Note: Descriptions are shown in the official language in which they were submitted.
21 80348
~ 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
21 80348
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.
=
21 80348
~ 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
21 80348
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
_ _ _ _
21 80348
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:
21 80348
W0 95/18636 . ~ . ~
/0
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
2 1 80348
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:
2 1 80348
~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
0348
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
21 80348
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.
21 80348
W0 95/18636 ~ 5
/6
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
21 80348
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
-
21 80348
~ 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
WO 95/18636
~ ;~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
_, _ . ~ , . . .
2 1 80348
~ 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
21 80348
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
2 1 80348
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
21 80348
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
.
21 80348
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 '
2 1 80348
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
2 1 80348
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.
21 80348
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.
-
2 1 80348
~ ~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.
21 80348
O 95/18636
,~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
21 80348
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.
21 80348
~ 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
2 1 80348
WO 95118636 . _11. ' ~L O
7G
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).
80348
~ 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
2 1 80348
~ 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.
21 80348
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
-- PAD 37
- PLGA 16
GABA 2
araA glutarate PLL 5
glutarate PLL (10 Kd) 14
succinate PLL 5
phosphak PLL 7
AMCC 4
ACV glutarate PLL 4
succinate PLL 5
phosphate PLL
ddC glutarate PLL 12
glutarate PLL (10 Kd) 17
PAD 3 1
DP - 2
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.
21 80348
~W0 95/18636 P~
Y3
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
21 80348
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,
2 1 80348
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.
21 80348
~ 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
21 80348
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.
