U.S. patent application number 14/140223 was filed with the patent office on 2014-07-31 for compositions and methods for enhancing drug delivery across and into epithelial tissues.
This patent application is currently assigned to KAI PHARMACEUTICALS, INC.. The applicant listed for this patent is KAI PHARMACEUTICALS, INC.. Invention is credited to Thorsten A. Kirschberg, Leo P. McGrane, Jonathan B. Rothbard, Lalitha V.S. Sista, Paul A. Wender.
Application Number | 20140213532 14/140223 |
Document ID | / |
Family ID | 25157022 |
Filed Date | 2014-07-31 |
United States Patent
Application |
20140213532 |
Kind Code |
A1 |
Rothbard; Jonathan B. ; et
al. |
July 31, 2014 |
COMPOSITIONS AND METHODS FOR ENHANCING DRUG DELIVERY ACROSS AND
INTO EPITHELIAL TISSUES
Abstract
This invention provides compositions and methods for enhancing
delivery of drugs and other agents across epithelial tissues,
including the skin, gastrointestinal tract, pulmonary epithelium,
ocular tissues and the like. The compositions and methods are also
useful for delivery across endothelial tissues, including the blood
brain barrier. The compositions and methods employ a delivery
enhancing transporter that has sufficient guanidine or amidino
sidechain moieties to enhance delivery of a compound conjugated to
the reagent across one or more layers of the tissue, compared to
the non-conjugated compound. The delivery-enhancing polymers
include, for example, poly-arginine molecules that are preferably
between about 6 and 25 residues in length.
Inventors: |
Rothbard; Jonathan B.;
(Cupertino, CA) ; Wender; Paul A.; (Menlo Park,
CA) ; McGrane; Leo P.; (Mountain View, CA) ;
Sista; Lalitha V.S.; (Sunnyvale, CA) ; Kirschberg;
Thorsten A.; (Mountain View, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
KAI PHARMACEUTICALS, INC. |
South San Francisco |
CA |
US |
|
|
Assignee: |
KAI PHARMACEUTICALS, INC.
South San Francisco
CA
|
Family ID: |
25157022 |
Appl. No.: |
14/140223 |
Filed: |
December 24, 2013 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12816358 |
Jun 15, 2010 |
8623833 |
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14140223 |
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11542278 |
Oct 2, 2006 |
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12816358 |
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10740365 |
Dec 17, 2003 |
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11542278 |
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09792480 |
Feb 23, 2001 |
6669951 |
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10740365 |
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09648400 |
Aug 24, 2000 |
6593292 |
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09792480 |
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60150510 |
Aug 24, 1999 |
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Current U.S.
Class: |
514/20.5 ;
514/179; 514/254.07; 514/291; 514/449 |
Current CPC
Class: |
A61P 27/06 20180101;
A61K 47/55 20170801; A61P 23/00 20180101; A61K 49/085 20130101;
A61K 47/64 20170801; A61K 47/645 20170801; A61K 47/665 20170801;
A61P 43/00 20180101; A61K 47/54 20170801; A61K 49/0056 20130101;
A61P 5/00 20180101; A61P 25/02 20180101; A61P 37/06 20180101; A61P
33/02 20180101; A61P 5/38 20180101; A61P 31/04 20180101; A61K 47/62
20170801; A61P 31/10 20180101; A61P 9/00 20180101; A61P 31/12
20180101; A61K 47/555 20170801; C07K 7/64 20130101; A61K 38/13
20130101; A61K 47/557 20170801; A61K 31/337 20130101; A61K 31/573
20130101; A61P 35/00 20180101; A61K 31/155 20130101; A61K 38/00
20130101; A61P 29/00 20180101; A61P 27/02 20180101; A61P 27/12
20180101; A61K 31/436 20130101; A61K 49/0043 20130101; A61K 49/146
20130101; C07K 7/06 20130101; A61K 31/496 20130101; A61P 21/00
20180101; B82Y 5/00 20130101; A61P 1/04 20180101; A61P 1/10
20180101 |
Class at
Publication: |
514/20.5 ;
514/179; 514/449; 514/254.07; 514/291 |
International
Class: |
A61K 47/48 20060101
A61K047/48; A61K 31/436 20060101 A61K031/436; A61K 31/337 20060101
A61K031/337; A61K 31/496 20060101 A61K031/496; A61K 38/13 20060101
A61K038/13; A61K 31/573 20060101 A61K031/573 |
Claims
1. A method of targeting a compound to a gastrointestinal
epithelium of an animal, the method comprising administering to the
gastrointestinal epithelium a conjugate comprising the compound and
a delivery-enhancing transporter, wherein: i. the compound is
attached to the delivery-enhancing transporter through a linker;
and ii. the delivery-enhancing transporter comprises fewer than 50
subunits and comprises at least 5 guanidine or amidino moieties,
thereby increasing delivery of the conjugate into the
gastrointestinal epithelium compared to delivery of the compound in
the absence of the delivery-enhancing transporter.
2. A. method for enhancing delivery of a compound into and across
one or more layers of an animal ocular epithelial tissue, the
method comprising: administering to the ocular epithelial tissue a
conjugate comprising the compound and a delivery-enhancing
transporter, wherein: i. the compound is attached to the
delivery-enhancing transporter through a linker, and ii. the
delivery-enhancing transporter comprises fewer than 50 subunits and
comprises at least 5 guanidino or amidino moieties, thereby
increasing delivery of the conjugate into the gastrointestinal
epithelium compared to delivery of the compound in the absence of
the delivery-enhancing transporter.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. patent
application Ser. No. 12/816,358, filed Jun. 15, 2010, now allowed,
which is a continuation of U.S. patent application Ser. No.
11/542,278, filed Oct. 2, 2006, now abandoned, which is a
continuation of U.S. patent application Ser. No. 10/740,365, filed
Dec. 17, 2003, now abandoned, which is a continuation of U.S.
patent application Ser. No. 09/792,480, filed Feb. 23, 2001, now
U.S. Pat. No. 6,669,951, which is a continuation-in-part
application of U.S. patent application Ser. No. 09/648,400, filed
Aug. 24, 2000, now U.S. Pat. No. 6,593,292, issued Jul. 15, 2003,
which claims priority to U.S. Provisional Patent Application No.
60/150,510, filed Aug. 24, 1999, all of which are incorporated
herein by reference in their entirety for all purposes.
REFERENCE TO SEQUENCE LISTING
[0002] A Sequence Listing is being submitted electronically via EFS
in the form of a text file, created Dec. 24, 2013, and named
"0915088100US10seqlist.txt" (40,960 bytes), the contents of which
are incorporated herein by reference in their entirety.
BACKGROUND OF THE INVENTION
[0003] This invention pertains to the field of compositions and
methods that enhance the delivery of drugs and other compounds
across the dermal and epithelial membranes, including, for example,
skin, the gastrointestinal epithelium and the bronchial
epithelium.
BACKGROUND
[0004] Transdermal or transmucosal drug delivery is an attractive
route of drug delivery for several reasons. Gastrointestinal drug
degradation and the hepatic first-pass effect are avoided. In
addition, transdermal and transmucosal drug delivery is well suited
to controlled, sustained delivery (see, e.g., Elias, In
Percutaneous Absorption: Mechanisms-Methodology-Drug Delivery,
Bronaugh & Maibach, Eds., pp 1-12, Marcel Dekker, New York,
1989). For many applications, traditional methods of administering
drugs are not optimal because of the very large initial
concentration of the drug. Transdermal delivery could allow a more
uniform, slower rate of delivery of a drug. Moreover, patient
compliance is encouraged because such delivery methods are easy to
use, comfortable, convenient and non-invasive.
[0005] These advantages of transdermal and transmucosal delivery
have not led to many clinical applications because of the low
permeability of epithelial membranes, the skin in particular, to
drugs. The difficulties in delivering drugs across the skin result
from the barrier property of skin. Skin is a structurally complex
thick membrane that represents the body's border to the external
hostile environment. The skin is composed of the epidermis, the
dermis, the hypodermis, and the adenexal. structures (epidermal
appendages) The epidermis, the outermost epithelial tissue of the
skin, is itself composed of several layers--the stratum corneum,
the stratum granulosum, the stratum spinosum, and the stratum
basale.
[0006] Compounds that move from the environment into and through
intact skin must first penetrate the stratum corneum, the outermost
layer of skin, which is compact and highly keratinized. The stratum
corneum is composed of several layers of keratin-filled skin cells
that are tightly bound together by a "glue" composed of cholesterol
and fatty acids. The thickness of the stratum corneum varies
depending upon body location. It is the presence of stratum corneum
that results in the impermeability of the skin to pharmaceutical
agents. The stratum corneum is formed naturally by cells migrating
from the basal layer toward the skin surface where they are
eventually sloughed off. As the cells progress toward the surface,
they become progressively more dehydrated and keratinized. The
penetration across the stratum corneum layer is generally the
rate-limiting step of drug permeation across skin. See, e.g.,
Flynn, G. L., In Percutaneous Absorption:
Mechanisms-Methodology-Drug Delivery, supra, at pages 27-53.
[0007] After penetration through the stratum corneum layer,
systemically acting drug molecules then must pass into and through
the epidermis; the dermis, and finally through the capillary walls
of the bloodstream. The epidermis, which lies under the stratum
corneum, is composed of three layers. The outermost of these layers
is the stratum granulosum, which lies adjacent to the stratum
corneum, is composed of cells that are differentiated from basal
cells and keratinocytes, which make up the underlying layers,
having acquired additional keratin and a more flattened shape. The
cells of this layer of the epidermis, which contain granules that
are composed largely of the protein filaggrin. This protein is
believed to bind to the keratin filaments to form the keratin
complex. The cells also synthesize lipids that function as a
"cement" to hold the cells together. The epidermis, in particular
the stratum granulosum, contains enzymes such as
aminopeptidases.
[0008] The next-outermost layer of the epidermis is the stratum
spinosum, the principal cells of which are keratinocytes, which are
derived from basal cells that comprise the basal cell layer.
Langerhans cells, which are also found in the stratum spinosum, are
antigen-presenting cells and thus are involved in the mounting of
an immune response against antigens that pass into the skin. The
cells of this layer are generally involved in contact sensitivity
dermatitis.
[0009] The innermost epidermal layer is the stratum basale, or
basal cell layer, which consists of one cell layer of cuboidal
cells that are attached by hemi-desmosomes to a thin basement
membrane which separates the basal cell layer from the underlying
dermis. The cells of the basal layer are relatively
undifferentiated, proliferating cells that serve as a progenitor of
the outer layers of the epidermis. The basal cell layer includes,
in addition the basal cells, melanocytes.
[0010] The dermis is found under the epidermis, which is separated
from the dermis by a basement membrane that consists of
interlocking rete ridges and dermal papillae. The dermis itself is
composed of two layers, the papillary dermis and the reticular
dermis. The dermis consists of fibroblasts, histiocytes,
endothelial cells, perivascular macrophages and dendritic cells,
mast cells, smooth muscle cells, and cells of peripheral nerves and
their end-organ receptors. The dermis also includes fibrous
materials such as collagen and reticulin, as well as a ground
substance (principally glycosaminoglycans, including hyaluronic
acid, chondroitin sulfate, and dermatan sulfate).
[0011] Several methods have been proposed to enhance transdermal
transport of drugs. For example, chemical enhancers (Burnette, R. R
In Developmental Issues and Research Initiatives; Hadgraft J., Ed.,
Marcel Dekker: 1989; p.p. 247-288), iontophoresis, and others have
been used. However, in spite of the more than thirty years of
research that has gone into delivery of drugs across the skin in
particular, fewer than a dozen drugs are now available for
transdermal administration in, for example, skin patches.
[0012] Transport of drugs and other molecules across the
blood-brain barrier is also problematic. The brain capillaries that
make up the blood-brain barrier are composed of endothelial cells
that form tight junctions between themselves (Goldstein et al.,
Scientific American 255:74-83 (1986); Pardridge, W. M., Endocrin.
Rev. 7:314-330 (1986)). The endothelial cells and the tight
intercellular junctions that join the cells form a barrier against
the passive movement of many molecules from the blood to the brain.
The endothelial cells of the blood-brain barrier have few
pinocytotic vesicles, which in other tissues can allow somewhat
unselective transport across the capillary wall. Nor is the
blood-brain barrier interrupted by continuous gaps or channels that
run through the cells, thus allowing for unrestrained passage of
drugs and other molecules.
[0013] Thus, a need exists for improved reagents and methods for
enhancing delivery of compounds, including drugs, across epithelial
tissues and endothelial tissues such as the skin, the
gastrointestinal tract, the eye and the blood-brain barrier. The
present invention fulfills this and other needs.
SUMMARY OF THE INVENTION
[0014] The present invention provides methods of targeting a
compound to a gastrointestinal epithelium of an animal. The methods
involve administering to the gastrointestinal epithelium a
conjugate that includes the compound and a delivery-enhancing
transporter. The delivery-enhancing transporters, which are also
provided by the invention, have sufficient guanidine guanidino
moieties to increase delivery of the conjugate into the
gastrointestinal epithelium or ocular tissues compared to delivery
of the compound in the absence of the delivery-enhancing
transporter. In some embodiments, delivery of the conjugate into
the gastrointestinal epithelium or ocular tissue is increased at
least two-fold compared to delivery of the compound in the absence
of the delivery-enhancing transporter. In more preferred
embodiments, delivery of the conjugate into the gastrointestinal
epithelium is increased at least ten-fold compared to delivery of
the compound in the absence of the delivery-enhancing transporter.
The delivery-enhancing transporter and the compound are typically
attached through a linker. In addition, the conjugate can comprise
two or more delivery-enhancing transporters linked to the
compound.
[0015] Typically, the delivery-enhancing transporters comprise
fewer than 50 subunits and comprise at least 6 guanidine or amidino
moieties. In some embodiments, the subunits are amino acids. In
some embodiments, the delivery-enhancing transporters have from 6
to 25 guanidine or amidino moieties, and more preferably between 7
and 15 guanidine moieties and still more preferably, at least six
contiguous guanidino and/or amidino moieties. In some embodiments,
the delivery-enhancing transporters consist essentially of 5 to 50
subunits, at least 50 of which comprise guanidine or amidino
residues. In some of these embodiments, the subunits are natural or
non-natural amino acids. For example, in some embodiments, the
delivery-enhancing transporter comprises 5 to 25 arginine residues
or analogs thereof. For example, the transporter can comprise seven
contiguous D-arginines.
[0016] In some embodiments, the delivery-enhancing transporter
comprises 7-15 arginine residues or analogs of arginine. The
delivery-enhancing transporter can have at least one arginine that
is a D-arginine and in some embodiments, all arginines are
D-arginine. In some embodiments, at least 70% of the amino acids
are arginines or arginine analogs. In some embodiments, the
delivery-enhancing transporter comprises at least 5 contiguous
arginines or arginine analogs. The delivery-enhancing transporters
can comprise non-peptide backbones. In addition, in some aspects,
the transporter is not attached to an amino acid sequence to which
the delivery-enhancing molecule is attached in a naturally
occurring protein.
[0017] The delivery-enhancing transporters and methods of the
invention are useful for delivering drugs, diagnostic agents, and
other compounds of interest to the gastrointestinal epithelium. The
methods and compositions of the invention can be used not only to
deliver the compounds to the particular site of administration, but
also provide systemic delivery. In some embodiments, the conjugate
is administered bucally or as a suppository. The compounds of the
conjugate can be a therapeutic for a disease such as inflammatory
bowel disease, colon cancer, ulcerative colitis, gastrointestinal
ulcers, constipation and imbalance of salt and water absorption.
Thus, the compounds can include immunosuppressives, ascomycins,
corticosteroids, laxatives, antibiotics or anti-neoplastic agents.
In some aspects of the invention, the compound is targeted to the
iliem and/or colon.
[0018] The delivery-enhancing transporters and methods of the
invention are useful for delivering drugs, diagnostic agents, and
other compounds of interest to the eye and other ocular tissues. In
some embodiments, the conjugate is administered as eye drops or as
an injection. The compounds of the conjugate include therapeutics
for a disease such as conjunctivitis, bacterial infections, viral
infections, dry eye and glaucoma. Thus, the compounds can include
antibacterial compounds, antiviral compounds, cyclosporin,
ascomycins and corticosteroids.
[0019] As discussed above, the compound to be delivered can be
connected to the delivery-enhancing transporter by a linker. In
some embodiments, the linker is a releasable linker which releases
the compound, in biologically active form, from the
delivery-enhancing transporter after the compound has passed into
and/or through one or more layers of the epithelial and/or
endothelial tissue. In some embodiments, the compound is released
from the linker by solvent-mediated cleavage. The conjugate is, in
some embodiments, substantially stable at acidic pH but the
compound is substantially released from the delivery-enhancing
transporter at physiological pH. In some embodiments, the half-life
of the conjugate is between 5 minutes and 24 hours upon contact
with the skin or other epithelial or endothelial tissue. For
example, the half-life can be between 30 minutes and 2 hours upon
contact with the skin or other epithelial or endothelial
tissue.
[0020] Examples of conjugate structures of the invention include
those having structures such as 3, 4, or 5, as follows:
##STR00001##
wherein R.sup.1 comprises the compound; X is a linkage formed
between a functional group on the biologically active compound and
a terminal functional group on the linking moiety; Y 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, acyl, or allyl; R.sup.3 comprises the
delivery-enhancing transporter; R.sup.4 is S, O, NR.sup.6 or
CR.sup.7R.sup.6; R.sup.5 is H, OH, SH or NHR.sup.6; R.sup.6 is
hydrogen, alkyl, aryl, acyl or allyl; k and m are each
independently selected from 1 and 2; and n is 1 to 10.
[0021] Preferably, X is 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 phosphinate, and CR.sup.7R.sup.8, wherein R.sup.7 and
R.sup.8 are each independently selected from the group consisting
of H and alkyl. In some embodiments, R.sup.4 is S; R.sup.5 is
NHR.sup.6; and R.sup.6 is hydrogen, methyl, allyl, butyl or phenyl.
In some embodiments, R.sup.2 is benzyl; k, m, and n are each 1, and
X is O. In some embodiments, the conjugate. comprises structure 3,
Y is N, and R.sup.2 is methyl, ethyl, propyl, butyl, allyl, benzyl
or phenyl. In some embodiments, R.sup.2 is benzyl; k, m, and n are
each 1, and X is --OC(O)--. In some embodiments, the conjugate
comprises structure 4; R.sup.4 is S; R.sup.5 is NHR.sup.6; and
R.sup.6 is hydrogen, methyl, allyl, butyl or phenyl. In some
embodiments, the conjugate comprises structure 4; R.sup.5 is
NHR.sup.6; R.sup.6 is hydrogen, methyl, allyl, butyl or phenyl; and
k and m are each 1.
[0022] The invention also provides conjugates in which the release
of the linker from the biological agent involves a first,
rate-limiting intramolecular reaction, followed by a faster
intramolecular reaction that results in release of the linker. The
rate-limiting reaction can, by appropriate choice of substituents
of the linker, be made to be stable at a pH that is higher or lower
than physiological pH. However, once the conjugate has passed into
and across one or more layers of an epithelial or endothelial
tissue, the linker will be cleaved from the agent. An example of a
compound that has this type of linker is structure 6, as
follows:
##STR00002##
wherein R.sup.1 comprises the compound; X is a linkage formed
between a functional group on the biologically active compound and
a terminal functional group on the linking moiety; Y is a linkage
formed from a functional group on the transport moiety and a
functional group on the linking moiety; Ar is an aryl group having
the attached radicals arranged in an ortho or para configuration,
which aryl group can be substituted or unsubstituted; R.sup.3
comprises the livery-enhancing transporter; 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 selected from hydrogen or alkyl; and
k and m are each independently selected from 1 and 2.
[0023] In preferred embodiments, the compositions of the invention
comprise a linker susceptible to solvent-mediated cleavage. For
example, a preferred linker is substantially stable at acidic pH
but is substantially cleaved at physiological pH.
BRIEF DESCRIPTION OF THE FIGURES
[0024] FIG. 1 shows a reaction scheme for the preparation of an
a-chloroacetyl cyclosporin A derivative.
[0025] FIG. 2 shows a general procedure for the coupling of
cysteine-containing peptides to the .alpha.-chloro acetyl
cyclosporin A derivative.
[0026] FIG. 3 shows a reaction scheme for the coupling of the
cyclosporin A derivative to a biotin-labeled peptide.
[0027] FIG. 4 shows a reaction scheme for coupling of a cyclosporin
A derivative to an unlabeled peptide.
[0028] FIG. 5 A-H show various types of cleavable linkers that can
be used to link a delivery-enhancing transporter to a biologically
active agent or other molecule of interest. FIG. 5A shows an
example of a disulfide linkage. FIG. 5B shows a photocleavable
linker which is cleaved upon exposure to electromagnetic radiation.
FIG. 5C shows a modified lysyl residue used as a cleavable linker.
FIG. 5D shows a conjugate in which the delivery-enhancing
transporter T is linked to the 2'-oxygen of the anticancer agent,
paclitaxel. The linking moiety 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. FIG. 5E
a linkage of a delivery-enhancing transporter to a biologically
active agent, e.g., paclitaxel, by an aminoalkyl carboxylic acid; a
linker amino group is joined to a delivery-enhancing transporter by
an amide linkage and to a paclitaxel moiety by an ester linkage.
FIGS. 5F and G show chemical structures and conventional numbering
of constituent backbone atoms for paclitaxel and "TAXOTERE.TM."
(R'=H, R''=BOC). FIG. 5G shows the general chemical structure and
ring atom numbering for taxoid compounds.
[0029] FIG. 6 displays a synthetic scheme for a chemical conjugate
between a heptamer of L-arginine and cyclosporin A (panel A) and
its pH dependent chemical release (panel B). The .alpha.-chloro
ester (6i) was treated with benzylamine in the presence of sodium
iodide to effect substitution, giving the secondary amine (6ii).
Amine (6ii) was treated with anhydride (6) and the resultant crude
acid (6iii) was converted to its corresponding NHS ester (6iv).
Ester (6iv) was then coupled with the amino terminus of
hepta-L-arginine, giving the N-Boc protected CsA conjugate (6v).
Finally, removal of the Boc protecting group with formic acid
afforded the conjugate (6vi) as its octatrifluoroacetate salt after
HPLC purification.
[0030] FIG. 7 displays inhibition of inflammation in murine contact
dermatitis by releasable R7 CsA. Balb/c (6-7 weeks) mice were
painted on the abdomen with 100 .mu.l of 0.7% DNFB in acetone olive
oil (95:5). Three days later both ears of the animals were
restimulated with 0.5% DNFB in acetone. Mice were treated one,
five, and twenty hours after restimulation with either vehicle
alone, 1% R7 peptide alone, 1% CsA, 1% nonreleasable R7 CsA,
0.01%/0.1%/1.0% releasable R7 CsA, and the fluorinated steroid
positive control 0.1% triamcinolone acetonide. Ear inflammation was
measured 24 hours after restimulation using a spring loaded
caliper. The percent reduction of inflammation was calculated using
the following formula (t-n)/(u-n), where t=thickness of the treated
ear, n=the thickness of a normal untreated ear, and u=thickness of
an inflamed ear without any treatment. N=20 animals in each
group.
[0031] FIG. 8 shows a procedure for the preparation of a copper
diethylene-triaminepentaacetic acid complex (Cu-DTPA).
[0032] FIG. 9 shows a procedure for linking the Cu-DTPA to a
transporter through an aminocaproic acid.
[0033] FIG. 10 shows a reaction for the acylation of hydrocortisone
with chloroacetic anhydride.
[0034] FIG. 11 shows a reaction for linking the acylated
hydrocortisone to a transporter.
[0035] FIG. 12 shows a reaction for preparation of C-2' derivatives
of taxol.
[0036] FIG. 13 shows a schematic of a reaction for coupling of a
taxol derivative to a biotin-labeled peptide.
[0037] FIG. 14 shows a reaction for coupling of an unlabeled
peptide to a C-2' derivative of taxol.
[0038] FIG. 15A-C shows a reaction scheme for the formation of
other C-2' taxol-peptide conjugates.
[0039] FIG. 16 shows a general strategy for synthesis of a
conjugate in which a drug or other biological agent is linked to a
delivery-enhancing transporter by a pH-releasable linker.
[0040] FIG. 17 shows a schematic diagram of a protocol for
synthesizing a taxol 2'-chloroacetyl derivative.
[0041] FIG. 18 shows a strategy by which a taxol2'-chloroacetyl
derivative is linked to an arginine heptamer delivery-enhancing
transporter.
[0042] FIG. 19 shows three additional taxol-r7 conjugates that can
be made using the reaction conditions illustrated in FIG. 18.
[0043] FIG. 20 shows the results of a 3 day MIT cytotoxicity assay
using taxol and two different linkers.
[0044] FIG. 21 shows the FACS cellular uptake assay of truncated
analogs of Tat.sub.49-57 (F1-ahx-RKKRRQRRR): Tat.sub.49-56
(F1-ahx-RKKRRQRR), Tat.sub.49-55 (F1-ahx-RKKRRQR), Tat.sub.50-57
(F1-ahx-KKRRQRRR), and Tat.sub.51-57 (F1-ahx-KRRQRRR). Jurkat cells
were incubated with varying concentrations (12.5 .mu.M shown) of
peptides for 15 min at 23.degree. C.
[0045] FIG. 22 shows FACS cellular uptake assay of
alanine-substituted analogs of Tat.sub.49-57: A-49
(F1-ahx-AKKRRQRRR), A-50 (F1-ahx-RAKRRQRRR), A-51
(F1-ahx-RKARRQRRR), A-52 (F1-ahx-RKKARQRRR), A-53
(F1-ahx-RKKRAQRRR), A-54 (F1-ahx-RKKRAQRRR), A-55
(F1-ahx-RKKRRQRRR), A-56 (F1-ahx-RKKRRQRRR), and A-57
(F1-ahx-RKKRRQRRR). Jurkat cells were incubated with varying
concentrations (12.5 .mu.M shown) of peptides for 12 min at
23.degree. C.
[0046] FIG. 23 shows the FACS cellular uptake assay of d- and
retro-isomers of Tat.sub.149-57: d-Tat.sub.49-57 (F1-ahx-rkkrrqrr),
Tat.sub.57-49 (F1-ahx-RRRQRRKKR), and d-Tat.sub.57-49
(F1-ahx-rrqrrkkr). Jurkat cells were incubated with varying
concentrations (12.5 .mu.M shown) of peptides for 15 min at
23.degree. C.
[0047] FIG. 24 shows the FACS cellular uptake of a series of
arginine oligomers and Tat.sub.49-57: R5 (F1-ahx-RRRRR), R6
(F1-ahx-RRRRRR), R7 (F1-ahx-RRRRRRR), R8 (F1-ahx-RRRRRRRR), R9
(F1-ahx-RRRRRRRRR), r5 (F1-ahx-rrrr), r6 (F1-ahx-rrrrrr), r7
(F1-ahx-rrrrrr), r8 (F1-ahx-rrrrr), r9 (F1-ahx-rrrrr). Jurkat cells
were incubated with varying concentrations (12.5 .mu.M shown) of
peptides for 4 min at 23.degree. C.
[0048] FIG. 25 displays the preparation of guanidine-substituted
peptoids.
[0049] FIG. 26 displays the FACS cellular uptake of polyguanidine
peptoids and d-arginine oligomers. Jurkat cells were incubated with
varying concentrations (12.5 .mu.M shown) of peptoids and peptides
for 4 min at 23.degree. C.
[0050] FIG. 27 displays the FACS cellular uptake of d-arginine
oligomers and polyguanidine peptoids. Jurkat cells were incubated
with varying concentrations (12.5 .mu.M shown) of fluorescently
labeled peptoids and peptides for 4 min at 23.degree. C.
[0051] FIG. 28 displays the FACS cellular uptake of and d-arginine
oligomers and N-hxg peptoids. Jurkat cells were incubated with
varying concentrations (6.3 .mu.M shown) of fluorescently labeled
peptoids and peptides for 4 min at 23.degree. C.
[0052] FIG. 29 shows the FACS cellular uptake of d-arginine
oligomers and N-chg peptoids. Jurkat cells were incubated with
varying concentrations (12.5 .mu.M shown) of fluorescently labeled
peptoids and peptides for 4 min at 23.degree. C.
[0053] FIG. 30 shows a general strategy for attaching a
delivery-enhancing transporter to a drug that includes a triazole
ring structure.
[0054] FIG. 31A and FIG. 31B show synthetic schemes for making
conjugates in which FK506 is attached to a delivery-enhancing
transporter.
[0055] FIG. 32 show that short oligomers of arginine, but not
lysine, effectively enter Caco-2 cells. Caco-2 cells were incubated
with varying concentrations with each of the peptides shown,
washed, and analyzed by flow cytometry. Uptake could be inhibited
with preincubation with sodium azide, demonstrating that it was
energy dependent.
[0056] FIG. 33 displays the measured fluorescence of Caco-2 cells
exposed to fluorescent taxol or fluorescent, nonreleasable taxol
conjugates with either heptamers (r7) or decamers (r10) of
D-arginine.
[0057] FIG. 34 demonstrates Caco-2 monolayer integrity by the
measurement of a stable transepithlial electric resistance of
greater than 100 ohm cm.sup.2 for the duration of the experiments
described in this report.
[0058] FIG. 35 shows the accumulation of either F1 aca r5, F1 aca
r9, Lucifer Yellow, or hydrocortisone in the basolateral chamber of
a diffusion apparatus after transport through a Caco-2 cell
monolayer after one hour.
[0059] FIG. 36 displays the blood levels of CsA in rats measured by
LC MS MS at thirty minute intervals after intracolonic injection.
CsA was administered in Cremophor E1:ethanol, 1:1, whereas the CsA
conjugates were administered in PBS. The half life in PBS, pH 7.4
at 37.degree. C., of the CsA conjugate (CGC1072) was 1.5 hours.
[0060] FIG. 37 displays the blood levels of taxol in rats measured
by LC MS MS at thirty minute intervals after intracolonic
injection. Taxol was administered in Cremophor El:ethanol, 1:1,
whereas the two taxol conjugates were administered in PBS. The half
life in PBS, pH 7.4 at 37.degree. C., of the slower releasing
conjugate (14) was 5 hours, while that of the faster conjugate (13)
was ten minutes. See, Example 18.
[0061] FIG. 38 displays the blood levels of taxol in rats measured
by LC MS MS at thirty minute intervals after buccal delivery. Taxol
was administered in Cremophor El:ethanol, 1:1, whereas the taxol
conjugate was administered in PBS. The half life in PBS, pH 7A at
37.degree. C., of the conjugate (13) was ten minutes.
[0062] FIG. 39 illustrates the conjugation of acyclovir to r7-amide
via an N-terminal cysteine group. Conjugation with a
biotin-containing transporter is also shown.
[0063] FIG. 40 illustrates the conjugate formed between a retinal
and a r9 (shown without spacing amino acids).
[0064] FIG. 41 illustrates the use of a cleavable linker in
preparing a retinoic acid-r9 conjugate.
DETAILED DESCRIPTION
Definitions
[0065] An "epithelial tissue" is 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.
[0066] The term "trans-epithelial" delivery or administration
refers to the delivery or administration of agents by permeation
through 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 transmucosal administration. Delivery
can be to a deeper layer of the tissue for example, and/or delivery
to the bloodstream.
[0067] "Delivery enhancement, "penetration enhancement" or
"permeation enhancement" as used herein relates to an increase in
amount and/or rate of delivery of a compound that is delivered into
and across one or more layers of an epithelial or endothelial
tissue. An enhancement of delivery can be observed by measuring the
rate and/or amount of the compound that passes through one or more
layers of animal or human skin or other tissue. Delivery
enhancement also can involve an increase in the depth into the
tissue to which the compound is delivered, and/or the extent of
delivery to one or more cell types of the epithelial or other
tissue (e.g., increased delivery to fibroblasts, immune cells, and
endothelial cells of the skin or other tissue). Such measurements
are readily obtained by, for example, using a diffusion cell
apparatus as described in U.S. Pat. No. 5,891,462.
[0068] The amount or rate of delivery of an agent across and/or
into skin or other epithelial or endothelial membrane is sometimes
quantitated in terms of the amount of compound passing through a
predetermined area of skin or other tissue, which is a defined area
of intact unbroken living skin or mucosal tissue. That area will
usually be in the range of about 5 cm.sup.2 to about 100 cm.sup.2
more usually in the range of about 10 cm.sup.2 to about 100
cm.sup.2, still more usually in the range of about 20 cm.sup.2 to
about 60 cm.sup.2.
[0069] The terms "guanidyl," guanidinyl" and "guanidine" are used
interchangeably to refer to a moiety having the formula
--HN.dbd.C(NH.sub.2)NH (unprotonated form). As an example, arginine
contains a guanidyl (guanidine) moiety, and is also referred to as
2-amino-5-guanidinovaleric acid or
.alpha.-amino-.delta.-guanidinovaleric acid. "Guanidium" refers to
the positively charged conjugate acid form. The term "guanidine
moiety" includes, for example, guanidine, guanidinium, guanidine
derivatives such as (RNHC(NH)NHR'), monosubstituted guanidines,
monoguanides, biguanides, biguanide derivatives such as
(RNHC(NH)NHC(NH)NHR'), and the like. In addition, the term
"guanidine moiety" encompasses any one or more of a guanide alone
or a combination of different guanides.
[0070] "Amidinyl" and "amidino" refer to a moiety having the
formula --C(.dbd.NH)(NH.sub.2). "Amidinium" refers to the
positively charged conjugate acid form.
[0071] The term "trans-barrier concentration" or "trans-tissue
concentration" refers to the concentration of a compound present on
the side of one or more layers of an epithelial or endothelial
barrier tissue that is opposite or "trans" to the side of the
tissue to which a particular composition has been added. For
example, when a compound is applied to the skin, the amount of the
compound measured subsequently across one or more layers of the
skin is the trans-barrier concentration of the compound.
[0072] "Biologically active agent" or "biologically active
substance" refers to a chemical substance, such as a small
molecule, macromolecule, or metal ion, that causes 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 or decreased rate of synthesis of a metabolite,
increased or decreased cell proliferation, and the like.
[0073] The terms "therapeutic agent", "therapeutic composition",
and "therapeutic substance" refer, without limitation, to any
composition that can be used to the benefit of a mammalian species.
Such agents may take the form of ions, small organic molecules,
peptides, proteins or polypeptides, oligonucleotides, and
oligosaccharides, for example.
[0074] The term "macromolecule" as used herein 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.
[0075] "Small organic molecule" refers to a carbon-containing agent
having a molecular weight (MW) of less than or equal to 1000
daltons.
[0076] The terms "non-polypeptide agent" and "non-polypeptide
therapeutic agent, refer to the portion of a conjugate that does
not include the delivery-enhancing transporter, and that is a
biologically active agent other than a polypeptide. An example of a
non-polypeptide agent is an anti-sense oligonucleotide, which can
be conjugated to a poly-arginine peptide to form a conjugate for
enhanced delivery into and across one or more layers of an
epithelial or endothelial tissue.
[0077] A "subunit," as used herein, is a monomeric unit that are
joined to form a larger polymeric compound. The set of amino acids
are an example of subunits. Each amino acid shares a common
backbone (--C--C--N--), and the different amino acids differ in
their sidechains. The backbone is repeated in a polypeptide. A
subunit represents the shortest repeating pattern of elements in a
polymer backbone. For example, two amino acids of a peptide are not
considered a peptide because two amino acids would not have the
shortest repeating pattern of elements in the polymer backbone.
[0078] The term "polymer" refers to a linear chain of two or more
identical or non-identical subunits joined by covalent bonds. A
peptide is an-example of a polymer; peptides can be composed of
identical or non-identical amino acid subunits that are joined by
peptide linkages (amide bonds).
[0079] The term "peptide" as used herein 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., R7 represents a heptamer that consists of
L-arginine residues).
[0080] The term "protein" as used herein 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.
[0081] "Polypeptide" as used herein refers to a polymer of at least
two amino acid residues and which contains one or more peptide
bonds. "Polypeptide" encompasses pep-tides and proteins, regardless
of whether the polypeptide has a well-defined conformation.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0082] The present invention provides compositions and methods that
enhance the transfer of compounds, including drugs and other
biologically active compounds, into and across one or more layers
of an animal epithelial or endothelial tissue. The methods involve
contacting the tissue with a conjugate that includes the compound
of interest linked to a delivery-enhancing transporter. The
delivery enhancing transporters provided by the invention are
molecules that include sufficient guanidino or amidino moieties to
increase delivery of the conjugate into and across one or more
intact epithelial and endothelial-tissue layers. The methods and
compositions are useful for trans-epithelial and trans-endothelial
delivery of drugs and other biologically active molecules, and also
for delivery of imaging and diagnostic molecules. The methods and
compositions of the invention are particularly useful for delivery
of compounds that require trans-epithelial or trans-endothelial
transport to exhibit their biological effects, and that by
themselves (without conjugation to a delivery-enhancing
transporters or some other modification), are unable, or only
poorly able, to cross such tissues and thus exhibit biological
activity.
[0083] The delivery-enhancing transporters and methods of the
invention provide significant advantages over previously available
methods for obtaining trans-epithelial and trans-endothelial tissue
delivery of compounds of interest. The transporters make possible
the delivery of drugs and other agents across tissues that were
previously impenetrable to the drug. For example, while delivery of
drugs across skin was previously nearly impossible for all but a
few compounds, the methods of the invention can deliver compounds
not only into cells of a first layer of an epithelial tissue such
as skin, but also across one or more layers of the skin. The blood
brain barrier is also resistant to transport of drugs and other
diagnostic and therapeutic reagents; the methods and transporters
of the invention provide means to obtain such transport.
[0084] The delivery-enhancing transporters increase delivery of the
conjugate into and across one or more intact epithelial or
endothelial tissue layers compared to delivery of the compound in
the absence of the delivery-enhancing transporter. The
delivery-enhancing transporters can, in some embodiments, increase
delivery of the conjugate significantly over that obtained using
the tat protein of HIV-1 (Frankel et al. (1991) PCT Pub. No. WO
91/09958). Delivery is also increased significantly over the use of
shorter fragments of the tat protein containing the tat basic
region (residues 49-57 having the sequence RKKRRQRRR) (Barsoum et
al. (1994) WO 94/04686 and Fawell et al. (1994) Proc. Nat.'l. Acad.
Sci. USA 91: 664-668). Preferably, delivery obtained using the
transporters of the invention is increased more than 2-fold, still
more preferably six-fold, still more preferably ten-fold, and still
more preferably twenty-fold, over that obtained with tat residues
49-57. In some embodiments, the compositions of the invention do
not include tat residues 49-57.
[0085] Similarly, the delivery-enhancing transporters of the
invention can provide increased delivery compared to a 16 amino
acid peptide-cholesterol conjugate derived from the Antennapedia
homeodomain that is rapidly internalized by cultured neurons
(Brugidou et al. (1995) Biochem. Biophys. Res. Commun. 214:
685-93). This region, residues 43-58 at minimum, has the amino acid
sequence RQIKIWFQNRRMKWKK. The Herpes simplex protein VP22, like
tat and the Antennapedia domain, was previously known to enhance
transport into cells, but was not known to enhance transport into
and across endothelial and epithelial membranes (Elliot and O'Hare
(1997) Cell 88: 223-33; Dilber et al. (1999) Gene Ther. 6: 12-21;
Phelan et al. (1998) Nature Biotechnol. 16: 440-3). In presently
preferred embodiments, the delivery-enhancing transporters provide
significantly increased delivery compared to the Antennapedia
homeodomain and to the VP22 protein. In some embodiments, the
compositions of the invention do not include the Antennapedia
homeodomain, the VP22 protein or eight contiguous arginines.
[0086] Structure of Delivery-Enhancing Transporters
[0087] The delivery-enhancing transporters of the invention are
molecules that have sufficient guanidine and/or amidino moieties to
increase delivery of a compound to which the delivery-enhancing
transporter is attached into and across one or more layers of an
epithelial tissue (e.g., skin or mucous membrane) or an endothelial
tissue (e.g., the blood-brain barrier). The delivery-enhancing
transporters generally include a backbone structure to which is
attached the guanidine and/or amidino sidechain moieties. In some
embodiments, the backbone is a polymer that consists of subunits
(e.g., repeating monomer units), at least some of which subunits
contain a guanidine or amidino moiety.
[0088] A. Guanidino and/or Amidino Moieties
[0089] The delivery-enhancing transporters typically display at
least 5 guanidine and/or amidino moieties, and more preferably 7 or
more such moieties. Preferably, the delivery-enhancing transporters
have 25 or fewer guanidine and/or amidino moieties, and often have
15 or fewer of such moieties. In some embodiments, the
delivery-enhancing transporter consists essentially of 50 or fewer
subunits, and can consist essentially of 25 or fewer, 20 or fewer,
or 15 or fewer subunits. The delivery-enhancing transporter can be
as short as 5 subunits, in which case all subunits include a
guanidino amidino sidechain moiety. The delivery-enhancing
transporters can have, for example, at least 6 subunits, and in
some embodiments have at least 7 or 10 subunits. Generally, at
least 50% of the subunits contain a guanidine or amidino sidechain
moiety. More preferably, at least 70% of the subunits, and
sometimes at least 90% of the subunits in the delivery-enhancing
transporter contain a guanidine or amidino sidechain moiety.
[0090] Some or all of the guanidine and/or amidino moieties in the
delivery-enhancing transporters can be contiguous. For example, the
delivery-enhancing transporters can include from 6 to 25 contiguous
guanidine and/or amidino-containing subunits. Seven or more
contiguous guanidine and/or amidino-containing subunits are present
in some embodiments. In some embodiments, each subunit that
contains a guanidine moiety is contiguous, as exemplified by a
polymer containing at least six contiguous arginine residues.
[0091] The delivery-enhancing transporters are exemplified by
peptides. Arginine residues or analogs of arginine can constitute
the subunits that have a guanidine moiety. Such an
arginine-containing peptide can be composed of either all D-, all
L- or mixed D and L-amino acids, and can include additional amino
acids, amino acid analogs, or other molecules between the arginine
residues. Optionally, the delivery-enhancing transporter can also
include a non-arginine residue to which a compound to be delivered
is attached, either directly or through a linker. The use of at
least one D-arginine in the delivery-enhancing transporters can
enhance biological stability of the transporter during transit of
the conjugate to its biological target. In some cases the
delivery-enhancing transporters are at least about 50% D-arginine
residues, and for even greater stability transporters in which all
of the subunits are D-arginine residues are used. If the delivery
enhancing transporter molecule is a peptide, the transporter is not
attached to an amino acid sequence to which the amino acids that
make up the delivery enhancing transporter molecule are attached in
a naturally occurring protein.
[0092] Preferably, the delivery-enhancing transporter is linear. In
a preferred embodiment, an agent to be delivered into and across
one or more layers of an epithelial tissue is attached to a
terminal end of the delivery-enhancing transporter. In some
embodiments, the agent is linked to a single transport polymer to
form a conjugate. In other embodiments, the conjugate can include
more than one delivery-enhancing transporter linked to an agent, or
multiple agents linked to a single delivery-enhancing
transporter.
[0093] More generally, it is preferred that each subunit contains a
highly basic sidechain moiety which (i) has a pKa of greater than
11, more preferably 125 or greater, and (ii) contains, in its
protonated state, at least two geminal amino groups (NH.sub.2)
which share a resonance-stabilized positive charge, which gives the
moiety a bidentate character.
[0094] The guanidine or amidino moieties extend away from the
backbone by virtue of being linked to the backbone by a sidechain
linker. The sidechain atoms are preferably provided as methylene
carbon atoms, although one or more other atoms such as oxygen,
sulfur or nitrogen can also be present. For example, a linker that
attaches a guanidine moiety to a backbone can be shown as:
##STR00003##
In these formulae, n is preferably at least 2, and is preferably
between 2 and 7. In some embodiments, n is 3 (arginine for
structure 1). In other embodiments, n is between 4 and 6; most
preferably n is 5 or 6. Although the sidechain in the exemplified
formulae is shown as being attached to a peptide backbone (i.e., a
repeating amide to which the sidechain is attached to the carbon
atom that is a to the carbonyl group, subunit 1) and a peptoid
backbone (i.e., a repeating amide to which the sidechain is
attached to the nitrogen atom that is .beta. to the carbonyl group,
subunit 2), other non-peptide backbones are also suitable, as
discussed in more detail herein. Thus, similar sidechain linkers
can be attached to nonpeptide backbones (e.g., peptoid
backbones).
[0095] In some embodiments, the delivery-enhancing transporters are
composed of linked subunits, at least some of which include a
guanidino and/or amidino moiety. Examples of suitable subunits
having guanidine and/or amidino moieties are described below.
[0096] Amino Acids.
[0097] In some embodiments, the delivery-enhancing transporters are
composed of D or L amino acid residues. The amino acids can be
naturally occurring or non-naturally occurring amino acids.
Arginine (.alpha.-amino-.beta.-guanidinovaleric acid) and
.alpha.-amino-.epsilon.-amidino-hexanoic acid (isosteric amidino
analog) are examples of suitable guanidine- and amidino-containing
amino acid subunits. The guanidinium group in arginine has a pKa of
about 12.5. In some preferred embodiments the transporters are
comprised of at least six contiguous arginine residues.
[0098] Other amino acids, such as
.alpha.-amino-.beta.-guanidino-propionic acid,
.alpha.-amino-.gamma.-guanidino-butyric acid, or
.alpha.-amino-.epsilon.-guanidino-caproic acid (containing 2, 3 or
5 sidechain linker atoms, respectively, between the backbone chain
and the central guanidinium carbon) can also be used.
[0099] D-amino acids can also be used in the delivery enhancing
transporters. Compositions containing exclusively D-amino acids
have the advantage of decreased enzymatic degradation. However,
they can also remain largely intact within the target cell. Such
stability is generally not problematic if the agent is biologically
active when the polymer is still attached. For agents that are
inactive in conjugate form, a linker that is cleavable at the site
of action (e.g., by enzyme- or solvent-mediated cleavage within a
cell) should be included within the conjugate to promote release of
the agent in cells or organelles.
[0100] In addition, the transport moieties are amino acid oligomers
of the following formulae: (ZYZ).sub.nZ, (ZY).sub.nZ, (ZYY).sub.nZ
and (ZYYY).sub.nZ. See, U.S. Patent Application No. 60/269,627
filed Feb. 16, 2001 (Attorney docket No. 019801-001000US. "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.
[0101] 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
.delta.-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.
[0102] 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.
[0103] In yet another group of preferred embodiments, the transport
moiety has the formula (ZY).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.
[0104] In still another group of preferred embodiments, the
transport moiety has the formula (ZYYY)nZ, wherein each "Y" is
preferably selected from glycine, P-alanine, y-amino butyric acid
and E-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.
[0105] 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.
[0106] In other embodiments, each of the Y groups will be selected
to enhance certain desired properties of the transport moiety. For
example, when transport moieties 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).
[0107] 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).
[0108] Other Subunits.
[0109] Subunits other than amino acids can also be selected for use
in forming transport polymers. Such subunits can include, but are
not limited to, hydroxy amino acids, N-methyl-amino acids amino
aldehydes, and the like, which result in polymers with reduced
peptide bonds. Other subunit types can be used, depending on the
nature of the selected backbone, as discussed in the next
section.
[0110] B. Backbones
[0111] The guanidine and/or amidino moieties that are included in
the delivery-enhancing transporters are generally attached to a
linear backbone. The backbone can comprise a variety of atom types,
including carbon, nitrogen, oxygen, sulfur and phosphorus, with the
majority of the backbone chain atoms typically consisting of
carbon. A plurality of sidechain moieties that include a terminal
guanidine or amidino group are attached to the backbone. Although
spacing between adjacent sidechain moieties is typically
consistent, the delivery-enhancing transporters used in the
invention can also include variable spacing between sidechain
moieties along the backbone.
[0112] A more detailed backbone list includes N-substituted amide
(CONR replaces CONH linkages), esters (CO.sub.2), keto-methylene
(COCH.sub.2) reduced or methyleneamino (CH.sub.2NH), thioamide
(CSNH), phosphinate (PO.sub.2RCH.sub.2), phosphonamidate and
phosphonamidate ester (PO.sub.2RNH), retropeptide (NHCO),
trans-alkene (CR.dbd.CH), fluoroalkene (CF.dbd.CH), dimethylene
(CH.sub.2CH.sub.2), thioether (CH.sub.2S), hydroxyethylene
(CH(OH)CH.sub.2), methyleneoxy (CH.sub.2O), tetrazole (CN.sub.4),
retrothioamide (NHCS), retroreduced (NHCH.sub.2), sulfonamide
(SO.sub.2NH), methylenesulfonamido (CHRSO.sub.2NH),
retrosulfonamide (NHSO.sub.2), and peptoids (N-substituted amides),
and backbones with malonate and/or gem-diamino-alkyl subunits, for
example, as reviewed by Fletcher et al. ((1998) Chem. Rev. 98:763)
and detailed by references cited therein. Many of the foregoing,
substitutions result in approximately isosteric polymer backbones
relative to backbones formed from .alpha.-amino acids.
[0113] As mentioned above, in a peptoid backbone, the sidechain is
attached to the backbone nitrogen atoms rather than the carbon
atoms. (See e.g., Kessler (1993) Angew. Chem. Int. Ed. Engl.
32:543; Zuckerman et al. (1992) Chemtracts-Macromol. Chem. 4:80;
and Simon et al. (1992) Proc. Nat'l. Acad. Sci. USA 89:9367). An
example of a suitable peptoid backbone is
poly-(N-substituted)glycine (poly-NSG). Synthesis of peptoids is
described in, for example, U.S. Pat. No. 5,877,278. As the term is
used herein, transporters that have a peptoid backbone are
considered "non-peptide" transporters, because the transporters are
not composed of amino acids having naturally occurring sidechain
locations. Non-peptide backbones, including peptoid backbones,
provide enhanced biological stability (for example, resistance to
enzymatic degradation in vivo).
[0114] C. Synthesis of Delivery-Enhancing Transporters
[0115] Delivery-enhancing transporters are constructed by any
method known in the art. Exemplary peptide polymers can be produced
synthetically, preferably using a peptide synthesizer (e.g., an
Applied Biosystems Model433) or can be synthesized recombinantly by
methods well known in the art. Recombinant synthesis is generally
used when the delivery enhancing transporter is a peptide which is
fused to a polypeptide or protein of interest.
[0116] N-methyl and hydroxy-amino acids can be substituted for
conventional amino acids in solid phase peptide synthesis. However,
production of delivery-enhancing transporters with reduced peptide
bonds requires synthesis of the dimer of amino acids containing the
reduced peptide bond. Such dimers are incorporated into polymers
using standard solid phase synthesis procedures. Other synthesis
procedures are well known and can be found, for example, in
Fletcher et al. (1998) Chem. Rev. 98:763, Simon et al. (1992) Proc.
Nat'l. Acad. Sci. USA 89:9367, and references cited therein.
[0117] The delivery-enhancing transporters of the invention can be
flanked by one or more non-guanidino/non-amidino subunits (such as
glycine, alanine, and cysteine, for example), or a linker (such as
an aminocaproic acid group), that do not significantly affect the
rate of trans-tissue layer transport of the corresponding
delivery-enhancing transporter-containing conjugates. Also, any
free amino terminal group can be capped with a blocking group, such
as an acetyl or benzyl group, to prevent ubiquitination in
vivo.
[0118] Where the transporter is a peptoid polymer, one synthetic
method involves the following steps: 1) a peptoid polyamine is
treated with a base and pyrazole-1-carboxamidine to provide a
mixture; 2) the mixture is heated and then allowed to cool; 3) the
cooled mixture is acidified; and 4) the acidified mixture is
purified. Preferably the base used in step 1 is a carbonate, such
as sodium carbonate, and heating step 2 involves heating the
mixture to approximately 50.degree. C. for between about 24 hours
and about 48 hours. The purification step preferably involves
chromatography (e.g., reverse-phase HPLC).
[0119] D. Attachment of Transport Polymers to Biologically Active
Agents
[0120] The agent to be transported can be linked to the
delivery-enhancing transporter according to a number of
embodiments. In one embodiment, the agent is linked to a single
delivery-enhancing transporter, either via linkage to a terminal
end of the delivery-enhancing transporter or to an internal subunit
within the reagent via a suitable linking group.
[0121] In a second embodiment, the agent is attached to more than
one delivery-enhancing transporter, in the same manner as above.
This embodiment is somewhat less preferred, since it can lead to
crosslinking of adjacent cells.
[0122] In a third embodiment, the conjugate contains two agent
moieties attached to each terminal end of the delivery-enhancing
transporter. For this embodiment, it is presently preferred that
the agent has a molecular weight of less than 10 kDa.
[0123] With regard to the first and third embodiments just
mentioned, the agent is generally not attached to one any of the
guanidino or amidino sidechains so that they are free to interact
with the target membrane.
[0124] The conjugates of the invention can be prepared by
straightforward synthetic schemes. Furthermore, the conjugate
products are usually substantially homogeneous in length and
composition, so that they provide greater consistency and
reproducibility in their effects than heterogeneous mixtures.
[0125] According to an important aspect of the present invention,
it has been found by the applicants that attachment of a single
delivery-enhancing transporter to any of a variety of types of
biologically active agents is sufficient to substantially enhance
the rate of uptake of an agent into and across one or more layers
of epithelial and endothelial tissues, even without requiring the
presence of a large hydrophobic moiety in the conjugate. In fact,
attaching a large hydrophobic moiety can significantly impede or
prevent cross-layer transport due to adhesion of the hydrophobic
moiety to the lipid bilayer of cells that make up the epithelial or
endothelial tissue. Accordingly, the present invention includes
conjugates that do not contain substantially hydrophobic moieties,
such as lipid and fatty acid molecules.
[0126] Delivery-enhancing transporters of the invention can be
attached covalently to biologically active agents by chemical or
recombinant methods.
[0127] 1. Chemical Linkages
[0128] Biologically active agents such as small organic molecules
and macromolecules can be linked to delivery-enhancing transporters
of the invention via a number of methods known in the art (see, for
example, Wong, S. S., Ed., Chemistry of Protein Conjugation and
Cross-Linking, CRC Press, Inc., Boca Raton, Fla. (1991), either
directly (e.g., with a carbodiimide) or via a linking moiety. In
particular, carbamate, ester, thioether, disulfide, and hydrazone
linkages are generally easy to form and suitable for most
applications. Ester and disulfide linkages are preferred if the
linkage is to be readily degraded in the cytosol, after transport
of the substance across the cell membrane.
[0129] Various functional groups (hydroxyl, amino, halogen, etc.)
can be used to attach the biologically active agent to the
transport polymer. Groups which are not known to be part of an
active site of the biologically active agent are preferred,
particularly if the polypeptide or any portion thereof is to remain
attached to the substance after delivery.
[0130] Polymers, such as peptides produced as described in PCT
application US98/10571 (Publication No. WO 9852614), are generally
produced with an amino terminal protecting group, such as FMOC. For
biologically active agents which can survive the conditions used to
cleave the polypeptide from the synthesis resin and deprotect the
sidechains, the FMOC may be cleaved from the N-terminus of the
completed resin-bound polypeptide so that the agent can be linked
to the free N-terminal amine. In such cases, the agent to be
attached is typically activated by methods well known in the art to
produce an active ester or active carbonate moiety effective to
form an amide or carbamate linkage, respectively, with the polymer
amino group. Of course, other linking chemistries can also be
used.
[0131] To help minimize side-reactions, guanidino and amidino
moieties can be blocked using conventional protecting groups, such
as carbobenzyloxy groups (CBZ), di-t-BOC, PMC, Pbf, N--NO.sub.2,
and the like.
[0132] Coupling reactions are performed by known coupling methods
in any of an array of solvents, such as N,N-dimethyl formamide
(DMF), N-methylpyrrolidinone, dichloromethane, water, and the like.
Exemplary coupling reagents include, for example,
O-benzotriazolyloxy tetramethyluronium hexafluorophosphate (HATU),
dicyclohexyl carbodiimide, bromo-tris(pyrrolidino) phosphonium
bromide (PyBroP), etc. Other reagents can be included, such as
N,N-dimethylamino pyridine (DMAP), 4-pyrrolidino pyridine,
N-hydroxy succinimide, N-hydroxy benzotriazole, and the like.
[0133] 2. Fusion Polypeptides
[0134] Delivery-enhancing transporters of the invention can be
attached to biologically active polypeptide agents by recombinant
means by constructing vectors for fusion proteins comprising the
polypeptide of interest and the delivery-enhancing transporter,
according to methods well known in the art. Generally, the
delivery-enhancing transporter component will be attached at the
C-terminus or N-terminus of the polypeptide of interest, optionally
via a short peptide linker.
[0135] 3. Releasable Linkers
[0136] The biologically active agents are, in presently preferred
embodiments, attached to the delivery-enhancing transporter using a
linkage that is specifically cleavable or releasable. The use of
such linkages is particularly important for biologically active
agents that are inactive until the attached delivery-enhancing
transporter is released. In some cases, such conjugates that
consist of a drug molecule that is attached to a delivery-enhancing
transporter can be referred to as prodrugs, in that the release of
the delivery-enhancing transporter from the drug results in
conversion of the drug from an inactive to an active form. As. used
herein, "cleaved" or "cleavage" of a conjugate or linker refers to
release of a biological agent from a transporter molecule, thereby
releasing an active biological agent. "Specifically cleavable" or
"specifically releasable" refers to the linkage between the
transporter and the agent being cleaved, rather than the
transporter being degraded (e.g., by proteolytic degradation).
[0137] In some embodiments, the linkage is a readily cleavable
linkage, meaning that it is susceptible to cleavage under
conditions found in vivo. Thus, upon passing into and through one
or more layers of an epithelial and/or endothelial tissue, the
agent is released from the delivery-enhancing transporter. Readily
cleavable linkages can be, for example, linkages that are cleaved
by an enzyme having a specific activity (e.g., an esterase,
protease, phosphatase, peptidase, and the like) or by hydrolysis.
For this purpose, linkers containing carboxylic acid esters and
disulfide bonds are sometimes preferred, where the former groups
are hydrolyzed enzymatically or chemically, and the latter are
severed by disulfide exchange, e.g., in the presence of
glutathione. The linkage can be selected so it is cleavable by an
enzymatic activity that is known to be present in one or more
layers of an epithelial or endothelial tissue. For example, the
stratum granulosum of skin has a relatively high concentration of
N-peptidase activity.
[0138] A specifically cleavable linker can be engineered onto a
transporter molecule. For example, amino acids that constitute a
protease recognition site, or other such specifically recognized
enzymatic cleavage site, can be used to link the transporter to the
agent. Alternatively, chemical or other types of linkers that are
cleavable by, for example, exposure to light or other stimulus can
be used to link the transporter to the agent of interest.
[0139] A conjugate in which an agent to be delivered and a
delivery-enhancing transporter are linked by a specifically
cleavable or specifically releasable linker will have a half-life.
The term "half-life" in this context refers to the amount of time
required after applying the conjugate to an epithelial or
endothelial membrane for one half of the amount of conjugate to
become dissociated to release the free agent. The half-life for
some embodiments is typically between 5 minutes and 24 hours, and
more preferably is between 30 minutes and 2 hours. The half-life of
a conjugate can be "tuned" or modified, according to the invention,
as described below.
[0140] In some embodiments, the cleavage rate of the linkers is pH
dependent. For example, a linker can form a stable linkage between
an agent and a delivery-enhancing transporter at an acidic pH
(e.g., pH 6.5 or less, more preferably about 6 or less, and still
more preferably about 5.5 or less). However, when the conjugate is
placed at physiological pH (e.g., pH 7 or greater, preferably about
pH 7.4), the linker will undergo cleavage to release the agent.
Such pH sensitivity can be obtained by, for example, including a
functional group that, when protonated (i.e., at an acidic pH),
does not act as a nucleophile. At a higher (e.g., physiological)
pH, the functional group is no longer protonated and thus can act
as a nucleophile. Examples of suitable functional groups include,
for example, N and S. One can use such functional groups to
fine-tune the pH at which self-cleavage occurs.
[0141] 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.
[0142] Examples of conjugates containing self-immolating linking
moieties (e.g., biologically active agent-1-transport moiety
conjugates) are represented by structures 3, 4 and 5:
##STR00004##
[0143] 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; y 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.5; 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 n is an integer ranging from 1 to 10. Non-limiting
examples of the X and Y 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, --SO2NH--, --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, Y
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 Y) can contain substituted (e.g., N-alkyl or N-acyl)
linkages as well.
[0144] Turning first to linking groups illustrated by structure 3,
an example and preferred embodiment is illustrated for formula
3a:
##STR00005##
[0145] 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 20 (FIG. 6). In this Example and-FIG. 6,
cyclosporin A is treated with chloroacetic anhydride to form the
chloroacetate ester 6i (numbering in FIG. 6) which is then combined
with benzylamine to form the N-benzyl glycine conjugate 6ii.
Condensation of the glycine conjugate with Boc-protected diglycine
anhydride provides the acid 6iii which is converted to the more
reactive N-hydroxy succinimide ester 6iv 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 3a, to provide an appropriate steric or
spatial orientation that allows the linkage to be cleaved in vivo
(see FIG. 6B).
[0146] Accordingly, for structure 3, the following substituents are
preferred: A is N; R.sup.2 is benzyl; k, m and n are 1; X is
--OC(O) and Y is --C(O)NH--.
[0147] Linkages of structure 4, are exemplified by formula 4a:
##STR00006##
[0148] 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 4a are shown in Examples 20-22. In Example 20 (see
scheme in FIG. 39), acyclovir is acylated with .alpha.-chloroacetic
anhydride to form the .alpha.-chloroacetate ester 39i. Reaction of
39i with a heptamer of D-arginine having an N-terminal cysteine
residue, provides the thioether product 39ii. Alternatively,
acyclovir can be attached to the C-terminus of a transport moiety
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:
##STR00007##
[0149] Accordingly, in one group of preferred embodiments, the
conjugate is represented by formula 5, in which X is --OC(O)--; Y
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)--; Y 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.
[0150] Linking groups represented by the conjugates shown in
formula 6 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. For structure 6, 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 Y is --C(O)NH--.
[0151] 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.
[0152] For a conjugate having the structure 3, 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 3 is preferably methyl, ethyl, propyl, butyl, allyl,
benzyl or phenyl.
[0153] 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 3 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.
[0154] 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.
[0155] Structure 6 is an example of a two-step, self-immolating
moiety that is incorporated into a transport moiety-biologically
active compound conjugate:
##STR00008##
[0156] 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, 0,
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 Ware independently hydrogen or alkyl; and, k and m are
independently either 1 or 2.
[0157] An example of a suitable linking group to produce a
conjugate of formula 6 is:
##STR00009##
[0158] The construction of a conjugate containing a linking group
of formula 6a is provided in Example 24 (see also FIG. 14). In this
example (and Figure), the .alpha.-chloroacetate ester of
2,4-dimethyl-4-hydroxymethylphenol (41i) is coupled to retinoic
acid (41ii) using dicyclohexylcarbodiimide (DCC) and
4-dimethylaminopyridine (DMAP) to provide the intermediate 41iii.
Subsequent coupling of 41iii with a cysteine residue present on the
N-terminus of an arginine heptamer transport moiety provides the
target conjugate 41 iv.
[0159] Preferably, the linking groups used in the conjugates of
formula 6, 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 Y is --C(O)O-- or --C(O)NH--. More
preferably, R.sup.6 is hydrogen or acetyl.
[0160] 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.
[0161] 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.
[0162] 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).
[0163] 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.
[0164] In another approach, the conjugate includes a photocleavable
linker that is cleaved upon exposure to electromagnetic radiation.
Application of this methodology is provided for a related system in
FIG. 5B of POT 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.
[0165] 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.
[0166] 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 ex-amino group, and the 5-fluorouracil is
linked via the ex-carbonyl. The lysyl .epsilon.-amino group has
been modified to a carbamate ester of o-hy oxymethyl 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 .alpha.-amino group with the amide
linkage to the 5-fluorouracil group leads to cyclization with
release of the 5-fluorouracil group.
[0167] 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.
[0168] 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
(n=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.
[0169] 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.
[0170] Uses of Delivery-Enhancing Transporters
[0171] The delivery-enhancing transporters find use in therapeutic,
prophylactic and diagnostic applications. The delivery-enhancing
transporters can carry a diagnostic or biologically active reagent
into and across one or more layers of skin or other epithelial
tissue (e.g., gastrointestinal, lung, ocular and the like), or
across endothelial tissues such as the blood brain barrier. This
property makes the reagents useful for treating conditions by
delivering agents that must penetrate across one or more tissue
layers in order to exert their biological effect.
[0172] Moreover, the transporters of the present invention can also
be used alone, or in combination with another therapeutic or other
compound, as a furin inhibitor. For example, in addition to various
poly-arginine transporters, the synthetic transporters described
herein, including peptoid and those transporters comprising
non-naturally occurring amino acids can be used to inhibit furins.
See, e.g., Cameron et al., J. Biol. Chem. 275(47): Furins are
proteases that convert a variety of pro-proteins to their active
components. Inhibition of furins is useful, for instance, for
treating infections by viruses that rely on furin activity for
virulence or replication. See, e.g., Molloy, et al., T. Cell Biol.
9:28-35 (1999).
[0173] Similarly, the transporters of the invention are useful
inhibitors of capthesin C. For example, certain poly arginine
compounds are inhibitors of capthesin C. See, e.g., Hom, et al.,
Eur. J. Biochem. 267(11):3330-3336 (2000). Similarly, the
transporters of the invention, including those comprising synthetic
amino acids, are useful to inhibit capthesin C.
[0174] In one aspect of the invention, a furin inhibition assay can
be used to screen for additional transporters. For example,
candidate transporter compounds can be tested for their ability to
compete with poly arginine for their ability to bind furins or
capthesin C using standard competition assays. Alternatively,
candidate transporters can be screened for their ability to inhibit
furin protease activity as discussed in Cameron et al., supra, and
Horn et al., supra. Particularly active candidates can then be
further tested for their ability to act as transporters into and/or
across tissues such as the epithelium.
[0175] Compositions 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.
[0176] Administration of the compounds 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, 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, eye drops, or the like, preferably in unit dosage forms
suitable for simple administration of precise dosages.
[0177] 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).
[0178] 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.
[0179] 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.
[0180] 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 PEG4000 [4%]).
[0181] Liquid compositions can be prepared by dissolving or
dispersing compound (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.
[0182] 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.
[0183] 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.
[0184] 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.
[0185] Generally, the compounds 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.
[0186] Stability of the conjugate can be further controlled by the
composition and stereochemistry of the backbone and sidechains of
the delivery-enhancing transporters. For polypeptide
delivery-enhancing transporters, D-isomers are generally resistant
to endogenous proteases, and therefore have longer half-lives in
serum and within cells. D-polypeptide polymers are therefore
appropriate when longer duration of action is desired.
L-polypeptide polymers have shorter half-lives due to their
susceptibility to proteases, and are therefore chosen to impart
shorter acting effects. This allows side-effects to be averted more
readily by withdrawing therapy as soon as side-effects are
observed. Polypeptides comprising mixtures of D and L-residues have
intermediate stabilities. Home-D-polymers are generally
preferred.
[0187] A. Application to Skin
[0188] The delivery-enhancing transporters of the invention make
possible the delivery of biologically active and diagnostic agents
across the skin. Surprisingly, the transporters 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.
[0189] 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).
[0190] In some embodiments, the agent 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.
[0191] In other embodiments, the delivery-enhancing transporters
are useful for delivering cosmetics and agents that can treat skin
conditions. Target cells in the skin that are of interest include,
for example, fibroblasts, epithelial cells and immune cells. For
example, the transporters provide the ability to deliver compounds
such as anti-inflammatory agents to immune cells found in the
dermis.
[0192] Glucocorticoids (adrenocorticoid steroids) are among the
compounds for which delivery across skin can be enhanced by the
delivery-enhancing transporters of the invention. Conjugated
glucocorticoids of the invention are useful for treating
inflammatory skin diseases, for example. Exemplary glucocorticoids
include, e.g., hydrocortisone, prenisone (deltasone) and
predrisonlone (hydeltasol). Examples of particular conditions
include eczema (including atopic dermatitis, contact dermatitis,
allergic dermatitis), bullous disease, collagen vascular diseases,
sarcoidosis, Sweet's disease, pyoderma gangrenosum, Type I reactive
leprosy, capillary hemangiomas, lichen planus, exfoliative
dermatitis, erythema nodosum, hormonal abnormalities (including
acne and hirsutism), as well as toxic epidermal necrolysis,
erythema multiforme, cutaneous T-cell lymphoma, discoid lupus
erythematosus, and the like.
[0193] Retinoids are another example of a biologically active agent
for which one can use the delivery-enhancing transporters of the
invention to enhance delivery into and across one or more layers of
the skin or other epithelial or endothelial tissue. Retinoids that
are presently in use include, for example retinol, tretinoin,
isotretinoin, etretinate, acitretin, and arotinoid. Conditions that
are treatable using retinoids conjugated to the delivery-enhancing
transporters of the invention include, but are not limited to,
acne, keratinization disorders, skin cancer, precancerous
conditions, psoriasis, cutaneous aging, discoid lupus
erythematosus, scleromyxedema, verrucous epidermal nevus,
subcorneal pustular dermatosis, Reiter's syndrome, warts, lichen
planus, acanthosis nigricans, sarcoidosis, Grover's disease,
porokeratosis, and the like.
[0194] Cytotoxic and immunosuppressive drugs constitute an
additional class of drugs for which the delivery-enhancing
transporters of the invention are useful. These agents are commonly
used to treat hyperproliferative diseases such as psoriasis, as
well as for immune diseases such as bullous dermatoses and
leukocytoclastic vasculitis. Examples of such compounds that one
can conjugate to the delivery-enhancing transporters of the
invention include, but are not limited to, antimetabolites such as
methotrexate, azathioprine, fluorouracil, hydroxyurea,
6-thioquanine, mycophenolate, chlorambucil, vinicristine,
vinblastine and dactinomycin. Other examples are alkylating agents
such as cyclophosphamide, mechloroethamine hydrochloride,
carmustine, taxol, tacrolimus and vinblastine are additional
examples of useful biological agents, as are dapsone and
sulfasalazine. Immunosuppressive drugs such as cyclosporin and
Ascomycins, such as FK506 (tacrolimus), and rapamycin (e.g., U.S.
Pat. No. 5,912,253) and analogs of such compounds are of particular
interest (e.g., Mollinson et al., Current Pharm. Design
4(5):367-380 (1998); U.S. Pat. Nos. 5,612,350; 5,599,927;
5,604,294; 5,990,131; 5,561,140; 5,859,031; 5,925,649; 5,994,299;
6,004,973 and 5,508,397). Cyclosporins include cyclosporin A, B, C,
D, G and M. See, e.g., U.S. Pat. Nos. 6,007,840; and 6,004,973. For
example, such compounds are useful in treating psoriasis, eczema
(including atopic dermatitis, contact dermatitis, allergic
dermatitis) and alopecia areata.
[0195] The delivery-enhancing transporters can be conjugated to
agents that are useful for treating conditions such as lupus
erythematosus (both discoid and systemic), cutaneous
dermatomyositis, porphyria cutanea tarda and polymorphous light
eruption. Agents useful for treating such conditions include, for
example, quinine, chloroquine, hydroxychloroquine, and
quinacrine.
[0196] The delivery-enhancing transporters of the invention are
also useful for transdermal delivery of antiinfective agents. For
example, antibacterial, antifungal and antiviral agents can be
conjugated to the delivery-enhancing transporters. Antibacterial
agents are useful for treating conditions such as acne, cutaneous
infections, and the like. Antifungal agents can be used to treat
tinea corporis, tinea pedis, onychomycosis, candidiasis, tinea
versicolor, and the like. Because of the delivery-enhancing
properties of the conjugates, these conjugates are useful for
treating both localized and widespread infections. Antifungal
agents are also useful for treating onychomycosis. Examples of
antifungal agents include, but are not limited to, azole
antifungals such as itraconazole, myconazole and fluconazole.
Examples of antiviral agents include, but are not limited to,
acyclovir, famciclovir, and valacyclovir. Such agents are useful
for treating viral diseases, e.g., herpes.
[0197] Another example of a biologically active agent for which
enhancement of delivery by conjugation to the delivery-enhancing
transporters of the invention is desirable are the antihistamines.
These agents are useful for treating conditions such as pruritus
due to urticaria, atopic dermatitis, contact dermatitis, psoriasis,
and many others. Examples of such reagents include, for example,
terfenadine, astemizole, lorotadine, cetirizine, acrivastine,
temelastine, cimetidine, ranitidine, famotidine, nizatidine, and
the like. Tricyclic antidepressants can also be delivered using the
delivery-enhancing transporters of the invention.
[0198] Topical antipsoriasis drugs are also of interest. Agents
such as corticosteroids, calcipotriene, and anthralin can be
conjugated to the delivery-enhancing transporters of the invention
and applied to skin.
[0199] The delivery-enhancing transporters of the invention are
also useful for enhancing delivery of photochemotherapeutic agents
into and across one or more layers of skin and other epithelial
tissues. Such compounds include, for example, the psoralens, and
the like. Sunscreen components are also of interest; these include
p-aminobenzoic acid esters, cinnamates and salicylates, as well as
benzophenones, anthranilates, and avobenzone.
[0200] Pain relief agents and local anesthetics constitute another
class of compounds for which conjugation to the delivery-enhancing
transporters of the invention can enhance treatment. Lidocaine,
bupibacaine, novocaine, procaine, tetracaine, benzocaine, cocaine,
and the opiates, are among the compounds that one can conjugate to
the delivery-enhancing transporters of the invention. Application
of pain relief agents to the joints or skin near the joints, e.g.,
in patients suffering from rhematoid arthritis, is also
contemplated.
[0201] Other biological agents of interest include, for example,
minoxidil, keratolytic agents, destructive agents such as
podophyllin, hydroquinone, capsaicin, masoprocol, colchicine, and
gold.
[0202] Treatment of inflamed joints such as occurs in rheumatoid
arthritis can also be treated with compounds useful for such
treatments conjugated to the transporters of the invention.
[0203] B. Gastrointestinal Administration
[0204] The delivery-enhancing transporters 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.
[0205] Among the gastrointestinal conditions that are treatable
using appropriate reagents conjugated to the delivery-enhancing
transporters are inflammatory bowel disease such as Crohn's disease
(e.g., cyclosporin and ascomycins such as FK506; aminosalicylates,
e.g., aspirin-like drugs, which include sulfasalazine and
mesalamine; corticosteroids, e.g., prednisone and
methylprednisolone; immune modifiers, e.g. azathioprine, 6 MP,
methotrexate; and antibiotics, e.g., metronidazole, ampicillin,
ciprofloxacin, and others). Other treatable gastrointestinal
conditions include ulcerative colitis, gastrointestinal ulcers,
peptic ulcer disease, imbalance of salt and water absorption (can
lead to constipation, diarrhea, or malnutrition), abnormal
proliferative diseases, and the like. Ulcer treatments include, for
example, drugs that reduce gastric acid secretion, such as H2
histamine inhibitors (e.g., cymetidine and ranitidine) and
inhibitors of the proton-potassium ATPase (e.g., lansoprazle and
omeprazle), and antibiotics directed at Helicobacter pylori.
[0206] Compounds useful for the treatment of constipation can also
be used in conjunction with the transporters of the invention.
Useful compounds for treating constipation include, e.g.,
surfactant laxatives such as docusate sodium, poloxamer 188,
dehydrochloric acid and ricinoleic acid. Exemplary stimulant
laxatives include, e.g., phenolphthalein, bisacodyl and
anthraquinone laxatives such as danthron.
[0207] Antibiotics are among the biologically active agents that
are useful when conjugated to the delivery-enhancing transporters
of the invention, particularly those that act on invasive bacteria,
such as Shigella, Salmonella, and Yersinia. Such compounds include,
for example, norfloxacin, ciprofloxacin, trimethoprim,
sulfamethyloxazole, and the like.
[0208] Anti-neoplastic agents, for example, for the treatment of
colon cancer, can also be conjugated to the delivery-enhancing
transporters of the invention and administered by the
gastrointestinal route. These include, for example, cisplatin,
methotrexate, taxol, fluorouracil, mercaptopurine, donorubicin,
bleomycin, streptozocin, mitomycin and the like.
[0209] For gastrointestinal and colonic delivery of orally
administered transporters or active compounds, it can be beneficial
to coat or encapsulate the compounds so that the compounds are not
released until they are delivered to the gastrointestinal (GI)
tract or colon. Methods and composition useful for delivery to the
GI tract or colon are described in, e.g., U.S. Pat. Nos. 6,183,466
and 6,120,803.
[0210] C. Respiratory Tract Administration
[0211] The delivery-enhancing transporters of the invention can
also be 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 transporters 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.
[0212] The transporters of the invention can be used to deliver
biological agents that are useful for treatment of pulmonary
conditions. Examples of conditions treatable by nasal
administration include, for example, asthma. These compounds
include anti-inflammatory agents, such as corticosteroids,
cromolyn, and nedocromil, bronchodialators such as
.beta.2-selective adrenergic drugs and theophylline, and
immunosuppressive drugs (e.g., cyclosporin and FK506). Other
conditions include, for example, allergic rhinitis (which can be
treated with glucocorticoids), and chronic obstructive pulmonary
disease (emphysema). Other drugs that act on the pulmonary tissues
and can be delivered using the transporters of the invention
include beta-agonists, mast cell stabilizers, antibiotics,
antifungal and antiviral agents, surfactants, vasoactive drugs,
sedatives and hormones.
[0213] Respiratory tract administration is useful not only for
treatment of pulmonary conditions, but also for delivery of drugs
to distant target organs via the circulatory system. A wide variety
of such drugs and diagnostic agents can be administered through the
respiratory tract after conjugation to the delivery-enhancing
transporters of the invention.
[0214] D. Delivery of Agents Across the Blood Brain Barrier
[0215] The delivery-enhancing transporters are also useful for
delivering biologically active and diagnostic agents 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.
[0216] E. Diagnostic Imaging and Contrast Agents
[0217] The delivery-enhancing transporters of the invention are
also useful for delivery of diagnostic imaging and contrast agents
into and across one or more layers of an epithelial and/or
endothelial tissue. Examples of diagnostic agents include
substances that are labeled with radioactivity, such as .sup.99 mTc
glucoheptonate, or substances used in magnetic resonance imaging
(MRI) procedures such as gadolinium doped chelation agents (e.g.
Gd-DTPA). Other examples of diagnostic agents include marker genes
that encode proteins that are readily detectable when expressed in
a cell (including, but not limited to, .beta.-galactosidase, green
fluorescent protein, luciferase, and the like). A wide variety of
labels may be employed, such as radionuclides, fluors, enzymes,
enzyme substrates, enzyme cofactors, enzyme inhibitors, ligands
(particularly haptens), etc.
[0218] F. Ocular Administration
[0219] The delivery-enhancing transporters of the invention can
also be used to enhance administration of drugs through the tissues
of the eye and other related tissues such as the eye lid. The
ocular tissues include the cornea, iris, lens, vitreus, vitreus
humor, the optic nerve and the eyelid.
[0220] Examples of conditions treatable with the compositions of
the invention include the following. Conjunctivitis, sometimes
called pink eye, is an inflammation of the blood vessels in the
conjunctiva, the membrane that covers the sclera and inside of the
eyelids. Conjunctivitis may be caused by bacteria or viruses.
[0221] Antibacterial and antiviral compounds useful for treating
bacterial or viral infections of the eye are well known. See, e.g.,
Hardman and Limbird, supra. Exemplary compounds include
chloramphenicol, Ciproflaxacin, polymyxin B and tetracycline.
Exemplary antiviral compounds include idoxuridine, acyclovir and
ganciclovir.
[0222] Styes are noncontagious, bacterial infections of one of the
sebaceous glands of the eyelid. A stye looks like a small, red bump
either on the eye lid or on the edge of the eyelid.
[0223] Dry eye, or "Sjogren's syndrome," is an immune system
disorder characterized by inflammation and dryness of the mouth,
eyes, and other mucous membranes, damages the lacrimal glands, and
this damage affects tear production. Dry eye can be treated with
immunosuppressive compounds such as cyclosporin as well as with
ascomycins or steroids.
[0224] Glaucoma is a condition in which the normal fluid pressure
inside the eyes (intraocular pressure, or IOP) slowly rises as a
result of the fluid aqueous humor, which normally flows in and out
of the eye, not being able to drain properly. Instead, the fluid
collects and causes pressure damage to the optic nerve and loss of
vision. Useful compounds to treat glaucoma, blindness, and other
eye disorders include, e.g., timolol, levobunolol and
phenylepherine. Growth factors such as nerve growth factor (NGF)
(see, e.g., Bennett, et al. Mol Ther 1(6):501-5 (2000)) are also
useful for treating glaucoma and other ocular disorders.
[0225] Therapeutic compounds for treatment of ocular diseases, such
as those discussed above, are well known to those of skill in the
art. Typically, administration of the composition of the invention
to the ocular tissues is in the form of an eye drop. Alternatively,
for example, the compositions can be injected into the eye or
applied as an ointment.
[0226] Eye drops including the compounds of the invention can also
include an isotonic agent added to sterilized purified water, and
if required, a preservative, a buffering agent, a stabilizer, a
viscous vehicle and the like are added to the solution and
dissolved therein. After dissolution, the pH is adjusted with a pH
controller to be within a range suitable for use as an ophthalmic
medicine, preferably within the range of 4.5 to 8.
[0227] Sodium chloride, glycerin, mannitol or the like may be used
as the isotonic agent; p-hydroxybenzoic acid ester, benzalkonium
chloride or the like as the preservative; sodium hydrogenphosphate,
sodium dihydrogenphosphate, boric acid or the like as the buffering
agent; sodium edetate or the like as the stabilizer; polyvinyl
alcohol, polyvinyl pyrrolidone, polyacrylic acid or the like as the
viscous vehicle; and sodium hydroxide, hydrochloric acid or the
like as the pH controller
[0228] Biologically Active and Diagnostic Molecules useful with the
Delivery-enhancing transporters
[0229] The delivery-enhancing transporters can be conjugated to a
wide variety of biologically active agents and molecules that have
diagnostic use.
[0230] A. Small Organic Molecules
[0231] Small organic molecule therapeutic agents can be
advantageously attached to linear polymeric compositions as
described herein, to facilitate or enhance transport across one or
more layers of an epithelial or endothelial tissue. For example,
delivery of highly charged agents, such as levodopa
(L-3,4-dihydroxy-phenylalanine; L-DOPA) may benefit by linkage to
delivery-enhancing transporters as described herein. Peptoid and
peptidomimetic agents are also contemplated (e.g., Langston (1997)
DDT 2:255; Giannis et al. (1997) Advances Drug Res. 29:1). Also,
the invention is advantageous for delivering small organic
molecules that have poor solubilities in aqueous liquids, such as
serum and aqueous saline. Thus, compounds whose therapeutic
efficacies are limited by their low solubilities can be
administered in greater dosages according to the present invention,
and can be more efficacious on a molar basis in conjugate form,
relative to the non-conjugate form, due to higher uptake levels by
cells.
[0232] Since a significant portion of the topological surface of a
small molecule is often involved, and therefore required, for
biological activity, the small molecule portion of the conjugate in
particular cases may need to be severed from the attached
delivery-enhancing transporter and linker moiety (if any) for the
small molecule agent to exert biological activity after crossing
the target epithelial tissue. For such situations, the conjugate
preferably includes a cleavable linker for releasing free drug
after passing through an epithelial tissue.
[0233] FIG. 5D and FIG. 5E are illustrative of another aspect of
the invention, comprising taxane- and taxoid anticancer conjugates
which have enhanced trans-epithelial tissue transport rates
relative to corresponding non-conjugated forms. The conjugates are
particularly useful for inhibiting growth of cancer cells. Taxanes
and taxoids are believed to manifest their anticancer effects by
promoting polymerization of microtubules (and inhibiting
depolymerization) to an extent that is deleterious to cell
function, inhibiting cell replication and ultimately leading to
cell death.
[0234] The term "taxane" refers to paclitaxel (FIG. 5F, R'=acetyl,
R''=benzyl) also known under the trademark "TAXOL") and naturally
occurring, synthetic, or bioengineered analogs having a backbone
core that contains the A, B, C and D rings of paclitaxel, as
illustrated in FIG. 5G. FIG. 5F also indicates the structure of
"TAXOTERE.TM." (R'=H, R''=BOC), which is a somewhat more soluble
synthetic analog of paclitaxel sold by Rhone-Poulenc. "Taxoid"
refers to naturally occurring, synthetic or bioengineered analogs
of paclitaxel that contain the basic A, B and C rings of
paclitaxel, as shown in FIG. 5H. Substantial synthetic and
biological information is available on syntheses and activities of
a variety of taxane and taxoid compounds, as reviewed in Suffness
(1995) Taxol: Science and Applications, CRC Press, New York, N.Y.,
pp. 237-239, particularly in Chapters 12 to 14, as well as in the
subsequent paclitaxel literature. Furthermore, a host of cell lines
are available for predicting anticancer activities of these
compounds against certain cancer types, as described, for example,
in Suffness at Chapters 8 and 13.
[0235] The delivery-enhancing transporter is conjugated to the
taxane or taxoid moiety via any suitable site of attachment in the
taxime or taxoid. Conveniently, the transport polymer is linked via
a C2'-oxygen atom, C7-oxygen atom, using linking strategies as
above. Conjugation of a transport polymer via a C7-oxygen leads to
taxane conjugates that have anticancer and antitumor activity
despite conjugation at that position. Accordingly, the linker can
be cleavable or non-cleavable. Conjugation via the C2'-oxygen
significantly reduces anticancer activity, so that a cleavable
linker is preferred for conjugation to this site. Other sites of
attachment can also be used, such as C10.
[0236] It will be appreciated that the taxane and taxoid conjugates
of the invention have improved water solubility relative to taxol
(.apprxeq.0.25 .mu.g/mL) and taxotere (6-7 .mu.g/mL). Therefore,
large amounts of solubilizing agents such as "CREMOPHOR EL"
(polyoxyethylated castor oil), polysorbate 80 (polyoxyethylene
sorbitan monooleate, also known as "TWEEN 80"), and ethanol are not
required, so that side-effects typically associated with these
solubilizing agents, such as anaphylaxis, dyspnea, hypotension, and
flushing, can be reduced.
[0237] B. Metals
[0238] Metals can be transported into and across one or more layers
of epithelial and endothelial tissues using chelating agents such
as texaphyrin or diethylene triamine pentacetic acid (DTPA),
conjugated to a delivery-enhancing transporter of the invention, as
illustrated by Example. These conjugates are useful for delivering
metal ions for imaging or therapy. Exemplary metal ions include Eu,
Lu, Pr, Gd, Tc99m, Ga67, In111, Y90, Cu67, and Co57. Preliminary
membrane-transport studies with conjugate candidates can be
performed using cell-based assays such as described in the Example
section below. For example, using europium ions, cellular uptake
can be monitored by time-resolved fluorescence measurements. For
metal ions that are cytotoxic, uptake can be monitored by
cytotoxicity.
[0239] C. Macromolecules
[0240] The enhanced transport methods of the invention are
particularly suited for enhancing transport into and across one or
more layers of an epithelial or endothelial tissue for a number of
macromolecules, including, but not limited to proteins, nucleic
acids, polysaccharides, and analogs thereof. Exemplary nucleic
acids include oligonucleotides and polynucleotides formed of DNA
and RNA, and analogs thereof, which have selected sequences
designed for hybridization to complementary targets (e.g.,
antisense sequences for single- or double-stranded targets), or for
expressing nucleic acid transcripts or proteins encoded by the
sequences. Analogs include charged and preferably uncharged
backbone analogs, such as phosphonates (preferably methyl
phosphonates), phosphoramidates (N3' or N5'), thiophosphates,
uncharged morpholino-based polymers, and protein nucleic acids
(PNAs). Such molecules can be used in a variety of therapeutic
regimens, including enzyme replacement therapy, gene therapy, and
anti-sense therapy, for example.
[0241] By way of example, protein nucleic acids (PNA) are analogs
of DNA in which the backbone is structurally homomorphous with a
deoxyribose backbone. The backbone consists of
N-(2-aminoethyl)glycine units to which the nucleobases are
attached. PNAs containing all four natural nucleobases hybridize to
complementary oligonucleotides obeying Watson-Crick base-pairing
rules, and is a true DNA mimic in terms of base pair recognition
(Egholm et al. (1993) Nature 365:566-568. The backbone of a PNA is
formed by peptide bonds rather than phosphate esters, making it
well-suited for anti-sense applications. Since the backbone is
uncharged, PNA/DNA or PNA/RNA duplexes that form exhibit greater
than normal thermal stability. PNAs have the additional advantage
that they are not recognized by nucleases or proteases. In
addition, PNAs can be synthesized on an automated peptides
synthesizer using standard t-Boc chemistry. The PNA is then readily
linked to a transport polymer of the invention.
[0242] Examples of anti-sense oligonucleotides whose transport into
and across epithelial and endothelial tissues can be enhanced using
the methods of the invention are described, for example, in U.S.
Pat. No. 5,594,122. Such oligonucleotides are targeted to treat
human immunodeficiency virus (HIV). Conjugation of a transport
polymer to an anti-sense oligonucleotide can be effected, for
example, by forming an amide linkage between the peptide and the
5'-terminus of the oligonucleotide through a succinate linker,
according to well-established methods. The use of PNA conjugates is
further illustrated in Example 11 of PCT Application
PCT/US98/10571. FIG. 7 of that application shows results obtained
with a conjugate of the invention containing a PNA sequence for
inhibiting secretion of gamma-interferon (.gamma.-IFN) by T cells,
as detailed in Example 11. As can be seen, the anti-sense PNA
conjugate was effective to block .gamma.-IFN secretion when the
conjugate was present at levels above about 10 .mu.M. In contrast,
no inhibition was seen with the sense-PNA conjugate or the
non-conjugated antisense PNA alone.
[0243] Another class of macromolecules that can be transported
across one or more layers of an epithelial or endothelial tissue is
exemplified by proteins, and in particular, enzymes. Therapeutic
proteins include, but are not limited to replacement enzymes.
Therapeutic enzymes include, but are not limited to, alglucerase,
for use in treating lysozomal glucocerebrosidase deficiency
(Gaucher's disease), alpha-L-iduronidase, for use in treating
mucopolysaccharidosis I, alpha-N-acetylglucosamidase, for use in
treating sanfilippo B syndrome, lipase, for use in treating
pancreatic insufficiency, adenosine deaminase, for use in treating
severe combined immunodeficiency syndrome, and triose phosphate
isomerase, for use in treating neuromuscular dysfunction associated
with triose phosphate isomerase deficiency.
[0244] In addition, and according to an important aspect of the
invention, protein antigens may be delivered to the cytosolic
compartment of antigen-presenting cells (APCs), where they are
degraded into peptides. The peptides are then transported into the
endoplasmic reticulum, where they associate with nascent HLA class
I molecules and are displayed on the cell surface. Such "activated"
APCs can serve as inducers of class I restricted antigen-specific
cytotoxic T-lymphocytes (CTLs), which then proceed to recognize and
destroy cells displaying the particular antigen. APCs that are able
to carry out this process include, but are not limited to, certain
macrophages, B cells and dendritic cells. In one embodiment, the
protein antigen is a tumor antigen for eliciting or promoting an
immune response against tumor cells. The transport of isolated or
soluble proteins into the cytosol of APC with subsequent activation
of CTL is exceptional, since, with few exceptions, injection of
isolated or soluble proteins does not result either in activation
of APC or induction of CTLs. Thus, antigens that are conjugated to
the transport enhancing compositions of the present invention may
serve to stimulate a cellular immune response in vitro or in
vivo.
[0245] In another embodiment, the invention is useful for
delivering immunospecific antibodies or antibody fragments to the
cytosol to interfere with deleterious biological processes such as
microbial infection. Recent experiments have shown that
intracellular antibodies can be effective antiviral agents in plant
and mammalian cells (e.g., Tavladoraki et al. (1993) Nature
366:469; and Shaheen et al. (1996) J. Virol. 70:3392. These methods
have typically used single-chain variable region fragments (scFv),
in which the antibody heavy and light chains are synthesized as a
single polypeptide. The variable heavy and light chains are usually
separated by a flexible linker peptide (e.g., of 15 amino acids) to
yield a 28 kDa molecule that retains the high affinity ligand
binding site. The principal obstacle to wide application of this
technology has been efficiency of uptake into infected cells. But
by attaching transport polymers to scFv fragments, the degree of
cellular uptake can be increased, allowing the immunospecific
fragments to bind and disable important microbial components, such
as HN Rev, HIV reverse transcriptase, and integrase proteins.
[0246] D. Peptides
[0247] Peptides to be delivered by the enhanced transport methods
described herein include, but should not be limited to, effector
polypeptides, receptor fragments, and the like. Examples include
peptides having phosphorylation sites used by proteins mediating
intra-cellular signals. Examples of such proteins include, but are
not limited to, protein kinase C, RAF-1, p21Ras, NF-.sub.KB, C-JUN,
and cytoplasmic tails of membrane receptors such as IL-4 receptor,
CD28, CTLA-4, V7, and MHC Class I and Class II antigens.
[0248] When the delivery-enhancing transporter is also a peptide,
synthesis can be achieved either using an automated peptide
synthesizer or by recombinant methods in which a polynucleotide
encoding a fusion peptide is produced, as mentioned above.
EXAMPLES
[0249] The following examples are offered to illustrate, but not to
limit the present invention.
Example 1
Penetration of Biotinylated Polymers of D-Arginine into the Skin of
Nude Mice
[0250] This Example demonstrates that poly-arginine heptamers can
deliver conjugated biotin into and across layers of the skin, both
follicularly and interfollicularly, and into the dermis.
Methods
[0251] Biotinylated 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. Fastmoc
cycles were used with
O-(7-aiabenzotriazol-1-yl)-1,1,3,3-tetramethyluronium
hexfluorophosphate (HATU) substituted for HBTU/HOBt as the coupling
reagent. Prior to the addition of biotin to the amino terminus of
the peptide, amino caproic acid (aca) was conjugated and acted as a
spacer. The peptides were cleaved from the resin using 96%
trifluoroacetic acid, 2% triisopropyl silane, and 2% phenol for
between 1 and 12 hours. The longer reaction times were necessary to
completely remove the Pbf protecting groups from the polymers of
arginine. The peptides subsequently were filtered from the resin,
precipitated using diethyl ether, purified using HPLC reverse phase
columns (Alltech Altima, Chicago, Ill.) and characterized using
either electrospray or matrix assisted laser desorption mass
spectrometry (Perceptive Biosystems, Boston, Mass.).
[0252] Varying concentrations (1 mM-10 .mu.M) of a heptarner of
D-arginine with biotin Covalently attached to the amino terminus
using an amino caproic acid spacer (bio r7), dissolved in phosphate
buffered saline (PBS), were applied to the back of anesthetized
nude mice. Samples (100 .mu.l) were applied as a liquid without
excipient, prevented from dispersing by a Vaseline.TM. barrier, and
allowed to penetrate for fifteen minutes. At the end of this period
the animal was sacrificed, the relevant sections of skin were
excised, embedded in mounting medium (OCT), and frozen. Frozen
sections (5 microns) were cut using a cryostat, collected on
slides, and stained with fluorescently labeled streptavidin (Vector
Laboratories, Burlingame, Calif.). The slides were fixed in acetone
at 4.degree. C. for ten minutes, air dried, soaked in PBS for five
minutes, blocked with normal goat serum for five minutes, and
washed with PBS for five minutes. The section was stained by
incubation with fluorescently labeled streptavidin at 30 .mu.g/ml
for thirty minutes, washed with PBS, counterstained with propidium
iodide (1 .mu.g/ml) for two minutes, and the section was mounted
with Vectashield.TM. mounting media. Slides were analyzed by
fluorescent microscopy. Parallel studies were done using
streptavidin-horse radish peroxidase rather than
fluorescein-streptavidin. The biotinylated peptide was visualized
by treatment of the sections with the horseradish peroxidase
substrate diaminobenzadine, and visualization with light
microscopy.
Results
[0253] Biotinylated arginine heptamer crossed into and across the
epidermis and into the dermis. The cytosol and nuclei of all cells
in the field were fluorescent, indicating penetration into
virtually every cell of the nude mouse skin in the section. The
staining pattern was consistent with unanticipated transport that
was both follicular and interfollicular. In addition, positive
cells were apparent in papillary and reticular dermis. In contrast,
no staining was apparent in mice treated with biotin alone, or
phosphate buffered saline alone.
Example 2
Penetration of Biotinylated Polymers of D-Arginine into the Skin of
Normal Balb/C Mice
[0254] Varying concentrations (1 mM-100 .mu.M) of a heptamer of
D-arginine with biotin covalently attached to the amino terminus
using an amino caproic acid spacer (bio r7), dissolved in PBS, were
applied to a skin of the groin of an anesthetized Balb/C mice.
Sample (100 .mu.l) was applied as a liquid within excipient and
prevented from dispersing by a Vaseline.TM. barrier and allowed to
penetrate for thirty minutes. At the end of this period animal was
sacrificed, the relevant section of skin was excised, embedded in
mounting medium (OCT) and frozen. Frozen sections were cut using a
cryostat, collected on slides, and stained with fluorescently
labeled streptavidin (Vector Laboratories, Burlingame, Calif.) as
described in Example 1. Slides were analyzed by fluorescent
microscopy.
Results
[0255] As with the skin from nude mice, biotinylated arginine
heptamer crossed into and across the epidermis and into the dermis.
The cytosol and nuclei of all cells in the field were fluorescent,
indicating penetration into virtually every cell of the nude mouse
skin in the section. The staining pattern was consistent with
unanticipated transport that was both follicular and
interfollicular. In addition, positive cells were apparent in
papillary and reticular dermis. In contrast, no staining was
apparent in mice treated with biotin alone, or phosphate buffered
saline alone.
Example 3
Penetration of Biotinylated Polymers of D-Arginine into Normal
Human Skin Grafted onto Nude Mice
[0256] Varying concentrations (1 mM-100 .mu.M) of a heptamer of
D-arginine with biotin covalently attached to the amino terminus
using an amino caproic acid spacer (bio r7), dissolved in PBS, were
applied to human foreskin grafts on the back of SCID mice (see,
e.g., Deng et al. (1997) Nature Biotechnol. 15: 1388-1391; Khavari
et al. (1997) Adv. Clin. Res. 15:27-35; Choate and Khavari (1997)
Human Gene Therapy 8:895-901). Samples (100 .mu.l) were applied as
a liquid within excipient and prevented from dispersing by a
Vaseline.TM. barrier and allowed to penetrate for fifteen minutes.
At the end of this period animal was sacrificed, the relevant
section of skin was excised, embedded in mounting medium (OCT) and
frozen. Frozen sections were cut using a cryostat, collected on
slides, and stained with fluorescently labeled streptavidin (Vector
Laboratories, Burlingame, Calif.) as described in Example 1. Slides
were analyzed by fluorescent microscopy.
Results
[0257] As with the skin from nude and normal mice, biotinylated
arginine heptamer crossed into and across the epidermis and into
the dermis of the human skin. The cytosol and nuclei of all cells
in the field were fluorescent, indicating penetration into and
through the epidermis and dermis. Intense staining was seen at both
20.times. and 40.times. magnification. The staining pattern was
consistent with unanticipated transport that was both follicular
and interfollicular. In addition, positive cells were apparent in
papillary and reticular dermis. In contrast, no staining was
apparent in mice treated with biotin alone, or phosphate buffered
saline alone, and very little staining was observed with the
biotinylated arginine pentamer conjugate, either at low or high
magnification.
Example 4
Increased Penetration of Biotinylated Polymers of D-Arginine into
Skin of Nude Mouse Using Plastic Wrap or a Lotion Excipient
[0258] Varying concentrations (1 mM-100 .mu.M) of a heptamer of
D-arginine with biotin covalently attached to the amino terminus
using an amino caproic acid spacer (bio r7), dissolved in PBS, and
mixed with an equal volume of Lubriderrn.TM.. The lotion mixture
Was then applied to the back of nude mice and allowed to penetrate
for thirty, sixty, and 120 minutes. Alternatively, sample (100
.mu.l) was applied as a liquid without excipient and prevented from
evaporating by wrapping plastic wrap over the sample sealed with
Vaseline.TM.. The samples were allowed to penetrate for thirty,
sixty, and 120 minutes. At the end of this period animal was
sacrificed, the relevant section of skin was excised, embedded in
mounting medium (OCT) and frozen. Frozen sections were cut using a
cryostat, collected on slides, and stained with fluorescently
labeled streptavidin (Vector Laboratories, Burlingame, Calif.) as
described in Example 1. Slides were analyzed by fluorescent
microscopy.
Results
[0259] Both lotion and plastic wrap resulted in increased uptake
compared with staining without excipient. Lotion was more effective
than plastic wrap in enhancing uptake of the conjugate.
Biotinylated arginine pentamers crossed into and across several
skin layers, reaching both the cytosol and nuclei of epidermal cell
layers, both follicular and interfollicular. In addition, positive
cells were apparent in papillary and reticular dermis.
Example 5
Penetration of Cyclosporin Conjugated to a Biotinylated Pentamer,
Heptamer, and Nonamer of D-Arginine into the Skin of Nude Mice
Methods
[0260] A. Linking Cyclosporin to Delivery-Enhancing
Transporters
[0261] 1. Preparation of the .alpha.-Chloroacetyl Cyclosporin a
Derivative.
[0262] The .alpha.-chloroacetyl cyclosporin A derivative was
prepared as shown in FIG. 1. Cyclosporin A (152.7 mg, 127 .mu.mol)
and chloroacetic acid anhydride (221.7 mg; 1300 .mu.mol) were
placed into a dry flask under N.sub.2-atrnosphere. Pyridine (1.0
mL) was added and the solution was heated to 50.degree. C. (oil
bath). After 16 hours the reaction was cooled to room temperature
and quenched with water (4.0 mL). The resulting suspension was
extracted with diethyl ether (E 15 mL). The combined organic layers
were dried over MgSO.sub.4. Filtration and evaporation of solvents
in vacuo delivered a yellow oil, which was purified by flash
chromatography on silica gel (eluent: EtOAc/hexanes: 40%-80%)
yielding 136 mg (106.4 .mu.mol, 83%) of the desired product.
[0263] 2. Coupling to Transporter Molecules
[0264] A general procedure for the coupling of cysteine containing
peptides to the .alpha.-chloro acetyl Cyclosporin A derivative is
shown in FIG. 2.
[0265] a. Labeled Peptides
[0266] The cyclosporin A derivative and the labeled peptide (1
equivalent) were dissolved in DMF (.about.10 mmol of Cyclosporin A
derivative/mL DMF) under an N.sub.2-atmosphere.
Diisopropylethylamine (10 equivalents) was added and stirring at
room temperature was continued until all starting material was
consumed (usually after 16 hours) (FIG. 3). The solvents were
removed in vacuo and the crude reaction product was dissolved in
water and purified by reversed phase high pressure liquid
chromatography (RP-HPLC) (eluent: water/MeCN*TFA). The products
were obtained in the following yields: [0267]
B-aca-r5-Ala-Ala-Cys-O-acyl-Cyclosporin A: 47% [0268]
B-aca-r7-Cys-O-acyl-Cyclosporin A: 43% [0269]
B-aca-r9-Cys-O-acyl-Cyclosporin A: 34% [0270]
B-aca-Cys-O-acyl-Cyclosporin A: 55%
[0271] b. Unlabeled Peptides
[0272] The peptide (34.7 mg, 15.3 .mu.mol) and the Cyclosporin A
derivative (19.6 mg, 15.3 .mu.mol) were dissolved in DMF (1.0 mL)
under an N.sub.2-atmosphere (FIG. 4). Diisopropylethylamine (19.7
mg, 153 .mu.mol) was added and stirring at room temperature was
continued. After 12 hours the solvent was removed in vacuo. The
crude material was dissolved in water and purified by RP-HPLC
(eluent: water/MeCN*TFA) yielding the pure product (24.1 mg, 6.8
mmol, 44%).
[0273] B. Analysis of Transport Across Skin
[0274] Varying concentrations (1 mM-100 .mu.M) of cyclosporin
conjugated to either biotinylated pentamer, heptamer, or nonamers
of D-arginine (bio r5, r7, or r9), dissolved in PBS, were applied
to the back of nude mice. Samples (100 .mu.l) were applied as a
liquid within excipient and prevention from dispersing by a
Vaseline.TM. barrier and allowed to penetrate for thirty, sixty,
and 120 minutes. At the end of this period animal was sacrificed,
the relevant section of skin was excised, embedded in mounting
medium (OCT) and frozen. Frozen sections were cut using a cryostat,
collected on slides, and stained with fluorescently labeled
streptavidin (Vector Laboratories, Burlingame, Calif.) as described
in Example 1. Slides were analyzed by fluorescent microscopy.
Results
[0275] The conjugates of cyclosporin with biotinylated heptamers
and nonamers of D-arginine effectively entered into and across the
epidermis and into the dermis of the skin of nude mice. In
contrast, very little uptake was seen using a conjugate between a
pentamer of arginine and cyclosporin, and no staining was seen with
a PBS control. The cytosol and nuclei of all cells in the field
were fluorescent, indicating penetration into and through the
epidermis and dermis. The staining pattern was consistent with
unanticipated transport that was both follicular and
interfollicular. In addition, positive cells were apparent in
papillary and reticular dermis. These results demonstrate
remarkable uptake only when sufficient guanidinyl groups are
included in the delivery-enhancing transporter.
Example 6
Demonstration that a D-Arginine Heptamer can Penetrate Human
Skin
[0276] Human and murine skin differ significantly in a number of
ways, with human epidermis being considerably thicker. To determine
if the D-arginine heptamers/cyclosporin A (r7 CsA) conjugate could
also penetrate human skin, biotin r7 CsA was applied to full
thickness human skin grafted onto the back of a SCID mouse. As in
murine skin, conjugated cyclosporin A penetrated human epidermis
and dermis. Fluorescence was observed in both the cytosol and the
nuclei of cells in tissue exposed to biotinylated peptides alone,
but in sections stained with biotin r7 CsA the majority of
fluorescence was cytosolic, consistent with r7 CsA binding to
cyclosporin A's known cytoplasmic targets.
Example 7
Demonstration that Cyclosporin A-Transporter Conjugates Enter T
Cells in the Dermis
Methods
[0277] Inhibition of IL-2 secretion by releasable R7-CsA conjugate.
Jurkat cells (5.times.10.sup.4) were incubated with varying
concentrations of a non releasable or releasable R7-CsA conjugate
or CsA overnight at 37.degree. C. to allow for the release of the
active form of CsA prior to stimulation with PMA and ionomycin. T
cells subsequently were stimulated to produce IL-2 by addition of
10 ng/ml PMA (Sigma, St. Louis, Mo.) and 1 .mu.M ionomycin
(CalBiochem, San Diego, Calif.). Cultures were incubated overnight
at 37.degree. C. and supernatants were collected and IL-2 was
measured using a fluorescent ELISA. Briefly, plates were coated
with 4 .mu.g/ml anti-human IL-2 antibody (BD Pharmingen, San Diego,
Calif.), blocked with PBS containing 10% FBS for 1 hour at room
temperature, washed, and supernatants added and incubated for 1
hour. Media was removed and biotinylated anti-human IL-2 (1.6
.mu.g/ml), was added for one hour. The plates were washed, and then
europium labeled streptavidin (0.04 ng/ml) was added for one hour.
After another wash, enhancement solution was added and the
resulting fluorescence was measured using a Wallac plate reader
(Wallac, Turku, Finland).
Results
[0278] To determine whether biotinylated D-arginine
heptamer-cyclosporin (r7 CsA) conjugate would reach infiltrating T
cells within inflamed skin in vivo, biotin r7 CsA was applied to
the site of inflammation on the back of a mouse with experimentally
induced contact dermatitis. Inflamed skin was stained with
rhodamine labeled goat anti-mouse CD3 to localize T-cells and with
fluorescein labeled streptavidin to localize the biotin r7 CsA.
Biotin r7 CsA was found in all CD3[+] T cells in the tissue in
addition to a variety of other cells that probably represent other
inflammatory cells as well as resident fibroblasts. These data
indicate that biotin r7 CsA penetrates inflamed skin to reach key
target T lymphocytes.
Example 8
Synthesis, In Vitro and In Vivo Activity of a Releasable Conjugate
of a Short Oligomer of Arginine and CsA
[0279] Modification of the 2.degree. alcohol of Cyclosporin A
results in significant loss of its biological activity. See, e.g.,
R. E. Handschumacher et al., Science 226, 544-7 (1984).
Consequently, to ensure release of free Cyclosporin A from its
conjugate after transport into cells, Cyclosporin A was conjugated
to an oligo-arginine transporter through a pH sensitive linker as
shown in FIG. 10. The resultant conjugate is stable at acidic pH
but at pH>7 it undergoes an intramolecular cyclization involving
addition of the free amine to the carbonyl adjacent to Cyclosporin
A (FIG. 6), which results in the release of unmodified Cyclosporin
A
[0280] Another modification in the design of the releasable
conjugate was the use of L-arginine (R), and not D-arginine (r) in
the transporter. While the oligo-D-arginine transporters were used
for the histological experiments to ensure maximal stability of the
conjugate and therefore accuracy in determining its location
through fluorescence, oligomers of L-arginine were incorporated
into the design of the releasable conjugate to minimize its
biological half-life. Consistent with its design, the resultant
releasable conjugate was shown to be stable at acidic pH, but
labile at physiological pH in the absence of serum. This releasable
Cyclosporin A conjugate's half-life in pH 7.4 PBS was 90
minutes.
Results
[0281] The releasable guanidino-heptamer conjugate of Cyclosporin A
was shown to be biologically active by inhibiting IL-2 secretion by
the human T-cell line, Jurkat, stimulated with PMA, and ionomycin
in vitro. See R. Wiskocil, et al., J Immunol 134, 1599-603 (1985).
The conjugate was added 12 hours prior to the addition of
PMA/ionomycin and dose dependent inhibition was observed by the
releasable R7 CsA conjugate. This inhibition was not observed with
a nonreleasable analog (FIG. 6) that differed from the releasable
conjugate by retention of the t-Boc protecting group, which
prevented cyclization and resultant release of the active drug. The
EC.sub.50 of the releasable R7 cyclosporin conjugate was
approximately two fold higher than CsA dissolved in alcohol and
added at the same time as the releasable conjugate.
[0282] The releasable R7 CsA conjugate was assayed in vivo for
functional activity using a murine model of contact dermatitis.
Treatment with the 1% releasable R7 CSA conjugate resulted in
73.9%.+-.4.0 reduction in ear inflammation (FIG. 7). No reduction
in inflammation was seen in the untreated ear, indicating that the
effect seen in the treated ear was local and not systemic. Less
inhibition was observed in the ears of mice treated with 0.1 and
0.01% R7-CsA (64.8%.+-.4.0 and 40.9%.+-.3.3 respectively),
demonstrating that the effect was titratable. Treatment with the
fluorinated corticosteroid positive control resulted in reduction
in ear swelling (34.1%.+-.6.3), but significantly less than that
observed for 0.1% releasable R7 CsA (FIG. 7). No reduction of
inflammation was observed in any of the mice treated with
unmodified Cyclosporin A, vehicle alone, R7, or nonreleasable R7
CsA.
Example 9
The Penetration of Copper and Gadolinium-DTPA-r7 Complexes into the
Skin of Nude Mice
Methods
[0283] 1. Preparation of Metal Complexes
Step 1
Preparation of copper-diethylenetriaminepentaacetic Acid Complex
(Cu-DTPA)
[0284] Copper carbonate (10 mmol) and diethylenetriaminpentacetic
acid (10 mmol) were dissolved in water (150 mL) (FIG. 8). After 18
h, the solution was centrifuged to remove any solids. The blue
solution was decanted and lyophilized to provide a blue powder
(yields>90%).
Step 2
Preparation of DTPA Transporter
[0285] The Cu-DTPA was linked to a transporter through an
aminocaproic acid spacer using a PE Applied Biosystems Peptide
Synthesizer (ABI 433A) (FIG. 9). The material was cleaved from the
resin by treatment with trifluoroacetic acid (TFA) (40 mL),
triisopropyl silane (100 .mu.L) and phenol (100 .mu.L} for 18 h.
The resin was filtered off and the peptide was precipitated by
addition of diethyl ether (80 mL). The solution was centrifuged and
the solvent decanted off. The crude solid was purified by
reverse-phase HPLC using a water/acetonitrile gradient. Treatment
with TFA resulted in loss of Cu.sup.2+ ion which needed to be
reinserted.
[0286] DTPA-aca-R7-CO.sub.2H (10 mg, 0.0063 mmol) and copper
sulfate (1.6 mg, 0.0063 mmol) were dissolved in water (1 mL). Let
gently stir for 18 hand lyophilized to provide product as a white
powder (10 mg).
[0287] 2. Analysis of Transport Across Skin
[0288] Metal diethylenetriaminepentaacetic acid (DTPA) complexes
were formed by mixing equimolar amounts of metal salts with DTPA in
water for 18 hours. At the end of this time, the solutions were
centrifuged, frozen and lyophilized. The dried powder was
characterized by mass spectrometry and used in solid phase peptide
synthesis. The metal-DTPA complexes were attached to polymers of D-
or L-arginine that were still attached to solid-phase resin used in
peptide synthesis. The metal-DTPA complexes were attached using an
aminocaproic acid spacer. The solid phase peptide synthesis
techniques were described in Example 1, with the exception that
cleavage of the peptide-DTPA-metal complex in trifluoroacetic acid
released the metal. The metal is replaced after HPLC purification
and lyophilization of the peptide-DTPA complex. Replacement of the
metal involved incubation of equimolar amounts of the metal salt
with the peptide-aminocaproic acid-DTPA complex and subsequent
lyophilization.
[0289] Varying concentrations (1 .mu.M to 1 mM) of the
Cu-DTPA-aca-r7 complex were applied to the abdominal region of nude
mice for 15, 30 and 45 minutes. As controls, an equimolar amount of
the Cu-DTPA complex was spotted onto the abdominal region. At the
end of the incubation period, the samples were simply wiped off and
intense blue color was apparent on the skin where the Cu DTPA
aca-r7 complex was spotted and not where the Cu-DTPA alone was
spotted. In the case of the application of 1 mM, visible blue dye
was seen for three days, decreasing with time, but being apparent
for the full period.
[0290] Varying concentrations (1 .mu.M to 1 mM) of the
Gd-DTPA-aca-r7 complex are injected into the tail vein of BALB/c
mice in 100 .mu.l. Distribution of the Gd is observed in real time
using magnetic resonance imaging. Distribution of the dye is
apparent throughout the bloodstream, entering liver, spleen,
kidney, and heart. When injected into the carotid artery of
rabbits, the dye is seen to cross the blood brain barrier.
Example 10
Penetration of Hydrocortisone Conjugated to a Biotinylated
Pentamer, Heptamer, and Nonamer of D-Arginine into the Skin of Nude
Mice
Methods
[0291] A. Linking of Hydrocortisone to Delivery-Enhancing
Transporters
Step 1
Acylation of Hydrocortisone with Chloroacetic Anhydride
[0292] A solution of hydrocortisone (200 mg, 0.5.5 mmol) and
chloroacetic anhydride (113 mg, 0.66 mmol) in pyridine (5 mL) was
stirred at room temperature for 2 h (FIG. 10). The solvent was
evaporated off and the crude product was chromatographed on silica
using 50% hexanes/ethyl acetate as the eluent. Product isolated a
whites solid (139 mg, 58%).
Step 2
Linking to Transporter
[0293] A solution of the chloroacetic ester of hydrocortisone
(0.0137 mmol), a transporter containing a cysteine residue (0.0137)
and diisopropylethylamine (DIEA) (0.0274 mmol) in dimethylformamide
(DMF) (1 mL) was stirred at room temperature for 18 h (FIG. 11).
The material was purified via reverse-phase HPLC using a
water/acetonitrile gradient and lyophilized to provide a white
powder.
[0294] r5 conjugate--12 mg obtained (29% isolated yield)
[0295] r7 conjugate--22 mg obtained (55% isolated yield)
[0296] R7 conjugate--13 mg obtained (33% isolated yield)
[0297] B. Analysis of Transport Across Skin
[0298] Varying concentrations (1 mM-100 .mu.M) of hydrocortisone
conjugated to either biotinylated pentamer, heptamer, or nonamers
of D-arginine (bio r5, r7, or r9), dissolved in PBS, were applied
to the back of nude mice. Samples (100 .mu.l) were applied as a
liquid within excipient and prevented from dispersing by a
Vaseline.TM. barrier and allowed to penetrate for thirty, sixty,
and 120 minutes. At the end of this period animal was sacrificed,
the relevant section of skin was excised, embedded in mounting
medium (OCT) and frozen. Frozen sections were cut using a cryostat,
collected on slides, and stained with fluorescently labeled
streptavidin (Vector Laboratories, Burlingame, Calif.) as described
in Example 1. Slides were analyzed by fluorescent microscopy.
Results
[0299] The conjugation of hydrocortisone with biotinylated
heptarners of D-arginine effectively entered into and across the
epidermis and into the dermis of the skin of nude mice. In
contrast, very little uptake was seen using a conjugate between a
pentamer of arginine and hydrocortisone, and no staining was seen
with a PBS control. The cytosol and nuclei of all cells in the
field were fluorescent, indicating penetration into and through the
epidermis and dermis. The staining pattern was consistent with
unanticipated transport that was both follicular and
interfollicular. In addition, positive cells were apparent in
papillary and reticular dermis. These results demonstrate
remarkable uptake only when sufficient guanidinyl groups are
included in the delivery-enhancing transporter.
Example 11
Penetration of Taxol Conjugated to a Biotinylated Pentamer,
Heptamer, and Nonamer of D-Arginine into the Skin of Nude Mice
Methods
[0300] 1 Conjugation of C-2' Activated Taxol Derivatives to
Biotin-Labeled Peptides
Synthesis of C-2' Derivatives
[0301] Taxol (48.7 mg, 57.1 .mu.mol) was dissolved in
CH.sub.2Cl.sub.2 (3.0 mL) under an N.sub.2-atmosphere. The solution
was cooled to 0.degree. C. A stock solution of the chloroformate of
benzyl-(p-hydroxy benzoate) (200 mmol, in 2.0 mL CH.sub.2Cl.sub.2--
freshly prepared from benzyl-(p-hydroxy benzoate) and diphosgene)
was added at 0.degree. C. and stirring at that temperature was
continued for 5 hours, after which the solution was warmed to room
temperature (FIG. 12). Stirring was continued for additional 10
hours. The solvents were removed in vacuo and the crude material
was purified by flash chromatography on silica gel (eluent:
EtOAc/hexanes 30%-70%) yielding the desired taxol C-2' carbonate
(36.3 mg, 32.8 .mu.mol, 57.4%).
[0302] Coupling to Biotin-Labeled Peptides.
[0303] A procedure for coupling to biotin-labeled peptides is shown
in FIG. 13. The taxol derivative and the biotin labeled peptide
(1.2 equivalents) were dissolved in DMF (.about.10 .mu.mol/mL DMF)
under an N.sub.2-atmosphere. Stock solutions of
diisopropykthylamine (1.2 equivalents in DMF) and DMAP (0.3
equivalents in DMF) were added and stirring at room temperature was
continued until all starting material was consumed. After 16 hours
the solvent was removed in vacuo. The crude reaction mixture was
dissolved in water and purified by RP-HPLC (eluent: water/MeCN*TFA)
yielding the conjugates in the indicated yields:
[0304] B-aca-r5-K-taxol: 3.6 mg, 1.32 mmol, 20%.
[0305] B-aca-r7-K-taxol: 9.8 mg, 3.01 mmol, 44%
[0306] B-aca-r9-K-taxol: 19.4 mg, 5.1 mmol, 67%.
[0307] Unlabeled C-2' Carbamates:
[0308] The taxol derivative (12.4 mg, 11.2 mol) and the unlabeled
peptide (27.1 mg, 13.4 .mu.mol) were dissolved in DMF (1.5 mL)
under an N.sub.2-atmosphere (FIG. 14). Diisopropylethylamine (1.7
mg, 13.4 .mu.mol) was added as a stock solution in DMF, followed by
DMAP (0.68 mg, 5.6 .mu.mol) as a stock solution in DMF. Stirring at
room temperature was continued until all starting material was
consumed. After 16 hours the solvent was removed in vacuo. The
crude material was dissolved in water and purified by RP-HPLC
(eluent: water/MeCN*TFA) yielding the desired product (16.5 mg, 5.9
.mu.mol, 53%).
[0309] Other C-2' Conjugates
[0310] The taxol derivative (8.7 mg, 7.85 .mu.mol) was dissolved in
EtOAc (2.0 mL). Pd/C (10%, 4.0 mg) was added and the reaction flask
was purged with H.sub.2 five times (FIG. 15A). Stirring under an
atmosphere of hydrogen was continued for 7 hours. The Pd/C was
filtered and the solvent was removed in vacuo. The crude material
(6.7 mg, 6.58 .mu.mol, 84%) obtained in this way was pure and was
used in the next step without further purification.
[0311] The free acid taxol derivative (18.0 mg, 17.7 mol) was
dissolved in CH.sub.2Cl.sub.2 (2.0 mL). Dicyclohexylcarbodiimide
(4.3 mg, 21.3 .mu.mol) was added as a stock solution in
CH.sub.2Cl.sub.2 (0.1 mL). N-Hydroxysuccinimide (2.0 mg, 17.7
.mu.mol) was added as a stock solution in DMF (0.1 mL) (FIG. 15B).
Stirring at room temperature was continued for 14 hours. The
solvent was removed in vacuo and the resultant crude material was
purified by flash chromatography on silica gel (eluent:
EtOAc/hexanes 40%-80%) yielding the desired product (13.6 mg, 12.2
.mu.mol, 69%).
[0312] The activated taxol derivative (14.0 mg, 12.6 .mu.mol) and
the peptide (30.6 mg, 15.1 .mu.mol) were dissolved in DMF (3.0 mL)
under an N.sub.2-atrnosphere (FIG. 15C). Diisopropylethylamine
(1.94 mg, 15.1 .mu.mol) was added as a stock solution in DMF (0.1
mL), followed by DMAP (0.76 mg, 6.3 .mu.mol) as a stock solution in
DMF 0.1 mL). Stirring at room temperature was continued until all
the starting material was consumed. After 20 hours the solvent was
removed in vacuo. The crude material was dissolved in water and
purified by RP-HPLC (eluent: water/MeCN*TFA) yielding the two
depicted taxol conjugates in a ratio of 1:6 (carbonate vs
carbamate, respectively).
[0313] 2. Analysis of Transport Across Skin
[0314] Varying concentrations (1 mM-100 .mu.M) of taxol conjugated
to either biotinylated pentamer, heptamer, or nonamers of
D-arginine (bio r5, r7, or r9), dissolved in PBS, were applied to
the back of nude mice. Samples (100 .mu.l) were applied as a liquid
within excipient and prevented from dispersing by a Vaseline.TM.
barrier and allowed to penetrate for thirty, sixty, and 120
minutes. At the end of this period animal was sacrificed, the
relevant section of skin was excised, embedded in mounting medium
(OCT) and frozen. Frozen sections were cut using a cryostat,
collected on slides, and stained with fluorescently labeled
streptavidin (Vector Laboratories, Burlingame, Calif.) as described
in Example 1. Slides were analyzed by fluorescent microscopy.
Results
[0315] The conjugates of taxol with biotinylated heptamers and
nonamers of D-arginine effectively entered into and across the
epidermis and into the dermis of the skin of nude mice. In
contrast, very little uptake was seen using a conjugate between a
pentamer of arginine and taxol, and no staining was seen with a PBS
control. The cytosol and nuclei of all cells in the field were
fluorescent, indicating penetration into and through the epidermis
and dermis. The staining pattern was consistent with unanticipated
transport that was both follicular and interfollicular. In
addition, positive cells were apparent in papillary and reticular
dermis. These results demonstrate remarkable uptake only when
sufficient guanidinyl groups are included in the delivery-enhancing
transporter.
Example 12
Conjugate of Taxol and Delivery-Enhancing Transporter with
pH-Releasable Linker
[0316] This Example demonstrates the use of a general strategy for
synthesizing prodrugs that have a delivery-enhancing transporter
linked to a drug by a linker that releases the drug from the
delivery-enhancing transporter upon exposure to physiological pH.
In general, a suitable site on the drug is derivatized to carry an
.alpha.-chloroacetyl residue. Next, the chlorine is displaced with
the thiol of a cysteine residue that carries an unprotected amine.
This scheme is shown in FIG. 16.
Methods
[0317] Synthesis of Taxol-2'-chloroacetyl Taxol (89.5 mg, 104.9
.mu.mol) was dissolved in CH.sub.2Cl.sub.2 (3.5 mL). The solution
was cooled to 0.degree. C. under an N.sub.2-atmosphere
.alpha.-Chloroacetic anhydride (19.7 mg, 115.4 .mu.mol) was added,
followed by DIEA (14.8 mg, 115.4 .mu.mol). The solution was allowed
to warm to room temperature. After thin layer chromatography (tlc)
analysis indicated complete consumption of starting material, the
solvent was removed in vacuo and the crude material was purified by
flash chromatography on silica gel (eluent: EtOAC/Hex 20%-50%)
yielding the desired material (99.8 mg, quantitative) (FIG.
18).
[0318] .sup.1H-NMR (CDCl.sub.3): .delta.=8.13 (d, J=7.57 Hz, 2H),
7.72 (d, J=7.57 Hz, 2H), 7.62-7.40 (m, 11H), 6.93 (d, J=9.14 Hz,
1H), 6.29-6.23 (m, 2H), 6.01 (d, J=7.14 Hz, 1H), 5.66 (d, J=6.80
Hz, 1H), 5.55 (d, J=2.24 Hz, 1H), 4.96 (d, J=8.79 Hz, 1H), 4.43 (m,
1H), 4.30 (d, J=8.29 Hz, 1H), 4.20-4.15 (m, 2H), 3.81 (d, J=6.71
Hz, 1H), 2.56-2.34 (m, 3H), 2.45 (s, 3H), 2.21 (s, 3H), 2.19 (m,
1H), 1.95-1.82 (m, 3H), 1.92 s, (3H), 1.67 (s, 3H), 1.22 (s, 3H),
1.13 (s, 3H) ppm.
[0319] .sup.13C-NMR (CDCl.sub.3): .delta.=203.6, 171.1, 169.7,
167.3, 167.0, 166.9, 166.3, 142.3, 136.4, 133.6, 133.5, 132.9,
132.0, 130.1, 129.2, 121.1, 128.7, 128.6, 127.0, 126.5, 84.3, 81.0,
79.0, 76.3, 75.4, 75.2, 75.0, 72.2, 72.0, 58.4, 52.7, 45.5, 43.1,
40.1, 35.5, 26.7, 22.6, 22.0, 20.7, 14.7, 9.5 ppm.
[0320] Linkage of Taxol to Delivery-Enhancing Transporter
[0321] The peptide (47.6 mg, 22.4 .mu.mol) was dissolved in DMF
(1.0 mL) under an N.sub.2-atmosphere. DIEA (2.8 mg, 22.4 .mu.mol)
was added. A solution of taxol-2'-chloroacetate (2.0.8 mg, 22.4
.mu.mol) in DMF (1.0 mL) was added. Stirring at room temperature
was continued for 6 hours. Water containing 0.1% TFA (1.0 mL) was
added, the sample was frozen and the solvents were lyophilized. The
crude material was purified by RP-HPLC (eluent: water/MeCN*0.1%
TFA: 85%-15%). A schematic of this reaction is shown in FIG.
18.
Synthesis of Related Conjugates
[0322] Using the conjugation conditions outlined above, the three
additional conjugates shown in were synthesized.
Cytotoxicity Assay
[0323] The taxol conjugates were tested for cytotoxicity in a
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium-bromide (MTT)
dye reduction. Results, which are shown in FIG. 20, demonstrate
that the taxol conjugated to r7 with a readily pH-releasable linker
(CG 1062; R.dbd.Ac in the structure shown in FIG. 19) is
significantly more cytotoxic than either taxol alone or taxol
conjugated to r7 with a less-readily pH-releasable linker (CG 1040;
R.dbd.H in the structure shown in FIG. 19).
Example 13
Structure-Function Relationships of Fluorescently-Labeled Peptides
Derived from Tat49-57
Methods
[0324] General.
[0325] Rink amide resin and Boc.sub.2O were purchased from
Novabiochem. Diisopropylcarbodiimide, bromoacetic acid, fluorescein
isothiocyanate (FITC-NCS), ethylenediamine, 1,3-diaminopropane,
1,4-diaminobutane, 1,6-diaminohexane, trans-1,6-diaminocyclohexane,
and pyrazole-1-carboxamidine were all purchased from Aldrich.RTM..
All solvents and other reagents were purchased from commercial
sources and used without further purification. The mono-Boc amines
were synthesized from the commercially available diamines using a
literature procedure (10 equiv. of diamine and 1 equiv. of
Boc.sub.2O in chloroform followed by an aqueous work up to remove
unreacted diamine) (34).
[0326] N-tert-butoxycarbonyl-1,6-trans-diaminocyclohexane. Mp
159-161.degree. C.; .sup.1H NMR (CDCl.sub.3) .delta. 4.35 (br s,
1H), 3.37 (br s, 1H), 2.61 (br s, 1H), 1.92-2.02 (m, 2H), 1.81-1.89
(m, 2H), 1.43 (s, 9H), 1.07-1.24 (m, 4H) ppm; .sup.13C NMR
(D.sub.6-DMSO) .delta. 154.9, 77.3, 49.7, 48.9, 35.1, 31.4, 28.3
ppm; ES-MS (M+I) calcd 215.17. found 215.22.
[0327] General Procedure for Peptide Synthesis.
[0328] Tat.sub.49-57 (RKKRRQRRR), truncated and alanine-substituted
peptides derived from Tat.sub.49-57, Antennapedia.sub.43-58
(RQIKIWFQNRRMKWKK), and homopolymers of arginine (R5-R9) and
d-arginine (r5-r9) were prepared with an automated peptide
synthesizer (ABI433) using standard solid-phase Fmoc chemistry (35)
with HATU as the peptide coupling reagent. The fluorescein moiety
was attached via a aminohexanoic acid spacer by treating a
resin-bound peptide (1.0 mmol) with fluorescein isothiocyanate (1.0
mmol) and DIEA (5 mmol) in DMF (10 mL) for 12 h. Cleavage from the
resin was achieved using 95:5 TFA/triisopropylsilane. Removal of
the solvent in vacuo gave a crude oil which was triturated with
cold ether. The crude mixture thus obtained was centrifuged, the
ether was removed by decantation, and the resulting orange solid
was purified by reverse-phase HPLC (H.sub.2O/CH.sub.3CN in 0.1%
TFA). The products were isolated by lyophilization and
characterized by electrospray mass spectrometry. Purity of the
peptides was >95% as determined by analytical reverse-phase HPLC
(H.sub.2O/CH.sub.3CN in 0.1% TFA).
[0329] All peptides and peptoids synthesized contain an
aminohexanoic (ahx) acid moiety attached to the N-terminal amino
group with a fluorescein moiety (Fl) covalently linked to the amino
group of the aminohexanoic acid spacer. The carboxyl terminus of
every peptide and peptoid is a carboxamide.
[0330] Cellular Uptake Assay.
[0331] The arginine homopolymers and guanidine-substituted peptoids
were each dissolved in PBS buffer (pH 7.2) and their concentration
was determined by absorption of fluorescein at 490 nm
(.epsilon.=67,000). The accuracy of this method for determining
concentration was established by weighing selected samples and
dissolving them in a known amount of PBS buffer. The concentrations
determined by UV spectroscopy correlated with the amounts weighed
out manually. Jurkat cells (human T cell line), murine B cells
(CH27), or human PBL cells were grown in 10% fetal calf serum and
DMEM and each of these were used for cellular uptake experiments.
Varying amounts of arginine and oligomers of guanidine substituted
peptoids were added to approximately 3.times.10.sup.6 cells in 2%
FCS/PBS (combined total of 200 .mu.L) and placed into microtiter
plates (96 well) and incubated for varying amounts of time at
23.degree. C. or 4.degree. C. The microtiter plates were
centrifuged and the cells were isolated, washed with cold PBS
(3.times.250 .mu.L), incubated with 0.05% trypsin/0.53 mM EDTA at
37.degree. C. for 5 min, washed with cold PBS, and resuspended in
PBS containing 0.1% propidium iodide. The cells were analyzed using
fluorescent flow cytometry (FACScan, Becton Dickinson) and cells
staining with propidium iodide were excluded from the analysis. The
data presented is the mean fluorescent signal for the 5000 cells
collected.
[0332] Inhibition of Cellular Uptake with Sodium Azide.
[0333] The assays were performed as previously described with the
exception that the cells used were preincubated for 30 min with
0.5% sodium azide in 2% FCS/PBS buffer prior to the addition of
fluorescent peptides and the cells were washed with 0.5% sodium
azide in PBS buffer. All of the cellular uptake assays were run in
parallel in the presence and absence of sodium azide.
[0334] Cellular Uptake Kinetics Assay.
[0335] The assays were performed as previously described except the
cells were incubated for 0.5, 1, 2, and 4 min at 4.degree. C. in
triplicate in 2% FCS/PBS (50 .mu.l) in microtiter plates (96 well).
The reactions were quenched by diluting the samples into 2% FCS/PBS
(5 mL). The assays were then worked up and analyzed by fluorescent
flow cytometry as previously described.
Results
[0336] To determine the structural requirements for the cellular
uptake of short arginine-rich peptides, a series of fluorescently
labeled truncated analogues of Tat.sub.49-57 were synthesized using
standard solid phase chemistry. See, e.g., Atherton, E. et al.
SOLID-PHASE PEPTIDE SYNTHESIS (IRL: Oxford, Engl. 1989). A
fluorescein moiety was attached via an aminohexanoic acid spacer on
the amino termini. The ability of these fluorescently labeled
peptides to enter Jurkat cells was then analyzed using fluorescent
activated cell sorting (FACS). The peptide constructs tested were
Tat.sub.49-57 (Fl ahx-RKKRRQRRR); Tat.sub.49-56 (Fl-ahx-RKKRRQRR),
Tat.sub.49-55 (Fl-ahx-RKKRRQR), Tat.sub.50-57 (Fl-ahx-KKRRQRRR),
and Tat.sub.51-57 (Fl-ahx-KRRQRRR). Differentiation between cell
surface binding and internalization was accomplished throughout by
running a parallel set of assays in the presence and absence of
sodium azide. Because sodium azide inhibits energy-dependent
cellular uptake but not cell surface binding, the difference in
fluorescence between the two assays provided the amount of
fluorescence resulting from internalization.
[0337] Deletion of one arginine residue from either the amine
terminus (Tat.sub.50-57) or the carboxyl terminus (Tat.sub.49-56)
resulted in an 80% loss of intracellular fluorescence compared to
the parent sequence (Tat.sub.49-57). From the one amino acid
truncated analogs, further deletion of R-56 from the carboxyl
terminus (Tat.sub.49-55) resulted in an additional 60% loss of
intracellular fluorescence, while deletion of K-50 from the amine
terminus (Tat.sub.51-57) did not further diminish the amount of
internalization. These results indicate that truncated analogs of
Tat.sub.49-57 are significantly less effective at the transcellular
delivery of fluorescein into Jurkat cells, and that the arginine
residues appear to contribute more to cellular uptake than the
lysine residues.
[0338] To determine the contribution of individual amino acid
residues to cellular uptake, analogs containing alanine
substitutions at each site of Tat.sub.49-57 were synthesized and
assayed by FACS analysis (FIG. 22). The following constructs were
tested: A-49 (F1-ahx-AKKRRQRRR), A-50 (F1-ahx-RAKRRQRRR), A-51
(F1-ahx-RKARRQRRR), A-52 (F1-ahx-RKKARQRRR), A-53
(F1-ahx-RKKRAQRRR), A-54 (F1-ahx-RKKRRARRR), A-55
(F1-ahx-RKKRRQARR), A-56 (F1-ahx-RKKRRQRAR), and A-57
(F1-ahx-RKKRRQRRA). Substitution of the non-charged glutamine
residue of Tat.sub.49-57 with alanine (A-54) resulted in a modest
decrease in cellular internalization. On the other hand, alanine
substitution of each of the cationic residues individually produced
a 70-90% loss of cellular uptake. In these cases, the replacement
of lysine (A-50, A-51) or arginine (A-49, A-52, A-55, A-56, A-57)
with alanine had similar effects in reducing uptake.
[0339] To determine whether the chirality of the transporter
peptide was important, the corresponding d-(d-Tat.sub.49-57),
retro-I-(Tat.sub.57-49), and retro-inverso isomers
(d-Tat.sub.57-49) were synthesized and assayed by FACS analysis
(FIG. 23). Importantly, all three analogs were more effective at
entering Jurkat cells then Tat.sub.49-57. These results indicated
that the chirality of the peptide backbone is not crucial for
cellular uptake. Interestingly, the retro-I isomer (Tat.sub.57-49)
which has three arginine residues located at the amine terminus
instead of one arginine and two lysines found in Tat.sub.49.57
demonstrated enhanced cellular uptake. Thus residues at the amine
terminus appear to be important and that arginines are more
effective than lysines for internalization. The improved cellular
uptake of the unnatural d-peptides is most likely due to their
increased stability to proteolysis in 2% FCS (fetal calf serum)
used in the assays. When serum was excluded, the d- and i-peptides
were equivalent as expected.
[0340] These initial results indicated that arginine content is
primarily responsible for the cellular uptake of Tat.sub.49-57.
Furthermore, these results were consistent with our previous
results where we demonstrated that short oligomers of arginine were
more effective at entering cells then the corresponding short
oligomers of lysine, ornithine, and histidine. What had not been
established was whether arginine homo-oligomers are more effective
than Tat.sub.49-57. To address this point, Tat.sub.49-57 was
compared to the 1-arginine (R5-R9) and d-arginine (r5-r9)
oligomers. Although Tat.sub.49-57 contains eight cationic residues,
its cellular internalization was between that of R6 and R7 (FIG.
24) demonstrating that the presence of six arginine residues is the
most important factor for cellular uptake. Significantly,
conjugates containing 7-9 arginine residues exhibited better uptake
than Tat.sub.49-57.
[0341] To quantitatively compare the ability of these arginine
oligomers and Tat.sub.49-57 to enter cells, Michaelis-Menton
kinetic analyses were performed. The rates of cellular uptake were
determined after incubation (3.degree. C.) of the peptides in
Jurkat cells for 30, 60, 120, and 240 seconds (Table 1). The
resultant K.sub.m values revealed that r9 and R9 entered cells at
rates approximately 100-fold and 20-fold faster than Tat.sub.49-57
respectively. For comparison, Antennapedia.sub.43-58 was also
analyzed and was shown to enter cells approximately 2-fold faster
than Tat.sub.49-57, but significantly slower than r9 or R9.
TABLE-US-00001 TABLE 1 Michaelis-Menton kinetics:
Antennapedia.sub.43-58 (F1-ahx- RQIKIWFQNRRMKWKK). peptide Km (JIM)
Vmax Ta.sub.49-57 770 0.38 Antennapedia.sub.43-58 427 0.41 R9 44
0.37 r9 7.6 0.38
Example 14
Design and Synthesis of Peptidomimetic Analogs of Tat49-57
Methods
[0342] General Procedure for Peptoid Polyamine Synthesis.
[0343] Peptoids were synthesized manually using a fritted glass
apparatus and positive nitrogen pressure for mixing the resin
following the literature procedure developed by Zuckermann. See,
e.g., Murphy, J. E. et al., Proc. Natl. Acad. Sci. USA 95,
1517-1522 (1998); Simon, R. J. et al., Proc. Natl. Acad. Sci. USA
89, 9367-9371 (1992); Zuckermann, R. N. et al., J. Am. Chem. Soc.
114, 10646-10647 (1992). Treatment of Fmoc-substituted Rink amide
resin (0.2 mmol) with 20% piperidine/DMF (5 mL) for 30 min
(2.times.) gave the free resin-bound amine which was washed with
DMF (3.times.5 mL). The resin was treated with a solution of
bromoacetic acid (2.0 mmol) in DMF (5 mL) for 30 min. This
procedure was repeated. The resin was then washed (3.times.5 mL
DMF) and treated with a solution of mono-Boc diamine (8.0 mmol) in
DMF (5 mL) for 12 hrs. These two steps were repeated until an
oligomer of the required length was obtained (Note: the solution of
mono-Boc diamine in DMF could be recycled without appreciable loss
of yield). The resin was then treated with N-Fmoc-aminohexanoic
acid (2.0 mmol) and DIC (2.0 mmol) in DMF for 1 h and this was
repeated. The Fmoc was then removed by treatment with 20%
piperidine/DMF (5 mL) for 30 min. This step was repeated and the
resin was washed with DMF (3.times.5 mL). The free amine resin was
then treated with Fluorescein isothiocyanate (0.2 mmol) and DIEA
(2.0 mmol) in DMF (5 mL) for 12 hrs. The sin was then washed with
DMF (3.times.5 mL) and dichloromethane (5.times.5 mL). Cleavage
from the resin was achieved using 95:5 TFA/triisopropylsilane (8
mL). Removal of the solvent in vacuo gave a crude oil which was
triturated with cold ether (20 mL). The crude mixture thus obtained
was centrifuged, the ether was removed by decantation, and the
resulting orange solid was purified by reverse-phase HPLC
(H.sub.2O/CH.sub.3CN in 0.1% TFA). The products were isolated by
lyophilization and characterized by electrospray mass spectrometry
and in selected cases by .sup.1H NMR spectroscopy.
[0344] General Procedure for Perguanidinylation of Peptoid
Polyamines.
[0345] A solution of peptoid amine (0.1 mmol) dissolved in
deionized water (5 mL) was treated with sodium carbonate (5
equivalents per amine residue) and pyrazole-1-carboxamidine (5
equivalents per amine residue) and heated to 50.degree. C. for
24-48 hr. The crude mixture was then acidified with TFA (0.5 mL)
and directly purified by reverse-phase HPLC (H.sub.2O/CH.sub.3CN in
0.1% TFA). The products were characterized by electrospray mass
spectrometry and isolated by lyophilization and further purified by
reverse-phase HPLC. The purity of the guanidine-substituted
peptoids was >95% as determined by analytical reverse-phase HPLC
(H.sub.2O/CH.sub.3CN in 0.1% TFA).
Results
[0346] Utilizing the structure-function relationships that had been
determined for the cellular uptake of Tat.sub.49-57, we designed a
set of polyguanidine peptoid derivatives that preserve the 1,4
backbone spacing of side chains of arginine oligomers, but have an
oligoglycine backbone devoid of stereogenic centers. These peptoids
incorporating arginine-like side chains on the amide nitrogen were
selected because of their expected resistance to proteolysis, and
potential ease and significantly lower cost of synthesis (Simon et
al., Proc. Natl. Acad. Sci. USA 89:9367-9371 (1992); Zuckermann, et
al., J. Am. Chem. Soc. 114:10646-10647 (1992). Furthermore,
racemization, frequently encountered in peptide synthesis, is not a
problem in peptoid synthesis; and the "sub-monomer" peptoid
approach allows for facile modification of side-chain spacers.
Although the preparation of an oligurea and peptoid-peptide hybrid
(Hamy, et al, Proc. Natl. Acad. Sci. USA 94:3548-3553 (1997))
derivatives of Tat.sub.49-57 have been previously reported, their
cellular uptake was not explicitly studied.
[0347] The desired peptoids were prepared using the "sub-monomer"
approach (Simonet et al.; Zuckermann et al.) to peptoids followed
by attachment of a fluorescein moiety via an aminohexanoic acid
spacer onto the amine termini. After cleavage from the solid-phase
resin, the fluorescently labeled polyamine peptoids thus obtained
were converted in good yields (60-70%) into polyguanidine peptoids
by treatment with excess pyrazole-1-carboxamidine (Bernatowicz, et
al., J. Org. Chem. 57:2497-2502 (1992) and sodium carbonate (as
shown in FIG. 25). Previously reported syntheses of peptoids
containing isolated N-Arg units have relied on the synthesis of
N-Arg monomers (5-7 steps) prior to peptoid synthesis and the use
of specialized and expensive guanidine protecting groups (Pmc, Pbf)
(Kruijtzer, et al., Chem. Eur. J. 4:1570-1580 (1998); Heizmann, et
al. Peptide Res. 7:328-332 (1994). The compounds reported here
represent the first examples of polyguanidinylated peptoids
prepared using a perguanidinylation step. This method provides easy
access to polyguanidinylated compounds from the corresponding
polyamines and is especially useful for the synthesis of
perguanidinylated homooligomers. Furthermore, it eliminates the use
of expensive protecting groups (Pbf, Pmc). An additional example of
a perguanidinylation of a peptide substrate using a novel
triflyl-substituted guanylating agent has recently been reported
(Feichtinger, et al., J. Org. Chem. 63:8432-8439 (1998)).
[0348] The cellular uptake of fluorescently labeled polyguanidine
N-arg-5,7,9 peptoids was compared to the corresponding d-arginine
peptides r-5,7,9 (similar proteolytic properties) using Jurkat
cells and FACS analysis. The amount of fluorescence measured inside
the cells with N-arg-5,7,9 was proportional to the number of
guanidine residues: N-arg9>N-arg7>N-arg5 (FIG. 26), analogous
to that found for r-5,7,9. Furthermore, the N-arg-5,7,9 peptoids
showed only a slightly lower amount of cellular entry compared to
the corresponding peptides, r5,7,9. The results demonstrate that
the hydrogen bonding along the peptide backbone of Tat.sub.49-57 or
arginine oligomers is not a required structural element for
cellular uptake and oligomeric guanidine-substituted peptoids can
be utilized in place of arginine-rich peptides as molecular
transporters. The addition of sodium azide inhibited
internalization demonstrating that the cellular uptake of peptoids
was also energy dependent.
Example 15
The Effect of Side Chain Length on Cellular Uptake
[0349] After establishing that the N-arg peptoids efficiently
crossed cellular membranes, the effect of side chain length (number
of methylenes) on cellular uptake was investigated. For a given
number of guanidine residues (5,7,9), cellular uptake was
proportional to side chain length. Peptoids with longer side chains
exhibited more efficient cellular uptake. A nine-mer peptoid analog
with a six-methylene spacer between the guanidine head groups and
the backbone (N-hxg9) exhibited remarkably higher cellular uptake
than the corresponding d-arginine oligomer (r9). The relative order
of uptake was N-hxg9 (6 methylene)>N-btg9 (4 methylene)>r9 (3
methylene)>N-arg9 (3 methylene)>N-etg9 (2 methylene) (FIG.
27). Of note, the N-hxg peptoids showed remarkably high cellular
uptake, even greater than the corresponding d-arginine oligomers.
The cellular uptake of the corresponding heptamers and pentamers
also showed the same relative trend. The longer side chains
embodied in the N-hxg peptoids improved the cellular uptake to such
an extent that the amount of internalization was comparable to the
corresponding d-arginine oligomer containing one more guanidine
residue (FIG. 28). For example, the N-hxg7 peptoid showed
comparable cellular uptake to r8.
[0350] To address whether the increase in cellular uptake was due
to the increased length of the side chains or due to their
hydrophobic nature, a set of peptoids was synthesized containing
cyclohexyl side chains. These are referred to as the N-chg-5,7,9
peptoids. These contain the same number of side chain carbons as
the N-hxg peptoids but possess different degrees of freedom.
Interestingly, the N-chg peptoid showed much lower cellular uptake
activity than all of the previously assayed peptoids, including the
N-etg peptoids (FIG. 29). Therefore, the conformational flexibility
and sterically unencumbered nature of the straight chain alkyl
spacing groups is important for efficient cellular uptake.
Discussion
[0351] The nona-peptide, Tat.sub.49-57, has been previously shown
to efficiently translocate through plasma membranes. The goal of
this research was to determine the structural basis for this effect
and use this information to develop simpler and more effective
molecular transporters. Toward this end, truncated and alanine
substituted derivatives of Tat.sub.49-57 conjugated to a
fluorescein label was prepared. These derivatives exhibited greatly
diminished cellular uptake compared to Tat.sub.49-57, indicating
that all of the cationic residues of Tat.sub.49-57 are required for
efficient cellular uptake. When compared with our previous studies
on short oligomers of cationic oligomers, these findings
suggested-that an oligomer of arginine might be superior to
Tat.sub.49-57 and certainly more easily and cost effectively
prepared. Comparison of short arginine oligomers with Tat.sub.49-57
showed that members of the former were indeed more efficiently
taken into cells. This was further quantified for the first time by
Michaelis-Menton kinetics analysis which showed that the R9 and r9
oligomers had Km values 30-fold and 100-fold greater than that
found for Tat.sub.49-57.
[0352] Given the importance of the guanidine head group and the
apparent insensitivity of the oligomer chirality revealed in our
peptide studies, we designed and synthesized a novel series of
polyguanidine peptoids. The peptoids N-arg-5,7,9, incorporating the
arginine side chain, exhibited comparable cellular uptake to the
corresponding d-arginine peptides r5,7,9, indicating that the
hydrogen bonding along the peptide back one and backbone chirality
are not essential for cellular uptake. This observation is
consistent with molecular models of these peptoids, arginine
oligomers, and Tat.sub.49-57, all of which have a deeply embedded
backbone and a guanidinium dominated surface. Molecular models
further reveal that these structural characteristics are retained
in varying degree in oligomers with different alkyl spacers between
the peptoid backbone and guanidino head groups. Accordingly, a
series of peptoids incorporating 2-(N-etg), 4-(N-btg), and 6-atom
(N-hxg) spacers between the backbone and side chain were prepared
and compared for cellular uptake with the N-arg peptoids (3-atom
spacers) and d-arginine oligomers. The length of the side chains
had a dramatic effect on cellular entry. The amount of cellular
uptake was proportional to the length of the side chain with
N-hxg>N-btg>N-arg>N-etg. Cellular uptake was improved when
the number of alkyl spacer units between the guanidine head group
and the backbone was increased. Significantly, N-hxg9 was superior
to r9, the latter being 100-fold better than Tat.sub.49-57. This
result led us to prepare peptoid derivatives containing longer
octyl spacers (N-ocg) between the guanidino groups and the
backbone. Issues related to solubility prevented us from testing
these compounds.
[0353] Because both perguanidinylated peptides and
perguanidinylated peptoids efficiently enter cells, the guanidine
head group (independent of backbone) is apparently the critical
structural determinant of cellular uptake. However, the presence of
several (over six) guanidine moieties on a molecular scaffold is
not sufficient for active transport into cells as the N-chg
peptoids did not efficiently translocate into cells. Thus, in
addition to the importance of the guanidine head group, there are
structure/conformational requirements that are significant for
cellular uptake.
[0354] In summary, this investigation identified a series of
structural characteristics including sequence length, amino acid
composition, and chirality that influence the ability of
Tat.sub.49-57 to enter cells. These characteristics provided the
blueprint for the design of a series of novel peptoids, of which 17
members were synthesized and assayed for cellular uptake.
Significantly, the N-hxg9 transporter was found to be superior in
cell uptake to r9 which was comparable to N-btg9. Hence, these
peptoid transporters proved to be substantially better than
Tat.sub.49-57. This research established that the peptide backbone
and hydrogen bonding along that backbone are not required for
cellular uptake, that the guanidino head group is superior to other
cationic subunits, and most significantly, that an extension of the
alkyl chain between the backbone and the head group provides
superior transporters. In addition to better uptake performance,
these novel peptoids offer several advantages over Tat.sub.49-57
including cost-effectiveness, ease of synthesis of analogs, and
protease stability. These features along with their significant
water solubility (>100 mg/mL) indicate that these novel peptoids
could serve as effective transporters for the molecular delivery of
drugs, drug candidates, and other agents into cells.
Example 16
Synthesis of Itraconazole-Transporter Conjugate
[0355] This Example provides one application of a general strategy
for attaching a delivery-enhancing transporter to a compound that
includes a triazole structure. The scheme, using attachment of
itraconazole to an arginine (r7) delivery-enhancing transporter as
an example, is shown in FIG. 30. In the scheme, R is H or alkyl, n
is 1 or 2, and X is a halogen.
[0356] The reaction involves making use of quaternization of a
nitrogen in the triazole ring to attach an acyl group that has a
halogen (e.g., Br, F1, I) or a methyl ester. Compound 3 was
isolated by HPLC. Proton NMR in D.sub.2O revealed itraconazole and
transporter peaks.
[0357] The methyl ester provided yields of 70% and greater, while
yields obtained using the Br-propionic acid/ester pair were 40-50%.
The acyl-derivative is then reacted with the amine of the
delivery-enhancing transporter to form the conjugate.
Alternatively, the halogenated acyl group can first be attached to
the transporter molecule through an amide linkage, after which the
reaction with the drug compound is conducted.
Example 17
Preparation of FK506 Conjugates
[0358] This Example describes the preparation of conjugates in
which FK506 is attached to a delivery-enhancing transporter. Two
different linkers were used, each of which released FK506 at
physiological pH (pH 5.5 to 7.5), but had longer half-lives at more
acidic pH. These schemes are diagrammed in FIGS. 31A and B.
Linker 1: 6-maleimidocaproic hydrazide trifkuroacetate (Scheme I
and II)
[0359] A solution of FK506 (1) (0.1 g, 124.4 .mu.mol),
6-maleiimidocaproic hydrazide trifluoroacetate (2) (0.126 g, 373.2
.mu.mol) and trifluoroacetic acid (catalytic, 1 .mu.L) in anhydrous
methanol (5 mL) was stirred at room temperature for 36 h. The
reaction was monitored by thin layer chromatography that showed
almost complete disappearance of the starting material. [TLC
solvent system--dichloromethane (95): methanol (5), R.sub.f=0.3].
The reaction mixture was concentrated to dryness and dissolved in
ethyl acetate (20 mL). The organic layer was washed with water and
10% sodium bicarbonate solution and then dried over sodium sulfate,
filtered and concentrated. The residue was purified by column
chromatography using dichloromethane (96): methanol (4) as eluent
to give the hydrazone 3 (0.116 g, 92%).
[0360] A solution of the above hydrazone (3) (0.025 g, 24.7
.mu.mol), transporter (1.times., Bacar.sub.9CCONH.sub.2.9TFA,
Bacar.sub.7CCONH.sub.2.7TFA, BacaCCONH.sub.2,
NH.sub.2r.sub.7CCONH.sub.2.8TFA, NH.sub.2R.sub.7CCONH.sub.2.8TFA)
and diisopropylethylamine (1.times.) in anhydrous dimethylformamide
(1 mL) were stirred under nitrogen at room temperature for 36 h
when TLC indicated the complete disappearance of the starting
hydrazone. Solvent was evaporated from the reaction mixture and the
residue purified by reverse phase HPLC using trifluoroacetic acid
buffered water and acetonitrile. [0361] Yields of conjugates with
various transporters: [0362] Conjugate with
Bacar.sub.9CCONH.sub.2.9TFA (4)--73% [0363]
Bacar.sub.7CCONH.sub.2.7TFA (5)--50% [0364] BacaCCONH.sub.2
(6)--52.9% [0365] NH.sub.2r.sub.7CCONH.sub.2.8TFA (7)--43.8% [0366]
NH.sub.2R.sub.7CCONH.sub.2.8TFA (8)--62.8%
[0367] Structures of all the products were confirmed by 1H-NMR
spectra and TOF MS analysis.
Linker 2: 2-(2-pyridinyldithio)ethyl hydrazine carboxylate (Scheme
III and IV)
[0368] A solution of FK506 (1) (0.1 g, 124.4 .mu.mol),
2-(2-pyridinyldithio)ethyl hydrazine carboxylate (9) (0.091 g,
373.2 .mu.mol) and trifluoroacetic acid (catalytic, 1 .mu.L) in
anhydrous methanol (5 mL) was stirred at room temperature for 16 h.
The reaction was monitored by thin layer chromatography that showed
almost complete disappearance of the starting material. [TLC
solvent system--ethyl acetate R.sub.f=0.5]. The reaction mixture
was concentrated to dryness and dissolved in ethyl acetate (20 mL).
The organic layer was washed with water and 10% sodium bicarbonate
solution and then dried over sodium sulfate, filtered and
concentrated. The residue was purified by column chromatography
using dichloromethane (97): methanol (3) as eluent to give the
hydrazone 10 (0.091 g, 71%)
Example 18
Differential Uptake of Transporters in the Gastrointestinal
Tract
Methods
Gastrointestinal Absorption Protocol
[0369] Experiments were performed on 8- to 10-week-old female Swiss
Webster mice purchased from Taconic (Germantown, N.Y.). Mice were
anesthetized with Nembutal and a midline incision was made along
the abdomen. Intestines were measured, tied off at both ends of the
desired section with sutures, and biotinylated peptides were
injected into the lumen (approximately 100.lamda./inch). After a
fifteen minute incubation, the tissue was removed and the lumen was
gently washed with PBS.
[0370] To determine whether CellGate transporters could enter the
squamous epithelia of the oral cavity, mice were anesthetized with
Nembutal, their heads tipped to one side and solutions of the
biotinylated peptides were placed in their mouths. After fifteen
minutes the liquid was removed by pipette.
Preparation of Histological Sections of Regions of the
Gastrointestinal Tract
[0371] Immediately following incubation with biotinylated peptides,
the anesthetized rodents were sacrificed by cervical dislocation
and the tied off sections of the GI tract were removed. The lumens
of the various sections were filled with OCT using a plastic tipped
syringe, immersed in OCT filled boats, and snap frozen in a
2-methyl-butarie/dry ice solution. Frozen sections were allowed to
warm slightly and 2 mm thick sagital cuts were made using a steel
razor blade. The cuts were placed into OCT molds and snap frozen in
a 2-methyl-butane/dry ice solution. Sections (5 .mu.n) were cut on
the cryostat, fixed in acetone at 4.degree. C. for 10 minute, and
allowed to air dry. After rehydration in PBS for 5 minutes,
sections were blocked with normal horse serum, washed, incubated
with streptavidin-FITC (20).mu.g/ml) for 30 minutes, washed, and
mounted with mounting medium containing propidium iodide (PI).
Tissue Culture
[0372] Caco-2 cells were acquired from ATCC, thawed, and grown in
DMEM containing penicillin, streptomycin, glutamine, and 10% fetal
bovine serum for 3-5 days to confluency (>250,000
cells/cm.sup.2) and then passaged at 60,000 cells/cm.sup.2 for
several passage cycles. Passage numbers 25-29 were plated on
Snapwell 12 mm diameter 0.4-micron pore size polycarbonate
membranes (Coming Costar, Coming, N.Y.) at 60,000 cells per
membrane and allowed to grow for at least 21 days, with media
changed every other day.
Cellular Uptake in Caco-2 Cells
[0373] To analyze the penetration of fluorescent oligomers of
D-arginine into the cells when in suspension, the monolayers were
treated with trypsin (4.5 ml of a 0.05% solution Gibco, Grand
Rapids, Mich.) and the individual cell suspension was spun down.
Cells were resuspended, counted and treated with varying
concentrations of F1 aca r5, F1 aca r7, F1 aca r9 and F1 aca k9
(from 50-0.8 .mu.M) for five minutes, washed, resuspended in
400.lamda., PBS, 2% FBS, 40 ngPI/ml and analyzed by flow
cytometry.
[0374] To analyze the ability of the fluorescent peptides to enter
Caco-2 cells when part of a monolayer, the cells were seeded in
lab-tek flaskette microscope slides (Nalge Nunc Int., Naperville,
Ill.) in 4 ml at a density of 60,000 cells/ml and grown for 21 days
with the media being changed every other day. Once the monolayer
was established, it was incubated with 100 .mu.M F1 aca r9
CONH.sub.2 for a five minutes. The monolayer was subsequently
washed with PBS/2% FBS twice to remove labeled peptide and analyzed
using fluorescent microscopy.
Transport Across Monolayers of Caco-2 Cells
[0375] The experiments analyzing whether short oligomers of
D-arginine could cross monolayers of Caco-2 cells were performed
using a voltage clamp amplifier and Easymount side-by-side
horizontal diffusion chamber (Physiologic Instruments, San Diego,
Calif.) with a 95% oxygen 5% carbon dioxide gas lift system
connected to a recirculating water bath.
[0376] Current measurements (I.sub.2-I.sub.1) were taken at five
minute intervals, including 10 minutes prior to the addition of the
test compounds to insure monolayers were intact. At zero time test
compounds were added at the proper concentrations and measurements
recorded. Measurements were taken for 60 minutes at 5 minute
intervals; transepithelia electrical resistance (TEER) and short
circuit current (Isc) were subsequently calculated from these
values.
Synthetic Chemistry
[0377] Cyclosporin A
[0378] The details of the CsA conjugate used in this study have
been described previously (Rothbard et al. Nature Medicine 6, 1253
2000). See also, FIG. 6. Briefly, CsA was conjugated to a heptamer
of D-arginine through a pH sensitive linker as shown in FIG. 6A.
The resultant conjugate is stable at acidic pH but at pH>7 it
undergoes an intramolecular cyclization involving addition of the
free amine to the carbonyl adjacent to CsA (FIG. 6B), which results
in the release of unmodified CsA.
[0379] Taxol
[0380] Taxol was treated with .alpha.-chloro acetic anhydride
delivering the C-2' chloro acetyl derivative 12 in essentially
quantitative yield.
##STR00010##
[0381] The halogen was displaced by the thiol of the N-terminal (L)
cysteine containing heptamer of D-arginine. Conjugations were
performed at room temperature in DMF in the presence of DIEA. The
final products were isolated by RP-HPLC and lyophilized to yield
TFA salts, which were very hygroscopic and readily dissolve in
water.
[0382] Compound 13 was designed to release taxol 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.
Intracolonic Injections
[0383] Wistar rats, females of approximately 200-300 g (Simonsen,
Gilroy, Calif.), were anesthetized, their abdomens were shaved,
midline incisions were made, and a one-half inch section of the
ascending colon in each animal was tied off with sutures. Taxol and
cyclosporin (5 mg/kg) were injected as solutions in a 1:1 v/v
Cremophor EL:ethanol mixture, whereas equivalent molar amounts of
the r7 conjugates were injected in PBS. In all cases, the volume
injected was approximately 500.lamda.. The colon was placed back
into the cavity, and the incision was closed using sutures.
[0384] Blood samples were taken from the tail vein at time zero
(prior to drug injection), and every thirty minutes for the
duration of the experiment, which was empirically determined, with
the exception of one animal that expired after ninety minutes.
Clotting was inhibited by transferring the blood to glass tubes
containing 100.lamda., of 0.5% EDTA, and the blood was frozen.
Drug Extraction from Whole Blood and HPLC MS MS Analysis
[0385] Either taxol or cyclosporin was extracted from the whole
blood using a modification of literature procedures. Briefly, whole
blood (100.lamda.) was transferred to a screw capped glass tube
containing five mls of diethyl ether. The sample was vortexed
vigorously for two minutes, centrifuged, and frozen in dry
ice/methanol. The ethereal layer was transferred to another glass
tube and the ether was evaporated. In the case of cyclosporin, the
residue was resuspended in 1.5 mls of methanol, water, acetonitrile
(3:2:1), while for taxol the residue was resuspended in 1.5 mls
methanol:acetonitrile (1:1). Samples were placed into a
Perkin-Elmer series 200 autosampler and sequentially injected onto
a CI8 reverse column at 70.degree. C. connected to a Shimadzu HPLC
system, eluted with 70% methanol, 30% aqueous ammonium formate
buffer, and the effluent was analyzed on a PE Sciex API 3000 tandem
mass spectrometer. Known amounts of either cyclosporin A (10-1000
ng/ml) or taxol (1-1000 ng/ml) were added to whole blood and
extracted as previously described to generate standard curves.
Cyclosporin A was monitored by the two transitions from both 1220
to 1203 daltons and 1220 to 100 daltons. The 1220 species
corresponds to the cyclosporin A+ ammonia, the 1203 is protonated
cyclosporin, while 100 is a known fragment. Tasol was monitored by
the two transitions from both 872 to 855 daltons and 872 to 110
daltons. The 872 species corresponds to the ammonium adduct of
taxol, the 855 is the protonated parent compound, whereas 110 is
the predominant fragment seen in the second quadrupole.
[0386] The total amount of cyclosporin A and taxol the samples was
determined by comparing the integrated area of the appropriate peak
with values established by the standard curves.
Results
Differential Uptake of Transporters in the Gastrointestinal
Tract
[0387] Short oligomers of D-arginine have been shown to cross
rapidly and efficiently the plasma membrane of a large variety of
cell lines grown in suspension. In addition, they have been shown
to penetrate multiple layers of the skin when applied topically,
multiple layers of endothelial and smooth muscle cells of veins and
arteries when injected intravenously, and multiple cell layers of
lung tissue when inhaled. To determine whether these compounds
could enter the nontkeratinized epithelia of the gastrointestinal
tract, sections of the small and large intestines of fasted mice
were tied off and solutions of biotinylated nonamers of D-arginine,
bio aca r9 CONH.sub.2, (100 .mu.M) were injected. After fifteen
minutes of exposure, the relevant section of tissue was dissected,
frozen, sectioned, and stained with Streptavidin fluorescein to
define the location of the biotinylated peptide, and propidium
iodide to counterstain all the nuclei in the section.
[0388] When injected into the lumen of murine duodenum no
detectable staining over background was observed. Poor staining
also was seen when the biotinylated peptide was injected into the
jejunum. If multiple sections were scanned detectable staining was
seen on the tips of some villi. The first sign of uniform staining
was observed when sections of the ileum were analyzed. The staining
was localized to the tips of the microvilli and did not extend into
the crypt cells. Although seen throughout all sections of the
ileum, the observed fluorescence did not approach the level of
intensity previously observed in the skin, lungs, or the
endothelial cells of arteries or veins.
[0389] The relatively poor staining of the small intestine markedly
differed from that seen when regions of the colon were examined. In
both the ascending and transverse sections of the large intestine,
biotinylated nonamers of arginine stained all surface areas of the
villi, and penetrated several cell layers, reminiscent of the
intense staining of the epidermis and dermis when applied
topically. In addition, the crypt cells were heavily stained in the
colonic sections and evidence for penetration of the full thickness
of the section was observed in several areas.
[0390] The histological analysis demonstrated that the uptake of
bio r9 into the nonkeratinized epithelia layers of the GI tract
varied significantly, with uptake increasing with distance from the
gastric pylorus. The staining observed in the ileum was greater
than that seen in the jejunum, which was greater than the duodenum.
The greatest staining was seen in the ascending and transverse
regions of the colon. Without intending to limit the invention to a
particular theory or mechanism, one theory is that the composition
and amount of mucus lining the epithelia might be an important
factor in the differential staining.
[0391] Cellular Uptake into Caco-2 Cells
[0392] When Caco-2 cells, commercially-available human colon cells,
were incubated in suspension with varying amounts of fluorescently
labeled pentamers, heptamers, and nonamers of D-arginine (F1 aca
r5, F1 aca r7, F1 aca r9) or nonamers of lysine (F1 aca k7), and
analyzed by flow cytometry, a pattern similar to that previously
seen in a variety of other suspension cells (Mitchell et al.
Peptide Research 56, 318 (2000)) was observed (FIG. 32). Uptake of
the fluorescent peptides increased with arginine content with r9
being more effective than r7, which was more effective than r5. All
the polymers of arginine entered cells more effectively than the
nonamers of lysine. Both the rate, the relative amount of
fluorescence, and the lack of apparent efflux of the internalized
fluorescent peptides over an extended period of time (several
hours) were reminiscent of earlier experiments with
lymphocytes.
[0393] To confirm that the rapid penetration of the short oligomers
of arginine into Caco-2 cells was not an artifact seen only when
the cells were in suspension, F1 aca r9 CONH.sub.2 (50 .mu.M) was
incubated for five minutes with Caco-2 cells grown as a monolayer
on a microscope slide, washed, and analyzed by fluorescent
microscopy. Consistent with the flow cytometry analysis of the
suspension cells, virtually every cell in the monolayer was
fluorescent after five minutes.
[0394] The ability of the peptides to rapidly enter Caco-2 cells
was firmly established by placing a monolayer of Caco-2 cells as a
membrane in a diffusion chamber and exposing it to fluorescent
transporter drug conjugates between taxol and oligomers of arginine
of different length. Varying concentrations of the taxol conjugates
(50-0.08 .mu.M) were added to the apical side and exposed to the
monolayer of Caco-2 cells for three minutes. The membrane was
removed from the apparatus, the cells trypsinized, and the
resulting cell suspension was analyzed by flow cytometry (FIG. 33).
As with the peptides alone, the drug conjugates quickly and
efficiently entered the cells composing the monolayer with those
containing more arginine subunits being more effective.
[0395] Without intending to limit the theory of the invention,
these experiments provide strong support for the hypothesis that
short oligomers of arginine, either conjugated to fluorescein or
therapeutic drugs, such as taxol, rapidly enter Caco-2 cells both
in suspension and when grown in monolayers.
Transport Across Caco-2 Monolayers
[0396] Crossing the luminal membrane of the gut epithelia is
necessary to increase blood levels of a delivered (e.g., buccal
administered) drug. To determine whether the transporters could
cross the gut epithelia, monolayers of Caco-2 cells were grown in
culture and placed a membrane in a commercially available diffusion
chamber. The integrity of the membranes was established by
demonstrating that the transepithelial electronic resistance (TEER)
was always greater than 100 ohm cm.sup.2 (FIG. 34). Such a pattern
of stable resistance only is observed when the membrane is intact
with no significant spaces between the cells.
[0397] Additional evidence that the membrane was both viable and
contiguous was that Lucifer Yellow (200 .mu.M) was not transported
across the monolayer, whereas hydrocortisone was transported at
amounts consistent with published reports (FIG. 35).
[0398] When a variety of fluorescent oligomers of D- or L-arginine,
ranging from four to 15 subunits, were placed in the apical chamber
in multiple experiments with a large number of membranes, none were
significantly transported into the basolateral chamber (FIG.
35).
[0399] Taken together, the data presented herein are consistent
with the model that short oligomers of arginine either conjugated
to delivered compounds such as fluorescein or taxol rapidly enter
but are not transported across Caco-2 cell monolayers. This model
also implies that the transporters of the invention do not enter
Caco-2 cells by endosomes, which are transported across colonic
epithelium and excreted on the basolateral side by a well
understood pathway. CellGate transporters appear to enter the
cytosol directly and do not have a high rate of efflux on the
basolateral side.
Measurement of Drug Blood Levels after Intracolonic Injections
[0400] Although short oligomers of arginine labeled with
fluorescein, either alone, or when conjugated to either taxol or
cyclosporin, were unable to cross a monolayer of Caco-2 cells in
vitro, the cell line may not precisely mimic the in vivo behavior
of the colon. Furthermore, the pattern of fluorescence seen in
colon tissue after incubation with short oligomers of arginine
demonstrated that the peptides penetrated into layers known to be
vascularized.
[0401] To determine whether the transporters of the invention could
enhance the delivery of orally administered drugs, releasable
conjugates of cyclosporin A and taxol were injected
intracolonically and the resulting blood levels of the released
drugs were measured by LC MS MS.
[0402] The first experiment was designed to measure blood levels of
CsA after intracolonic injection of 5 mg/kg of CsA in Cremophor
EL:ethanol compared with an equimolar amount of a releasable r7
conjugate of CsA dissolved in phosphate buffered saline. In the
case of the parent drug, blood levels rose to approximately 25
ng/ml of CsA after thirty minutes, and then rapidly fell off to
levels close to baseline levels (FIG. 36). No further data was
obtained because after 90 minutes the animal died. In contrast,
when an equimolar amount of CsA-r7 conjugate was injected,
detectable levels of CsA in the blood were observed only after 3
hours (FIG. 36). To determine whether the altered pharmacokinetics
of CsA when conjugated to a short oligomer of arginine was
reproducible, a third rat was injected with 10 mg/kg equivalent of
the water soluble, releasable conjugate.
[0403] The rate of uptake of CsA in the blood of this animal
resembled the animal injected with 5 mg/kg of the conjugate. With
more conjugate administered a small increase was seen at 30
minutes, but larger amounts appeared in the blood only after two
hours, with blood levels approaching 45 ng/ml after three hours.
The overall pattern was similar in the two animals injected with
conjugate. In both cases the overall amount of CsA measured in the
circulation was significantly greater than observed when CsA was
injected.
[0404] The half-life of the CsA conjugate was approximately ninety
minutes, which was consistent with the delay in the appearance of
CsA in the blood relative to the parent compound. This fact
combined with the histological data demonstrating rapid and
efficient uptake in the columnar epithelium of the colon and the
failure of nonreleasable conjugates of CsA or taxol to cross
monolayers of colonic cells in vitro, leads to a sensible and
simple model describing the phenomena. Without intending to limit
the invention to a particular mechanism or theory, it appears that
the conjugates enter the columnar epithelia of the colon with
greater efficiency and more rapidly than CsA injected in Cremophor,
but were retained in the epithelial cells and did not cross the
endothelial cells surrounding the capillaries until the conjugate
hydrolyzed. Once released, the CsA freely diffused, or was actively
transported, into the blood.
[0405] To test this hypothesis, taxol and two different r7-taxol
conjugates were injected into the colon and blood levels of the
drug were measured at thirty minute intervals. The two r7-taxol
conjugates had significantly different half-lives (10 minutes and 5
hours) and were used to test the hypothesis that the hydrolysis of
the drug conjugate was the rate limiting step-in appearance of the
drug in the circulation. If true, the premise predicted that the
r7-taxol conjugate with the ten minute half life would release
taxol in the epithelia so that it could be detected in the
circulation at the thirty minute time point. In contrast, taxol
should not be released from the more stable r7 conjugate
(t.sub.1/2=5 hours) and should not be detected in the blood samples
taken during the experiment.
[0406] This premise was supported by the appearance of taxol in the
blood (FIG. 37). Detectable levels of taxol did not appear in the
blood when injected in the colon until 2.5 hours with subsequent
waves at 4 and 5.5 hours. The oscillating levels of taxol in the
blood as a function of time were consistent with published studies
with the pattern being rationalized to the ability of Cremophor to
sequester some of the material and act as a timed release vehicle.
In contrast, when a labile, water soluble r7 conjugate of taxol
(13) was administered, greater than 200 ng/ml of taxol was observed
at the earliest time point (30 minutes) which continued to increase
up to 1 hour, at which point the levels slowly diminished out to
five hours. As predicted, injection of the more stable, water
soluble r7 conjugate of taxol (14) did not result in significant
blood levels of taxol within five hours. These data support the
hypothesis that transporters of the invention enter, but do not
transverse the colonic epithelium. They can improve oral
bioavailability of drugs both by dramatically improving water
solubility and by increasing the rate of uptake of the conjugate in
the colon. Ultimate delivery of the drug into the blood stream
appears to depend on the drug's inherent ability to diffuse through
biological membranes and the rate of release from the
conjugate.
Discussion
[0407] A significant discovery in the experiments described herein
is the failure of the peptides to enter the epithelia of the
duodenum. This represents the first example of the transporters not
entering a cell type or tissue. The pattern of increasing uptake
the further the tissue extended from the gastric pylorus was
intriguing, most likely due to a gradient of an inhibitor, such as
a negatively charged mucus. The failure to stain the upper regions
of the small intestine was in stark contrast with the intense
staining in the colon, leading to the speculation that transporters
of the present invention could be used for selective delivery of
therapeutics to the colon. Not only were all luminal surfaces of
the colon highly fluorescent, but the staining pattern also
revealed that the biotinylated compounds were able to penetrate
multiple cell layers and reach vascularized regions of the tissue,
suggesting that the transporters should enhance transport into the
bloodstream.
[0408] However, separate studies using monolayers of the colonic
cell line, Caco-2, suggests an alternative mechanism. In the Caco-2
system the transporters rapidly entered, but did not traverse the
monolayer. There are several examples of the transporters
exhibiting high rates of transport into, but limited rates of
efflux from cells and tissue. This is the case for all suspension
cell lines examined to date, the best studied being the human T
cell line, Jurkat. Short oligomers of D-arginine rapidly enter
these cells and exhibit very low exit rates, losing less than 5% of
the fluorescent signal after one hour of incubation at 37.degree.
C. An even more relevant example of this phenomenon is the low
levels of CsA measured in the blood stream of mice receiving
multiple topical treatments of a releasable CsA r7 conjugate. As in
the case for the colon, staining patterns in sections of skin
treated with a biotinylated analog of the CsA conjugate established
that the drug penetrated into highly vascularized regions of the
dermis. In addition, staining with monoclonal antibodies
established that one of the prominent cell types in the dermis that
were highly fluorescent were the endothelial cells of the
capillaries. Nevertheless, detectable levels of CsA in the blood of
these animals were never observed even after ten days of treatment
with a 4% ointment applied twice a day. This observation, although
anecdotal, is consistent with results better studied in this
report.
[0409] The ability of the peptides to enter and subsequently exit
multiple layers of cells in the skin, the lungs, and the
cardiovascular system is in marked contrast to their inability to
exit from lymphocytes or Caco-2 cells. Without intending to limit
the present invention to a particular theory or mechanism, one
difference is the cells through which the peptides rapidly
penetrate are connected by tight junctions and other membrane
structures inherent in tissue architecture, whereas the individual
cells in suspension and perhaps the side of the membrane of
endothelial cells contacting the bloodstream lack these features.
If structures such as tight or gap junctions modify the surrounding
lipid to permit exit of the transporters, then they should be able
to diffuse rapidly throughout a tissue, such as skin or the colon,
but not be transported into the bloodstream. Consistent with this
hypothesis is the model constructed to explain the variations in
the rates of appearance of taxol and CsA in the circulation in this
report. In this model, the short oligomers of arginine greatly
promoted uptake into columnar epithelium of the colon or the
squamous epithelium of the cheek, but did not transport the drug
into the bloodstream. The blood levels observed were the result of
the diffusion, or active transport, of the drug out of the
epithelium into the circulation after hydrolysis of the
conjugate.
Example 18
Buccal Delivery of Transporter Conjugates
[0410] Buccal delivery of taxol and CsA involved adding a
concentrated solution (250.lamda. of 5 mg/kg) to the oral cavity of
an anesthetized rat lying on its side. Blood samples were taken
from the tail vein at time zero (prior to drug injection), and
every thirty minutes for the duration of the experiment, which was
empirically determined. Clotting was inhibited by transferring the
blood to glass tubes containing 100.lamda., of 0.5% EDTA, and the
blood was frozen.
[0411] The capacity of the transporters to enter the squamous
epithelial layers of the oral cavity was examined. A mouse was
anesthetized, its head was tipped so that a solution of
biotinylated r9 could be administered. The animal was kept in this
position for fifteen minutes, at which time it was sacrificed, and
the tongue and cheek were dissected, frozen, sectioned, stained
with streptavidin-fluorescein, and counterstained with propidium
iodide. In both the cheek and the tongue the biotinylated peptides
quickly and efficiently penetrated multiple layers of the epithelia
and penetrated deep into the interior layers of the tissue,
reminiscent of the staining of both the epidermis and dermis of the
skin.
[0412] In a second experiment, rats were anesthetized and solutions
of both taxol and CsA (5 mg/kg) in Cremophor EL:ethanol 1:1 or
equimolar amounts of the corresponding r7 conjugates of these drugs
in PBS were simply incubated in the cheek pouch of the animal for
the duration of the experiment. LC MS was used to determine the
blood levels of only taxol and the fast releasing r7 conjugate.
Both the amount and the kinetics of appearance of taxol in the
blood when administered in the oral cavity (FIG. 38) differed from
when it was injected into the colon. Buccal administration of taxol
conjugates appeared to be less effective, with less than one eighth
of the amount of taxol being observed in the circulation compared
with intracolonic injection. Another difference was the rate of
appearance of the unmodified drug. When administered in the oral
cavity, taxol appeared in the blood stream by the first time point,
whereas in the colonic injection detectable amounts of taxol did
not appear for several hours. Even though there was no difference
between taxol and the r7-taxol conjugate in the appearance of taxol
in the circulation in buccal administration, approximately twice as
much material. reached the circulation when the conjugated was
used.
Example 19
Ocular Delivery of Transporter Conjugates
[0413] The ability of the transporters of the invention to
penetrate the tissues of the eye was examined. Biotinylated r8 was
both injected into the eyes of rabbits and also applied as eyedrops
to the outside of the eye.
[0414] Briefly, 5 drops of a 1 mM solution of biotinylated r8 in
PBS was applied to both eyes of a rabbit and allowed to incubate 15
minutes. The animal was sacrificed and one eye was dissected intact
with adjacent tissue, whereas the other was separated into each of
its component parts, frozen and separately sectioned, stained with
streptavidin-fluorescein and counterstained with propidium iodide.
Results demonstrated staining in the cornea and eyelid, but not the
lens.
[0415] 50 microliters of a 10 mM solution of biotinylated r8 in PBS
was injected into the vitreus humor of another animal and the
animal was sacrificed 30 minutes later and the injected eye was
dissected. Again one orbital was frozen intact while the other was
dissected and the components separately frozen. Results from the
injection experiments demonstrated that all interior surfaces of
the orb were stained.
Example 20
[0416] This example illustrates the conjugation of cyclosporin to a
transport moiety using a pH sensitive linking group (see FIGS. 6A
and 9B).
[0417] In this example, cyclosporin is converted to its
.alpha.-chloroacetate ester using chloroacetic anhydride to provide
6i (see FIG. 6). The ester 6i is then treated with benzylamine to
provide 6ii. Reaction of the amine with Boc-protected iminodiacetic
acid anhydride provides the acid 6iii which is then converted to an
activated ester (6iv) with N-hydroxy succinimide. Coupling of 6iv
with L-Arginine heptamer provides the BOC-protected conjugate 6v,
which can be converted to conjugate 6vi by removal of the BOC
protecting group according to established methods.
[0418] 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 and in the following examples that show
oligoarginine transport groups.
Example 21
[0419] This example illustrates the conjugation of acyclovir to a
transport moiety.
[0420] a. Conjugation of acyclovir to r.sub.7CONH.sub.2
[0421] The example illustrates the conjugation of acyclovir to
r.sub.7CONH.sub.2 via the linking group:
##STR00011##
i) Preparation of acyclovir .alpha.-chloroester
##STR00012##
[0423] 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.sup.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.13CNMR (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].
ii) Conjugation of acyclovir .alpha.-chloro ester to
H.sub.2N--C-r7-CONH.sub.2
##STR00013##
[0425] 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 dimethylfohnamide
(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 m91 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].
[0426] 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].
[0427] b. Conjugation of acyclovir to a Biotin-Containing
Derivative of r.sub.5-Cys-CONH.sub.2
##STR00014##
[0428] Reactions were carried out as illustrated above, using the
synthetic techniques provided in the examples above.
[0429] 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].
[0430] Similarly,
[0431] 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 22
[0432] This example illustrates the conjugation of hydrocortisone
to a transport moiety.
[0433] a. Conjugation of hydrocortisone to r7CONH.sub.2
i) Preparation of hydrocortisone .alpha.-chloroester
##STR00015##
[0435] 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 NHC1 and brine. The organic
phase was collected, dried (Na.sub.2S0.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, 3 13H); .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).
[0436] (Reference for acetylation--Zhao, H.; Pendri, A.; Greenwald,
R. B. J. Org. Chem. 1998, 63, 7559-7562).
[0437] ii) Coupling to R'NH-Cys-r.sub.7-CONH.sub.2
##STR00016##
[0438] 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.3CNH.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].
[0439] The use of 10 molar equivalents of diisopropylethylainine
rather than 1.2 molar equivalents provided the desired product as a
yellow powder (52% yield). TOF-MS (m/z): 887.0 [(M+ITFA)/2], 830.6
[(M+H)/2], 553.7 [(M+H)/3], 415.5 [(M+H)/4].
b. Conjugation of Hydrocortisone to a Biotin-Containing Derivative
of r.sub.5-CYS-CONH.sub.2
##STR00017##
[0440] Reactions were carried out as illustrated above, using the
synthetic techniques provided in the examples above.
[0441] 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].
[0442] ii) Biotin-anzinocaproic
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 23
[0443] This example illustrates the conjugation of taxol to a
transport moiety.
[0444] a. Conjugation of Taxol to r.sub.7CONH.sub.2
[0445] This example illustrates the application of methodology
outlined above to the preparation of a taxol conjugate (see FIG.
12).
[0446] i) Preparation of a taxol .alpha.-chloroacetate ester
##STR00018##
[0447] Taxol was treated with .alpha.-chloro acetic anhydride
providing the C-2' chloro acetyl derivative 12i in essentially
quantitative yield.
[0448] ii) Formation of Taxol Conjugate
##STR00019##
[0449] 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.
[0450] 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 hygioscopic and readily dissolve in water.
[0451] 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 24
[0452] 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.
[0453] a. Linkage Between a Biological Agent Having an Aldehyde
Functional Group
[0454] 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. 40 provides a
schematic presentation of the reactions. As seen in FIG. 40,
condensation of either retinal with
H.sub.2N-r.sub.9-CO.sub.2H.10TFA in MeOH in the presence of 4A
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.
[0455] b. Conjugation of Retinoic Acid to r.sub.7-CONH.sub.2
[0456] This example illustrates the preparation of a conjugate
between retinoic acid and r.sub.7-CONH.sub.2 using the linking
group
##STR00020##
[0457] Here, preparation of the conjugate follows the scheme
outlined in FIG. 41. In this scheme, retinoic acid (41ii) is first
combined with the chloroacetate ester of
4-hydroxymethyl-2,6-dimethylphenol (41i) to provide the conjugate
shown as 41iii. Combination of 41i with retinoic acid in methylene
chloride in the presence of dicyclohexylcarbodiimide and a
catalytic amount of 4-dimethylaminopyridine provided the retinoid
derivative 41iii in 52-57% yield. Condensation of 41iii 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 41iv.
[0458] It is understood that the examples and embodiments described
herein are for illustrative purposes only and that various
modifications or changes in light thereof will be suggested to
persons skilled in the art and are to be included within the spirit
and purview of this application and scope of the appended claims.
All publications, patents, and patent applications cited herein are
hereby incorporated by reference for all purposes.
Sequence CWU 1
1
5715PRTArtificial SequenceArg homopolymer R5, Arg oligomer 1Arg Arg
Arg Arg Arg1 526PRTArtificial SequenceArg homopolymer R6, Arg
oligomer 2Arg Arg Arg Arg Arg Arg1 537PRTArtificial SequenceArg
homopolymer R7, heptamer of L-Arg, hepta-L-Arg, Arg oligomer 3Arg
Arg Arg Arg Arg Arg Arg1 548PRTArtificial SequenceArg homopolymer
R8, Arg oligomer 4Arg Arg Arg Arg Arg Arg Arg Arg1 559PRTArtificial
SequenceArg homopolymer R9, Arg oligomer 5Arg Arg Arg Arg Arg Arg
Arg Arg Arg1 567PRTArtificial Sequenceheptamer of L-Arg chemical
after pH dependent chemical release 6Xaa Arg Arg Arg Arg Arg Arg1
5710PRTArtificial Sequenceunlabeled peptide 7Arg Arg Arg Arg Arg
Arg Arg Gly Gly Xaa1 5 1089PRTArtificial Sequenceanalog of HIV-1
tat protein basic region Tat-49-57 8Xaa Lys Lys Arg Arg Gln Arg Arg
Arg1 598PRTArtificial SequenceTat-49-56 truncated analog of HIV-1
tat protein basic region Tat-49-57 9Xaa Lys Lys Arg Arg Gln Arg
Arg1 5107PRTArtificial SequenceTat-49-55 truncated analog of HIV-1
tat protein basic region Tat-49-57 10Xaa Lys Lys Arg Arg Gln Arg1
5118PRTArtificial SequenceTat-50-57 truncated analog of HIV-1 tat
protein basic region Tat-49-57 11Xaa Lys Arg Arg Gln Arg Arg Arg1
5127PRTArtificial SequenceTat-51-57 truncated analog of HIV-1 tat
protein basic region Tat-49-57 12Xaa Arg Arg Gln Arg Arg Arg1
5139PRTArtificial SequenceA-49 Ala-substituted analog of HIV-1 tat
protein basic region Tat-49-57 13Xaa Lys Lys Arg Arg Gln Arg Arg
Arg 1 5149PRTArtificial SequenceA-50 Ala-substituted analog of
HIV-1 tat protein basic region Tat-49-57 14Xaa Ala Lys Arg Arg Gln
Arg Arg Arg1 5159PRTArtificial SequenceA-51 Ala-substituted analog
of HIV-1 tat protein basic region Tat-49-57 15Xaa Lys Ala Arg Arg
Gln Arg Arg Arg1 5169PRTArtificial SequenceA-52 Ala-substituted
analog of HIV-1 tat protein basic region Tat-49-57 16Xaa Lys Lys
Ala Arg Gln Arg Arg Arg1 5179PRTArtificial SequenceA-53
Ala-substituted analog of HIV-1 tat protein basic region Tat-49-57
17Xaa Lys Lys Arg Ala Gln Arg Arg Arg1 5189PRTArtificial
SequenceA-54 Ala-substituted analog of HIV-1 tat protein basic
region Tat-49-57 18Xaa Lys Lys Arg Arg Ala Arg Arg Arg1
5199PRTArtificial SequenceA-55 Ala-substituted analog of HIV-1 tat
protein basic region Tat-49-57 19Xaa Lys Lys Arg Arg Gln Ala Arg
Arg1 5209PRTArtificial SequenceA-56 Ala-substituted analog of HIV-1
tat protein basic region Tat-49-57 20Xaa Lys Lys Arg Arg Gln Arg
Ala Arg1 5219PRTArtificial SequenceA-57 Ala-substituted analog of
HIV-1 tat protein basic region Tat-49-57 21Xaa Lys Lys Arg Arg Gln
Arg Arg Ala1 5229PRTArtificial SequenceTat-57-49 retro-isomer of
HIV-1 tat protein basic region Tat-49-57 22Xaa Arg Arg Gln Arg Arg
Lys Lys Arg1 5235PRTArtificial SequenceArg oligomer R5, Arg
homopolymer 23Xaa Arg Arg Arg Arg1 5246PRTArtificial SequenceArg
oligomer R6, Arg homopolymer 24Xaa Arg Arg Arg Arg Arg1
5257PRTArtificial SequenceArg oligomer R7, Arg homopolymer 25Xaa
Arg Arg Arg Arg Arg Arg1 5268PRTArtificial SequenceArg oligomer R8,
Arg homopolymer 26Xaa Arg Arg Arg Arg Arg Arg Arg1
5279PRTArtificial SequenceArg oligomer R9, Arg homopolymer 27Xaa
Arg Arg Arg Arg Arg Arg Arg Arg1 5289PRTArtificial SequenceHIV-1
tat protein basic region Tat-49-57 28Arg Lys Lys Arg Arg Gln Arg
Arg Arg1 52916PRTArtificial SequenceAntennapedia homeodomain,
Antennapedia-43-58 29Arg Gln Ile Lys Ile Trp Phe Gln Asn Arg Arg
Met Lys Trp Lys Lys1 5 10 153016PRTArtificial SequenceAntennapedia
homeodomain, Antennapedia-43-58 30Xaa Gln Ile Lys Ile Trp Phe Gln
Asn Arg Arg Met Lys Trp Lys Lys1 5 10 15317PRTArtificial
SequenceDescription of Artificial Sequencepeptide- aminocaproic
acid-DTPA complex 31Xaa Arg Arg Arg Arg Arg Arg1 5327PRTArtificial
SequenceDescription of Artificial SequenceNH-2R-7CCONH-2.8TFA
transporter conjugate 32Arg Arg Arg Arg Arg Arg Xaa1
5337PRTArtificial Sequenceamino acid oligomer delivery enhancing
transport moiety 33Arg Xaa Arg Arg Xaa Arg Arg1 53410PRTArtificial
Sequenceamino acid oligomer delivery enhancing transport moiety
34Arg Xaa Arg Arg Xaa Arg Arg Xaa Arg Arg1 5 103513PRTArtificial
Sequenceamino acid oligomer delivery enhancing transport moiety
35Arg Xaa Arg Arg Xaa Arg Arg Xaa Arg Arg Xaa Arg Arg1 5
103616PRTArtificial Sequenceamino acid oligomer delivery enhancing
transport moiety 36Arg Xaa Arg Arg Xaa Arg Arg Xaa Arg Arg Xaa Arg
Arg Xaa Arg Arg1 5 10 15379PRTArtificial Sequenceamino acid
oligomer delivery enhancing transport moiety 37Arg Xaa Arg Xaa Arg
Xaa Arg Xaa Arg1 53811PRTArtificial Sequenceamino acid oligomer
delivery enhancing transport moiety 38Arg Xaa Arg Xaa Arg Xaa Arg
Xaa Arg Xaa Arg1 5 103913PRTArtificial Sequenceamino acid oligomer
delivery enhancing transport moiety 39Arg Xaa Arg Xaa Arg Xaa Arg
Xaa Arg Xaa Arg Xaa Arg1 5 104015PRTArtificial Sequenceamino acid
oligomer delivery enhancing transport moiety 40Arg Xaa Arg Xaa Arg
Xaa Arg Xaa Arg Xaa Arg Xaa Arg Xaa Arg1 5 10 154117PRTArtificial
Sequenceamino acid oligomer delivery enhancing transport moiety
41Arg Xaa Arg Xaa Arg Xaa Arg Xaa Arg Xaa Arg Xaa Arg Xaa Arg Xaa1
5 10 15Arg4219PRTArtificial Sequenceamino acid oligomer delivery
enhancing transport moiety 42Arg Xaa Arg Xaa Arg Xaa Arg Xaa Arg
Xaa Arg Xaa Arg Xaa Arg Xaa1 5 10 15Arg Xaa Arg4321PRTArtificial
Sequenceamino acid oligomer delivery enhancing transport moiety
43Arg Xaa Arg Xaa Arg Xaa Arg Xaa Arg Xaa Arg Xaa Arg Xaa Arg Xaa1
5 10 15Arg Xaa Arg Xaa Arg 204413PRTArtificial Sequenceamino acid
oligomer delivery enhancing transport moiety 44Arg Xaa Xaa Arg Xaa
Xaa Arg Xaa Xaa Arg Xaa Xaa Arg1 5 104516PRTArtificial
Sequenceamino acid oligomer delivery enhancing transport moiety
45Arg Xaa Xaa Arg Xaa Xaa Arg Xaa Xaa Arg Xaa Xaa Arg Xaa Xaa Arg1
5 10 154619PRTArtificial Sequenceamino acid oligomer delivery
enhancing transport moiety 46Arg Xaa Xaa Arg Xaa Xaa Arg Xaa Xaa
Arg Xaa Xaa Arg Xaa Xaa Arg1 5 10 15Xaa Xaa Arg4722PRTArtificial
Sequenceamino acid oligomer delivery enhancing transport moiety
47Arg Xaa Xaa Arg Xaa Xaa Arg Xaa Xaa Arg Xaa Xaa Arg Xaa Xaa Arg1
5 10 15Xaa Xaa Arg Xaa Xaa Arg 204825PRTArtificial Sequenceamino
acid oligomer delivery enhancing transport moiety 48Arg Xaa Xaa Arg
Xaa Xaa Arg Xaa Xaa Arg Xaa Xaa Arg Xaa Xaa Arg1 5 10 15Xaa Xaa Arg
Xaa Xaa Arg Xaa Xaa Arg 20 254928PRTArtificial Sequenceamino acid
oligomer delivery enhancing transport moiety 49Arg Xaa Xaa Arg Xaa
Xaa Arg Xaa Xaa Arg Xaa Xaa Arg Xaa Xaa Arg1 5 10 15Xaa Xaa Arg Xaa
Xaa Arg Xaa Xaa Arg Xaa Xaa Arg 20 255031PRTArtificial
Sequenceamino acid oligomer delivery enhancing transport moiety
50Arg Xaa Xaa Arg Xaa Xaa Arg Xaa Xaa Arg Xaa Xaa Arg Xaa Xaa Arg1
5 10 15Xaa Xaa Arg Xaa Xaa Arg Xaa Xaa Arg Xaa Xaa Arg Xaa Xaa Arg
20 25 305117PRTArtificial Sequenceamino acid oligomer delivery
enhancing transport moiety 51Arg Xaa Xaa Xaa Arg Xaa Xaa Xaa Arg
Xaa Xaa Xaa Arg Xaa Xaa Xaa1 5 10 15Arg5221PRTArtificial
Sequenceamino acid oligomer delivery enhancing transport moiety
52Arg Xaa Xaa Xaa Arg Xaa Xaa Xaa Arg Xaa Xaa Xaa Arg Xaa Xaa Xaa1
5 10 15Arg Xaa Xaa Xaa Arg 205325PRTArtificial Sequenceamino acid
oligomer delivery enhancing transport moiety 53Arg Xaa Xaa Xaa Arg
Xaa Xaa Xaa Arg Xaa Xaa Xaa Arg Xaa Xaa Xaa1 5 10 15Arg Xaa Xaa Xaa
Arg Xaa Xaa Xaa Arg 20 255429PRTArtificial Sequenceamino acid
oligomer delivery enhancing transport moiety 54Arg Xaa Xaa Xaa Arg
Xaa Xaa Xaa Arg Xaa Xaa Xaa Arg Xaa Xaa Xaa1 5 10 15Arg Xaa Xaa Xaa
Arg Xaa Xaa Xaa Arg Xaa Xaa Xaa Arg 20 255533PRTArtificial
Sequenceamino acid oligomer delivery enhancing transport moiety
55Arg Xaa Xaa Xaa Arg Xaa Xaa Xaa Arg Xaa Xaa Xaa Arg Xaa Xaa Xaa1
5 10 15Arg Xaa Xaa Xaa Arg Xaa Xaa Xaa Arg Xaa Xaa Xaa Arg Xaa Xaa
Xaa 20 25 30Arg5637PRTArtificial Sequenceamino acid oligomer
delivery enhancing transport moiety 56Arg Xaa Xaa Xaa Arg Xaa Xaa
Xaa Arg Xaa Xaa Xaa Arg Xaa Xaa Xaa1 5 10 15Arg Xaa Xaa Xaa Arg Xaa
Xaa Xaa Arg Xaa Xaa Xaa Arg Xaa Xaa Xaa 20 25 30Arg Xaa Xaa Xaa Arg
355741PRTArtificial Sequenceamino acid oligomer delivery enhancing
transport moiety 57Arg Xaa Xaa Xaa Arg Xaa Xaa Xaa Arg Xaa Xaa Xaa
Arg Xaa Xaa Xaa1 5 10 15Arg Xaa Xaa Xaa Arg Xaa Xaa Xaa Arg Xaa Xaa
Xaa Arg Xaa Xaa Xaa 20 25 30Arg Xaa Xaa Xaa Arg Xaa Xaa Xaa Arg 35
40
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