Note: Descriptions are shown in the official language in which they were submitted.
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TETRAPARTATE PRODRUGS
TECHNICAL FIELD
The present invention relates to tetrapartate prodrugs. In particular, the
invention relates to polymer conjugates that provide tetrapartate prodrugs
that
deliver active agents linlced to uptalce enhancing moieties effective to
provide
enhanced efficacy, e.g_, as antitumor agents or the like.
to BACKGROUND OF THE INVENTION
Over the years, several methods of administering biologically-effective
materials to mammals have been proposed. Many biologically-effective
materials,
~, including medicinal agents and the like, are available as water-soluble
salts
and can be included in pharmaceutical formulations relatively easily. Problems
15 arise when the desired biologically-effective material is either insoluble
in aqueous
fluids or is rapidly degraded iy2 vivo. For example, alkaloids are often
especially
difficult to solubilize.
One way to solubilize biologically-effective materials) is to include them
as part of a soluble prodrug. Thus, prodrugs include chemical derivatives of a
2o biologically-active material, or parent compound which, upon
administration,
eventually liberate the parent compound zya vivo. Procli-ugs allow the artisan
to
modify the onset and/or duration of action of an agent zfz vzvo and can modify
the
transportation, distribution or solubility of a drug in the body. Furthermore,
prodrug formulations often reduce the toxicity and/or otherwise overcome
25 difficulties encountered when administering pharmaceutical preparations.
Typical examples of prodxugs include organic phosphates or esters of
alcohols or thioalcohols. See Remin~ton's Pharmaceutical Sciences, 16th Ed.,
A.
Osol, Ed. (1980), the disclosure of which is incorporated by reference herein.
Prodrugs are, by definition, forms of the parent or active compound. The .
30 rate of release of the active drug, typically, but not exclusively, by
hydrolysis of
the prodrug, is influenced by several factors, but especially by the type of
bond
joining the active drug to the modifier. Care must be taken to avoid preparing
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prodrugs which are eliminated through the kidney or reticular endothelial
system,
etc., before a sufficient amount of the parent compound is released. By
incorporating a polymer as part of the prodrug system, one can increase the
circulating half-life of the drug. However, in some situations, such as with
alkaloids, it has been determined that when only one or two polymers of less
than
about 10,000 daltons are conjugated thereto, the resulting conjugates are
rapidly
eliminated ire vivo especially if a somewhat hydrolysis-resistant linlcage is
used. In
fact, such conjugates are so rapidly cleared from the body that even if a
hydrolysis-
prone ester linlcage is used, not enough of the parent molecule is regenerated
in
vivo. This is often not a concern with moieties such as proteins, enzymes and
the
lilce, even when hydrolysis-resistant linlcages are used. In those cases
multiple
polymer strands, each having a molecular weight of about 2-5 kDa, are used to
further increase the molecular weight and circulating half life.
One way in which these problems have been addressed is described, for
example, by co-owned patent applications Serial Nos. 09/183,557, filed October
30, 1998 and 08/992,435, filed on December 17, 1997. These teach double
prodrugs, i.e., tripartate, that comprise polymer conjugates of various
biologically-
effective materials, and methods of making these conjugates. The double
prodrug
linkages are selected to hydrolyze ih vivo at a rate which generates
sufficient
amounts of the "second" and more reactive prodrug compound within a suitable
time after administration by, ,e.~., a 1,4-aryl or 1,6-aryl (e.g_, benzyl)
elimination
reaction, providing improved control of the pharmacokinetics of a number of
small
molecule drugs, agents and the like. However, further opportunities for
particularly selective targeting of diagnostic and/or therapeutic agents to
tissues or
cells of interest, by means of a rationally designed prodrug conjugate remain.
One particularly desirable target tissue for prodrugs is tumor tissue. It is
well known that tumors generally exhibit abnormal vascular permeability
characterized by enhanced permeability and retention ("EPR effect"). This EPR
effect advantageously allows biologically-effective materials, in the form of
3o macromolecules, ~, protein(s) such as enzymes and/or antibodies and
derivatives
or fragments thereof, or the like, to readily enter tumor interstitial tissue
space (see,
2
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for example, the review article by Maeda et al., 2000, J. of Controlled
Release,
65:271-284, incorporated by reference herein). Certain other tissues, in
addition to
tumors, can exhibit this same EPR effect, under conditions of inflammation,
and
the like.
In brief, and without being bound by any theory or hypothesis as to the
worlung of the EPR effect, it is believed that the EPR effect allows
penetration of
large molecule or macromolecule substances, including polymer-based delivery
systems. This provides a substantially selective delivery of polymer
conjugates
into tumor tissue space, e.g_, tumor interstitial space. Thereafter, however,
the
same EPR effect is believed to allow the released prodrug(s) andlor any newly
released relatively low molecular weight, biologically-effective materials, to
rapidly diffuse out of the extracellular tissue space of the targeted tissue.
It is
believed that if the released active agent fails to be taken up by the
surrounding
cells at a sufficient rate, they diffuse away from the release site in the
ongoing
blood or lymphatic flow.
Thus, there continues to be a need to provide additional technologies for
forming prodrugs which would benefit from the multiple level prodrug concept
and
compensate or control for the EPR effect by allowing for more rapid update or
transport of the released biologically-effective materials into tumor cells
andlor
cells of other tissues of interest that exhibit the EPR effect.
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SUMMARY OF THE INVENTION
Broadly, the invention provides fox a tetrapartate prodrug in the form of a
compound of Formula I:
LRzJr fRsls
R9 Y3 Ri Y4
(I) Rm C L1 C YZ ~ C Y1-C -Z-[D~y
15 R1o m P R4
IRelc Ar
RS~u
wherein:
Ll is a bifunctional linking moiety.
20 Broadly, D is a moiety that is a leaving group, or a residue of a compound
to be delivered into a cell. More particularly, D is a residue of an active
biological
material, or H and (y) is a positive integer equal to 1 or greater.
Preferably, (y)
ranges from 1 to about 5. When (y) is greater than l, each D moiety is
independently selected.
25 D can be any biologically active material that it is desired to deliver
into a
target cell or cells of an animal in need of such treatment, including anti-
inflammatory agents, detoxifying agents, anticancer agents, and diagnostics
for any
of these or other conditions.
Preferably, D is an anticancer agent, an anticancer prodrug, a detectable
30 tag, and combinations thereof. Any anticancer agent or suitable tag that
can be
linked to the tetrapartate prodrug is contemplated. Simply by way of example,
these include an anthracycline compound, a topoisomerase I inhibitor,
daunorubicin, doxorubicin; p-aminoaniline to name but a few.
When D is a leaving group, D can be, e.~., N-hydroxybenzotriazolyl,
35 halogen, N-hydroxyphthalimidyl, p-nitrophenoxy, imidazolyl,
N-hydroxysuccinimidyl andlor thiazolidinyl thione.
Z is covalently linked to [D]y, wherein Z is a moiety that is actively
transported into a target cell, a hydrophobic moiety, and combinations
thereof.
Optionally, Z is monovalent, multivalent, or more preferably, bivalent,
wherein (y)
40 is 1 or 2. Z itself optionally includes an amino acid residue, a sugar
residue, a fatty
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acid residue, a peptide residue, a C6_lg alkyl, a substituted aryl, a
heteroaryl,
--C(=O), --C(=S), and --C(=NR16), where R16 is as defined below.
Wheri Z includes at least one amino acid residue, the ariuno acid is, e. g_,
alanine, valine, leucine, isoleucine, glycine, serine, threonine, methionine,
cysteine,
phenylalanine, tyrosine, tryptophan, aspaxtic acid, glutamic acid, lysine,
arginine,
histidine, proline, andlor a combination thereof, to name but a few. When Z
includes a peptide, the peptide ranges in size, for instance, from about 2 to
about
amino acid residues. In one preferred embodiment, the peptide is Gly-Phe-Leu-
Gly or Gly-Phe-Leu.
to . In addition, Yl through Y4 are independently O, S, or NR12 ; and Rl l is
a
mono- or divalent polymer residue.
RI, R4, R9, Rio, Rrz and R16 are independently hydrogen, C1_6 alkyl, C3_iz
branched alkyl, C3_$ cycloalkyl, Cl_6 substituted alkyl, C3_$ substituted
cycloalkyl,
aryl, substituted aryl, arallcyl, Cl~ heteroalkyl, and/or substituted C1_6
heteroalkyl.
R2, R3, RS and R6 are independently hydrogen, C 1 _6 alkyl, C 1 _6 allcoxy,
phenoxy, C1_$ heteroalkyl, Cl_$ heteroalkoxy, substituted C1_6 alkyl, C3_g
cycloallcyl,
C3_g substituted cycloalkyl, aryl, substituted aryl, aralkyls, halo-, vitro-
and cyano-,
carboxy-, C1_6 carboxyalkyl and/or substituted Cl_6 alkylcarbonyl.
Ar is a moiety which, when included in Formula (17,~ forms a multi-
2o substituted aromatic hydrocarbon or a mufti-substituted heterocyclic group;
wherein (m), (r), (s), (t), and (u) are independently zero or one; .(p) is
zero or a
positive integer. In certain preferred embodiments, (p) is 1.
Ll is independently one of the following: .
~14 '
5
' -C ~' b
' Re
~ is and
a
i6 Rm
-C C rrRlg
i .
Ris
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wherein:
M is X or Q; where X is an electron withdrawing group;
Y3
Q is a moiety containing a free electron pair positioned three to six atoms
from -C-.
Simply by way of example, Q is one of the following: CZ_4 alkyls, cycloalkyls,
aryls, and aralkyl groups substituted with a member of the group consisting of
NH,
to O, S, -CHZ-C(O)-N(H)-, and/or ortho-substituted phenyls;
Y6 Rm
X is, for instance any one of O, NR2o, -C-N-, S, SO and SOZ;
(a) and (n) are independently zero or a positive integer; (b) is zero or one;
(g) is a
positive integer of 1 or greater;
(q) is three or four;
R~, Rs, R14, Rls, Rl~, Ris and R2o are independently selected from the same
group as that which defines Rl; and YS and Y~ are independently O, S, or NR12.
It
will be appreciated that when (y) is greater than 1, each of the D moieties
are the
same or different, respectively.
Preferably, (g) ranges from 1 to about 20, or more, but more typically
ranges from 1 to about 10.
Y3
Optionally, -[ L1-C]- comprises an amino acid residue, either naturally
occurring
or non-naturally occurring.
Rll is a mono- or bivalent polymer, e.g_, having a number average
molecular weight of from about 2,000 to about 100,000 daltons.
Methods of preparing the tetrapartate prodrugs of the invention are also
3o provided. In one embodiment, the method includes reacting a compound of
formula (III): LRsls
LR2lr
(III) R9 Ys O ~1 Yi C4 B
Rll C L1 C Y2
R10 p ~. R4
m
~R6~t a _i
6
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with a compound of the formula (IV):
(IV) Lx - Z - [D]Y;
wherein B is a leaving group for Formula (~ and is defined as above for
when D is a leaving group.
Lx is a leaving group for Formula (IV) and is defined as above for when D
is a leaving group.
L1, Ar, Z, D, Rl - R6, R~ - Rll , Yi-Y3, and integers are defined as above.
The reaction between (III) and (IV) is preferably conducted in the presence of
a
solvent and a base. The solvent is, for example, chloroform, methylene
chloride,
toluene, dimethylformamide and/or combinations thereof. Dimethylformamide is
generally preferred. The base is, fox example, dimethylaminopyridine,
diisopropylethylamine, pyridine, triethylamine and/or combinations thereof.
Yet another method of preparing a tetrapartate prodrug of the invention is
conducted by reacting a compound of formula (V)
~R2~r ~R3~s
R1 Yq.
(V) R9 Yg ~ II
R11 C L1 C Y2 O -C-Y1~C -Z-La
R1o m P Ar R4
rn ,
with a biologically active material; wherein
La is a leaving group as defined when D is a leaving group.
L1, Ar, Z, D, Rz - R~, R9 - Rll , Yl-Y3, and integers are defined as above.
The reaction between (V) and the biologically active material is conducted
in the presence of a coupling agent, ~, 1,3-diisopropylcarbodiimide, a dialkyl
carbodiimides, 2-halo-1-alkyl-pyridinium halide, 1-(3-dimethylamino-propyl)-3-
ethyl carbodiimide, 1-propanephosphonic acid cyclic anhydride, phenyl
3o dichlorophosphates, andlor combinations thereof. The reaction between (V)
and
the biologically active material is conducted in the presence of a solvent and
a
base, e.~., as described above for the previous synthetic method.
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Methods of using the inventive tetrapartate prodrugs are also provided, ~,
by treating a disease or disorder in an animal, by administering a
pharmaceutically
acceptable composition comprising an effective amount of a compound of
Formula I, to an animal in need thereof. In particular, a method is provided
of
delivering a biologically active material D, into a cell in need of treatment
therewith, by a process of:
administering a compound of Formula I to an animal wherein the cell is
present, and wherein Formula I is hydrolyzed ire vivo extracellularly to
yield:
LR2~r ~R3~s
Formula I-(i) Rl y4
Y* ~ C Yl C Z [
R4
ru ,~ Ar
wherein Y* is the remainder of YZ, and is independently selected from the
group
consisting of HO-, HS-, or HNR12 - ;
and Formula I-(i) then spontaneously hydrolyzes to
LR2~r ~R3~s
R1
Formula I-(ii)
Y** Q~=C
n4
~R6~t Ar RS~u
and COZ ,
and Formula I-(iii) Z-[D]y ;
wherein Y** is the remainder of Y*, and is independently selected from the
group
consisting of O , S , or NR12 and
Z-[D]y crosses the membrane of the cell, and is hydrolyzed therein to release
D.
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BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1: Illustrates a summary of the reaction scheme for the preparation of
the
tetrapartate prodrugs of doxorubicin by the methods of Examples 1-5, wherein
the
aromatic is a benzyl derivative.
Figure 2A: Illustrates a general reaction scheme for the degradation of the
tetrapartate prodrug prodrug by sequential hydrolysis of the compound of
Formula
I. Thus, variables of Fig. 1 are defined as for Formula I, supra. Symbols for
reaction steps: (a): controllable rate in vivo cleavage; (b) "first" prodrug;
(c) fast
to reaction in presence of water; (d) Z-D is the "second" prodrug and it is
released
substantially into extracellular space; (e) uptalee of Z-D into cells and
intracellular
enzymatic hydrolysis releases D.
Figure 2B: Illustrates a specific reaction schemes for the degradation of
tetrapartate prodrug compound identified herein as compounds 14 (top scheme)
and 17 (bottom scheme) wherein both schemes result in a final product of
doxorubicin substantially released within a cell. Symbols for reaction steps
are
analogous to those of Figure 2A, so that (a), (b), (c), (d) and (e) are the
analogous
reaction steps for the in vivo degradation of compound 14, and that (a'),
(b'), (c'),
(d') and (e') are the analogous reaction steps for the ire vivo degradation of
2o compound 17. The products of step (e') are D and Z, but the figure
illustrates that
much of Z further degrades into a C12 acid and leucine.
Figure 3: Illustrates a schematic of the synthesis of compound 2 as described
by
Method 1 of Example 1.
Figure 4: Illustrates the synthesis of compound 2, as described by Method 2 of
Example 1.
Figure 5: Illustrates the synthesis of compound 6, as described by Example 2.
Figure 6: Illustrates a schematic of the synthesis of compound 8, as described
by
Example 3.
Figure 7A: Illustrates a schematic of the synthesis of compound 10, as
described
3o by Example 4.
9
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Figure 7B: Illustrates a schematic of the synthesis of compound 12, as
described
by Example 5.
Figure 8: Illustrates a schematic of the synthesis of compound 14, as
described by
Example 6.
Figure 9: Illustrates a schematic of the synthesis of compound 17, as
described by
Example 7.
DETAILED DESCRIPTION OF THE INVENTION
Accordingly, the invention provides triple prodrugs compositions,
hereinafter, "tetrapartate" prodrugs for delivering a biologically active
material
into certain important target cells, such as, for instance, tumor cells, as
well as
methods of making and using the same. The tetrapartate prodrug compositions of
the present invention contain hydrolyzable linkages between the polymer
portion
and a biologically active material. The biologically active material is, for
example,
a moiety derived from a biologically active nucleophile, i.e., a native or
unmodified drug or diagnostic tag. These linkages are preferably ester and/or
amide linkages designed to hydrolyze at a rate which generates sufficient
amounts
of the biologically active parent compound in a suitable time. The term
"sufficient
amounts" for purposes of the present invention shall mean an amount which
2o achieves a therapeutic effect.
The present invention is broadly based upon the principle that biologically
active materials suitable for incorporation into the polymer-based prodrug
conjugates, ~, the double prodrug compositions as discussed supra, may
themselves be substances/compounds which are not active after hydrolytic
release
from the linlced composition, but which will become active after undergoing a
further chemical process/reaction, thus providing triple-acting prodrugs.
These
triple acting prodrugs are referred to herein as "tetrapartate" prodrugs
because the
inventive conjugates are provided in essentially four parts.
With the tetrapartate prodrugs of the invention, a therapeutic or diagnostic
agent that is delivered to the bloodstream by the above-described double
prodrug
transport system, will remain inactive until entering or being actively
transported
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into a target cell of interest, whereupon it is activated by intracellular
chemistry,
e.g_, by an enzyme or enzyme system present in that tissue or cell.
In particular, it has now been discovered that when certain types of
additional moieties are linked to the biologically active material as part of
the
above described double prodrug conjugates, the effectiveness of many such
biologically active materials is markedly increased, relative to the
effectiveness
seen with analogous prodrugs that lack such additional moiety. The
tetrapartate
prodrug conjugates of the invention are thought to provide enhanced
effectiveness,
e. g_, for therapeutic and/or diagnostic activity, in the delivery and
activity of
l0 certain biologically active materials, e.~.,, particularly small molecule
therapeutic
and diagnostic agents. The tetrapartate prodrugs of the invention prepared so
that
in vivo hydrolysis of the polymer-based conjugate cleaves the conjugate so as
to
release the active biological material into extracellular fluid, while still
linked to
the additional moiety. The biologically active materials are preferably, but
not
exclusively, small molecule therapeutic andlor diagnostic agents. As
exemplified
below, in one preferred embodiment these are small molecule anticancer agents,
and the tissue to be treated is tumor tissue.
Without intending to be bound by any theory or hypothesis as to how the
invention might operate, it is believed that, depending upon the additional
moiety
2o selected as a transport enhancer, the rate of transport of a biologically
active
material into tumor cells is by the delivery of a biologically active material
into
extracellular tissue space, e.~., of a tissue exhibiting an EPR effect, in a
protected
andlor transport-enhanced form. For convenience in description, the
"additional
moiety(s)" as mentioned are described herein as, "transport enhancers."
However, in providing this convenient descriptive term, it is not intended to
limit the scope of the invention solely to added moieties that solely enhance
transport of biologically active materials into targeted cells, since it is
believed that
additional or alternative mechanisms, such as protection of the Z-[D]y from
extracellular hydrolytic enzyme activity, may contribute to the advantages of
the
3o inventive tetrapartate prodrug.
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In a further still option, the transport enhancer is selected from among
known substrates for a cell membrane transport system. Simply by way of
example, cells are known to actively transport certain nutrients and endocrine
factors, and the like, and such nutrients, or analogs thereof, are readily
employed to
enhance active transport of a biologically effective material into target
cells.
Examples of these nutrients include amino acid residues, peptides, e.g_, short
peptides ranging in size from about 2 to about 10 residues or more, simple
sugars
and fatty acids, endocrine factors, and the like.
Desirable amino acid residues include all of the known naturally-occurring
L-amino acids. For example, L-isoleucine as a transport enhancer is
exemplified
in the Examples provided below. Surprisingly, it has also been discovered that
D-
amino acids are useful as transport enhancers, e.g., both D and L-alanine, and
other
analogous amino acid optical isomers, show the same activity. Derivatives and
analogs of the naturally occurring amino acids, as well as various art-known
non-
naturally occurnng amino acids (D or L), hydrophobic or non-hydrophobic, are
also contemplated to be within the scope of the invention. Simply by way of
example, amino acid analogs and derivates include: 2-aminoadipic acid,
3-aminoadipic acid, beta-alanine, beta-aminopropionic acid, 2-aminobutyric
acid,
4-aminobutyric acid, piperidinic acid, 6-aminocaproic acid, 2-aminoheptanoic
acid,
2-aminoisobutyric acid, 3-aminoisobutyric acid, 2-aminopimelic acid,
2,4-diaminobutyric acid, desmosine, 2,2-diaminopimelic acid,
2,3-diaminopropionic acid, n-ethylglycine, N-ethylasparagine, 3-
hydroxyproline,
4-hydroxyproline, isodesmosine, allo-isoleucine, N-methylglycine, sarcosine,
N-methylisoleucine, 6-N-methyllysine, N-methylvaline, norvaline, norleucine,
ornithine, and others too numerous to mention, that are listed in 63 Fed.
Rep.,
29620, 29622, incorporated by reference herein.
Short peptides are, for example, peptides ranging from 2 to about 10, or
more, amino acid residues, as mentioned supra. In this embodiment of the
invention, it is believed that such peptide transport enhancers need not be
hydrophobic, but are thought to function in other ways to enhance uptake
and/or to
protect the linked small molecule agents from premature hydrolysis in the
general
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v. .."~. .. , .~.. .V",, "",., ..,.,.,. . ,. w,~ ~:;.,.:~"~, k-
bloodstream. For instance, peptide transport enhancers, and other transport
enhancers of similar molecular weight ranges, are thought to sterically hinder
cleavage from the biologically active agent by plasma-based hydrolytic
enzymes,
but are then cleaved within a target cell by various peptides and/or
proteases, such
as cathepsins.
Preferably, the transport enhancer is a hydrophobic moiety. Without
meaning to be bound to any theory or hypothesis as to how hydrophobicity
contributes to efficacy, it is believed that a hydrophobic moiety inhibits the
extracellular cleavage of the transport enhancer away from the active
biological
to agent, by inhibiting the attach of hydrolytic enzymes, etc. present in the
extracellular tissue space, e.g_, in the plasma. Thus, preferred transport
enhancers
include, e.g_, . hydrophobic amino acids such as alanine, valine, leucine,
isoleucine, methionine, proline, phenylalanine, tyrosine, and tryptophane, as
well
as non-naturally occurring derivatives and analogs thereof, as mentioned
supra.
Tn a further option, the transport enhancer is a hydrophobic organic moiety.
Simply by way of example, the organic moiety is a C~_l8, or larger, alkyl,
aryl or
heteroaryl-substituted or nonsubstituted. The organic moiety transport
enhancer is
also contemplated to encompass and include organic functional groups
including,
~, - C(=S) and/or - C(=Y3)~
In order to appreciate the nature of the invention, several definitions and
explanations are provided as follows. The term, "tetrapartate" refers to
prodrug
conjugates, and in particular, to conjugates incorporating the features of the
double
prodrugs, as discussed supra, and an additional moiety serving as a transport
enhancer positioned between the residue of the biologically active compound
and
the polymer moiety to form a 4-part structure wherein the biologically active
agent
is the fourth part of the conjugate. This structure provides that the residue
of the
biologically active compound is optimized for transport and release
substantially
into a target cell. The fourth element of the "tetrapartate" is therefore the
residue
of the biologically active compound, itself. Further, diagnostic tetrapartate
conjugates incorporating detectable tags are also contemplated, and the use of
the
terms, "tetrapartate prodrug," or simply, "prodrug" herein, with reference to
the
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inventive conjugates, broadly also includes conjugates and methods of making
and
delivering diagnostic reagents, including tagged drugs, as well, unless
otherwise
specified or distinguished.
For purposes of the present invention, the terms, "biologically active
material," and "biologically active compound," and/or "biologically active
agent,"
etc., are used interchangeably unless otherwise stated. These terms refer, for
example, to a drug or pharmaceutical, and/or a diagnostic agent or reagent,
such as
a detectable label or marker. The terms "drug," "agent," "medicinal agent,"
and
"active agent" herein refer to compounds) with useful activity, particularly
when
l0 administered to an animal, ih vivo, and/or to precursors of the same,
unless
otherwise stated.
As noted in the previous lines, biological activity is any property of such a
material or compound that is useful in an animal or person, ~., for medical,
and/or diagnostic purposes. Preferably, the biological activity is manifested
in the ,
intracellular space, i.e., the drug or diagnostic agent preferably but not
exclusively
is useful once delivered/released into the cytoplasm and/or nucleus of one or
more
types of target cell of interest.
For purposes of the present invention, the use of the singular or plural is
not
meant to be limiting of the numerical number of the referenced item or object.
2o Thus, the use of the singular to refer to a cell, polymer or drug does not
imply that
only one cell is treated, only one molecule is prepared or employed, and/or
only
one drug is employed, and the use of the plural does not exclude application
to a
single referenced item, unless expressly stated. Further to this point, for
purposes
of the present invention, the terms, "cell," "cell type," "target cell," and
etc., are
used interchangeably unless otherwise specified and refer to both singular and
plural cells, however organized into a tissue, tissues or other system or
component,
normal or pathological, of an animal or patient to be treated.
For purposes of the present invention, the term "residue" shall be
understood to mean that portion of a biologically active compound which
remains
3o attached by modification of e.g_, an available hydroxyl or amino group, to
form,
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after it has undergone a reaction in which the prodrug carrier portion has
been for
example, an ester or amide group, respectively.
For purposes of the present invention, the term "alkyl" shall be understood
to include, e.,~., straight, branched, substituted C1_12 alkyls, including
alkoxy,
C3_$ cycloalkyls or substituted cycloalkyls, etc.
When the prodrugs of the present invention include the double prodrugs
taught by co-owned Serial Nos. 09/832,557 and 08/992,435, it is generally
preferred that the polymeric portion is first released by hydrolysis and then
the
resultant "second prodrug" moiety undergoes a 1,4- or 1,6-aryl e.(~., benzyl)
l0 elimination reaction to regenerate, for example, a moiety comprising a
further
prodrug. Thereafter, the released moiety diffuses and/or is transported into
target
cells, where a substantial proportion of the incorporated remainder of the
prodrug
is further cleaved or hydrolyzed by intracellular enzymes to release the
biologically
active compound.
In addition, the terms "cancer" or "tumor" are clinically descriptive terms
which encompass a myriad of diseases characterized by cells that exhibit
unchecked and abnormal cellular proliferation. The term "tumor", when applied
to
tissue, generally refers to any abnormal tissue growth, i~e., excessive and
abnormal
cellular proliferation. The term "cancer" is an older term which is generally
used to
2o describe a malignant tumor or the disease state arising therefrom.
Alternatively,
the art refers to an abnormal growth as a neoplasm, and to a malignant
abnormal
growth as a malignant neoplasm. These general clinical terms, when used with
reference to cells, tissues, and/or one or more conditions characterized as a
disease
or disorder, as used herein, are intended to be interchangeable and
synonymous,
unless otherwise specified.
A. FORMULA (1)
In one aspect of the invention, there are provided compounds of formula (I):
LR2~r ~R3~s
R9 Y3 ,R1 1'4
(I) i .. ~ i
R1t C L1,C p Y2 O -R_Yt_C_Z_~DIY
Rlo m Ar 4
~Rs~t ~Rs~u
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WO 02/43663 PCT/USO1/45127
wherein:
Ll is a bifunctional linking moiety; for example, Ll is independently one of
the following:
R~ R
',14
-M C-
- Y
~CH2~ Re n ~ s b
R1s
and
i 6 R14
X18
Rls
g
Simply by way of example, in one optional embodiment, Ll is
NH
O
In a further embodiment, Z is a transport enhancer and/or protector that is
covalently linked to D, wherein Z is selected to enhance or improve
intracellular
delivery of Z-[D]y into a cell; relative to the intracellular delivery of D
without Z.
In certain embodiments, Z optionally includes one of the following: an
amino acid residue, a sugar residue, a fatty acid residue, a peptide residue,
a C6_ls
allcyl, a substituted aryl, a heteroaryl, -C(=O), -C(=S), and -C(=NR16),
and/or
combinations thereof.
D is a moiety that is a leaving group, or a residue of a compound to be
delivered into a cell. Preferably but not exclusively, D is an active
biological
material, or H.
The artisan will appreciate that each D can be selected independently, so
that there can be as many as five, or more, different types of moieties linked
to Z
16
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WO 02/43663 PCT/USO1/45127
for delivery into a target cell of interest. Preferably, D is a therapeutic
agent or
drug, but D is also optionally a diagnostic agent.
Simply by way of illustration, and in certain additional optional
embodiments, y is 2 and Z is divalent, D can be two moieties, including both a
therapeutic agent and a diagnostic tag for delivery into the same cell type or
into
the cells of a tissue type of interest. Further still, such plural D moieties
will
comprise multiple different therapeutic agents, preferably targeted to a
single type
of cell, where when delivered and released together, the different agents act
synergistically to achieve a desired therapeutic effect. In one preferred
optional
embodiment, D is one or more anticancer agents) and/or an anticancer prodrug,
or
residue thereof.
In certain further embodiments, D is
Yi
H, or -C-B; where
B is H, a leaving group, a residue of an amine-containing moiety, or a
residue of a hydroxyl-containing moiety;
Yi_6 are independently O, S or NR12;
M is X or Q; where
X is an electron withdrawing group;
Q is a moiety containing a free electron pair positioned three to six
Ys
atoms from -C-;
Rl, R4, R~, R8, R~, Rlo, R12, R14, Ris, Rm and Rl$ are independently selected
from the group consisting of hydrogen, Cl_6 alkyls, C3_12 branched allcyls,
C3_$
cycloalkyls, Cl_G substituted alkyls, C3_8 substituted cycloalkyls, aryls,
substituted
aryls, arallcyls, Cl_6 heteroalkyls and substituted C1_6 heteroalkyls;
R2, R3, RS and R6 are independently selected from the group consisting of
hydrogen, Cl_6 alkyls, C1_6 alkoxys, phenoxy, Cl_$ heteroalkyls, Cl_g
heteroalkoxy,
substituted C1_6 alkyls, C3_$ cycloalkyls, C3_$ substituted cycloalkyls,
aryls,
17
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WO 02/43663 PCT/USO1/45127
substituted aryls, aralkyls, halo-, nitro- and cyano-, carboxy-, Cl_6
carboxyalkyls
and Cl_6 alkylcarbonyls;
Ar is a moiety which when included in Formula (I) forms a multi-
substituted aromatic hydrocarbon or a mufti-substituted heterocyclic group;
(b), (m), (r), (s), (t), and (u) are independently zero or one;
(a) and (n) are independently zero or a positive integer;
(p) is zero or a positive integer;
(q) is three or four; and
Rl l is a polymer such as a polyalkylene oxide.
B. DESCRIPTION OF THE Ar MOIETY
Referring to Formula (I), it can be seen that the Ar is a moiety, which when
included in Formula (I), forms a mufti-substituted aromatic hydrocarbon or a
mufti-substituted heterocyclic group. A key feature is that the Ar moiety is
aromatic in nature. Generally, to be aromatic, the oc-electrons must be shared
within a "cloud" both above and below the plane of a cyclic molecule.
Furthermore, the number of a electrons must satisfy the Huclde rule (4n+2).
Those of ordinary skill will realize that a myriad of moieties will satisfy
the
aromatic requirement of the moiety and thus are suitable for use herein.
2o Preferred aromatic hydrocarbon moieties include, without limitation:
, OZ ,
E Z E N
O , OO ,
v a v
~J J
Z ~~~ ~ .
\,
OZ, O
Z ~ ~Z~ ' ~N
18
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WO 02/43663 PCT/USO1/45127
000,0 ,~
O
~_.~. ~~~~p.
fR2l
and
fR~l
to In the above-listed aromatic moieties, J is O, S, or N-Rl~, E and Z are
independently C-Rl~ or N-Rl~; and Rl~ is independently selected from the same
group as that which defines R~ in Formula (I) e.~., hydrogen, C1_6 alkyls,
etc.
Isomers of the five and six-membered rings are also contemplated as well as
benzo- and dibenzo- systems and their related congeners are also contemplated.
It
15 will also be appreciated by the artisan of ordinary shill that the aromatic
rings can
optionally be substituted with hetero-atoms such as O, S, NR13, etc. so long
as
Huckel's rule is obeyed. Furthermore, the aromatic or heterocyclic structures
may
optionally be substituted with halogens) and/or side chains as those terms are
commonly understood in the art. However, all structures suitable for Ar
moieties
20 of the present invention are capable of allowing the Y3 and C(Rl)(R4)
moieties to
be in a para or an ortho arrangement with the same plane as shown in Formulas
I-
A and I-B, below.
R2 As s f l
L Rz' r
25 I-A /~ Y3 O ~I /l I B ' r
/~ Ys ~Ra~s
n~ Ra
Ar
R6 \Rs1
R6 t
wherein all variables are as defined above for Formula (I).
3o When the Ar moiety includes a para arrangement of the Y3 and C(Rl)(R~.)
moieties, preferred aspects of the present invention define (r), (s), (t), and
(u) as
19
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WO 02/43663 PCT/USO1/45127
one and RZ and R6 as being independently selected from the group consisting of
metlryl, Cl_6 alkyls, methyl, C1_6 alkoxys, and methoxy. More preferably, R2
and R6
are either both methyl or methoxy moieties. Furthermore, R3 and RS are
preferably
both hydrogen, Rl and R4 are preferably either hydrogen, CH3 or CH2CH3. Y1
through Y4 i.e.,Yl~) are preferably O or NR12 where R12 is H or a C1_6 alkyl
or
substituted alkyl. More preferably, Yl and Y4 are O.
For purposes of the present invention, substituted alkyls include
carboxyalkyls, aminoalkyls, dialkylarninos, hydroxyalkyls and mercaptoalkyls;
substituted cycloalkyls include moieties such as 4-chlorocyclohexyl; aryls
include
to moieties such as napthyl; substituted aryls include moieties such as 3-
bromophenyl; aralkyls include moieties such as toluyl; heteroalkyls include
moieties such as ethylthiophene; substituted heteroalkyls include moieties
such as
3-methoxy-thiophene; allcoxy includes moieties such as methoxy; and phenoxy
includes moieties such as 3-nitrophenoxy. Halo- shall be understood to include
15 fluoro, chloro, iodo and bromo.
C. LINKER MOIETY Ll
As shown above, the invention includes bifunctional linking moiety Li
Y3
2o II
which when combined with -C-, forms an amino acid residue linker, or when (p)
is greater than one, a peptide residue linker.
Suitable amino acid residues can be selected from naturally-occurring or
synthetic, i.e. non-naturally-occurring, amino acids including alanine,
valine,
25 leucine, isoleucine, glycine, serine, threonine, methionine, cysteine,
phenylalanine,
tyrosine, tryptophan, aspartic acid, glutamic acid, lysine, arginine,
histidine or
proline. Some preferred peptide residues include Gly-Phe-Leu-Gly and Gly-Phe-
Leu. It is noted that the terminal amino group of the amino acid or peptide
residue
will be proximal to Rl l (i.e. polymer). Peptides can be readily synthesized
or
30 obtained from commercial sources for inclusion herein.
In alternative embodiments, LI includes the moiety (M) which is either an
electron withdrawing group (designated herein as X), or a moiety containing a
free
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WO 02/43663 PCT/USO1/45127
Y3
electron paix positioned three to six atoms from the -C- (designated herein as
Q).
In a particularly preferred embodiment, the tetrapartate conjugates of the
invention
are based upon an aromatic moiety that is a substituted benzyl, and the first
two
breaks in the prodrug, in vivo, are based on a 1,4 or 1,6 benzyl elimination
mechanism. This embodiment provides a conjugate as illustrated by Figure 1. In
Figure 1, an overview of the genexal reaction scheme followed in synthesis of
the
compounds produced in Examples 1-5, provided herein below, is set forth.
to Precursor compound (Fig. 1, compound a) is reacted in the presence of
leucine
doxorubicin ("leu-dox"), dimethylaminopyridine and dimethyformamide to form
(Fig. 1, compound b), wherein the variables are set forth by Table 1, below.
TABLE 1
Compound No.
Variables
2 L1, Y2=O; R2, R6=H
6 Ll = NH; Y2 = O; R2, R6 = H
8 Ll = NHCOCHZCH2NH; YZ = O; R2, R6 = H
Ll = CH2 ; YZ = O; R2, R6 = CH3
12 Ll, YZ = O; R2, R6 = CH3
D. THE DOUBLE PRODRUG LINKAGE PORTION
The first labile bond of the tetrapartate prodrug system, which joins the Ll
Y3
to -C-, is selected to hydrolyze, such as via an esterase catalyzed hydrolysis
in vivo
at a rate which generates sufficient amounts of the "second" prodrug compound
within a suitable time after administration. The term "sufficient amounts" for
purposes of the present invention shall mean an amount which may later undergo
sufficient 1,4 or 1,6 -benzyl elimination ira viva to release the native
compound and
achieve a desired effect. Preferably, (n) is an integer from 1 to about 12.
More
preferably, (n) is 1 or 2.
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WO 02/43663 PCT/USO1/45127
1. The Electron Withdrawing Group X
In those aspects of Formula (I) where Ll includes M, the moiety may be an
electron withdrawing group, designated herein as X. For purposes of the
present
invention, "electron withdrawing groups" are groups which tend to pull shared
s electrons toward themselves thereby making carbon more electro-positive.
This, in
turn, destabilizes the carbonyl moiety, causing more rapid hydrolysis. Thus,
when
X is in the cc position to the ester, it modulates the rates of hydrolysis and
enzymatic cleavage.
Y6 Rm
to II I
In particular, X can be moieties such as O, NR2o, -C-N-, S, SO and S02
where Y~ is the same as that defined by Yl, R12 and Rl~ are the same as
defined
above i.e., H, Cl_~ alkyls, branched alkyls, aryls, etc. R1 is the same as
defined by
Formula I, supra. Preferably, however, when X is NR2o, R2o is H, a Cl_6 alkyl
15 such as methyl or ethyl or substituted C1_~ alkyl. It is preferred that X
is either O or
~20~
2. O Portion of L1
20 Alternatively, when Ll includes Q, which is a moiety containing a free
Y3
II
electron pair positioned three to six atoms from the -C- moiety, the polymer,
Rll, is
preferably attached to Q via a heteroatom such as oxygen. In a preferred
25 embodiment, the free electron pair is five atoms from this oxygen. Q can be
selected from the non-limiting list of C2_4 alkyls or cycloalkyls, aryls or
arallcyl
groups substituted with a member of the group consisting of O, S and NR12. The
free electron pair can be anywhere along the Q moiety as long as the defined
spacing between the free electron pair and Y4 is maintained.
3o In these embodiments, Rll is attached to Q via NRl2a O, or S. Thus, Q
assists hydrolysis of the prodrug linkage by anchimeric assistance because the
free
electron pair moiety can generate a three- to six-membered, but preferably
five-
membered, ring by-product upon hydrolysis of the preferably ester linkage.
22
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WO 02/43663 PCT/USO1/45127
Q can also be selected from the group consisting of CZ_4 alkyls, cycloalkyls,
aryls, aralkyl groups substituted with a member of the group consisting of NH,
O,
S, -CHZ-C(O)-N(H)-, and ortho-substituted phenyls such as
and
NN/
RZi-N-C
wherein R21 is selected from the same group as that which defines R12
supYa and T is any moiety linked to Q.
3. Drug Generation Via Hydrolysis of the Prodru~
The prodrug compounds of the present invention are designed so that the
t1,2 of hydrolysis is less than the t1,2 of elimination in plasma. The
linkages
included in the compounds have in-vivo hydrolysis rates, in plasma, that are
short
enough to allow sufficient amounts of the transport enhanced conjugate with
parent
compounds, i-e., the amino- or hydroxyl-containing biologically active
compound,
to be released prior to elimination. Some preferred compounds of the present
invention, i-e., those in which (n) is 1, have a tli2 for hydrolysis in plasma
ranging
from about 5 minutes to about 12 hours. Preferably, the compositions have a
plasma tli2 of hydrolysis ranging from about 0.5 to about 8 hours and most
2o preferably from about 1 to about 6 hours.
4. 1,4 or 1,6 -Benzyl Elimination;
Release of Native Drug-Linked to TJptake Enhancer and
Intracellular Release of Native Drug from Transport Enhancer
Once the hydrolysis of the double prodrug portion of the conjugate has
taken the place i~c vivo, usually via esterase activity or pH moderated
activity or
cyclization reaction, the polymeric residue is cleaved and the resultant
second
prodrug moiety remains.
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WO 02/43663 PCT/USO1/45127
Without meaning to be bound by any theory or hypothesis as to how the
tetrapartate conjugates or prodrugs of the invention operate, it is believed
that once
the biologically effective material as linked to the uptake, enters a target
cell,
various intracellular peptidases and/or proteases, including ~, cathepsins,
cleave,
~, by enzymatic hydrolysis, the transport enhancer moiety to release the
biologically effective material within the target cell. The following
degradation
scheme is provided for illustrative purposes and is not intended to limit the
scope
of the invention.
With reference to Figures 2A and 2B, when Ar is a benzyl derivative, a 1,4
Io or 1,6-benzyl elimination (or an analogous reaction with other aromatic
moieties)
occurs if2 vivo and produces the desired transport-enhanced native compound by
electron migration, causing irreversible decomposition, which releases the
transport-enhanced native compound.
Thus, Fig. 2A illustrates, in overview, an in vivo degradation reaction is
shown wherein the variables of the starting tetrapartate compound are defined
as
described supra for Formula I. With reference to Fig. 2, the illustrated
tetrapartate
prodrug undergoes a controllable rate cleavage, ih vivo, labeled as (a) to
remove
Rll. The remaining compound (b) immediately undergoes a fast hydrolysis (c) in
the presence of water to separate (b) and release Z-D, the enhancer-prodrug is
released (d) mostly into the extracellular tissue space as Z-D. Z-D is, in
turn, taken
up by surrounding cells and hydrolyzed intracellularly (e) to release D. Any D
that
might be released in the intracellular tissue space is thought to be rapidly
removed
by blood or plasma flow, and is believed to provide little or no added benefit
in
terms of a therapeutic or diagnostic function.
In somewhat more detail, Fig. 2B provides schematics of in vivo
degradation reactions thought to occur for compounds 14 and 17 (see, e.g_, the
Examples below for synthetic details). Analogously to the scheme of Fig. 2A,
compound 14 is degraded by a controllable rate cleavage, iya vivo, labeled as
(a) to
remove PEG. The remaining compound (b) immediately undergoes a fast
3o hydrolysis (c) in the presence of water to separate (b) and release inter
alia, (d)
which is analogous to Z-D of Fig. 2A. The enhancer-doxorubicin prodrug is
24
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WO 02/43663 PCT/USO1/45127
released mostly into the extracellular tissue space (d). The enhancer-
doxorubicin
prodrug is, in turn, talcen up by surrounding cells and hydrolyzed
intracellularly (e)
to release active doxorubicin. An analogous degradation process is described
in
Fig. 2B for compound 27, wherein the analogous process steps are labeled as
(a'),
(b'), (c'), (d') and (e'). Reaction (f) and (f ') refers to respective side
reactions
wherein the aromatic remainder that is cleaved from the prodrug is converted
to a
water soluble hydroxyl derivative.
E. SUBSTANTIALLY NON-ANTIGENIC POLYMERS
The " tetrapartate prodrug" compositions of the present invention include
water-soluble polymer, Rll. Optionally, RIZ includes a capping group A.
Capping
group A includes, for example, hydrogen, Cr_~ alkyl moieties, carboxyallcyl,
dialkyl
acyl urea alkyls, and/or a compound of formula (II) shown below, which forms a
bis-system:
Ya Y3
G' Z-C Y Y2 C L1
P
~io
m
(B)
wherein G' is the.same as D or another member of the group defined by D, v is
0 or
1, and the remaining variables are as set forth above with regard to Formula
(I).
2o Suitable examples of such polymers include polyallcylene oxides such as
polyethylene glycols which are also preferably substantially non-antigenic.
The
general formula for PEG and its derivatives, i.e. A'-O-(CHZCH20)X (CH~,)n A,
where (x) represents the degree of polymerization (i.e. 10-2,300) or number of
repeating units in the polymer chain and is dependent on the molecular weight
of
the polymer; (n) is zero or a positive integer; A is a capping group as
defined
herein, i.e. an -H, amino, carboxy, halo, Cl_6 alkyl or other activating group
and A'
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WO 02/43663 PCT/USO1/45127
is the same as A or another A moiety. Also useful are polypropylene glycols,
branched PEG derivatives such as those described in commonly-assigned U.S.
Patent No. 5,643,575, "star-PEG's" and mufti-armed PEG's such as those
described in Shearwater Polymers, Inc. catalog "Polyethylene Glycol
Derivatives
1997-1998"; the disclosure of each is incorporated herein by reference. It
will be
understood that the water-soluble polymer will be functionalized for
attachment to
the linkage via M, X or Q herein. As an example, the PEG portion of the
prodrugs
can be the following non-limiting compounds:
-C(=Y~)-(CH2)n O-(CH2CH20)X A,
to -C(=Y~)-Y~-(CHZ)n O-(CH2CH20)X A and
-C(=Y~)-NR12-(CH2)n O-(CH2CH20)X-A,
where Y~ is O or S; and A, R12, (n) and (x) are as defined above.
In many aspects of the present invention, polyethylene glycols (PEG's),
mono-activated, Cl_4 alkyl-terminated PAO's such as mono-methyl-terminated
polyethylene glycols (mPEG's) are preferred when mono-substituted polymers are
desired; bis-activated polyethylene oxides are preferred when di-substituted
prodrugs are desired.
In order to provide the desired hydrolyzable linkage, mono- or di-acid
activated polymers such as PEG acids or PEG diacids can be used as well as
mono-
or di-PEG amines and mono- or di-PEG diols. Suitable PAO acids can be
synthesized by first converting mPEG-OH to an ethyl ester followed by
saponification. See also Gehrhardt, H., et al. Polymer Bulletin 18: 487 (1987)
and
Veronese, F.M., et al., J. Controlled Release 10; 145 (1989). Alternatively,
the
PAO-acid can be synthesized by converting mPEG-OH into a t-butyl ester
followed by acid cleavage. See, for example, commonly assigned U.S. Patent No.
5,605,976. The disclosures of each of the foregoing are incorporated by
reference
herein.
Although PAO's and PEG's can vary substantially in number average
molecular weight, polymers ranging from about 2,000 to about 100,000 daltons
are
usually selected for the purposes of the present invention. Molecular weights
of
from about 20,000 to about 50,000 are preferred, and 20,000 to about 40,000
are
- 26
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WO 02/43663 PCT/USO1/45127
particularly preferred. The number average molecular weight of the polymer
selected for inclusion in the " tetrapartate prodrug" must be sufficient so as
to
provide sufficient circulation of the " tetrapartate prodrug" before removal
of the
transport enhancer. Within the ranges provided above, polymers having
molecular
weight ranges of at least 20,000 are preferred in some aspects for
chemotherapeutic
and organic moieties. In the case of some nucleophiles such as certain
proteins,
enzymes and the like, polymers having a molecular weight range of from about
12,000 to about 20,000 are preferred.
The polymeric substances included herein are preferably water-soluble at
room temperature. A non-limiting list of such polymers include polyalkylene
oxide homopolymers such as polyethylene glycol (PEG) or polypropylene glycols,
polyoxyethylenated polyols, copolymers thereof and block copolymers thereof,
provided that the water solubility of the block copolymers is maintained.
As an alternative to PAO-based polymers, effectively non-antigenic
materials such as dextran, polyvinyl alcohols, carbohydrate-based polymers,
hydroxypropylmethacrylamide (HPMA), and copolymers thereof, etc. and the like
can be used if the same type of activation is employed as described herein for
PAO's such as PEG. Those of ordinary skill in the art will realize that the
foregoing list is merely illustrative and that all polymeric materials having
the
2o qualities described herein are contemplated. For purposes of the present
invention,
"effectively non-antigenic" means all polymeric materials understood in the
art as
being nontoxic and not eliciting an appreciable immune response in mammals.
F. POLYMERIC TETRAPARTATE TRANSPORT SYSTEM SYNTHESIS
Synthesis of representative, specific prodrugs is set forth in the Examples.
Generally, however, the transport enhanced prodrugs of the present invention
can
be prepared in several fashions. Thus, one method includes reacting a compound
of formula (TIT).
27
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WO 02/43663 PCT/USO1/45127
LR2lr ~R3~s
(III) R R1 Yq.
~'3 ~ II
Rll C L1 C Y2 O C Y1-C-B
R10 m p Ar R4
~R6~t RS~u
with a compound of the formula (IV): Lx - Z - [D]y
In this embodiment, Ll is a bifunctional linking moiety;
B and Lx are independently selected leaving groups and are defined as
l0 above for when D is a leaving group;
Z is a moiety that is actively transported into a target cell, a hydrophobic
moiety and combinations thereof. Preferably, Z is selected from the group
consisting of an amino acid residue, a C6_lg alkyl, a substituted aryl, a
hetero aryl,
-C(=Y4), -C(=S), -C(=NR16) and combinations thereof, wherein R16 is
selected from the same group as Rlz;
Rl, R4, R9, Rlo and R12 are independently selected from the group
consisting of hydrogen, C1_6 alkyls, C3_12 branched alkyls, C3_$ cycloalkyls,
C1_6 substituted alkyls, C3_$ substituted cycloalkyls, aryls, substituted
aryls,
aralkyls, C1_6 heteroalkyls and substituted C1_6 heteroalkyls;
R2, R3, RS and R6 are independently selected from the group consisting of
hydrogen, C1_6 alkyls, C1_6 allcoxys, phenoxy, C1_$ heteroalkyls, C1_$
heteroalkoxys,
substituted C1_6 alkyls, C3_$ cycloalkyls, C3_$ substituted cycloalkyls,
aryls,
substituted aryls, aralkyls, halo-, nitro- and cyano-, carboxy-, C1_6
carboxyalkyls
and CI_6 alkylcarbonyls;
Ar is a moiety which when included in Formula (III), and subsequently in
Formula (I), forms a multi-substituted aromatic hydrocarbon or a multi-
substituted
heterocyclic group;
(m), (r), (s), (t), and (u) are independently zero or one;
(p) is zero or a positive integer;
Yl.~ are independently O, S, or NRIZ, wherein the definition of R12 is
defined as per formula I, supra; (y) is one or two; and Rl l is a monovalent
or
divalent polymer residue.
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WO 02/43663 PCT/USO1/45127
Typically, the reaction between (III) and (IV) is conducted in the presence
of a solvent and a base. The solvent is preferably an inert solvent, i.e.,
inert with
respect to the reactants and products. Exemplary solvents include, simply by
way
of example, chloroform, toluene, methylene chloride, dimethylformamide and
combinations thereof. Generally, dimethylformamide is preferred. Exemplary
bases solvents include, simply by way of example, dimethylaminopyridine,
diisopropylethylamine, pyridine, triethylamine and combinations thereof.
Generally, dimethylanunopyridine is preferred.
Optionally, the tetrapartate prodrugs of the invention can also be prepared
io by reacting a compound of formula (V)
~R2~r ~R3~s
R9 Y3 R1 Y~.
(V) Rll C L1 C Y2 ~ 'C-Y1-C -Z~-La
P ~ Rq.
R10 m ~n ,
with a biologically effective material, e.g_, a biologically active compound
such as
a drug or diagnostic tag. In this embodiment,
La is a leaving group for Formula V as defined for D when D is a leaving
group.
L1 is a bifunctional linl~ing moiety;
Z is a moiety that is actively transported into a target cell, a hydrophobic
moiety and combinations thereof. Preferably, Z is selected from the group
consisting of an amino acid residue, a CG_l8 alleyl, a substituted aryl, a
hetero aryl,
-C(=Y4), -C(=S), -C(=NR16) and combinations thereof;
Ri, R4, R~, Rlo, Ri2 and R16 are independently selected from the group
consisting of hydrogen, Cl_6 alkyls, C3_12 branched allcyls, C3_8
cycloallcyls,
C1_G substituted allcyls, C3_$ substituted cycloalkyls, aryls, substituted
aryls,
aralkyls, Cl_6 heteroalkyls and substituted Cl_6 heteroalkyls;
3o RZ, R3, RS and R6 are independently selected from the group consisting of
hydrogen, Cl_~ allcyls, Cl_6 alkoxys, phenoxy, Cl_8 heteroalkyls, Cl_8
heteroalkoxys,
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WO 02/43663 PCT/USO1/45127
substituted Cl_6 alkyls, C3_8 cycloalkyls, C3_8 substituted cycloalkyls,
aryls,
substituted aryls, aralkyls, halo-, nitro- and cyano-, carboxy-, Cl_6
carboxyalkyls
and Cl_~ allcylcarbonyls.
Ar is a moiety which when included in Formula (V) forms a multi
substituted aromatic hydrocarbon or a multi-substituted heterocyclic group.
(m), (r), (s), (t), and (u) are independently zero or one.
(p) is zero or a positive integer.
Yi_4 are independently O, S, or NR12, wherein the definition of R12 is
defined as per formula I, supra; and Rll is a monovalent or divalent polymer
to residue.
Optionally, the reaction between (V) and the biologically effective material
is conducted in the presence of a coupling agent, including, for example, 1,3-
diiso-
propylcarbodiimide, a dialkyl carbodiimides, 2-halo-1-alkyl-pyridinium halide,
1-(3-dimethylamino-propyl)-3-ethyl carbodiimide, 1-propanephosphonic acid
cyclic anhydride, phenyl dichlorophosphates, and combinations thereof. In
addition, the reaction between (V) and the biologically active material is
typically
conducted in the presence of a solvent. and a base, each of which are defined
as
described supra, for the reaction between formulas (III) and (IV). In
addition, the
base is preferably dimethylamino pyridine.
2o Biologically effective materials for the tetrapartate prodrug are discussed
below.
G. LEAVING GROUPS OR RESIDUE PORTION "D"
1. Leaving Groups
In those aspects where B is a leaving group and further with reference to
the Lz and/or La leaving groups, as described above, suitable leaving groups
include, without limitations, moieties such as N-hydroxybenzotriazolyl,
halogen,
N-hydroxyphthalimidyl, p-nitrophenoxy, imidazolyl, N-hydroxysuccinimidyl;
thiazolidinyl throne, or other good leaving groups as will be apparent to
those of
3o ordinary shill. The synthesis reactions used and described herein will be
understood by those of ordinary skill without undue experimentation.
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For example, the acylated intermediate compound (III) can be reacted with
such as 4-nitrophenyl chloroformate, disuccinimidyl carbonate (DSC),
carbonyldiimidazole, thiazolidine thione, etc. to provide the desired
activated
derivative.
The acylation of the p-hydroxybenzyl alcohol or the p-aminobenzyl alcohol
and the o-hydroxbenzyl alcohol or the o-aminobenzyl alcohol can be carried out
with, for example, thiazolidine thione activated polymers, succinimidyl
carbonate
activated polymers, carboxylic acid activated polymers, blocked amino acid
derivatives.
Once in place, the "activated" form of the PEG prodrug (or bloclced prodrug)
is ready for conjugation with an amine- or hydroxyl-containing compound. Some
preferred activated transport forms are shown below.
2. Residues of Biolo i~, cally Active Materials
Broadly, the only limitations on the types of biologically effective materials
suitable for inclusion herein is that there is available at least one site for
covalent
attachment to the uptake enhancer moiety. Simply by way of example, this can
be
a (primary or secondary) amine-containing position or functional group which
can
react and link with a carrier portion, e.g., by forming an amide bond. Other
sites
2o for covalent attachment to the uptake enhancer moiety include, e.g_, a
hydroxyl
functional group, to form, ~, an ester linkage. Of course, the artisan will
appreciate that the selected linkage between the biologically effective
material of
interest, and the uptake enhancer is such that there is no substantial loss of
bioactivity after the double prodrug portion of the conjugate releases the
parent
compound in linkage with a transport enhancer.
After conjugation, the remaining amine-containing compound is refeiTed to
as the residue of the unconjugated compound.
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I. ~ Residues of Amine-containing Compounds
In some aspects of the invention, e.g,, after the prodrug transport has been
formed, D is a residue of an amine-containing organic compound. Organic
compounds include, without limitation, moieties such as anthracycline
compounds
including daunorubicin, doxorubicin; p-aminoaniline mustard, melphalan, Ara-C
(cytosine arabinoside) and related anti-metabolite compounds, ~, gemcitabine,
etc.
Also included herein is any portion of a polypeptide, nucleic acid, peptide
nucleic acids and any combinations thereof, ranging in size from about 50
daltons
to through about 2,500 daltons, or greater, demonstrating in vzvo bioactivity.
This
includes, e.g_, peptides, nucleic acids (DNA, RNA) with at least one amine
functional group, e.g_, peptide nucleic acids and the like.
Thus, in a preferred aspect of the invention, biologically effective material
is a biologically active compound that is suitable for medicinal or diagnostic
use in
the treatment of animals, e.g_, avians andlor mammals, including humans, for
conditions for which such treatment is desired. The foregoing list of
biological
materials is meant to be illustrative and not limiting for the compounds which
can
be modified. Those of ordinary skill will realize that other such compounds
can be
similarly modified without undue experimentation. It is to be understood that
2o those biologically active materials not specifically mentioned but having
suitable
amino-groups are also intended and are within the scope of the present
invention.
II. Residues of Hydrox~-Containing Compounds
a. Camptothecin and Related Topoisomerase I Inhibitors
Camptothecin is a water-insoluble cytotoxic alkaloid produced by
Camptotheca accuminata trees indigenous to China and nothapodytes foetida
trees
indigenous to India. Camptothecin and related compounds and analogs are also
known to be potential anticancer or antitumor agents and have been shown to
exhibit these activities ifz vitro and in vivo. Camptothecin and related
compounds
3o are also candidates for conversion to the tetrapartate prodrugs of the
present .
invention.
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Camptothecin and certain related analogues share the structure:
From this core structure, several known analogs have been prepared. For
example, the A ring in either or both of the 10- and 11-positions can be
substituted
with an OH. The A ring can also be substituted in the 9-position with a
straight or
branched C1_3o alkyl or Cl_l~ allcoxy, optionally linked to the ring by a
heteroatom
i.e.- O or S. The B ring can be substituted in the 7-position with a straight
or
branched Cl-so allcyl or substituted alkyl-, CS_g cycloalkyl, Cl_3o alkoxy,
phenyl
allcyl, etc., alkyl carbamate, alkyl carbazides, phenyl hydrazine derivatives,
amino-,
aminoallcyl-, aralkyl, etc. Other substitutions are possible in the C, D and E
rings.
See, for example, U.S. Patent Nos. 6,111,107; 5,004,758; 4,943,579; Re
32,518, the contents of which are incorporated herein by reference. Such
derivatives can be made using known synthetic techniques without undue
experimentation. Preferred camptothecin derivatives for use herein include
those
which include a 20-OH or another OH moiety which is capable of reacting
directly
with activated forms of the polymer transport systems described herein or to
the
linl~ing moiety intermediates, e.g. iminodiacetic acid, etc., which are then
,attached
to a polymer such as PEG.
Reference to camptothecin analogs herein has been made for purposes of
illustration and not limitation.
b. Taxanes and Paclitaxel Derivatives
One class of compounds included in the tetrapartate prodrug compositions of
the present invention is taxanes. For purposes of the present invention, the
term
"taxane" includes all compounds within the taxane family of terpenes. Thus,
taxol
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(paclitaxel), 3'-substituted tent-butoxy-carbonylTamine derivatives
(taxoteres) and
the like as well as other analogs which are readily synthesized using standard
organic techniques or are available
from commercial sources such as Sigma Chemical of St. Louis, Missouri are
within the scope of the present invention. Representative taxanes are shown
below.
to Paclitaxel: R'1 = C6H5; R'2 = CH3C0; Taxotere: R'1= (CH3)3C0; R'2 = H
These derivatives have been found to be effective anti-cancer agents.
Numerous studies indicate that the agents have activity against several
malignancies. To date, their use has been severely limited by, among other
things,
their short supply, poor water solubility and hypersensitivity. It is to be
understood
that other taxanes including the 7-aryl-carbamates and 7-carbazates disclosed
in
commonly assigned U.S. Patent Nos. 5,622,986 and 5,547,981 can also be
included in the tetrapartate prodrugs of the present invention. The contents
of the
foregoing U.S. patents are incorporated herein by reference. The only
limitation
on the taxane is that it must be capable of undergoing a hydroxyl based
substitution
reaction such as at the 2' position. Paclitaxel, however, is a preferred
taxane.
c. Additional Biologically-Active Moieties
In addition to the foregoing molecules, the tetrapartate prodrug
formulations of the present invention can be prepared using many other
compounds. For example, drugs such as gemcitabine, etoposide, triazole-based
antifungal agents such as fluconazole andlor ciclopirox can be used.
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The parent compounds selected for tetrapartate prodrug forms need not be
substantially water-insoluble, although the polymer-based tetrapartate
prodrugs of
the present invention are especially well suited for delivering such water-
insoluble
compounds. Other useful parent compounds include, for example, certain low
molecular weight biologically active proteins, enzymes and peptides, including
peptido glycans, as well as other anti-tumor agents; cardiovascular agents
such as
forslcolin; anti-neoplastics such as combretastatin, vinblastine, doxorubicin,
ara-C,
maytansine, etc.; anti-infectives such as vancomycin, erythromycin, etc.; anti-
fungals such as nystatin, amphoteracin B, triazoles, papulocandins,
l0 pneumocandins, echinocandins, polyoxins, nikkomycins, pradimicins,
benanomicins, etc. see, "Antibiotics That Inhibit Fungal Cell Wall
Development"
Annu. Rev. Microbiology, 1994, 48:471-97, the contents of which are
incorporated
herein by reference; anti-anxiety agents, gastrointestinal agents, central
nervous
system-activating agents, analgesics, fertility or contraceptive agents, anti-
inflammatory agents, steroidal agents, anti-urecemic agents, cardiovascular
agents,
vasodilating agents, vasoconstricting agents and the like.
After conjugation, the remaining amine-or hydroxyl-containing compound
is referred to, as the residue of the unconjugated compound.
2o 3. Polymeric Hybrids
In another aspect of the invention there are provided hybrid types of the
polymeric tetrapartate prodrug transport system described herein. In
particular, the
hybrid system includes not only the reversible double prodrug system described
above but also a second polymeric transport system based on more permanent
types of linkages. The hybrids can be prepared by at Ieast two methods. For
example, the benzyl-elimination-based prodrug can be synthesized first and
then
PEGylated using any art-recognized activated polymer such as thiazolidinyl
thione-or succinimidyl carbonate-activated PEG. Alternatively, the more
permanent conjugation reaction can be performed first and the resultant
conjugates
3o can be used to form the double prodrug portion of the tetrapartate
conjugates
described herein. It will be understood that the hybrid systems will be better
suited
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for proteins, enzymes and the like where multiple amino groups are available
for
attachment of the polymeric transport forms. For purposes of the present
invention, "activated polymers" will be understood to include polymers
containing
one or more terminal groups which are capable of reacting with one or more of
a-
amino groups, ~-amino groups, histidine nitrogens, carboxyl groups, sulfhydryl
groups, etc. found on enzymes, proteins, etc., as well as such groups found on
synthetically prepared organic compounds. It will further be appreciated that
the
activating groups described below can also be used to form the activated
transport
forms described above.
1o The activating terminal moiety can be any group which facilitates
conjugation of the polymers with the biologically active material, i-e.,
protein,
enzyme, etc. either before of after the double prodrug transport system of the
present invention has been synthesized. See, for example, U.S. Patent No.
4,179,337, the disclosure of which is hereby incorporated by reference. Such
activating groups can be a moiety selected from:
I. Functional groups capable of reacting with an amino group such as:
a) carbonates such as the p-nitrophenyl, or succinimidyl; see, for
example, U.S. Patent No. 5,122,614, the disclosure of which is
hereby incorporated by reference;
b) carbonyl imidazole;
c) azlactones; see, for example, U.S. Patent No. 5,321,095, the
disclosure of which is hereby incorporated by reference;
d) cyclic imide thiones see, for example, U.S. Patent No. 5,349,001,
the disclosure of which is hereby incorporated by reference;
e) isocyanates or isothiocyanates; or
f) active esters such as N-hydroxy-succinimidyl or N-
hydroxybenzotriazolyl.
II. Functional groups capable of reacting with carboxylic acid groups and
reactive carbonyl groups such as:
3o a) primary amines; or.
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b) hydrazine and hydrazide functional groups such as the acyl
hydrazides, carbazates, semicarbamates, thiocarbazates, etc.
III. Functional groups capable of reacting with mercapto or sulfhydryl
groups such as phenyl glyoxals; see, for example, U.S. Patent No. 5,093,531,
the
disclosure of which is hereby incorporated by reference;
IV. Functional groups capable of reacting with hydroxyl groups such as
(carboxylic) acids or other nucleophiles capable of reacting with an
electrophilic
center. A non-limiting list includes, for example, hydroxyl, amino, carboxyl,
thiol
groups, active methylene and the like.
l0 The activating moiety can also include a spacer moiety located proximal to
the polymer. The spacer moiety may be a heteroalkyl, alkoxy, alkyl containing
up
to 18 carbon atoms or even an additional polymer chain. The spacer moieties
can
added using standard synthesis techniques.
is H. METHODS OF TREATMENT
Broadly, another aspect of the present invention provides methods for
delivering biologically active materials, such as therapeutic or diagnostic
agents
into cells where such biological activity is desired. While the tetrapartate
prodrugs of the invention are readily employed to deliver biologically active
20 materials into a wide variety of cells, found throughout the animal body,
certain
applications are preferred. For example, the tetrapartate prodrugs of the
invention
are particularly useful in delivering biologically active materials, such as
drugs
and/or diagnostics, into cells present in tissues exhibiting the above-
discussed EPR
effect. A number of tissue types exhibiting EPR occur in different diseases
and
25 disorders, including tissues undergoing inflammation, toxic reactions of
various
kinds, as well as solid tumors.
Thus, the broad method includes contacting living tissue with the inventive
tetrapartate prodrugs. Preferably the tissue exhibits the EPR effect, so that
polymer linked conjugates preferably enter such tissues. Of course, the
artisan will
3o appreciate that an agent, once delivered into a target cell and activated,
can then be
released by that cell and provide biological activity in other tissue spaces.
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Simply by way of example, a non-active prodrug of the invention that is
delivered into an exocrine cell of the liver or pancreas under suitable
conditions,
e.g_, during a disease process that causes inflammation, and results in an EPR
effect, can be activated within the cytoplasm of the target cell, and then the
activated drug or diagnostic agent can then be secreted into the
gastrointestinal
("G.I") tract fluid space for therapeutic and/or diagnostic purposes. In this
instance, treatment and/or diagnosis of certain diseases or disorders of the
G.I. tract
by means of the targeted delivery of appropriate agents, including anti-cancer
or
antiviral agents, is therefore facilitated. Analogous methods of treatment and
delivery of biologically active materials is readily contemplated for other
organ
and/or tissue systems.
In one preferred embodiment, the tissues are tumor or cancer tissues, and
the tetrapal-tate prodrugs of in the invention comprise agents suitable for
treatment
and/or diagnosis of such conditions. Thus, the tetrapartate prodrug
compositions
are useful for, among other things, treating diseases which are similar to
those
which are treated with the parent compound(s), e.g, including compounds
suitable
for treating neoplastic disease, reducing tumor burden, inhibiting metastasis
of
tumors or neoplasms and preventing recurrences of tumorlneoplastic growths in
mammals. The treated animals are preferably mammals, and more preferably
human patients. While veterinary use of the prodrugs of the invention will
typically be employed in mammalian species, it is further contemplated that
the
prodrugs can also be readily employed in other species generally within the
veterinary practice and animal husbandry arts, e.~,, including highly valued
non-
mammalian exotic animals.
The amount of the prodrug and/or diagnostic tetrapartate tag that is
administered will depend upon the amount of the parent molecule included
therein.
Generally, the amount of tetrapartate prodrug used in the treatment methods is
that
amount which effectively achieves the desired therapeutic or diagnostic result
in
mammals. Naturally, the dosages of the various prodrug compounds will vary
somewhat depending upon the parent compound, rate of ifa vivo hydrolysis,
molecular weight of the polymer, etc. In general, tetrapartate prodrug
polymeric
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WO 02/43663 PCT/USO1/45127
derivatives are administered in amounts ranging from about 5 to about 500
mg/m2
per day, based on the native drug. The range set forth above is illustrative
and
those skilled in the art will determine the optimal dosing of the prodrug
selected
based on clinical experience and the treatment indication. Actual dosages will
be
apparent to the artisan without undue experimentation.
The compositions, including prodrugs, of the present invention can be
included in one or more suitable pharmaceutical compositions for
administration to
an animal in need thereof. The pharmaceutical compositions may be in the form
of
a solution, suspension, tablet, capsule or the lilce, prepared according to
methods
1o well known in the art. Tt is also contemplated that administration of such
compositions may be by the oral and/or parenteral routes depending upon the
needs of the artisan. A solution and/or suspension of the composition may be
utilized, for example, as a carrier vehicle for injection or infiltration of
the
composition by any art known methods, e.g., by intravenous, intramuscular,
subdermal injection and the like.
Such administration may also be by infusion into a body space or cavity, as
well as by inhalation and/or intranasal routes. In preferred aspects of the
invention,
however, the prodrugs are parenterally administered to animals in need
thereof.
The novel methods of treatment or administration according to the
2o invention further includes the multi-step cleavage of the prodrug,
resulting in
release of the biologically active material, such as a drug or tag, within a
target
cell.
I. IN VIVO DIAGNOSTICS
A further aspect of the invention provides the tetrapartate conjugates of the
invention optionally prepared with a diagnostic tag linked to the transport
enhancer
described above, wherein the tag is selected for diagnostic or imaging
purposes.
Thus, a suitable tag is prepared by linking any suitable moiety, e. g;, an
amino acid
residue, to any art-standard emitting isotope, radio-opaque label, magnetic
resonance label, or other non-radioactive isotopic labels suitable for
magnetic
resonance imaging, fluorescence-type labels, labels exhibiting visible colors
and/or
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capable of fluorescing under ultraviolet, infrared or electrochemical
stimulation, to
allow for imaging tumor tissue during surgical procedures, and so forth.
Optionally, the diagnostic tag is incorporated into andlor linked to a
conjugated
therapeutic moiety, allowing for monitoring of the distribution of a
therapeutic
biologically active material within an animal or human patient.
In a still further aspect of the invention, the inventive tagged tetrapartate
conjugates are readily prepared, by art-known methods, with any suitable
label,
including, e.g_, radioisotope labels. Simply by way of example, these include
isllodine, lzsIodine, 9~I"Technetium and/or 111Indium to produce
to radioimmunoscintigraphic agents for selective uptake into tumor cells, in
vivo. For
instance, there are a number of art-known methods of linl~ing peptide to Tc-
99m,
including, simply by way of example, those shown by U.S. Patent Nos.
5,328,679;
5,888,474; 5,997,844; and 5,997,845, incorporated by reference herein.
Broadly, for anatomical localization of tumor tissue in a patient, the
15 tetrapartate conjugate tag is administered to a patient or animal suspected
of having
a tumor. After sufficient time to allow the labeled immunoglobulin to localize
at
the tumor site(s), the signal generated by the label is detected, for
instance,
visually, by X-ray radiography, computerized transaxial tomography, MRI, by
instrumental detection of a luminescent tag, by a photo scanning device such
as a
2o gamma camera, or any other method or instrument appropriate for the nature
of the
selected tag.
The detected signal is then converted to an image or anatomical and/or
physiological determination of the tumor site. The image makes it possible to
locate the tumor iyZ vivo and to devise an appropriate therapeutic strategy.
In those
25 embodiments where the tagged moiety is itself a therapeutic agents, the
detected
signal provides evidence of anatomical localization during treatment,
providing a
baseline for follow-up diagnostic and therapeutic interventions.
J. EXAMPLES
3o It should be noted for all of the compounds that were produced by the
following examples, and as illustrated by Figures 1-9, that "PEG" is:
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~O w0 Jn
although other art-lcnown variations are readily employed, as mentioned supYa.
In addition, the PEG employed in the following examples had a molecular weight
of about 40 kDa.
The following examples serve to provide further appreciation of the
invention but are not meant in any way to restrict the effective scope of the
invention.
to EXAMPLE 1
Synthesis of Compound 2
Compound 2 (Fig. 3) was prepared by one of the following methods:
Method 1
Leucine-doxorubicin (130 mg, 0.198 mmol) and dimethylaminopyridine
("DMAP," 121 mg, 0.99 mmol) were added to a solution of compound 1, having a
PEG chain of about 40 kDa (2.4 g, 0.060 mmol, Fig. 1) in 40 mL of anhydrous
dimethyformamide ("DMF"). The mixture was stirred at room temperature
2o overnight. Ethyl ether (~ 200 mL) was added to the reaction mixture to
precipitate
PEG derivatives, and the solid filtered and recrystallized twice from from 80
mL 2-
propanol ("IPA") to give pure product of compound 2 (1.85 g, 75 °70,
Fig. 3).
Method 2
(Step 1) To a solution of compound 1 (4.5 g, 0.111 mmol, Fig. 4) in 80 mL of
anhydrous methylene chloride, was added leucine t-butyl ester (248 mg, 1.11
mmol)
and DMAP (136 mg, 1.11 mmol). The reaction mixture was stirred for 18 hours at
room temperature. The mixture was evaporated under reduced pressure, and the
residue was re-crystallized from IPA to give compound 3 (4.5 g, 99 %, Fig. 4).
The
3o structure was confirmed by 13C NMR (67.8 MHz, CDCl3) with peaks at ~
171.55,
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155.24, 152.88, 150.24, 133.79, 128.68, 120.48, 81.05, 69.0-72.5 (PEG), 52.51,
41.26, 27.40, 24.19, 22.27, 21.44.
(Step 2) compound 3 (4.70 g, 0.114 mmol) was dissolved in 22.5 mL
trifluoroacetic acid and 45 mL of methylene chloride and stirred at room
temperature
for 2 hours. Ethyl ether was added to precipitate the PEG derivative. The
crude
product was filtered and washed with ethyl ether to yield compound 4 (4.4 g,
94 %,
Fig. 4). The structure was confirmed by 13C NMR (67.8 MHz, CDC13) with pealcs
at
8173.30, 155.36, 152.94, 150.24, 133.81, 128.67, 120.52, 69.0-72.5 (PEG),
52.65,
41.20, 24.18, 22.44, 21.31.
to (Step 3) Doxorubicin (57 mg, 0.0984 mmol) and DMAP (42 mg, 0.344
mmol) were added to a solution of compound 4 (1.0 g, 0.0246 mmol, Fig. 4) in
anhydrous methylene chloride (20 mL) at 0°C for 20 minutes. 1-[3-
(dimethylamino)
propyl]-3-ethylcarbodiimide hydrochloride ("EDC," 28 mg, 0.148 mmol) was
added,
and the reaction mixture gradually warmed to room temperature and stirred
overnight. The solution was filtered by gravity, and the solvent evaporated.
The
residual solid was recrystallized from IPA (50 mL) to give pure compound 2
(0.656
g, 67 %).
The structure was confirmed by 13C NMR (67.8 MHz, CDCl3) with peaks at 8
213.56, 186.82, 186.50, 171.27, 160.76, 155.93, 155.50, 153.21, 150.51,
135.55,
135.22, 133.84, 133.47, 129.05, 120.81, 119.53, 118.24, 111.26, 111.00,
100.40,
69.0-72.5 (PEG), 68.43, 67.46, 67.16, 65.86, 56.41, 53.39, 45.15, 41.40,
35.23, 29.21,
24.35, 22.72, 21.57, 16.61.
EXAMPLE 2
Synthesis of Compound 6
Compound 6 (Fig. 5), was prepared by adding leucine-doxorubicin (130 mg,
0.198 mrnol, Fig. 3) and DMAP (121 mg, 0.99 mmol) to a solution of compound 5
(2.2 g, 0.054 mmol, Fig. 5) in anhydrous DMF (30 mL). After stirring at room
temperature overnight, ethyl ether (~ 100 mL) was added to the reaction
mixture and
3o the precipitated solid filtered. The solid was recrystallized twice from
IPA (70 mL) to
give pure product (2.09 g, 93 %). .
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The structure was confirmed by 13C NMR (67.8 MHz, CDC13) with peals at:
S 213.40, 186.56, 186.23, 171.23, 160.61, 155.83, 155.65, 155.23, 154.25,
150.51,
135.41, 134.99, 133.43, 133.25, 132.79, 128.85, 121.26, 120.40, 119.36,
118.17,
111.06, 110.88, 100.34, 69.0-72.5 (PEG), 68.33, 67.10, 65.89, 65.08, 56.28,
53.27,
45.03, 41.20, 40.61, 40.41, 35.25, 33.50, 29.07, 24.23, 22.65, 21.40, 16.52.
EXAMPLE 3
Synthesis of Compound 8
to Compound 8 (Fig. 6), was prepared by the methods described in Example 2,
above: by adding leucine-doxorubicin (80mg, 0. 122 mmol ) to a solution of
compound 7 (1.6 g, 0.41 mmol, Fig. 4) in 30 mL of anhydrous DMF, and reacting
the
mixture to give the product 8, (1.44 g, 85 %) which was recrystallized from
1PA.
The structure was confirmed by 13C NMR (67.8 MHz, CDC13) with peaks at:
15 b 213.64, 186.80, 186.42, 171.32, 171.21, 160.76, 155.97, 155.41, 154.30,
150.68,
135.54, 135.19, 133.50, 133.38, 129.06, 127.52, 121.41, 119.54, 118.25,
111.26,
111.06, 100.47, 69.0-72.5 (PEG), 68.54, 67.20, 66.13, 65.25, 56.43, 53.48,
45.11,
41.24, 38.97, 37.10, 35.40, 35.06, 33.64, 29.25, 24.39, 22.75, 21.55, 16.64.
2o EXAMPLE 4
Synthesis of Compound 10
Compound 10 (Fig. 7A) was prepared by the methods described in Example
2, above: by adding leucine-doxorubicin (97mg, 0.147 mmol, Fig. 7A) to a
solution
25 of compound 9 (2.0g, 0.049mmo1, Fig. 7A) in 30 mL of anhydrous DMF. Pure
compound 10 was obtained by recrystallization from IPA (1.70 g, 83 %).
The structure was confirmed by 13C NMR (67.8 MHz, CDC13) with peals at:
8 213.71, 186.93, 186.55, 171.35, 168.14, 160.88, 156.03, 155.51, 135.61,
135.31,
133.67, 133.50, 130.08, 128.38, 120.69, 119.64, 118.31, 111.38, 111.19,
100.50,
30 69.0-72.5 (PEG), 68.64, 67.13, 66.28, 65.33, 56.51, 53.60, 45.23, 41.43,
35.50, 33.80,
29.47, 24.49, 22.79, 21.68, 16.69.
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EXAMPLE 5
Synthesis of Comuound 12
Compound 12 (Fig. 7B), was prepared by the methods described in Example
2, above: by adding leucine-doxorubicin (134 mg, 0.204 mmol, Fig. 7B) to a
solution
of compound 11 (2.0 g, 0.049 mmol) ) in 30 mL of anhydrous DMF. Compound 12
was purified by recrystallization from IPA (1.69 g, 82 %).
EXAMPLE 6
1o Synthesis of Compound 14
Compound 14 (Fig 8), was prepared as follows.
(Step 1) To a solution of compound 1 (6.88 g, 0.170 mmol, Fig. 8), in 90 mL
anhydrous DMF, was added 12-aminododecanoic acid (0.15 g, 0.680 mmol) and
DMAP (0.114 g, 0.934 mmol). The resulting reaction mixture was stirred for 18
15 hours at a temperature between 50 to 60°C. The mixture was filtered,
the filtrate
evaporated under reduced pressure, and the residue recrystallized from IPA to
give
compound 13 (6.2 g, 90). The structure was confirmed by 13C NMR (67.8 MHz,
CDC13) with peaks at 8174.58, 155.79, 152.97, 150.20, 134.19, 128.77, 127.31,
120.52, 69.0-72.5 (PEG), 40.54, 33.32, 29.42, 28.90, 28.70, 28.59, 26.17,
24.87,
20 24.36.
(Step 2) To a solution of compound 13 (3.0 g, 0.074 mmol) as obtained in
Step 1, above, doxorubicin hydrochloride (256 mg, 0.441 mmol), 4-
methylmorpholine ("NMM", 130 uL, 1.18 mmol), and 1-hydroxybenzotriazole
hydrate ("HOBT", 60 mg, 0.441 mmol) in 80 mL anhydrous DMF / methylene
25 chloride (1 :1) was EDC (113 mg, 0:589 mmol), and the mixture stirred at
room
temperature overnight. The solvent was evaporated under reduced pressure and
the
residue was recrystallized from IPA (100 mL) to give pure compound 14 (2.80 g,
91
%)).
The structure was confirmed by 13C NMR (67.8 MHz, CDC13), with pealcs at
3o b 213.40, 186.56, 186.18, 172.11, 160.56, 155.97, 155.83, 155.22, 153.04,
150.26,
135.41, 134.96, 134.23, 133.45, 133.26, 128.83, 127.38, 120.60, 120.38,
118.14,
111.00, 110.81, 100.50, 69.0-72.5 (PEG), 68.28,67.28, 67.04, 65.89, 65.02,
56.26,
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WO 02/43663 PCT/USO1/45127
44.61, 40.60, 40.41, 35.88, 35.26, 33.43, 29.46, 29.30, 28.93, 28.86, 26.20,
25.12,
16.55.
EXAMPLE 7
Synthesis of Compound 17
Compound 17 was prepared as illustrated by Fig. 9.
(Step 1) To a solution of compound 13 (5.20 g, 0.128 mmol) in 50 mL
anhydrous methylene chloride was added leucine t-butyl ester (0.287 g, 1.28
mmol),
DMAP (0.281 g, 2.30 mmol), and EDC (0.197 g, 1.02 mmol). The reaction mixture
1o was stirred at room temperature for 18 hours. Solvent was removed under
reduced
pressure, and the residue recrystallized from IPA to yield 15 (4.8 g, 92 %,
Fig. 9).
The structure was confirmed by 13C NMR (67.8 MHz, CDC13) with peaks at $
172.22, 171.87, 155.79, 152. 98, 150.22, 134. 20, 128.78, 120.54, 69.0-72.5
(PEG),
50.62, 41.37, 40.55, 35.96, 29.43, 28.88, 28.73, 27.49, 26.19, 25.05, 24.89,
24.39,
15 22.32, 21.65.
(Step 2) compound 15 (4.70 g, 0.114 mmol) was dissolved in 25 mL
trifluoroacetic acid and 50 mL methylene chloride and stirred at room
temperature for
2 hours. Ethyl ether was added to precipitate PEG product. The solid was
filtered
and washed with additional ethyl ether to yield compound 16 (4.4 g, 94 %, Fig.
9).
2o The structure was confirmed by 13C NMR (67.8 MHz, CDCl3) with peaks at 8
173.50, 172.50, 155.83, 153.01, 150.22, 134. 20, 128.80, 120.55, 69.0-72.5
(PEG),
49.85, 41.16, 40.57, 35.91, 29.43, 28.84, 28.66, 26.17, 25.04, 24.35, 24.47,
21.52.
(Step 3) To a solution of compound 16 (3.3 g, 0.080 mmol), doxorubicin
hydrochloride (280 mg, 0.480 mmol), NMM (130 uL, 1.18 mmol), and HOBT (74
25 mg, 0.480 mmol) in 90 mL of anhydrous DMF and methylene chloride (1 : 1)
was
added EDC (120 mg, 0.640 mmol), and the mixture stirred at room temperature
overnight. The solvent was evaporated under reduced pressure and the residual
solid
was recrystallized from lPA (100 mL) to give pure compound 17 (2.90 g, 85 %).
The structure was confirmed by 13C NMR (67.8 MHz, CDCl3) with peaks at
30 8 213.30, 186.44, 186.16, 172.77, 171.23, 160.55, 155.75, 155.13, 153.04,
150.26,
135.37, 134.91, 134.22, 133.38, 133.19, 128.81, 127.38, 120.58, 120.38,
119.27,
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WO 02/43663 PCT/USO1/45127
118.14, 110.97, 110.79, 100.24, 69.0-72.5 (PEG), 68.26, 67.27, 67.01, 65.10,
56.21,
51.11, 45.08, 41.02, 40.60, 35.91, 35.23, 33.39, 29.43, 28.90, 28.69, 26.22,
25.05,
24.23, 22.58, 21.61, 16.48.
EXAMPLE 8
Confirmation of Efficac~of Tetrapartate Prodru~ Relative to Doxorubicin
The efficacy of PEG-leu-doxorubicin analogs against a subcutaneous
to human ovarian carcinoma (A2780) injected into nude mice was determined as
follows.
Following at least one week of acclimation, tumors were established in nude
mice by injecting 1 x lOG (100,000) harvested A2780 ovarian carcinoma cells in
a
single subcutaneous site, on the left axillary flank region of each animal.
The tumor
15 injection site was observed twice weeldy and measured once palpable. The
tumor
volume for each mouse was determined by measuring two dimensions with calipers
and calculated using the formula: tumor volume = (length x width2)/2). When
tumors
reached the average volume of approximately 80 mm3, the mice were divided into
their respective experimental groups, which consists of vehicle (20 m M sodium
2o phosphate in 0.6 % NaCI) controls, Leu-Doxorubicin, compound 1 of Example
1,
compound 10 of Example 4, and compound 12 of Example 5). .The mice were
sorted to evenly distribute tumor size, grouped into 6 mice/cage, and ear
punched for
permanent identification. Drugs were dosed intravenously via the tail vein
once per
week for three weeks (Qd7 x 3). Mouse weight and tumor sizes were measured at
the
25 beginning of study and twice weekly through week 5.
The overall growth of tumors was calculated as the mean tumor volume at
one week following the end of the treatment. A percent treatment over control
(T/C) value was also calculated when the control group's median tumor size
reached approximately 800-1100 mm3 and again at one week following treatment.
30 The T/C value in percent is a non-quantitative indication of anti-tumor
effectiveness.
Data is presented in Table 2, below.
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TABLE 2
PEG-Leu-Doxorubicin Treatment of a
Human Ovarian Carcinoma (A2780) Xeno~raft in Nude Mice a
Compound Treatment Tumor Volume T/C (%)R T/C (alo)a
Schedule (mean sem) At 1000 At Day 21
(mg/kg/dose)Day 21 mm3
Control 0 4238 356 - -
Leu-Dox Qd7 x3 (30) 659 173 26.3 19.7
Com ound Qd7 x3 (30) 619 231 13.2 7.4
2
Compound Qd7 x3 (30) 507 173 13.0 8.8
Com ound Qd7 x3 (30) 379 134 14.3 6.1
12
5 a - Intravenous treatment in nude mice bearing established tumors (~80 mm3).
n=6/group.
~i - The median tumor volume of treatment and control groups were measured and
compared when the control group's median tumor volume reached approximately
1000 mm3 and one week after final dosage (day 21). T/C < 42°70 at 1000
mm3 is
to considered significant anti-tumor activity by the Drug Evaluation Branch of
the
NCI.
Thus, as can be appreciated from the data presented by Table 2, above, the
PEG conjugate forms of leu-doxorubicin were more effective than the non-
conjugated parent compound.
EXAMPLE 9
Synthesis of Compound 19
(Step 1) TFA~Alanine-Camptothecin 18 was prepared as described in
commonly assigned United States Patent No. 6,127,355, Example 21, the contents
of
which are incorporated herein by reference.
(Step 2) 18 (185 mg, 0.36 mmol), was added to solution containing 9 (4 g,
0.0982 mmol) in 50 mL anhydrous methylene chloride and DMAP (49 mg, 0.40
rnmol). The mixture was stirred at room temperature overnight. The reaction
mixture was evaporated to dryness and the residual solid recrystallized two
times
from 2-propanol (80 mL) to give pure compound 19 (3.5 g, 86 °70). 13C
NMR (67.8
MHz, CDCl3) 8170.80, 167.30, 166.08, 156.33, 151.53, 147.99, 146.72, 145.53,
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WO 02/43663 PCT/USO1/45127
142.07, 133.55, 130.46, 129.61, 129.27, 128.89, 127.94, 127.44, 127.02,
119.09,
107.16, 95.35, 69.74-65.23 (PEG), 59.13, 49.17, 39.32, 30.76, 21.02, 16.45,
15.36,
6.69.
EXAMPLE 10
Synthesis of Compound 20
TFA~Alanine-Camptothecin (185 mg, 0.36 mmol) 18 was added to a solution
of 11 (4.0 g, 0.0983 mmol) in 50 mL anhydrous methylene chloride and DMAP (49
mg, 0.40 mmol). The mixture was stirred at room temperature overnight, the
reaction
mixture evaporated to dryness, and the residual solid was recrystallized from
2-
to propanol two times (80 mL) to give pure 20 (3.5 g, 86 %). 13C NMR (67.8
MHz,
CDCl3) 8170.83, 166.23, 156.50, 155.21, 152.10, 151.39, 148.20, 145.70,
144.62,
130.75, 130.00, 129.76, 129.04, 128.25, 128.03, 127.54, 127.19, 119.45,
107.70,
95.44, 69.91-65.25 (PEG), 49.31, 44.90, 30.98, 16.82, 15.31, 15.20, 6.81.
EXAMPLE 11
Synthesis of Compound 21
TFA~Alanine-Camptothecin (102 mg, 0.179 mmol) 18 and DMAP (37 mg,
0.30 mmol) were added to a solution of 5 (2.0 g, 0.0493 mmol) in 40 mL
anhydrous
methylene chloride. The mixture was stirred at room temperature overnight, the
2o reaction mixture evaporated to dryness, and the residual solid was
recrystallized twice
from 2-propanol (60 mL) to give pure 21(1.77 g, 87 %).13C NMR (67.8 MHz,
CDCl3) ~ 171.00, 166.00, 161.07, 156.09, 154.00, 151.00, 148.00, 145.50,
135.75,
130.55, 129.56, 128.81, 127.75, 127.41, 127.02, 126.69, 120.70, 119.00,
107.70,
72.69-67.00 (PEG), 50.70, 48.38, 40.06, 36.86, 30.32, 16.20, 6.65.
EXAMPLE 12
Synthesis of Compound 22
A solution of compound 4 (5 g, 0.123 mmol) in toluene (75 mL) is
azeotroped with the removal of 25 mL of distillate. The reaction mixture is
cooled
3o to 30 °C, followed by the addition of oxalyl chloride (0.031 g,
0.246 mmol) and
one drop of dimethylformamide. This mixture is stirred for 3 hours at 30-40
°C,
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WO 02/43663 PCT/USO1/45127
followed by the addition of 2-mercaptothizoline (0.044 g, 0.369 mmol). The
reaction mixture is refluxed for 1 hour, followed by filtration and removal of
the
solvent ifz vacuo. The crude residue is recrystallized from IPA (100 mL) to
yield
compound 22 (4 g, 90 °70). The structure is comfirmed by 13C NMR.
EXAMPLE 13
Synthesis of Compound 23
Native bovine hemoglobin (bHb) in 100 mM sodium phosphate (pH 8.4)/65
mM NaCl buffer is modified to form the conjugated compound 23 as follows. In a
l0 polypropylene container, 20 mL of 22 (0.8 g dissolved in 20 mM sodium
phosphate / 65 mM NaCl buffer at 4 °C) is added to 20 mL of bHb at
4°C (22.2 g
at 11 mg/mL) with gentle stirring. The pH of the reaction is monitored. The
mixture is stirred for 1 hour, the reaction is quenched by the addition of
glycine,
and stirring is continued for an additional 15 minutes. Cysteine (dissolved in
100
mM sodium phosphate / 65 mM NaCl buffer at 4°C, 30 mM final
concentration) is
added to reduce oxidized hemoglobin (met-Hb) formation, and the reaction
mixture is stirred for 16 hours at 4°C. The PEG-Hb is diluted and
diafiltered into
formulation buffer (5 mM sodium bicarbonate, 4 mM Na~HP04, 1mM NaH2P04,
150 mM NaCI, pH 7.4) to remove the unreacted PEG and/or PEG-glycine
conjugate, followed by concentration to 60 mg/mL of compound 23. The purity of
the PEG-Hb is determined by size exclusion HPLC.
49
