyl derivative. Preferred acyl derivatives are glutaryl and succinyl derivatives. Carboxyacyl derivatives of therapeutic agents (e.g., nucleoside analogs) can be prepared as previously described (see for example Erlanger, B.F., et al. (1967) Methods Immun. ~ 144) and as detailed in Examples I and 2. The derivative 25 ~,a~bu~ya~.yl group of the therapeutic agent functions as a crosslinker to allow c.... j ~;,.l ;..., of the therapeutic agent to a ligand or carrier-ligand complex. 'rhe calbuA.9a~,yl derivative of the agent is activated (for example, with N h~u~ I ) to form an active ester. The actived ~albu~yal,yl compound is then reacted with a ligand or a carrier (e.g., in a carrier-ligand complex, such as polylysine-ASOR) having functional amino groups to form amide 30 bonds between the ~,albvA~a._yl crosslinker and the amino groups of the carrier or ligand, thereby coupling the agent to the carrier or ligand. The ~albu~ya~,yl derivative carl be conjugated to a carrier or ligand as described in detail in Examples I and 2. A preferred polymeric carrier molecule to which carboxyæyl derivatives of therapeutic agents can be conjugated is a polyamino acid with reactive amino groups, such as polylysine or35 IJuly~ ' The polymeric carrier molecule can first be coupled to a ligand, such as ASOR, as described in Example I and then the ca l,u~ya~,yl-derivative of the therapeutic agent can be conjugated to the carrier-ligand comples. For example, a ~buAya~yl derivative of the nucleoside analog araC can be conjugated to polylysine-ASOR as follows:

~I WO 95/lN636 f3 HN ~ 1 ) ~1 !, , ~N~ 2) ~ rJolylysine-ASOR ¢¦~ r ~ ASOR
HO~ HO~J
HO n=1: araC-succinate n=1: araC-succinyl- l~ ASOR
n=2: araC-slutarate HO n=2: araC-slutaryl-polylysine-ASOR
Aminnq~yl Cr~qqlin~ rc An amino derivative of ~I therapeutic agent can be conjugated to a ligand or carrier-ligand complex having reactive carboxyl groups through formation of amide bonds between the arnino group of the derivatized therapeutic agent and the carboxyl groups of the ligand or carrier. The amino derivative of the theMpeutic agent is conjugated to the ligand or 10 carrier by . I,o~ coupling. A preferred amino derivative of a therapeutic agent is an a~ninoacylderivative. Forexarnple,an.u..;.v~ Lllyl~ ' ' ' ylor4-a~ vbuiylyl derivative of the therapeutic agent can ~e prepared and coupled to a ligand or carrier-ligand as described in detail in Example 4. To prepare the aminoacyl derivative of the therapeutic agent, the amino group of an alllillv~,albu~ylic acid (e.g.,: ' yL,~ u~ylic 15 acid (AMCC) or I - acid (GABA)) is protected, for exemple by Schotten-Baumar~ ca l,allwylaLion, and the protec ted ~ l/v~ylic acid is reæted with the therapeutic agent by - I,o-l;, . . - -1 -IJlVlllU.~d - '' ; r~ ." Following this reaction, the p}otecting group is removed by llydlu~,llOly~;~ (Brown, C.A. and Brovin, H.C. (1966) ~
Org. Chem. ~1:3989-3995) and the aminoacyl derivative of the agent is coupled to a ligand or 20 carrier-ligand complex havmg reætive carboxyl groups by ~budi;llli~e coupling (e.g., with (3-dilll~Lhy~ r u~yl)-3-ethylcal~vdii~llide4~dlu~lllvli~e)~ Forexample,an ~VII.~ yl (AM:CC)-derivative of a generic nucleoside analog can be conjugated to ASOR as follo~,vs:

H2N ~ O
--~o_~O B ~H--)~ ~ o_~
nucleoside-AMCC + ASOR nu~ icl., /`~'CG-ASOR X Y

wo 9~/18636 2 1 8 P~./.,~. ~ ~ I s /~
The aminoacyl group can be coupled to the therapeutic agent through a reactive hydroxyl group (e.g., the 5' -OH of a nucleoside analog as shov~n above) or through a reactive amino group (e.g., the N4 group of cytosine-derived nucleoside analogs such as araC
S and ddC). If the therapeutic agent has both a reactive hydroxyl group and a reactive amino group, one group can be protected during o~ ua~,yl~lliull. For example, the 5' -OH of a cytosine nucleoside analog can be protected with a trityl group during ~ Liu.~ and then detritylated (see Example 4).

B. Ph.~nh~tl Crocclinkr-rc A phosphate derivative of a therapeutic agent can be conjugated to a ligand or carrier-ligand complex having reactive amino groups through formation of l ' , ' - ' 15 bonds between the phosphate group of the therapeutic agent and amino groups of the ligand or carrier. A therapeutic agent can be Is' . ' ~' ' by starldard procedures or a phosphate derivative of the agent can be obtained, ~, .. . :_lI~r . For exarnple, 5' IIIUIIU~
derivativesofcertainnucleosideanalogscanbeobtained rrmm~rri~lly (e.g.,araA-...."...~)l~r,~l3,..~ araC--- ,' .' ). Proceduressuitablefor pllu~llulyl~Lill~ nucleoside analogs are described in Fiume, L., et al. (1989) Nulu~ ,- h-,~: , 76:74-76 and Sowa, T.
and Ouchi, S. (1975) Bulletln of the Chemical Society of Japan 48(7):2084-2090 and in Example 3. The phosphate derivative of the therapeutic agent can then be conjugated to a ligand or carrier-ligand complex (e.g~, pul~ ,-ASOR) which has reactive amino groups.
For example, polyamino acids such as polylysine and p~ ' can be used as carrier molecules. The phosphate derivative of the therapeutic agent is coupled to the ligand or carrier-ligand complex by ~1--,,1;; - ;~1~ coupling (such as with 1-(3-dilll.,;ll,y' ' 11.71UIJ,yl)-3-~:UIy~ u~ lulid~, EDC) to form ~ ' , ' ' bonds as described in detail in Example 3. For example, a 5' ~ 1~ ' derivative of a generic nucleoside analogcan be conjugated to polylysine-ASOR (prepared as described in Example 1) as follows:

1l B
o~ ~/ EDC ASOR-pûlylysinc.~N r o~O¦
polylysin~ASOR O
X Y X~y (B=nucleûside base) nucleûside-pl lur~pl ,~ , polylysine-ASOR

O W0 95118636 1 ~
/~
C. pP,otiAP C - '' ' A preferred type of crosslinker for use in the conjugates is a peptideS crosslinker which can be hydrolyzed ;..~ rlli 1- Iy (e.g., by lysosomal enzymes) to release the therapeutic agent from the conjugat~. Drug conjugates prepared wjth a peptide crosslinker have been found to be stable in serum in vivo and to release the drug in active form i. ,~ r ~ ly through the action of lysosomal hydrolases (Trouet, A., et al., (1982) Proc. NatL Acad Sci. US,~ 626-629~. A preferred peptide contains the amino acid 10 sequence leucine ~ ' -h,u~ e (LAL ) and is at least a tripeptide or a tr~tr~rrrtirip Structurally, a peptide has both a reactilre amino group (i.e., the N-terminal end) and a reactive carboxy group (i.e, the C-terminal end). In a preferred ~ vll;~ the peptide is used as a crosslinker between a therapelltic agent amd a ligand (or carrier-ligamd) in a C-terminal to N-terminal orientation (i.e., the C-terminal end of the peptide is coupled to the 15 therapeutic agent amd the N-terminal end is coupled to the ligamd or carrier-ligand).
Accordingly, a peptide can be coupled to a therapeutic agent which has a reactive amino group by formation of al~ amide bond between the amino group of the therapeutic agent and the C-terrninal calboxyl group of the peptide as described in detail in 20 Example 5. For example, a peptide cam be coupled to the the N4 group of cytosine-derived nucleoside amalogs such as araC and ddC. Other reactive groups on the therapeutic agent and peptide (e.g., the N-terminal amino grollp) can be prevented from reacting by use of protecting groups. For example, the amino group of the peptide can be protected as a carbamate using standard techniques known in the art and reactive hydroxy groups of a 25 nucleoside analog (e.g., 3' amd 5' -OH groups) can be protected with tertbuty~ ilyl groups. The peptide amd therapeutic age,nt can be coupled by active ester coupling or . - I ~v~ . couplmg to create a peptide derivative of the therapeutic agent. The peptide derivative of the therapeutic agent cam be conjugated to a ligamd or carrier-ligamd complex having reactive calboxyl groups througll formation of amide bonds between the (deprotected) 30 N-terminal amino group of the peptide ~md the carboxyl groups of the ligand or carrier. The peptide derivative of the therapeutic agent can be conjugated to the ligamd or ca~rier with carboxyl groups by - bv~ coupling as described in detail in Example 5. For example, a leucmc-. ' -1~,.-.,;.~ (LAL) tripeptide derivative of a therapeutic agent can be conjugated to ASOR or to a carrier-ASOR complex, such as a pvl ~ acid-ASOR or pvl r~ Li-35 acid-ASOR complex. Additionally, a F~eptide-derivative of a therapeutic agent can be conjugated to an aldehyde containing ligand or carrier-ligand complex (e.g. pvly ' ' ' ~J~
dextran-ASOR) by reductive amination.

W0 95/18636 ~ 5 Altematively, the peptide could be used as a crosslinker between a therapeutic agent and a ligand (or carrier-ligand) in an N-temlinal to C-temlinal orientation (i.e., the N-temmina~ end of the peptide is coupled to the therapeutic agent and the C-temminal end is coupled to the ligand or carrier-ligand), such as when the therapeutic agent has a reactive 5 carboxy group and the ligand or carrier has reætive arnino groups (e.g., polylysine).
Additionally, a diamine peptide or a dh,al~u~ylic acid peptide could be used with appropriate therapeutic agents, ligands ard carriers.
In a preferred ~ ,o~ , the peptide contains the amino acid sequence Leu-10 Ala-Leu. Additional amino acid residues can be added to the N-temninal or C-terminal end of this tripeptide. For example, Leu-Ala-Leu-Lys could be used. The side chains of atnino acids contained within the peptide can also be used for coupling purposes. For example, the amino group of the side chain of Lys contained within a peptide can be used for coupling to carboxy groups (e.g. on polyglutatnic acid) or the carboxy group of the side chain of Glu 15 contained within a peptide can be used for coupling to atnino groups (e.g., on polylysine).
D. P~ tiYely-Ls~hile ~`roccli~ rc A reductively-labile crosslinker can be coupled to a therapeutic agent and then 20 this complex cam be coupled to a ligand for ASGR through sulfhydryl groups of amino acid side chains of the ligamd to form disulfide bonds between the crosslinker and the ligand as described in detail in Example 6. For example, a 3-(2-~!ylid~ ' ' - )propionyl (PDP) derivative of the therapeutic agent cam be prepared through a reætive amino or hydroxy group present on the therapeutic agent (e.g. the N4 amino or 5' hydroxy group of a nucleoside 25 analog). The PDP derivative of the therapeutic agent is then coupled to an ASGR ligand or a derivative thereof which reacts with thiol groups. For example, the PDP derivative of a generic nucleoside amalog can be coupled to ASO~ as follows:
O O
~;~ ~S~o~\<OYB ASOR~ `S~O~\~B
)~ + SH~ASOR ~ X Y
nucleoside-PDP nucleoside-DP-ASOR
A thiol-derivative of a therapeutic agent cam also be coupled to a Iyru~ iul~dl carrier molecule with reactive thiol groups. For example, thiolated derivates WO 95/lX636 , ~
of a polyamino acid (e.g., polylysine or pol~ .,) can be prepared by reacting the polyamino acid with SPDP to form a PDP-derivative of the polyamino acid. The PDP-derivative of the polyamino acid can th~n be coupled to the PDP-derivative of the therapeutic agent by reduction of one of the PDP-derivatives followed by a thiol exchange reaction.
S Alternatively, a thiolized ~uly~ ridt, such as a thiolized dextran, can be used as a polyru~ Livllal carrier molecule with a Ihiol-containing crosslinker.
F. rolyr~.,. I;..,.~C7~i.~rswifhl\/' "i'-A~ rol~c A polyrulll,livl~l carrier molecule with multiple amino groups can be conjugated to an ASGR ligand by C~ub~ coupling to form amide bonds between the amino groups of the carrier and carbox~l side chairls or C-terminus of the ligand.
, the pOlyrl ~liv~l~l carriel molecule or ligand can be derivatized to allow coupling of the carrier and the ligand via functional groups other than the amino groups of the 15 carrier and the carboxyl groups of the ligand. For example, a thiol derivative of the carrier can be made and coupled to the ligand ~y a thio-ether linkage. Alternatively, a hydrazide derivative can be used.
Preferred carrier molecules with multiple amino groups are polyamino acids 20 such as polylysine and poly. ' P'referably, the amino acids of the polymer are the naturally-occurring L amino acids (e.g., poly-L-amino acids such as poly-L-lysine or poly-L-ornithine). For example, poly-L-lysine can be conjugated to ASOR as described in Example I and in U. S. Patent Application Serial No. 08/043,008 by Findeis et al., i.,...,~ .1 herein by reference. A carrier-ligand complex can be reæted with a therapeutic agent or with a 25 crosslinker-therapeutic agent complex ~which reæts with amino groups in order to conjugate the therapeutic agent to the ligand via the carrier. For example, a phosphate, glut7rate or succinate derivative of a therapeutic age.nt can be conjugated to a carrier having multiple amino groups as described above and in Examples 1-3.
Preferably, the pOI~rull~,livl~l carrier molecule with multiple amino groups is a polymer, such as a poly amino acid wl.th repeating amino æid residues. The carrying capæity of a polymeric carrier molecul~: (i.e., the number of molecules which can be coupled to the carrier) is a function of the numb( r of reactive groups present on the molecule, which increases as the size of the polymer increases. Therefore, the carrying capæity of a carrier molecule can be increased by using a laLger (i.e., greater molecular weight) carrier. For example, poly-L-lysine of about 4000 d~ltons can be used in conjugates of the invention, or for a greater carrying capacity, poly-L-l~sine of 10,000 daltons can be used. Poly-L-lysine of up to about 60,000 daltons can be used in the conjugates. Thus, the molar s~lhctiflltion ratio ` 21 80348 WO95/18636 J~ I/u.,. I ~
/~
of a carrier-containing conjugate can be increased by increasing the siæ of the catrier (see Example 9, Table 1).
F. Pvlyr~ rriP.~ with Ml.ltil.lP ~'~rbnyyl ~ro~,ne A pvl~ ~ ' carrier molecule with multiple carboxyl groups can also be used in the conjugate of the invention. Preferred carrier molecules with multiple carboxyl grvups are polyamino acids such as polyglutamic æid and ,uul.~ a~ , acid. Preferably, the polyamino acids are poly-L-amino æids, such as poly-L-glutamic acid or poly-L-aspartic 10 acid. For example, a therapeutic agent with reactive amino groups (e.g., araC) can be conjugated to a poly-L-glutamic acid carrier and the therapeutic agent-PLGA complex can be conjugated to ASOR as described in l~xample 7. A ~vlyrull~ivllal carrier molecule with multiple carboxy groups can be conjugated to an ASGR ligand by ~ . coupling to form amide bonds between the carboxyl groups of the carrier and amino side chains of the5 ligand. Alternatively, a crosslinker which reæts with carboxyl groups can be used as an y between the therapeutic agent and the car~ier with multiple reactive carboxy groups. For example, an aminoacyl or peptide derivative of a therapeutic agent cam be conjugated to a carrier having multiple carboxyl groups.
2û As discussed above for carriers with multiple amino groups, the carrying capacity of a carrier molecule with multiple carboxyl groups c m be increased by using a larger (i.e., greater molecular weight) carrier and thus the molar ratio of a carrier-containing conjugate can be increased by increasing the size of the car~ier. For example, poly-L-glutamic æid of about 14,000 daltons can be used in conjugate and a molar~lhstitl~tinn ratio (~L Ug.Uall;~.l) of 29 can be achieved with this size carrier (see Example 7).
Poly-L-glut~mic acid of up to about 60,000 daltons can be used in the conjugates.
n Polyrl~ C:~riP~.~ with ~' "i ' AlAPh,y~lP ('..o.~c Certain polymeric carrier molecules allow for: ; . ,, of both the therapeutic agent and the ligand directly to the catrier molecule without the need for a crosslinker molecule as a bridging agent between the agent and the carrier. For example, a therapeutic agent with a reactive amino group can be conjugated to a carrier with multiple aldehyde residues by reductive amination. Additionally, a therapeutic agent with a hydrazone or hydrazide group can be coupled to a carrier with multiple aldehyde residues. A ligand car~
also be conjugated to the carrier through amino groups present on the ligand (e.g., atnino groups of Iysine side chains of the ~ ,vlJIut~hl, such as ASOR) with ~oly ' ' ' yd~ residues on the carrier by reductive amination. A preferred polymeric carrier molecule with polyaldehyde groups is a,uvl~,a~,lla.;dt, such as polyaldehyde dextran. rvly~ dt -~ W0 95/18636 r~ . t l~
/~
dextran can be prepared from dextran by standard procedures (Bernstein, K, et al., (1978) J.
Na~L Cancer Inst. ~iQa):379-384; Foster, RL. (1975) Experientia, 772-773) as described in Example 7. A therapeutic agent, such as a nucleoside analog, and a ligand, such as ASOR, can be then be conjugated to polyaldeh yde dextran as described in Example 8. For example, 5 araC and ASOR can be conjugsted to polyaldehyde dextran as follows:
-f_o ~0 HO~ l'AD l~do ~ \ ~R
araC
HO

Alternative to directly ~ , _ ,, the therapeutic agent to the carrier with multiple aldehyde groups, a crosslinker can be used as an ' y between the therapeutic agent and the carrier. For c~:ample, a derivative of the therapeutic agent which provides a reactive amino group (i.e., a~ amino derivative such as an aminoacyl compound), 15 a reactive hydrazine group or a reactive hydrazide group can be used to crosslink the therapeutic agent to a carrier with multil~le reactive aldehyde groups.
IV. Conl?lin~ S
~rhe particular coupling strategy used to prepare a conjugate of the invention, that is, the particular crosslinker amd/or l~olyrul~ iu~lal carrier molecule used to conjugate a therapeutic agent to a ligand for ASGR ~e.g., ASOR), will depend in part on the chemical structure of the therapeutic agent to be conjugated and thus can vary with different therapeutic agents. However, the coupling strategies used in the invention can be applied to a wide range of therapeutic agents. Therapeutic agents with a reactive annino, hydroxy, carboxyl, II~IIUnYIa~ IO~ hydrazo or sulfhydryl group can be conjugated to a crosslinker or carrier molecule according to one or mo~re of the coupling strategies described in the invention. When a tnerapeutic agent has multiple reactive groups, protecting agents can be ~ 21 80348 ~ ;~o used (as described above and in the Examples) to direct a coupling reaction to a particular reactive group and then the protecting agent can be removed. When the ASGR ligand to be used in the conjugate is a ~Iyuu~.u,u;l,, e.g., ASOE~, the ligand possesses reactive amino groups and carboxyl groups, and possibly reactive sulfhydryl groups, from the side chains ûf 5 amino acids and the N- amd C-terminal ends of the ~Iy~,u~ulu~ . Thus, wuaal;l~tla which react with any of these functional groups can be coupled tû the ligand. Likewise, Uùlyru~ iu~lal carrier molecules with either multiple amino groups, multiple carboxyl groups or multiple aldehyde groups can be coupled to the ligand (for example, ASOR can be conjugated to polylysine, puly~,lu~lu~, acid or pulyalJ~,lly~ dextran as described in the 10 Examples). When both a crûsslirlker and a carrier molecule are used in the conjugate, an appropriate ~ nn of crosslinxer and carrier are chosen. For example, a crosslinker which reacts with amino groups is used with a carrier molecule having multiple amino groups. An appropriate C ~ can be selected from the groups of crosslinkers and carriers shown below:

Alninn_~ r,tiV" Crncclinkprs pol~ rriPrc ,qlhu~a~,y; (e.g., glutarate; succinate) poly-L-lysine phosphate poly-L-ornithine Alternatively, a crosslinker which reacts with carboxyl groups is used with a carrier molecule having multiple carboxyl groups. An appropriate . ' can be selected from the groups of ~., " ' and carriers shown below:
r~r~r~yl-~P -rtive Crr,cqlinl~prc r,~ r~iPr5 25aminoacyl (e.g., AMCC, GABA) poly-L-glutamic acid peptide (e.g., Leu-Ala-Leu) poly-L-aspartic acid V. Artiviw of the C~
The activity of the conjugates of the invention has essentially two ,r~ the targeting activity of the ligand and the therapeutic activity of the therapeutic agent. The targeting activity of the ligand carl be assessed in vivo by ' ~ the conjugate ;llila~lùu~l~ into a subject (e.g., a mammal) and then measuring the rate of 35 clearance of the conjugate from the circulation and/or measuring the association of the conjugate with target cells which express ~ V~IU~.~;II receptors, for example liver cells. The clearance of the conjugate from the circulation and association of the conjugate with target cells can be compared relative to a non-conjugated therapeutic agent and relative to the association of the conjugate with other organs. A conjugate can be directly detected by _, _ . ~ , . . .

~ WO 9~/18636 1 ~l/U.. ~
~ /
labeling it with a detectable substance, for example a radioactive isotope, to follow its distibution in a subject. For example, a therapeutic agent can be labeled with a radioactive isotope such as tritium or 14c or the ligand can be labeled with 1251 Alternatively, the distribution of a conjugate can be assessed by it~c ability to cvlll,u~,~iLi ~ ~,ly inhibit the binding of amather ' ~o~/cul~lut~,;.. to ASGR (see for example Keenan-Rogers, V. and Wu, G.Y. (1990) Cancer Cl~emother. Pharmacol. 26:93-96). In this case, an unlabeled conjugate is ù~ ' cd with labeled ~ .,u,ulutc;ll. For exa[nple, an unlabeled ASOR-containing conjug~,te can be . . . -- l . ., ' t. cJ with a labeled ~ ~fpfllin lû The clearance ûf the labeled: ' ' , '~ JlVtC;.l from the circulation and/or the association of the labeled ' ol~,u~lut~ with the lliver is measured with and without c.~
of the conjugate. A conjugate which is effectively targeted to liver cells will decrease the rate of clearance of the labeled ' "~-,U,U:U.~,;II from the circulation and decrease the association of the labeled: ~ .,U~lVt~;ll v~ith the liver by competing with the labeled 5 : ' '~ vulv;c;ll for binding to asialoOly~u~lut~.;ll receptors on liver cells.
Additionally, the amoun~ of a conjugate in the circulation can be assessed by ;... ~.. I~lhO;. -I or chemical methods. For example, plasma samples can be collected at various times following illL-~ UU:i injection of the conjugate and the amount of conjugate 20 present therein can be determined by Hl~LC or by an i. . ~ol~ assay, such as a .,.. 1;.. ;" .. `~.. J or ELISA (for example, using an antibody against the ligand or carrier portionoftheconjugate). Ful~ ulc~the/1ictnhllt~ oftheconiugatecanbeassessedby ' -~ , ' - or nuclear imaging methods using a ~ conjugate (for instance a conjugate in which the ligand is labeled with 125I).
The activity of the therapeutic agent in a conjugate can be assessed by measuring the therapeutic ~rf; ~ of the conjugate agamst a disease or disorder to be treated by the therapeutic agent using ar~ appropriate assay. For example, the antiviral activity of an anti-viral agent c~m be determined by measuring the amount of viral DNA
30 replication or viral particle (or marker) I~roduction which occurs in the presence or absence of the conjugate relative to the ~ ;, ' therapeutic agcnt. The effect of a conjugate on viral DNA replication can be assessed i~7 vitro using a virally-infected cell line which expresses ' ,,~y.,u~ulvtc;ll receptors. For example, a hepatocyte cell line can be used.
Hepatocyte cell lines have been transfected with hepatitis B virus DNA to create stable cell 35 lines wbich trmscribe HBV genes, translate HBV proteins and accumulate HBV DNA
replicative ;.. . ,., ~1: f~ ~ Such cell lines can be used to assess the anti-viral activity of conjugates. Appropriate HBV DNA-col1taining cell lines which can be used include the human ~ 1 ' (HepG2)-derive~l cell line, 2.2.15 (Sells, M.A., et al., (1987) Proc.
~Vatl Acad Sci. USA 84:1005-1009; Sells, M.A., et al., (1988) J. ViroZ ~Z:2336-2344) and 21 8~348 WO g~/18636 r~l~uv.
the human I . I ' (Huh 6)-derived cell line HB 611 (Tsurimoto, T., et al. (1987)Proc. NatL Acad Sci. US~ 84:444-448).
Viral DNA-containing cells in vitro can be treated with various, 5 of a conjugate and the cu~ therapeutic agent and the effect of the treatments on in~ r~ r and/or . ~ viral DNA production can be ~1rtrtmin~
TntrP~ llPr DNA can be isolated from cells and l~Ytrpr~ llrr DNA can be isolated from the culture medium. The DNA can then be analyzed by a hybridization procedure (e.g., dot blot hybridization, Southern blot etc.) or other appropriate DNA analysis procedure. For example, 10 assays such as those described by Korba, B.E. and Gerin, J.L. ((1992) Anti-viral Research 1.:55-70) and Ueda et al. ((1989)Virolo~,v 169.213-216) c~m be used. In the case of hepatitis B virus, the effect of the free and conjugated agent on different forms of HBV DNA can be measured. For example, the r ' " of relaxed circular DNA, replicative and integrated HBV DNA can be determined as described in Example 10. Since tbe amount 15 of integrated (i.e, non-replicating) HBV DNA should not change upon treatment with either the free or conjugated agent, this DNA can be used as an internal control. The IDso (i.e., dose necessary to inhibit 50 % of the viral DNA replication) can be determined for the conjugated and ~ ; ~ ' agent to assess the therapeutic ~ ,;,a of the conjugate.
Additionally, production of viral antigens in vitro can be assessed to determine the 20 therapeutic ~ Li~ a ofthe conjugate.
The ability of the conjugates of the invention to target a therapeutic agent to a cell expressing ~ah~ ly~,uulut~,;ll receptors can be assessed using cells in culture by comparing the ;y ~u~u~i~,;Ly of the conjugates for ASGR+ cells to the ~;y~u~u~d~ y of the5 conjugates for ASGR- cells. (This can then be compared to the uy ~uLv~i.,;~y of the b~ ~ I therapeutic agent for ASGR+ and ASGR- cells as a control). At a given dosage, a conjugate that is effectively targeted to ASGR+ cells will be taken up to a greater extent by ASGR+ cells th~m by ASGR- cells. Thus, a conjugate which effectively targets a therapeutic agent to ASGR+ cells will be cytotoxic for ASGR+ cells at a lower dosage than is 30 needed to kill ASGR- cells. The cytoxicity of the conjugates of the invention can be measured using ASGR+ and ASGR- cells as described in Example 10.
An appropriate animal model of a viraT human disease can also be used to assess the anti-viral activity of anti-viraT agent conjugates in vivo. For example, the effect of 35 conjugated anti-viral agents cam be assessed in Ectromelia virus-infected mice (for example, see Fiume, L., et aT. (1981) FEBS Letters 129:261-264). Appropriate animal models exist for human hepatitis virus infection. For example, one animal model system for human hepatitis is woodchuck hepatitis virus (WHV)-infected ~ oo~l~hl-rk~ Similar to humans, the Eastern woodchuck (Marmora monax) can be chronically infected with WHV. The genomic .

- 21 ~0348 W0 95/18636 P~ 3 flr~?--i7:1tif/n of WHV is identical to H13V and the virological ~ of the two diseases are similar (Summers, J., et al (1975) Proc. I`~atl. Acad Sci. USA ~:4533-4537;
Galibert, F., et al. (1982) ~ Virol. 41:51-65; Wong, D.C., et al., (1982) J. Clin. Microbiol.
15:484-490; Pon_etto, A., et al. ( 1984) J: Virol. 52:70-76; Pon_etto, A., et al. (1985) Virus S Res. ~:301-315). Virally-infected anir~als cam be injected . ~,uuu~ly with a conjugate o}
the CUIIC~ '- ~ free drug. Plasma levels of the conjugate can be measured as described above. The effect of the conjugated ve~sus ~ ,.... j y,. ~I agent on viral DNA replication can be 1' 1, for example, by measuring serum levels of viral DNA. Other appropriate animal models for human hepatitis virus infection include duck hepatitis virus infection (Civitico, G., et al. (1990) J. Med Virol. 31:90-97), ground squirrel hepatitis virus infection (Marion, P.L. et al. (1983) ~epatolo~v _:519-527) and, most preferably, infection of with human hepatitis virus (Thung, S.N. et al. (1981) Am. J. Pathology 105:328-332; Shouval, D, et al. (1980) Proc. ~atL Acad Sci. USA lI:6147-6151).
The therapeutic activity of conjugates can also be assessed in vivo in human subjects. For example, humans chronically infected with HBV can be treated with a conjugate or the f ~ r ~ ~ Ull~,UII; Ll~ ,d agent. The effect of the conjugate on HBV
infection can be determined by measuring the effect of the conjugate on one or more HBV
markers, such as HBsAg, anti-HBs, HBeAg, anti-HBe, anti-HBc or HBV DNA during the 20 course of treatment.
Vl. UsP~ f~f thr C~
The conjugates of the invention can be used to target a therapeutic agent to a 25 cell of interest, i.e., a cell which expresses ' ~I~I,U~ ' receptors and to which delivery of the tberapeutic agent is desired for th~erapeutic purposes. A ' ~ ,U,U~U..,;II receptors are expressed on ' . ~ ,.,. and thus a co]ljugate can target a therepeutic agent to ~
Galactosyl receptors have been reported to be present on rat testicular cells (Abdullah, M., et al.(l989)~ CellBioL 108:367-375)butthesereceptorsarethoughttodifferstructurally 30 from hepatic ASGRs (i.e, be only partial receptors). Thus, the conjugates of the rnvention are not likely to be targeted to testicular cells. The conjugates of the invention therefore can be used to target a therapeutic agent selectiively to I . ~. For example, conjugates comprising an anti-viral drug can be targeted to virally-infected L~.IJalu~ .,. Alternatively, a cell can be engineered to express ASGR, for example by introducing into the cell a nucleic 35 acid encoding the asialo~;ly.,u,ulu.~,.h- ref,eptor in a form suitable for expression of ASGR on the cell surface, to convert the cell into a target cell for the conjugates of the invention.
The conjugates can also be used to elicit a desired therapeutic effect in a subject. For example, a conjugate coml1rising an anti-viral drug can be used to treat a viral WO 95/18636 1 ~
,ty infection, such as to decrease replication of viral DNA, inhibit viral palticle replication and production, reduce symptoms of viral infection, etc. Because the conjugates are targeted to a tissue of interest and away from unaffected tissue (e.g., non-hepatic tissue), the non-specific toxicity of the therapeutic agent is diminished compared to the I . v ' agent.
5 Additionally, because ~ ;, . of a therapeutic agent to a targeting ligand results in increased delivery of the agent to the cell(s) of interest compared to I ~ ' agent, the therapeutic index of the agent is mcreased, thereby providing Ih ~ ly effective dosages at c.. " ,. . . -1;.,, ,~ lower than is needed with the I ~ ~ ' agent.
l O The conjugates of the invention are ' ~I to subjects in a biologically compatible form suitable for~l --"._....1;..1 i~.l.";":~,.l;t~n in vivo to target the therapeutic agent to cells expressing ~ yc~ V~;ll receptors. By "biologically compatible form suitable for ~ ;. - in vivo" is meant a form of the conjugate to be ad.~ t~,lcd in which any toxic effects are outweighed by the therapeutic effects of the conjugate. The term lS subject is intended to include living organisms in which a therapeutic agent can be targeted to cells expressing asialo~ ,v~ t~,;.. receptors, e.g., marnmals. Examples of subjects include humans, ~.,,,~.1.1".. ~ dogs, cats, mice, rats, andtransgenic speciesthereof. ~1,.,;..;~1.,.1;~", of a conjugate as described herein can be in any p~ I form including a Ih. .,.~ active amount of conjugate alone or in c~nmhinRtinn with another therapeutic 20 agent and a ~ t;~ y acceptable carrier. For example, a conjugate of the invention can be ~o I -;..: .t. ~J with another therapeutic agent effective against a particular disease or condition to be treated. For example, a conjugate containing an anti-viral agent (e.g., a nucleoside analog) which is effective against hepatitis B virus can be R~mini~fr~ together with an interferon, since interferons have also shown therapeutic activity against hepatitis B
25 virus infection.
,A~' ' ' ' of a ll~ ; -lly active arnount of the coniugates of the invention is deflned as an effective amount, at dosages and for periods of time, necessary to achieve the desired result. For example, a 11.. ' "1'` - ; ~IIY active arnount of a conjugate may vary 30 according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the conjugate to elicit a desired response in the individual. Dosage regimens may be adjusted to provide the optimum therapeutic response. For example, several divided doses may be dlll .Il.,t~,lcd daily or the dose may be ~ Jul ~ivllally reduced as indicated by the exigencies of the therapeutic situation.
The active compound (e.g., conjugate) is preferably ,..~., .:-~. .~;i IVU~Iy (e.g., by injection). The active conjugate may be coated with or C.n~ l.";": ~. .~ul with a material to protect the conjugate from the action of enzymes, acids and other natural conditions which may inactivate the conjugate. For example, a conjugate may W0 9V/18636 I ~
be d~LI;IuD~I~d to an individual in an appropriate carrier or diluent and co-a.LI,;.~ lc.i widh enzyrne inhibitors. F' Ily acceptable diluents include saline and aqueous buffersolutions. Dispersions can also be prepared in glycerol, liquid polyedhylene g~ycols, and mixtures dhereof and in oils. Under ordinary conditions of storave amd use, dhese lu~ Lul~D
S may contain a IJl~DI..I V_l; V' to prevent dle growth of Ill;~,lVI v r~ - suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for dhe ..... ,.I.. A"~v~ preparation of sterile ilnjectable solutions or dispersion. In all cases, dhe 10 ~ must be sterile and must be fluid to dhe extent dhat easy syringability exists. It must be stable under dle conditions of I~ ul c and storage and must be preserved against dhe, . .. ,1_.,, ;. .AI; l v action of III;~.IU~ v ' such as bacteria and fimgi. iAhe carrier can be a solvent or dispersion medium containing, for example, water, edlanol, polyol (for example, glycerol, propylene glycol, and liquid p~ ,LI~h~ glycol, and dhe like), and suitable 15 mixtures dhereof. Al he proper fluidity can be mo;A~oin~rl for example, by dhe use of a coating such as lecidlin, by dhe of the required pa~ticle size in the case of dispersion and by dhe use of surfact~mts. Prevention of dhe action of UUI~_I;DIIID can be achieved by various .. ~ ;AI and antifimgal agents, for example, parabens, ~ lulvvu~lvl, phenol, asorbic acid, dlimerosal, and dhe like. Al`he osmolarity ûf dhe . ~ can be maintained 20 in a physiological r~mge by inclusion of appropriate amoumts of compoumds such as sugars, pol~ - (e.g, mannitol or sorbitol) or sodium chloride in dhe . Prolonged absorption of dhe nnjectable c. ~ can be brought about by including in dhe an agent which delays absorption, for example, aluminum mnnn and gelatin.
Sterile nnjectable solutiolls can be prepared by . v dhe conjugate in dhe required amount in an appropriate solvent with one or a ' of ingredients I above, as requnred, followeli by filtered ~ Generally, dispersions are prepared by; ~ Av dhe active compûumd into a sterile vehicle which contains a basic 30 dispersion mediurn amd dhe required odher ingredients from dhose ~ ' above. In dhe case of sterile powders for dhe preparation of sterile injectable solutions, dhe preferred medlods of preparation are vacuum drying and freeze-drying which yields a powder of dhe active ingredient (e.g., conjugate) plus any additional desired ingredient from a previously sterile-filtered solution dhereof.
It is especially ad~ v to formulate parenteral . in dosage unit form for ease of r ' ' ' ' ' ' and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for dhe 1,, ""AI;A,, subjects to be treated; each unit containing a ~ ' ' quantity of active compound calculated to WO 95118636 ~ I ~
,%6 produce the desired therapeutic effect in association with the required ~ f i. -I carrier.
The ~ . . ri A~ for the dosage unit forms of the invention are dictated by and directly dependent on (a) the unique I~ of the active compound and the particular therapeutic effect to be achieved, and (b) the limitations inherent in the art Of ~ .v~
5 such an active compound for the treatment of sensitivity in ulJ; v ' This invention is further illustrated by the following examples wmch should not be construed as limiting. The contents of all patents, references and published patent Al ~ cited 10 throughout this application are hereby il~culuul~ by reference.
The follûwing general ", II~ Y was used in the Examples.
~lPnPrAAl Mptho~lc Thin layer clu~ O , ' y (I~C) was performed using glass backed 15 BakerTM SI250F silica gel plates. Visualization was by ultraviolet irradiation or by dipping in an aqueous solution of 4.8 % ammonium molybdate, 0.2 % ceric ammonium nitrate, and 10 % sulfuric acid, followed by heating. Melting points were determined in capillary tubes using a Mel-Temp apparatus and are ullcul.c~,tcJ. Dialysis was carried out in Spectrapor 12,000-14,000 MWCO tubing at 4 C unless otherwise indicated. Ultraviolet/visible spectra 20 were acquired on a Beckman DU70 ~.u~ . Olu~u~llucoid was purified from outdated plasma obtained from the American Red Cross Blood Bank. It was desialated at 80 C, pH 1-2 for 60 min. Gel el~ u~llul~ was carried out on a Novex Xcell llrM mini-gel system using 12 % SDS-PAGE denaturing l~oluc.luc;llg gels, unless otherwise indicated.
25 Polylysine, dextran, EDC, and the antiviral drugs araA, a~aC, ddC, acyclovir, araAMP, araCMP and AZT were obtained from Sigma. Other reagents were obtained from Aldrich.
The following abbreviations are used in the Examples and throughout the rl.l .l i. ~l ;....
30 ACV acyclovir ACVMP acyclovir r~ A 1 '`i AMCC ~u~;llVI~ dc~ bu~ylic acid araA adenosine ~r:lhinr~ 1p araAMP adenosine arabinoside ~llul~ul l . ' 35 araC cytosine arabinoside ASOR I - ' u ~v~u.,u;d DCC .1;~.,lVII~ b~ I;;,., IP
ddC dideoxycytidine DMAP d;lll~,lll~l~ll;~lu,u~l;dine .

WO 95118636 1~ I
DMF ," ylr~
DMSO diu~ Jl~ulru~-de EDC 1-(3~ lyllllulv~Jlu~Jyl)-3-et~lylru~hn~ m~ L~llv~,lllulil~
EDTA ~IIIyl l;-.l;., t~ ; æid 5FABMS fastatom~ ' ' mass~.~ ulll.~,ly HBV hepatitis B virus MES 2-[~v-MA~rrh~ Ar~ r acid MSR molar ~ . ratio MWCO molecular weight cutûff 10PAD l,vl~ . ' ' ' y~ dextran PAGE l.oly~l~l~ll;l~ gel cl.,~,llv~llul~,s;a PBS phûsphate buffered saline PLL poly-L-lysine PLGA poly-L-glutamic acid 15PMS phenazinem~th SDS sodium dodecyl sulfate SPDP lV-Su.,~u ull;dyl 3-(2-~uyl;Jykl;Lll;o)propionate TLC thin layer chr.-,m-t.-gr_rhy Tris tris(LrlL
20XTT 2,3-bis(2-methoxy4-nitro-5 ~ l) S [(pll~llyl-luu~.~)carbollyl]-2H ~ vl;
hydroxide 25F.X~MPI,~ l Pl ., of D~rug -'~ _ using a Glutarate Crosslinker and a Polylysine C~rrier In tbis example, asialoulu~vlllu~.u;d was prepared from Ul~ ' and tben conjugated to a pùly ' carrier molecule with reactive amino groups, polylysine.
30 Glutarate derivatives of different nucleoside analogs were prepared to provide a crosslinker which reæts with amino groups to enable CUII; _ " of the nucleoside analûgs to the polylysinc ~;dIOUIV:~VIIIU~VhI complex. The glutarate derivatives of the nucleoside analogs were conjugated to the pOIyl~ ~LI.,-L~ ~VVI- ' by ætive ester co~pling. Glutarate derivatives of the nucleoside analogs araC and araA were prepared by . ,~ ; r~ "c of 35 previously described procedures and coupled to polyl~ ., V~VIIIUI~V;d. Glut rate derivatives of the nucleoside analogs ddC, ACV and AZT were prepared as described herein and coupled to pVIyly ~-a5 ~uul1 ' W095/18636 r,l~l 5 tlE O
~Y
C ~ ~ a-l-a~`iA ~ . tl 1,), Olu~ulll~oid (OR) was isolated from human plasma.Human plasma was obtained from the American Red Cross Blood Services, Farmington, CT.
Pooled humam plasma (4 units, - I . I L) was transfcrred to dialysis tubing (12-14 Kd MWCO) and dialyzed ûver~ught at 4 CC against 20 L of Buffer I (Buffer 1: 0.05M NaOAc, pH 4.5).
The dialyzed plasma was then centrifuged at 10,000 rpm (15,000 x g) for 10 minutes at 4 C.
The supernat~mt was then filtered through Whatman #I paper and the precipitate was discarded. the dialyzed and filtered plasma was applied to the DEAE-cellulose column. The column was prepared by suspending DEAE-cellulose (84g) in water, allowing it to swell for 2 h, and then washing ~u~ .,ly with 0.5 N HCI, 0.5 N NaOH, and 0.01 M EDTA. The DEAE-cellulose was poured to a bed volume of 5 cm x 25 cm in a Waters AP-5 column.
Using a peristaltic pump (10 mL/min flow rate) the column was ~ I with Buffer I
until the pH of the column eluate was 4.5. After the dialyzed amd filtered plasma was applied to the column, the column was then washed with Buffer I umtil the eluate has an absorbance at 280 nm of less than 0. 10. The column was then eluted with Buffer 2 (Buffer 2: 0. 1 0 M
NaAc, pH 4.0). The eluate was collected starting when the A280 began to increase and ending after the A280 had peaked amd was < 0.10. After the ulu ~u~ ,uid-rich fraction had be2n eluted and collected, the column was washed with Buffer 3 (Buffer 3: 0.05 M NaAc, pH
3.0; I L), and rPPq~lil ' ' with Buffer 1.
The Ul~ ' ' rich eluate was brought to 50 % saturation with ammonium sulfate (31 3g/L of eluate) and stirred overnight at 4 C. This solution was then centrifuged (14,000 rpm x 15 min, 4 C) and the supernatant retained. Ammonium sulfate (320 g/L of 50 % saturation ~ ) was slowly added to bring the solution to 92 % saturation. This solution was then stirred for at least 4 h at 4 C and then centrifuged (10,000 rpm x 30 mm,

4 C). The pellet was retained and dissolved in a minimal volume of water and tr msferred to dialysis tubing, leaving a 3-fold volume for expamsion of the dialysate, and dialyæd for 2 days at 4 C agairlst 20 L of water (the water was changed after I day). The resulting dialysate was Iyophiliæd and stored at -20 C. The OR was run on SDS-PAGE and showed a single band at MW = 44 Kd (OR has a MW of 41,000 but runs on SDS-PAGE with an increased apparent MW) by staining with Coomasie blue. The typical yield of Iyophiliæd salt-free OR using tbis procedure is 350-400 mg.
A ' ~ ' A ' u~u~llu~,u;d was prepared from OR which was isolated as described above. ~R was dissolved in water (10 mg/ml) and an equal volume of 0.1 N
H2SO4 was added to the OR solution and the resulting mixture was heated at 80 C for I h in a water bath to hydrolyze sialic acids from the protein. The acidolysis mixture was remûved from the water bath, neutralized with NaOH, dialyæd against water for 2 days and then Iyophiliæd. The ~ acid assay of Warren was then used to verify desialation of the OR (Sambrook, J., et al. (1989) IvlolPr~ r Cl ~ 2rl~1 F~l Cold Spring HarborLaboratory Press: Cold Spring Harbor. Chapter 6). Targetability of ASOR samples was W0 95118636 r~ u...~ F
verified by labeling with 125I measurillg liver uptake in rats/mice (Cristiano, R.J., et al.
(1993) Proc NatL Acad. Sci. USA, 90:2122-2126.).
Poiy-L-lysin~ m7rljl~t~ (PLT~-A~OR;)~ Thepoly-L-lysine-~ ' uav~ ,v;J conjugate (PLL-ASOR) was prepared by .,~ubodiillJ;~c coupling as follows. A ' ~ ' (200 mg, prepared as described above) and poly-L-lysine (160 mg) were dissolved in water (20 mL) and the pH adjusted to 7.5 with sodium hydroxide.
EDC (94 mg) was added and the pH again adjusted to 7.5. The solution was stirred 16 h at 25 C. Conjuages made using 4 Kd polylysine were then dialyzed sequentially against 4 L of I M guanidine, 4 L of I M sodium chloride, and 2 x 20 L of water, and Iyophilized.
Conjugates made using 10 Kd polylysine were purified by preparative acid-urea gel ,llU~llVI~,a;a and then dialyzed agaillst 4 L of I M sodium chloride, 20 L of water and Iyophilized.
(N4 (4-C~b~ ylyl)-l-~-D-~ r~ yh,yl~ (araCell-t~rPtr). araCglutarate was prepared according to Ishida, T., et al., U.S. Patent No. 3,991,045. Briefly, 300 mg of l-,~-D-,rl" ~ yl~ va;lA. was dissolve~ in 1.6 ml of water, and 5 ml of dioxane ws added, followed by further addition of 415 mg of glutaric arlhydride. The mixture was stirred at room ~ for 48 hours. The reaction mixture was ~ ' at reduced pressure at 60 C to obtain a solid residue. The re~;idue was dried ir~ a vacuum desiccator to obtain a colorless transparent jelly-like substance. CL. . ~"y~ y on flash silica gel using ethyl acetate-methanol-acetic acid afforded the product (30 %). IH-NMR (CD30D) o l.76 (m, 2H), 2.10 (t, 2H), 2.35 (t, 2H), 4.05 (s, 2H), 4.28 (m, 3H), 5.91 (d, IH), 6.08 (d, IH), 7.56 (d, IH).
ara('~ ~t~-PTiT -A~OR. aro('glllta~lt~ (10 mg, 0.03 mmol; prepared as described above) was then dissolved in DMSO (0.25 mL). N-llydlu~ y ~ (4 mg, 0.03 mmol) and EDC (7 mg, 0.036 mmol) were added and the solution was stirred for 16 h. The solution was then applied directly to a flash silica gel column (10 mm diameter) and eluted with ~ vl~r(~"~ h .-1(85:15). ThepartiaTlypureN-l~yvlv~y~ llidylester(TLC, meth mol-acetic acid, 80:15:5, Rf = 0.24) thus obtained (20 ,umol) was dissolvedin DMSO (300 IlL) and added dropwise to a solution of PLL-ASOR (10 mg in 400 ~LL, pH
adusted to 7.5 with I N NaOH) at 4 C with vigorous stirring. After 4 h the solution was applied to a Sephadex G25 colun~n and eluted with PBS, pH = 6.8. The absorbance of the eluent was monitored at 260 rim, and the first peak to elute was dialyzed against water (2 x 2 L) and Iyophilized. An aliquot of the Iyophilized product was clissolved to I mg/mL in water and amalyzed by L ~v;GI~;/V;a;bl~ absorption ~,u~llui~,uuy. ;l~ma~ 305, 285, 250 nm.

WO 95/18636 ~ L
;~0

5~~0~13~~ )-9-~-D-~ lhin~ aroA-~ ). araA-glutarate was prepared according to Fiume, L. et al. (1980) FEBS letters 116:185-188, using a slightly modified procedure. araA (489 mg, 1.8 mmol) was dissolved in DMF (15 ml) with warming.
Glutaric arlhydride (261 mg, 2.3 mmol) and DMAP(22 mg, 0.2 mmol) were added and the 5 solution was stirred for 18 h at 25 C. The solvent was removed in vacuo, and the oil thus obtained was ,lu~ <1 on flash silica gel with chloroform-methanol-acetic acid (80:20:5) to afford 232 mg (33 %) of araAglutarate. Analysis: IH-NMR (DMSO-d6) o 1.41 (m, 2H), 1.92 (m, 2H), 2.04 (m, 2H), 2.11 (m, 2H), 2.52 (s, IH), 3.70 (m, 2H), 3.87 (m, IH), 4.49 (m, IH), 5.13 (m, IH) 5.22 (m, IH), 5.28 (d, IH), 6.45 (d, IH), 7.29 (s, IH), 8.13 (s, 1 0 I H), 8.26 (s, IH); 13C-NMR (DMSO-d6) d 1 8, 30, 3 1 , 59, 70, 76, 79, 81 , 11 7, 1 38, 1 47, 1 5 1 , 154, 170, 172.
arn~-~' DT T -A~OR. araA-glutarate (20 ~ng, 0.05 mmol) was then dissolved in DMSO (0 7 mL). N-HydluA.~ (6 mg, 0.05 mmol) and EDC (31 mg, 0.16 rnmol) were added and the solution was stirred 16 h. The solution was then applied directly to a flash silica gel column (10 mm diameter) and eluted with ~ 1 (80 20)~ A
portion of the partially pure IV ~ Lu~ yl ester (TLC, chloroform-methanol-aceticacid, 80:20:5, Rf = 0.56) thus obtained (13 umol) in DMSO (170 ~L) was added dropwise to a solution of PLL-ASOR (10 mg in 378 IlL, pH adusted to 7.5 with I N NaOH) at 4 C with vigorous stirring. After 4 h the solution was applied to a Sephadex G25 column and eluted with PBS, pH = 6.8. The absorbance ofthe eluent was monitored at 260 nm. Fractions containing conjugate were pooled, dialyzed against water (2 x 2 L) and Iyophiliæd. An aliquot of the Iyophilized product was dissolved to I mglmL in water and analyzed by ulllaviul~llv;~;bl~ absorption a~ llu~,u~uy. ~ma~; 262 nm.
N4-(4-t'~ ycyti~line ~ ). AArgll~t -~tf- was prepared as follows. Glutaric anhydride (123 mg, 1.08 mmol) was added to a solution of 2',3'-didc~ ;d;lA~ (189 mg, 0.90 mmol) in DMF (8 mL), and the solution was stirred for 16 h at 25 C. The solvent was removed in vacuo, affording a crystalline gum. Crystallization from 95 % ethanol afforded 157 mg of the product along with a small amount of impurity, as determined by TLC R~ aLiull gave 129 mg (44 %) of pure N4-(4-~afl~w~ybulyl-yl)-23'-dideoxycytidine. Analysis: TLC, Rf (chloroform ~ ~I ncetic acid, 80:20:5): 0.26;
Melting point: 149 - 151 C; IH-NMR (DMSO-d6/D2O) o 1.75 (t, J = 7.4 Hz, 2H), 2.23 (t, J
= 7.4 Hz, 2H), 2.42 (t, J = 7.4 Hz, 2H), 3.58 (dd, J= 3.5 Hz, 12.1 Hz), 3.75 (dd, J = 3.0 Hz, 12.1 Hz), 4.11 (m, IH), 5.92 (dd, J = 1.7 Hz, 6.5 Hz, IH), 7.20 (d, J = 7.4 Hz, IH), 8.47 (d, 7.5 Hz, IH); IR: 3408, 2933,1730,1696,1641, 1583, 1506,1394, 1321,1278, 1097, 821, 792 cm~l; W: ~ma~ 310, 273 nm.

-~ ~V095118636 I~,l/L_ I~I';E
~/
riri~ ~ DLT -A ~OR. To a solution of N4-~4-Gubu~ y. y l)-.li.i~A ~ (7 mg, 2û
llmol),preparedasdescribedabove,imDMSO(18011L)wasaddedNh,~dlu,~---. ..;.";if(3 mg, 0.03 mmol) arld EDC (6 mg, 0.03 mmol). The solution was stirred for 2 h, then added directly to a solution of PLL-ASOR, prepared as described above, (28 mg in 400 IlL water, 5 pH adjusted to 7.5 with 0.1 N NaOH). The coupling reaction was allowed to proceed overnight at 4 C, then diluted to 2 mL and dialyzed against 2 x 2 L of PBS, followed by I x 3 L of water. The product was Iyophilized and analyzed by ultraviolet/visible absorption `1'`''~"` Vl~Y. ~max 295, 245 mm.
9-(2-(4-(`~ ,vb~ ,v)-r~ vll~ ' (,ACvel!ltslrAtf)~ Glutaricarlhydride(196 mg, 1.70 mmol) and DMAP (13 mg, 0.11 mmol) were added to a suspension of 9-(2-Lu~ u~-yl~l.,lllyl)guarline (189 mg, 0.90 mmol) in DMF (22 mL), and the suspension stirred for 16 h at 50 C. An additional 10 mg (0.1 mmol) of DMAP was added and the mixture was heated to 65 C, at which point a clear solution formed. The reaction was allowed to continue for 18 h, then the ~;olvent was removed in vacuo, to afford a thick oil.
The oil was suspended in hot eth~mûl, ~hen chilled, filtered, and washed with cold etharlol yielding a white solid 300 mg (81 ~/~). Analysis: TLC: Rf ( ' ' . ' ' I acetic acid, 80:20:5), 0.41; Melting point: 200 - 202 C; IH-NMR (DMSO-d6/D2O) o 1.69 (t, J = 7.3 Hz, 2H), 2.21 (t, J = 7.1 Hz, 2H), 2.29 (t, J = 7.5 Hz, 2H), 3.36 (bs, 2H), 4.08 (bs, 2H), 5.36 (s,2H),7.83(s,IH); IR(KBr): 3318,3142,2960,2646,1731,1413,1213,1178,1136, 1104,752,693cm~l; UV: ~ma,~270,255nm.
ACVe' ' pT.I .-A~QR. Toasolutionof9-(2-(4-1.~bu~yb~ ylu~y)-~LI.~u,.yll.~,;Lyl)guanine (40 mg, 0.12 mmol), prepared æ described above, im DMF (1.0 mL) was added N-l~u~ ~ ' (16.3 mg, 0.15 mmol) amd DCC (35.6 mg, 0.17 mmol).
The solution was stirred 18 h, filtered, and added to a solution of PLL-ASOR, prepared as described above, (10 mg in 208 IlL water, pH adjusted to 7.5 with 0.1 N NaOH). The coupling reaction was allowed to proceed 3 h, then purified on a Sephadex G25 column with PBS, pH = 6.8. The absorbance of the eluent was monitored at 260 nm. Fractions containing conjugate were pooled and analyzed b~ ~ ' v;vL,;/v; ,;blc absorption ,I~ u ,~,u~: ~ma~ 275, 245 nm.
5'-~-(4-C-l,..~b~ v)-3~ -3~ ,V~ If (~7.T ' ). Asolutionof3'-azido-3'-~".yllly.ll;li.lc (100 mg, 0.37 mmol), glutaric anhydride (94 mg, 0.83 mmol), and DMAP (45 mg, 0.37 mmol) in DMF (3 mL) was stirred for 24 h at 25 C. The solvent was removed in vacuo and the resulting oil ~.1--. " I ' ' on a 20 mm column of flasb silica gel with ~,hlul~ r methanol (9:1) as oluent. The product was isolated as an oil in 41 %
yield. TLC. chloroform-methanol (8:2), Rf 0.56; IH-NMR (DMSO-d6/D20) ~ 1.73 (t, 2H), 1.80 (s, 3H), 2. 17 (t, IH), 2.39 (m, 3H), 3.15 (s, IH), 3.97 (dd, IH), 4.25 (m, 2H), 4.45 (dd, 21 ~0348 WO 95/l8636 ~ L
lH), 6.12 (~t, IH), 7.45 (s, IH); IR: 3500, 3250, 2924, 2109,1702,1560, 1461,1408, 1262, 1096, 801 cm-l; W~ 267 nm.
~ 7.T~F' nT T .-A~OR. To a solution of A_Tglutarate (11 mg, 30 llmol) in DMSO (250 5 ,uL), prepared as described above, was added N-llydlu~... :..."..~1 (4 mg, 30 llmol) and EDC (7 mg, 36 ,umol). The solution was stirred 18 h, then purified on a column of flash silica gel with .,I.Iu,. r methanol (98:2) as eluent. The active ester thus obtained (Rf 0.35) was dissûlved in 225 IlL of DMSO and added dropwise to a solution of PL-ASOR (10 mg in 400 ~LL of water, pH adjusted to 7.5 with 0.1 N NaOH). The coupling reaction was allowed 10 to proceed 4 h at 4 C, then diluted and purified by dialysis against 6 L of PBS, pH = 6.8, followed by I x 20 L of water. The dialyzate was filtered tbrough a 0.45 ,u nylon membrane and analyzed by ~ ' viOl~t/v;a;l,l~, absorption a,u~,~,llua~ u,uy . ~aY 267 mm.
15 li`.~AMPl .T~'. 2: Pl ~. ' of Drug Conjugates using a Succirlate Crosslinker and a Pobbsine Carrier In this example, polylysine was conjugated to ' uavlllu~,Oid as described in Example 1. Succinate derivatives of different nucleoside analogs were prepared to provide 20 a crosslinker which reacts with amino groups to enable ~ of the nucleoside amalogs to the polylysine-as:L'~ul, ' complex. The succinate derivatives of the nucleoside analogs were conjugated to the polylysinc- ~uOluaulllu~uid by active ester coupling. The succinate derivative ûf the nucleoside analog ACV was prepared be a . "~ , of a previously described procedures and coupled to polylysine-: ' - Jauu~ uid. The 25 succinate derivative of the nucleoside analog araA was prepared as described herein and coupled to polylysinc-~ '- . I
9~ ~v~u~ ACVs ). ACVsuccinatewas preparedbya".~ ;..lloftheproceduredescribedinSchaefferetal.(1980)U.S.Patent No. 4, 199,574. Acyclovir (210 mg, 0.93 mmol), succinic anhydride (139 mg, 10.09 mmol) and DMAP (19 mg, 0.16 mmol) were combmed in DMF (18 mL). Tbe suspension was heated to 65 C, at which point all A ' went into solution, and $irred for 16 h. The solvent was removed in l~acuo and the resulting oil ~lu~ on flash silica gel with .,llul~ r methamol-acetic acid (80:20:5) as eluent, affording ACVsuccmate (186 mg, 61 %). Analysis: TLC: Rf (chloroform-methanol-acetic acid, 80:20:5), 0.33; Melting point: 194-196 C; IH-NMR (DMSO-d6/D20) o 2.50 (m, 4H), 3.66 (m, 2H), 4.09 (m, 2H), 5.35 (s, 2H), 7.82 (s, IH); UV: ~maX 270, 252 nm.

,~3 ACV! -PJ T -A!~OR. To a solution of ACVsuccinate (37.5 mg, 0.12 mmol; prepared as described above) in DMF (1.0 mL) was added N-l~v~u~ ' (16 mg, 0.15 mmol) and DCC (34 mg, 0.16 mmol). The salution was stirred 18 h, filtered, and added to a solution of PLL-ASOR (10 mg in I mL water, pH adjusted to 7.5 with 0.1 N NaOH). The coupling 5 reaction was allowed to proceed 3 h, tllen purified on a Sephadex G25 column with PBS, pH = 6.8. The absorbance of the eluer t was monitored at 260 ran. Fractions containing conjugate were pooled and analyzed b y ultravioleVvisible absorption au~,~,llUa~,U,u,v . ~ma~ 277, 250 nm.
5'-0-~3-C- l~ yy,~ ,v1)-9-,~-D-~ in~ 4ellrrin:1tP) araAsuccinate was prepared as follows. To a solution of 9-,~-D z . r .V~ I ' (825 mg, 3.10 mmol) in DMF (25 mL) was added succinic anhydride (342 mg, 3.42 mmol) and DMAP (38 mg, 0.31 mmol). The reaction mixture was stirred 22 h, additional succinic anhydride (342 mg) and DMAP (3 8 mg) were added, zlnd the reaction stirred 18 h. The solvent was removed 15 in vacuo and the resulting oil .,LI..,ll ~ fA on flash silica gel using chloroform-methanol-acetic acid (80:20:5). The oily residue thus obtained was dissolved in methanol and ,Ul~ , ' in ether, affording an off-white, lly~lua~,ulJ;c powder (320 mg, 28 %).
Analysis: TLC: Rf (chloroform-meth~mol-acetic acid, 80:20:5), 0.46; MP = 145-147 C;
IR(KBr): 2932(br),1701,1420,1310,1201,921,639cm-1; W: ~ma~ 259nm.
ar~A ' ~ -Pl T .-A~OR. To a solution of 5'-0-(3-c~bunyulu,u;(lllyl)-9-~-D-~.,,.l. .,,.r~ Vlai~. u,e (19 mg, 51 ~umol), prepared as described above, in DMF (475 ,uL) was added N-hv~u~ - ";Af (7.6 mg, 65 llmol) and DCC (16.2 mg, 78 ,umol). The solution was stirred 18 h, filtered, and added to a solution of PLL-ASOR (5 mg in 475 ,uL
25 water, pH adjusted to 7.5 with 0.1 N NaOH). The coupling reaction was allowed to proceed 3 h, then purified on a Sephadex G25 column with PBS, pH = 6.8. The absorbance of the eluent was monitored at 260 mn. Fraclions containing conjugate were pooled and aualyzed by ulL~Iv;~l~,l/v;a;b ~ absorption alu~ a~,u,uy: ~ma~ 262 nm.

T~'XAMPLT~' 3 ~ " of Drug Conjugates using a Pbosphate C~rosslinker and a Polylysine Carrier In this example, polylysine was conjugated to ' . ~ as described 35 in Example I . Mr -~ ~F' , ' derivatives of different nucieoside analogs were obtained or prepared to provide a crosslinker which reacts with amino groups to enable conjugation of the nucleoside analogs to the polylysine-aa;alou,. ' complex. The . ' , ' derivatives of the nucleoside analogs were conjugated to the polylysine-. I - uavlllu~.uid by UalbOd;;lllidf coupling. The )~ ' I ' derivatives of the nucleoside analogs araC

W0 95/18636 F~l/~m, ;~
and araA were obtained cu~ ly (Sigma). ACVIlwllul ' , ' was prepared as described below.
9-(2-L~ u~ u~l ~ ~' ' (ACVMP). Phosphorusu~y.,LIul;de (200 IlL, 2.1 mmol), water (23 ,uL, 1.3 mmol) and pyridine (200 ~L, 2.3 mrnol) were combined in acetonitrile (0.5 mL) at 0-4 C. Acyclovir (100 mg, 0.44 mmol) was added and the solution was stirred 4 h at 0-4 C. The solution was then added to ice-water and stirred an additional hour. The pH was ædjusted to 2 and the solution was applied to a prewashed column of charcoal-celite (2 g each). The column was further washed with 50 mL of water and the nucleoside products eluted with 50 mL of ethanol " . n~n~nil-m hydroxide(10:9:1). Thisfrætionwasevaporatedtodrynessandredissolvedin200mLofwater. The pH was ædjusted to 4 and the solution was applied to a ?5 mL column of BioRad AG1-X8 resin, formate salt. The column was washed I 11~, with 0.1 M, I M and 2 M formicacid. The IM formic acid fractions contained pure ACVMP (65 mg; 48 %). IH-NMR
(DMSO-d6/D2O) o 3.66 (m, 2H), 3.90 (m, 2H), 5.39 (s, 2H), 8.10 (s, lH).
arat',.,.",..~ -PLT.-A!~OR. I-~-D-A~ r~ v~ , ' (3.3 mg, 10 llmol; obtained ~;ullllll~ ,;ally from Sigma) and PLL-ASOR (10 mg) were dissolved in 168 ,uL water with pH adjustment to 7.5. The reætion was initiated at 4 C by addition of EDC (2.7 mg, 14 ,umol). After 2 h an additional 2.7 mg of EDC was added, and the reaction was left stirring at 4 C 16 h. The solution was diluted to 2 mL, dialyzed against I x 3 L of 0 9 % NaCI followed by I x 3 L of water, Iyophilized, and analyzed by I ' Yi~L,L/v; ,ibl~
absorption i~v~ u~ ulJy: ~ma~ 275 nm.
arn~ ' ' DL-A~OR. 9-~-D-Ar~hin' ~l~v~ ' (4 mg, 10 mol; obtained ~ullllll~ ;ally from Sigma) and PLL-ASOR (10 mg) were dissolved in 520 ~lL of water with pH adjustment to 7.5. The reætion was initiated at 4 C by addition of EDC (4 mg, 20 ,umol). After 7 h the solution was diluted to 2 mL and dialyzed against 2 x 3 L of 0.9 % NaCI followed by I x 3 L of water. The product was Iyophilized (14 mg), and analyzed by ~ " vi~h.l/~;,;'vl~ absorption S~IlU:~,U~ ma~; 260 mm.
ACV--- ' ' DLT -A~OR. ACV- , ' , ' (4 mg, 13 ,umol), prepared as described above, and PLL-ASOR (22 mg) were dissolved in 600 IlL of water with pHadjustment to 7.5. The reaction was initiated at 4 C by æddition of EDC (4.5 mg, 23 llmol).
After 7 h the solution was diluted to 2 mL and dialyzed against 2 x 3 L of 0.9 % NaC1 followed by I x 3 L of water. The product was Iyophilized (18 mg), and analyzed by av;vl~,L/v;:~;lvl~ absorption ~.,~IlU~U~).y. ~ma~ 260 mm.

~ WO 9S118636 1 ~
3~
AMpl~F~ 4: PIC;A - o~rDrug~ nsinganAminoacylCrosslinker In this example, aminoacyl derivatives of nucleoside analogs were prepared to provide a crosslinker which reacts with carboxyl groups to enable, _ of the 5 nucleoside analogs to carboxyl groups on ~ ' , ' The aminoacyl .,lu~alil.h~
used were derived from ~llillU....,~I~,yl~ yli~, acid (AMCC) and u~ acid (GABA). ~rhe aminoacyl derivatives of the nucleoside analogs were conjugated to ' ' by ~bUViil~l;Vt~ coupling.
5'-O-(trAnc-4-n",;".,.\, ll~,ylcy '-' ' ')-9-13-D Al~;""r~ ' (arn4.-~, The benzyl carbamate of r-~ ' y;(,yl ' ' ' Yl;C acid was prepared by treatment ûf ~ U~YIi~ æid (I g, 6.4 mmol) dissolved in 5 mL of 2N NaOH with b.,llL~ IUAY~ )U~IYI chloride (I mL, 7.0 mmol) slowly added with concurrent addition of an additional I mL of 2N ~aOH at 5 C. The mixture was stirred I h, then diluted with 25 mL water, and washed with 3 x 15 mL of ether. The aqueous solution was then acidified to a pH < 2.5. A vulu~ lv~ white precipitate formed, which was filtered and washed with 500 mL of cold water arld dried in vacuo. A portion of the product (27 mg, 0.1 mmol) was added to a solution of ara~ (27 mg, 0.1 mmol) dissolved in DMF (0.6 rnL).
DCC (29 nng, 0.14 mmol) amd DMAP (2.5 mg, 0.02 mmol) were added and the solution20 stirred 16 h. PLu;rl~tiu~l by flash silica gel clh".~ ~ ~L~ y afforded Z-AMCC-araA (13 mg, 25 %). Hydlu~ ~lol~ of Z-AMCC-araA (185 mg, 0.34 mmol) afforded compound AMCC-araA (117 mg, 0.28 mmol). ~,nalysis: IH-NMR (CDC13) d 0.64 - 0.99 (m, 3H), 1.01- 1.22 ( m, 2H), 1.29 -1.48 (m, 3H), 1.62 -1.77 (m, 4H), 1.79 - 1.93 (s, IH), 1.96 - 2.09 (m, IH), 2.61 - 2.73 (d, 2H), 3.82 - 4.01 (m, 3H), 4.63 - 4.69 (yt, J = 6.6 Hz, IH), 5.38 - 5.43 (~t, J = 6.2, IH), 6.51 - 6.57 (d, J = 6.1, IH, Hl'), 8.17 (s, IH, H8), 8.31 (s, IH, H2); W:
~ma,c 260 nm.
ar~7~-AM(~C-A~OR I araA-AMCC (8 mg, 0.02 mmol), prepared as described above, dissolved in DMSO (60 IlL) was added to ASOR (10 mg) in 200 juL of MES, 0. IM, pH = 5.6. EDC (16 mg, 0.08 mmol) was added and the solution was stirred 3.5 h at 4 C.
The solution was then Ul~ on Sephadex G25 with PBS æ eluent. The first peal~
was collected and analyzed by h'L,,v;oh,L/v;~;l,lc absorption ~ llu~u~y. ~max 263 nm.
~L(4-,~ ' ~ V-l-~-I~ - AI; ",~",.- ~ "~ araC-(~AF~) To a solution of S'-O-trityl-l-,~-D-A~ rl~ lcytosine (833 mg, 1.72 mmol) in DMF (12.5 mL) was added 4-lw~y~.A~bu~y;A l.hlo)-butsmoic acid (1.38 g, 5.82 mmol) and DCC (365 mg, 1.76 mmol). The solution was stirred 5 h, then the solvent was removed in vacuo and the resulting oil ~ d on a column of fl~lsh silica using chloroform-methanol (9:1 ) as eluent .
A 238 mg aliquot of the product (Rf 0.38) thus obtained was detritylated by warming at WO 95118636 . _11. ' ~L O

90 C in 50 % acetic acid. Cbl, O , ' y using the same eluent afforded pure araC-GABA-Z (137 mg, 0.3 mmol). IH-NMR (CD30D) o 1.85 (quin, 2H, COCH2C~I2CH2NH), 2.48 (t, 2H, Coc~2cH2cH2NH)~ 3.18 (t, 2H, COCH2CH~CE12NH), 3.82 (m, 2H, H5 & 5'), 4.02 (m, IH), 4.10 (~t, J = 2.3 Hz, IH), 4.25 (dd, J - 2.2 Hz, 3.5 Hz, IH), 5.05 (s, 2H, 5 benzylic), 6.19 (d, J = 3.7 Hz, IH, Hl'), 7.32 (m, 5H, phenyl), 7.42 (d, J = 7.5 Hz, lH), 8.23 (d, J = 7.5, IH). This compound was dissolved in 5 mL of ~a~ul... . acetic acid (3:2:0.5) and ~ VD~ Oly~A~d over 10 % Pd/C using tbe procedure of Brown (Brown, C.A.
and Brown, H.C. (1966) J. Org Cl~em. ~1:3989-3995) until the starting material was no longer detectable by TLC and a non-migrating, ninhydrin pûsitive spot appeared (about 30 10 min). The mixt~Are was filtered tbrough Celite, the etbanol remûved at 20 - 30 mm Hg, and the resulting solution Iyophilized to afford a white crystalline material. Melting point: 120 -122 C; IR (KBr): 3408(br), 2355, 1654, 1560, 1498, 1406, 1314, 1114, 1054, 804 cm-l;
W: ~ 305, 275 nm.
araC-GA.R~-A~OR. araC-GARA (10 mg, 30 llmol) in DMSO (100 IlL) was addea to a solutionûfASOR(21 mg)in420~LLofO.1 MMES,pH5.6. EDC(50mg,0.26mmol)was added and the solution stirred for 3 h at 25 C. The solution was cl.. ", ~ l on a Sephadex G25 column with PBS, pH 6.5 as eluent. The first peak to elute was dialyzed against water and Iyophilized.
EXAMPI .F. 5: P. ., of Drug (~ D ' using a ~eptide Crosslinker In tbis example, a tripeptide derivative of a nucleoside analog was prepared to provide a crosslinker wbich reacts with carboxyl groups to enable ~ ., on of the25 nucleoside analog to carboxyl groups on: ' - v~u...~vid. The tripeptide derivative was conjugated to ~ VUll 1 by c~ d;..,,;d~ coupling.
3'. 5'-Di-O-~ ~ h ' '- ''1, ' lVV-I-~-~ Al: l, r~ (TRnM~-araC). This compound was prepared accordmg to Wipf, P. et al. (1991) Bioorganic & Medicinal C}~em.
Lettersl:745-750. Asolutionofl-~-D-~ r~ ~l~; (I g,4.2mmol),imidazole (2.2 g, 34 mmol), TBDMSCI (2.5 g, 17 mmol), and DMAP (53 mg, 0.4 mmol) was stirred 24 h at 25 C. The mixture was then diluted with 50 mL of water and extracted with 3 x 50 mL
of ether. The combined organic extracts were dried over magnesium sulfate, filtered, and ' in vacuo. Flash silica gel ~.1.., ~, , ' ~ of the resulting oil afforded TBDMS-35 araC. lH-NMR (CDCI3) o 0.11, 0.12, 0.13, 0.15 (4s, 12H, methyl), 0.90, 0.96 (2s, 18H, tbutyl), 3.85 (m, 2H), 3.95 (m, lH), 4.18 (m, IH), 4.40 (m, IH), 5.46 (d, J = 7.3 Hz, IH, Hl'),

6.14 (d, J = 5.1 Hz, IH), 7.78 (d, J = 7.3 Hz, IH).

~ WO95/18636 2 ~ IE
3~
N~ .. ~I)-T ~ lqT f ~ -T AT -OT~. To a solution of N-(B~ lu~y~1ul,~ 1)-LeuAlaOH (2 g, 6 rAmol), N-mellly~ ul~ul~olille (0.67 mL, 6 mmol) and ~SVlJuLyl~ ull ' ' (0.82mL,6mmol)inDMF(10mL)wasaddedasolutionof Leu(OtBu) HCI (1.3 g, 6 mmol) a nd ~fi~Lllyl~ (0.87 mL, 6 mmol) in THF (25 mL) at 5 -5 C. The mixTure was allowed to come to room i , , the solids removed by filtration, and washed with THF. Ethyl acetate was added, and the combirled organic ph. ses washed with 30 mL portiors of water, saturated sodium l~ , water, I % HCI . nd 2 x water. The organic phase was dried over ,, sulfate, filtered and ~ ' in vacuo to afford a white foam (1.5 g, 50 %). FABMS [M + Hl+ 506. To an aliquot of 300 mg (0.59 mmol) of this material in dh,hlulu~ (3 mL) was add I '' acid (1.5 mL). After 1.5 h the starting material was consumed as judged by TLC with chloroform-methanol (9:1). Removal of the solvents followed by filtration through a short columTI of flash silica gel with the s. me solvent afforded Z-LAL-OH (216 mg, 80 %). FABMS [M
H]+ 450.
N4 a q~. AIAT f'~ .>-D-~ ;. .. ,rl.. A111 ~V I~"y t- ~ - r ~raC-LeuAIAT ell). To a solution of Z-LAL-OH(100mg,0.22mmol;preparedasdescribedabove),N-.~ --"~ l;". (25IlL, 0.22 mmol) . nd io~u~yl~,lllulurulll._~, (30 IlL, 0.22 mmol) in THF (0.6 mL) was added a solution of TBDMS-araC (79 mg, 0.22 mmol; prep, red as described above) in THF (0.5 mL) at 0 C. After 30 min stirring the soluti~n was diluted with ether (15 mL) and extracted with 3 x 10 mL of water. The organic phase was dried over rA~u~ eillm sulfate, filtered aTld I in vacuo. The resulting oil was ulll, " . ' ' on flash silica gel with 4 %
methanol-chloroform to afford 80 mg (72 %) of TBDMS-araC-(LeuAlaLeu-Z). A portion of this product (16 mg, 0.02 mmol) in THF (0.5 mL) was desilylated by treatment with ~.I_bu~y' fluoride (10 mg, 0.04 mmol) for I h. Flash silica gel "Ll, ~ , ' y with 13 % methanol-chloroform afforde~ araC-(LAL-Z) (10 mg, 74 %). IH-NMR (CD30D) I llA. ,.. ,~ . ;~1.. signals: o 0.91 (m, 12H, I,eu C~3), 3.80 - 4.55 (8H, arabinose & peptide a H's), 5.08 (2H, ben.,ylic), 6.19 (IH, Hl'), 7.30 (m, 4H, phenyl & I cytosine), 7.40 (IH, phenyl), 8.25 (IH, cytosine).
To remove the Z protecting group, this compound is dissolved in 5 mL of e~ ùl~ ~ ~ - . l . acetic acid (3 :2:0.5) and 1l~Lu~ u4 ~l over 10 % Pd/C using the procedure of Brown (Brown, C.A. and Brown, H.C. (1966) J. Org Chem. 31:3989-3995) urltil the startmg material is no longer detectable by TLC and a non-migrating, ninhydrin positive spot appears (about 30 min). The mixture is filtered through Celite, the ethanol removed at 20 -30 mm Hg, and the resulting solution Iyophilized.
arP12-T.~ PT eu-A~OR. To prepare araC-LeuAlaLeu-ASOR, TBDMS-araC-LeuAlaLeu (30 llmol; prepared as described above) in DMSO (100 ~L) is added to a solution of ASOR
(21 mg) in 420 IlL of 0.1 M MES, pH 5.6. EDC (50 mg, 0.26 mmol) is added and the WO 95118636 2 1 8 0 3 4 8 ~ E
solution stirred for 3 h at 25 C. The solution is ~,Iu~ on a Sephadex G25 column with PBS, pH 6.5 as eluent. The first peak to elute is dialyzed against water and Iyophilized.
S EXAMPl.~.6: P~, . ' of DrugConjugatesusinga P~ Labile Crosslinker In this example, a nucleoside amalog was conjugated to ~ ~l~ulu ~ull~u~,ù;d via a reductively-labile crosslinker.
1!~(3-(~-pyridy~ fhio)rrh~ ro~ pAlxycyti~iine (~C-PDP) Did.,~)~yl,yLi~ e (40 mg, 0.2 mmol) ar~d SPDP (60 mg, 0.2 mmol) in DMF (I mL) were stirred 17 h at 25 C. Purification on a 20 mm flash silica gel column yielded ddC-PDP (12 mg, 16 %). Analysis: IH-NMR
(DMSO-d6) ~ signals: o 5.91 (IH, Hl'), 7.18, 8.49 (2H, cytosine), 7.25, 7.80, 8.47 (4H, pyridyl); UV: ~max 295, 245; treatment with ~ threithl resulted in theIUIJIII~..: of an absorbance maximum at 340 nm" ~ of the liberation of pyridine-2-thione.
ti~iC-DP-A~OR. ASOR (4 mg), in 200 ~LL of aegassed 50 mM borate, I mM EDTA, pH 8.5, was reacted for l .S h at 2 C with 32 ~1L of a 9 mg/mL solution of 2~ .f in thesame buffer. The protein was separated from urreacted 2-;l--;....ll.;hlA..f using a PD10 column, eluted with degassed PBS, lmM EDTA, pH 7Ø Fractions (0.5 mL) 6-8 were pooled and ~ . 1 to 150 IlL using a CentriconlOTM. An 80 IlL aliquot was retained as a reference. To the remainder of the protein solution ddC-PDP (2 mg in 100 IlL of DMF) 25 was added dropwise with stirring at 4 C. After 1.5.h at 4 C the conjugate was separated from unreacted ddC-DP on a PDI 0 column as above. Analysis: PAGE, 45 % of the protein stained with coomassie blue migrated as a single band of Mr 36,000, 13 % with an Mr 80,000, and 14 % with an Mr > 100,000; UV: il,ma~ 280 nm.
30 E~AMPl F. 7: Pl.l ' of Drug C~ O ' Using P~ ~' L ' ' Acid as a Carrier In this example, a nucleoside analog was crosslinked to polyglutamic acid and then conjugated to ASOR by ~Aul,ùdiilllkle coupling.
araC-PLG~. araC was coupled to polyglutar~uc acid (PLGA; 14KD) according to the literature procedure (Kato, Y., et al. ( 1984) Cancer Research 9_ :25-30). Briefly, LUI)U~YIU7~Y~A btJI~yl chloride (556 mg) and Lli~,lly' - (391 mg) were added to a solution of PLGA (500 mg) in dry DMF (40 ml) U -8 to -5 C, and the mixture was stirred at this ~ Wo 95/18636 ~ ,r 3f"
h~ ulc for I hour. To the resultin solution was added a solution of araC (942 mg) in dry DMF (20 ml) and L~ lly' (391 mg), and the reaction was allowed to proceed at4 C for three days and at room ~c.~ aluuc for 4 hours. The reaction mixture was poured into cold 0.4 M phosphate buffer, pH 8.10 (20 ml), and any insoluble material was removed by 5 filtration. The filt~ate was dialyzed against 3 % NaCI solulion and against water.
araC-PLGA-ACOR. A 10 mg aliquot of araC-PLGA (prepared as described above) was combined with ASOR (15 mg) in water (0.5 mL). The pH was adjusted to 6.0, EDC (11 mg) was added, and the solution was stirred 16 h at 25 C. The mixture was separated on a 2.5 x 100 cm Sephacryl S100 column with PBS, pH 6.7 as eluent at 0.3 mL/min.. Fractions of 8 mL were collected and fractions 28-32 were pooled and Iyophilized (6 mg). Analysis: Non-reducing PAGE, Mr 46,000; UV: ~maX 295 nm, 248 nm; ~1,,,~,ll..1 .l...l..., ,. :i ;. _lly determined ron~ ntrAfif n of 97 llg araC per mg of conjugate.
EXAMPlli'8: P.~, . " of DrngConjugatesusinga r. ~ Dextran Carrier In this example, the polyruucliul~l carrier molecule polyzl ;1,hydc dextran (PAD) was prepared frvm dextran. ~ ' u~vlllu~oid was conjugated to PAD together with a cytosine-contarning nucleoside analog, araC or ddC, by reductive amination.
polyq~ hy~ trAn (PAI )). rul~ ' ' ' yvc dextr~m was synthesized following literature precedence (Bernstein, K., et al. (1978) v~ IVatL Cancer Insf. ~Q:379-384, Foster, RL. (1975) ~perienfia, 772-773)usinga1:1molarratioof~ u~ monomer. Briefly,Ig (û. 108 mrnol) of dextran (average molecular weight 9300) was dissolved in 186 mL of 0.03 M sodium periodate and stirred for 18 h at 25 C in the absence of light. The solution was then dialyzed, in 3500 MWCO dialysis bubing~ against 3 x 20 L followed by I x 5 L of water, amd Iyophilized.
arac-pAn-AcoR~ To PAD (16 mg in 300 ~LL of PBS, pH 7.2), prepared as described above, was added araC (10 mg in 75 ~LL of PBS). The solution was stured for 20 h at 25 C after which bime ASOR (6 mg in 200 ,uL of PBS) was added followed by am additional 20 h of reaction bime. Sodium c~ ~ ul~ydlhl~ (6 mg, 90 ,umol) was bhen added, amd the solution stirred for 1.5 h at 37 C. The conjugate was then separated from unreacted drug on a Sephadex G25 column with PBS as eluellt. The first peak to elute was dialyzed against 4 L of water, Iyophiliæd (16 mg), and analyzed by ulbravioleVvisible absorption q~ .,u~y.
~maX 278 nm.

W0 95/18636 2 1 ~ 0 3 4 8 ~ 0 yo ~l~C-PAn-A'~OR. To PAD (15 mg in 275 ,uL of PBS, pH 7.2), prepared as described above, was added ddC (10 mg, 47 ,umol). The solution was stirred 20 h at 25 C after which time ASOR (6 mg in 200 IlL of PBS) was added followed by an additional 20 h of reaction time.
Sodium ~"~ L ul~ Lid~ (6 mg, 90 llmol) was then added, and the solution stirred 1.5 h at 37 C. The reaction mixture was then separated from unreacted drug on a Sephadex G25 =.
column with PBS as eluent. The first peak to elute was dialyzed against 4 L of water, Iyûphilized (17 mg), amd analyæd by ultraviolet/visible absorption ~ u~.u~ ma~ 277 nm.

li XAMPl .li' 9: Molar " ~. Ratios of Drug Cor jugates D 1- ,.,;.,~1;,.,.of ~Ir~ r Suhctitl-tion P s~tinc The~ ;....ofdrugonconjugateswas determined ~ t~ y using the equation:
D Atotal~ (Eprotein x C/d) EdrUgX d x 1000/C
20 where D is the ..~..~ ...1,, l;.... of drug on the conjugate in ~lg/mL; ~ are in (mg/mL)-I; C is mg/mL of conjugate in the stock solution used; and d is the dilution of stock in the cuvette (I
cm path length).
The following ~ were made: 1) the extinction coefficient of bound drug is the 25 same as that of free drug; 2) the dry weight of the conjugate is the amount of protein and polymer (the weight of drug contributes less than 10 %); 3) the molecular weight of ASOR-polylysine conjugates is 40 Kd (amino acid analysis of sampled ASOR-polylysine conjugates supports a 1:1 ratio of ASOR (36 Kd):polylysine (4 Kd)). Extinction c.~Pffi.iPnt~ for the drugs used irt the conjugates were determined by standard techniques.
The average molar sllh~titllti~n ratios for drug conjugates prepared in Examples 1-8 are shown in Table 1.

W0 95/18636 P~
Y/
Table I
Average MSRI of Drug-Conjugates on ASOR.
Drug Crosslink~ r Carrier MSR
araC glutarate PLL~ 6 glutarate PLL (10 Kd) 19 phosphate PLL 3 araA glutarate PLL 5 glutarate PLL (10 Kd) 14 succinate PLL 5 phosphak PLL 7 ACV glutarate PLL 4 succinate PLL 5 phosphate PLL
ddC glutarate PLL 12 glutarate PLL (10 Kd) 17 AZT glutarate PLL 16 IMSR expressed in terms of mol drug:mol ASOR.
2Poly-L-lysine (PLL) is 4 Kd unless otherwise indicated.
The molar ' ' ~n ra~tio for araC-GABA-ASOR was determined by HPLC. HPLC was carried out on a Waters 600~ solvent delivery system v.~ith a 486absorbance detector and using an Appli~ d Biosystems 4.6 mm x 100 mm Cl 8 column. With a 25 min, I mL/min linear gradient of water to acetonitrile (each containing 0.1 % acetic acid), araC eluted with a retention time of 12 min. Detection was at 260 mm. A Waters 743 Data 15 module was used to quantitate peaks. Liberation of araC by alkaline hydrolysis of the iV-butyryl bond of the crosslinker was effe~:ted in 0.1 M, pH 9.5 borate buffer. A 1 mglmL
portion of the araC-GABA-ASOR conjllgate in this buffer was incubated at 37 C.
Injections of 20 IlL aliquots were made iinitially, amd after I, 2, and 5 days. The amount of free araC detected in the conjugate initi~llly was < 3 llg/mg. After 24 h 20 llg of free drug 20 had been released per mg of conjugate. A maximum of 25 ,ug/mg (UUllC~.UUlldillg to an MSR = 2) was detected after 5 days.
.. . . . ........... ... ..

WO 95/18636 2 1 8 0 3 4 8 P~ LI.. 5 ~ E
F.XAMPl ,~.10: Inhibition of HBV DNA ~ . " ' by Drug Conjug~tes The HBV-DNA transfected human l . ' ' ~ (HepG2)-derived cell line, 2.2.15 (Sells, M.A., et al., (1987) Proc. Natl. Acad Sci. USA 84:1005-1009; Sells, M.A. et al., (1988) J: Virol. ~:2336-2344), was used to evaluate the antiviral activity ofthe drug conjugates. Cells were either untreated, treated with a free drug, or treated with a conjugated drug (i.e., drug-PLL-ASOR). After exposure of 2.2.15 cel~s to the drug conjugate, the presence of HBV DNA ~-Ytr~ ly (i.e., DNA in virions released from the cells) ; " ~ r ll~ m the form of relaxed circular DNA, replication " (single-stranded DNA and partial relaxed circles), and integrated HBV DNA was measured to determine the effect of the conjugate on viral DNA replication. The IDsos (dose necessary to inhibit 50% of the viral DNA replication) for free and conjugated araA, acyclovir, araC, AZT, and ddC were determined relative to the untreated control. The CDs0 (dose of drug required to kill 50% of the cells) was determined for free araC and ddC and for one of the conjugates (ACVMP-PLL-ASOR), using both 2.2.15 cells and SKHepl cells. In addition, to measure the level of clearance of the drug conjugates to the liver when ad~ Ltl c~d in vivo, Balb/C mice were tail-vein injected with I o6 cpm/llg of 125 l-r~ drug conjugate and their liversexcised and assayed for the presence of labeled drug conjugate after five minutes.
The results from the HBV antiviral assays (IDs0s) are ~ below in Table 2. Also shown in Table 2 are the results from the liver clearance assay (% to liver) and the CDso Il~ ,.IL~ using both 2.2.15 (ASGR +) cells and SKHep I (ASGR -) cells.
The ~ ;",. .,1..1 methods are also described in detail below.

~W0 95/18636 P~

T~ble 2 HBV HBV
In hibition Inhibition DrugCrosslinker % to CD50 CD50 Type Liver extrnctllu18r intrscellul5~r (ID50) DNA 2.2.15 SKHepl HB(lDso) (ASGR +) (ASGR -) araAfree drug 30011M
glutarate 97 ] 5 IlM
phosphate 96 30 IlM
acyclovir free drug >300 ,uM >I mM ~3 mM ~3 mM
glutarate 98 30,uM
succinate 98 30,uM
phosphate 96 3 ,uM 23 IlM 170 ,uM >1 mM
aminoacyl 98 70,uM
ddCfree drug ~0 IlM 0.1 IlM 3.5 mM 190 IlM
N-glutarate 99 >60,uM
PAD98 1 IlM 0.1 ,uM 3.5 ,uM
,4Z~freedrug > 3 mM
glutarate 99 150 uM
araCfree drug 0.4 IlM 3.5 IlM 3.5 ,uM 4.7 IlM
N-glutarate 96 6 ,uM 0.1-0.2 ,uM 2.4 ~M
PAD >6uM
s F~,.;,.... ~ 1 ~/lr~thr~rlc To assay HBV antiviral a!ctivity for drug conjugates containing araA-glutarate-PLL-ASOR, araA~ -PLL-ASOR, acyclovir-glutarate-PLL-ASOR, acyclovir-succinate-PLL-ASOR, acyclovir-pllGa~ PLL-ASOR, 10 acyclovir-aminoacyl-PLL-ASOR, ddC-glutarate-PLL-ASOR, ddC-PAD-ASOR, .4ZT-glutarate-PLL-ASOR, araC-glutarate-PLL-ASOR, and araC-PAD-ASOR, stock cultures of 2.2.15 cells were maintained irl RPMI 1640 , . ' i with 5 % fetal bovine serum and 2 mM L-glutamine. The cells were incubated at 37 C in a moist atmosphere containing 5 % CO2. For antiviral treatment, 2.2.1 5 cells were seeded onto collagen-coated 24-well plates at a density of 4x104 cells/cm2. Confluent cultures (6-8 days post-seeding) were incubated in RPMI containing 2 % fetal bovine serum vl.l,l .. , . ~1 with increasmg . ~.. ,r. .,~ of either conjugate or free drug. The drug-containing medium was added on WO 95/18636 r~ t :E O
YY
day I (post-confluence) and replæed every 2 days (days 3, 5, 7 and 9) with medium containing fresh drug conjugate or free drug. On day 10, the medium and cells were collected for intT~rP~ r and eYtr~P~ r HBV DNA analysis.
Ten days after drug treatment, the cells were Iysed and total DNA was isolated. Total nucleic acids were extracted from conjugate-treated 2.2.15 cultures and HBV DNA was analyzed as follows. Cells were washed two times with excess Tris-Buffered Saline. The monolayer was Iysed in 400 1ll of Iysis buffer (0.6 % SDS, 10 mM
EDTA, 10 mM Tris-CI pH 7.4) containing 20 llg/ml of RN~tse A and incubated at 3 7 C for 30-60 minutes. The Iysate was transferred to a microfuge tube, proteinase K was added to a final ~ of 100 llg/ml and incubated at 50 C for at least 2 hours. The Iysate was then adjusted to contain 300 mM sodium acetate, extracted once with phenol/chloroform/
isoamyl alcohol (25:24:1 v/v) and once with chloroform/isoamyl alcohol (24:1 v/v). The DNA was ~ ' by ethanol IJIC~ ;tdtiU~ c~ J~I in 50 ul of 10 mM Tris HCI, I
mM EDTA, pH 8.0, and digested with the restriction enzyme Hind Ill. For Southernblotting, a third of the digested DNA was eL,~ ,,ul.u.c~l in a 0.8 % agarose gel amd tramsferred to Micron SPr~r~tifnc M~gnA('r~rh~9 nylon membrane by overnight capillary transfer using 1 0X SSC transfer buffer. Hyblid;~dtiull was performed at 68 C with a [32P]dCTP-labeled EcoRI fragment of pADW-HTD (provided by T.J. Liang) containing the full length 3.2 genome of HBV. All labeling reactions were carried out with the Random Primers DNA Labeling System (BRL, Life T ~ lg;P~). Levels of integrated DNA, relaxed circle DNA and replication ' were quantitated using a Packard Instant Imager and were graphed as a percentage of the umtreated control.
The presence of HBV DNA; ~ rl~ as relaxed circle, replication ' (single-stranded DNA and partial relaxed circle), and integrated HBV DNA wasquantitatively compared. The relative amounts of relaxed circle DNA and replicative ' were normalized to the amounts of integrated DNA because the levels of integrated DNA should not be affected by antiviral treatment. The data for the araC-glutarate-PL-ASOR conjugate in particular are plotted as a percent of the umtreated control in Figure 1. The araC-glutdrate-PL-ASOR conjugate had a dose dependent inhibitory effect on intrA~pll~ r HBV relaxed circle ~ ;" The IDso (dose necessary to inhibit 50 % ofthe viral' DNA replication) for the relaxed circle DNA (final product) ~ ';. ", was at about 0.1 IlM (30 ng/ml), whereas the replication " continued to accumulate even35 athigh~". .:,,l;.",c(e.g.,0.5mM(lOOng/ml))ofdrug. ThelDsoforfreearaCwasabout 3.5 IlM (750 ng/ml). However, this is the same value we determined to be the CDso (dose of drug required to kill 50 % of the cells). Therefore, the increase of antiviral activity for the free araC is probably due to cell death, rather than specific viral inhibition. Others have d- - ' that free araC is not an inhibitor of HBV replication in 2.2.15 cells (B. Korba, e W0 95ll8636 r~ "~
~/J
personal ) Thus, by talgeting araC to cells via the ~ "u,u.~;
receptor, the antiviral activity of the drug may be enhanced.
Figure 2 shows the HB~ antiviral activity of the acyclovir-l ' ~ . ' PLL-5 ASOR conjugate on ;., l l ,.. . ll l l - HBV DNA, plotted as a percent of the untreated control.
The conjugate inhibited at an IDso Of 23 IlM (7 llg/ml), wherea s free acyclovir had liffle effect on in~ Pll ' HBV DNA L ' '' (ID50 of>l Mm (>300 ,ug/ml)). These results .' - that acyclovir becomes a much more potent inhibitor of HBV replication when t. rgeted to cells via the ' ~ V~JlUt~, .. receptor.
Figure 4 shows the effect of the ddC-PAD-ASOR conjugate on intr~l-Pl~ qr HBV DNA, plotted as a percent of the luntreated control. Both the free ddC a nd the ddC-PAD-ASOR conjugate had an inhibitor~ effect on intrqnPll ' HBV relaxed circle and replication;"t~l.~ 1; ' d~,.,l ' '' v~ithanIDsoofabout0.1,uM(20ng/ml) The effect of the drug conjugates on PYtrq.~Plllllqr HBV DNA ( DNA in virions released from the cells) was also evaluated. For the analysis of PYtrq~
DNA, the culture medium from the conjugate-treated 2.2.15 cells were centrifuged in a microfuge for 2 minutes to remove cellular debris. To denature the PYtrq~Pll~lqr DNA, 400~1 20 of the clarifled medium was incubated for 20 minutes at room i l ~lul~ (25 C) in I M
NaOH.lOX SSC (IX SSC is 0.15 M NaCW.015 M Sodium Citrate, pH 7.2). The samples were directly applied to nylûn mrnnhrqnqc (Micron Sep. rations Systems Mq~qrJ-qrh~) presoaked in 20X SSC using a slot blot .lpparatus (BRL). To neutralize the bound DNA, slots were washed twice with 0.5ml of I M Tris, pH 7.2/2M NaCI and once with 0.5 ml of 25 20X SSC. The filters were removed, washed briefly in 2X SSC and W crosslinked(Stratalinker, Strategene) prior to ~.~ Ul;ViLnliUII with the full length HBV probe (described above). The results are shown in Table :2 (above) and again ~IPm~ that by t. rgeting drugs to I , 3~ via the ~ ,ulJIut~,;.. receptor, their antiviral activity against HBV
can be ~j3 r ~ enh. nced, as measuled by eYtr ~rPIllllqr HBV DNA L ' "
In particular, as shown in Table 2, both conjugates of araA (i.e., the glutqrateconjugate and the phosphate conjugate) showed inhibition of HBV PYt.,q.,~Plllllqr DNA at greater thqn ten fold lower: than did free araA. Likewise, all acyclovir conjugates hdd IDsos far lower than free acyclovir. The most potent acyclovir conjugate, 35 acyclovir-phosphate-PLL-ASOR, inhibited production of HBV PYt-qrPIll.lqr DNA at a greater than one hundred fold lower ~UlI~.~.llU.:L~iUII thqn did the free drug. The effect of the acyclovir-phosphate-PLL-ASOR conjugate on eYtrq~Pll ' HBV DNA production is plotted as a percent of the untreated control in Figure 3. When the levels of PYt-q~Plllllqr DNA
(I~,UI~ DNA in virions released from the cells) were measured, it was found that the W0 95118636 2 1 8 0 3 4 8 ~ " ~
conjugate inhibited at an IDso of less than 3 IlM (I ~g/ml), whereas free ac,vclovir had linle effect on eYtr~ ~P~ qr HBV DNA . ' For example, at 300 IlM (100 ~Lg/ml), the inhibition was only 40% (see Figure 3). Similarly, as shown in Table 2, the ddC-PAD-ASOR
conjugate and the ~ glutarate-ASOR conjugate both inhibited HBV more than one orda of magnitude greater than did the free drugs. These results clearly fl.. ~ . that by targeting antiviral agents to cells via the asiah,~ . u~ ,t.;ll receptor, their efficacy, as measured by pYrr~ p~ or HBV ~ ;..,. is greatly increased compared to the free drugs.
The ~ tulut~ y of selected free and conjugated drugs were determined as 10 follows. 2.2.15andSKHeplcellswereseededona96wellmicrotiterplateatadensityof 3.75x103 cells/well. SK Hepl cells do not have the receptor for ASOR and therefore serve as a control. Twenty four hours after seeding, increasing ..~. r . ,l. ,.l;. . ~ of free and conjugated drugs were added to the plates. Twenty four hours after drug addition, the drug was removed by a mediuln change. Seventy two hours after the initial drug ~rrli~ s~ti~n the plates were 15 stained with a ~ . ' ;"of the tetrazolium reagent XTT which is metabolically reduced in viable cells to a water soluble formazan product and PMS which markedly enhances cellular reduction of XTT and allows direct absorbance readrngs. Staining was done according to Scudiero, D.A., et al., (1988) Cancer Research, ~:4827~833. The absorbance was read at 450 nm. The percent survival was calculated by dividing the absorbance of each well by the 100% survival absorbance (no drug added) and multiplying by 100. The results are shown in Table 2. Less than 5-fold tbe amount of acyclovir-~ -PLL-ASOR was needed to killthe ASGR-expressing 2.2.15 cells than was needed to kill the ASGR negative SK Hepl cells.
To measure the percent clearance of the drug conjugates to the liver, in vivo targeting assays were performed on mice as follows. Conjugates were iodinated using the -Tprocedure(Woodetal.(1981)J:ClinChem.Clin.Biochem.19:1051-1056).
Balb/C mice were tail-vein injected with I o6 cpm/llg of 1251-drug conjugate in 0.5 ml PBS.
The average specific activity was lo6 cpm/llg. Animals were sacrificed by cervical dislocation at 5 minutes post injection. Five major organs (liver, spleen, kidneys, heart, and lungs) were excised and counted in the gamma counter to deterrnine targeting of conjugate. As shown in Table 2, greater than 95% of all drug conjugates cleared to the liver, , ' g that the drug conjugates are effectively delivered to liver cells in vivo.

~ WO 95/18636 1~
FXAMPI.F ll: r,. of Drug~ ~ Colchicineanda 1~. ' -.~1~ Labile Crosslinlier Drug conjugates of colchicine liniced to ASOR were prepared as follows:
N-~3-¢2-Pyl;~lyl~ yl~ yl-,ul 1~; ;, SPDP(29mg,0.092mmol)andDMAP
(11 mg, 0.092 mmol) were added to J~ ly' ' ' (33 mg, 0.092 mmol) in Ji~,Llululll~,llall~ (I mL), and the solut;on stirred for 2 hours at 23C. The solution was then .,L, " , ' ' on flash siiica gel with 6% methanol in chio}oform as a mobile phase. The first eluted colchicine derivative (21 mg, 0.038 mmol) was determined to be the correct product by lH-NMR (CDC13) ~ 1.25 I'S, IH), 1.56 (s, 2H), 1.87 (m, IH), 2.30 (m, IH), 2.46 (m,lH), 2.53 (m, IH), 2.67 (q, 2H), 3.~\3 (m, 2H), 3.65, 3.90, 3.94, 3.98 (4s, 12H), 4.68 (m, IH), 6.53 (s, IH), 6.80 (d, J = 10.8, IH), 7.15 (m, IH), 7.30 (m, IH), 7.44 (s, lH, H8), 7.56 (d, J = 8.2, IH), 7.63 (m, IH), 8.50 (d, J = 4.4 Hz I H).
Col~hirin~ -DP-A~OR 5 mg of SPDlF' in 0.06 mL of DMSO was added to 8 mg of ASOR in 0.5 mL of HEPES, 0.1 m, pH 7.5. The reaction mixture was stirred vigorously for 2 hours at 0 - 4C. The mixture was then microfuged at 3000 rpm, 10 min. The supernatant was .,L,. ,.,~ .l on a PD10 column ~ith sodium acetate, sodium chloride, 0.1 M pH 4.5 and the Illa~,~vll~O~ ulal fraction ~ ' to 0.25 mL using a Centricon 10. Dithiothreitol (6 mg) in 0.25 mL of the same buffer was added and the solution stirred 30 min at 23C. The solution was then ~,lu~ ~, , ' ' on a PD10 column with degassed PBS containing I mM
EDTA and 0.02% sodium a~ide. To the Illa~"ulllGic~llal fraction was added N-(3-(2-pyridyldithio)~l u,uiv.lyl icac~lylcolchicine (2 mg) dissolved in 0.05 mL of DMSO. The mixturewasstirredinitiallyat04C,tllenat23Cfor17hours. N-E~ily' ' ' (Img) was added and the mixture stirred an additional I hour. The mixture was then micrvfuged at 5000 rpm, 10 min, and the ~Tnqt~f ULUVII._ V ~àull~,~ on a PDI 0 column in PBS. Tile ~,lulllOlc~ula~ fraction was anaiyzed for colchicine by measuring absorbance at 353 nm, and for protein using the BioRad protein assay and PAGE. The conjugate contained 2 mol colchicine per mol ASOR.
The colchicine-DP-ASOR conjllgates can be used in c~ ^ti~n with other drug-containing conjugates ofthe invention (e.g., those described above in Examples 1-10), or with nucleic acid-containing conjugates, to increase delivery of the targeted drugs or nucleic acids to cells. It is believed that colchicine inhibits the Irqn~l~u qti~n and/or fusion of endosomes to Iysosomes. Therefore, when co-; ~ r 1 into am endosome of a cell, aiong with other drug or nucleic acid-containing conjugates, the colchicine-DP-ASOR conjugate may prevent the ,1, ~,, ..,l~1;", . of the druLr or nucleic acid-containing conjugates by Iysosomes.
Accordingly, in one,, ~ ,v~ 1: . . ,. of the invention, conjugates including colchicine or other WO 9~118636 r~
y8~
agents which inhibit the trS~clnr~inn and/or fusion of endosomes to lysosomes. and ASOR
can be used to increase the antiviral activity or the level of expression of nucleic acids targeted to I, yt~
s EQUIVAI FNTS
Those skilled in the art will recognize, or be able to ascertain using no more than routine, , many equivalents to the specific ~ " of the invention described herein. Such equivalents are intended to be ,...1,~ i by the following claims.

Claims

-49-1. (Amended) A conjugate for targeting a therapeutic agent to a cell expressing an asialoglycoprotein receptor, the conjugate comprising a general formula A-B-C-D, wherein:
A is a therapeutic agent selected from the group consisting of antiviral agents,anti-tumor agents, DNA binding agents, hormones, growth factors, vitamins, and agents which inhibit the translocation and/or fusion of endosomes to lysosomes, which is covalently bonded to a polyfunctional carrier molecule;
B is a crosslinker which is covalently bonded to the therapeutic agent and to a polyfunctional carrier molecule;
C is a polyfunctional carrier molecule; and D is a ligand for the asialoglycoprotein receptor selected from the group consisting of asialoorosomucoid, arabinogalactan and a Tris-(N-acetyl galactosamine aminohexyl glycoside) amide of tyrosyl(glutamyl)glutamate, wherein the ligand is covalently bonded to the polyfunctional carrier molecule such that the ligand can bind to the asialoglycoprotein receptor.
2. (Amended) The conjugate of claim 1 wherein the antiviral agent is selected from the group consisting of nucleoside analogs, reverse transcriptase inhibitors, topoisomerase inhibitors, DNA gyrase inhibitors and DNA binding agents.
3. The conjugate of claim 2 wherein the antiviral agent is effective against a hepatotropic virus.
4. The conjugate of claim 3 wherein the hepatotropic virus is selected from a group consisting of hepatitis A, hepatitis B, hepatitis C and hepatitis D.
5. (Amended) The conjugate of claim 2 wherein the nucleoside analog is selected from the group consisting of 9-.beta.-D-arabinofuranosyladenine(araA), 9-.beta.-D-arabinofuranosylcytosine (araC),2',3'-dideoxycytidine (ddC),3'-azido-3'-deoxythymidine (AZT),9-(2-hydroxyethyoxymethyl)guanine(ACV), gancyclovir, famcyclovir, pencyclovir, bromovinyldeoxyuridine, phosphonoformate, 2',3'-dideoxynucleosides of adenosine -49.1-(ddA), inosine (ddI). guanosine (ddG), thymidine (ddT) and uracil (ddU). 9-.beta.-D-arabinofuranosyladenine-erythro-9-(2-hydroxynonyl)adenine (AraA-EHNA),2'-fluoro-1-.beta.-D-arabinofuranourosyl-5-methyluracil(FMAU),2'-fluoro-1-.beta.-D-arabinofuranourosyl-5-ethyluracil(FEAU),2'-fluoro-1-.beta.-D-arabinofuranosyl-5-iodouracil(FIAU),2'-fluoro-1-.beta.-D-arabinofuranosyl-5-iodocytidine (FIAC),3'-fluoro-ddC,5-chloro-ddC,3'-fluoro-5-chloro-ddC,3'-azido-5-chloro-ddC,3'-fluoro-ddT,3'-fluoro-ddU,3'-fluoro-5-chloro-ddU,3'-azido-ddU,3'-azido-5-chloro-ddU,2'-6'-diaminopurine2',3'-dideoxyriboside (ddDAPR) and carbocylic analogs of deoxyguanosine(2'-CDG).
6. The conjugate of claim 5 wherein the nucleoside analog is selected from a group consisting of 9-.beta.-D-arabinofuranosyladenine (araA),9-.beta.-D-arabinofuranosylcytosine (araC), dideoxycytidine (ddC), 9-(2-hydroxyethyoxymethyl)guanine (acyclovir; ACV) and 3'-azido-3'-deoxythymidine (AZT).
7. The conjugate of claim 1 wherein the crosslinker is covalently bonded to the polyfunctional carrier molecule through an amide bond.
8. The conjugate of claim 7 wherein the crosslinker is derived from a carboxyacyl compound.

9 The conjugate of claim 8 wherein the carboxyacyl compound is glutarate or succinate 10. The conjugate of claim 7 wherein the crosslinker is derived from an aminoacyl compound.
11. The conjugate of claim 10 wherein the aminoacyl compound is trans-4-aminomethylcyclohexanecarboxylate or 4-aminobutyrate.
12. The conjugate of claim 7 wherein the crosslinker is a peptide which is hydrolyzable intracellularly.
13. The conjugate of claim 12 wherein the peptide comprises an amino acid seqeunce Leu-Ala-Leu.
14. The conjugate of claim 1 wherein the crosslinker is covalently bonded to thepolyfunctional carrier molecule through a phosphoamide bond.
15. The conjugate of claim 14 wherein the crosslinker is phosphate.
16. The conjugate of claim 1 wherein the polyfunctional carrier molecule is a poly-amino acid.
17. The conjugate of claim 16 wherein the poy-amino acid is polylysine or polyomithine.
18. The conjugate of claim 16 wherein the poly-amino acid polyglutamic acid or polyaspartic acid.
19. The conjugate of claim 1 wherein the polyfunctional carrier molecule is polyaldehyde dextran.
20. The conjugate of claim 1, wherein the cell is a hepatocyte.

- 50.1 -21. (Amended) A conjugate for targeting a therapeutic agent to a cell expressing an asialoglycoprotein receptor, the conjugate comprising a general formula A-B-C-D, wherein:
A is a nucleoside analog is selected from a group consisting of 9-.beta.-D-arabinofuranosyladenine (araA), 9-.beta.-D-arabinofuranosylcylosine (araC), dideoxycytidine (ddC), 9-(2-hydroxyethyoxymethyl)guanine (acyclovir;
ACV) and 3'-azido-3'-deoxythymidine (AZT);

B is a crosslinker which is covalently bonded to the therapeutic agent and to a polyfunctional carrier molecule;
C is a polyfunctional carrier molecule; and D is asialoorosomucoid, wherein asialoorosomucoid is covalently bonded to the polyfunctional carrier molecule such that asialoorosomucoid, can bind to the asialoglycoprotein receptor.
22. (Amended) A conjugate for targeting a therapeutic agent to a cell expressing an asialoglycoprotein receptor, the conjugate comprising a general formula A-C-D, wherein:
A is a therapeutic agent selected from the group consisting of antiviral agents,anti-tumor agents, DNA binding agents, hormones, growth factors, vitamins, and agents which inhibit the translocation and/or fusion of endosomes to lysosomes, which is covalently bonded to a polyfunctional carrier molecule, C is a polyfunctional carrier molecule; and D is a ligand for an asialoglycoprotein receptor, wherein the ligand iscovalently bonded to the polyfunctional carrier molecule such that the ligand can bind to the asialoglycoprotein receptor.
23. (Amended) The conjugate of claim 22 wherein the antiviral agent is selectedfrom the group consisting of nucleoside analogs, reverse transcriptase inhibitors, topoisomerase inhibitors, DNA gyrase inhibitors and DNA binding agents.
24. The conjugate of claim 23 wherein the antiviral agent is effective against ahepatotropic virus.
25. The conjugate of claim 24 wherein the hepatotropic virus is selected from a group consisting of hepatitis A, hepatitis B, hepatitis C and hepatitis D.
26. (Amended) The conjugate of claim 23 wherein the nucleoside analog is selected from the group consisting of 9-.beta.-D-arabinofuranosyladenine (araA), 9-.beta.-D--51.1-arabinofuranosylcytosine (araC).2'.3'-dideoxycytidine (ddC),3'-azido-3'-deoxythymidine (AZT),9-(2-hydroxyethyoxymethyl)guanine (ACV), gancyclovir, famcyclovir. pencyclovir, bromovinyldeoxyuridine, phosphonoformate, 2',3'-dideoxynucleosides of adenosine (ddA), inosine (ddI), guanosine (ddG), thymidine (ddT) and uracil (ddU), 9-.beta.-D-arabinofuranosyladenine-erythro-9-(2-hydroxynonyl)adenine (AraA-EHNA),2'-fluoro-1-.beta.-D-arabinofuranourosyl-5-methyluracil(FMAU),2'-fluoro-1-.beta.-D-arabinofuranourosyl-5-ethyluracil(FEAU),2'-fluoro-1-.beta.-D-arabinofuranosyl-5-iodouracil (FIAU), 2'-fluoro-1-.beta.-D-arabinofuranosyl-5-iodocytidine (FIAC),3'-fluoro-ddC,5-chloro-ddC,3'-fluoro-5-chloro-ddC, 3'-azido-5-chloro-ddC,3'-fluoro ddT,3'-fluoro-ddU,3'-fluoro-5-chloro-ddU,3'-azido-ddU,3'-azido-5-chloro-ddU,2'-6'-diaminopurine,2',3'-dideoxyriboside (ddDAPR) andcarbocylic analogs of deoxyguanosine(2'-CDG).
27. The conjugate of claim 26 wherein the nucleoside analog is selected from a group consisting of 9-.beta.-D-arabinofuranosyladenine (araA), 9-.beta.-D-arabinofuranosylcytosine (araC), dideoxycytidine (ddC), 9-(2-hydroxyethyoxymethyl)guanine (acyclovir; ACV) and 3'-azido-3'-deoxythymidine(AZT).
28. The conjugate of claim 22 wherein the polyfunctional carrier molecule has reactive aldehyde groups.

29. The conjugate of claim 28 wherein the polyfunctional carrier molecule is polyaldehyde dextran.
30. The conjugate of claim 22 wherein the polyfunctional carrier molecule is a poly-amino acid.
31. The conjugate of claim 22 wherein the ligand for the asialoglycoprotein receptor is asialoorosomucoid.
32. The conjugate of claim 22 wherein the ligand for the asialoglycoprotein receptor is arabinogalactan or a Tris-(N-acetyl galactosamine aminohexyl glycoside) amide of tyrosyl(glutamyl)glutamate.
33. The conjugate of claim 22 wherein the cell is a hepatocyte.
34. A conjugate for targeting a therapeutic agent to a cell expressing an asialoglycoprotein receptor, the conjugate comprising a general formula A-B-D, wherein:
A is a therapeutic agent selected from the group consisting of nucleoside analogs, reverse transcriptase inhibitors, topoisomerase inhibitors, DNA
gyrase inhibitors and DNA binding agents;
B is a crosslinker which is covalently bonded to the therapeutic agent and asialoorosomucoid, and D is asialoorosomucoid, wherein asialoorosomucoid is covalently bonded to the crosslinker such that asialoorosomucoid can bind to the asialoglycoprotein receptor.
35. The conjugate of claim 34 wherein the therapeutic agent is effective against a hepatotropic virus.
36. The conjugate of claim 35 wherein the hepatotropic virus is selected from agroup consisting of hepatitis A, hepatitis B, hepatitis C and hepatitis D.
37. The conjugate of claim 34 wherein the nucleoside analog is selected from a group consisting of 9-.beta.-D-arabinofuranosyladenine(araA), 9-.beta.-D-arabinofuranosylcytidine (araC),dideoxycytidine(ddC),9-(2-hydroxyethyoxymethyl)guaanine(acyclovir; ACV) and 3'-azido-3'-deoxythymidine (AZT).
38. The conjugate of claim 34 wherein the crosslinker is covalently bonded to asialoorosomucoid through an amide bond.
39. The conjugate of claim 38 wherein the crosslinker is derived from an aminoacyl compound.
40. The conjugate of claim 39 wherein the aminoacyl compound is trans-4-aminomethylcyclohexanecarboxylate or 4-aminobutyrate.
41. The conjugate of claim 38 wherein the crosslinker is a peptide which is hydrolyzable intracellularly, 42. The conjugate of claim 41 wherein the peptide comprises an amino acid seqeunce Leu-Ala-Leu.
43. The conjugate of claim 34 wherein the crosslinker is covalently bonded to asialoorosomucoid through a disulfide bond.
44. The conjugate of claim 43 wherein the crosslinker is derived from (3-(2-pyridyldithio)propionate.
45. The conjugate of claim 34, wherein the cell is a hepatocyte.
46. A conjugate comprising 9-.beta.-D-arabinofuranosylcytosine (araC) and a ligand for an asialoglycoprotein receptor, wherein araC is conjugated to the ligand by a crosslinker, a polyfunctional carrier molecule or a crosslinker and a polyfunctional carrier molecule.
47. The conjugate of claim 46 wherein the ligand is asialoorosomucoid.
48. The conjugate of claim 46 wherein the ligand is arabinogalactan or a Tris-(N-acetyl galactosamine aminohexyl glycoside) amide of tyrosyl(glutamyl)glutamate.
49. The conjugate of claim 47, wherein araC is covalently bonded to a crosslinker selected from the group consisting of phosphate, glutarate and succinate, the crosslinker is covalently bonded to araC and to a polyfunctional carrier molecule selected from the group consisting of polylysine and polyornithine and the polyfunctional carrier molecule is covalently bonded to asialoorosomucoid.

50. The conjugate of claim 47 wherein araC and asialoorosomucoid are covalently bonded to polyaldehyde dextran.
51. The conjugate of claim 47, wherein araC is covalently bonded to an aminoacyl crosslinker, the crosslinker is covalently bonded to araC and to a polyfunctional carrier molecule selected from the group consisting of polyglutamic acid, polyaspartic acid and polyaldehyde dextran, and the polyfunctional carrier molecule is covalently bonded to asialoorosomucoid.
52. The conjugate of claim 51 wherein the aminoacyl crosslinker is derived from trans-4-aminomethylcyclohexanecarboxylate or 4-aminobutyrate.
53. The conjugate of claim 51 wherein the aminoacyl crosslinker is a peptide comprising an amino acid sequence Leu-Ala-Leu.
54. A pharmaceutical composition comprising a solution of the conjugate of claim47 and a physiologically acceptable carrier.
55. A conjugate comprising 9-(2-hydroxyethyoxymethyl)guanine (acyclovir;
ACV) and a ligand for an asialoglycoprotein receptor wherein ACV is conjugated to the ligand by a polyfunctional carrier molecule or a crosslinker and a polyfunctional carrier molecule.
56. The conjugate of claim 55 wherein the ligand is asialoorosomucoid.
57. The conjugate of claim 55 wherein the ligand is arabinogalactan or a Tris-(N-acetyl galactosamine aminohexyl glycoside) amide of tyrosyl(glutamyl)glutamate.
58. The conjugate of claim 56, wherein ACV is covalently bonded to a crosslinker selected from the group consisting of phosphate, glutarate and succinate, the crosslinker is covalently bonded to ACV and to a polyfunctional carrier molecule selected from the group consisting of polylysine and polyornithine, and the polyfunctional carrier molecule is covalently bonded to asialoorosomucoid.
59. The conjugate of claim 56, wherein ACV is covalently bonded to an aminoacyl crosslinker, the crosslinker is covalently bonded to ACV and to a polyfunctional carrier molecule selected from the group consisting of polyglutamic acid, polyaspartic acid and polyaldehyde dextran, and the polyfunctional carrier molecule is covalently bonded to asialoorosomucoid.
60. The conjugate of claim 59 wherein the aminoacyl crosslinker is derived from trans-4-aminomethylcyclohexanecarboxylate or 4-aminobutyrate.
61. The conjugate of claim 59 wherein the aminoacyl crosslinker is a peptide comprising an amino acid sequence Leu-Ala-Leu.
62. A pharmaceutical composition comprising a solution of the conjugate of claim56 and a physiologically acceptable carrier.
63. A conjugate comprising dideoxycytidine (ddC) and a ligand for an asialoglycoprotein receptor wherein ddC is conjugated to the ligand by a crosslinker, a polyfunctional carrier molecule or a crosslinker and a polyfunctional carrier molecule.
64. The conjugate of claim 63 wherein the ligand is asialoorosomucoid.
65. The conjugate of claim 63 wherein the ligand is arabinogalactan or a Tris-(N-acetyl galactosamine aminohexyl glycoside) amide of tyrosyl(glutamyl)glutamate.
66. The conjugate of claim 64, wherein ddC is covalently bonded to a crosslinker selected from the group consisting of phosphate, glutarate and succinate, the crosslinker is covalently bonded to ddC and to a polyfunctional carrier molecule selected from the group consisting of polylysine and polyornithine, and the polyfunctional carrier molecule is covalently bonded to asialoorosomucoid.
67. The conjugate of claim 64 wherein ddC and asialoorosomucoid are covalently bonded to polyaldehyde dextran.
68. The conjugate of claim 64, wherein ddC is covalently bonded to an aminoacyl crosslinker, the crosslinker is covalently bonded to ddC and to a polyfunctional carrier molecule selected from the group consisting of polyglutamic acid, polyaspartic acid and polyaldehyde dextran, and the polyfunctional carrier molecule is covalently bonded to asialoorosomucoid.

-55.1-69. The conjugate of claim 68 wherein the aminoacyl crosslinker is derived from trans4-aminomethylcyclohexanecarboxylate or 4-aminobutyrate.

70. The conjugate of claim 68 wherein the aminoacyl crosslinker is a peptide comprising an amino acid sequence Leu-Ala-Leu.
71. The conjugate of claim 64 wherein the crosslinker is derived from (3-(2-pyridyldithio)propionate.
72. A pharmaceutical composition comprising a solution of the conjugate of claim64 and a physiologically acceptable carrier.
73. A conjugate comprising 9-.beta.-D-arabinofuranosyladenine (araA) and a ligand for an asialoglycoprotein receptor selected from the group consisting of asialoorosomucoid, arabinogalactan and a Tris-(N-acetyl galactosamine aminohexyl glycoside) amide of tyrosyl(glutamyl)glutamate, wherein araA is conjugated to the ligand by a crosslinker, a polyfunctional carrier molecule or a crosslinker and a polyfunctional carrier molecule.
74. The conjugate of claim 73 wherein the ligand is asialoorosomucoid.
75. The conjugate of claim 74 wherein araA is covalently bonded to a crosslinkerselected from the group consisting of phosphate, glutarate and succinate, the crosslinker is covalently bonded to araA and to a polyfunctional carrier molecule selected from the group consisting of polylysine and polyornithine, and the polyfunctional carrier molecule is covalently bonded to asialoorosomucoid.
76. The conjugate of claim 74 wherein araA is is covalently bonded to an aminoacyl crosslinker, the crosslinker is covalently bonded to araA and to a polyfunctional carrier molecule selected from the group consisting of polyglutamic acid, polyaspartic acid and polyaldehyde dextran, and the polyfunctional carrier molecule is covalently bonded to asialoorosomucoid.
77. The conjugate of claim 76 wherein the aminoacyl crosslinker is derived from trans-4-aminomethylcyclohexanecarboxylate or 4-aminobutyrate.
78. The conjugate of claim 70 wherein the aminoacyl crosslinker is a peptide comprising an amino acid sequence Leu-Ala-Leu.
79. A pharmaceutical composition comprising a solution of the conjugate of claim74 and a physiologically acceptable carrier.

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B is a crosslinker which is covalently bonded to the therapeutic agent and to a polyfunctional carrier molecule, C is a polyfunctional carrier molecule, D is a ligand for the asialoglycoprotein receptor selected from the group consisting of asialoorosomucoid, arabinogalactan and a Tris-(N-acetyl galactosamine aminohexyl glycoside) amide of tyrosyl(glutamyl)glutamate, wherein the ligand is covalently bonded to the polyfunctional carrier molecule such that the ligand can bind to the asialoglycoprotein receptor; and (b) administering the conjugate in a physiologically acceptable carrier to the subject.
89. A method for targeting a therapeutic agent to a cell with asialoglycoprotein receptors in a subject, comprising:
(a) forming a conjugate comprising a general formula A-C-D, wherein:
A is a therapeutic agent, which is covalently bonded to a polyfunctional carrier molecule, C is a polyfunctional carrier molecule, D is a ligand for an asialoglycoprotein receptor, wherein the ligand is covalently bonded to the polyfunctional carrier molecule such that the ligand can bind to the asialoglycoprotein receptor; and (b) administering the conjugate in a physiologically acceptable carrier to the subject.
90. A method for targeting a therapeutic agent to a cell with asialoglycoprotein receptors in a subject, comprising:
(a) forming a conjugate comprising a general formula A-B-D, wherein:

A is a therapeutic agent selected from the group consisting of nucleoside analogs, reverse transcriptase inhibitors, topoisomerase inhibitors, DNA
gyrase inhibitors and DNA binding agents, B is a crosslinker which is covalently bonded to the therapeutic agent and to asialoorosomucoid, D is asialoorosomucoid, wherein asialoorosomucoid is covalently bonded to the crosslinker such that asialoorosomucoid can bind to the asialoglycoprotein receptor; and (b) administering the conjugate in a physiologically acceptable carrier to the subject.
91. A method for targeting 9-.beta.-D-arabinofuranosylcytosine (araC) to a cell with asialoglycoprotein receptors in a subject comprising administering to the subject the pharmaceutical composition of claim 54.
92. A method for targeting 9-(2-hydroxyethyoxymethyl)guanine (ACV) to a cell with asialoglycoprotein receptors in a subject comprising administering to the subject the pharmaceutical composition of claim 62.
93. A method for targeting dideoxycytidine (ddC) to a cell with asialoglycoprotein receptors in a subject comprising administering to the subject the pharmaceutical composition of claim 72.
94. A method for targeting 9-.beta.-D-arabinofuranosyladenine (araA) to a cell with asialoglycoprotein receptors in a subject comprising administering to the subject the pharmaceutical composition of claim 79.
95. A method for targeting 3'-azido-3'-deoxythymidine (AZT) to a cell with asialoglycoprotein receptors in a subject comprising administering to the subject the pharmaceutical composition of claim 87.
96. A conjugate for targeting an agent that inhibits the translocation and/or fusion of endosomes to lysosomes in a cell expressing an asialoglycoprotein receptor, the conjugate comprising a general formula A-B-D, wherein:

A is am agent that inhibits the translocation and/or fusion of endosomes to lysosomes;
B is a reductively labile crosslinker which is covalently bonded to the agent that inhibits the translocation and/or fusion of endosomes to lysosomes; and D is a ligand for the asialoglycoprotein receptor, wherein the ligand is covalently bonded to the crosslinker such that the ligand can bind to the asialoglycoprotein receptor.
97. The conjugate of claim 96, wherein the agent that inhibits the translocation and/or fusion of endosomes to lysosomes is colchicine.
98. The conjugate of claim 97, wherein the reductively labile crosslinker is dithiopropionyl.
99. The conjugate of claim 96, wherein the ligand for the asialoglycoprotein receptor is an asialoglycoprotein.
100. The conjugate of claim 99, wherein the asialoglycoprotein is asialoorosomucoid.
101. A conjugate for targeting colchicine to a cell expressing an asialoglycoprotein receptor, the conjugate comprising a general formula A-B-D, wherein:
A is colchicine;
B is a reductively labile crosslinker which is covalently bonded to thecolchicine, and D is a ligand for the asialoglycoprotein receptor, wherein the ligand is covalently bonded to the crosslinker such that the ligand can bind to the asialoglycoprotein receptor.
102. The conjugate of claim 101, wherein the reductively labile crosslinker is dithiopropionyl.
103. The conjugate of claim 101, wherein the ligand for the asialoglycoprotein receptor is an asialoglycoprotein.

104. The conjugate of claim 103, wherein the asialoglycoprotein is asialoorosomucoid.