U.S. patent application number 12/553649 was filed with the patent office on 2010-04-01 for transporters comprising spaced arginine moieties.
Invention is credited to Erik L. Kreider, Jonathan B. Rothbard, Christopher L. VanDeusen, Paul A. Wender, Lee Wright.
Application Number | 20100081618 12/553649 |
Document ID | / |
Family ID | 23028023 |
Filed Date | 2010-04-01 |
United States Patent
Application |
20100081618 |
Kind Code |
A1 |
Wender; Paul A. ; et
al. |
April 1, 2010 |
Transporters Comprising Spaced Arginine Moieties
Abstract
The present invention provides compositions and methods for
enhancing transport of biologically active compounds across
biological membranes and across and into animal epithelial or
endothelial tissues. The composition includes a biologically active
agent and a transport moiety. The transport moiety includes a
structure selected from the group consisting of (ZYZ).sub.nZ,
(ZY).sub.nZ, (ZYY).sub.nZ and (ZYYY).sub.nZ. Subunit "Z" is
L-arginine or D-arginine, and subunit "Y" is an amino acid that
does not comprise an amidino or guanidino moiety. Subscript "n" is
an integer ranging from 2 to 10. The method for enhancing transport
involves the administration of the aforementioned composition.
Inventors: |
Wender; Paul A.; (Menlo
Park, CA) ; Rothbard; Jonathan B.; (Cupertino,
CA) ; Wright; Lee; (Redwood City, CA) ;
Kreider; Erik L.; (Sunnyvale, CA) ; VanDeusen;
Christopher L.; (Columbus, OH) |
Correspondence
Address: |
MINTZ, LEVIN, COHN, FERRIS, GLOVSKY AND POPEO, P.C
One Financial Center
BOSTON
MA
02111
US
|
Family ID: |
23028023 |
Appl. No.: |
12/553649 |
Filed: |
September 3, 2009 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
10078247 |
Feb 14, 2002 |
7585834 |
|
|
12553649 |
|
|
|
|
60269627 |
Feb 16, 2001 |
|
|
|
Current U.S.
Class: |
514/1.1 |
Current CPC
Class: |
A61P 35/00 20180101;
A61K 47/64 20170801; A61P 31/12 20180101 |
Class at
Publication: |
514/12 ; 514/17;
514/16; 514/15; 514/14; 514/13 |
International
Class: |
A61K 38/16 20060101
A61K038/16; A61K 38/10 20060101 A61K038/10; A61K 38/08 20060101
A61K038/08; A61P 43/00 20060101 A61P043/00 |
Goverment Interests
STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSORED
RESEARCH AND DEVELOPMENT
[0002] This invention was made with the support of NIH grants
numbered CA31841, CA31845, and CA65237. Accordingly, the U.S.
Government has certain rights in this invention.
Claims
1. A composition comprising a biologically active compound and a
transport moiety, wherein the transport moiety comprises a
structure selected from the group consisting of (ZYZ).sub.nZ,
(ZY).sub.nZ, (ZYY).sub.nZ and (ZYYY).sub.nZ, wherein each Z is
L-arginine or D-arginine, and each Y is independently an amino acid
that does not comprise an amidino or guanidino moiety, and wherein
n is an integer of from 2 to 10.
2. The composition according to claim 1, wherein each Y is
independently selected from the group consisting of alanine,
cysteine, aspartic acid, glutamic acid, phenylalanine, glycine,
histidine, isoleucine, lysine, leucine, methionine, asparagine,
proline, glutamine, serine, threonine, valine, tryptophan,
hydroxyproline, tyrosine, .gamma.-amino butyric acid,
.beta.-alanine, sarcosine and .epsilon.-amino caproic acid.
3. The composition according to claim 1, wherein the transport
moiety comprises the structure (ZYZ).sub.nZ, and wherein n is an
integer ranging from 2 to 5.
4. The composition according to claim 1, wherein the transport
moiety comprises the structure (ZY).sub.nZ, and wherein n is an
integer ranging from 4 to 10.
5. The composition according to claim 1, wherein the transport
moiety comprises the structure (ZYY).sub.nZ, and wherein n is an
integer ranging from 4 to 10.
6. The composition according to claim 1, wherein the transport
moiety comprises the structure (ZYYY).sub.nZ, and wherein n is an
integer ranging from 4 to 10.
7. The composition according to claim 1, wherein the transport
moiety is attached to the biologically active compound by a linking
moiety to form a conjugate.
8. The composition according to claim 1, wherein Y is a
gene-encoded amino acid.
9. The composition according to claim 1, wherein Y is an amino acid
other than a gene-encoded amino acid.
10. The composition according to claim 3, wherein each Y is
independently selected from the group consisting of glycine,
.gamma.-amino butyric acid, .beta.-alanine and .epsilon.-amino
caproic acid, and n is 3 or 4.
11. The composition according to claim 4, wherein each Y is
independently selected from the group consisting of glycine,
.gamma.-amino butyric acid, .beta.-alanine and .epsilon.-amino
caproic acid, and n is 6, 7 or 8.
12. The composition according to claim 5, wherein each Y is
independently selected from the group consisting of glycine,
.gamma.-amino butyric acid, .beta.-alanine and .epsilon.-amino
caproic acid, and n is 6, 7 or 8.
13. The composition according to claim 6, wherein each Y is
independently selected from the group consisting of glycine,
.gamma.-amino butyric acid, .beta.-alanine and .epsilon.-amino
caproic acid, and n is 6, 7 or 8.
14. The composition according to claim 7, wherein the conjugate has
the following structure: ##STR00017## wherein: R.sup.1 is the
biologically active compound; X is a linkage between a functional
group on the biologically active compound and a functional group on
the linker between R.sup.1 and R.sup.3; Y is a linkage between a
functional group on the transport moiety and a functional group on
the linker between R.sup.1 and R.sup.3; A is N or CH; R.sup.2 is
hydrogen, alkyl, aryl, arylalkyl, acyl or allyl; R.sup.3 is a
transport moiety; k and m are independently either 1 or 2; and n is
an integer of from 1 to 10.
15. The composition according to claim 14, wherein each of X and Y
is independently selected from the group consisting of --C(O)O--,
--C(O)NH--, --OC(O)NH--, --S--S--, --C(S)O--, --C(S)NH--,
--NHC(O)NH--, --SO.sub.2NH--, --SONH--, phosphate, phosphonate and
phosphinate.
16. The composition according to claim 14, wherein each of X and Y
is independently selected from the group consisting of --C(O)O--,
--C(O)NH--, --OC(O)NH-- and --NHC(O)NH--.
17. The composition according to claim 7, wherein the conjugate has
the following structure: ##STR00018## wherein: R.sup.1 is the
biologically active compound; X is a linkage between a functional
group on the biologically active compound and a functional group on
the linker between R.sup.1 and R.sup.3; Y is a linkage between a
functional group on the transport moiety and a functional group on
the linker between R.sup.1 and R.sup.3; R.sup.3 is a transport
moiety; R.sup.4 is S, O, NR.sup.6 or CR.sup.7R.sup.8; R.sup.5 is
OH, SH or NHR.sup.6; R.sup.6 is hydrogen, alkyl, aryl, arylalkyl,
acyl or allyl; R.sup.7 and R.sup.8 are independently hydrogen,
alkyl or arylalkyl; and k and m are independently either 1 or
2.
18. The composition according to claim 17, wherein each of X and Y
is independently selected from the group consisting of --C(O)O--,
--C(O)NH--, --OC(O)NH--, --S--S--, --C(S)O--, --C(S)NH--,
--NHC(O)NH--, --SO.sub.2NH--, --SONH--, phosphate, phosphonate and
phosphinate.
19. The composition according to claim 17, wherein each of X and Y
is independently selected from the group consisting of --C(O)O--,
--C(O)NH--, --OC(O)NH-- and --NHC(O)NH--.
20. The composition according to claim 7, wherein the conjugate has
the following structure: ##STR00019## wherein: R.sup.1 is the
biologically active compound; X is a linkage between a functional
group on the biologically active compound and a functional group on
the linker between R.sup.1 and R.sup.3; Y is a linkage between a
functional group on the transport moiety and a functional group on
the linker between R.sup.1 and R.sup.3; R.sup.3 is the transport
moiety; R.sup.5 is H, OH, SH or NHR.sup.6; R.sup.6 is hydrogen,
alkyl, aryl, arylalkyl, acyl or allyl; and k is 1 or 2.
21. The composition according to claim 20, wherein each of X and Y
is independently selected from the group consisting of --C(O)O--,
--C(O)NH--, --OC(O)NH--, --S--S--, --C(S)O--, --C(S)NH--,
--NHC(O)NH--, --SO.sub.2NH--, --SONH--, phosphate, phosphonate and
phosphinate.
22. The composition according to claim 20, wherein each of X and Y
is independently selected from the group consisting of --C(O)O--,
--C(O)NH--, --OC(O)NH-- and --NHC(O)NH--.
23. The composition according to claim 7, wherein the conjugate has
the following structure: ##STR00020## wherein: R.sup.1 is the
biologically active compound; X is a linkage between a functional
group on the biologically active compound and a functional group on
the linker between R.sup.1 and R.sup.3; Y is a linkage between a
functional group on the transport moiety and a functional group on
the linker between R.sup.1 and R.sup.3; Ar is a substituted or
unsubstituted aryl group, wherein the methylene and oxygen
substituents are either ortho or para to one another; R.sup.3 is
the transport moiety; R.sup.4 is S, O, NR.sup.6 or CR.sup.7R.sup.8;
R.sup.5 is H, OH, SH, CONHR.sup.6 or NHR.sup.6; R.sup.6 is
hydrogen, alkyl, aryl, arylalkyl, acyl or allyl; R.sup.7 and
R.sup.8 are independently hydrogen or alkyl; and, k and m are
independently either 1 or 2.
24. The composition according to claim 23, wherein each of X and Y
is independently selected from the group consisting of --C(O)O--,
--C(O)NH--, --OC(O)NH--, --S--S--, --C(S)O--, --C(S)NH--,
--NHC(O)NH--, --SO.sub.2NH--, --SONH--, phosphate, phosphonate and
phosphinate.
25. The composition according to claim 23, wherein each of X and Y
is independently selected from the group consisting of --C(O)O--,
--C(O)NH--, --OC(O)NH-- and --NHC(O)NH--.
26. The composition according to claim 12, wherein A is N, R.sup.2
is benzyl, k, m and n are 1, and X is --C(O)O--.
27. The composition according to claim 13, wherein R.sup.4 is S,
R.sup.5 is NHR.sup.6, R.sup.6 is hydrogen, methyl, allyl, butyl or
phenyl, k and m are 1 and X is --C(O)O--.
28. The composition according to claim 14, wherein R.sup.5 is
NHR.sup.6, R.sup.6 is hydrogen, methyl, allyl, butyl or phenyl, k
is 2 and X is --C(O)O--.
29. The composition according to claim 15, wherein Ar is an
unsubstituted aryl group, R.sup.4 is S, R.sup.5 is NHR.sup.6,
R.sup.6 is hydrogen, methyl, allyl, butyl or phenyl, k and m are 1
and X is --C(O)O--.
30. A method for increasing the transport of a biologically active
compound across a biological membrane comprising: administering a
composition comprising a biologically active compound and a
transport moiety, wherein the transport compound comprises a
structure selected from the group consisting of (ZYZ).sub.nZ,
(ZY).sub.nZ, (ZYY).sub.nZ and (ZYYY).sub.nZ, wherein Z is
L-arginine or D-arginine, and wherein Y is an amino acid that does
not comprise an amidino or guanidino moiety, and wherein n is an
integer ranging from 2 to 10, wherein transport of the biologically
active compound across the biological membrane is increased
relative to transport of the biologically active compound in the
absence of said transport moiety.
31. The method according to claim 20, wherein the biologically
active compound is attached to the transport moiety by a linking
moiety to form a conjugate.
32. The method of claim 21, wherein the conjugate has the following
structure: ##STR00021## wherein: R.sup.1 is the biologically active
compound; X is a linkage between a functional group on the
biologically active compound and a functional group on the linker
between R.sup.1 and R.sup.3; Y is a linkage between a functional
group on the transport moiety and a functional group on the linker
between R.sup.1 and R.sup.3; A is N or CH; R.sup.2 is hydrogen,
alkyl, aryl, arylalkyl, acyl or allyl; R.sup.3 is a transport
moiety; k and m are independently either 1 or 2; and n is an
integer of from 1 to 10.
33. The method of claim 21, wherein the conjugate has the following
structure: ##STR00022## wherein: R.sup.1 is the biologically active
compound; X is a linkage between a functional group on the
biologically active compound and a functional group on the linker
between R.sup.1 and R.sup.3; Y is a linkage between a functional
group on the transport moiety and a functional group on the linker
between R.sup.1 and R.sup.3; R.sup.3 is a transport moiety; R.sup.4
is S, O, NR.sup.6 or CR.sup.7R.sup.8; R.sup.5 is OH, SH or
NHR.sup.6; R.sup.6 is hydrogen, alkyl, aryl, arylalkyl, acyl or
allyl; R.sup.7 and R.sup.8 are independently hydrogen, alkyl or
arylalkyl; and k and m are independently either 1 or 2.
34. The method of claim 21, wherein the conjugate has the following
structure: ##STR00023## wherein: R.sup.1 is the biologically active
compound; X is a linkage between a functional group on the
biologically active compound and a functional group on the linker
between R.sup.1 and R.sup.3; Y is a linkage between a functional
group on the transport moiety and a functional group on the linker
between R.sup.1 and R.sup.3; R.sup.3 is the transport moiety;
R.sup.5 is H, OH, SH or NHR.sup.6; R.sup.6 is hydrogen, alkyl,
aryl, arylalkyl, acyl or allyl; and k is 1 or 2.
35. The method of claim 21, wherein the conjugate is of the
following structure: ##STR00024## wherein: R.sup.1 is the
biologically active compound; X is a linkage between a functional
group on the biologically active compound and a functional group on
the linker between R.sup.1 and R.sup.3; Y is a linkage between a
functional group on the transport moiety and a functional group on
the linker between R.sup.1 and R.sup.3; Ar is a substituted or
unsubstituted aryl group, wherein the methylene and oxygen
substituents are either ortho or para to one another; R.sup.3 is
the transport moiety; R.sup.4 is S, O, NR.sup.6 or CR.sup.7R.sup.8;
R.sup.5 is H, OH, SH, CONHR.sup.6 or NHR.sup.6; R.sup.6 is
hydrogen, alkyl, aryl, arylalkyl, acyl or allyl; R.sup.7 and
R.sup.8 are independently hydrogen or alkyl; and, k and m are
independently either 1 or 2.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit of U.S. Ser. No.
10/078,247, filed Feb. 14, 2002, which claims the benefit of U.S.
Ser. No. 60/269,627, filed Feb. 16, 2001. The disclosures of the
aforementioned applications are incorporated in their entireties by
reference herein.
BACKGROUND OF THE INVENTION
[0003] There are a number of natural barriers that exist in living
organisms. The barriers most likely developed over time as a
mechanism to exclude harmful stimuli, such as toxic chemicals, from
sensitive biochemical pathways. Cellular membranes, epithelial
tissues and endothelial tissues are examples of such barriers.
[0004] Unfortunately, barriers such as cellular membranes function
by excluding chemical compounds possessing particular physical
characteristics rather than by specifically repelling harmful
compositions. This can be problematic in the field of drug
discovery where one is targeting a biopolymer encased in the
barrier. Cellular membranes, for instance, are particularly
intractable to highly charged compounds such as polynucleotides.
When one administers a highly charged compound (e.g.,
oligonucleotide) to an organism, its medicinal utility is hindered
by its inability to efficiently access its intracellular
target.
[0005] Researchers have attempted to develop technologies for
enhancing the transport of chemical compounds across organismal
barriers. For instance, Ryser et al. discusses the use of high
molecular weight lysine polymers for increasing the transport of
various molecules across cellular membranes. See, Ryser, H. J. P.,
PCT Pub. No. WO 79/00515 (1979). Frankel et al. reports the
conjugation of selected molecules to HIV tat protein, which
increased cellular uptake of the molecules. See, Frankel et al.,
PCT Pub. No. WO 91/09958 (1991). Barsoum discusses the use of the
HIV tat sequence RKKRRQRRR (SEQ ID NO:1) in enhancing cellular
membrane transport. See, Barsoum et al., PCT Pub. No. WO 94/04686
(1994). Brugidou et al. report the rapid internalization of a 16
amino acid peptide-cholesterol conjugate derived from the
Antennapedia homeodomain by cultured neurons. See, Brugidou, J., et
al. Biochem. Biophys. Res. Comm. 214(2):685-693 (1995).
[0006] Several methods have also been proposed to enhance the
transport of compounds across epithelial tissues. For example,
chemical enhancers (Burnette, R.R. In Developmental Issues and
Research Initiatives; Hadgraft J., Ed., Marcel Dekker: 1989, pp.
247-288) and iontophoresis have been used for the transdermal
transport of drugs. These efforts have produced fewer than a dozen
drugs for transdermal administration.
[0007] More recently, methods and compositions have been described
for transporting drugs and macromolecules across biological
membranes in which the drug or macromolecule is covalently attached
to a transport polymer consisting of from 6 to 25 subunits, at
least 50% of which contain a guanidino or amidino side chain. The
transport polymers are preferably polyarginine peptides composed of
all D-, all L- or mixtures of D- and L-arginine subunits (see
PCT/US/10571, WO/52614, published 26 Nov. 1998).
[0008] Despite the above, a need exists for new compositions and
methods for enhancing the transport of compounds across biological
barriers. The present invention fulfills this and other needs.
SUMMARY OF THE INVENTION
[0009] The present invention provides compositions and methods for
enhancing transport of biologically active compounds across
biological membranes and across and into animal epithelial or
endothelial tissues. The composition includes a biologically active
agent and a transport moiety, either linked covalently by a bond or
an optional linking group. The transport moiety includes a
structure selected from the group consisting of (ZYZ).sub.nZ,
(ZY).sub.nZ, (ZYY).sub.nZ and (ZYYY).sub.nZ. Subunit "Z" is
L-arginine or D-arginine, and subunit "Y" is an amino acid that
does not comprise an amidino or guanidino moiety. Subscript "n" is
an integer ranging from 2 to 10. The method for enhancing transport
involves the administration of the aforementioned composition.
BRIEF DESCRIPTION OF THE DRAWINGS
[0010] FIG. 1 is a bar graph that illustrates the increase in
cellular uptake of a fluorescence agent when a transport oligomer
of spaced arginine residues is used relative to the uptake when an
R.sup.7 homopolymer is the transport oligomer. Uptake increased
with increased spacing (glycine insert<aminobutyric acid
insert<aminocaproic acid insert).
[0011] FIG. 2 is a bar graph that illustrates a general increase in
cellular uptake of a fluoresence agent when a transport oligomer of
spaced arginine residues is used relative to the uptake when an
R.sup.7 homopolymer is the transport oligomer. The spacing groups
are naturally-occurring amino acids.
[0012] FIG. 3 is a bar graph as in FIG. 2 in which the
concentrations of the various transport compositions has been
reduced, illustrating that the increase in cellular uptake does not
change at lower concentrations.
[0013] FIG. 4 is a bar graph illustrating the cellular uptake of
the compositions used in FIGS. 2 and 3 at 6.25 micromolar. At this
concentration, the uptake of Fl-aca-R.sup.7CONH.sub.2 begins to
drop off, while the compositions of the present invention maintain
a significant uptake advantage.
[0014] FIG. 5 is a bar graph showing the increase in cellular
uptake of the series (Raca).sub.xR at 50 .mu.M when the subscript x
is 4, 5 and 6. For brevity, the attached Fl-aca in each of the
transport agents is not shown.
[0015] FIG. 6 is a bar graph showing the uptake (as determined by
mean fluorescence) of (RX).sub.6R transport oligomers versus
homopolymers of arginine (r19 and r13). In this Figure the symbol X
represents aminocaproic acid, gly-gly, or gly. For brevity, the
attached Fl-aca in each of the transport agents is not shown.
[0016] FIG. 7 illustrates the sixty permutations of aca-spaced
arginine heptamers that were prepared and evaluated (results shown
in FIG. 8).
[0017] FIG. 8 is a bar graph showing the relative uptake of R aca
spaced analogs relative to r7.
[0018] FIG. 9 illustrates the conjugation of cyclosporin to r7-acid
using a self-immolating linker (see 9A). The mechanism for cleavage
of the linker and release of cyclosporine as the pH decreases to
5.5 is shown in FIG. 9B.
[0019] FIG. 10 illustrates the conjugation of acyclovir to r7-amide
via an N-terminal cysteine group. Conjugation with a
biotin-containing transporter is also shown.
[0020] FIG. 11 illustrates the conjugation of hydrocortisone to
r7-amide via an N-terminal cysteine group. Conjugation with a
biotin-containing transporter is also shown.
[0021] FIG. 12 illustrates the conjugation of taxol to r7-amide via
an N-terminal cysteine group.
[0022] FIG. 13 illustrates the conjugate formed between a retinal
and a r9 (shown without spacing amino acids).
[0023] FIG. 14 illustrates the use of a cleavable linker in
preparing a retinoic acid-r9 conjugate.
DETAILED DESCRIPTION OF THE INVENTION
A. Abbreviations and Definitions
[0024] The following abbreviations are used herein: abu,
.gamma.-aminobutyric acid; aca, .epsilon.-aminocaproic acid; Fl,
fluorescein; Fmoc, fluorenylmethoxycarbonyl. Single letter or three
letter abbreviations are used for many amino acid herein in
accordance with acceptable convention. Lower case single letter
abbreviations for amino acids are intended to denote the D-isomer,
also in accordance with conventional use.
[0025] The term "alkyl" refers to unsubstituted or substituted
linear, branched, cyclic, or combinations of alkyl carbon chains of
up to 15 carbon atoms. Linear alkyl groups include, for example,
methyl, ethyl, n-propyl, n-butyl, n-pentyl, n-hexyl, n-heptyl and
n-octyl. Branched alkyl groups include, for example, iso-propyl,
sec-butyl, iso-butyl, tert-butyl and neopentyl. Cyclic alkyl groups
include, for example, cyclopropyl, cyclobutyl, cyclopentyl and
cyclohexyl. Combinations of alkyl groups are also within the scope
of the present definition and invention (e.g., cyclopropylmethyl,
cyclohexylmethyl, cyclobutylpropyl, and the like). The alkyl groups
can also be substituted with one or more substituents, preferably
from zero to three substituents. Nonlimiting examples of such
substituents include NH.sub.2, SH, NO.sub.2, ONO.sub.2, F, Cl, Br,
I, OH, CO.sub.2H, CO.sub.2CH.sub.3, CN, alkoxy, alkylthio,
alkylamino, dialkylamino, aryl and heteroaryl.
[0026] The term "aryl" refers to an unsubstituted or substituted
aromatic, carbocyclic group. Aryl groups are either single ring or
multiple condensed ring compounds. A phenyl group, for example, is
a single ring, aryl group. An aryl group with multiple condensed
rings is exemplified by a naphthyl group. Aryl groups can be
substituted with one or more substituents, preferably from zero to
three substitutents. Nonlimiting examples of such substituents
include NH.sub.2, SH, NO.sub.2, ONO.sub.2, F, Cl, Br, I, OH,
CO.sub.2H, CO.sub.2CH.sub.3, CN, alkoxy, alkylthio, alkylamino,
dialkylamino, aryl and heteroaryl.
[0027] The term "heteroaryl" refers to those aryl groups in which
one or more heteroatoms (e.g., O, S, N) occupies a position held by
a carbon atom or pair of carbon atoms. Examples of suitable
heteroaryl groups include pyridine, thiophene, oxazole,
benzimidazole, pyrrole, furan, quinoline, quinazoline, and the
like.
[0028] An "epithelial tissue" refers to the basic tissue that
covers surface areas of the surface, spaces, and cavities of the
body. Epithelial tissues are composed primarily of epithelial cells
that are attached to one another and rest on an extracellular
matrix (basement membrane) that is typically produced by the cells.
Epithelial tissues include three general types based on cell shape:
squamous, cuboidal, and columnar epithelium. Squamous epithelium,
which lines lungs and blood vessels, is made up of flat cells.
Cuboidal epithelium lines kidney tubules and is composed of cube
shaped cells, while columnar epithelium cells line the digestive
tract and have a columnar appearance. Epithelial tissues can also
be classified based on the number of cell layers in the tissue. For
example, a simple epithelial tissue is composed of a single layer
of cells, each of which sits on the basement membrane. A
"stratified" epithelial tissue is composed of several cells stacked
upon one another; not all cells contact the basement membrane. A
"pseudostratified" epithelial tissue has cells that, although all
contact the basement membrane, appear to be stratified because the
nuclei are at various levels.
[0029] The term "trans-epithelial" delivery or administration
refers to the delivery or administration of agents by permeation
through and/or into one or more layers of a body surface or tissue,
such as intact skin or a mucous membrane, by topical
administration. Thus, the term is intended to include both
transdermal (e.g., percutaneous adsorption, and including
intradermal) and transmucosal administration. Delivery can be to a
deeper layer of the tissue, for example, and/or delivery to the
bloodstream.
[0030] "Transport enhancement," refers to an increase in amount
and/or rate of delivery of a biologically active compound across a
biological barrier. The term is also meant to include altering
tissue distribution, localization and release of a biologically
active compound or agent. The amount of transport enhancement is
determined by comparing the delivery of the compound or agent that
is not attached to a transport moiety to the delivery of one that
is.
[0031] "Biological barrier" refers to a physiological barrier to
the delivery of a biologically active compound to its intended
target site. It includes, for example, biological membranes,
epithelial tissue and endothelial tissue.
[0032] "Biological membrane" refers to a lipid-containing barrier
that separates cells or groups of cells from extracellular
space.
[0033] "Biologically active compound" refers to a therapeutic
compound or a diagnostic agent, as well as lead compounds in a
research and development setting. Still further the term is meant
to include various probes (e.g., oligonucleotides alone or those
having attached imaging agents).
[0034] The term "therapeutic compound" refers, without limitation,
to any composition that can be used to the benefit of a mammalian
species. A number of such agents cause an observable change in the
structure, function or composition of a cell upon uptake by the
cell. Observable changes include increased or decreased expression
of one or more mRNAs, increased or decreased expression of one or
more proteins, phosphorylation of a protein or other cell
component, inhibition or activation of an enzyme, inhibition or
activation of binding between members of a binding pair, an
increased rate of synthesis of a metabolite, increased or decreased
cell proliferation and the like. Other agents exert therapeutic
effects when present in a tissue, even in the absence of cellular
entry.
[0035] The term "diagnostic agent" refers to both diagnostic
imaging agents and contrast agents. The following are non-limiting
examples of diagnostic agents: radio-labeled substances such as
.sup.99mTc glucoheptonate; substances used in magnetic resonance
imaging such as gadolinium doped chelation agents (e.g., Gd-DTPA);
metals bound to chelating agents such as Eu, Lu, Pr, Gd,
Tc.sup.99m, Ga.sup.67.sub., In.sup.111, Y.sup.90, Cu.sup.67 and
Co.sup.57; and proteins such as .beta.-galactosidase, green
fluorescent protein and luciferase. Other diagnostic agents include
molecular sensors.
[0036] The term "macromolecule" refers to large molecules (MW
greater than 1000 daltons) exemplified by, but not limited to,
peptides, proteins, oligonucleotides and polynucleotides of
biological or synthetic origin.
[0037] "Small organic molecule" refers to a carbon-containing agent
having a molecular weight (MW) of less than or equal to 1000
daltons.
[0038] The term "peptide" refers to a compound made up of a single
chain of D- or L-amino acids or a mixture of D- and L-amino acids
joined by peptide bonds. Generally, peptides contain at least two
amino acid residues and are less than about 50 amino acids in
length. D-amino acids are represented herein by a lower-case
one-letter amino acid symbol (e.g., r for D-arginine), whereas
L-amino acids are represented by an upper case one-letter amino
acid symbol (e.g., R for L-arginine). Homopolymer peptides are
represented by a one-letter amino acid symbol followed by the
number of consecutive occurrences of that amino acid in the
peptide--(e.g., R.sup.7 represents a heptamer that consists of
L-arginine residues).
[0039] The term "protein" refers to a compound that is composed of
linearly arranged amino acids linked by peptide bonds, but in
contrast to peptides, has a well-defined conformation. Proteins, as
opposed to peptides, generally consist of chains of 50 or more
amino acids.
[0040] "Polypeptide" refers to a oligomer of at least two amino
acid residues and which contains one or more peptide bonds.
"Polypeptide" encompasses peptides and proteins, regardless of
whether the polypeptide has a well-defined conformation.
[0041] The term "half-life" generally refers to the time required
for half the quantity of a compound to be destroyed, degraded or
eliminated. In the context of self-immolating linkers, "half-life"
refers to the time required for half the quantity of a transport
moiety-biologically active compound conjugate containing such a
linker to undergo intramolecular cleavage.
B. General
[0042] The present invention relates to the surprising discovery
that arginine residues provide an enhanced transport of drugs and
other agents across biological membranes when the residues are part
of a polypeptide that provides suitable spacing between the
arginines. This is in contrast to the previously described polymers
of, for example, arginine, in which the guanidino moieties are
present on essentially all subunits of the transport polymer. Thus,
the transport oligomers of the present invention can be viewed in
one group of embodiments as polypeptides in which arginine residues
are present, but separated by other amino acids such that, at most,
two arginine residues are adjacent. Additionally, the remaining
amino acids residues can be selected to provide side chains that
enhance solubility and which do not encumber the conformational
freedom of the transport oligomer. Alternatively, one of the
remaining amino acids can provide a site for attachment to either a
linking group or the therapeutic agent of interest (e.g., a serine
hydroxyl group, a lysine amino group or a cysteine thiol).
C. Transport Moiety-Biologically Active Compound Conjugates
[0043] As noted above, the present invention provides compositions
and methods that enhance the transport of biologically active
compounds across biological membranes and across and into animal
epithelial or endothelial tissue. The compositions are represented
by the formula R.sup.1-L-R.sup.3, wherein R.sup.1 is a biologically
active compound (i.e., a therapeutic compound, diagnostic agent,
lead compound or a probe), R.sup.3 is a transport moiety and "L" is
an optional linking moiety.
[0044] The transport moieties are amino acid oligomers of the
following formulae: (ZYZ).sub.nZ, (ZY).sub.nZ, (ZYY).sub.nZ and
(ZYYY).sub.nZ. "Z" in the formulae is D or L-arginine. "Y" is an
amino acid that does not contain a guanidyl or amidinyl moiety. The
subscript "n" is an integer ranging from 2 to 25.
[0045] In the above transport moiety formulae, the letter "Y"
represents a natural or non-natural amino acid. The amino acid can
be essentially any compound having (prior to incorporation into the
transport moiety) an amino group (NH.sub.2 or NH-alkyl) and a
carboxylic acid group (CO.sub.2H) and not containing either a
guanidyl or amidinyl moiety. Examples of such compounds include D
and L-alanine, D and L-cysteine, D and L-aspartic acid, D and
L-glutamic acid, D and L-phenylalanine, glycine, D and L-histidine,
D and L-isoleucine, D and L-lysine, D and L-leucine, D and
L-methionine, D and L-asparagine, D and L-proline, D and
L-glutamine, D and L-serine, D and L-threonine, D and L-valine, D
and L-tryptophan, D and L-hydroxyproline, D and L-tyrosine,
sarcosine, .beta.-alanine, .gamma.-amino butyric acid and
.epsilon.-amino caproic acid. In each of the above formulae, each Y
will be independent of any other Y present in the transport moiety,
though in some embodiments, all Y groups can be the same.
[0046] In one group of preferred embodiments, the transport moiety
has the formula (ZYZ).sub.nZ, wherein each "Y" is independently
selected from glycine, .beta.-alanine, .gamma.-amino butyric acid
and .epsilon.-amino caproic acid, "Z" is preferably L-arginine, and
n is preferably an integer ranging from 2 to 5. More preferably,
each "Y" is glycine or .epsilon.-amino caproic acid and n is 3.
Within this group of embodiments, the use of glycine is preferred
for those compositions in which the transport moiety is fused or
covalently attached directly to a polypeptide biological agent such
that the entire composition can be prepared by recombinant methods.
For those embodiments in which the transport moiety is to be
assembled using, for example, solid phase methods, .epsilon.-amino
caproic acid is preferred.
[0047] In another group of preferred embodiments, the transport
moiety has the formula (ZY).sub.nZ, wherein each "Y" is preferably
selected from glycine, .epsilon.-alanine, .gamma.-amino butyric
acid and .epsilon.-amino caproic acid, "Z" is preferably
L-arginine, and n is preferably an integer ranging from 4 to 10.
More preferably, each "Y" is glycine or .epsilon.-amino caproic
acid and n is 6. As with the above group of specific embodiments,
the use of glycine is preferred for those compositions in which the
transport moiety is fused or covalently attached directly to a
polypeptide biological agent such that the entire composition can
be prepared by recombinant methods. For solution or solid phase
construction of the transport moiety, .epsilon.-amino caproic acid
is preferred.
[0048] In yet another group of preferred embodiments, the transport
moiety has the formula (ZYY).sub.nZ, wherein each "Y" is preferably
selected from glycine, .beta.-alanine, .gamma.-amino butyric acid
and .epsilon.-amino caproic acid, "Z" is preferably L-arginine, and
n is preferably an integer ranging from 4 to 10. More preferably,
each "Y" is glycine or .epsilon.-amino caproic acid and n is 6.
[0049] In still another group of preferred embodiments, the
transport moiety has the formula (ZYYY).sub.nZ, wherein each "Y" is
preferably selected from glycine, .beta.-alanine, .gamma.-amino
butyric acid and .epsilon.-amino caproic acid, "Z" is preferably
L-arginine, and n is preferably an integer ranging from 4 to 10.
More preferably, "Y" is glycine and n is 6. In other embodiments,
each of the Y groups will be selected to enhance certain desired
properties of the transport moeity. For example, when transport
moeities having a more hydrophobic character are desired, each Y
can be selected from those naturally occurring amino acids that are
typically grouped together as hydrophobic amino acids (e.g.,
phenylalanine, phenylglycine, valine, leucine, isoleucine).
Similarly, transport moieties having a more hydrophilic character
can be prepared when some or all of the Y groups are hydrophilic
amino acids (e.g., lysine, serine, threonine, glutamic acid, and
the like).
[0050] One of skill in the art will appreciate that the transport
moiety can be a polypeptide fragment within a larger polypeptide.
For example, the transport moiety can be of the formula
(ZYY).sub.nZ yet have additional amino acids which flank this
moiety (e.g., X.sub.m(ZYY).sub.nZ-X.sub.p wherein the subscripts m
and p represent integers of zero to about 10 and each X is
independently a natural or non-natural amino acid).
[0051] Thus, the transport moiety can be viewed as a peptide or
having certain peptide character in the sense that it has a carboxy
terminus and an amino terminus. One of the termini is either
covalently attached to a biologically active compound (R.sup.1) or,
alternatively, to a linking moiety (L) that is part of a linking
moiety-active compound conjugate (e.g., R.sup.1-L). For those
embodiments in which the transport moiety is covalently attached
directly to a biologically active compound, the point of attachment
is preferably the carboxy terminus. For those embodiments in which
the transport moiety is covalently attached to a linking moiety,
the point of attachment is preferably the amino terminus. In still
other embodiments, the biologically active compound can be attached
to the transport moiety via a linking moiety that is in turn
attached to an amino acid side chain functional group (e.g., the
hydroxy group of a serine residue, the amino group of a lysine
residue, the carboxylic acid group of a glutamic acid residue, and
the like).
[0052] Turning next to the biologically active compound (R.sup.1),
the present invention finds broad application to essentially any
therapeutic or diagnostic agent. Examples of therapeutic compounds
include, without limitation, the following: topical antipsoriasis
drugs such as corticosteroids, calcipotriene and anthralin;
photochemotherapeutic agents such as psoralens; phorphyrins and
enlarged phorphyrins; sunscreen components such as p-aminobenzoic
esters, cinnamates, salicylates, benzophenones, anthranilates and
avobenzone; pain relief agents and local anesthetics such as
lidocaine, novocaine, procaine, tetracaine, benzocaine, cocaine and
the opiates; biological agents such as minoxidil, keratolytic
agents; destructive agents such as podophyllin, hydroquinone,
masoprocol, colchicine and gold; drugs for gastrointestinal
conditions such as cyclosporin, FK506, H.sub.2 histamine inhibitors
(e.g., cymetidine and ranitidine), ATPase proton-potassium
inhibitors (e.g., lansoprazole and omeprazle); antiobiotics such as
norfloxacin, ciprofloxacin, trimethoprim and sulfamethyloxazole;
antineoplastic agents such as cisplatin, methotrexate, taxol,
fluorouracil, mercaptopurine, donorubicin and bleomycin;
antiinflammatory agents such as corticosteroids, cromolyn and
nedocromil; immunosuppressive drugs such as cyclosporin and FK506;
drugs acting on pulmonary tissues such as beta-agonists, mast cell
stabilizers, antibiotics, antifungal agents, antiviral agents,
vasoactive drugs, sedatives and hormones; CNS agents such as
anti-apoptotics, neurotransmitters, morphine, opiates and
5-hydroxy-tryptamine receptor antagonists; small molecules such as
L-3,4-dihydroxy phenylalanine (L-DOPA; taxanes such as paclitaxel;
proteins such as alglucerase, alpha-L-iduronidase,
alpha-N-acetylglucosamidase, lipase, adenosine deaminase and triose
phosphate isomerase; peptides having phosphorylation sites used by
proteins mediating intracellular signals such as protein kinase C,
RAF-1, p21Ras, NF-.kappa.B and C-JUN; cytoplasmic tails of membrane
receptors such as IL-4 receptor, CD28, CTLA-4, V7, MHC Class I
antigens and MHC Class II antigens; oligonucleotides and
polynucleotides formed of DNA and RNA; oligonucleotide analogs such
as phosphonates (e.g., methyl phosphonates), phosphoramidates (N3'
or N5'), thiophosphates, uncharged morpholino-based polymers and
protein nucleic acids (PNAs); and, polysaccharides and
polysaccharide analogs.
[0053] Diagnostic agents include both diagnostic imaging agents and
contrast agents. The following are non-limiting examples of
diagnostic agents: radio-labeled substances such as .sup.99mTc
glucoheptonate; substances used in magnetic resonance imaging such
as gadolinium doped chelation agents (e.g., Gd-DTPA); metals bound
to chelating agents such as Eu, Lu, Pr, Gd, Tc.sup.99m, Ga.sup.67,
In.sup.111, Y.sup.90, Cu.sup.67 and Co.sup.57; and, proteins such
as .beta.-galactosidase, green fluorescent protein and luciferase.
Still other useful agents include dyes such as, for example,
fluorescein.
[0054] In certain embodiments, the transport moiety is attached to
the biologically active compound through a linking moiety (L). Such
a linking moiety has two termini, one that covalently bonds to the
transport moiety and one that covalently bonds to the biologically
active compound. The termini each contain a functional group that
serves as a facile point of attachment. Examples of such groups
include, without limitation, carboxylic acids, carboxylic acid
derivatives, alcohols, amines and thiols. For example,
HO.sub.2CCH.sub.2CH.sub.2OH is a linking moiety having a carboxylic
acid at one terminus and an alcohol at the other. One formula
representing its use in attaching the transport moiety to the
biologically active compound is as follows:
R.sup.1--O.sub.2CCH.sub.2CH.sub.2O--R.sup.3.
[0055] The linking moiety is preferably cleaved in vivo. "Cleaved"
in this case refers to separation of a linking moiety terminus from
the biologically active agent. The separation is effected through
dissociation of a covalent bond. For instance, where a carboxylic
acid terminus of a linking moiety is attached to an alcohol
functionality on a biologically active agent (i.e., ester linkage),
cleavage results in the formation of a carboxylic acid (linking
moiety terminus) and a free active agent.
[0056] In one embodiment, the linking moiety is cleaved through
hydrolysis. Such hydrolytic cleavages are typically pH dependent.
For instance, an acid mediated hydrolysis occurs at a faster rate
at pH 6.0 than at pH 7.4.
[0057] In another embodiment, the linking moiety is cleaved through
self-immolation. Such linking moieties in a transport
moiety-biologically active compound conjugate contain a nucleophile
(e.g., oxygen, nitrogen and sulfur) distal to the biologically
active compound and a cleavable group (e.g., ester, carbonate,
carbamate and thiocarbamate) proximal to the biologically active
compound. Intramolecular attack of the nucleophile on the cleavable
group results in the scission of a covalent bond, thereby releasing
the linking moiety from the biologically active compound.
[0058] Examples of conjugates containing self-immolating linking
moieties (e.g., biologically active agent-L-transport moiety
conjugates) are represented by structures 1, 2 and 3:
##STR00001##
wherein: R.sup.1 is the biologically active compound; X is a
linkage formed between a functional group on the biologically
active compound and a terminal functional group on the linking
moiety; Q is a linkage formed from a functional group on the
transport moiety and a functional group on the linking moiety; A is
N or CH; R.sup.2 is hydrogen, alkyl, aryl, arylalkyl, acyl or
allyl; R.sup.3 is the transport moiety; R.sup.4 is S, O, NR.sup.6
or CR.sup.7R.sup.8; R.sup.5 is H, OH, SH or NHR.sup.6; R.sup.6 is
hydrogen, alkyl, aryl, acyl or allyl; R.sup.7 and R.sup.8 are
independently hydrogen or alkyl; k and m are independently either 1
or 2; and n1 is an integer ranging from 1 to 10. Non-limiting
examples of the X and Q linkages are (in either orientation):
--C(O)O--, --C(O)NH--, --OC(O)NH--, --S--S--, --C(S)O--,
--C(S)NH--, --NHC(O)NH--, --SO.sub.2NH--, --SONH--, phosphate,
phosphonate and phosphinate. One of skill in the art will
appreciate that when the biological agent has a hydroxy functional
group, then X will preferably be --OC(O)-- or --OC(O)NH--.
Similarly, when the linking group is attached to an amino terminus
of the transport moiety, Q will preferably be --C(O)NH--,
--NHC(O)NH--, --SO.sub.2NH--, --SONH-- or --OC(O)NH-- and the like.
In each of the groups provided above, NH is shown for brevity, but
each of the linkages (X and Q) can contain substituted (e.g.,
N-alkyl or N-acyl) linkages as well.
[0059] Turning first to linking groups illustrated by structure 1,
an example and preferred embodiment is illustrated for formula
1a:
##STR00002##
wherein the wavy lines indicate points of attachment to the
transport moiety and to the biologically active compound.
Preparation of a conjugate containing this linking group is
illustrated in Example 4 (FIG. 9). In this Example and FIG. 9,
cyclosporin A is treated with chloroacetic anhydride to form the
chloroacetate ester 9i (numbering in FIG. 9) which is then combined
with benzylamine to form the N-benzyl glycine conjugate 9ii.
Condensation of the glycine conjugate with Boc-protected diglycine
anhydride provides the acid 9iii which is converted to the more
reactive N-hydroxy succinimide ester 9iv and then combined with the
amino terminus of a transport moiety to form an amide linkage. One
of skill in the art will appreciate that the N-benzyl group can be
replaced with other groups (e.g., alkyl, aryl, allyl and the like)
or that methylene groups can be replaced with, for example,
ethylene, propylene and the like. Preferably, the methylene groups
are retained as shown in 1a, to provide an appropriate steric or
spatial orientation which allows the linkage to be cleaved in vivo
(see FIG. 9B).
[0060] Accordingly, for structure 1, the following substituents are
preferred: A is N; R.sup.2 is benzyl; k, m and n1 are 1; X is
--OC(O)-- and Q is --C(O)NH--.
[0061] Linkages of structure 2, are exemplified by formula 2a:
##STR00003##
wherein, as above, the wavy lines indicate the point of attachment
to each of the transport moiety and the biologically active agent.
The preparation of conjugates having linking groups of formula 2a
are shown in Examples 5-7. In Example 5 (see scheme in FIG. 10),
acyclovir is acylated with .alpha.-chloroacetic anhydride to form
the .alpha.-chloroacetate ester 10i. Reaction of 10i with a
heptamer of D-arginine having an N-terminal cysteine residue,
provides the thioether product 10ii. Alternatively, acyclovir can
be attached to the C-terminus of a transport moiety (see Example
5b) using a similar linkage formed between acyclovir
.alpha.-chloroacetate ester and a heptamer of D-arginine having an
C-terminal cysteine residue. In this instance, the cysteine residue
is provided on the r.sub.7 transport moiety as a C-terminal amide
and the linkage has the form:
##STR00004##
[0062] Accordingly, in one group of preferred embodiments, the
conjugate is represented by formula 2, in which X is --OC(O)--; Q
is --C(O)NH--; R.sup.4 is S; R.sup.5 is NHR.sup.6; and the
subscripts k and m are each 1. In another group of preferred
embodiments, the conjugate is represented by formula 2, in which X
is --OC(O)--; Q is NHC(O)--; R.sup.4 is S; R.sup.5 is CONH.sub.2;
and the subscripts k and m are each 1. Particularly preferred
conjugates are those in which R.sup.6 is hydrogen, methyl, allyl,
butyl or phenyl.
[0063] Linking groups represented by the conjugates shown in
formula 3 are generally of the heterobifunctional type (e.g,
.epsilon.-aminocaproic acid, serine, homoserine,
.gamma.-aminobutyric acid, and the like), although suitably
protected dicarboxylic acids or diamines are also useful with
certain biological agents.
[0064] For structure 3, the following substituents are preferred:
R.sup.5 is NHR.sup.6, wherein R.sup.6 is hydrogen, methyl, allyl,
butyl or phenyl; k is 2; X is --C(O)O--; and Q is --C(O)NH--.
[0065] Self-immolating linkers typically undergo intramolecular
cleavage with a half-life between about 10 minutes and about 24
hours in water at 37.degree. C. at a pH of approximately 7.4.
Preferably, the cleavage half-life is between about 20 minutes and
about 4 hours in water at 37.degree. C. at a pH of approximately
7.4. More preferably, the cleavage half-life is between about 30
minutes and about 2 hours in water at 37.degree. C. at a pH of
approximately 7.4.
[0066] For a conjugate having the structure 1, one can adjust the
cleavage half-life by varying the R.sup.2 substituent. By using an
R.sup.2 of increased or decreased size, one can obtain a conjugate
having a longer or shorter half-life respectively. R.sup.2 in
structure 1 is preferably methyl, ethyl, propyl, butyl, allyl,
benzyl or phenyl.
[0067] Where there is a basic or acidic group in a self-immolating
linker, one can oftentimes adjust cleavage half-life according to
the pH of the conjugate solution. For instance, the backbone amine
group of structure 1 is protonated at acidic pH (e.g., pH 5.5). The
amine cannot serve as a nucleophile inducing intramolecular
cleavage when it is protonated. Upon introduction of the conjugate
into a medium at physiological pH (7.4), however, the amine is
unprotonated a significant portion of the time. The cleavage
half-life is correspondingly reduced.
[0068] In one embodiment, cleavage of a self-immolating linker
occurs in two steps: intramolecular reaction of a nucleophilic
group resulting in the cleavage of a portion of the linking moiety;
and, elimination of the remaining portion of the linking moiety.
The first step of the cleavage is rate-limiting and can be
fine-tuned for pH sensitivity and half-life.
[0069] Structure 4 is an example of a two-step, self-immolating
moiety that is incorporated into a transport moiety-biologically
active compound conjugate:
##STR00005##
wherein: R.sup.1 is the biologically active compound; X represents
a linkage between a functional group on the biologically active
compound and a functional group on the linking moiety; Ar is a
substituted or unsubstituted aryl group, wherein the methylene
substituent and phenolic oxygen atom are either ortho or para to
one another; R.sup.3 is the transport moiety; R.sup.4 is S, O,
NR.sup.6 or CR.sup.7R.sup.8; R.sup.5 is H, OH, SH or NHR.sup.6;
R.sup.6 is hydrogen, alkyl, aryl, arylalkyl, acyl or allyl; R.sup.7
and R.sup.8 are independently hydrogen or alkyl; and, k and m are
independently either 1 or 2.
[0070] An example of a suitable linking group to produce a
conjugate of formula 4 is:
##STR00006##
[0071] The construction of a conjugate containing a linking group
of formula 4a is provided in Example 8 (see also FIG. 14). In this
example (and Figure), the .alpha.-chloroacetate ester of
2,4-dimethyl-4-hydroxymethylphenol (141) is coupled to retinoic
acid (141i) using dicyclohexylcarbodiimide (DCC) and
4-dimethylaminopyridine (DMAP) to provide the intermediate 14iii.
Subsequent coupling of 14iii with a cysteine residue present on the
N-terminus of an arginine heptamer transport moiety provides the
target conjugate 14iv. Preferably, the linking groups used in the
conjugates of formula 4, are those in which Ar is an substituted or
unsubstituted phenylene group; R.sup.4 is S; R.sup.5 is NHR.sup.6,
wherein R.sup.6 is hydrogen, methyl, allyl, butyl, acetyl or
phenyl; k and m are 1; X is --C(O)O--; and Q is --C(O)O-- or
--C(O)NH--. More preferably, R.sup.6 is hydrogen or acetyl.
[0072] While linking groups above have been described with
reference to conjugates containing arginine heptamers, one of skill
in the art will understand that the technology is readily adapted
to conjugates with the "spaced" arginine transport moieties of the
present invention.
[0073] Still other useful linking groups for use in the present
invention have been described in copending PCT applications. See,
for example PCT applications US98/10571 (Publication No. WO
9852614) and US00/23440 (Publication No. WO 01/13957) which
describe linking groups for similar compositions, e.g., conjugates
of biologically active agents and transport oligomers. The linking
technology described therein can be used in the present
compositions in a similar manner.
[0074] Thus, in one group of embodiments, the linking moiety
contains a first cleavable group distal to the biologically active
compound and a second cleavable group proximal to the biologically
active compound. Cleavage of the first cleavable group yields a
nucleophile capable of reacting intramolecularly with the second
cleavable group, thereby cleaving the linking moiety from the
biologically active compound. Examples of methods by which the
first group is cleaved include photo-illumination and enzyme
mediated hydrolysis. This methodology has been illustrated for
various related small molecule conjugates discussed in PCT
application US98/10571 (Publication No. WO 9852614).
[0075] In one approach, the conjugate can include a disulfide
linkage, as illustrated in FIG. 5A of PCT application US00/23440
(Publication No. WO 01/13957), (see also, PCT application
US98/10571 (Publication No. WO 9852614)), which shows a conjugate
(I) containing a transport polymer T which is linked to a cytotoxic
agent, 6-mercaptopurine, by an N-acetyl-protected cysteine group
which serves as a linker. Thus, the cytotoxic agent is attached by
a disulfide bond to the 6-mercapto group, and the transport polymer
is bound to the cysteine carbonyl moiety via an amide linkage.
Cleavage of the disulfide bond by reduction or disulfide exchange
results in release of the free cytotoxic agent. A method for
synthesizing a disulfide-containing conjugate is provided in
Example 9A of PCT application US98/10571. The product described
therein contains a heptamer of Arg residues which is linked to
6-mercaptopurine by an N-acetyl-Cys-Ala-Ala linker, where the Ala
residues are included as an additional spacer to render the
disulfide more accessible to thiols and reducing agents for
cleavage within a cell. The linker in this example also illustrates
the use of amide bonds, which can be cleaved enzymatically within a
cell.
[0076] In another approach, the conjugate includes a photocleavable
linker which is cleaved upon exposure to electromagnetic radiation.
Application of this methodology is provided for a related system in
FIG. 5B of PCT application US00/23440 (Publication No. WO 01/13957)
which shows a conjugate (II) containing a transport polymer T which
is linked to 6-mercaptopurine via a meta-nitrobenzoate linking
moiety. Polymer T is linked to the nitrobenzoate moiety by an amide
linkage to the benzoate carbonyl group, and the cytotoxic agent is
bound via its 6-mercapto group to the p-methylene group. The
compound can be formed by reacting 6-mercaptopurine with
p-bromomethyl-m-nitrobenzoic acid in the presence of
NaOCH.sub.3/methanol with heating, followed by coupling of the
benzoate carboxylic acid to a transport polymer, such as the amino
group of a .gamma.-aminobutyric acid linker attached to the polymer
(see also, e.g., Example 9B of PCT application US98/10571).
Photo-illumination of the conjugate causes release of the
6-mercaptopurine by virtue of the nitro group that is ortho to the
mercaptomethyl moiety. This approach finds utility in phototherapy
methods as are known in the art, particularly for localizing drug
activation to a selected area of the body.
[0077] In one group of preferred embodiments, the cleavable linker
contains first and second cleavable groups that can cooperate to
cleave the oligomer from the biologically active agent, as
illustrated by the following approaches. That is, the cleavable
linker contains a first cleavable group that is distal to the
agent, and a second cleavable group that is proximal to the agent,
such that cleavage of the first cleavable group yields a
linker-agent conjugate containing a nucleophilic moiety capable of
reacting intramolecularly to cleave the second cleavable group,
thereby releasing the agent from the linker and oligomer.
[0078] Reference is again made to co-owned and copending PCT
application US00/23440 (Publication No. WO 01/13957), in which FIG.
5C shows a conjugate (III) containing a transport polymer T linked
to the anticancer agent, 5-fluorouracil (5FU). In that figure, the
linkage is provided by a modified lysyl residue. The transport
polymer is linked to the .alpha.-amino group, and the
5-fluorouracil is linked via the .alpha.-carbonyl. The lysyl
s-amino group has been modified to a carbamate ester of
o-hydroxymethyl nitrobenzene, which comprises a first, photolabile
cleavable group in the conjugate. Photo-illumination severs the
nitrobenzene moiety from the conjugate, leaving a carbamate that
also rapidly decomposes to give the free .alpha.-amino group, an
effective nucleophile. Intramolecular reaction of the
.epsilon.-amino group with the amide linkage to the 5-fluorouracil
group leads to cyclization with release of the 5-fluorouracil
group.
[0079] Still other linkers useful in the present invention are
provided in PCT application US00/23440 (Publication No. WO
01/13957). In particular, FIG. 5D of US00/23440 illustrates a
conjugate (IV) containing a delivery-enhancing transporter T linked
to 2'-oxygen of the anticancer agent, paclitaxel. The linkage is
provided by a linking moiety that includes (i) a nitrogen atom
attached to the delivery-enhancing transporter, (ii) a phosphate
monoester located para to the nitrogen atom, and (iii) a
carboxymethyl group meta to the nitrogen atom, which is joined to
the 2'-oxygen of paclitaxel by a carboxylate ester linkage.
Enzymatic cleavage of the phosphate group from the conjugate
affords a free phenol hydroxyl group. This nucleophilic group then
reacts intramolecularly with the carboxylate ester to release free
paclitaxel, fully capable of binding to its biological target.
Example 9C of PCT application US98/10571 describes a synthetic
protocol for preparing this type of conjugate.
[0080] Still other suitable linkers are illustrated in FIG. 5E of
PCT application US00/23440 (Publication No. WO 01/13957). In the
approach provided therein, a delivery-enhancing transporter is
linked to a biologically active agent, e.g., paclitaxel, by an
aminoalkyl carboxylic acid. Preferably, the linker amino group is
linked to the linker carboxyl carbon by from 3 to 5 chain atoms
(n1=3 to 5), preferably either 3 or 4 chain atoms, which are
preferably provided as methylene carbons. As seen in FIG. 5E, the
linker amino group is joined to the delivery-enhancing transporter
by an amide linkage, and is joined to the paclitaxel moiety by an
ester linkage. Enzymatic cleavage of the amide linkage releases the
delivery-enhancing transporter and produces a free nucleophilic
amino group. The free amino group can then react intramolecularly
with the ester group to release the linker from the paclitaxel.
[0081] In another approach, the conjugate includes a linker that is
labile at one pH but is stable at another pH. For example, FIG. 6
of PCT application US00/23440 (Publication No. WO 01/13957)
illustrates a method of synthesizing a conjugate with a linker that
is cleaved at physiological pH but is stable at acidic pH.
Preferably, the linker is cleaved in water at a pH of from about
6.6 to about 7.6. Preferably the linker is stable in water at a pH
from about 4.5 to about 6.5.
D. Synthesis of Transport Moieties and Compositions
[0082] The transporters of the present invention can be constructed
using a variety of conventional synthetic methods. For example, the
amino acid oligomers can be produced synthetically, preferably
using a peptide synthesizer (e.g., and Applied Biosystems Model
433) or can be synthesized recombinantly using methods well-known
in the art. Recombinant synthesis is generally used when the
biologically active compound attached to the transport moiety is a
peptide or protein.
[0083] In other embodiments, the transport oligomers can be
prepared using solid phase peptide synthesis which is extensively
described and used in the art to prepare peptides. The transport
oligomers can also be prepared using liquid phase synthetic
methods, which are also well known in the art. See, M. Bodanszky
and A. Bodanszky, The Practice of Peptide Synthesis, 2nd ed.,
Springer-Verlag, New York, N.Y. (1994).
E. Uses of the Present Invention
[0084] The transport moiety-biologically active compound conjugates
of the present invention find use in therapeutic, prophylactic and
diagnostic applications. The conjugates readily penetrate
biological membranes, move across and/or into one or more layers of
skin or other epithelial tissue (e.g., gastrointestinal, lung and
the like) and traverse endothelial tissues (e.g., blood-brain
barrier). This property makes the conjugates useful where transport
compounds must penetrate such membranes or tissues to exert their
effect.
[0085] Conjugates and methods of the present invention have
particular utility in the area of human and veterinary
therapeutics. Generally, administered dosages will be effective to
deliver picomolar to micromolar concentrations of the therapeutic
composition to the effector site. Appropriate dosages and
concentrations will depend on factors such as the therapeutic
composition or drug, the site of intended delivery, and the route
of administration, all of which can be derived empirically
according to methods well known in the art. Further guidance can be
obtained from studies using experimental animal models for
evaluating dosage, as are known in the art.
[0086] Administration of the conjugates of the invention with a
suitable pharmaceutical excipient as necessary can be carried out
via any of the accepted modes of administration. Thus,
administration can be, for example, intravenous, topical,
subcutaneous, transcutaneous, intramuscular, oral, intra joint,
parenteral, peritoneal, intranasal, transocular, sublingual, or by
inhalation. Suitable sites of administration thus include, but are
not limited to, skin, bronchial, gastrointestinal, anal, vaginal,
eye, and ear. The formulations may take the form of solid,
semi-solid, lyophilized powder, or liquid dosage forms, such as,
for example, tablets, pills, capsules, powders, solutions,
suspensions, emulsions, suppositories, retention enemas, creams,
ointments, lotions, aerosols or the like, preferably in unit dosage
forms suitable for simple administration of precise dosages.
[0087] The compositions typically include a conventional
pharmaceutical carrier or excipient and may additionally include
other medicinal agents, carriers, adjuvants, and the like.
Preferably, the composition will be about 5% to 75% by weight of a
compound or compounds of the invention, with the remainder
consisting of suitable pharmaceutical excipients. Appropriate
excipients can be tailored to the particular composition and route
of administration by methods well known in the art, e.g.,
REMINGTON'S PHARMACEUTICAL SCIENCES, 18TH ED., Mack Publishing Co.,
Easton, Pa. (1990).
[0088] For oral administration, such excipients include
pharmaceutical grades of mannitol, lactose, starch, magnesium
stearate, sodium saccharine, talcum, cellulose, glucose, gelatin,
sucrose, magnesium carbonate, and the like. The composition may
take the form of a solution, suspension, tablet, pill, capsule,
powder, sustained-release formulation, and the like.
[0089] In some embodiments, the pharmaceutical compositions take
the form of a pill, tablet or capsule, and thus, the composition
can contain, along with the biologically active conjugate, any of
the following: a diluent such as lactose, sucrose, dicalcium
phosphate, and the like; a disintegrant such as starch or
derivatives thereof; a lubricant such as magnesium stearate and the
like; and a binder such a starch, gum acacia, polyvinylpyrrolidone,
gelatin, cellulose and derivatives thereof.
[0090] The active compounds of the formulas may be formulated into
a suppository comprising, for example, about 0.5% to about 50% of a
compound of the invention, disposed in a polyethylene glycol (PEG)
carrier (e.g., PEG 1000 [96%] and PEG 4000 [4%]).
[0091] Liquid compositions can be prepared by dissolving or
dispersing conjugate (about 0.5% to about 20%), and optional
pharmaceutical adjuvants in a carrier, such as, for example,
aqueous saline (e.g., 0.9% w/v sodium chloride), aqueous dextrose,
glycerol, ethanol and the like, to form a solution or suspension,
e.g., for intravenous administration. The active compounds may also
be formulated into a retention enema.
[0092] If desired, the composition to be administered may also
contain minor amounts of non-toxic auxiliary substances such as
wetting or emulsifying agents, pH buffering agents, such as, for
example, sodium acetate, sorbitan monolaurate, or triethanolamine
oleate.
[0093] For topical administration, the composition is administered
in any suitable format, such as a lotion or a transdermal patch.
For delivery by inhalation, the composition can be delivered as a
dry powder (e.g., Inhale Therapeutics) or in liquid form via a
nebulizer.
[0094] Methods for preparing such dosage forms are known or will be
apparent to those skilled in the art; for example, see Remington's
Pharmaceutical Sciences, supra., and similar publications. The
composition to be administered will, in any event, contain a
quantity of the pro-drug and/or active compound(s) in a
pharmaceutically effective amount for relief of the condition being
treated when administered in accordance with the teachings of this
invention.
[0095] Generally, the conjugates of the invention are administered
in a therapeutically effective amount, i.e., a dosage sufficient to
effect treatment, which will vary depending on the individual and
condition being treated. Typically, a therapeutically effective
daily dose is from 0.1 to 100 mg/kg of body weight per day of drug.
Most conditions respond to administration of a total dosage of
between about 1 and about 30 mg/kg of body weight per day, or
between about 70 mg and 2100 mg per day for a 70 kg person.
F. Delivery Methods Using the Present Invention
[0096] The delivery-enhancing transport moieties of the invention
make possible or enhance the delivery of biologically active
compounds across the skin. Surprisingly, the transport moieties can
deliver an agent across the stratum corneum, which previously had
been a nearly impenetrable barrier to drug delivery. The stratum
corneum, the outermost layer of the skin, is composed of several
layers of dead, keratin-filled skin cells that are tightly bound
together by a "glue" composed of cholesterol and fatty acids. Once
the agents are delivered through the stratum corneum by the
transporters of the invention, the agents can enter the viable
epidermis, which is composed of the stratum granulosum, stratum
lucidum and stratum germinativum which, along with the stratum
corneum, make up the epidermis. Delivery in some embodiments of the
invention is through the epidermis and into the dermis, including
one or both of the papillary dermis and the reticular dermis.
[0097] This ability to obtain penetration of one or more layers of
the skin can greatly enhance the efficacy of compounds such as
antibacterials, antifungals, antivirals, antiproliferatives,
immunosuppressives, vitamins, analgesics, hormones, and the like.
Numerous such compounds are known to those of skill in the art
(see, e.g., Hardman and Limbird, Goodman & Gilman's The
Pharmacological Basis of Therapeutics, McGraw-Hill, New York,
1996).
[0098] In some embodiments, the biologically active compound is
delivered into a blood vessel that is present in the epithelial
tissue, thus providing a means for delivery of the agent
systemically. Delivery can be either intrafollicular or
interfollicular, or both. Pretreatment of the skin is not required
for delivery of the conjugates.
[0099] The delivery-enhancing transport moieties of the invention
are also useful for delivery of conjugated drugs by
gastrointestinal administration. Gastrointestinal administration
can be used for both systemically active drugs, and for drugs that
act in the gastrointestinal epithelium.
[0100] The delivery-enhancing transport moieties of the invention
can also used to enhance administration of drugs through the
respiratory tract. The respiratory tract, which includes the nasal
mucosa, hypopharynx, and large and small airway structures,
provides a large mucosal surface for drug absorption. The enhanced
penetration of the conjugated agents into and across one or more
layers of the epithelial tissue that is provided by the
delivery-enhancing transport moieties of the invention results in
amplification of the advantages that respiratory tract delivery has
over other delivery methods. For example, lower doses of an agent
are often needed to obtain a desired effect, a local therapeutic
effect can occur more rapidly, and systemic therapeutic blood
levels of the agent are obtained quickly. Rapid onset of
pharmacological activity can result from respiratory tract
administration. Moreover, respiratory tract administration
generally has relatively few side effects.
[0101] The delivery-enhancing transport moieties are also useful
for delivering biologically active compounds across the blood brain
barrier. The agents are useful for treating ischemia (e.g., using
an anti-apoptotic drug), as well as for delivering
neurotransmitters and other agents for treating various conditions
such as schizophrenia, Parkinson's disease, pain (e.g., morphine,
the opiates). The 5-hydroxytryptamine receptor antagonist is useful
for treating conditions such as migraine headaches and anxiety.
G. Examples
Example 1
[0102] This example illustrates the synthesis of various peptides,
including those having attached linking groups which are natural or
non-natural amino acids.
[0103] Peptides were synthesized using solid phase techniques and
commercially available Fmoc amino acids, resins, and reagents (PE
Biosystems, Foster City Calif., and Bachem Torrence, Calif.) on a
Applied Biosystems 433 peptide synthesizer. Fmoc amino caproic acid
and Fmoc amino butyric acid were purchased from NovaBiochem (San
Diego, Calif.). Fastmoc cycles were used with
O-(7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluronium
hexyluorophosphate (HATU) substituted for HBTU/HOBt as the coupling
reagent. The peptides were cleaved from the resin using 96%
trifluoroacetic acid, 2% triisopropyl silane, and 2% phenol for 12
hours. The longer reaction times were necessary to completely
remove the Pbf protecting groups from the multiple arginines in the
peptides. The peptides subsequently were filtered from the resin,
precipitated using diethyl ether, purified using C-18 reverse phase
columns (Alltech Altima, Chicago, Ill.) and characterized using
either electrospray or matrix assisted laser desorption mass
spectrometry (Perceptive Biosystems, Boston, Mass.).
[0104] Fluorescent analogs were synthesized by coupling fluorescein
isothiocyanate (Aldrich Milwaukee, Wis.) to the amino terminus of
the peptide while still attached to the resin.
[0105] Table 1 provides a number of transport moieties as their
fluorescent conjugates that were prepared and evaluated for
cellular uptake.
TABLE-US-00001 TABLE 1 Sequence Seq. I.D. No. Fl-aca-R7CONH.sub.2 2
Fl-aca-R10CONH.sub.2 3 Fl-aca-RGRRGRRGRR-CONH.sub.2 4
Fl-aca-RabuRRabuRRabuRR-CONH.sub.2 5
Fl-aca-RacaRRacaRRacaRR-CONH.sub.2 6 Fl-aca-RARRARRARR-CONH.sub.2 7
Fl-aca-RDRRDRRDRR-CONH.sub.2 8 Fl-aca-RERRERRERR-CONH.sub.2 9
Fl-aca-RFRRFRRFRR-CONH.sub.2 10 Fl-aca-RHRRHRRHRR-CONH.sub.2 11
Fl-aca-RIRRIRRIRR-CONH.sub.2 12 Fl-aca-RKRRKRRKRR-CONH.sub.2 13
Fl-aca-RLRRLRRLRR-CONH.sub.2 14 Fl-aca-RMRRMRRMRR-CONH.sub.2 15
Fl-aca-RNRRNRRNRR-CONH.sub.2 16 Fl-aca-RPRRPRRPRR-CONH.sub.2 17
Fl-aca-RQRRQRRQRR-CONH.sub.2 18 Fl-aca-RSRRSRRSRR-CONH.sub.2 19
Fl-aca-RTRRTRRTRR-CONH.sub.2 20 Fl-aca-RVRRVRRVRR-CONH.sub.2 21
Fl-aca-RYRRYRRYRR-CONH.sub.2 22 Fl-aca-(Raca).sub.4R-CONH.sub.2 23
Fl-aca-(Raca).sub.5R-CONH.sub.2 24 Fl-aca-(Raca).sub.6R-CONH.sub.2
25 Fl-aca-(RGG).sub.6R-CONH.sub.2 26 Fl-aca-(RG).sub.6R-CONH.sub.2
27 r13 28 r19 29 Fl-aca-(RGGG).sub.6R-CONH.sub.2 30
Example 2
[0106] This example illustrates a cellular uptake assay which can
be used to evaluate the conjugates of the present invention.
[0107] The fluorescently labeled peptides were dissolved in PBS, pH
7.2, and their concentrations were determined by absorption of
fluorescein at 490 nm (c=67,000). The human T cell line, Jurkat,
grown in 10% fetal calf serum in DMEM, was used for all the
cellular uptake experiments. Varying amounts of the peptides were
added to 3.times.10.sup.5 cells in a total of 200.lamda., in wells
of microtiter plates, and incubated for varying periods of time,
ranging from three to five minutes. The cells were spun, washed
three times with cold PBS, incubated with 0.05% trypsin/0.53 mM
EDTA (Gibco, Grand Island, N.Y.) at 37.degree. C. for five minutes,
washed with PBS, and resuspended in PBS containing 0.1% propidium
iodide. The cells were analyzed either using confocal microscopy or
fluorescent flow cytometry (FACScan, Becton Dickinson, Milpitas,
Calif.). Cells staining with propidium iodide were excluded from
the analysis. Data presented is the mean fluorescent signal for the
5000 cells collected. To prevent cellular uptake of the peptides,
the cells were treated with sodium azide (1.0%) for 25 minutes
prior to exposure to the peptides. Intracellular staining was
calculated by subtracting the signal seen on cells treated with
sodium azide from that observed on untreated cells.
Example 3
[0108] This example provides results of arginine spacing in the
cellular uptake assay of Example 2.
[0109] 3A--Comparison of R7 Homopolymer with Single Amino Acid
Spacing (no Side Chains)
[0110] A series of arginine containing conjugates was prepared as
described in Example 1 and evaluated as described in Example 2.
Spacing between seven arginine residues was provided by glycine
(see Seq. I.D. No. 4), .gamma.-aminobutyric acid (see Seq. I.D. No.
5), and .epsilon.-aminocaproic acid (see Seq. I.D. No. 6). FIG. 1
shows the results of cellular uptake for the "spaced" arginine
transport moieties when tethered to fluorescein. As can be seen
from this figure, cellular uptake generally increases when spacing
increases (.epsilon.-aminocaproic acid>.gamma.-aminobutyric
acid>glycine.
[0111] 3B--Comparison of R7 Homopolymer with Naturally-Occurring
Single Amino Acid Spacing
[0112] A series of arginine containing conjugates was prepared as
described in Example 1 and evaluated as described in Example 2.
Spacing between seven arginine residues was provided by naturally
occurring (gene-encoded) amino acids (see Seq. I.D. Nos. 7 to 22).
FIG. 2 shows the results of cellular uptake for these "spaced"
arginine transport moieties when tethered to fluorescein. As can be
seen from this figure, an increase in cellular uptake was found
(relative to the homopolymer R.sup.7 having no "spacing") for those
compositions containing the amino acids Alanine, Phenylalanine,
Glycine, Histidine, Isoleucine, Lysine, Methionine, Asparagine,
Proline, Glutamine, Serine, Threonine and Valine. Reduced, or
comparable uptake was seen with those compositions containing
Aspartic acid, Glutamic acid, and Tyrosine. Without intending to be
bound by theory, it is thought that the carboxylic acid residues
(for Asp and Glu) and the phenolic hydroxy group of Tyr may cancel
or coordinate with the positively charged Arginine residue and lead
to a reduction in the number of positively charged Arginines that
are available to provide enhanced penetration into the cell. FIGS.
3 and 4 illustrate the same general results at different
concentrations indicating that these results are essentially
independent of concentration. At the lower concentration of 6.25
.mu.M, the uptake of Fl-aca-R.sup.7--CONH.sub.2 begins to drop off
relative to the spaced arginine transport moieties.
[0113] 3C--Comparison of (R-aca).sub.xR Transport Moieties
[0114] A series of Fl-aca-(R-aca).sub.xR compositions was prepared
wherein x is 4, 5 or 6, as described in Example 1 and evaluated for
cellular uptake as described in Example 2. The results are shown in
FIG. 5. Here, an increase in uptake was found as overall arginine
content increased (x=6>x=5>x=4).
[0115] 3D--Comparison of (RX).sub.6R Versus Homopolymers of
D-Arginine (r13 and r19)
[0116] A series of Fl-aca-(R--X).sub.6R compositions was prepared
wherein x is aca, gly or glygly, as described in Example 1 and
evaluated for cellular uptake as described in Example 2. A
comparison to homopolymers of D-arginine (Fl-aca-r19-CONH.sub.2 and
Fl-aca-r13-CONH.sub.2) was made and the results are shown in FIG.
6. The homopolymers, r13 and r19, were selected as being of
equivalent length to (RG).sub.6R and (RGG).sub.6R, respectively.
Surprisingly, the homopolymers were significantly less effective in
entering the cells, despite the increased number of
positively-charged arginine groups.
[0117] 3E--Evaluation of Number and Position of Spacing Groups
(aca)
[0118] A entire series of Fl-aca-R.sup.7 oligomers having aca
insertions within the arginine homopolymer portion was prepared as
described in Example 1. The 60 peptide sequences are shown in FIG.
7. Evaluation of each compositions for cellular uptake was carried
out as described in Example 2. FIG. 8 shows the results of the
cellular uptake and indicates that, in general, a greater number of
insertions leads to a greater uptake.
Example 4
[0119] This example illustrates the conjugation of cyclosporine to
a transport moiety using a pH sensitive linking group (see FIGS. 9A
and 9B).
[0120] In this example, cyclosporine is converted to its
.alpha.-chloroacetate ester using chloroacetic anhydride to provide
9i (see FIG. 9). The ester 9i is then treated with benzylamine to
provide 9ii. Reaction of the amine with Boc-protected iminodiacetic
acid anhydride provides the acid 9iii which is then converted to an
activated ester (9iv) with N-hydroxy succinimide. Coupling of 9iv
with L-Arginine heptamer provides the BOC-protected conjugate 9v,
which can be converted to conjugate 9vi by removal of the BOC
protecting group according to established methods.
[0121] Transport moieties having arginine groups separated by, for
example, glycine, .epsilon.-aminocaproic acid, or
.gamma.-aminobutyric acid can be used in place of the arginine
heptamer in this an in the following examples that show
oligoarginine transport groups.
Example 5
[0122] This example illustrates the conjugation of acyclovir to a
transport moiety.
[0123] a. Conjugation of Acyclovir to r.sub.7CONH.sub.2
[0124] This example illustrates the conjugation of acyclovir to
r.sub.7CONH.sub.2 via the linking group:
##STR00007##
[0125] i) Preparation of Acyclovir .alpha.-Chloroester:
##STR00008##
[0126] A solution of acyclovir (100 mg, 0.44 mmol),
dimethylaminopyridine (5.4 mg, 0.044 mmol) and chloroacetic
anhydride (226 mg, 1.32 mmol) in dimethylformamide (9 mL) was
stirred at room temperature for 18 h. The dimethylformamide was
removed by evaporation. The crude product was purified by
reverse-phase HPLC (22 mm.times.250 mm C-18 column, a 5-25%
CH.sub.3CN/H.sub.2O gradient with 0.1% trifluoroacetic acid, 214
and 254 nm UV detection) and lyophilized. The product was obtained
as a white powder (62 mg, 47%). .sup.1H NMR (300 MHz, DMSO-d.sub.6)
.delta. 10.67 (s, 1H), 7.88 (s, 1H), 6.53 (s, 1H), 5.27 (s, 2H),
4.35 (s, 2H), 4.21 (t, J=3 Hz, 2H), 3.70 (t, J=3 Hz, 2H); .sup.13C
NMR (75 MHz, DMSO-d.sub.6) .delta. 168.1, 157.6, 154.8, 152.3,
138.6, 117.1, 72.7, 67.1, 65.2, 41.8; TOF-MS (m/z): 302.0
[M+H].
[0127] ii) Conjugation of Acyclovir .alpha.-Chloro Ester to
H.sub.2N--C-r7-CONH.sub.2
##STR00009##
[0128] A solution of acyclovir .alpha.-chloroester (7 mg, 0.024
mmol), H.sub.2N--C-r7-CONH.sub.2 (50 mg, 0.024 mmol) and
diisopropylethylamine (6.4 .mu.L, 0.036 mmol) in dimethylformamide
(1 mL) was stirred for 18 h. The dimethylformamide was removed by
evaporation. The crude product was purified by reverse-phase HPLC
(22 mm.times.250 mm C-18 column, a 5-25% CH.sub.3CN/H.sub.2O
gradient with 0.1% trifluoroacetic acid, 214 and 254 nm UV
detection) and lyophilized. The desired product was obtained as a
white powder (24 mg, 69%). TOF-MS (m/z): 494.6 [(M+H)/3], 371.0
[(M+H)/4].
[0129] The yield could be increased by using 10 molar equivalents
of diisopropylethylamine rather than 1.5 molar equivalents. Product
was again obtained as a white powder (79%). TOF-MS (m/z): 508.7
[(M+H)/3], 381.5 [(M+H)/4], 305.5 [(M+H)/5].
[0130] b. Conjugation of Acyclovir to a Biotin-Containing
Derivative of r.sub.5-Cys-CONH.sub.2
##STR00010##
[0131] Reactions were carried out as illustrated above, using the
synthetic techniques provided in the examples above.
[0132] i) Biotin-aminocaproic acid-[5-Cys(acyclovir)-CONH.sub.2 was
obtained as a white powder (36%). TOF-MS (m/z): 868.2 {(M+2
TFA)/2], 811.2 [(M+1 TFA)/2], 754.1 [(M+1 TFA)/3], 503.0 [(M+H)/3],
377.4 [(M+H)/4].
[0133] Similarly,
[0134] ii) Biotin-aminocaproic acid-r7-C(acyclovir)-CONH.sub.2--
was obtained as a white powder (33%). TOF-MS (m/z): 722.1 [(M+3
TFA)/3], 684.6 [(M+2 TFA)/3], 607.1 [(M+H)/3], 455.5 [(M+H)/4],
364.8 [(M+H)/5], 304.3 [(M+H)/6].
Example 6
[0135] This example illustrates the conjugation of hydrocortisone
to a transport moiety.
[0136] a. Conjugation of Hydrocortisone to r.sub.7CONH.sub.2
[0137] i) Preparation of Hydrocortisone .alpha.-Chloroester:
##STR00011##
[0138] To a solution of hydrocortisone (500 mg, 1.38 mmol),
scandium triflate (408 mg, 0.83 mmol) and chloroacetic anhydride
(708 mg, 4.14 mmol) in dry THF was added dimethylaminopyridine (506
mg, 4.14 mmol). The solution turned bright yellow upon addition of
dimethylaminopyridine. After 30 min the solvent was evaporated off
and the crude material taken up into ethyl acetate (100 mL). The
ethyl acetate layer was washed with 1.0 N HCl and brine. The
organic phase was collected, dried (Na.sub.2SO.sub.4) and
evaporated to provide the product as a white solid (533 mg, 88%).
.sup.1H NMR (300 MHz, DMSO-d.sub.6) .delta. 5.56 (s, 1H), 5.46 (s,
1H), 5.20 (d, J=18 Hz, 1H), 4.85 (d, J=18 Hz, 1H), 4.51 (s, 2H),
4.37 (br s, 1H), 4.27 (br s, 1H), 2.54-2.33 (m, 2H), 2.22-2.03 (m,
3H), 1.99-1.61 (m, 8H), 1.52-1.24 (m, 5H), 1.02-0.98 (d, J=12 Hz,
1H), 0.88-0.85 (d, J=9 Hz, 1H), 0.77 (s, 3H); .sup.13C NMR (75 MHz,
DMSO-d.sub.6) .delta. 205.4, 198.8, 173.0, 167.6, 122.3, 89.5,
69.7, 67.3, 56.4, 52.4, 47.8, 41.6, 39.7, 35.0, 34.3, 34.0, 33.6,
32.3, 32.0, 24.2, 21.3, 17.4; TOF-MS (m/z): 439.1 (M+H).
[0139] (Reference for acetylation--Zhao, H.; Pendri, A.; Greenwald,
R. B. J. Org. Chem. 1998, 63, 7559-7562.)
[0140] ii) Coupling to R'NH-Cys-r.sub.7-CONH.sub.2
##STR00012##
[0141] A solution of hydrocortisone .alpha.-chloroester (31 mg,
0.071 mmol), H.sub.2N--C-r7-CONH.sub.2 (150 mg, 0.071 mmol) and
diisopropylethylamine (15 .mu.L, 0.085 mmol) in dimethylformamide
(1 mL) was stirred for 18 h. The dimethylformamide was evaporated
off. The crude product purified by reverse-phase HPLC (22
mm.times.250 mm C-18 column, a 5-30% CH.sub.3CN/H.sub.2O gradient
with 0.1% trifluoroacetic acid, 214 and 254 nm UV detection) and
lyophilized. The desired product was obtained as a white powder (25
mg, 14%). TOF-MS (m/z): 1037.4 [(M+4 TFA)/2], 616.1 [(M+2 TFA)/3],
578.3 [(M+1 TFA)/3], 540.5 [(M+H)/3], 405.7 [(M+H)/4], 324.5
[(M+H)/5].
[0142] The use of 10 molar equivalents of diisopropylethylamine
rather than 1.2 molar equivalents provided the desired product as a
yellow powder (52% yield). TOF-MS (m/z): 887.0 [(M+1TFA)/2], 830.6
[(M+H)/2], 553.7 [(M+H)/3], 415.5 [(M+H)/4].
[0143] b. Conjugation of Hydrocortisone to a Biotin-Containing
Derivative of r.sub.5-Cys-CONH.sub.2
##STR00013##
[0144] Reactions were carried out as illustrated above, using the
synthetic techniques provided in the examples above.
[0145] i) Biotin-aminocaproic
acid-r5-C(hydrocortisone)-CONH.sub.2-- Used 10 molar equivalents of
diisopropylethylamine rather than 1.2 molar equivalents. Product a
white powder (65%). TOF-MS (m/z): 880.7 [(M+1 TFA)/2], 548.7
[(M+H)/3].
[0146] ii) Biotin-aminocaproic
acid-r7-C(hydrocortisone)-CONH.sub.2-- Used 10 molar equivalents of
diisopropylethylamine rather than 1.2 molar equivalents. Product a
white powder (36%). TOF-MS (m/z): 692.3 [(M+1 TFA)/3], 652.8
[(M+H)/3], 520.0 [(M+1 TFA)/4], 490.0 [(M+H)/4], 392.5
[(M+H)/5].
Example 7
[0147] This example illustrates the conjugation of taxol to a
transport moiety.
[0148] a. Conjugation of Taxol to r.sub.7-CONH.sub.2
[0149] This example illustrates the application of methodology
outlined above to the preparation of a taxol conjugate (see FIG.
12).
[0150] i) Preparation of a Taxol .alpha.-Chloroacetate Ester
##STR00014##
[0151] Taxol was treated with .alpha.-chloro acetic anhydride
providing the C-2' chloro acetyl derivative 121 in essentially
quantitative yield.
[0152] ii) Formation of Taxol Conjugate
##STR00015##
[0153] The halogen atom of the chloroacetate ester was displaced by
the thiol of an N-terminal (L) cysteine containing heptamer of
arginine. To avoid degradation of the transporter entity by
proteases in-vivo, D-arginine was used as the building unit.
Conjugation reactions were performed at room temperature in DMF in
the presence of diisopropylethylamine. The final products were
isolated by RP-HPLC and lyophilized to white powders. It is
important to note that the native conjugate (R.dbd.H) is isolated
as its TFA salt at the cysteine primary amine. The conjugates are
generally quite hygroscopic and readily dissolve in water.
[0154] The conjugate wherein R.dbd.H was designed to release the
parent drug via a nucleophilic attack of the N-terminal nitrogen
onto the C2' ester carbonyl. The protonation state of this nitrogen
is crucial for this mechanism, since only the free amine will be
capable of this release. Additionally, both conjugates share a
common .alpha.-hetero atom substituted acetate moiety making them
susceptible to simple ester hydrolysis. This offers an additional
release pathway.
Example 8
[0155] This example illustrates two methods of linking active
agents to transport moieties. Illustration is provided for retinoic
acid derivatives linked to poly-D-Arg derivatives but can be
applied to linkages between other biological agents and the
transport moieties of the present invention.
[0156] a. Linkage Between a Biological Agent Having an Aldehyde
Functional Group
[0157] This example illustrates the preparation of a conjugate
between a nonamer of D-arginine (H.sub.2N-r.sub.9-CO.sub.2H.10TFA)
and either all trans-retinal or 13-cis-retinal. FIG. 13 provides a
schematic presentation of the reactions. As seen in FIG. 13,
condensation of either retinal with
H.sub.2N-r.sub.9-CO.sub.2H.10TFA in MeOH in the presence of 4 .ANG.
molecular seives at room temperature for four hours results in the
formation of a Schiff base-type linkage between the retinal
aldehyde and the amino terminal group. Purification of the
conjugate can be accomplished by filtering the molecular sieves and
removing methanol under reduced pressure.
[0158] b. Conjugation of Retinoic Acid to r.sub.7-CONH.sub.2
[0159] This example illustrates the preparation of a conjugate
between retinoic acid and r.sub.7-CONH.sub.2 using the linking
group
##STR00016##
[0160] Here, preparation of the conjugate follows the scheme
outlined in FIG. 14. In this scheme, retinoic acid (141i) is first
combined with the chloroacetate ester of
4-hydroxymethyl-2,6-dimethylphenol (14i) to provide the conjugate
shown as 14iii. Combination of 14i with retinoic acid in methylene
chloride in the presence of dicyclohexylcarbodiimide and a
catalytic amount of 4-dimethylaminopyridine provided the retinoid
derivative 14iii in 52-57% yield. Condensation of 14iii with
H.sub.2NCys-r.sub.7CONH.sub.2.8TFA in the presence of
diisopropylethylamine (DMF, room temperature, 2 h) provides the
desired conjugated product 14iv.
[0161] All publications and patent applications cited in this
specification are herein incorporated by reference as if each
individual publication or patent application were specifically and
individually indicated to be incorporated by reference. Although
the foregoing invention has been described in some detail by way of
illustration and example for purposes of clarity of understanding,
it will be readily apparent to those of ordinary skill in the art
in light of the teachings of this invention that certain changes
and modifications may be made thereto without departing from the
spirit or scope of the appended claims.
* * * * *