U.S. patent application number 15/419173 was filed with the patent office on 2017-11-16 for binding ligand linked drug delivery conjugates of tubulysins.
The applicant listed for this patent is ENDOCYTE, INC.. Invention is credited to Christopher P. LEAMON, Iontcho R. VLAHOV, Kevin Yu WANG.
Application Number | 20170327537 15/419173 |
Document ID | / |
Family ID | 39588060 |
Filed Date | 2017-11-16 |
United States Patent
Application |
20170327537 |
Kind Code |
A1 |
VLAHOV; Iontcho R. ; et
al. |
November 16, 2017 |
BINDING LIGAND LINKED DRUG DELIVERY CONJUGATES OF TUBULYSINS
Abstract
Described herein are compounds, pharmaceutical compositions and
methods for treating pathogenic cell populations. The compounds
described herein include conjugates of tubulysins and vitamin
receptor binding ligands. The conjugates also include a releasable
bivalent linker.
Inventors: |
VLAHOV; Iontcho R.; (West
Lafayette, IN) ; LEAMON; Christopher P.; (West
Lafayette, IN) ; WANG; Kevin Yu; (Zionsville,
IN) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
ENDOCYTE, INC. |
West Lafayette |
IN |
US |
|
|
Family ID: |
39588060 |
Appl. No.: |
15/419173 |
Filed: |
January 30, 2017 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12530952 |
Sep 11, 2009 |
9555139 |
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PCT/US2008/056824 |
Mar 13, 2008 |
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15419173 |
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60911551 |
Apr 13, 2007 |
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60894901 |
Mar 14, 2007 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61P 43/00 20180101;
A61K 47/55 20170801; A61P 35/00 20180101; C07K 5/06078 20130101;
C07K 7/06 20130101; C07K 5/021 20130101; A61K 47/551 20170801; A61K
47/6811 20170801; A61K 47/65 20170801; A61K 51/0497 20130101; A61K
47/64 20170801 |
International
Class: |
C07K 7/06 20060101
C07K007/06; C07K 5/02 20060101 C07K005/02; A61K 51/04 20060101
A61K051/04; A61K 47/64 20060101 A61K047/64; A61K 47/55 20060101
A61K047/55; C07K 5/065 20060101 C07K005/065; A61K 47/68 20060101
A61K047/68 |
Claims
1-36. (canceled)
37. A drug delivery conjugate of the formula ##STR00168## wherein D
is a tubulysin; or a pharmaceutically acceptable salt thereof.
38. The drug delivery conjugate of claim 37, wherein D is of the
formula ##STR00169## wherein n is an integer from 1 to 3; V is
selected from H, OR.sup.2, and halo, and W is selected from H,
OR.sup.2, and alkyl, wherein R.sup.2 is independently selected from
H, alkyl, and C(O)R.sup.3, wherein R.sup.3 is selected from alkyl,
cycloalkyl, alkenyl, aryl, and arylalkyl, providing that R.sup.2 is
not H when both V and W are OR.sup.2; or V and W are taken together
with the attached carbon to form a carbonyl; X is selected from H,
C.sub.1-4 alkyl, alkenyl, each of which is optionally substituted,
and CH.sub.2QR.sup.9; where Q is selected from --NH--, --O--, and
--S--; R.sup.9 is selected from H, C.sub.1-4 alkyl, alkenyl, aryl,
and C(O)R.sup.10; and R.sup.10 is selected from C.sub.1-6 alkyl,
alkenyl, aryl, and heteroaryl; Z is alkyl or C(O)R.sup.4, where
R.sup.4 is selected from alkyl, CF.sub.3, and aryl; R.sup.1 is H,
or R.sup.1 represents 1 to 3 substituents selected from halo,
nitro, carboxylate or a derivative thereof, cyano, hydroxyl, alkyl,
haloalkyl, alkoxy, haloalkoxy, phenol protecting groups, prodrug
moieties, and OR.sup.6, where R.sup.6 is selected from alkyl, aryl,
COR.sup.7, P(O)(OR.sup.8).sub.2, and SO.sub.3R.sup.8, wherein
R.sup.7 and R.sup.8 are independently selected from H, alkyl,
alkenyl, cycloalkyl, heterocyclyl, aryl, and arylalkyl, each of
which is optionally substituted, or R.sup.8 is a metal cation; and
* represents the point of attachment of D to the rest of the
conjugate; or a pharmaceutically acceptable salt thereof.
39. The drug delivery conjugate of claim 37, wherein D is of the
formula ##STR00170## wherein n is an integer from 1 to 3; V is
selected from H, OR.sup.2, and halo, and W is selected from H,
OR.sup.2, and alkyl, wherein R.sup.2 is independently selected from
H, alkyl, and C(O)R.sup.3, wherein R.sup.3 is selected from alkyl,
cycloalkyl, alkenyl, aryl, and arylalkyl, providing that R.sup.2 is
not H when both V and W are OR.sup.2; or V and W are taken together
with the attached carbon to form a carbonyl; X is selected from H,
C.sub.1-4 alkyl, alkenyl, each of which is optionally substituted,
and CH.sub.2QR.sup.9; where Q is selected from --NH--, --O--, and
--S--; R.sup.9 is selected from H, C.sub.1-4 alkyl, alkenyl, aryl,
and C(O)R.sup.10; and R.sup.10 is selected from C.sub.1-6 alkyl,
alkenyl, aryl, and heteroaryl; Z is alkyl or C(O)R.sup.4, where
R.sup.4 is selected from alkyl, CF.sub.3, and aryl; T is H or
OR.sup.6, wherein R.sup.6 is selected from H, alkyl, aryl,
COR.sup.7, P(O)(OR.sup.8).sub.2, and SO.sub.3R.sup.8, wherein
R.sup.7 and R.sup.8 are independently selected from H, alkyl,
alkenyl, cycloalkyl, heterocyclyl, aryl, and arylalkyl, each of
which is optionally substituted, or R.sup.8 is a metal cation; S1
and U are each independently selected from the group consisting of
H, halo, nitro, cyano, alkyl, haloalkyl, alkoxy, and haloalkoxy;
and * represents the point of attachment of D to the rest of the
conjugate; or a pharmaceutically acceptable salt thereof.
40. The drug delivery conjugate of claim 37, wherein D is of the
formula ##STR00171## ##STR00172## wherein n is an integer from 1 to
3; V is selected from H, OR.sup.2, and halo, and W is selected from
H, OR.sup.2, and alkyl, wherein R.sup.2 is independently selected
from H, alkyl, and C(O)R.sup.3, wherein R.sup.3 is selected from
alkyl, cycloalkyl, alkenyl, aryl, and arylalkyl, providing that
R.sup.2 is not H when both V and W are OR.sup.2; or V and W are
taken together with the attached carbon to form a carbonyl; Q is
selected from --NH--, --O--, and --S--; R.sup.9 is selected from H,
C.sub.1-4 alkyl, alkenyl, aryl, and C(O)R.sup.10; and R.sup.10 is
selected from C.sub.1-6 alkyl, alkenyl, aryl, and heteroaryl;
R.sup.12 represents 1 or more substituents selected from alkyl,
alkenyl, cycloalkyl, aryl, and arylalkyl, each of which is
optionally substituted; and R.sup.13 is selected from C(O)R.sup.10,
C(O)OR.sup.10, and CN; Z is alkyl or C(O)R.sup.4, where R.sup.4 is
selected from alkyl, CF.sub.3, and aryl; T is H or OR.sup.6,
wherein R.sup.6 is selected from H, alkyl, aryl, COR.sup.7,
P(O)(OR.sup.8).sub.2, and SO.sub.3R.sup.8, wherein R.sup.7 and
R.sup.8 are independently selected from H, alkyl, alkenyl,
cycloalkyl, heterocyclyl, aryl, and arylalkyl, each of which is
optionally substituted, or R.sup.8 is a metal cation; S1 and U are
each independently selected from the group consisting of H, halo,
nitro, cyano, alkyl, haloalkyl, alkoxy, and haloalkoxy; and *
represents the point of attachment of D to the rest of the
conjugate; or a pharmaceutically acceptable salt thereof.
41. The drug delivery conjugate of claim 37, wherein D is of the
formula ##STR00173## wherein n is an integer from 1 to 3; V is
selected from H, OR.sup.2, and halo, and W is selected from H,
OR.sup.2, and alkyl, wherein R.sup.2 is independently selected from
H, alkyl, and C(O)R.sup.3, wherein R.sup.3 is selected from alkyl,
cycloalkyl, alkenyl, aryl, and arylalkyl, providing that R.sup.2 is
not H when both V and W are OR.sup.2; or V and W are taken together
with the attached carbon to form a carbonyl; X.sup.3 is selected
from halogen, OS(O).sub.2R.sup.10, OP(O)(OR.sup.10a)R.sup.10 and
OP(O)(OR.sup.10a).sub.2; where R.sup.10 and R.sup.10a are each
independently selected from the group consisting of H, alkyl,
alkenyl, cycloalkyl, aryl, and arylalkyl, each of which is
optionally substituted, or R.sup.10a is a metal cation; Z is alkyl
or C(O)R.sup.4, where R.sup.4 is selected from alkyl, CF.sub.3, and
aryl; T is H or OR.sup.6, wherein R.sup.6 is selected from H,
alkyl, aryl, COR.sup.7, P(O)(OR.sup.8).sub.2, and SO.sub.3R.sup.8,
wherein R.sup.7 and R.sup.8 are independently selected from H,
alkyl, alkenyl, cycloalkyl, heterocyclyl, aryl, and arylalkyl, each
of which is optionally substituted, or R.sup.8 is a metal cation;
S1 and U are each independently selected from the group consisting
of H, halo, nitro, cyano, alkyl, haloalkyl, alkoxy, and haloalkoxy;
and * represents the point of attachment of D to the rest of the
conjugate; or a pharmaceutically acceptable salt thereof.
42. The drug delivery conjugate of claim 37, wherein D is a
naturally occurring tubulysin, or an analog or derivative thereof;
or a pharmaceutically acceptable salt thereof.
43. The drug delivery conjugate of claim 37, wherein D is of the
formula ##STR00174## wherein R.sup.10 is selected from C.sub.1-6
alkyl, alkenyl, aryl, and heteroaryl; R.sup.1 is H, or R.sup.1
represents 1 to 3 substituents selected from halo, nitro,
carboxylate or a derivative thereof, cyano, hydroxyl, alkyl,
haloalkyl, alkoxy, haloalkoxy, phenol protecting groups, prodrug
moieties, and OR.sup.6, where R.sup.6 is selected from alkyl, aryl,
COR.sup.7, P(O)(OR.sup.8).sub.2, and SO.sub.3R.sup.8, wherein
R.sup.7 and R.sup.8 are independently selected from H, alkyl,
alkenyl, cycloalkyl, heterocyclyl, aryl, and arylalkyl, each of
which is optionally substituted, or R.sup.8 is a metal cation; and
* represents the point of attachment of D to the rest of the
conjugate; or a pharmaceutically acceptable salt thereof.
44. The drug delivery conjugate of claim 38, wherein Z is methyl;
or a pharmaceutically acceptable salt thereof.
45. The drug delivery conjugate of claim 38, wherein R.sup.1 is H;
or a pharmaceutically acceptable salt thereof.
46. The drug delivery conjugate of claim 38, wherein R.sup.1 is
OR.sup.6, where R.sup.6 is selected from H, alkyl, and COR.sup.7;
wherein R.sup.7 is selected from H, alkyl, alkenyl, cycloalkyl,
heterocyclyl, aryl, and arylalkyl, each of which is optionally
substituted; or a pharmaceutically acceptable salt thereof.
47. The drug delivery conjugate of claim 38, wherein V is H, and W
is OC(O)R.sup.3, wherein R.sup.3 is selected from alkyl,
cycloalkyl, alkenyl, aryl, and arylalkyl; or a pharmaceutically
acceptable salt thereof.
48. The drug delivery conjugate of claim 38, wherein X is
CH.sub.2OC(O)R.sup.10 and R.sup.10 is selected from C.sub.1-6
alkyl, alkenyl, aryl, and heteroaryl; or a pharmaceutically
acceptable salt thereof.
49. A pharmaceutical composition comprising a therapeutically
effective amount of the drug delivery conjugate claim 37 or a
pharmaceutically acceptable salt thereof, and a pharmaceutically
acceptable carrier, diluent, or excipient therefore, or a
combination thereof.
50. A method of eliminating a population of pathogenic cells in a
host animal harboring the population of pathogenic cells wherein
the members of the pathogenic cell population have an accessible
binding site for a vitamin, or a vitamin receptor binding analog or
a derivative thereof, and wherein the binding site is uniquely
expressed, overexpressed, or preferentially expressed by the
pathogenic cells, said method comprising the step of administering
to the host a therapeutically effective amount of the drug delivery
conjugate of claim 37 or a pharmaceutically acceptable salt
thereof, or a pharmaceutical composition thereof.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit of priority under 35
U.S.C. .sctn.119(e) of U.S. provisional patent application Ser. No.
60/911,551 filed Apr. 13, 2007, and U.S. provisional patent
application Ser. No. 60/894,901 filed 14 Mar. 2007, the disclosures
of which are incorporated herein by reference in their
entirety.
TECHNICAL FIELD
[0002] The present invention relates to compositions and methods
for use in targeted drug delivery. More particularly, the invention
is directed to cell-surface receptor binding drug delivery
conjugates for use in treating disease states caused by pathogenic
cell populations and to methods and pharmaceutical compositions
that use and include such conjugates.
BACKGROUND
[0003] The mammalian immune system provides a means for the
recognition and elimination of tumor cells, other pathogenic cells,
and invading foreign pathogens. While the immune system normally
provides a strong line of defense, there are many instances where
cancer cells, other pathogenic cells, or infectious agents evade a
host immune response and proliferate or persist with concomitant
host pathogenicity. Chemotherapeutic agents and radiation therapies
have been developed to eliminate, for example, replicating
neoplasms. However, many of the currently available
chemotherapeutic agents and radiation therapy regimens have adverse
side effects because they work not only to destroy pathogenic
cells, but they also affect normal host cells, such as cells of the
hematopoietic system. The adverse side effects of these anticancer
drugs highlight the need for the development of new therapies
selective for pathogenic cell populations and with reduced host
toxicity.
[0004] Researchers have developed therapeutic protocols for
destroying pathogenic cells by targeting cytotoxic compounds to
such cells. Many of these protocols utilize toxins conjugated to
antibodies that bind to antigens unique to or overexpressed by the
pathogenic cells in an attempt to minimize delivery of the toxin to
normal cells. Using this approach, certain immunotoxins have been
developed consisting of antibodies directed to specific antigens on
pathogenic cells, the antibodies being linked to toxins such as
ricin, Pseudomonas exotoxin, Diptheria toxin, and tumor necrosis
factor. These immunotoxins target pathogenic cells, such as tumor
cells, bearing the specific antigens recognized by the antibody
(Olsnes, S., Immunol. Today, 10, pp. 291-295, 1989; Melby, E. L.,
Cancer Res., 53(8), pp. 1755-1760, 1993; Better, M. D., PCT
Publication Number WO 91/07418, published May 30, 1991).
[0005] Another approach for targeting populations of pathogenic
cells, such as cancer cells or foreign pathogens, in a host is to
enhance the host immune response against the pathogenic cells to
avoid the need for administration of compounds that may also
exhibit independent host toxicity. One reported strategy for
immunotherapy is to bind antibodies, for example, genetically
engineered multimeric antibodies, to the surface of tumor cells to
display the constant region of the antibodies on the cell surface
and thereby induce tumor cell killing by various immune-system
mediated processes (De Vita, V. T., Biologic Therapy of Cancer, 2d
ed. Philadelphia, Lippincott, 1995; Soulillou, J. P., U.S. Pat. No.
5,672,486). However, these approaches have been complicated by the
difficulties in defining tumor-specific antigens.
[0006] Tubulysins are a group of potent inhibitors of tubulin
polymerization. Tubulysins are useful in treating diseases and
disease states that include pathogenic cell populations, such as
cancer. Two particular species of mycobacteria synthesize
tubulysins in high titer during fermentation. One species,
Archangium gephyra, produces as the main component factors
tubulysins A, B, C, G, and I, each of which is characterized by a
including the tubutyrosine (Tut, an analog of tyrosine) residue. In
contrast, another species, Angiococcus disciformis, produces as the
main component factors tubulysins D, E, F, and H, each of which is
characterized by a including the tubuphenylalanine (Tup, an analog
of phenylalanine) residue. Such bacterial fermentations are
convenient sources of tubulysins.
SUMMARY OF THE INVENTION
[0007] In one illustrative embodiment of the invention, conjugates
of tubulysins having the formula
B-L-D
are described where B is a binding or targeting ligand, L is a
relesable linker, and D is a tubulysin, or an analog or derivative
thereof. It is to be understood that as used herein, the term
tubulysin refers both individually and/or collectively to naturally
occurring tubulysins, synthetically prepared tubulysins, and
analogs and derivatives of such compounds.
[0008] In another embodiment, conjugates of tubulysin comprising a
binding or targeting ligand B, a polyvalent releasable linker L,
and one or more drugs D are described, where at least one drug D is
a first tubulysin, and where B and D are each covalently bonded to
L.
[0009] In another embodiment, conjugates of tubulysins of the
formula
##STR00001##
and pharmaceutical salts thereof are described herein, where B is a
binding or targeting ligand, L is a relesable linker,
[0010] n is 1-3;
[0011] V is H, OR.sup.2, or halo, and W is H, OR.sup.2, or alkyl,
where R.sup.2 is independently selected in each instance from H,
alkyl, and C(O)R.sup.3, where R.sup.3 is alkyl, cycloalkyl,
alkenyl, aryl, or arylalkyl, each of which is optionally
substituted; providing that R.sup.2 is not H when both V and W are
OR.sup.2; or V and W are taken together with the attached carbon to
form a carbonyl;
[0012] X.dbd.H, C.sub.1-4 alkyl, alkenyl, each of which is
optionally substituted, or CH.sub.2QR.sup.9; where Q is --N--,
--O--, or --S--; R.sup.9.dbd.H, C.sub.1-4 alkyl, alkenyl, aryl, or
C(O)R.sup.10; and R.sup.10.dbd.C.sub.1-6 alkyl, alkenyl, aryl, or
heteroaryl, each of which is optionally substituted;
[0013] Z is alkyl and Y is O; or Z is alkyl or C(O)R.sup.4, and Y
is absent, where R.sup.4 is alkyl, CF.sub.3, or aryl;
[0014] R.sup.1 is H, or R.sup.1 represents 1 to 3 substituents
selected from halo, nitro, carboxylate or a derivative thereof,
cyano, hydroxyl, alkyl, haloalkyl, alkoxy, haloalkoxy, phenol
protecting groups, prodrug moieties, and OR.sup.6, where R.sup.6 is
optionally substituted aryl, C(O)R.sup.7, P(O)(OR.sup.8).sub.2, or
SO.sub.3R.sup.8, where R.sup.7 and R.sup.8 are independently
selected in each instance from H, alkyl, alkenyl, cycloalkyl,
heterocyclyl, aryl, and arylalkyl, each of which is optionally
substituted, or R.sup.8 is a metal cation; and
[0015] R is OH or a leaving group, or R forms a carboxylic acid
derivative.
[0016] In another embodiment, conjugates of naturally occurring
tubulysins are described herein, where the tubulysins are
conjugated to a binding or targeting ligand via an optional
releasable linker L.
[0017] In another embodiment, the conjugates described herein are
included in pharmaceutical compositions in amounts effective to
treat diseases and disease states associated with pathogenic
populations of cells.
[0018] In another embodiment, the conjugates described herein, and
pharmaceutical compositions containing them are used in methods for
treating diseases and disease states associated with pathogenic
populations of cells.
BRIEF DESCRIPTION OF THE DRAWINGS
[0019] FIG. 1 shows that EC0305 exhibited dose-responsive behavior
and specificity for the folate receptor after a 2 hour pulse and a
72 hour chase against KB cells: ( ) EC0305 (IC.sub.50.about.1.5
nM); (0) EC0305+excess folic acid.
[0020] FIG. 2 shows that EC0305 exhibited low serum binding in
various species: (a) human, (b) dog, (c) Balb/c mouse, (d) rat, (e)
rabbit, and (f) fetal calf serum. Human serum binding was 67%.
[0021] FIG. 3 shows that EC0305 tested in human serum for stability
exhibited a half life of about 20 hours.
[0022] FIG. 4 shows the relative affinity assay results in 10%
serum/FDRPMI for EC0305: ( ) folic acid, relative affinity=1;
(.box-solid.) EC0305, relative affinity=0.96.
[0023] FIG. 5 shows the activity of EC305 against KB tumors dosed
TIW on a two week schedule at various doses, as compared to
controls: ( ) PBS treated control; (0) EC0305 2 .mu.mol/kg TIW (5/5
complete responses); (.box-solid.) EC0305 1 .mu.mol/kg TIW (5/5
complete responses); (.tangle-solidup.) EC0305 0.5 .mu.mol/kg TIW
(1/5 complete responses); (.quadrature.) EC0305 1 .mu.mol/kg
TIW+EC-20 (rhenium) 40 .mu.mol/kg TIW (0/5 complete responses). The
vertical dotted line indicates the last day of dosing.
[0024] FIG. 6 shows the measure of percent weight change in treated
animals, as compared to controls: ( ) PBS treated control;
(.largecircle.) EC0305 2 .mu.mol/kg TIW; (.box-solid.) EC0305 1
.mu.mol/kg TIW; (.tangle-solidup.) EC0305 0.5 .mu.mol/kg TIW;
(.quadrature.) EC0305 1 .mu.mol/kg TIW+EC-20 (rhenium) 40
.mu.mol/kg TIW. The vertical dotted line indicates the last day of
dosing.
[0025] FIG. 7 shows the activity of EC305 against M109 tumors dosed
at 2 .mu.mol/kg TIW on a two week schedule at various doses, as
compared to controls: ( ) PBS treated control; (.box-solid.) EC0305
(5/5 complete responses). The vertical dotted line indicates the
last day of dosing.
[0026] FIG. 8 shows the measure of percent weight change in treated
animals, as compared to controls: ( ) PBS treated control;
(.box-solid.) EC0305. The vertical dotted line indicates the last
day of dosing.
[0027] FIG. 9 shows that absence of efficacy (0/5 complete or
partial responses) of unconjugated tubulysin B at both tolerable
and highly toxic dose levels, as compared to controls: ( ) PBS
treated control; (.largecircle.) 0.5 .mu.mol/kg TIW;
(.tangle-solidup.) 0.2 .mu.mol/kg TIW; (.box-solid.) 0.1 .mu.mol/kg
TIW.
[0028] FIG. 10 shows the percent weight change of animals treated
with both tolerated and highly toxic dose levels of unconjugated
tubulysin B, as compared to controls: ( ) PBS treated control;
(.largecircle.) 0.5 .mu.mol/kg TIW; (.tangle-solidup.) 0.2
.mu.mol/kg TIW; (.box-solid.) 0.1 .mu.mol/kg TIW.
[0029] FIG. 11 shows the relative activity of tubulysin conjugate
EC0305 compared to vinca alkaloid conjugate EC145, each dosed at 2
.mu.mol/kg TIW on a two-week schedule, and as compared to controls:
( ) PBS treated control; (.largecircle.) EC145 (2/5 complete
responses); (.box-solid.) EC0305 (5/5 complete responses). The
vertical dotted line indicates the last day of dosing.
[0030] FIG. 12 shows the relative activity of tubulysin conjugates,
EC0305 and EC0436, on M109 tumors, each dosed at 2 .mu.mol/kg three
times per week for two weeks, as compared to controls: (a) PBS
treated control; (b) EC0305 (4/5 complete responses); (c) EC0436
(5/5 complete responses). The vertical dotted line indicates the
last day of dosing.
[0031] FIG. 13 shows the measure of percent weight change in
treated animals, as compared to controls: ( ) PBS treated control;
(.ident.) is EC0305 (TIW 2 .mu.mol/kg, 2 wks); (.box-solid.) is
EC0436 (TIW 2 .mu.mol/kg, 2 wks). The vertical dotted line
indicates the last day of dosing.
[0032] FIG. 14 shows the percentage body weight change of Balb/c
mice treated intravenously three times in a week for one week with
EC0436 and EC0305 at various doses, as compared to controls: ( )
PBS treated control; (.tangle-solidup.) 2 .mu.mol/kg TIW EC0436; ()
2.5 .mu.mol/kg TIW EC0436; (.box-solid.) 3 .mu.mol/kg TIW EC0436;
(.DELTA.) 2 .mu.mol/kg TIW EC0305; (.gradient.) 2.5 .mu.mol/kg TIW
EC0305; (.quadrature.) 3 .mu.mol/kg EC0305. The vertical dotted
line indicates the last day of dosing.
DETAILED DESCRIPTION
[0033] Drug delivery conjugates are described herein consisting of
a binding ligand (B), a bivalent linker (L), and a tubulysin (D),
including analogs and derivatives thereof. The binding ligand (B)
is covalently attached to the bivalent linker (L), and the
tubulysin, or analog or derivative thereof, is also covalently
attached to the bivalent linker (L). The bivalent linker (L)
comprises one or more spacer linkers and/or releasable linkers, and
combinations thereof, in any order. In one variation, releasable
linkers, and optional spacer linkers are covalently bonded to each
other to form the linker. In another variation, a releasable linker
is directly attached to the tubulysin, or analog or derivative
thereof. In another variation, a releasable linker is directly
attached to the binding ligand. In another variation, either or
both the binding ligand and the tubulysin, or analog or derivative
thereof, is attached to a releasable linker through one or more
spacer linkers. In another variation, each of the binding ligand
and the tubulysin, or analog or derivative thereof, is attached to
a releasable linker, each of which may be directly attached to each
other, or covalently attached through on e or more spacer linkers.
From the foregoing, it should be appreciated that the arrangement
of the binding ligand, and the tubulysin, or analog or derivative
thereof, and the various releasable and optional spacer linkers may
be varied widely. In one aspect, the binding ligand, and the
tubulysin, or analog or derivative thereof, and the various
releasable and optional spacer linkers are attached to each other
through heteroatoms, such as nitrogen, oxygen, sulfur, phosphorus,
silicon, and the like. In variations, the heteroatoms, excluding
oxygen, may be in various states of oxidation, such as N(OH), S(O),
S(O).sub.2, P(O), P(O).sub.2, P(O).sub.3, and the like. In other
variation, the heteroatoms may be grouped to form divalent
radicals, such as for example hydroxylamines, hydrazines,
hydrazones, sulfonates, phosphinates, phosphonates, and the
like.
[0034] In one aspect, the receptor binding ligand (B) is a vitamin,
or analog or derivative thereof, or another vitamin receptor
binding compound.
[0035] As used herein, tubulysins refer generally to tetrapeptide
compounds of the formula
##STR00002##
and pharmaceutical salts thereof, where
[0036] n is 1-3;
[0037] V is H, OR.sup.2, or halo, and W is H, OR.sup.2, or alkyl,
where R.sup.2 is independently selected in each instance from H,
alkyl, and C(O)R.sup.3, where R.sup.3 is alkyl, cycloalkyl,
alkenyl, aryl, or arylalkyl, each of which is optionally
substituted; providing that R.sup.2 is not H when both V and W are
OR.sup.2; or V and W are taken together with the attached carbon to
form a carbonyl;
[0038] X.dbd.H, C.sub.1-4 alkyl, alkenyl, each of which is
optionally substituted, or CH.sub.2QR.sup.9; where Q is --N--,
--O--, or --S--; R.sup.9.dbd.H, C.sub.1-4 alkyl, alkenyl, aryl, or
C(O)R.sup.10; and R.sup.10.dbd.C.sub.1-6 alkyl, alkenyl, aryl, or
heteroaryl, each of which is optionally substituted;
[0039] Z is alkyl and Y is O; or Z is alkyl or C(O)R.sup.4, and Y
is absent, where R.sup.4 is alkyl, CF.sub.3, or aryl;
[0040] R.sup.1 is H, or R.sup.1 represents 1 to 3 substituents
selected from halo, nitro, carboxylate or a derivative thereof,
cyano, hydroxyl, alkyl, haloalkyl, alkoxy, haloalkoxy, phenol
protecting groups, prodrug moieties, and OR.sup.6, where R.sup.6 is
optionally substituted aryl, C(O)R.sup.7, P(O)(OR.sup.8).sub.2, or
SO.sub.3R.sup.8, where R.sup.7 and R.sup.8 are independently
selected in each instance from H, alkyl, alkenyl, cycloalkyl,
heterocyclyl, aryl, and arylalkyl, each of which is optionally
substituted, or R.sup.8 is a metal cation; and
[0041] R is OH or a leaving group, or R forms a carboxylic acid
derivative.
[0042] Conjugates of each of the foregoing tubulysins are described
herein. In one variation, Z is methyl. In another variation,
R.sup.1 is H. In another variation, R.sup.1 is OR.sup.6 at C(4),
where R.sup.6 is H, alkyl, or COR.sup.7. In another variation, V is
H, and W is OC(O)R.sup.3.
[0043] In another embodiment, conjugates of tubulysins of the
following general formula are described
##STR00003##
and pharmaceutical salts thereof, where
[0044] n is 1-3;
[0045] V is H, OR.sup.2, or halo, and W is H, OR.sup.2, or alkyl,
where R.sup.2 is independently selected in each instance from H,
alkyl, or C(O)R.sup.3, where R.sup.3 is alkyl, alkenyl or aryl,
providing that R.sup.2 is not H when both V and W are OR.sup.2; or
V and W are taken together with the attached carbon to form a
carbonyl;
[0046] X.dbd.H, C.sub.1-4 alkyl, alkenyl, each of which is
optionally substituted, or CH.sub.2QR.sup.9; where Q is --N--,
--O--, or --S--; R.sup.9.dbd.H, C.sub.1-4 alkyl, alkenyl, aryl, or
C(O)R.sup.10; and R.sup.10.dbd.C.sub.1-6 alkyl, alkenyl, aryl, or
heteroaryl, each of which is optionally substituted;
[0047] Z is alkyl or C(O)R.sup.4, where R.sup.4 is alkyl, CF.sub.3,
or aryl;
[0048] T is H or OR.sup.6, where R.sup.6 is H, alkyl, aryl,
COR.sup.7, P(O)(OR.sup.8).sub.2, or SO.sub.3R.sup.8, where R.sup.7
and R.sup.8 are independently selected in each instance from H,
alkyl, alkenyl, cycloalkyl, heterocyclyl, aryl, and arylalkyl, each
of which is optionally substituted, or R.sup.8 is a metal cation,
or R.sup.6 is a phenol protecting group, or a prodrug moiety;
[0049] S and U are each independently selected from the group
consisting of H, halo, nitro, cyano, alkyl, haloalkyl, alkoxy, and
haloalkoxy; and
[0050] R is OH or a leaving group, or R forms a carboxylic acid
derivative.
[0051] In one variation, Z is methyl or C(O)R.sup.4.
[0052] Natural tubulysins are generally linear tetrapeptides
consisting of N-methyl pipecolic acid (Mep), isoleucine (Ile), an
unnatural aminoacid called tubuvalin (Tuv), and either an unnatural
aminoacid called tubutyrosine (Tut, an analog of tyrosine) or an
unnatural aminoacid called tubuphenylalanine (Tup, an analog of
phenylalanine). In another embodiment, naturally occurring
tubulysins, and analogs and derivatives thereof, of the following
general formula are described
##STR00004##
and pharmaceutical salts thereof, where R, R.sup.1, and R.sup.10
are as described in the various embodiments herein. Conjugates of
each of the foregoing tubulysins are described herein.
[0053] In another embodiment, conjugates of naturally occurring
tubulysins of the following general formula are described
TABLE-US-00001 ##STR00005## Factor R.sup.10 R.sup.1 A
(CH.sub.3).sub.2CHCH.sub.2 OH B CH.sub.3(CH.sub.2).sub.2 OH C
CH.sub.3CH.sub.2 OH D (CH.sub.3).sub.2CHCH.sub.2 H E
CH.sub.3(CH.sub.2).sub.2 H F CH.sub.2CH.sub.3 H G
(CH.sub.3).sub.2C.dbd.CH OH H CH.sub.3 H I CH.sub.3 OH
and pharmaceutical salts thereof.
[0054] In another embodiment, conjugates of tubulysins of the
following formula are described:
##STR00006##
and pharmaceutical salts thereof, where n is 1-3; T is H or
OR.sup.6, where R.sup.6 is H, alkyl, aryl, COR.sup.7,
P(O)(OR.sup.8).sub.2, or SO.sub.3R.sup.8, where R.sup.7 and R.sup.8
are independently selected in each instance from H, alkyl, alkenyl,
cycloalkyl, heterocyclyl, aryl, and arylalkyl, each of which is
optionally substituted, or R.sup.8 is a metal cation, or R.sup.6 is
a phenol protecting group, or a prodrug moiety; Z is alkyl or
C(O)R.sup.4, where R.sup.4 is alkyl, CF.sub.3, or aryl; and R is OH
or a leaving group, or R forms a carboxylic acid derivative.
Illustrative examples of such compounds, and their preparation are
described in J. Med. Chem. 10.1021/jm701321p (2008), the disclosure
of which is incorporated herein by reference.
[0055] In another embodiment, conjugates of tubulysins of the
following formula are described:
##STR00007##
and pharmaceutical salts thereof, where n, S, T, U, V, W, Z, R, and
R.sup.10 are as described in the various embodiments herein.
[0056] In another embodiment, conjugates of tubulysins of the
following formula are described:
##STR00008##
and pharmaceutical salts thereof, where n, S, T, U, V, W, Z,
QR.sup.9, and R are as described in the various embodiments herein.
In one variation, Q is --N--, --O--, or --S--; and R.sup.9 is H,
alkyl, alkenyl, cycloalkyl, aryl, or arylalkyl, each of which is
optionally substituted. In another variation, QR.sup.9 are taken
together to form C(O)R.sup.10, S(O).sub.2R.sup.10,
P(O)(OR.sup.10a).sub.2, where R.sup.10 and OR.sup.10a are
independently selected in each instance from the group consisting
of H, alkyl, alkenyl, cycloalkyl, aryl, and arylalkyl, each of
which is optionally substituted, or R.sup.10a is a metal
cation.
[0057] In another embodiment, conjugates of tubulysins of the
following formula are described:
##STR00009##
and pharmaceutical salts thereof, where R.sup.12 represents 1 or
more substituents selected from alkyl, alkenyl, cycloalkyl, aryl,
and arylalkyl, each of which is optionally substituted; and where
n, S, T, U, V, W, Z, and R are as described in the various
embodiments herein. It is to be understood that other olefins may
form by isomerization, depending on the conditions of the reaction
and the identity of R.sup.1. For example, when R.sup.1 is alkyl, it
is appreciated that under the reaction conditions, the double bond
can migrate to other carbon atoms along the alkenyl chain,
including to form the terminal or .omega.-olefin.
[0058] In another embodiment, conjugates of tubulysins of the
following formula are described:
##STR00010##
and pharmaceutical salts thereof, where R.sup.13 is C(O)R.sup.10,
C(O)OR.sup.10 or CN; and where n, S, T, U, V, W, Z, R, and R.sup.10
are as described in the various embodiments herein, where R.sup.10
is independently selected in each instance.
[0059] In another embodiment, conjugates of tubulysins of the
following formula are described:
##STR00011##
and pharmaceutical salts thereof, where n, S, T, U, V, W, Z, and R
are as described in the various embodiments herein.
[0060] In another embodiment, conjugates of tubulysins of the
following formula are described:
##STR00012##
and pharmaceutical salts thereof, where X.sup.3 is halogen,
OS(O).sub.2R.sup.10, OP(O)(OR.sup.10a)R.sup.10, or
OP(O)(OR.sup.10a).sub.2; where R.sup.10 and R.sup.10a are
independently selected in each instance from the group consisting
of H, alkyl, alkenyl, cycloalkyl, aryl, and arylalkyl, each of
which is optionally substituted, or R.sup.10a is a metal cation;
and where n, S, T, U, V, W, Z, and R are as described in the
various embodiments herein.
[0061] Additional tubulysins useful in preparing the conjugates
described herein are described in US patent application publication
Nos. 2006/0128754 and 2005/0239713, the disclosures of which are
incorporated herein by reference. Additional tubulysins useful in
preparing the conjugates described herein are described in
co-pending U.S. provisional application Ser. Nos. 60/982,595 and
61/036,176, the disclosures of which are incorporated herein by
reference. Tubulysins may also be prepared are described in Peltier
et al., "The Total Synthesis of Tubulysin D," J. Am. Chem. Soc.
128:16018-19 (2006), the disclosure of which is incorporated herein
by reference.
[0062] In each of the foregoing embodiments, it is understood that
in one variation, the compounds of the various formulae have the
following absolute configuration:
##STR00013##
at the indicated asymmetric backbone carbon atoms.
[0063] It is to be understood that the conjugate of the tubulysin
or analog or derivative thereof may be formed at any position.
Illustratively, conjugates of tubulysins are described where the
bivalent linker (L) is attached to any of the following
positions:
##STR00014##
where the (*) symbol indicates optional attachment locations.
[0064] In another embodiment, the conjugates are formed from
carboxylic acid derivatives of the tubulysin, or analog or
derivative thereof. Illustrative carboxylic acid conjugate
derivatives of the tubulysin are represented by the following
general formula
##STR00015##
and pharmaceutical salts thereof, where
[0065] B is a binding ligand;
[0066] L is a linker; where L includes a heteroatom linker
covalently attached to the tubulysin, such as an oxygen, nitrogen,
or sulfur heteroatom;
[0067] n is 1-3;
[0068] V is H, OR.sup.2, or halo, and W is H, OR.sup.2, or alkyl,
where R.sup.2 is independently selected in each instance from H,
alkyl, or C(O)R.sup.3, where R.sup.3 is alkyl, alkenyl or aryl,
providing that R.sup.2 is not H when both V and W are OR.sup.2; or
V and W are taken together with the attached carbon to form a
carbonyl;
[0069] X.dbd.H, C.sub.1-4 alkyl, alkenyl, each of which is
optionally substituted, or CH.sub.2QR.sup.9; where Q is --N--,
--O--, or --S--; R.sup.9.dbd.H, C.sub.1-4 alkyl, alkenyl, aryl, or
C(O)R.sup.10, and R.sup.10.dbd.C.sub.1-6 alkyl, alkenyl, aryl, or
heteroaryl, each of which is optionally substituted;
[0070] Z is alkyl and Y is O; or Z is alkyl or C(O)R.sup.4, and Y
is absent, where R.sup.4 is alkyl, CF.sub.3, or aryl;
[0071] R.sup.1 is H, or R.sup.1 represents 1 to 3 substituents
selected from halo, nitro, carboxylate or a derivative thereof,
cyano, hydroxyl, alkyl, haloalkyl, alkoxy, haloalkoxy, phenol
protecting groups, prodrug moieties, and OR.sup.6, where R.sup.6 is
optionally substituted aryl, C(O)R.sup.7, P(O)(OR.sup.8).sub.2, or
SO.sub.3R.sup.8, where R.sup.7 and R.sup.8 are independently
selected in each instance from H, alkyl, alkenyl, cycloalkyl,
heterocyclyl, aryl, and arylalkyl, each of which is optionally
substituted, or R.sup.8 is a metal cation; and
[0072] R is OH or a leaving group, or R forms a carboxylic acid
derivative.
[0073] In another embodiment, illustrative carboxylic acid
conjugate derivatives of tubulysin of the following general formula
are described
##STR00016##
and pharmaceutical salts thereof, where
[0074] B is a binding ligand;
[0075] L is a linker; where L includes a heteroatom linker
covalently attached to the tubulysin, such as an oxygen, nitrogen,
or sulfur heteroatom;
[0076] n is 1-3;
[0077] V is H, OR.sup.2, or halo, and W is H, OR.sup.2, or alkyl,
where R.sup.2 is independently selected in each instance from H,
alkyl, or C(O)R.sup.3, where R.sup.3 is alkyl, alkenyl or aryl,
providing that R.sup.2 is not H when both V and W are OR.sup.2; or
V and W are taken together with the attached carbon to form a
carbonyl;
[0078] X.dbd.H, C.sub.1-4 alkyl, alkenyl, each of which is
optionally substituted, or CH.sub.2QR.sup.9; where Q is --N--,
--O--, or --S--; R.sup.9.dbd.H, C.sub.1-4 alkyl, alkenyl, aryl, or
C(O)R.sup.10; and R.sup.10.dbd.C.sub.1-6 alkyl, alkenyl, aryl, or
heteroaryl, each of which is optionally substituted;
[0079] Z is alkyl or C(O)R.sup.4, where R.sup.4 is alkyl, CF.sub.3,
or aryl;
[0080] T is H or OR.sup.6, where R.sup.6 is H, alkyl, aryl,
COR.sup.7, P(O)(OR.sup.8).sub.2, or SO.sub.3R.sup.8, where R.sup.7
and R.sup.8 are independently selected in each instance from H,
alkyl, alkenyl, cycloalkyl, heterocyclyl, aryl, and arylalkyl, each
of which is optionally substituted, or R.sup.8 is a metal cation,
or R.sup.6 is a phenol protecting group, or a prodrug moiety;
[0081] S and U are each independently selected from the group
consisting of H, halo, nitro, cyano, alkyl, haloalkyl, alkoxy, and
haloalkoxy; and
[0082] R is OH or a leaving group, or R forms a carboxylic acid
derivative.
[0083] In another embodiment, illustrative carboxylic acid
conjugate derivatives of the following general formulae are
described
##STR00017## ##STR00018## ##STR00019##
and pharmaceutical salts thereof, where B, L, n, S, T, U, V, W, X,
Z, Q, R.sup.1, R.sup.9, R.sup.10, R.sup.12, R.sup.13, and X.sup.3
are as described herein in the various embodiments and aspects.
[0084] In another embodiment, illustrative carboxylic acid
conjugate derivatives of naturally occurring tubulysins such as
tubulysin A, tubulysin B, and tubulysin I, are described, and
pharmaceutical salts thereof.
[0085] In another embodiment, illustrative carboxylic acid
conjugate derivatives of the following tubulysin analogs and
derivative are described
[0086] Additional tubulysins that are useable in the conjugates
described herein include the following, including the IC50 for
inhibition of 3H thymidine update in 72 hour continuous assay of KB
cells:
TABLE-US-00002 ##STR00020## Conjugate X.sup.3 B-L-EC0313
--O--CH.sub.3 B-L-EC0346 --O--(CH.sub.2).sub.2--OH B-L-EC0356
--O--(CH.sub.2).sub.2CH(CH.sub.3).sub.2 B-L-EC0374
--S--(CH.sub.2).sub.2--SH B-L-EC0386 --OH B-L-EC0550
--(CH.sub.2).sub.2--CH.dbd.CH.sub.2 B-L-EC0560
--S--(CH.sub.2).sub.2--OH B-L-EC0575
--O--C(O)--(CH.dbd.CH)--CH.sub.2--Cl B-L-EC0585
--NH--C(O)--CH.sub.2CH(CH.sub.3).sub.2 B-L-EC0611
--O--(CH.sub.2).sub.2CH.sub.3 B-L-EC0623
--S--(CH.sub.2).sub.2CH.sub.3
and pharmaceutical salts thereof.
[0087] As described herein, the tubulysin compounds may be
inhibitors of tubulin polymerization, and also may be
DNA-alkylators. Accordingly, methods for treating diseases and
disease states including pathogenic cell populations, such as
cancer, are contemplated herein.
[0088] In another embodiment, the bivalent linker (L) is a chain of
atoms selected from C, N, O, S, Si, and P that covalently connects
the binding ligand (B) to the tubulysin (D). The linker may have a
wide variety of lengths, such as in the range from about 2 to about
100 atoms. The atoms used in forming the linker may be combined in
all chemically relevant ways, such as chains of carbon atoms
forming alkylene, alkenylene, and alkynylene groups, and the like;
chains of carbon and oxygen atoms forming ethers, polyoxyalkylene
groups, or when combined with carbonyl groups forming esters and
carbonates, and the like; chains of carbon and nitrogen atoms
forming amines, imines, polyamines, hydrazines, hydrazones, or when
combined with carbonyl groups forming amides, ureas,
semicarbazides, carbazides, and the like; chains of carbon,
nitrogen, and oxygen atoms forming alkoxyamines, alkoxylamines, or
when combined with carbonyl groups forming urethanes, amino acids,
acyloxylamines, hydroxamic acids, and the like; and many others. In
addition, it is to be understood that the atoms forming the chain
in each of the foregoing illustrative embodiments may be either
saturated or unsaturated, such that for example, alkanes, alkenes,
alkynes, imines, and the like may be radicals that are included in
the linker. In addition, it is to be understood that the atoms
forming the linker may also be cyclized upon each other to form
divalent cyclic structures that form the linker, including cyclo
alkanes, cyclic ethers, cyclic amines, arylenes, heteroarylenes,
and the like in the linker.
[0089] In another embodiment, the linker includes radicals that
form at least one releasable linker, and optionally one or more
spacer linkers. As used herein, the term releasable linker refers
to a linker that includes at least one bond that can be broken
under physiological conditions, such as a pH-labile, acid-labile,
base-labile, oxidatively labile, metabolically labile,
biochemically labile, or enzyme-labile bond. It is appreciated that
such physiological conditions resulting in bond breaking do not
necessarily include a biological or metabolic process, and instead
may include a standard chemical reaction, such as a hydrolysis
reaction, for example, at physiological pH, or as a result of
compartmentalization into a cellular organelle such as an endosome
having a lower pH than cytosolic pH.
[0090] It is understood that a cleavable bond can connect two
adjacent atoms within the releasable linker and/or connect other
linkers or V and/or D, as described herein, at either or both ends
of the releasable linker. In the case where a cleavable bond
connects two adjacent atoms within the releasable linker, following
breakage of the bond, the releasable linker is broken into two or
more fragments. Alternatively, in the case where a cleavable bond
is between the releasable linker and another moiety, such as an
additional heteroatom, a spacer linker, another releasable linker,
the tubulysin, or analog or derivative thereof, or the binding
ligand, following breakage of the bond, the releasable linker is
separated from the other moiety. Accordingly, it is also understood
that each of the spacer and releasable linkers are polyvalent, such
as bivalent.
[0091] Illustrative releasable linkers include methylene,
1-alkoxyalkylene, 1-alkoxycycloalkylene, 1-alkoxyalkylenecarbonyl,
1-alkoxycycloalkylenecarbonyl, carbonylarylcarbonyl,
carbonyl(carboxyaryl)carbonyl, carbonyl(biscarboxyaryl)carbonyl,
haloalkylenecarbonyl, alkylene(dialkylsilyl),
alkylene(alkylarylsilyl), alkylene(diarylsilyl),
(dialkylsilyl)aryl, (alkylarylsilyl)aryl, (diarylsilyl)aryl,
oxycarbonyloxy, oxycarbonyloxyalkyl, sulfonyloxy, oxysulfonylalkyl,
iminoalkylidenyl, carbonylalkylideniminyl, iminocycloalkylidenyl,
carbonylcycloalkylideniminyl, alkylenethio, alkylenearylthio, and
carbonylalkylthio, wherein each of the releasable linkers is
optionally substituted with a substituent X.sup.2, as defined
below.
[0092] In the preceding embodiment, the releasable linker may
include oxygen, and the releasable linkers can be methylene,
1-alkoxyalkylene, 1-alkoxycycloalkylene, 1-alkoxyalkylenecarbonyl,
and 1-alkoxycycloalkylenecarbonyl, wherein each of the releasable
linkers is optionally substituted with a substituent X.sup.2, as
defined below, and the releasable linker is bonded to the oxygen to
form an acetal or ketal. Alternatively, the releasable linker may
include oxygen, and the releasable linker can be methylene, wherein
the methylene is substituted with an optionally-substituted aryl,
and the releasable linker is bonded to the oxygen to form an acetal
or ketal. Further, the releasable linker may include oxygen, and
the releasable linker can be sulfonylalkyl, and the releasable
linker is bonded to the oxygen to form an alkylsulfonate.
[0093] In another embodiment of the above releasable linker
embodiment, the releasable linker may include nitrogen, and the
releasable linkers can be iminoalkylidenyl,
carbonylalkylideniminyl, iminocycloalkylidenyl, and
carbonylcycloalkylideniminyl, wherein each of the releasable
linkers is optionally substituted with a substituent X.sup.2, as
defined below, and the releasable linker is bonded to the nitrogen
to form an hydrazone. In an alternate configuration, the hydrazone
may be acylated with a carboxylic acid derivative, an orthoformate
derivative, or a carbamoyl derivative to form various acylhydrazone
releasable linkers.
[0094] Alternatively, the releasable linker may include oxygen, and
the releasable linkers can be alkylene(dialkylsilyl),
alkylene(alkylarylsilyl), alkylene(diarylsilyl),
(dialkylsilyl)aryl, (alkylarylsilyl)aryl, and (diarylsilyl)aryl,
wherein each of the releasable linkers is optionally substituted
with a substituent X.sup.2, as defined below, and the releasable
linker is bonded to the oxygen to form a silanol. In another
variation, the drug can include an oxygen atom, and the releasable
linker can be haloalkylenecarbonyl, optionally substituted with a
substituent X.sup.2, and the releasable linker is bonded to the
drug oxygen to form an ester.
[0095] In the above releasable linker embodiment, the drug can
include a nitrogen atom, the releasable linker may include
nitrogen, and the releasable linkers can be carbonylarylcarbonyl,
carbonyl(carboxyaryl)carbonyl, carbonyl(biscarboxyaryl)carbonyl,
and the releasable linker can be bonded to the heteroatom nitrogen
to form an amide, and also bonded to the drug nitrogen to form an
amide. In one variation, the drug can include a nitrogen atom, and
the releasable linker can be haloalkylenecarbonyl, optionally
substituted with a substituent X.sup.2, and the releasable linker
is bonded to the drug nitrogen to form an amide. In another
variation, the drug can include a double-bonded nitrogen atom, and
in this embodiment, the releasable linkers can be
alkylenecarbonylamino and 1-(alkylenecarbonylamino)succinimid-3-yl,
and the releasable linker can be bonded to the drug nitrogen to
form an hydrazone.
[0096] In another variation, the drug can include a sulfur atom,
and in this embodiment, the releasable linkers can be alkylenethio
and carbonylalkylthio, and the releasable linker can be bonded to
the drug sulfur to form a disulfide. Alternatively, the drug can
include an oxygen atom, the releasable linker may include nitrogen,
and the releasable linkers can be carbonylarylcarbonyl,
carbonyl(carboxyaryl)carbonyl, carbonyl(biscarboxyaryl)carbonyl,
and the releasable linker can form an amide, and also bonded to the
drug oxygen to form an ester.
[0097] The substituents X.sup.2 can be alkyl, alkoxy, alkoxyalkyl,
hydroxy, hydroxyalkyl, amino, aminoalkyl, alkylaminoalkyl,
dialkylaminoalkyl, halo, haloalkyl, sulfhydrylalkyl,
alkylthioalkyl, aryl, substituted aryl, arylalkyl, substituted
arylalkyl, heteroaryl, substituted heteroaryl, carboxy,
carboxyalkyl, alkyl carboxylate, alkyl alkanoate, guanidinoalkyl,
R.sup.4-carbonyl, R.sup.5-carbonylalkyl, R.sup.6-acylamino, and
R.sup.7-acylaminoalkyl, wherein R.sup.4 and R.sup.5 are each
independently selected from amino acids, amino acid derivatives,
and peptides, and wherein R.sup.6 and R.sup.7 are each
independently selected from amino acids, amino acid derivatives,
and peptides. In this embodiment the releasable linker can include
nitrogen, and the substituent X.sup.2 and the releasable linker can
form an heterocycle.
[0098] The heterocycles can be pyrrolidines, piperidines,
oxazolidines, isoxazolidines, thiazolidines, isothiazolidines,
pyrrolidinones, piperidinones, oxazolidinones, isoxazolidinones,
thiazolidinones, isothiazolidinones, and succinimides.
[0099] In another embodiment, the bivalent linker (L) includes a
disulfide releasable linker. In another embodiment, the bivalent
linker (L) includes at least one releasable linker that is not a
disulfide releasable linker.
[0100] In one aspect, the releasable and spacer linkers may be
arranged in such a way that subsequent to the cleavage of a bond in
the bivalent linker, released functional groups chemically assist
the breakage or cleavage of additional bonds, also termed
anchimeric assisted cleavage or breakage. An illustrative
embodiment of such a bivalent linker or portion thereof includes
compounds having the formulae:
##STR00021##
where X is an heteroatom, such as nitrogen, oxygen, or sulfur, or a
carbonyl group; n is an integer selected from 0 to 4;
illustratively 2; R is hydrogen, or a substituent, including a
substituent capable of stabilizing a positive charge inductively or
by resonance on the aryl ring, such as alkoxy and the like,
including methoxy; and the symbol (*) indicates points of
attachment for additional spacer, heteroatom, or releasable linkers
forming the bivalent linker, or alternatively for attachment of the
drug, or analog or derivative thereof, or the vitamin, or analog or
derivative thereof. In one embodiment, n is 2 and R is methoxy. It
is appreciated that other substituents may be present on the aryl
ring, the benzyl carbon, the alkanoic acid, or the methylene
bridge, including but not limited to hydroxy, alkyl, alkoxy,
alkylthio, halo, and the like. Assisted cleavage may include
mechanisms involving benzylium intermediates, benzyne
intermediates, lactone cyclization, oxonium intermediates,
beta-elimination, and the like. It is further appreciated that, in
addition to fragmentation subsequent to cleavage of the releasable
linker, the initial cleavage of the releasable linker may be
facilitated by an anchimerically assisted mechanism.
[0101] Illustrative examples of intermediates useful in forming
such linkers include:
##STR00022##
where X.sup.a is an electrophilic group such as maleimide, vinyl
sulfone, activated carboxylic acid derivatives, and the like,
X.sup.b is NH, O, or S; and m and n are each independently selected
integers from 0-4. In one variation, m and n are each independently
selected integers from 0-2. Such intermediates may be coupled to
drugs, binding ligands, or other linkers via nucleophilic attack
onto electrophilic group X.sup.a, and/or by forming ethers or
carboxylic acid derivatives of the. In one embodiment, the benzylic
hydroxyl group is converted into the corresponding activated
benzyloxycarbonyl compound with phosgene or a phosgene equivalent.
This embodiment may be coupled to drugs, binding ligands, or other
linkers via nucleophilic attack onto the activated carbonyl
group.
[0102] The releasable linker includes at least one bond that can be
broken or cleaved under physiological conditions (e.g., a
pH-labile, acid-labile, oxidatively-labile, or enzyme-labile bond).
The cleavable bond or bonds may be present in the interior of a
cleavable linker and/or at one or both ends of a cleavable linker.
It is appreciated that the lability of the cleavable bond may be
adjusted by including functional groups or fragments within the
bivalent linker L that are able to assist or facilitate such bond
breakage, also termed anchimeric assistance. In addition, it is
appreciated that additional functional groups or fragments may be
included within the bivalent linker L that are able to assist or
facilitate additional fragmentation of the vitamin receptor binding
drug conjugates after bond breaking of the releasable linker.
[0103] The lability of the cleavable bond can be adjusted by, for
example, substitutional changes at or near the cleavable bond, such
as including alpha branching adjacent to a cleavable disulfide
bond, increasing the hydrophobicity of substituents on silicon in a
moiety having a silicon-oxygen bond that may be hydrolyzed,
homologating alkoxy groups that form part of a ketal or acetal that
may be hydrolyzed, and the like.
[0104] Illustrative mechanisms for cleavage of the bivalent linkers
described herein include the following 1,4 and 1,6 fragmentation
mechanisms
##STR00023##
where X is an exogenous or endogenous nucleophile, glutathione, or
bioreducing agent, and the like, and either of Z or Z' is the
vitamin, or analog or derivative thereof, or the drug, or analog or
derivative thereof, or a vitamin or drug moiety in conjunction with
other portions of the polyvalent linker. It is to be understood
that although the above fragmentation mechanisms are depicted as
concerted mechanisms, any number of discrete steps may take place
to effect the ultimate fragmentation of the polyvalent linker to
the final products shown. For example, it is appreciated that the
bond cleavage may also occur by acid-catalyzed elimination of the
carbamate moiety, which may be anchimerically assisted by the
stabilization provided by either the aryl group of the beta sulfur
or disulfide illustrated in the above examples. In those variations
of this embodiment, the releasable linker is the carbamate moiety.
Alternatively, the fragmentation may be initiated by a nucleophilic
attack on the disulfide group, causing cleavage to form a thiolate.
The thiolate may intermolecularly displace a carbonic acid or
carbamic acid moiety and form the corresponding thiacyclopropane.
In the case of the benzyl-containing polyvalent linkers, following
an illustrative breaking of the disulfide bond, the resulting
phenyl thiolate may further fragment to release a carbonic acid or
carbamic acid moiety by forming a resonance stabilized
intermediate. In any of these cases, the releasable nature of the
illustrative polyvalent linkers described herein may be realized by
whatever mechanism may be relevant to the chemical, metabolic,
physiological, or biological conditions present.
[0105] Other illustrative mechanisms for bond cleavage of the
releasable linker include oxonium-assisted cleavage as follows:
##STR00024##
where Z is the vitamin, or analog or derivative thereof, or the
drug, or analog or derivative thereof, or each is a vitamin or drug
moiety in conjunction with other portions of the polyvalent linker,
such as a drug or vitamin moiety including one or more spacer
linkers and/or other releasable linkers. Without being bound by
theory, in this embodiment, acid catalysis, such as in an endosome,
may initiate the cleavage via protonation of the urethane group. In
addition, acid-catalyzed elimination of the carbamate leads to the
release of CO.sub.2 and the nitrogen-containing moiety attached to
Z, and the formation of a benzyl cation, which may be trapped by
water, or any other Lewis base.
[0106] Other illustrative linkers include compounds of the
formulae:
##STR00025##
where X is NH, CH.sub.2, or O; R is hydrogen, or a substituent,
including a substituent capable of stabilizing a positive charge
inductively or by resonance on the aryl ring, such as alkoxy and
the like, including methoxy; and the symbol (*) indicates points of
attachment for additional spacer, heteroatom, or releasable linkers
forming the bivalent linker, or alternatively for attachment of the
drug, or analog or derivative thereof, or the vitamin, or analog or
derivative thereof.
[0107] Illustrative mechanisms for cleavage of such bivalent
linkers described herein include the following 1,4 and 1,6
fragmentation mechanisms followed by anchimerically assisted
cleavage of the acylated Z' via cyclization by the hydrazide
group:
##STR00026##
where X is an exogenous or endogenous nucleophile, glutathione, or
bioreducing agent, and the like, and either of Z or Z' is the
vitamin, or analog or derivative thereof, or the drug, or analog or
derivative thereof, or a vitamin or drug moiety in conjunction with
other portions of the polyvalent linker. It is to be understood
that although the above fragmentation mechanisms are depicted as
concerted mechanisms, any number of discrete steps may take place
to effect the ultimate fragmentation of the polyvalent linker to
the final products shown. For example, it is appreciated that the
bond cleavage may also occur by acid-catalyzed elimination of the
carbamate moiety, which may be anchimerically assisted by the
stabilization provided by either the aryl group of the beta sulfur
or disulfide illustrated in the above examples. In those variations
of this embodiment, the releasable linker is the carbamate moiety.
Alternatively, the fragmentation may be initiated by a nucleophilic
attack on the disulfide group, causing cleavage to form a thiolate.
The thiolate may intermolecularly displace a carbonic acid or
carbamic acid moiety and form the corresponding thiacyclopropane.
In the case of the benzyl-containing polyvalent linkers, following
an illustrative breaking of the disulfide bond, the resulting
phenyl thiolate may further fragment to release a carbonic acid or
carbamic acid moiety by forming a resonance stabilized
intermediate. In any of these cases, the releasable nature of the
illustrative polyvalent linkers described herein may be realized by
whatever mechanism may be relevant to the chemical, metabolic,
physiological, or biological conditions present. Without being
bound by theory, in this embodiment, acid catalysis, such as in an
endosome, may also initiate the cleavage via protonation of the
urethane group. In addition, acid-catalyzed elimination of the
carbamate leads to the release of CO.sub.2 and the
nitrogen-containing moiety attached to Z, and the formation of a
benzyl cation, which may be trapped by water, or any other Lewis
base, as is similarly described herein.
[0108] In one embodiment, the polyvalent linkers described herein
are compounds of the following formulae
##STR00027##
[0109] where n is an integer selected from 1 to about 4; R.sup.a
and R.sup.b are each independently selected from the group
consisting of hydrogen and alkyl, including lower alkyl such as
C.sub.1-C.sub.4 alkyl that are optionally branched; or R.sup.a and
R.sup.b are taken together with the attached carbon atom to form a
carbocyclic ring; R is an optionally substituted alkyl group, an
optionally substituted acyl group, or a suitably selected nitrogen
protecting group; and (*) indicates points of attachment for the
drug, vitamin, imaging agent, diagnostic agent, other polyvalent
linkers, or other parts of the conjugate.
[0110] In another embodiment, the polyvalent linkers described
herein include compounds of the following formulae
##STR00028##
where m is an integer selected from 1 to about 4; R is an
optionally substituted alkyl group, an optionally substituted acyl
group, or a suitably selected nitrogen protecting group; and (*)
indicates points of attachment for the drug, vitamin, imaging
agent, diagnostic agent, other polyvalent linkers, or other parts
of the conjugate.
[0111] In another embodiment, the polyvalent linkers described
herein include compounds of the following formulae
##STR00029##
where m is an integer selected from 1 to about 4; R is an
optionally substituted alkyl group, an optionally substituted acyl
group, or a suitably selected nitrogen protecting group; and (*)
indicates points of attachment for the drug, vitamin, imaging
agent, diagnostic agent, other polyvalent linkers, or other parts
of the conjugate.
[0112] Another illustrative mechanism involves an arrangement of
the releasable and spacer linkers in such a way that subsequent to
the cleavage of a bond in the bivalent linker, released functional
groups chemically assist the breakage or cleavage of additional
bonds, also termed anchimeric assisted cleavage or breakage. An
illustrative embodiment of such a bivalent linker or portion
thereof includes compounds having the formula:
##STR00030##
where X is an heteroatom, such as nitrogen, oxygen, or sulfur, n is
an integer selected from 0, 1, 2, and 3, R is hydrogen, or a
substituent, including a substituent capable of stabilizing a
positive charge inductively or by resonance on the aryl ring, such
as alkoxy, and the like, and either of Z or Z' is the vitamin, or
analog or derivative thereof, or the drug, or analog or derivative
thereof, or a vitamin or drug moiety in conjunction with other
portions of the bivalent linker. It is appreciated that other
substituents may be present on the aryl ring, the benzyl carbon,
the carbamate nitrogen, the alkanoic acid, or the methylene bridge,
including but not limited to hydroxy, alkyl, alkoxy, alkylthio,
halo, and the like. Assisted cleavage may include mechanisms
involving benzylium intermediates, benzyne intermediates, lactone
cyclization, oxonium intermediates, beta-elimination, and the like.
It is further appreciated that, in addition to fragmentation
subsequent to cleavage of the releasable linker, the initial
cleavage of the releasable linker may be facilitated by an
anchimerically assisted mechanism.
[0113] In this embodiment, the hydroxyalkanoic acid, which may
cyclize, facilitates cleavage of the methylene bridge, by for
example an oxonium ion, and facilitates bond cleavage or subsequent
fragmentation after bond cleavage of the releasable linker.
Alternatively, acid catalyzed oxonium ion-assisted cleavage of the
methylene bridge may begin a cascade of fragmentation of this
illustrative bivalent linker, or fragment thereof. Alternatively,
acid-catalyzed hydrolysis of the carbamate may facilitate the beta
elimination of the hydroxyalkanoic acid, which may cyclize, and
facilitate cleavage of methylene bridge, by for example an oxonium
ion. It is appreciated that other chemical mechanisms of bond
breakage or cleavage under the metabolic, physiological, or
cellular conditions described herein may initiate such a cascade of
fragmentation. It is appreciated that other chemical mechanisms of
bond breakage or cleavage under the metabolic, physiological, or
cellular conditions described herein may initiate such a cascade of
fragmentation.
[0114] In another embodiment, the releasable and spacer linkers may
be arranged in such a way that subsequent to the cleavage of a bond
in the polyvalent linker, released functional groups chemically
assist the breakage or cleavage of additional bonds, also termed
anchimeric assisted cleavage or breakage. An illustrative
embodiment of such a polyvalent linker or portion thereof includes
compounds having the formula:
##STR00031##
where X is an heteroatom, such as nitrogen, oxygen, or sulfur, n is
an integer selected from 0, 1, 2, and 3, R is hydrogen, or a
substituent, including a substituent capable of stabilizing a
positive charge inductively or by resonance on the aryl ring, such
as alkoxy, and the like, and the symbol (*) indicates points of
attachment for additional spacer, heteroatom, or releasable linkers
forming the polyvalent linker, or alternatively for attachment of
the drug, or analog or derivative thereof, or the vitamin, or
analog or derivative thereof. It is appreciated that other
substituents may be present on the aryl ring, the benzyl carbon,
the alkanoic acid, or the methylene bridge, including but not
limited to hydroxy, alkyl, alkoxy, alkylthio, halo, and the like.
Assisted cleavage may include mechanisms involving benzylium
intermediates, benzyne intermediates, lactone cyclization, oxonium
intermediates, beta-elimination, and the like. It is further
appreciated that, in addition to fragmentation subsequent to
cleavage of the releasable linker, the initial cleavage of the
releasable linker may be facilitated by an anchimerically assisted
mechanism.
[0115] Another illustrative embodiment of the linkers described
herein, include releasable linkers that cleave under the conditions
described herein by a chemical mechanism involving beta
elimination. In one aspect, such releasable linkers include
beta-thio, beta-hydroxy, and beta-amino substituted carboxylic
acids and derivatives thereof, such as esters, amides, carbonates,
carbamates, and ureas. In another aspect, such releasable linkers
include 2- and 4-thioarylesters, carbamates, and carbonates.
[0116] In another illustrative embodiment, the linker includes one
or more amino acids. In one variation, the linker includes a single
amino acid. In another variation, the linker includes a peptide
having from 2 to about 50, 2 to about 30, or 2 to about 20 amino
acids. In another variation, the linker includes a peptide having
from about 4 to about 8 amino acids. Such amino acids are
illustratively selected from the naturally occurring amino acids,
or stereoisomers thereof. The amino acid may also be any other
amino acid, such as any amino acid having the general formula:
--N(R)--(CR'R'').sub.q--C(O)--
where R is hydrogen, alkyl, acyl, or a suitable nitrogen protecting
group, R' and R'' are hydrogen or a substituent, each of which is
independently selected in each occurrence, and q is an integer such
as 1, 2, 3, 4, or 5. Illustratively, R' and/or R'' independently
correspond to, but are not limited to, hydrogen or the side chains
present on naturally occurring amino acids, such as methyl, benzyl,
hydroxymethyl, thiomethyl, carboxyl, carboxylmethyl,
guanidinopropyl, and the like, and derivatives and protected
derivatives thereof. The above described formula includes all
stereoisomeric variations. For example, the amino acid may be
selected from asparagine, aspartic acid, cysteine, glutamic acid,
lysine, glutamine, arginine, serine, ornitine, threonine, and the
like. In one variation, the releasable linker includes at least 2
amino acids selected from asparagine, aspartic acid, cysteine,
glutamic acid, lysine, glutamine, arginine, serine, ornitine, and
threonine. In another variation, the releasable linker includes
between 2 and about 5 amino acids selected from asparagine,
aspartic acid, cysteine, glutamic acid, lysine, glutamine,
arginine, serine, ornitine, and threonine. In another variation,
the releasable linker includes a tripeptide, tetrapeptide,
pentapeptide, or hexapeptide consisting of amino acids selected
from aspartic acid, cysteine, glutamic acid, lysine, arginine, and
ornitine, and combinations thereof.
[0117] In another illustrative aspect of the vitamin receptor
binding drug delivery conjugate intermediate described herein, the
drug, or an analog or a derivative thereof, includes an alkylthiol
nucleophile.
[0118] In another embodiment, the spacer linker can be
1-alkylenesuccinimid-3-yl, optionally substituted with a
substituent X.sup.1, as defined below, and the releasable linkers
can be methylene, 1-alkoxyalkylene, 1-alkoxycycloalkylene,
1-alkoxyalkylenecarbonyl, 1-alkoxycycloalkylenecarbonyl, wherein
each of the releasable linkers is optionally substituted with a
substituent X.sup.2, as defined below, and wherein the spacer
linker and the releasable linker are each bonded to the spacer
linker to form a succinimid-1-ylalkyl acetal or ketal.
[0119] The spacer linkers can be carbonyl, thionocarbonyl,
alkylene, cycloalkylene, alkylenecycloalkyl, alkylenecarbonyl,
cycloalkylenecarbonyl, carbonylalkylcarbonyl,
1-alkylenesuccinimid-3-yl, 1-(carbonylalkyl)succinimid-3-yl,
alkylenesulfoxyl, sulfonylalkyl, alkylenesulfonylalkyl,
alkylenesulfonylalkyl, carbonyltetrahydro-2H-pyranyl,
carbonyltetrahydrofuranyl,
1-(carbonyltetrahydro-2H-pyranyl)succinimid-3-yl, and
1-(carbonyltetrahydrofuranyl)succinimid-3-yl, wherein each of the
spacer linkers is optionally substituted with a substituent
X.sup.1, as defined below. In this embodiment, the spacer linker
may include an additional nitrogen, and the spacer linkers can be
alkylenecarbonyl, cycloalkylenecarbonyl, carbonylalkylcarbonyl,
1-(carbonylalkyl)succinimid-3-yl, wherein each of the spacer
linkers is optionally substituted with a substituent X.sup.1, as
defined below, and the spacer linker is bonded to the nitrogen to
form an amide. Alternatively, the spacer linker may include an
additional sulfur, and the spacer linkers can be alkylene and
cycloalkylene, wherein each of the spacer linkers is optionally
substituted with carboxy, and the spacer linker is bonded to the
sulfur to form a thiol. In another embodiment, the spacer linker
can include sulfur, and the spacer linkers can be
1-alkylenesuccinimid-3-yl and 1-(carbonylalkyl)succinimid-3-yl, and
the spacer linker is bonded to the sulfur to form a
succinimid-3-ylthiol.
[0120] In an alternative to the above-described embodiments, the
spacer linker can include nitrogen, and the releasable linker can
be a divalent radical comprising alkyleneaziridin-1-yl,
carbonylalkylaziridin-1-yl, sulfoxylalkylaziridin-1-yl, or
sulfonylalkylaziridin-1-yl, wherein each of the releasable linkers
is optionally substituted with a substituent X.sup.2, as defined
below. In this alternative embodiment, the spacer linkers can be
carbonyl, thionocarbonyl, alkylenecarbonyl, cycloalkylenecarbonyl,
carbonylalkylcarbonyl, 1-(carbonylalkyl)succinimid-3-yl, wherein
each of the spacer linkers is optionally substituted with a
substituent X.sup.1, as defined below, and wherein the spacer
linker is bonded to the releasable linker to form an aziridine
amide.
[0121] The substituents X.sup.1 can be alkyl, alkoxy, alkoxyalkyl,
hydroxy, hydroxyalkyl, amino, aminoalkyl, alkylaminoalkyl,
dialkylaminoalkyl, halo, haloalkyl, sulfhydrylalkyl,
alkylthioalkyl, aryl, substituted aryl, arylalkyl, substituted
arylalkyl, heteroaryl, substituted heteroaryl, carboxy,
carboxyalkyl, alkyl carboxylate, alkyl alkanoate, guanidinoalkyl,
R.sup.4-carbonyl, R.sup.5-carbonylalkyl, R.sup.6-acylamino, and
R.sup.7-acylaminoalkyl, wherein R.sup.4 and R.sup.5 are each
independently selected from amino acids, amino acid derivatives,
and peptides, and wherein R.sup.6 and R.sup.7 are each
independently selected from amino acids, amino acid derivatives,
and peptides. In this embodiment the spacer linker can include
nitrogen, and the substituent X.sup.1 and the spacer linker to
which they are bound to form an heterocycle.
[0122] In one aspect of the various vitamin receptor binding drug
delivery conjugates described herein, the bivalent linker comprises
an a spacer linker and a releasable linker taken together to form
3-thiosuccinimid-1-ylalkyloxymethyloxy, where the methyl is
optionally substituted with alkyl or substituted aryl.
[0123] In another aspect, the bivalent linker comprises a spacer
linker and a releasable linker taken together to form
3-thiosuccinimid-1-ylalkylcarbonyl, where the carbonyl forms an
acylaziridine with the drug, or analog or derivative thereof.
[0124] In another aspect, the bivalent linker comprises an a spacer
linker and a releasable linker taken together to form
1-alkoxycycloalkylenoxy.
[0125] In another aspect, the bivalent linker comprises a spacer
linker and a releasable linker taken together to form
alkyleneaminocarbonyl(dicarboxylarylene)carboxylate.
[0126] In another aspect, the bivalent linker comprises a
releasable linker, a spacer linker, and a releasable linker taken
together to form 2- or 3-dithioalkylcarbonylhydrazide, where the
hydrazide forms an hydrazone with the drug, or analog or derivative
thereof.
[0127] In another aspect, the bivalent linker comprises a spacer
linker and a releasable linker taken together to form
3-thiosuccinimid-1-ylalkylcarbonylhydrazide, where the hydrazide
forms an hydrazone with the drug, or analog or derivative
thereof.
[0128] In another aspect, the bivalent linker comprises a spacer
linker and a releasable linker taken together to form 2- or
3-thioalkylsulfonylalkyl(disubstituted silyl)oxy, where the
disubstituted silyl is substituted with alkyl or optionally
substituted aryl.
[0129] In another aspect, the bivalent linker comprises a plurality
of spacer linkers selected from the group consisting of the
naturally occurring amino acids and stereoisomers thereof.
[0130] In another aspect, the bivalent linker comprises a
releasable linker, a spacer linker, and a releasable linker taken
together to form 3-dithioalkyloxycarbonyl, where the carbonyl forms
a carbonate with the drug, or analog or derivative thereof.
[0131] In another aspect, the bivalent linker comprises a
releasable linker, a spacer linker, and a releasable linker taken
together to form 3-dithioarylalkyloxycarbonyl, where the carbonyl
forms a carbonate with the drug, or analog or derivative thereof,
and the aryl is optionally substituted.
[0132] In another aspect, the bivalent linker comprises a spacer
linker and a releasable linker taken together to form
3-thiosuccinimid-1-ylalkyloxyalkyloxyalkylidene, where the
alkylidene forms an hydrazone with the drug, or analog or
derivative thereof, each alkyl is independently selected, and the
oxyalkyloxy is optionally substituted with alkyl or optionally
substituted aryl.
[0133] In another aspect, the bivalent linker comprises a
releasable linker, a spacer linker, and a releasable linker taken
together to form 2- or 3-dithioalkyloxycarbonylhydrazide.
[0134] In another aspect, the bivalent linker comprises a
releasable linker, a spacer linker, and a releasable linker taken
together to form 2- or 3-dithioalkylamino, where the amino forms a
vinylogous amide with the drug, or analog or derivative
thereof.
[0135] In another aspect, the bivalent linker comprises a
releasable linker, a spacer linker, and a releasable linker taken
together to form 2- or 3-dithioalkylamino, where the amino forms a
vinylogous amide with the drug, or analog or derivative thereof,
and the alkyl is ethyl.
[0136] In another aspect, the bivalent linker comprises a
releasable linker, a spacer linker, and a releasable linker taken
together to form 2- or 3-dithioalkylaminocarbonyl, where the
carbonyl forms a carbamate with the drug, or analog or derivative
thereof.
[0137] In another aspect, the bivalent linker comprises a
releasable linker, a spacer linker, and a releasable linker taken
together to form 2- or 3-dithioalkylaminocarbonyl, where the
carbonyl forms a carbamate with the drug, or analog or derivative
thereof, and the alkyl is ethyl.
[0138] In another aspect, the bivalent linker comprises a
releasable linker, a spacer linker, and a releasable linker taken
together to form 2- or 3-dithioarylalkyloxycarbonyl, where the
carbonyl forms a carbamate or a carbamoylaziridine with the drug,
or analog or derivative thereof.
[0139] In another embodiment, the polyvalent linker includes spacer
linkers and releasable linkers connected to form a polyvalent
3-thiosuccinimid-1-ylalkyloxymethyloxy group, illustrated by the
following formula
##STR00032##
where n is an integer from 1 to 6, the alkyl group is optionally
substituted, and the methyl is optionally substituted with an
additional alkyl or optionally substituted aryl group, each of
which is represented by an independently selected group R. The (*)
symbols indicate points of attachment of the polyvalent linker
fragment to other parts of the conjugates described herein.
[0140] In another embodiment, the polyvalent linker includes spacer
linkers and releasable linkers connected to form a polyvalent
3-thiosuccinimid-1-ylalkylcarbonyl group, illustrated by the
following formula
##STR00033##
where n is an integer from 1 to 6, and the alkyl group is
optionally substituted. The (*) symbols indicate points of
attachment of the polyvalent linker fragment to other parts of the
conjugates described herein. In another embodiment, the polyvalent
linker includes spacer linkers and releasable linkers connected to
form a polyvalent 3-thioalkylsulfonylalkyl(disubstituted silyl)oxy
group, where the disubstituted silyl is substituted with alkyl
and/or optionally substituted aryl groups.
[0141] In another embodiment, the polyvalent linker includes spacer
linkers and releasable linkers connected to form a polyvalent
dithioalkylcarbonylhydrazide group, or a polyvalent
3-thiosuccinimid-1-ylalkylcarbonylhydrazide, illustrated by the
following formulae
##STR00034##
where n is an integer from 1 to 6, the alkyl group is optionally
substituted, and the hydrazide forms an hydrazone with (B), (D), or
another part of the polyvalent linker (L). The (*) symbols indicate
points of attachment of the polyvalent linker fragment to other
parts of the conjugates described herein.
[0142] In another embodiment, the polyvalent linker includes spacer
linkers and releasable linkers connected to form a polyvalent
3-thiosuccinimid-1-ylalkyloxyalkyloxyalkylidene group, illustrated
by the following formula
##STR00035##
where each n is an independently selected integer from 1 to 6, each
alkyl group independently selected and is optionally substituted,
such as with alkyl or optionally substituted aryl, and where the
alkylidene forms an hydrazone with (B), (D), or another part of the
polyvalent linker (L). The (*) symbols indicate points of
attachment of the polyvalent linker fragment to other parts of the
conjugates described herein.
[0143] Additional illustrative spacer linkers include
alkylene-amino-alkylenecarbonyl,
alkylene-thio-carbonylalkylsuccinimid-3-yl, and the like, as
further illustrated by the following formulae:
##STR00036##
where the integers x and y are 1, 2, 3, 4, or 5:
[0144] The term cycloalkylene as used herein refers to a bivalent
chain of carbon atoms, a portion of which forms a ring, such as
cycloprop-1,1-diyl, cycloprop-1,2-diyl, cyclohex-1,4-diyl,
3-ethylcyclopent-1,2-diyl, 1-methylenecyclohex-4-yl, and the
like.
[0145] The term heterocycle as used herein refers to a monovalent
chain of carbon and heteroatoms, wherein the heteroatoms are
selected from nitrogen, oxygen, and sulfur, a portion of which,
including at least one heteroatom, form a ring, such as aziridine,
pyrrolidine, oxazolidine, 3-methoxypyrrolidine, 3-methylpiperazine,
and the like.
[0146] The term aryl as used herein refers to an aromatic mono or
polycyclic ring of carbon atoms, such as phenyl, naphthyl, and the
like. In addition, aryl may also include heteroaryl.
[0147] The term heteroaryl as used herein refers to an aromatic
mono or polycyclic ring of carbon atoms and at least one heteroatom
selected from nitrogen, oxygen, and sulfur, such as pyridinyl,
pyrimidinyl, indolyl, benzoxazolyl, and the like.
[0148] The term optionally substituted as used herein refers to the
replacement of one or more hydrogen atoms, generally on carbon,
with a corresponding number of substituents, such as halo, hydroxy,
amino, alkyl or dialkylamino, alkoxy, alkylsulfonyl, cyano, nitro,
and the like. In addition, two hydrogens on the same carbon, on
adjacent carbons, or nearby carbons may be replaced with a bivalent
substituent to form the corresponding cyclic structure.
[0149] The term iminoalkylidenyl as used herein refers to a
divalent radical containing alkylene as defined herein and a
nitrogen atom, where the terminal carbon of the alkylene is
double-bonded to the nitrogen atom, such as the formulae
--(CH).dbd.N--, --(CH.sub.2).sub.2(CH).dbd.N--,
--CH.sub.2C(Me)=N--, and the like.
[0150] The term amino acid as used herein refers generally to
aminoalkylcarboxylate, where the alkyl radical is optionally
substituted, such as with alkyl, hydroxy alkyl, sulfhydrylalkyl,
aminoalkyl, carboxyalkyl, and the like, including groups
corresponding to the naturally occurring amino acids, such as
serine, cysteine, methionine, aspartic acid, glutamic acid, and the
like. It is to be understood that such amino acids may be of a
single stereochemistry or a particular mixture of stereochemisties,
including racemic mixtures. In addition, amino acid refers to beta,
gamma, and longer amino acids, such as amino acids of the
formula:
--N(R)--(CR'R'').sub.q--C(O)--
where R is hydrogen, alkyl, acyl, or a suitable nitrogen protecting
group, R' and R'' are hydrogen or a substituent, each of which is
independently selected in each occurrence, and q is an integer such
as 1, 2, 3, 4, or 5. Illustratively, R' and/or R'' independently
correspond to, but are not limited to, hydrogen or the side chains
present on naturally occurring amino acids, such as methyl, benzyl,
hydroxymethyl, thiomethyl, carboxyl, carboxylmethyl,
guanidinopropyl, and the like, and derivatives and protected
derivatives thereof. The above described formula includes all
stereoisomeric variations. For example, the amino acid may be
selected from asparagine, aspartic acid, cysteine, glutamic acid,
lysine, glutamine, arginine, serine, ornitine, threonine, and the
like. In another illustrative aspect of the vitamin receptor
binding drug delivery conjugate intermediate described herein, the
drug, or an analog or a derivative thereof, includes an alkylthiol
nucleophile.
[0151] It is to be understood that the above-described terms can be
combined to generate chemically-relevant groups, such as
alkoxyalkyl referring to methyloxymethyl, ethyloxyethyl, and the
like, haloalkoxyalkyl referring to trifluoromethyloxyethyl,
1,2-difluoro-2-chloroeth-1-yloxypropyl, and the like, arylalkyl
referring to benzyl, phenethyl, methylbenzyl, and the like, and
others.
[0152] The term amino acid derivative as used herein refers
generally to an optionally substituted aminoalkylcarboxylate, where
the amino group and/or the carboxylate group are each optionally
substituted, such as with alkyl, carboxylalkyl, alkylamino, and the
like, or optionally protected. In addition, the optionally
substituted intervening divalent alkyl fragment may include
additional groups, such as protecting groups, and the like.
[0153] The term peptide as used herein refers generally to a series
of amino acids and/or amino acid analogs and derivatives covalently
linked one to the other by amide bonds.
[0154] Additional linkers are described in U.S. patent application
publication 2005/0002942, the disclosure of which is incorporated
herein by reference, and in Tables 1 and 2 below, where the (*)
atom is the point of attachment of additional spacer or releaseable
linkers, the drug, and/or the binding ligand.
TABLE-US-00003 TABLE 1 Illustrative spacer linkers. ##STR00037##
##STR00038## ##STR00039## ##STR00040## ##STR00041## ##STR00042##
##STR00043## ##STR00044## ##STR00045## ##STR00046## ##STR00047##
##STR00048## ##STR00049## ##STR00050## ##STR00051## ##STR00052##
##STR00053## ##STR00054## ##STR00055## ##STR00056## ##STR00057##
##STR00058## ##STR00059## ##STR00060## ##STR00061## ##STR00062##
##STR00063## ##STR00064## ##STR00065## ##STR00066## ##STR00067##
##STR00068## ##STR00069## ##STR00070## ##STR00071## ##STR00072##
##STR00073## ##STR00074## ##STR00075## ##STR00076## ##STR00077##
##STR00078## ##STR00079## ##STR00080## ##STR00081## ##STR00082##
##STR00083## ##STR00084##
TABLE-US-00004 TABLE 2 Illustrative releasable linkers.
##STR00085## ##STR00086## ##STR00087## ##STR00088## ##STR00089##
##STR00090## ##STR00091## ##STR00092## ##STR00093## ##STR00094##
##STR00095## ##STR00096## ##STR00097## ##STR00098## ##STR00099##
##STR00100## ##STR00101## ##STR00102## ##STR00103## ##STR00104##
##STR00105## ##STR00106## ##STR00107## ##STR00108## ##STR00109##
##STR00110## ##STR00111## ##STR00112## ##STR00113## ##STR00114##
##STR00115## ##STR00116## ##STR00117## ##STR00118##
##STR00119##
[0155] In another illustrative embodiment, bivalent linkers (L)
that include spacer linkers that substantially increase the water
solubility, biological transport, preferential renal clearance,
uptake, absorption, biodistribution, and/or bioavailability of the
conjugate are described herein. Illustrative spacer linkers that
include hydrophilic groups are described, such as compounds of the
formula
##STR00120##
where m is an integer independently selected in each instance from
1 to about 8; p is an integer selected 1 to about 10; and n is an
integer independently selected in each instance from 1 to about 3.
In one aspect, m is independently in each instance 1 to about 3. In
another aspect, n is 1 in each instance. In another aspect, p is
independently in each instance about 4 to about 6. Illustratively,
the corresponding polypropylene polyethers corresponding to the
foregoing are contemplated herein and may be included in the
conjugates as hydrophilic spacer linkers. In addition, it is
appreciated that mixed polyethylene and polypropylene polyethers
may be included in the conjugates as hydrophilic spacer linkers.
Further, cyclic variations of the foregoing polyether compounds,
such as those that include tetrahydrofuranyl, 1,3-dioxanes,
1,4-dioxanes, and the like are contemplated herein.
[0156] In another illustrative embodiment, the hydrophilic spacer
linkers described herein include a plurality of hydroxyl functional
groups, such as linkers that incorporate monosaccharides,
oligosaccharides, polysaccharides, and the like. It is to be
understood that the polyhydroxyl containing spacer linkers
comprises a plurality of --(CROH)-- groups, where R is hydrogen or
alkyl.
[0157] In another embodiment, the spacer linkers include one or
more of the following fragments:
##STR00121## ##STR00122##
wherein R is H, alkyl, cycloalkyl, or arylalkyl; m is an integer
from 1 to about 3; n is an integer from 1 to about 5, p is an
integer from 1 to about 5, and r is an integer selected from 1 to
about 3. In one aspect, the integer n is 3 or 4. In another aspect,
the integer p is 3 or 4. In another aspect, the integer r is 1.
[0158] In another embodiment, the spacer linkers include one or
more of the following fragments:
##STR00123## ##STR00124## ##STR00125##
wherein R is H, alkyl, cycloalkyl, or arylalkyl; m is an
independently selected integer from 1 to about 3; n is an integer
from 1 to about 6, p is an integer from 1 to about 5, and r is an
integer selected from 1 to about 3. In one variation, the integer n
is 3 or 4. In another variation, the integer p is 3 or 4. In
another variation, the integer r is 1.
[0159] In another embodiment, the spacer linker includes one or
more of the following cyclic polyhydroxyl groups:
##STR00126## ##STR00127##
wherein n is an integer from 2 to about 5, p is an integer from 1
to about 5, and r is an integer from 1 to about 4. In one aspect,
the integer n is 3 or 4. In another aspect, the integer p is 3 or
4. In another aspect, the integer r is 2 or 3. It is understood
that all stereochemical forms of such sections of the linkers are
contemplated herein. For example, in the above formula, the section
may be derived from ribose, xylose, glucose, mannose, galactose, or
other sugar and retain the stereochemical arrangements of pendant
hydroxyl and alkyl groups present on those molecules. In addition,
it is to be understood that in the foregoing formulae, various
deoxy compounds are also contemplated. Illustratively, compounds of
the following formulae are contemplated:
##STR00128##
wherein n is equal to or less than r, such as when r is 2 or 3, n
is 1 or 2, or 1, 2, or 3, respectively.
[0160] In another embodiment, the spacer linker includes a
polyhydroxyl compound of the following formula:
##STR00129##
wherein n and r are each an integer selected from 1 to about 3. In
one aspect, the spacer linker includes one or more polyhydroxyl
compounds of the following formulae:
##STR00130##
It is understood that all stereochemical forms of such sections of
the linkers are contemplated herein. For example, in the above
formula, the section may be derived from ribose, xylose, glucose,
mannose, galactose, or other sugar and retain the stereochemical
arrangements of pendant hydroxyl and alkyl groups present on those
molecules.
[0161] In another configuration, the hydrophilic linkers L
described herein include polyhydroxyl groups that are spaced away
from the backbone of the linker. Illustratively, such linkers
include fragments of the following formulae:
##STR00131##
wherein n, m, and r are integers and are each independently
selected in each instance from 1 to about 5. In one illustrative
aspect, m is independently 2 or 3 in each instance. In another
aspect, r is 1 in each instance. In another aspect, n is 1 in each
instance. In one variation, the group connecting the polyhydroxyl
group to the backbone of the linker is a different heteroaryl
group, including but not limited to, pyrrole, pyrazole,
1,2,4-triazole, furan, oxazole, isoxazole, thienyl, thiazole,
isothiazole, oxadiazole, and the like. Similarly, divalent
6-membered ring heteroaryl groups are contemplated. Other
variations of the foregoing illustrative hydrophilic spacer linkers
include oxyalkylene groups, such as the following formulae:
##STR00132##
wherein n and r are integers and are each independently selected in
each instance from 1 to about 5; and p is an integer selected from
1 to about 4.
[0162] In another embodiment, the hydrophilic linkers L described
herein include polyhydroxyl groups that are spaced away from the
backbone of the linker. Illustratively, such linkers include
fragments of the following formulae:
##STR00133##
wherein n is an integer selected from 1 to about 3, and m is an
integer selected from 1 to about 22. In one illustrative aspect, n
is 1 or 2. In another illustrative aspect, m is selected from about
6 to about 10, illustratively 8. In one variation, the group
connecting the polyhydroxyl group to the backbone of the linker is
a different functional group, including but not limited to, esters,
ureas, carbamates, acylhydrazones, and the like. Similarly, cyclic
variations are contemplated. Other variations of the foregoing
illustrative hydrophilic spacer linkers include oxyalkylene groups,
such as the following formulae:
##STR00134##
wherein n and r are integers and are each independently selected in
each instance from 1 to about 5; and p is an integer selected from
1 to about 4.
[0163] In another embodiment, the hydrophilic spacer linker is a
combination of backbone and branching side motifs such as is
illustrated by the following formulae
##STR00135##
wherein n is an integer independently selected in each instance
from 0 to about 3. The above formula are intended to represent 4,
5, 6, and even larger membered cyclic sugars. In addition, it is to
be understood that the above formula may be modified to represent
deoxy sugars, where one or more of the hydroxy groups present on
the formulae are replaced by hydrogen, alkyl, or amino. In
addition, it is to be understood that the corresponding carbonyl
compounds are contemplated by the above formulae, where one or more
of the hydroxyl groups is oxidized to the corresponding carbonyl.
In addition, in this illustrative embodiment, the pyranose includes
both carboxyl and amino functional groups and (a) can be inserted
into the backbone and (b) can provide synthetic handles for
branching side chains in variations of this embodiment. Any of the
pendant hydroxyl groups may be used to attach other chemical
fragments, including additional sugars to prepare the corresponding
oligosaccharides. Other variations of this embodiment are also
contemplated, including inserting the pyranose or other sugar into
the backbone at a single carbon, i.e. a spiro arrangement, at a
geminal pair of carbons, and like arrangements. For example, one or
two ends of the linker, or the agent A, or the binding ligand B may
be connected to the sugar to be inserted into the backbone in a
1,1; 1,2; 1,3; 1,4; 2,3, or other arrangement.
[0164] In another embodiment, the hydrophilic spacer linkers
described herein include are formed primarily from carbon,
hydrogen, and nitrogen, and have a carbon/nitrogen ratio of about
3:1 or less, or of about 2:1 or less. In one aspect, the
hydrophilic linkers described herein include a plurality of amino
functional groups.
[0165] In another embodiment, the spacer linkers include one or
more amino groups of the following formulae:
##STR00136##
where n is an integer independently selected in each instance from
1 to about 3. In one aspect, the integer n is independently 1 or 2
in each instance. In another aspect, the integer n is 1 in each
instance.
[0166] In another embodiment, the hydrophilic spacer linker is a
sulfuric acid ester, such as an alkyl ester of sulfuric acid.
Illustratively, the spacer linker is of the following formula:
##STR00137##
where n is an integer independently selected in each instance from
1 to about 3. Illustratively, n is independently 1 or 2 in each
instance.
[0167] It is understood, that in such polyhydroxyl, polyamino,
carboxylic acid, sulfuric acid, and like linkers that include free
hydrogens bound to heteroatoms, one or more of those free hydrogen
atoms may be protected with the appropriate hydroxyl, amino, or
acid protecting group, respectively, or alternatively may be
blocked as the corresponding pro-drugs, the latter of which are
selected for the particular use, such as pro-drugs that release the
parent drug under general or specific physiological conditions.
[0168] In each of the foregoing illustrative examples of linkers L,
there are also included in some cases additional spacer linkers
L.sub.S, and/or additional releasable linkers L.sub.R. Those spacer
linker and releasable linkers also may include asymmetric carbon
atoms. It is to be further understood that the stereochemical
configurations shown herein are merely illustrative, and other
stereochemical configurations are contemplated. For example in one
variation, the corresponding unnatural amino acid configurations
may be included in the conjugated described herein as follows:
##STR00138##
wherein n is an integer from 2 to about 5, p is an integer from 1
to about 5, and r is an integer from 1 to about 4, as described
above.
[0169] Additional linkers that include hydrophilic groups useful in
preparing the conjugates described herein are described in
co-pending U.S. provisional application Ser. Nos. 60/946,092 and
61/036,186, the disclosures of which are incorporated herein by
reference.
[0170] In another embodiment, multi-drug conjugates are described
herein. Several illustrative configurations of such multi-drug
conjugates are contemplated herein, and include the compounds and
compositions described in PCT international publication No. WO
2007/022494, the disclosure of which is incorporated herein by
reference. Illustratively, the polyvalent linkers may connect the
receptor binding ligand B to the two or more agents A, providing
that one agent is a tubulysin. Such polyvalent conjugates may be in
a variety of structural configurations, including but not limited
to the following illustrative general formulae:
##STR00139##
where B is the receptor binding ligand, each of (L.sup.1),
(L.sup.2), and (L.sup.3) is a polyvalent linker as described herein
comprising a hydrophilic spacer linker, and optionally including
one or more releasable linkers and/or additional spacer linkers,
and each of (A.sup.1), (A.sup.2), and (A.sup.3) is an agent A, or
an analog or derivative thereof. Other variations, including
additional agents A, or analogs or derivatives thereof, additional
linkers, and additional configurations of the arrangement of each
of (B), (L), and (A), are also contemplated herein.
[0171] In one variation, more than one receptor binding ligand B is
included in the delivery conjugates described herein, including but
not limited to the following illustrative general formulae:
##STR00140##
where each B is a receptor binding ligand, each of (L.sup.1),
(L.sup.2), and (L.sup.3) is a polyvalent linker as described herein
comprising a hydrophilic spacer linker, and optionally including
one or more releasable linkers and/or additional spacer linkers,
and each of (A.sup.1), (A.sup.2), and (A.sup.3) is an agent A, or
an analog or derivative thereof. Other variations, including
additional agents A, or analogs or derivatives thereof, additional
linkers, and additional configurations of the arrangement of each
of (B), (L), and (A), are also contemplated herein. In one
variation, the receptor binding ligands B are ligands for the same
receptor, and in another variation, the receptor binding ligands B
are ligands for different receptors.
[0172] In another illustrative embodiment, the additional agents
are selected based on activity against one or more populations of
pathogenic cells with a particular mechanism of action.
Illustrative mechanisms of action include alkylating agents, other
microtubule inhibitors, including those that stabilize and/or
destabilize microtubule formation, including beta-tubulin agents,
cyclin dependent kinase (CDK) inhibitors, topoisomerase inhibitors,
protein synthesis inhibitors, protein kinase inhibitors, including
Ras, Raf, PKC, PI3K, and like inhibitors, transcription inhibitor,
antifolates, heat shock protein blockers, and the like.
[0173] Illustrative alkylating agents include, but are not limited
to, mitomycins CBI, and the like. Illustrative cyclin dependent
kinase (CDK) inhibitors include, but are not limited to, CYC202,
seliciclib, R-roscovitine, AGM-1470, and the like. Illustrative
topoisomerase inhibitors include, but are not limited to,
doxorubicin, other anthracyclines, and the like. Illustrative
protein synthesis inhibitors include, but are not limited to,
bruceantin, and the like. Illustrative protein kinase inhibitors,
including Ras, Raf, PKC, PI3K, and like inhibitors, include but are
not limited to L-779,450, R115777, and the like. Illustrative
transcription inhibitors include, but are not limited to,
.alpha.-amanatin, actinomycin, and the like. Illustrative
antifolates include, but are not limited to, methotrexate, and the
like. Illustrative heat shock protein blockers include, but are not
limited to, geldanamycin, and the like.
[0174] Illustrative microtubule inhibitors, including those that
stabilize and/or destabilize microtubule formation, including
.beta.-tubulin agents, microtubule poisons, and the like.
Illustrative microtubule poisons that bind to selected receptors
include, but are not limited to, inhibitors biding to the vinca
binding site such as arenastatin, dolastatin, halichondrin B,
maytansine, phomopsin A, rhizoxin, ustiloxin, vinblastine,
vincristine, and the like, stabilizers binding to the taxol binding
site such as discodermalide, epothilone, taxol, paclitaxol, and the
like, inhibitors binding to the colchicine binding site such as,
colchicine, combretastatin, curacin A, podophyllotoxin,
steganacine, and the like, and others binding to undefined sites
such as cryptophycin, tubulysins, and the like.
[0175] In one embodiment, one of the agents is a tubulysin, or an
analog or derivative thereof, and at least one other of the agents
is a DNA alkylation agent. In one variation, at least one other of
the agents is an alkylating agent. In another variation, at least
one other of the drugs is a P-glycoprotein (PGP) inhibitor. In
another variation, at least one of the other agents is a vinca
alkaloid, or an analog or derivative thereof. Vinca alklaloids
described herein include all members of the vinca
indole-dihydroindole family of alkaloids, such as but not limited
to vindesine, vinblastine, vincristine, catharanthine, vindoline,
leurosine, vinorelbine, imidocarb, sibutramine, toltrazuril,
vinblastinoic acid, and the like, and analogs and derivatives
thereof.
[0176] The binding site for the binding ligand (B), such as a
vitamin, can include receptors for any binding ligand (B), or a
derivative or analog thereof, capable of specifically binding to a
receptor wherein the receptor or other protein is uniquely
expressed, overexpressed, or preferentially expressed by a
population of pathogenic cells. A surface-presented protein
uniquely expressed, overexpressed, or preferentially expressed by
the pathogenic cells is typically a receptor that is either not
present or present at lower concentrations on non-pathogenic cells
providing a means for selective elimination of the pathogenic
cells. The binding ligand drug delivery conjugates may be capable
of high affinity binding to receptors on cancer cells or other
types of pathogenic cells. The high affinity binding can be
inherent to the binding ligand or the binding affinity can be
enhanced by the use of a chemically modified ligand (e.g., an
analog or a derivative of a vitamin).
[0177] The binding ligand drug delivery conjugates described herein
can be formed from, for example, a wide variety of vitamins or
receptor-binding vitamin analogs/derivatives, linkers, and drugs.
The binding ligand drug delivery conjugates described herein are
capable of selectively targeting a population of pathogenic cells
in the host animal due to preferential expression of a receptor for
the binding ligand, such as a vitamin, accessible for ligand
binding, on the pathogenic cells. Illustrative vitamin moieties
that can be used as the binding ligand (B) include carnitine,
inositol, lipoic acid, pyridoxal, ascorbic acid, niacin,
pantothenic acid, folic acid, riboflavin, thiamine, biotin, vitamin
B.sub.12, and the lipid soluble vitamins A, D, E and K. These
vitamins, and their receptor-binding analogs and derivatives,
constitute an illustrative targeting entity that can be coupled
with the drug by a bivalent linker (L) to form a binding ligand (B)
drug delivery conjugate as described herein. The term vitamin is
understood to include vitamin analogs and/or derivatives, unless
otherwise indicated. Illustratively, pteroic acid which is a
derivative of folate, biotin analogs such as biocytin, biotin
sulfoxide, oxybiotin and other biotin receptor-binding compounds,
and the like, are considered to be vitamins, vitamin analogs, and
vitamin derivatives. It should be appreciated that vitamin analogs
or derivatives as described herein refer to vitamins that
incorporates an heteroatom through which the vitamin analog or
derivative is covalently bound to the bivalent linker (L).
[0178] Illustrative vitamin moieties include folic acid, biotin,
riboflavin, thiamine, vitamin B.sub.12, and receptor-binding
analogs and derivatives of these vitamin molecules, and other
related vitamin receptor binding molecules.
[0179] In one embodiment, the targeting ligand B is a folate, an
analog of folate, or a derivative of folate. It is to be understood
as used herein, that the term folate is used both individually and
collectively to refer to folic acid itself, and/or to such analogs
and derivatives of folic acid that are capable of binding to folate
receptors.
[0180] Illustrative embodiments of folate analogs and/or
derivatives include folinic acid, pteropolyglutamic acid, and
folate receptor-binding pteridines such as tetrahydropterins,
dihydrofolates, tetrahydrofolates, and their deaza and dideaza
analogs. The terms "deaza" and "dideaza" analogs refer to the
art-recognized analogs having a carbon atom substituted for one or
two nitrogen atoms in the naturally occurring folic acid structure,
or analog or derivative thereof. For example, the deaza analogs
include the 1-deaza, 3-deaza, 5-deaza, 8-deaza, and 10-deaza
analogs of folate. The dideaza analogs include, for example,
1,5-dideaza, 5,10-dideaza, 8,10-dideaza, and 5,8-dideaza analogs of
folate. Other folates useful as complex forming ligands include the
folate receptor-binding analogs aminopterin, amethopterin
(methotrexate), N.sup.10-methylfolate, 2-deamino-hydroxyfolate,
deaza analogs such as I-deazamethopterin or 3-deazamethopterin, and
3',5'-dichloro-4-amino-4-deoxy-N.sup.10-methylpteroylglutamic acid
(dichloromethotrexate). The foregoing folic acid analogs and/or
derivatives are conventionally termed folates, reflecting their
ability to bind with folate-receptors, and such ligands when
conjugated with exogenous molecules are effective to enhance
transmembrane transport, such as via folate-mediated endocytosis as
described herein. Other suitable binding ligands capable of binding
to folate receptors to initiate receptor mediated endocytotic
transport of the complex include antibodies to the folate receptor.
An exogenous molecule in complex with an antibody to a folate
receptor is used to trigger transmembrane transport of the
complex.
[0181] Additional analogs of folic acid that bind to folic acid
receptors are described in US Patent Application Publication Serial
Nos. 2005/0227985 and 2004/0242582, the disclosures of which are
incorporated herein by reference. Illustratively, such folate
analogs have the general formula:
##STR00141##
wherein X and Y are each--independently selected from the group
consisting of halo, R.sup.2, OR.sup.2, SR.sup.3, and
NR.sup.4R.sup.5;
[0182] U, V, and W represent divalent moieties each independently
selected from the group consisting of --(R.sup.6a)C.dbd., --N.dbd.,
--(R.sup.6a)C(R.sup.7a)--, and --N(R.sup.4a)--; Q is selected from
the group consisting of C and CH; T is selected from the group
consisting of S, O, N, and --C.dbd.C--;
[0183] A.sup.1 and A.sup.2 are each independently selected from the
group consisting of oxygen, sulfur, --C(Z)--, --C(Z)O--, --OC(Z)--,
--N(R.sup.4b)--, --C(Z)N(R.sup.4b)--, --N(R.sup.4b)C(Z)--,
--OC(Z)N(R.sup.4b)--, --N(R.sup.4b)C(Z)O--,
--N(R.sup.4b)C(Z)N(R.sup.5b)--, --S(O)--, --S(O).sub.2--,
--N(R.sup.4a)S(O).sub.2--, C(R.sup.6b)(R.sup.7b)--,
--N(C.ident.CH)--, --N(CH.sub.2C.ident.CH)--, C.sub.1-C.sub.12
alkylene, and C.sub.1-C.sub.12 alkyeneoxy, where Z is oxygen or
sulfur;
[0184] R.sup.1 is selected-from the group consisting of hydrogen,
halo, C.sub.1-C.sub.12 alkyl, and C.sub.1-C.sub.12 alkoxy; R.sup.2,
R.sup.3, R.sup.4, R.sup.4a, R.sup.4b, R.sup.5, R.sup.5b, R.sup.6b,
and R.sup.7b are each independently selected from the group
consisting of hydrogen, halo, C.sub.1-C.sub.12 alkyl,
C.sub.1-C.sub.12 alkoxy, C.sub.1-C.sub.12 alkanoyl,
C.sub.1-C.sub.12 alkenyl, C.sub.1-C.sub.12 alkynyl,
(C.sub.1-C.sub.12 alkoxy)carbonyl, and (C.sub.1-C.sub.12
alkylamino)carbonyl;
[0185] R.sup.6 and R.sup.7 are each independently selected from the
group consisting of hydrogen, halo, C.sub.1-C.sub.12 alkyl, and
C.sub.1-C.sub.12 alkoxy; or, R.sup.6 and R.sup.7 are taken together
to form a carbonyl group; R.sup.6a and R.sup.7a are each
independently selected from the group consisting of hydrogen, halo,
C.sub.1-C.sub.12 alkyl, and C.sub.1-C.sub.12 alkoxy; or R.sup.6a
and R.sup.7a are taken together to form a carbonyl group;
[0186] L is a divalent linker as described herein; and
[0187] n, p, r, s and t are each independently either 0 or 1.
[0188] As used herein, it is to be understood that the term folate
refers both individually to folic acid used in forming a conjugate,
or alternatively to a folate analog or derivative thereof that is
capable of binding to folate or folic acid receptors.
[0189] The vitamin can be folate which includes a nitrogen, and in
this embodiment, the spacer linkers can be alkylenecarbonyl,
cycloalkylenecarbonyl, carbonylalkylcarbonyl,
1-alkylenesuccinimid-3-yl, 1-(carbonylalkyl)succinimid-3-yl,
wherein each of the spacer linkers is optionally substituted with a
substituent X.sup.1, and the spacer linker is bonded to the folate
nitrogen to form an imide or an alkylamide. In this embodiment, the
substituents X.sup.1 can be alkyl, hydroxyalkyl, amino, aminoalkyl,
alkylaminoalkyl, dialkylaminoalkyl, sulfhydrylalkyl,
alkylthioalkyl, aryl, substituted aryl, arylalkyl, substituted
arylalkyl, carboxy, carboxyalkyl, guanidinoalkyl, R.sup.4-carbonyl,
R.sup.5-carbonylalkyl, R.sup.6-acylamino, and
R.sup.7-acylaminoalkyl, wherein R.sup.4 and R.sup.5 are each
independently selected from amino acids, amino acid derivatives,
and peptides, and wherein R.sup.6 and R.sup.7 are each
independently selected from amino acids, amino acid derivatives,
and peptides.
[0190] Illustrative embodiments of vitamin analogs and/or
derivatives also include analogs and derivatives of biotin such as
biocytin, biotin sulfoxide, oxybiotin and other biotin
receptor-binding compounds, and the like. It is appreciated that
analogs and derivatives of the other vitamins described herein are
also contemplated herein. In one embodiment, vitamins that can be
used as the binding ligand (B) in the drug delivery conjugates
described herein include those that bind to vitamin receptors
expressed specifically on activated macrophages, such as the folate
receptor, which binds folate, or an analog or derivative thereof as
described herein.
[0191] In addition to the vitamins described herein, it is
appreciated that other binding ligands may be coupled with the
drugs and linkers described and contemplated herein to form binding
ligand-linker-drug conjugates capable of facilitating delivery of
the drug to a desired target. These other binding ligands, in
addition to the vitamins and their analogs and derivatives
described, may be used to form drug delivery conjugates capable of
binding to target cells. In general, any binding ligand (B) of a
cell surface receptor may be advantageously used as a targeting
ligand to which a linker-drug conjugate can be attached.
Illustrative other ligands contemplated herein include peptide
ligands identified from library screens, tumor cell-specific
peptides, tumor cell-specific aptamers, tumor cell-specific
carbohydrates, tumor cell-specific monoclonal or polyclonal
antibodies, Fab or scFv (i.e., a single chain variable region)
fragments of antibodies such as, for example, an Fab fragment of an
antibody directed to EphA2 or other proteins specifically expressed
or uniquely accessible on metastatic cancer cells, small organic
molecules derived from combinatorial libraries, growth factors,
such as EGF, FGF, insulin, and insulin-like growth factors, and
homologous polypeptides, somatostatin and its analogs, transferrin,
lipoprotein complexes, bile salts, selectins, steroid hormones,
Arg-Gly-Asp containing peptides, retinoids, various Galectins,
.delta.-opioid receptor ligands, cholecystokinin A receptor
ligands, ligands specific for angiotensin AT1 or AT2 receptors,
peroxisome proliferator-activated receptor .lamda. ligands,
.beta.-lactam antibiotics such as penicillin, small organic
molecules including antimicrobial drugs, and other molecules that
bind specifically to a receptor preferentially expressed on the
surface of tumor cells or on an infectious organism, antimicrobial
and other drugs designed to fit into the binding pocket of a
particular receptor based on the crystal structure of the receptor
or other cell surface protein, binding ligands of tumor antigens or
other molecules preferentially expressed on the surface of tumor
cells, or fragments of any of these molecules. An example of a
tumor-specific antigen that could function as a binding site for a
binding ligand-drug conjugate include extracellular epitopes of a
member of the Ephrin family of proteins, such as EphA2. EphA2
expression is restricted to cell-cell junctions in normal cells,
but EphA2 is distributed over the entire cell surface in metastatic
tumor cells. Thus, EphA2 on metastatic cells would be accessible
for binding to, for example, an Fab fragment of an antibody
conjugated to a drug, whereas the protein would not be accessible
for binding to the Fab fragment on normal cells, resulting in a
binding ligand-drug conjugate specific for metastatic cancer
cells.
[0192] In another embodiment, methods for treating diseases caused
by or evidenced by pathogenic cell populations are described
herein. The binding ligand (B) drug delivery conjugates can be used
to treat disease states characterized by the presence of a
pathogenic cell population in the host wherein the members of the
pathogenic cell population have an accessible binding site for the
binding ligand (B), or analog or derivative thereof, wherein the
binding site is uniquely expressed, overexpressed, or
preferentially expressed by the pathogenic cells. The selective
elimination of the pathogenic cells is mediated by the binding of
the ligand moiety of the binding ligand (B) drug delivery conjugate
to a ligand receptor, transporter, or other surface-presented
protein that specifically binds the binding ligand (B), or analog
or derivative thereof, and which is uniquely expressed,
overexpressed, or preferentially expressed by the pathogenic cells.
A surface-presented protein uniquely expressed, overexpressed, or
preferentially expressed by the pathogenic cells is a receptor not
present or present at lower concentrations on non-pathogenic cells
providing a means for selective elimination of the pathogenic
cells.
[0193] For example, surface-expressed vitamin receptors, such as
the high-affinity folate receptor, are overexpressed on cancer
cells. Epithelial cancers of the ovary, mammary gland, colon, lung,
nose, throat, and brain have all been reported to express elevated
levels of the folate receptor. In fact, greater than 90% of all
human ovarian tumors are known to express large amounts of this
receptor. Accordingly, the binding ligand (B) drug delivery
conjugates described herein can be used to treat a variety of tumor
cell types, as well as other types of pathogenic cells, such as
infectious agents, that preferentially express ligand receptors,
such as vitamin receptors, and, thus, have surface accessible
binding sites for ligands, such as vitamins, or vitamin analogs or
derivatives. In one aspect, methods are described herein for
targeting binding ligand-linker-drug conjugates to maximize
targeting of the pathogenic cells for elimination.
[0194] The binding ligand (B) drug delivery conjugates described
herein can be used for both human clinical medicine and veterinary
applications. Thus, the host animal harboring the population of
pathogenic cells and treated with the binding ligand (e.g., a
vitamin) drug delivery conjugates can be human or, in the case of
veterinary applications, can be a laboratory, agricultural,
domestic, or wild animal. The methods described herein can be
applied to host animals including, but not limited to, humans,
laboratory animals such rodents (e.g., mice, rats, hamsters, etc.),
rabbits, monkeys, chimpanzees, domestic animals such as dogs, cats,
and rabbits, agricultural animals such as cows, horses, pigs,
sheep, goats, and wild animals in captivity such as bears, pandas,
lions, tigers, leopards, elephants, zebras, giraffes, gorillas,
dolphins, and whales.
[0195] The methods are applicable to populations of pathogenic
cells that cause a variety of pathologies in these host animals.
The term pathogenic cells refers to for example cancer cells,
infectious agents such as bacteria and viruses, bacteria- or
virus-infected cells, activated macrophages capable of causing a
disease state, and any other type of pathogenic cells that uniquely
express, preferentially express, or overexpress binding ligand
receptors, such as vitamin receptors or receptors that bind analogs
or derivatives of vitamins. Pathogenic cells can also include any
cells causing a disease state for which treatment with the binding
ligand drug delivery conjugates described herein results in
reduction of the symptoms of the disease. For example, the
pathogenic cells can be host cells that are pathogenic under some
circumstances such as cells of the immune system that are
responsible for graft versus host disease, but not pathogenic under
other circumstances.
[0196] Thus, the population of pathogenic cells can be a cancer
cell population that is tumorigenic, including benign tumors and
malignant tumors, or it can be non-tumorigenic. The cancer cell
population can arise spontaneously or by such processes as
mutations present in the germline of the host animal or somatic
mutations, or it can be chemically-, virally-, or
radiation-induced. The methods can be utilized to treat such
cancers as carcinomas, sarcomas, lymphomas, Hodgkin's disease,
melanomas, mesotheliomas, Burkitt's lymphoma, nasopharyngeal
carcinomas, leukemias, and myelomas. The cancer cell population can
include, but is not limited to, oral, thyroid, endocrine, skin,
gastric, esophageal, laryngeal, pancreatic, colon, bladder, bone,
ovarian, cervical, uterine, breast, testicular, prostate, rectal,
kidney, liver, and lung cancers.
[0197] In embodiments where the pathogenic cell population is a
cancer cell population, the effect of conjugate administration is a
therapeutic response measured by reduction or elimination of tumor
mass or of inhibition of tumor cell proliferation. In the case of a
tumor, the elimination can be an elimination of cells of the
primary tumor or of cells that have metastasized or are in the
process of dissociating from the primary tumor. A prophylactic
treatment with the binding ligand (B) drug delivery conjugate
(e.g., a vitamin used as the binding ligand) to prevent return of a
tumor after its removal by any therapeutic approach including
surgical removal of the tumor, radiation therapy, chemotherapy, or
biological therapy is also described. The prophylactic treatment
can be an initial treatment with the binding ligand (B) drug
delivery conjugate, such as treatment in a multiple dose daily
regimen, and/or can be an additional treatment or series of
treatments after an interval of days or months following the
initial treatment(s). Accordingly, elimination of any of the
pathogenic cell populations treated using the described methods
includes reduction in the number of pathogenic cells, inhibition of
proliferation of pathogenic cells, a prophylactic treatment that
prevents return of pathogenic cells, or a treatment of pathogenic
cells that results in reduction of the symptoms of disease.
[0198] In cases where cancer cells are being eliminated, the
methods can be used in combination with surgical removal of a
tumor, radiation therapy, chemotherapy, or biological therapies
such as other immunotherapies including, but not limited to,
monoclonal antibody therapy, treatment with immunomodulatory
agents, adoptive transfer of immune effector cells, treatment with
hematopoietic growth factors, cytokines and vaccination.
[0199] The methods are also applicable to populations of pathogenic
cells that cause a variety of infectious diseases. For example, the
methods are applicable to such populations of pathogenic cells as
bacteria, fungi, including yeasts, viruses, virus-infected cells,
mycoplasma, and parasites. Infectious organisms that can be treated
with the binding ligand (B) drug delivery conjugates described
herein are any art-recognized infectious organisms that cause
pathogenesis in an animal, including such organisms as bacteria
that are gram-negative or gram-positive cocci or bacilli. For
example, Proteus species, Klebsiella species, Providencia species,
Yersinia species, Erwinia species, Enterobacter species, Salmonella
species, Serratia species, Aerobacter species, Escherichia species,
Pseudomonas species, Shigella species, Vibrio species, Aeromonas
species, Campylobacter species, Streptococcus species,
Staphylococcus species, Lactobacillus species, Micrococcus species,
Moraxella species, Bacillus species, Clostridium species,
Corynebacterium species, Eberthella species, Micrococcus species,
Mycobacterium species, Neisseria species, Haemophilus species,
Bacteroides species, Listeria species, Erysipelothrix species,
Acinetobacter species, Brucella species, Pasteurella species,
Vibrio species, Flavobacterium species, Fusobacterium species,
Streptobacillus species, Calymmatobacterium species, Legionella
species, Treponema species, Borrelia species, Leptospira species,
Actinomyces species, Nocardia species, Rickettsia species, and any
other bacterial species that causes disease in a host can be
treated with the binding ligand drug delivery conjugates described
herein.
[0200] Of particular interest are bacteria that are resistant to
antibiotics such as antibiotic-resistant Streptococcus species and
Staphylococcus species, or bacteria that are susceptible to
antibiotics, but cause recurrent infections treated with
antibiotics so that resistant organisms eventually develop.
Bacteria that are susceptible to antibiotics, but cause recurrent
infections treated with antibiotics so that resistant organisms
eventually develop, can be treated with the binding ligand (B) drug
delivery conjugates described herein in the absence of antibiotics,
or in combination with lower doses of antibiotics than would
normally be administered to a patient, to avoid the development of
these antibiotic-resistant bacterial strains.
[0201] Viruses, such as DNA and RNA viruses, can also be treated
with the described methods. Such viruses include, but are not
limited to, DNA viruses such as papilloma viruses, parvoviruses,
adenoviruses, herpesviruses and vaccinia viruses, and RNA viruses,
such as arenaviruses, coronaviruses, rhinoviruses, respiratory
syncytial viruses, influenza viruses, picornaviruses,
paramyxoviruses, reoviruses, retroviruses, lentiviruses, and
rhabdoviruses.
[0202] The methods are also applicable to any fungi, including
yeasts, mycoplasma species, parasites, or other infectious
organisms that cause disease in animals. Examples of fungi that can
be treated with the methods and compositions include fungi that
grow as molds or are yeastlike, including, for example, fungi that
cause diseases such as ringworm, histoplasmosis, blastomycosis,
aspergillosis, cryptococcosis, sporotrichosis, coccidioidomycosis,
paracoccidio-idomycosis, mucormycosis, chromoblastomycosis,
dermatophytosis, protothecosis, fusariosis, pityriasis, mycetoma,
paracoccidioidomycosis, phaeohyphomycosis, pseudallescheriasis,
sporotrichosis, trichosporosis, pneumocystis infection, and
candidiasis.
[0203] The methods can also be utilized to treat parasitic
infections including, but not limited to, infections caused by
tapeworms, such as Taenia, Hymenolepsis, Diphyllobothrium, and
Echinococcus species, flukes, such as Fasciolopsis, Heterophyes,
Metagonimus, Clonorchis, Fasciola, Paragonimus, and Schitosoma
species, roundworms, such as Enterobius, Trichuris, Ascaris,
Ancylostoma, Necator, Strongyloides, Trichinella, Wuchereria,
Brugia, Loa Onchocerca, and Dracunculus species, ameba, such as
Naegleria and Acanthamoeba species, and protozoans, such as
Plasmodium, Trypanosoma, Leishmania, Toxoplasma, Entamoeba,
Giardia, Isospora, Cryptosporidium, and Enterocytozoon species.
[0204] The pathogenic cells to which the binding ligand drug
delivery conjugates described herein are directed can also be cells
harboring endogenous pathogens, such as virus-, mycoplasma-,
parasite-, or bacteria-infected cells, if these cells
preferentially express ligand receptors, such as vitamin
receptors.
[0205] In one embodiment, the binding ligand drug delivery
conjugates can be internalized into the targeted pathogenic cells
upon binding of the binding ligand moiety to a receptor,
transporter, or other surface-presented protein that specifically
binds the ligand and which is preferentially expressed on the
pathogenic cells. Such internalization can occur, for example,
through receptor-mediated endocytosis. If the binding ligand (B)
drug delivery conjugate contains a releasable linker, the binding
ligand moiety and the drug can dissociate intracellularly and the
drug can act on its intracellular target.
[0206] In an alternate embodiment, the binding ligand moiety of the
drug delivery conjugate can bind to the pathogenic cell placing the
drug in close association with the surface of the pathogenic cell.
The drug can then be released by cleavage of the releasable linker.
For example, the drug can be released by a protein disulfide
isomerase if the releasable linker is a disulfide group. The drug
can then be taken up by the pathogenic cell to which the binding
ligand (B) drug delivery conjugate is bound, or the drug can be
taken up by another pathogenic cell in close proximity thereto.
Alternatively, the drug could be released by a protein disulfide
isomerase inside the cell where the releasable linker is a
disulfide group. The drug may also be released by a hydrolytic
mechanism, such as acid-catalyzed hydrolysis, as described above
for certain beta elimination mechanisms, or by an anchimerically
assisted cleavage through an oxonium ion or lactonium ion producing
mechanism. The selection of the releasable linker or linkers will
dictate the mechanism by which the drug is released from the
conjugate. It is appreciated that such a selection can be
pre-defined by the conditions wherein the drug conjugate will be
used. Alternatively, the drug delivery conjugates can be
internalized into the targeted cells upon binding, and the binding
ligand and the drug can remain associated intracellularly with the
drug exhibiting its effects without dissociation from the vitamin
moiety.
[0207] In still another embodiment where the binding ligand is a
vitamin, the vitamin-drug delivery conjugate can act through a
mechanism independent of cellular vitamin receptors. For example,
the drug delivery conjugates can bind to soluble vitamin receptors
present in the serum or to serum proteins, such as albumin,
resulting in prolonged circulation of the conjugates relative to
the unconjugated drug, and in increased activity of the conjugates
towards the pathogenic cell population relative to the unconjugated
drug.
[0208] In one embodiment, the drugs for use in the methods
described herein remain stable in serum for at least 4 hours. In
another embodiment the drugs have an IC.sub.50 in the nanomolar
range, and, in another embodiment, the drugs are water soluble. If
the drug is not water soluble, the bivalent linker (L) can be
derivatized to enhance water solubility. The term drug also means
any of the drug analogs or derivatives described hereinabove. It
should be appreciated that a drug analog or derivative can mean a
drug that incorporates an heteroatom through which the drug analog
or derivative is covalently bound to the bivalent linker (L).
[0209] The binding ligand drug delivery conjugates can comprise a
binding ligand (B), a bivalent linker (L), a drug, and, optionally,
heteroatom linkers to link the binding ligand (B) receptor binding
moiety and the drug to the bivalent linker (L). In one illustrative
embodiment, it should be appreciated that a vitamin analog or
derivative can mean a vitamin that incorporates an heteroatom
through which the vitamin analog or derivative is covalently bound
to the bivalent linker (L). Thus, in this illustrative embodiment,
the vitamin can be covalently bound to the bivalent linker (L)
through an heteroatom linker, or a vitamin analog or derivative
(i.e., incorporating an heteroatom) can be directly bound to the
bivalent linker (L). In similar illustrative embodiments, a drug
analog or derivative is a drug, and a drug analog or derivative can
mean a drug that incorporates an heteroatom through which the drug
analog or derivative is covalently bound to the bivalent linker
(L). Thus, in these illustrative aspects, the drug can be
covalently bound to the bivalent linker (L) through an heteroatom
linker, or a drug analog or derivative (i.e., incorporating an
heteroatom) can be directly bound to the bivalent linker (L). The
bivalent linker (L) can comprise a spacer linker, a releasable
(i.e., cleavable) linker, and an heteroatom linker to link the
spacer linker to the releasable linker in conjugates containing
both of these types of linkers.
[0210] Generally, any manner of forming a conjugate between the
bivalent linker (L) and the binding ligand (B), or analog or
derivative thereof, between the bivalent linker (L) and the drug,
or analog or derivative thereof, including any intervening
heteroatom linkers, can be utilized. Also, any art-recognized
method of forming a conjugate between the spacer linker, the
releasable linker, and the heteroatom linker to form the bivalent
linker (L) can be used. The conjugate can be formed by direct
conjugation of any of these molecules, for example, through
complexation, or through hydrogen, ionic, or covalent bonds.
Covalent bonding can occur, for example, through the formation of
amide, ester, disulfide, or imino bonds between acid, aldehyde,
hydroxy, amino, sulfhydryl, or hydrazo groups.
[0211] In another embodiment, pharmaceutical compositions
comprising an amount of a binding ligand (B) drug delivery
conjugate effective to eliminate a population of pathogenic cells
in a host animal when administered in one or more doses are
described. The binding ligand drug delivery conjugate is preferably
administered to the host animal parenterally, e.g., intradermally,
subcutaneously, intramuscularly, intraperitoneally, intravenously,
or intrathecally. Alternatively, the binding ligand drug delivery
conjugate can be administered to the host animal by other medically
useful processes, such as orally, and any effective dose and
suitable therapeutic dosage form, including prolonged release
dosage forms, can be used.
[0212] Examples of parenteral dosage forms include aqueous
solutions of the active agent, in an isotonic saline, 5% glucose or
other well-known pharmaceutically acceptable liquid carriers such
as liquid alcohols, glycols, esters, and amides. The parenteral
dosage form can be in the form of a reconstitutable lyophilizate
comprising the dose of the drug delivery conjugate. In one aspect
of the present embodiment, any of a number of prolonged release
dosage forms known in the art can be administered such as, for
example, the biodegradable carbohydrate matrices described in U.S.
Pat. Nos. 4,713,249; 5,266,333; and 5,417,982, the disclosures of
which are incorporated herein by reference, or, alternatively, a
slow pump (e.g., an osmotic pump) can be used.
[0213] In one illustrative aspect, at least one additional
composition comprising a therapeutic factor can be administered to
the host in combination or as an adjuvant to the above-detailed
methodology, to enhance the binding ligand drug delivery
conjugate-mediated elimination of the population of pathogenic
cells, or more than one additional therapeutic factor can be
administered. The therapeutic factor can be selected from a
chemotherapeutic agent, or another therapeutic factor capable of
complementing the efficacy of the administered binding ligand drug
delivery conjugate.
[0214] In one illustrative aspect, therapeutically effective
combinations of these factors can be used. In one embodiment, for
example, therapeutically effective amounts of the therapeutic
factor, for example, in amounts ranging from about 0.1
MIU/m.sup.2/dose/day to about 15 MIU/m.sup.2/dose/day in a multiple
dose daily regimen, or for example, in amounts ranging from about
0.1 MIU/m.sup.2/dose/day to about 7.5 MIU/m.sup.2/dose/day in a
multiple dose daily regimen, can be used along with the binding
ligand drug delivery conjugates to eliminate, reduce, or neutralize
pathogenic cells in a host animal harboring the pathogenic cells
(MIU=million international units; m.sup.2=approximate body surface
area of an average human).
[0215] In another embodiment, chemotherapeutic agents, which are,
for example, cytotoxic themselves or can work to enhance tumor
permeability, are also suitable for use in the described methods in
combination with the binding ligand drug delivery conjugates. Such
chemotherapeutic agents include adrenocorticoids and
corticosteroids, alkylating agents, antiandrogens, antiestrogens,
androgens, aclamycin and aclamycin derivatives, estrogens,
antimetabolites such as cytosine arabinoside, purine analogs,
pyrimidine analogs, and methotrexate, busulfan, carboplatin,
chlorambucil, cisplatin and other platinum compounds, tamoxiphen,
taxol, paclitaxel, paclitaxel derivatives, Taxotere.RTM.,
cyclophosphamide, daunomycin, rhizoxin, T2 toxin, plant alkaloids,
prednisone, hydroxyurea, teniposide, mitomycins, discodermolides,
microtubule inhibitors, epothilones, tubulysin, cyclopropyl
benz[e]indolone, seco-cyclopropyl benz[e]indolone,
O--Ac-seco-cyclopropyl benz[e]indolone, bleomycin and any other
antibiotic, nitrogen mustards, nitrosureas, vincristine,
vinblastine, and analogs and derivative thereof such as
deacetylvinblastine monohydrazide, colchicine, colchicine
derivatives, allocolchicine, thiocolchicine, trityl cysteine,
Halicondrin B, dolastatins such as dolastatin 10, amanitins such as
.alpha.-amanitin, camptothecin, irinotecan, and other camptothecin
derivatives thereof, geldanamycin and geldanamycin derivatives,
estramustine, nocodazole, MAP4, colcemid, inflammatory and
proinflammatory agents, peptide and peptidomimetic signal
transduction inhibitors, and any other art-recognized drug or
toxin. Other drugs that can be used include penicillins,
cephalosporins, vancomycin, erythromycin, clindamycin, rifampin,
chloramphenicol, aminoglycoside antibiotics, gentamicin,
amphotericin B, acyclovir, trifluridine, ganciclovir, zidovudine,
amantadine, ribavirin, maytansines and analogs and derivatives
thereof, gemcitabine, and any other art-recognized antimicrobial
compound.
[0216] The therapeutic factor can be administered to the host
animal prior to, after, or at the same time as the binding ligand
drug delivery conjugates and the therapeutic factor can be
administered as part of the same composition containing the binding
ligand drug delivery conjugate or as part of a different
composition than the binding ligand drug delivery conjugate. Any
such therapeutic composition containing the therapeutic factor at a
therapeutically effective dose can be used.
[0217] Additionally, more than one type of binding ligand drug
delivery conjugate can be used. Illustratively, for example, the
host animal can be treated with conjugates with different vitamins,
but the same drug in a co-dosing protocol. In other embodiments,
the host animal can be treated with conjugates comprising the same
binding ligand linked to different drugs, or various binding
ligands linked to various drugs. In another illustrative
embodiment, binding ligand drug delivery conjugates with the same
or different vitamins, and the same or different drugs comprising
multiple vitamins and multiple drugs as part of the same drug
delivery conjugate could be used.
[0218] The unitary daily dosage of the binding ligand drug delivery
conjugate can vary significantly depending on the host condition,
the disease state being treated, the molecular weight of the
conjugate, its route of administration and tissue distribution, and
the possibility of co-usage of other therapeutic treatments such as
radiation therapy. The effective amount to be administered to a
patient is based on body surface area, patient weight, and
physician assessment of patient condition. In illustrative
embodiments, effective doses can range, for example, from about 1
ng/kg to about 1 mg/kg, from about 1 .mu.g/kg to about 500
.mu.g/kg, and from about 1 .mu.g/kg to about 100 .mu.g/kg.
[0219] In another illustrative aspect, any effective regimen for
administering the binding ligand drug delivery conjugates can be
used. For example, the binding ligand drug delivery conjugates can
be administered as single doses, or can be divided and administered
as a multiple-dose daily regimen. In other embodiments, a staggered
regimen, for example, one to three days per week can be used as an
alternative to daily treatment, and such intermittent or staggered
daily regimen is considered to be equivalent to every day treatment
and within the scope of the methods described herein. In one
embodiment, the host is treated with multiple injections of the
binding ligand drug delivery conjugate to eliminate the population
of pathogenic cells. In another embodiment, the host is injected
multiple times (preferably about 2 up to about 50 times) with the
binding ligand drug delivery conjugate, for example, at 12-72 hour
intervals or at 48-72 hour intervals. In other embodiments,
additional injections of the binding ligand drug delivery conjugate
can be administered to the patient at an interval of days or months
after the initial injections(s) and the additional injections
prevent recurrence of the disease state caused by the pathogenic
cells.
[0220] In one embodiment, vitamins, or analogs or derivatives
thereof, that can be used in the binding ligand drug delivery
conjugates include those that bind to receptors expressed
specifically on activated macrophages, such as the folate receptor
which binds folate, or an analog or derivative thereof. The
folate-linked conjugates, for example, can be used to kill or
suppress the activity of activated macrophages that cause disease
states in the host. Such macrophage targeting conjugates, when
administered to a patient suffering from an activated
macrophage-mediated disease state, work to concentrate and
associate the conjugated drug in the population of activated
macrophages to kill the activated macrophages or suppress
macrophage function. Elimination, reduction, or deactivation of the
activated macrophage population works to stop or reduce the
activated macrophage-mediated pathogenesis characteristic of the
disease state being treated. Exemplary of diseases known to be
mediated by activated macrophages include rheumatoid arthritis,
ulcerative colitis, Crohn's disease, psoriasis, osteomyelitis,
multiple sclerosis, atherosclerosis, pulmonary fibrosis,
sarcoidosis, systemic sclerosis, organ transplant rejection (GVHD)
and chronic inflammations. Administration of the drug delivery
conjugate is typically continued until symptoms of the disease
state are reduced or eliminated.
[0221] Illustratively, the binding ligand drug delivery conjugates
administered to kill activated macrophages or suppress the function
of activated macrophages can be administered parenterally to the
animal or patient suffering from the disease state, for example,
intradermally, subcutaneously, intramuscularly, intraperitoneally,
or intravenously in combination with a pharmaceutically acceptable
carrier. In another embodiment, the binding ligand drug delivery
conjugates can be administered to the animal or patient by other
medically useful procedures and effective doses can be administered
in standard or prolonged release dosage forms. In another aspect,
the therapeutic method can be used alone or in combination with
other therapeutic methods recognized for treatment of disease
states mediated by activated macrophages.
[0222] The drug delivery conjugates described herein can be
prepared by art-recognized synthetic methods. The synthetic methods
are chosen depending upon the selection of the optionally addition
heteroatoms or the heteroatoms that are already present on the
spacer linkers, releasable linkers. the drug, and/or or the binding
ligand. In general, the relevant bond forming reactions are
described in Richard C. Larock, "Comprehensive Organic
Transformations, a guide to functional group preparations," VCH
Publishers, Inc. New York (1989), and in Theodora E. Greene &
Peter G. M. Wuts, "Protective Groups ion Organic Synthesis," 2d
edition, John Wiley & Sons, Inc. New York (1991), the
disclosures of which are incorporated herein by reference.
EXAMPLES
Compound Examples
[0223] The compounds described herein may be prepared using the
process and syntheses described herein, as well as using general
organic synthetic methods. In particular, methods for preparing the
compounds are described in U.S. patent application publication
2005/0002942, the disclosure of which is incorporated herein by
reference.
[0224] General formation of folate-peptides. The folate-containing
peptidyl fragment Pte-Glu-(AA).sub.n-NH(CHR.sub.2)CO.sub.2H (3) is
prepared by a polymer-supported sequential approach using standard
methods, such as the Fmoc-strategy on an acid-sensitive
Fmoc-AA-Wang resin (1), as shown in the following Scheme:
##STR00142##
[0225] In this illustrative embodiment of the processes described
herein, R.sub.1 is Fmoc, R.sub.2 is the desired
appropriately-protected amino acid side chain, and DIPEA is
diisopropylethylamine. Standard coupling procedures, such as PyBOP
and others described herein or known in the art are used, where the
coupling agent is illustratively applied as the activating reagent
to ensure efficient coupling. Fmoc protecting groups are removed
after each coupling step under standard conditions, such as upon
treatment with piperidine, tetrabutylammonium fluoride (TBAF), and
the like. Appropriately protected amino acid building blocks, such
as Fmoc-Glu-OtBu, N.sup.10-TFA-Pte-OH, and the like, are used, as
described in the Scheme, and represented in step (b) by Fmoc-AA-OH.
Thus, AA refers to any amino acid starting material, that is
appropriately protected. It is to be understood that the term amino
acid as used herein is intended to refer to any reagent having both
an amine and a carboxylic acid functional group separated by one or
more carbons, and includes the naturally occurring alpha and beta
amino acids, as well as amino acid derivatives and analogs of these
amino acids. In particular, amino acids having side chains that are
protected, such as protected serine, threonine, cysteine,
aspartate, and the like may also be used in the folate-peptide
synthesis described herein. Further, gamma, delta, or longer
homologous amino acids may also be included as starting materials
in the folate-peptide synthesis described herein. Further, amino
acid analogs having homologous side chains, or alternate branching
structures, such as norleucine, isovaline, .beta.-methyl threonine,
.beta.-methyl cysteine, .beta.,.beta.-dimethyl cysteine, and the
like, may also be included as starting materials in the
folate-peptide synthesis described herein.
[0226] The coupling sequence (steps (a) & (b)) involving
Fmoc-AA-OH is performed "n" times to prepare solid-support peptide
(2), where n is an integer and may equal 0 to about 100. Following
the last coupling step, the remaining Fmoc group is removed (step
(a)), and the peptide is sequentially coupled to a glutamate
derivative (step (c)), deprotected, and coupled to TFA-protected
pteroic acid (step (d)). Subsequently, the peptide is cleaved from
the polymeric support upon treatment with trifluoroacetic acid,
ethanedithiol, and triisopropylsilane (step (e)). These reaction
conditions result in the simultaneous removal of the t-Bu, t-Boc,
and Trt protecting groups that may form part of the
appropriately-protected amino acid side chain. The TFA protecting
group is removed upon treatment with base (step (f)) to provide the
folate-containing peptidyl fragment (3).
##STR00143##
[0227] According to the general procedure described herein, Wang
resin bound 4-methoxytrityl (MTT)-protected Cys-NH.sub.2 was
reacted according to the following sequence: 1) a.
Fmoc-Asp(OtBu)-OH, PyBOP, DIPEA; b. 20% Piperidine/DMF; 2) a.
Fmoc-Asp(OtBu)-OH, PyBOP, DIPEA; b. 20% Piperidine/DMF; 3) a.
Fmoc-Arg(Pbf)-OH, PyBOP, DIPEA; b. 20% Piperidine/DMF; 4) a.
Fmoc-Asp(OtBu)-OH, PyBOP, DIPEA; b. 20% Piperidine/DIPEA; 5) a.
Fmoc-Glu-OtBu, PyBOP, DIPEA; b. 20% Piperidine/DMF; 6)
N.sup.10-TFA-pteroic acid, PyBOP, DIPEA. The MTT, tBu, and Pbf
protecting groups were removed with TFA/H.sub.2O/TIPS/EDT
(92.5:2.5:2.5:2.5), and the TFA protecting group was removed with
aqueous NH.sub.4OH at pH=9.3. Selected .sup.1H NMR (D.sub.2O)
.delta. (ppm) 8.68 (s, 1H, FA H-7), 7.57 (d, 2H, J=8.4 Hz, FA H-12
&16), 6.67 (d, 2H, J=9 Hz, FA H-13 &15), 4.40-4.75 (m, 5H),
4.35 (m, 2H), 4.16 (m, 1H), 3.02 (m, 2H), 2.55-2.95 (m, 8H), 2.42
(m, 211), 2.00-2.30 (m, 2H), 1.55-1.90 (m, 2H), 1.48 (m, 2H); MS
(ESI, m+H.sup.+) 1046.
##STR00144##
[0228] According to the general procedure described herein, Wang
resin bound 4-methoxytrityl (MTT)-protected Cys-NH.sub.2 was
reacted according to the following sequence: 1) a.
Fmoc-.beta.-aminoalanine(NH-MTT)-OH, PyBOP, DIPEA; b. 20%
Piperidine/DMF; 2) a. Fmoc-Asp(OtBu)-OH, PyBOP, DIPEA; b. 20%
Piperidine/DMF; 3) a. Fmoc-Asp(OtBu)-OH, PyBOP, DIPEA; b. 20%
Piperidine/DMF; 4) a. Fmoc-Asp(OtBu)-OH, PyBOP, DIPEA; b. 20%
Piperidine/DMF; 5) a. Fmoc-Glu-OtBu, PyBOP, DIPEA; b. 20%
Piperidine/DMF; 6) N.sup.10-TFA-pteroic acid, PyBOP, DIPEA. The
MTT, tBu, and TFA protecting groups were removed with a. 2%
hydrazine/DMF; b. TFA/H.sub.2O/TIPS/EDT (92.5:2.5:2.5:2.5).
[0229] The reagents shown in the following table were used in the
preparation:
TABLE-US-00005 Reagent (mmol) equivalents Amount
H-Cys(4-methoxytrityl)-2- 0.56 1 1.0 g chlorotrityl-Resin (loading
0.56 mmol/g) Fmoc-.beta.-aminoalanine(NH- 1.12 2 0.653 g MTT)-OH
Fmoc-Asp(OtBu)-OH 1.12 2 0.461 g Fmoc-Asp(OtBu)-OH 1.12 2 0.461 g
Fmoc-Asp(OtBu)-OH 1.12 2 0.461 g Fmoc-Glu-OtBu 1.12 2 0.477 g
N.sup.10TFA-Pteroic Acid 0.70 1.25 0.286 g (dissolve in 10 ml DMSO)
DIPEA 2.24 4 0.390 mL PyBOP 1.12 2 0.583 g
[0230] The coupling step was performed as follows: In a peptide
synthesis vessel add the resin, add the amino acid solution, DIPEA,
and PyBOP. Bubble argon for 1 hr. and wash 3.times. with DMF and
IPA. Use 20% piperidine in DMF for Fmoc deprotection, 3.times. (10
min), before each amino acid coupling. Continue to complete all 6
coupling steps. At the end wash the resin with 2% hydrazine in DMF
3.times. (5 min) to cleave TFA protecting group on Pteroic
acid.
[0231] Cleave the peptide analog from the resin using the following
reagent, 92.5% (50 ml) TFA, 2.5% (1.34 ml) H.sub.2O, 2.5% (1.34 ml)
Triisopropylsilane, 2.5% (1.34 ml) ethanedithiol, the cleavage step
was performed as follows: Add 25 ml cleavage reagent and bubble for
1.5 hr, drain, and wash 3.times. with remaining reagent. Evaporate
to about 5 mL and precipitate in ethyl ether. Centrifuge and dry.
Purification was performed as follows: Column-Waters NovaPak
C.sub.18 300.times.19 mm; Buffer A=10 mM Ammonium Acetate, pH 5;
B=CAN; 1% B to 20% B in 40 minutes at 15 ml/min, to 350 mg (64%);
HPLC-RT 10.307 min., 100% pure, .sup.1H HMR spectrum consistent
with the assigned structure, and MS (ES-): 1624.8, 1463.2, 1462.3,
977.1, 976.2, 975.1, 974.1, 486.8, 477.8.
##STR00145##
[0232] According to the general procedure described herein, Wang
resin bound MTT-protected Cys-NH.sub.2 was reacted according to the
following sequence: 1) a. Fmoc-Asp(OtBu)-OH, PyBOP, DIPEA; b. 20%
Piperidine/DMF; 2) a. Fmoc-Asp(OtBu)-OH, PyBOP, DIPEA; b. 20%
Piperidine/DMF; 3) a. Fmoc-Arg(Pbf)-OH, PyBOP, DIPEA; b. 20%
Piperidine/DMF; 4) a. Fmoc-Asp(OtBu)-OH, PyBOP, DIPEA; b. 20%
Piperidine/DMF; 5) a. Fmoc-Glu(.gamma.-OtBu)-OH, PyBOP, DIPEA; b.
20% Piperidine/DMF; 6) N.sup.10-TFA-pteroic acid, PyBOP, DIPEA. The
MTT, tBu, and Pbf protecting groups were removed with
TFA/H.sub.2O/TIPS/EDT (92.5:2.5:2.5:2.5), and the TFA protecting
group was removed with aqueous NH.sub.4OH at pH=9.3. The .sup.1H
NMR spectrum was consistent with the assigned structure.
##STR00146##
[0233] According to the general procedure described herein, Wang
resin bound MTT-protected D-Cys-NH.sub.2 was reacted according to
the following sequence: 1) a. Fmoc-D-Asp(OtBu)-OH, PyBOP, DIPEA; b.
20% Piperidine/DMF; 2) a. Fmoc-D-Asp(OtBu)-OH, PyBOP, DIPEA; b. 20%
Piperidine/DMF; 3) a. Fmoc-D-Arg(Pbf)-OH, PyBOP, DIPEA; b. 20%
Piperidine/DMF; 4) a. Fmoc-D-Asp(OtBu)-OH, PyBOP, DIPEA; b. 20%
Piperidine/DMF; 5) a. Fmoc-D-Glu-OtBu, PyBOP, DIPEA; b. 20%
Piperidine/DMF; 6) N.sup.10-TFA-pteroic acid, PyBOP, DIPEA. The
MTT, tBu, and Pbf protecting groups were removed with
TFA/H.sub.2O/TIPS/EDT (92.5:2.5:2.5:2.5), and the TFA protecting
group was removed with aqueous NH.sub.4OH at pH=9.3. The .sup.1H
NMR spectrum was consistent with the assigned structure.
##STR00147##
[0234] Similarly, EC089 was prepared as described herein.
##STR00148##
[0235] Preparation of tubulysin hydrazides. Illustrated by
preparing EC0347. N,N-Diisopropylethylamine (DIPEA, 6.1 .mu.L) and
isobutyl chloroformate (3.0 .mu.L) were added with via syringe in
tandem into a solution of tubulysin B (0.15 mg) in anhydrous EtOAc
(2.0 mL) at -15.degree. C. After stirring for 45 minutes at
-15.degree. C. under argon, the reaction mixture was cooled down to
-20.degree. C. and to which was added anhydrous hydrazine (5.0
.mu.L). The reaction mixture was stirred under argon at -20.degree.
C. for 3 hours, quenched with 1.0 mM sodium phosphate buffer (pH
7.0, 1.0 mL), and injected into a preparative HPLC for
purification. Column: Waters XTerra Prep MS C.sub.18 10 .mu.m,
19.times.250 mm; Mobile phase A: 1.0 mM sodium phosphate buffer, pH
7.0; Mobile phase B: acetonitrile; Method: 10% B to 80% B over 20
minutes, flow rate=25 mL/min. Fractions from 15.14-15.54 minutes
were collected and lyophilized to produce EC0347 as a white solid
(2.7 mg). The foregoing method is equally applicable for preparing
other tubulysin hydrazides by the appropriate selection of the
tubulysin starting compound.
##STR00149##
[0236] Synthesis of coupling reagent EC0311. DIPEA (0.60 mL) was
added to a suspension of
HOBt-OCO.sub.2--(CH.sub.2).sub.2--SS-2-pyridine HCl (685 mg, 91%)
in anhydrous DCM (5.0 mL) at 0.degree. C., stirred under argon for
2 minutes, and to which was added anhydrous hydrazine (0.10 mL).
The reaction mixture was stirred under argon at 0.degree. C. for 10
minutes and room temperature for an additional 30 minutes,
filtered, and the filtrate was purified by flash chromatography
(silica gel, 2% MeOH in DCM) to afford EC0311 as a clear thick oil
(371 mg), solidified upon standing.
##STR00150##
[0237] Preparation of tubulysin disulfides (stepwise process).
Illustrated for EC0312. DIPEA (36 .mu.L) and isobutyl chloroformate
(13 .mu.L) were added with the help of a syringe in tandem into a
solution of tubulysin B (82 mg) in anhydrous EtOAc (2.0 mL) at
-15.degree. C. After stirring for 45 minutes at -15.degree. C.
under argon, to the reaction mixture was added a solution of EC0311
in anhydrous EtOAc (1.0 mL). The resulting solution was stirred
under argon at -15.degree. C. for 15 minutes and room temperature
for an additional 45 minutes, concentrated, and the residue was
purified by flash chromatography (silica gel, 2 to 8% MeOH in DCM)
to give EC0312 as a white solid (98 mg). The foregoing method is
equally applicable for preparing other tubulysin derivatives by the
appropriate selection of the tubulysin starting compound.
##STR00151##
[0238] Hydroxydaunorubucin pyridyldisulfide. Similarly, this
compound was prepared as described herein in 65% yield, and
according to the foregoing scheme.
##STR00152##
[0239] Tubulysin B pyridyldisulfide. Similarly, this compound was
prepared as described herein.
##STR00153##
[0240] EC0488. This compound was prepared by SPPS according to the
general peptide synthesis procedure described herein starting from
H-Cys(4-methoxytrityl)-2-chlorotrityl-Resin, and the following SPPS
reagents:
TABLE-US-00006 Reagents mmol equivalent MW amount
H-Cys(4-methoxytrityl)-2- 0.10 0.17 g chlorotrityl-Resin (loading
0.6 mmol/g) EC0475 0.13 1.3 612.67 0.082 g Fmoc-Glu(OtBu)-OH 0.19
1.9 425.47 0.080 g EC0475 0.13 1.3 612.67 0.082 g Fmoc-Glu(OtBu)-OH
0.19 1.9 425.47 0.080 g EC0475 0.13 1.3 612.67 0.082 g
Fmoc-Glu-OtBu 0.19 1.9 425.47 0.080 g N.sup.10TFA-Pteroic Acid 0.16
1.6 408.29 0.066 g (dissolve in 10 ml DMSO) DIPEA 2.0 eq of AA
PyBOP 1.0 eq of AA
[0241] Coupling steps. In a peptide synthesis vessel add the resin,
add the amino acid solution, DIPEA, and PyBOP. Bubble argon for 1
hr. and wash 3.times. with DMF and IPA. Use 20% piperidine in DMF
for Fmoc deprotection, 3.times. (10 min), before each amino acid
coupling. Continue to complete all 9 coupling steps. At the end
treat the resin with 2% hydrazine in DMF 3.times. (5 min) to cleave
TFA protecting group on Pteroic acid, wash the resin with DMF
(3.times.), IPA (3.times.), MeOH (3.times.), and bubble the resin
with argon for 30 min.
[0242] Cleavage step. Reagent: 92.5% TFA, 2.5% H.sub.2O, 2.5%
triisopropylsilane, 2.5% ethanedithiol. Treat the resin with
cleavage reagent 3.times. (10 min, 5 min, 5 min) with argon
bubbling, drain, wash the resin once with cleavage reagent, and
combine the solution. Rotavap until 5 ml remains and precipitate in
diethyl ether (35 mL). Centrifuge, wash with diethyl ether, and
dry. About half of the crude solid (.about.100 mg) was purified by
HPLC.
[0243] HPLC Purification step. Column: Waters Xterra Prep MS C18 10
.mu.m 19.times.250 mm; Solvent A: 10 mM ammonium acetate, pH 5;
Solvent B: ACN; Method: 5 min 0% B to 25 min 20% B 26 mL/min.
Fractions containing the product was collected and freeze-dried to
give 43 mg EC0488 (51% yield). .sup.1H NMR and LC/MS (exact mass
1678.62) were consistent with the product.
##STR00154##
[0244] EC0351. Similarly, this compound was prepared as described
herein.
##STR00155##
[0245] General Synthesis of Disulfide Containing Tubulysin
Conjugates. Illustrated with pyridinyl disulfide derivatives of
certain naturally occurring tubulysins, where R.sup.1 is H or OH,
and R.sup.10, is alkyl or alkenyl. A binding ligand-linker
intermediate containing a thiol group is taken in deionized water
(ca. 20 mg/mL, bubbled with argon for 10 minutes prior to use) and
the pH of the suspension was adjusted by saturated NaHCO.sub.3
(bubbled with argon for 10 minutes prior to use) to about 6.9 (the
suspension may become a solution when the pH increased). Additional
deionized water is added (ca. 20-25%) to the solution as needed,
and to the aqueous solution is added immediately a solution of
EC0312 in THF (ca. 20 mg/mL). The reaction mixture becomes
homogenous quickly. After stirring under argon, e.g. for 45
minutes, the reaction mixture is diluted with 2.0 mM sodium
phosphate buffer (pH 7.0, ca 150 volume percent) and the THF is
removed by evacuation. The resulting suspension is filtered and the
filtrate may be purified by preparative HPLC (as described herein).
Fraction are lyophilized to isolate the conjugates. The foregoing
method is equally applicable for preparing other tubulysin
conjugates by the appropriate selection of the tubulysin starting
compound.
##STR00156##
[0246] General Method 2 for Preparing Conjugates (one-pot).
Illustrated with preparation of EC0543. DIPEA (7.8 .mu.L) and
isobutyl chloroformate (3.1 .mu.L) were added with the help of a
syringe in tandem into a solution of tubulysin A (18 mg) in
anhydrous EtOAc (0.50 mL) at -15.degree. C. After stirring for 35
minutes at -15.degree. C. under argon, to the reaction mixture was
added a solution of EC0311 (5.8 mg) in anhydrous EtOAc (0.50 mL).
The cooling was removed and the reaction mixture was stirred under
argon for an additional 45 minutes, concentrated, vacuumed, and the
residue was dissolved in THF (2.0 mL). Meanwhile, EC0488 (40 mg)
was dissolved in deionized water (bubbled with argon for 10 minutes
prior to use) and the pH of the aqueous solution was adjusted to
6.9 by saturated NaHCO.sub.3. Additional deionized water was added
to the EC0488 solution to make a total volume of 2.0 mL and to
which was added immediately the THF solution containing the
activated tubulysin. The reaction mixture, which became homogeneous
quickly, was stirred under argon for 50 minutes and quenched with
2.0 mM sodium phosphate buffer (pH 7.0, 15 mL). The resulting
cloudy solution was filtered and the filtrate was injected into a
preparative HPLC for purification. Column: Waters XTerra Prep MS
C.sub.18 10 .mu.m, 19.times.250 mm; Mobile phase A: 2.0 mM sodium
phosphate buffer, pH 7.0; Mobile phase B: acetonitrile; Method: 1%
B for 5 minutes, then 1% B to 60% B over the next 30 minutes, flow
rate=26 mL/min. Fractions from 20.75-24.50 minutes were collected
and lyophilized to afford EC0543 as a pale yellow fluffy solid (26
mg). The foregoing method is equally applicable for preparing other
tubulysin conjugates by the appropriate selection of the tubulysin
starting compound.
##STR00157##
[0247] EC0305. EC089 (86 mg) was suspended in deionized water (4.0
mL, bubbled with argon for 10 minutes prior to use) and the pH of
the suspension was adjusted by saturated NaHCO.sub.3 (bubbled with
argon for 10 minutes prior to use) to about 6.9 (the suspension
became a solution when the pH increased). Additional deionized
water was added to the solution to make a total volume of 5.0 mL
and to the aqueous solution was added immediately a solution of
EC0312 (97 mg) in THF (5.0 mL). The reaction mixture became
homogenous quickly. After stirring under argon for 45 minutes, the
reaction mixture was diluted with 2.0 mM sodium phosphate buffer
(pH 7.0, 15 mL) and the THF was removed on a Rotavapor. The
resulting suspension was filtered and the filtrate was injected
into a preparative HPLC for purification (Column: Waters XTerra
Prep MS C.sub.18 10 .mu.m, 19.times.250 mm; Mobile phase A: 2.0 mM
sodium phosphate buffer, pH 7.0; Mobile phase B: acetonitrile;
Method: 5% B to 80% B over 25 minutes, flow rate=25 mL/min).
Fractions from 10.04-11.90 minutes were collected and lyophilized
to give EC0305 as a pale yellow fluffy solid (117 mg).
##STR00158##
[0248] EC0352. Similarly, this compound was prepared as described
herein. EC0352 was prepared by forming a disulfide bond between
hydroxydaunorubucin pyridyldisulfide and EC0351 in 55% yield.
##STR00159##
[0249] EC0358. Similarly, this compound was prepared as described
herein. EC0358 was prepared by forming in DMF/DBU a disulfide bond
between EC0352 and tubulysin B pyridyldisulfide in 40% yield.
[0250] The following illustrative examples were also prepared using
the processes, syntheses, and tubulysins described herein.
##STR00160## ##STR00161## ##STR00162## ##STR00163##
##STR00164##
Method Examples
[0251] METHOD: Relative Affinity Assay. The affinity for folate
receptors (FRs) relative to folate was determined according to a
previously described method (Westerhof, G. R., J. H. Schornagel, et
al. (1995) Mol. Pharm. 48: 459-471) with slight modification.
Briefly, FR-positive KB cells were heavily seeded into 24-well cell
culture plates and allowed to adhere to the plastic for 18 h. Spent
incubation media was replaced in designated wells with folate-free
RPMI (FFRPMI) supplemented with 100 nM .sup.3H-folic acid in the
absence and presence of increasing concentrations of test article
or folic acid. Cells were incubated for 60 min at 37.degree. C. and
then rinsed 3 times with PBS, pH 7.4. Five hundred microliters of
1% SDS in PBS, pH 7.4, were added per well. Cell lysates were then
collected and added to individual vials containing 5 mL of
scintillation cocktail, and then counted for radioactivity.
Negative control tubes contained only the .sup.3H-folic acid in
FFRPMI (no competitor). Positive control tubes contained a final
concentration of 1 mM folic acid, and CPMs measured in these
samples (representing non-specific binding of label) were
subtracted from all samples. Notably, relative affinities were
defined as the inverse molar ratio of compound required to displace
50% of .sup.3H-folic acid bound to the FR on KB cells, and the
relative affinity of folic acid for the FR was set to 1.
[0252] The relative affinity assay results in 10% serum/FDRPMI for
EC0305 are shown in the FIG. 4. Compared to folic acid, EC0305
shown 96% relative affinity for the folate receptor.
[0253] METHOD: Inhibition of Cellular DNA Synthesis. The compounds
described herein were evaluated using an in vitro cytotoxicity
assay that predicts the ability of the drug to inhibit the growth
of folate receptor-positive KB cells. The compounds were comprised
of folate linked to a respective chemotherapeutic drug, as prepared
according to the protocols described herein. The KB cells were
exposed for up to 7 h at 37.degree. C. to the indicated
concentrations of folate-drug conjugate in the absence or presence
of at least a 100-fold excess of folic acid. The cells were then
rinsed once with fresh culture medium and incubated in fresh
culture medium for 72 hours at 37.degree. C. Cell viability was
assessed using a .sup.3H-thymidine incorporation assay. For
compounds described herein, dose-dependent cytotoxicity was
generally measurable, and in most cases, the IC.sub.50 values
(concentration of drug conjugate required to reduce
.sup.3H-thymidine incorporation into newly synthesized DNA by 50%)
were in the low nanomolar range. Furthermore, the cytotoxicities of
the conjugates were reduced in the presence of excess free folic
acid, indicating that the observed cell killing was mediated by
binding to the folate receptor.
[0254] For example, EC0305 exhibited dose-responsive behavior and
specificity for the folate receptor after a 2 hour pulse and a 72
hour chase, as shown in the FIG. 1. The IC.sub.50 for EC0305 was
about 1.5 nM. In addition, the cytotoxic activity of EC0305 was
blocked in the presence of an excess of folic acid, as also shown
in FIG. 1. Finally, EC0305 displayed no activity against
FR-negative cells. These results suggest that EC0305 is acting
through a folate selective or folate specific mechanism.
[0255] METHOD: In vitro test against the various cancer cell lines.
IC50 values were generated for various cell lines and the results
are shown in the table below. Cells are heavily seeded in 24-well
Falcon plates and allowed to form nearly confluent monolayers
overnight. Thirty minutes prior to the addition of the test
compound, spent medium is aspirated from all wells and replaced
with fresh folate-deficient RPMI medium (FFRPMI). A subset of wells
are designated to receive media containing 100 .mu.M folic acid.
The cells in the designated wells are used to determine the
targeting specificity. Without being bound by theory it is
suggested that the cytotoxic activity produced by test compounds in
the presence of excess folic acid, i.e. where there is competition
for FR binding, corresponds to the portion of the total activity
that is unrelated to FR-specific delivery. Following one rinse with
1 mL of fresh FFRPMI containing 10% heat-inactivated fetal calf
serum, each well receives 1 mL of medium containing increasing
concentrations of test compound (4 wells per sample) in the
presence or absence of 100 .mu.M free folic acid as indicated.
Treated cells are pulsed for 2 h at 37.degree. C., rinsed 4 times
with 0.5 mL of media, and then chased in 1 mL of fresh medium up to
70 h. Spent medium is aspirated from all wells and replaced with
fresh medium containing 5 .mu.Ci/mL.sup.3H-thymidine. Following a
further 2 h 37.degree. C. incubation, cells are washed 3 times with
0.5 mL of PBS and then treated with 0.5 mL of ice-cold 5%
trichloroacetic acid per well. After 15 min, the trichloroacetic
acid is aspirated and the cell material solubilized by the addition
of 0.5 mL of 0.25 N sodium hydroxide for 15 min. A 450 .mu.L
aliquot of each solubilized sample is transferred to a
scintillation vial containing 3 mL of Ecolume scintillation
cocktail and then counted in a liquid scintillation counter. Final
tabulated results are expressed as the percentage of
.sup.3H-thymidine incorporation relative to untreated controls.
[0256] Results for EC305 are shown in the following table:
TABLE-US-00007 Activity Blocked FR IC.sub.50 with Cell Model
Species Cancer Type Status (nM) Excess FA KB Human Nasophyngeal
Positive 1.5 Yes CA OVCAR-3 Human Ovarian CA Positive 1 Yes IGROV
Human Ovarian CA Positive 2 Yes RAW Mouse CML Positive 2.6 Yes
4T-1-FR Mouse Breast CA Positive 10 Yes 4T-1 Parent Mouse Breast
Negative >1000 n/a
Each of the cell lines is commercially available except for 4T-1
parent and 4T-1-FR, which were obtained from Rhone Poulenc
Rorer.
[0257] METHOD: Serum binding against different species. Compounds
are tested with 30K NMWL, subjected to Microcon filtration (10,000
g for 30 minutes), and compounds are detected by HPLC. EC0305 was
tested against various animal sera and exhibited low serum binding
in various species, as shown in the FIG. 2. In particular, EC0305
showed a low 67% binding in human serum.
[0258] METHOD: Human serum stability. EC0305 was tested in human
serum for stability and exhibited a half life of about 20 hours, as
shown in the FIG. 3.
[0259] METHOD: Inhibition of Tumor Growth in Mice. Four to seven
week-old mice (Balb/c or nu/nu strains) were purchased from Harlan
Sprague Dawley, Inc. (Indianapolis, Ind.). Normal rodent chow
contains a high concentration of folic acid (6 mg/kg chow);
accordingly, mice used were maintained on the folate-free diet
(Harlan diet #TD00434) for 1 week before tumor implantation to
achieve serum folate concentrations close to the range of normal
human serum. For tumor cell inoculation, 1.times.10.sup.6 M109
cells (Balb/c strain) or 1.times.10.sup.6 KB cells (nu/nu strain)
in 100 .mu.L were injected in the subcutis of the dorsal medial
area. Tumors were measured in two perpendicular directions every
2-3 days using a caliper, and their volumes were calculated as
0.5.times.L.times.W.sup.2, where L=measurement of longest axis in
mm and W=measurement of axis perpendicular to L in mm. Log cell
kill (LCK) and treated over control (T/C) values were then
calculated according to published procedures (see, e.g., Lee et
al., "BMS-247550: a novel epothilone analog with a mode of action
similar to paclitaxel but possessing superior antitumor efficacy"
Clin Cancer Res 7:1429-1437 (2001); Rose, "Taxol-based combination
chemotherapy and other in vivo preclinical antitumor studies" J
Natl Cancer Inst Monogr 47-53 (1993)). Dosing solutions were
prepared fresh each day in PBS and administered through the lateral
tail vein of the mice. Dosing was initiated when the s.c. tumors
had an average volume between 50-100 mm.sup.3 (t.sub.0), typically
8 days post tumor inoculation (PTI) for KB tumors, and 11 days PTI
for M109 tumors.
[0260] METHOD: Drug Toxicity determinations. Persistent drug
toxicity was assessed by collecting blood via cardiac puncture and
submitting the serum for independent analysis of blood urea
nitrogen (BUN), creatinine, total protein, AST-SGOT, ALT-SGPT plus
a standard hematological cell panel at Ani-Lytics, Inc.
(Gaithersburg, Md.). In addition, histopathologic evaluation of
formalin-fixed heart, lungs, liver, spleen, kidney, intestine,
skeletal muscle and bone (tibia/fibula) were conducted by
board-certified pathologists at Animal Reference Pathology
Laboratories (ARUP; Salt Lake City, Utah).
[0261] METHOD: General KB Tumor Assay. The anti-tumor activity of
the compounds described herein, when administered intravenously
(i.v.) to tumor-bearing animals, was evaluated in nu/nu mice
bearing subcutaneous KB tumors. Approximately 8 days post tumor
inoculation in the subcutis of the right axilla with
1.times.10.sup.6 KB cells (average tumor volume at t.sub.0=50-100
mm.sup.3), in mice (5/group) were injected i.v. three times a week
(TIW), for 3 weeks with 5 .mu.mol/kg of the drug delivery conjugate
or with an equivalent dose volume of PBS (control), unless
otherwise indicated. Tumor growth was measured using calipers at
2-day or 3-day intervals in each treatment group. Tumor volumes
were calculated using the equation V=a.times.b.sup.2/2, where "a"
is the length of the tumor and "b" is the width expressed in
millimeters.
[0262] METHOD: General M109 Tumors Assay. The anti-tumor activity
of the compounds described herein, when administered intravenously
(i.v.) to tumor-bearing animals, was evaluated in Balb/c mice
bearing subcutaneous M109 tumors (a syngeneic lung carcinoma).
Approximately 11 days post tumor inoculation in the subcutis of the
right axilla with 1.times.10.sup.6 M109 cells (average tumor volume
at t.sub.0=60 mm.sup.3), mice (5/group) were injected i.v. three
times a week (TIW), for 3 weeks with 1500 nmol/kg of the drug
delivery conjugate or with an equivalent dose volume of PBS
(control). Tumor growth was measured using calipers at 2-day or
3-day intervals in each treatment group. Tumor volumes were
calculated using the equation V=a.times.b.sup.2/2, where "a" is the
length of the tumor and "b" is the width expressed in
millimeters.
[0263] METHOD: General 4T-1 Tumor Assay. Six to seven week-old mice
(female Balb/c strain) were obtained from Harlan, Inc.,
Indianapolis, Ind. The mice were maintained on Harlan's folate-free
chow for a total of three weeks prior to the onset of and during
this experiment. Folate receptor-negative 4T-1 tumor cells
(1.times.10.sup.6 cells per animal) were inoculated in the subcutis
of the right axilla. Approximately 5 days post tumor inoculation
when the 4T-1 tumor average volume was .about.100 mm.sup.3, mice
(5/group) were injected i.v. three times a week (TIW), for 3 weeks
with 3 .mu.mol/kg of drug delivery conjugate or with an equivalent
dose volume of PBS (control), unless otherwise indicated herein.
Tumor growth was measured using calipers at 2-day or 3-day
intervals in each treatment group. Tumor volumes were calculated
using the equation V=a.times.b.sup.2/2, where "a" is the length of
the tumor and "b" is the width expressed in millimeters.
[0264] METHOD: Toxicity as Measured by Weight Loss. The percentage
weight change of the mice was determined in mice (5 mice/group) on
selected days post-tumor inoculation (PTI), and graphed.
[0265] METHOD: Alternate dosing schedule. Each of the foregoing
assays may be modified as follows: approximately 8 days post tumor
inoculation in the subcutis of the right axilla with
1.times.10.sup.6 KB cells (average tumor volume at t.sub.0=50-100
mm.sup.3), mice (5/group) are injected i.v. three times a week
(TIW), for 3 weeks with a drug delivery conjugate described herein,
or with an equivalent dose volume of PBS as control. Tumor growth
is measured using calipers at 2-day or 3-day intervals in each
treatment group. Tumor volumes were calculated using the equation
V=a.times.b.sup.2/2, where "a" is the length of the tumor and "b"
is the width expressed in millimeters.
[0266] METHOD: Alternate dosing schedule. Each of the foregoing
assays may be modified as follows: approximately 8 days post tumor
inoculation in the subcutis of the right axilla with
1.times.10.sup.6 KB cells (average tumor volume at t.sub.0=50-100
mm.sup.3), mice (5/group) are injected i.v. five times a week on
Monday through Friday for 2 or 3 weeks with a drug delivery
conjugate described herein, or with an equivalent dose volume of
PBS as control. Tumor growth is measured using calipers at 2-day or
3-day intervals in each treatment group. Tumor volumes were
calculated using the equation V=a.times.b.sup.2/2, where "a" is the
length of the tumor and "b" is the width expressed in
millimeters.
[0267] EC305 was tested at TIW on a two week schedule at various
doses, and showed complete responses in 5 of 5 animals tested at a
dose at or above 1 .mu.mol/kg, as shown in the FIG. 5. In FIG. 5,
the vertical dotted line indicates the last day of dosing. In
addition, no recurrence or regrowth of the tumors was observed
during the entire observation period for those doses in 5 of 5
animals, despite that the last administration of conjugate was
given more than 70 days earlier, as also shown in the FIG. 5. In
contrast, as also shown in the FIG. 5, the anti-tumor activity of
EC0305 was completely abolished (0/5 responses) in EC0305-treated
animals that were co-dosed with a competing but tumor inactive
folate-containing analog EC20 (rhenium complex). EC20 (rhenium
complex) is the compound of the formula
##STR00165##
chelated to Rhenium. The preparation of EC20 is described in U.S.
patent application publication no. US 2004/0033195 A1, the
synthetic procedure description of which is incorporated herein by
reference. It is believed that EC20 acts as a competitor of folate
targeted conjugates, such as EC0305 at folate receptors, and that
therefore the results show the specificity of the effects of EC0305
in targeting the folate receptor.
[0268] In addition, the observed activity occurred in the apparent
absence of weight loss or major organ tissue degeneration, as shown
in the FIG. 6, where the vertical dotted line indicates the last
day of dosing.
[0269] EC305 was repeat tested at 2 .mu.mol/kg TIW on a two week
schedule and again showed complete responses in 5 of 5 animals
tested. In addition, no recurrence or regrowth of the tumors was
observed in 5 of 5 animals during the entire observation period of
greater than 90 days, as shown in the FIG. 7. In addition, the
observed activity occurred in the apparent absence of weight loss
or major organ tissue degeneration, as shown in the FIG. 8.
[0270] EC0305 activity was evaluated against FR-positive tumors in
mice. Balb/c mice bearing subcutaneous M109 tumors were treated
intravenously with EC0305, and in this therapy, complete responses
were observed in 5 of 5 test animals. In addition, even after more
than 90 days post tumor implantation, and more than 70 days after
treatment was discontinued, 5 of 5 test animals remained free of
any measurable amounts of tumor. No recurrence or regrowth of the
tumors was observed in 5 of 5 animals. Moreover, this observed
activity in complete response and non-recurrence of disease,
occurred in the apparent absence of weight loss or major organ
tissue degeneration. The potency of EC0305 across tumor types was
confirmed in a human KB xenograft-nu/nu mice cancer model, as
described herein. EC0305 again displayed remarkable anti-tumor
activity (5/5 complete responses) in the apparent absence of weight
loss or major organ tissue degeneration.
[0271] In contrast to the results observed for the conjugates
described herein, the unconjugated tubulysin B free drug
##STR00166##
was found to be completely inactive (0/5 responses) at both
tolerable and highly toxic dose levels, as shown in the FIG. 9
(dosing was terminated early in each cohort due to excessive
toxicity of the unconjugated drug). FIG. 10 shows the dramatic
change in percent body weight of animals treated with unconjugated
tubulysin B, as compared to controls. As indicated in FIGS. 9 and
10, dosing was terminated early in each cohort due to excessive
toxicity of the unconjugated drug.
[0272] In addition, the tubulysin conjugate EC0305 was found to be
more efficacious than another folate targeted compound, EC145
having the following structure
##STR00167##
where the drug payload in the latter is a vinca alkaloid. Each was
dosed at 2 .mu.mol/kg TIW on a two-week schedule, where the
vertical line indicates the last day of dosing, as shown in the
FIG. 11. The vinca conjugate EC145 showed 2 of 5 complete responses
in treated animals, while the tubulysin conjugate EC0305 showed 5
of 5 complete responses. In addition, no recurrence or regrowth of
the tumors was observed in 5 of 5 animals treated with EC0305 over
the entire 90 plus day observation period.
[0273] FIG. 12 shows the relative activity of two different
tubulysin conjugates, EC0305 and EC0436, on M109 tumors compared to
controls. Treatment was initiated approximately 11 days after tumor
implantation, and each test animal received 2 .mu.mol/kg of EC0305
or EC0436 three times per week for two weeks. The vertical dotted
line in FIG. 12 shows that the last day of dosing was on day 20. As
shown in FIG. 12, both EC0305 and EC0436 showed complete responses
in all animals. However, near about day 35 PTI, the EC0305 treated
animals began to show tumor regrowth. In contrast, the EC0436
treated animals not only showed complete responses in 5 of 5
treated animals, but there was no tumor recurrence or regrowth
observed in the entire 60-plus day observation period. FIG. 13
shows the percent weight change in treated animals, as compared to
controls. In all treated animals, the observed efficacy was not
accompanied by any observed gross toxicity as determined by changes
in weight of the test animals.
[0274] FIG. 14 shows the relative toxicity of two different
tubulysin conjugates, EC0305 and EC0436, at doses above their
therapeutic doses, as compared to PBS treated controls ( ). Each
dose was administered three times, every other day, as indicated by
the arrows. EC0305 was administered at (.DELTA.) 2 .mu.mol/kg TIW;
(.gradient.) 2.5 .mu.mol/kg TIW; and (0) 3 .mu.mol/kg. EC0436 was
administered at (.tangle-solidup.) 2 .mu.mol/kg TIW; () 2.5
.mu.mol/kg TIW; and (.box-solid.) 3 .mu.mol/kg TIW. The data
suggests that EC0436 may have a higher therapeutic index than
EC0305. As shown in FIG. 12, EC0305 provides 4 of 5 complete
responses at 2 .mu.mol/kg, while EC0436 provides 5 of 5 complete
responses at the same dose. However, EC0305 begins to show
toxicity, as determined by changes in weight of the test animals
and as shown in FIG. 14, at doses at or above 2.5 .mu.mol/kg. In
contrast, no toxicity, as determined by changes in weight of the
test animals, was observed with EC0436 even at the highest dose of
3 .mu.mol/kg.
[0275] The foregoing exemplary embodiments are set forth to provide
a more detailed description of certain aspects of the invention
described herein. However, the foregoing are intended to be
illustrative and accordingly should not be construed as limiting
the invention in any way.
* * * * *