U.S. patent application number 10/338348 was filed with the patent office on 2003-08-28 for method and composition for enhancing transport across biological membranes.
Invention is credited to Rothbard, Jonathan B., Wender, Paul A..
Application Number | 20030162719 10/338348 |
Document ID | / |
Family ID | 21948441 |
Filed Date | 2003-08-28 |
United States Patent
Application |
20030162719 |
Kind Code |
A1 |
Rothbard, Jonathan B. ; et
al. |
August 28, 2003 |
Method and composition for enhancing transport across biological
membranes
Abstract
Methods and compositions for transporting drugs and
macromolecules across biological membranes are disclosed. In one
embodiment, the invention pertains to a method for enhancing
transport of a selected compound across a biological membrane,
wherein a biological membrane is contacted with a conjugate
containing a biologically active agent that is covalently attached
to a transport polymer. In a preferred embodiment, the polymer
consists of from 6 to 25 subunits, at least 50% of which contain a
guanidino or amidino sidechain moiety. The polymer is effective to
impart to the attached agent a rate of trans-membrane transport
across a biological membrane that is greater than the rate of
trans-membrane transport of the agent in non-conjugated form.
Inventors: |
Rothbard, Jonathan B.;
(Woodside, CA) ; Wender, Paul A.; (Menlo Park,
CA) |
Correspondence
Address: |
REED & EBERLE LLP
800 MENLO AVENUE, SUITE 210
MENLO PARK
CA
94025
US
|
Family ID: |
21948441 |
Appl. No.: |
10/338348 |
Filed: |
January 7, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10338348 |
Jan 7, 2003 |
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09396194 |
Sep 14, 1999 |
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09396194 |
Sep 14, 1999 |
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09083259 |
May 21, 1998 |
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6306993 |
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60047345 |
May 21, 1997 |
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Current U.S.
Class: |
514/44R ;
514/1.2; 514/19.3; 514/2.3 |
Current CPC
Class: |
C07B 2200/11 20130101;
A61K 31/785 20130101; A61K 48/00 20130101; A61P 43/00 20180101;
A61K 47/645 20170801; C40B 40/00 20130101; C07D 305/14 20130101;
A61K 47/62 20170801 |
Class at
Publication: |
514/14 ; 514/16;
514/17; 514/15 |
International
Class: |
A61K 038/10; A61K
038/08 |
Goverment Interests
[0002] This invention was made with the support of NIH grant number
CA 65237. Accordingly, the U.S. Government has certain rights in
this invention.
Claims
We claim:
1. A conjugate comprised of a biologically active agent covalently
attached to a polymeric carrier having a non-peptide backbone
composed of from about 6 to about 25 subunits, at least 50% of
which are substituted with a guanidino or amidino sidechain moiety,
wherein adjacent subunits are linked through a covalent linkage
selected from amido, N-substituted amido, ester, methylenecarbonyl,
methyleneimino, thioamido, phosphinate, phosphonamidate,
phosphonamidate ester, retropeptidyl, trans-alkenyl, fluoroalkenyl,
ethylene, thioether, hydroxyethylene, methyleneoxy, tetrazolyl,
retrothiamido, retromethyleneimino, sulfonamido,
methylenesulfonamido, retrosulfonamido, akylene, sulfonyl, azo, and
imino linkages, and combinations thereof, with the proviso that
when the subunits are amino acids, the covalent linkages are other
than amido linkages.
2. The conjugate of claim 1, wherein the subunits are selected from
amino acids, N-substituted amino acids, hydroxy amino acids, and
amino aldehydes.
3. The conjugate of claim 2, wherein the polymeric carrier is
composed of 7 to 15 subunits.
4. The conjugate of claim 3, wherein the subunits are N-substituted
amino acids.
5. The conjugate of claim 4, wherein the subunits are N-substituted
glycine residues.
6. The conjugate of claim 5, wherein adjacent subunits are linked
through N-substituted amide linkages, such that the polymeric
carrier is a peptoid.
7. The conjugate of claim 6, wherein the polymeric carrier is
composed of 7 to 15 N-substituted glycine residues.
8. The method of claim 3, wherein the subunits are amino acids.
9. The conjugate of claim 8, wherein the polymeric carrier is
composed of 7 to 15 amino acids.
10. The conjugate of claim 8, wherein the amino acids are arginine
residues.
11. The conjugate of claim 10, wherein at least one of the arginine
residues has a D-configuration.
12. The conjugate of claim 11, wherein all of the arginine residues
have a D-configuration.
13. The conjugate of claim 10, wherein the polymeric carrier is
composed of 7 to 15 arginine residues.
14. The conjugate of claim 1, wherein the carrier is effective to
increase the rate at which the conjugated biologically active agent
is transported through a biological membrane relative to the rate
at which the biologically active agent can be transported through
the biological membrane in unconjugated form.
15. The conjugate of claim 1, wherein the carrier is effective to
increase the amount of conjugated biologically active agent that is
transported through a biological membrane relative to the amount of
biologically active agent that can be transported through the
biological membrane in unconjugated form.
16. The conjugate of claim 1, wherein the carrier is effective to
increase the rate at which the conjugate is transported through a
biological membrane relative to the rate at which the biologically
active agent conjugated to a basic HIV tat peptide consisting of
residues 49-57 can be transported through the biological
membrane.
17. The conjugate of claim 1, wherein at least 70% of the subunits
contain a guanidino or amidino sidechain moiety.
18. The conjugate of claim 17, wherein at least 90% of the subunits
contain a guanidino or amidino sidechain moiety.
19. The conjugate of claim 18, wherein all of the subunits contain
a guanidino or amidino sidechain moiety.
20. The conjugate of claim 1, wherein the polymeric carrier
includes at least 6 contiguous subunits substituted with guanidino
sidechain moieties.
21. The conjugate of claim 1, wherein the guanidino sidechain
moieties have the structure 1and the amidino sidechain moieties
have the structure 2wherein n is 2, 3, 4 or 5.
22. The conjugate of claim 21, wherein n is 3.
23. The conjugate of claim 1, wherein the biologically active agent
is covalently attached to the polymeric carrier through a
linker.
24. The conjugate of claim 23, the linker contains a linkage that
is chemically or enzymatically cleaved in vivo.
25. The conjugate of claim 24, wherein the linkage is a carbamate,
ester, thioether, disulfide or hydrazone linkage.
26. The conjugate of claim 1, wherein the polymeric carrier is
attached at least one terminus to a flanking moiety that does not
significantly affect the delivery of the biologically active agent
across biological membranes.
27. The conjugate of claim 24, wherein the polymeric carrier is
attached at least one terminus to a flanking moiety that does not
significantly affect the delivery of the biologically active agent
across biological membranes.
28. The conjugate of claim 27, wherein the polymeric carrier has a
first terminus and a second terminus, with the first terminus
attached to a first flanking moiety and the second terminus
conjugated to the biologically active agent through the linker.
29. The conjugate of claim 28, wherein a second flanking moiety is
attached to the second terminus, such that the linker is attached
to the second flanking moiety.
30. The conjugate of claim 28, wherein the first flanking moiety
comprises one or more subunits that do not contain said guanidino
sidechain or said amidino sidechain.
31. The conjugate of claim 29, wherein the first flanking moiety
and the second flanking moiety comprise one or more subunits that
do not contain said guanidino sidechain or said amidino
sidechain.
32. The conjugate of claim 28, wherein the first flanking moiety is
a blocking group effective to prevent ubiquitination in vivo.
33. The conjugate of claim 32, wherein the blocking group is an
acetyl or benzyl group.
34. The conjugate of claim 1, wherein the biologically active agent
is a therapeutic compound whose efficacy in non-conjugated form is
limited by its solubility in aqueous liquid or its inability to
cross biological membranes to manifest biological activity.
35. The conjugate of claim 1, wherein the biologically active agent
is an antimicrobial agent.
36. The conjugate of claim 1, wherein the biologically active agent
is an anticancer agent.
37. The conjugate of claim 1, wherein the biologically active agent
is comprised of a metal.
38. The conjugate of claim 1, wherein the biologically active agent
is a macromolecule.
39. The conjugate of claim 38, wherein the macromolecule is
selected from the group consisting of nucleic acids,
oligonucleotides, polynucleotides, peptides, proteins, peptide
nucleic acids, and polysaccharides.
40. The conjugate of claim 39, wherein the macromolecule is a
protein.
41. The conjugate of claim 40, wherein the protein is an enzyme, an
antigen, an antibody, or an antibody fragment.
42. A pharmaceutical composition comprising the conjugate of claim
1 and a pharmaceutically acceptable carrier.
43. A pharmaceutical composition for administration, to a human
subject, of a biologically active agent whose efficacy in
non-conjugated form is limited by its aqueous solubility, said
composition comprising the conjugate of claim 34 and a
pharmaceutically acceptable carrier.
44. A method for enhancing transport of a selected biologically
active across a biological membrane, comprising contacting a
biological membrane with a conjugate comprised of the biologically
active agent and a polymeric carrier covalently attached thereto,
wherein the polymeric carrier has a non-peptide backbone composed
of from about 6 to about 25 subunits, at least 50% of which are
substituted with a guanidino or amidino sidechain moiety, wherein
adjacent subunits are linked through a covalent linkage selected
from amido, N-substituted amido, ester, methylenecarbonyl,
methyleneimino, thioamido, phosphinate, phosphonamidate,
phosphonamidate ester, retropeptidyl, trans-alkenyl, fluoroalkenyl,
ethylene, thioether, hydroxyethylene, methyleneoxy, tetrazolyl,
retrothiamido, retromethyleneimino, sulfonamido,
methylenesulfonamido, retrosulfonamido, alkylene, sulfonyl, azo,
and imino linkages, and combinations thereof, with the proviso that
when the subunits are amino acids, the covalent linkages are other
than amido linkages.
45. The method of claim 44, wherein the subunits are selected from
amino acids, N-substituted amino acids, hydroxy amino acids, and
amino aldehydes.
46. The method of claim 45, wherein the biological membrane is a
eukaryotic cell membrane.
47. The method of claim 45, wherein the biological membrane is a
prokaryotic cell membrane.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. Ser. No.
09/396,194, filed Sep. 14, 1999, which is a divisional of U.S. Ser.
No. 09/083,259, filed May 21, 1998, now U.S. Pat. No. 6,306,993,
which claims priority to U.S. provisional application Ser. No.
60/047,345, filed May 21, 1997. The aforementioned patent and
applications are incorporated herein by reference.
FIELD OF THE INVENTION
[0003] The present invention is directed to methods and
compositions that are effective to enhance transport of
biologically active agents, such as organic compounds,
polypeptides, oligosaccharides, nucleic acids, and metal ions,
across biological membranes.
BACKGROUND OF THE INVENTION
[0004] The plasma membranes of cells present a barrier to passage
of many useful therapeutic agents. In general, a drug must be
freely soluble in both the aqueous compartments of the body and the
lipid layers through which it must pass, in order to enter cells.
Highly charged molecules in particular experience difficulty in
passing across membranes. Many therapeutic macromolecules such as
peptides and oligonucleotides are also particularly intractable to
transmembrane transport. Thus, while biotechnology has made
available a greater number of potentially valuable therapeutics,
bioavailability considerations often hinder their medicinal
utility. There is therefore a need for reliable means of
transporting drugs, and particularly macromolecules, into
cells.
[0005] Heretofore, a number of transporter molecules have been
proposed to escort molecules across biological membranes. Ryser et
al. (PCT Pub. No. WO 79/00515) teaches the use of high molecular
weight polymers of lysine for increasing transport of various
molecules across cellular membranes, with very high molecular
weights being preferred. Although the authors contemplated polymers
of other positively charged residues such as ornithine and
arginine, operativity of such polymers was not shown.
[0006] Frankel et al. (PCT Pub. No. WO 91/09958) reported that
conjugating selected molecules to the tat protein of HIV can
increase cellular uptake of those molecules. However, use of the
tat protein has certain disadvantages, including unfavorable
aggregation and insolubility properties.
[0007] Barsoum et al. (PCT Pub. No. WO 94/04686) and Fawell et al.
(1994) (Proc. Natl. Acad. Sci. USA 91:664-668) proposed using
shorter fragments of the tat protein containing the tat basic
region (residues 49-57 having the sequence RKKRRQRRR (SEQ ID NO:
1). Barsoum et al. noted that moderately long polyarginine polymers
(MW 5000-15000 daltons) failed to enable transport of
.beta.-galactosidase across cell membranes (e.g., Barsoum on page
3), contrary to the suggestion of Ryser et al., supra.
[0008] Other studies have shown that a 16 amino acid
peptide-cholesterol conjugate derived from the Antennapedia
homeodomain is rapidly internalized by cultured neurons (Brugidou
et al. (1995) Biochem. Biophys. Res. Comm. 214(2):685-93). However,
slightly shorter versions of this peptide (15 residues) are not
effectively taken up by cells (Derossi et al. J. Biol. Chem.
269:10444-50).
[0009] The present invention is based in part on the applicants'
discovery that conjugation of certain polymers composed of
contiguous, highly basic subunits, particularly subunits containing
guanidyl or amidinyl moieties, to small molecules or macromolecules
is effective to significantly enhance transport of the attached
molecule across biological membranes. Moreover, transport occurs at
a rate significantly greater than the transport rate provided by a
basic HIV tat peptide consisting of residues 49-57 (SEQ ID NO:
1).
SUMMARY OF THE INVENTION
[0010] The present invention includes, in one aspect, a method for
enhancing transport of a selected compound across a biological
membrane. In the method, a biological membrane is contacted with a
conjugate containing a biologically active agent that is covalently
attached to at least one transport polymer. The conjugate is
effective to promote transport of the agent across the biological
membrane at a rate that is greater than the trans-membrane
transport rate of the biological agent in non-conjugated form.
[0011] In one embodiment, the polymer consists of from 6 to 25
subunits, at least 50% of which contain a guanidino or amidino
sidechain moiety, wherein the polymer contains at least 6, and more
preferably, at least 7 guanidino or amidino sidechain moieties. In
another embodiment, the polymer consists of from 6 to 20, 7 to 20,
or 7 to 15 subunits. More preferably, at least 70% of the subunits
in the polymer contain a guanidino or amidino sidechain moiety, and
more preferably still, 90%. Preferably, no guanidino or amidino
sidechain moiety is separated from another such moiety by more than
one non-guanidino or non-amidino subunit. In a more specific
embodiment, the polymer contains at least 6 contiguous subunits
each containing either a guanidino or amidino group, and preferably
at least 6 or 7 contiguous guanidino sidechain moieties.
[0012] In another embodiment, the transport polymer contains from 6
to 25 contiguous subunits, from 7 to 25, from 6 to 20, or
preferably from 7 to 20 contiguous subunits, each of which contains
a guanidino or amidino sidechain moiety, and with the optional
proviso that one of the contiguous subunits can contain a
non-arginine residue to which the agent is attached.
[0013] In one embodiment, each contiguous subunit contains a
guanidino moiety, as exemplified by a polymer containing at least
six contiguous arginine residues.
[0014] Preferably, each transport polymer is linear. In a preferred
embodiment, the agent is attached to a terminal end of the
transport polymer.
[0015] In another specific embodiment, the conjugate contains a
single transport polymer.
[0016] The transport-enhancing polymers are exemplified, in a
preferred embodiment, by peptides in which arginine residues
constitute the subunits. Such a polyarginine peptide may be
composed of either all D-, all L- or mixed D- and L-arginines, and
may include additional amino acids. More preferably, at least one,
and preferably all of the subunits are D-arginine residues, to
enhance biological stability of the polymer during transit of the
conjugate to its biological target.
[0017] The method may be used to enhance transport of selected
therapeutic agents across any of a number of biological membranes
including, but not limited to, eukaryotic cell membranes,
prokaryotic cell membranes, and cell walls. Exemplary prokaryotic
cell membranes include bacterial membranes. Exemplary eukaryotic
cell membranes of interest include, but are not limited to
membranes of dendritic cells, epithelial cells, endothelial cells,
keratinocytes, muscle cells, fungal cells, bacterial cells, plant
cells, and the like.
[0018] According to a preferred embodiment of the invention, the
transport polymer of the invention has an apparent affinity
(K.sub.m) that is at least 10-fold greater, and preferably at least
100-fold greater, than the affinity measured for tat (49-75)
peptide by the procedure of Example 6 when measured at room
temperature (23.degree. C.) or 37.degree. C.
[0019] Biologically active agents (which encompass therapeutic
agents) include, but are not limited to: metal ions, which are
typically delivered as metal chelates; small organic molecules,
such as anticancer (e.g., taxane) and antimicrobial molecules
(e.g., against bacteria or fungi such as yeast); and macromolecules
such as nucleic acids, peptides, proteins, and analogs thereof. In
one preferred embodiment, the agent is a nucleic acid or nucleic
acid analog, such as a ribozyme that optionally contains one or
more 2'-deoxy nucleotide subunits for enhanced stability.
Alternatively, the agent is a peptide nucleic acid (PNA). In
another preferred embodiment, the agent is a polypeptide, such as a
protein antigen, and the biological membrane is a cell membrane of
an antigen-presenting cell (APC). In another embodiment, the agent
is selected to promote or elicit an immune response against a
selected tumor antigen. In another preferred embodiment, the agent
is a taxane or taxoid anticancer compound. In another embodiment,
the agent is a non-polypeptide agent, preferably a non-polypeptide
therapeutic agent. In a more general embodiment, the agent
preferably has a molecular weight less than 10 kDa.
[0020] The agent may be linked to the polymer by a linking moiety,
which may impart conformational flexibility within the conjugate
and facilitate interactions between the agent and its biological
target. In one embodiment, the linking moiety is a cleavable
linker, e.g., containing a linker group that is cleavable by an
enzyme or by solvent-mediated cleavage, such as an ester, amide, or
disulfide group. In another embodiment, the cleavable linker
contains a photocleavable group.
[0021] In a more specific embodiment, the cleavable linker contains
a first cleavable group that is distal to the biologically active
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 polymer.
[0022] In another embodiment, the invention can be used to screen a
plurality of conjugates for a selected biological activity, wherein
the conjugates are formed from a plurality of candidate agents. The
conjugates are contacted with a cell that exhibits a detectable
signal upon uptake of the conjugate into the cell, such that the
magnitude of the signal is indicative of the efficacy of the
conjugate with respect to the selected biological activity. This
method is particularly useful for testing the activities of agents
that by themselves are unable, or poorly able, to enter cells to
manifest biological activity. In one embodiment, the candidate
agents are selected from a combinatorial library.
[0023] The invention also includes a conjugate library that is
useful for screening in the above method.
[0024] In another aspect, the invention includes a pharmaceutical
composition for delivering a biologically active agent across a
biological membrane. The composition comprises a conjugate
containing a biologically active agent covalently attached to at
least one transport polymer as described above, and a
pharmaceutically acceptable excipient. The polymer is effective to
impart to the agent a rate of trans-membrane transport that is
greater than the trans-membrane transport rate of the agent in
non-conjugated form. The composition may additionally be packaged
with instructions for using it.
[0025] In another aspect, the invention includes a therapeutic
method for treating a mammalian subject, particularly a human
subject, with a pharmaceutical composition as above.
[0026] These and other objects and features of the invention will
become more fully apparent when the following detailed description
of the invention is read in conjunction with the accompanying
drawings.
BRIEF DESCRIPTION OF THE DRAWINGS
[0027] FIGS. 1A and 1B are plots of cellular uptake of certain
polypeptide-fluorescein conjugates containing tat basic peptide
(49-57, SEQ ID NO: 1), poly-Lys (K9, SEQ ID NO:2), and poly-Arg
(R4-R9 and r4-r9, SEQ ID NO:3-8 and 12-17, respectively), as a
function of peptide concentration; FIG. 1C is a histogram of uptake
levels of the conjugates measured for conjugates at a concentration
of 12.5 .mu.M (Examples 2-3);
[0028] FIGS. 2A-2F show computer-generated images of confocal
micrographs (Example 4) showing emitted fluorescence (2A-2C) and
transmitted light (2D-2F) from Jurkat cells after incubation at
37.degree. C. for 10 minutes with 6.25 .mu.M of tat(49-57)
conjugated to fluorescein (panels) A and D), a 7-mer of
poly-L-arginine (R7) labeled with fluorescein (panels B and E), or
a 7-mer of poly-D-arginine (r7) labeled with fluorescein (panels C
and F);
[0029] FIG. 3 shows cellular uptake of certain poly-Arg-fluorescein
conjugates (r9, R9, R15, R20, and R25, SEQ ID NO: 17 and 8-11,
respectively) as a function of conjugate concentration (Example
5);
[0030] FIG. 4 shows a histogram of cellular uptake of
fluorescein-conjugated tat(49-57), and poly-Arg-fluorescein
conjugates (R9, R8, and R7, respectively) in the absence (four bars
on left) and presence (four bars on right) of 0.5% sodium azide
(Example 7);
[0031] FIGS. 5A-5C show plots of uptake levels of selected polymer
conjugates (K9, R9, r4, r5, r6, r7, r8 and r9) by bacterial cells
as a function of conjugate concentration; FIG. 5A compares uptake
levels observed for R9 and r9 conjugates as a function of conjugate
concentration, when incubated with E. coli HB 101 cells; FIG. 5B
shows uptake levels observed for K9 and r4 to r9 conjugates when
incubated with E. coli HB 101 cells; FIG. 5C compares uptake levels
of conjugates of r9 and K9 when incubated with Strep. Bovis
cells;
[0032] FIGS. 6A-6E show exemplary conjugates of the invention which
contain cleavable linker moieties; FIGS. 6F and 6G show chemical
structures and conventional numbering of constituent backbone atoms
for paclitaxel and "TAXOTERE"; FIG. 6H shows a general chemical
structure and ring atom numbering for taxoid compounds; and
[0033] FIG. 7 shows inhibition of secretion of gamma-interferon
(.gamma.-IFN) by murine T cells as a function of concentration of a
sense-PNA-r7 conjugate (SEQ ID NO: 6 conjugated to SEQ ID NO: 18),
antisense PNA-r7 conjugate (SEQ ID NO: 6 conjugated to SEQ ID NO:
19), and non-conjugated antisense PNA (SEQ ID NO: 19), where the
PNA sequences are based on a sequence from the gene for
gamma-interferon.
DETAILED DESCRIPTION OF THE INVENTION
[0034] I. Definitions
[0035] The term "biological membrane" as used herein refers to a
lipid-containing barrier that separates cells or groups of cells
from the extracellular space. Biological membranes include, but are
not limited to, plasma membranes, cell walls, intracellular
organelle membranes, such as the mitochondrial membrane, nuclear
membranes, and the like.
[0036] The term "transmembrane concentration" refers to the
concentration of a compound present on the side of a membrane that
is opposite or "trans" to the side of the membrane to which a
particular composition has been added. For example, when a compound
is added to the extracellular fluid of a cell, the amount of the
compound measured subsequently inside the cell is the transmembrane
concentration of the compound.
[0037] "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.
[0038] 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.
[0039] "Small organic molecule" refers to a carbon-containing agent
having a molecular weight (MW) of less than or equal to 1000
daltons.
[0040] 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.
[0041] The terms "non-polypeptide agent" and "non-polypeptide
therapeutic agent" refer to the portion of a transport polymer
conjugate that does not include the transport-enhancing polymer,
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 across biological
membranes.
[0042] 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 that can be composed of
identical or non-identical amino acid subunits that are joined by
peptide linkages.
[0043] 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.
[0044] 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.
[0045] "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 peptides and proteins, regardless
of whether the polypeptide has a well-defined conformation.
[0046] The terms "guanidyl", "guanidinyl", and "guanidino" 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 (guanidino) moiety, and is also referred to as
2-amino-5-guanidinovaleric acid or
.alpha.-amino-.delta.-guanidinovaleric acid. "Guanidinium" refers
to the positively charged conjugate acid form.
[0047] "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.
[0048] The term "poly-arginine" or "poly-Arg" refers to a polymeric
sequence composed of contiguous arginine residues; poly-L-arginine
refers to all L-arginines; poly-D-arginine refers to all
D-arginines. Poly-L-arginine is also abbreviated by an upper case
"R" followed by the number of L-arginines in the peptide (e.g., R8
represents an 8-mer of contiguous L-arginine residues);
poly-D-arginine is abbreviated by a lower case "r" followed by the
number of D-arginines in the peptide (r8 represents an 8-mer of
contiguous D-arginine residues).
[0049] Amino acid residues are referred to herein by their full
names or by standard single-letter or three-letter notations: A,
Ala, alanine; C, Cys, cysteine; D, Asp, aspartic acid; E, Glu,
glutamic acid; F, Phe, phenylalanine; G, Gly, glycine; H, His,
histidine; I, Ile, isoleucine; K, Lys, lysine; L, Leu, leucine; M,
Met, methionine; N, Asn, asparagine; P, Pro, proline; Q, Gln,
glutamine; R, Arg, arginine; S, Ser, serine; T, Thr, threonine; V,
Val, valine; W, Trp, tryptophan; X, Hyp, hydroxyproline; Y, Tyr,
tyrosine.
[0050] II. Structure of Polymer Moiety
[0051] In one embodiment, transport polymers in accordance with the
present invention contain short-length polymers of from 6 to up to
25 subunits, as described above. The conjugate is effective to
enhance the transport rate of the conjugate across the biological
membrane relative to the transport rate of the non-conjugated
biological agent alone. Although exemplified polymer compositions
are peptides, the polymers may contain non-peptide backbones and/or
subunits as discussed further below.
[0052] In an important aspect of the invention, the conjugates of
the invention are particularly useful for transporting biologically
active agents across cell or organelle membranes, when the agents
are of the type that require trans-membrane transport to exhibit
their biological effects, and that do not exhibit their biological
effects primarily by binding to a surface receptor, i.e., such that
entry of the agent does not occur. Further, the conjugates are
particularly useful for transporting biologically active agents of
the type that require trans-membrane transport to exhibit their
biological effects, and that by themselves (without conjugation to
a transport polymer or some other modification), are unable, or
only poorly able, to enter cells to manifest biological
activity.
[0053] As a general matter, the transport polymer used in the
conjugate preferably includes a linear backbone of subunits. The
backbone will usually comprise heteroatoms selected from carbon,
nitrogen, oxygen, sulfur, and phosphorus, with the majority of
backbone chain atoms usually consisting of carbon. Each subunit
contains a sidechain moiety that includes a terminal guanidino or
amidino group.
[0054] Although the spacing between adjacent sidechain moieties
will usually be consistent from subunit to subunit, the polymers
used in the invention can also include variable spacing between
sidechain moieties along the backbone.
[0055] The sidechain moieties extend away from the backbone such
that the central guanidino or amidino carbon atom (to which the
NH.sub.2 groups are attached) is linked to the backbone by a
sidechain linker that preferably contains at least 2 linker chain
atoms, more preferably from 2 to 5 chain atoms, such that the
central carbon atom is the third to sixth chain atom away from the
backbone. The chain atoms are preferably provided as methylene
carbon atoms, although one or more other atoms such as oxygen,
sulfur, or nitrogen can also be present. Preferably, the sidechain
linker between the backbone and the central carbon atom of the
guanidino or amidino group is 4 chain atoms long, as exemplified by
an arginine side chain.
[0056] The transport polymer sequence of the invention can be
flanked by one or more non-guanidino/non-amidino subunits, or a
linker such as an aminocaproic acid group, which do not
significantly affect the rate of membrane transport of the
corresponding polymer-containing conjugate, such as glycine,
alanine, and cysteine, for example. 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.
[0057] The agent to be transported can be linked to the transport
polymer according to a number of embodiments. In one preferred
embodiment, the agent is linked to a single transport polymer,
either via linkage to a terminal end of the transport polymer or to
an internal subunit within the polymer via a suitable linking
group.
[0058] In a second embodiment, the agent is attached to more than
one polymer, in the same manner as above. This embodiment is
somewhat less preferred, since it can lead to crosslinking of
adjacent cells.
[0059] In a third embodiment, the conjugate contains two agent
moieties attached to each terminal end of the polymer. For this
embodiment, it is preferred that the agent has a molecular weight
of less than 10 kDa.
[0060] 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.
[0061] 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.
[0062] According to an important aspect of the present invention,
it has been found by the applicants that attachment of a single
transport polymer to any of a variety of types of biologically
active agents is sufficient to substantially enhance the rate of
uptake of an agent across biological membranes, even without
requiring the presence of a large hydrophobic moiety in the
conjugate. In fact, attaching a large hydrophobic moiety may
significantly impede or prevent cross-membrane transport due to
adhesion of the hydrophobic moiety to the lipid bilayer.
Accordingly, the present invention includes conjugates that do not
contain large hydrophobic moieties, such as lipid and fatty acid
molecules. In another embodiment, the method is used to treat a
non-central nervous system (non-CNS) condition in a subject that
does not require delivery through the blood brain barrier.
[0063] A. Polymer Components
[0064] Amino acids. In one embodiment, the transport polymer is
composed of D or L amino acid residues. Use of naturally occurring
L-amino acid residues in the transport polymers has the advantage
that breakdown products should be relatively non-toxic to the cell
or organism. Preferred amino acid subunits are arginine
(.alpha.-amino-.delta.-guanidi- novaieric acid) and
.alpha.-amino-.epsilon.-amidinohexanoic acid (isosteric amidino
analog). The guanidinium group in arginine has a pKa of about
12.5.
[0065] More generally, it is preferred that each polymer subunit
contains a highly basic sidechain moiety which (i) has a pKa of
greater than 11, more preferably 12.5 or greater, and (ii)
contains, in its protonated state, at least two geminal amino
groups (N12) which share a resonance-stabilized positive charge,
which gives the moiety a bidentate character.
[0066] Other amino acids, such as
.alpha.-amino-.beta.-guanidino-propionic acid,
.alpha.-amino-.gamma.-guanidinobutyric acid, or
.alpha.-amino-.epsilon.-guanidinocaproic acid can also be used
(containing 2, 3 or 5 linker atoms, respectively, between the
backbone chain and the central guanidinium carbon).
[0067] D-amino acids may also be used in the transport polymers.
Compositions containing exclusively D-amino acids have the
advantage of decreased enzymatic degradation. However, they may
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.
[0068] Other Subunits. Subunits other than amino acids may also be
selected for use in forming transport polymers. Such subunits may
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.
[0069] B. Backbone Type
[0070] A variety of backbone types can be used to order and
position the sidechain guanidino and/or amidino moieties, such as
alkylene backbone moieties joined by thioethers or sulfonyl groups,
hydroxy acid esters (equivalent to replacing amido linkages with
ester linkages), peptidyl linkages in which the alpha carbon is
replaced with nitrogen to form an azo linkage, alkylene backbone
moieties joined by carbamate groups, polyethyleneimines (PEIs), and
amino aldehydes, which result in polymers composed of secondary
amines.
[0071] A more detailed backbone list includes N-substituted amido
(CONR replaces CONH linkages), esters (CO.sub.2), methylenecarbonyl
(COCH.sub.2) methyleneimino (CH.sub.2NH), thioamido (CSNH),
phosphinate (PO.sub.2RCH.sub.2), phosphonamidate and
phosphonamidate ester (PO.sub.2RNH), retropeptidyl (NHCO),
trans-alkenyl (CR.dbd.CH), fluoroalkenyl (CF.dbd.CH), ethylene
(CH.sub.2CH.sub.2), thioether (CH.sub.2S), hydroxyethylene
(CH(OH)CH.sub.2), methyleneoxy (CH.sub.2O), tetrazolyl (CN.sub.4),
retrothioamido (NHCS), retromethyleneimino (NHCH.sub.2),
sulfonamido (SO.sub.2NH), methylenesulfonamido (CHRSO.sub.2NH),
retrosulfonamido (NHSO.sub.2), and backbones with malonate and/or
gem-diaminoalkyl subunits, for example, as reviewed by Fletcher et
al. (1998) and detailed by references cited therein (Fletcher et
al. (1998) Chem. Rev. 98:763). Peptoid backbones (N-substituted
glycines) can also be used (e.g., Kessler et al. (1993) Angew.
Chem. Int. Ed. Engl. 32:543; Zuckermann et al. (1992)
Chemtracts-Macroinol. Chem. 4:80; and Simon et al. (1992) Proc.
Natl. Acad. Sci. 89:9367). Many of the foregoing substitutions
result in approximately isosteric polymer backbones relative to
backbones formed from a-amino acids.
[0072] Studies carried out in support of the present invention have
utilized polypeptides (e.g., peptide backbones). However, other
backbones, such as those described above, may provide enhanced
biological stability (for example, resistance to enzymatic
degradation in vivo).
[0073] C. Synthesis of Polymeric Transport Molecules
[0074] Polymers are constructed by any method known in the art.
Exemplary peptide polymers can be produced synthetically,
preferably using a peptide synthesizer (Applied Biosystems Model
433) or can be synthesized recombinantly by methods well known in
the art. Recombinant synthesis is generally used when the transport
polymer is a peptide that is fused to a polypeptide or protein of
interest.
[0075] N-methyl and hydroxy-amino acids can be substituted for
conventional amino acids in solid phase peptide synthesis. However,
production of polymers 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), supra, Simon et al. (1992), supra, and
references cited therein.
[0076] III. Attachment of Transport Polymers To Biologically Active
Agents
[0077] Transport polymers of the invention can be attached
covalently to biologically active agents by chemical or recombinant
methods.
[0078] A. Chemical Linkages
[0079] Biologically active agents such as small organic molecules
and macromolecules can be linked to transport polymers of the
invention via a number of methods known in the art (see, for
example, Wong, 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.
[0080] Various functional groups (hydroxyl, amino, halogen, etc.)
can be used to attach the biologically active agent to the
transport polymer. Groups that 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.
[0081] Polymers, such as peptides produced according to Example 1,
are generally produced with an amino terminal protecting group,
such as FMOC. For biologically active agents that 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.
[0082] 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-NO2, and the
like.
[0083] Coupling reactions are performed by known coupling methods
in any of an array of solvents, such as N,N-dimethyl formamide
(DMF), N-methyl pyrrolidinone, dichloromethane, water, and the
like. Exemplary coupling reagents include 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.
[0084] For biologically active agents that are inactive until the
attached transport polymer is released, the linker is preferably a
readily cleavable linker, meaning that it is susceptible to
enzymatic or solvent-mediated cleavage in vivo. For this purpose,
linkers containing carboxylic acid esters and disulfide bonds are
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.
[0085] In one preferred embodiment, 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 polymer. This embodiment is
further illustrated by the various small molecule conjugates
discussed below.
[0086] B. Fusion Polypeptides
[0087] Transport peptide polymers 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 transport peptide, according to methods well
known in the art. Generally, the transport peptide component will
be attached at the C-terminus or N-terminus of the polypeptide of
interest, optionally via a short peptide linker.
[0088] IV. Enhanced Transport of Biologically Active Agents Across
Biological Membranes
[0089] A. Measuring Transport Across Biological Membranes
[0090] Model systems for assessing the ability of polymers of the
invention to transport biomolecules and other therapeutic
substances across biological membranes include systems that measure
the ability of the polymer to transport a covalently attached
fluorescent molecule across the membrane. For example, fluorescein
(.about.376 MW) can serve as a model for transport of small organic
molecules (Example 2). For transport of macromolecules, a transport
polymer can be fused to a large polypeptide such as ovalbumin
(molecular weight 45 kDa; e.g., Example 14). Detecting uptake of
macromolecules may be facilitated by attaching a fluorescent tag.
Cellular uptake can also be analyzed by confocal microscopy
(Example 4).
[0091] B. Enhanced Transport Across Biological Membranes
[0092] In experiments carried out in support of the present
invention, transmembrane transport and concomitant cellular uptake
was assessed by uptake of a transport peptide linked to
fluorescein, according to methods described in Examples 2 and 3.
Briefly, suspensions of cells were incubated with fluorescent
conjugates suspended in buffer for varying times at 37.degree. C.,
23.degree. C., or 3.degree. C. After incubation, the reaction was
stopped and the cells were collected by centrifugation and analyzed
for fluorescence using fluorescence-activated cell sorting
(FACS).
[0093] Under the conditions used, cellular uptake of the conjugates
was not saturable. Consequently, ED.sub.50 values could not be
calculated for the peptides. Instead, data are presented as
histograms to allow direct comparisons of cellular uptake at single
conjugate concentrations.
[0094] FIGS. 1A-1C show results from a study in which polymers of
L-arginine (R; FIG. 1A) or D-arginine (r; FIG. 1B) ranging in
length from 4 to 9 arginine subunits were tested for ability to
transport fluorescein into Jurkat cells. For comparison, transport
levels for an HIV tat residues 49-57 ("49-57") and a nonamer of
L-lysine (K9) were also tested. FIG. 1C shows a histogram of uptake
levels for the conjugates at a concentration of 12.5 .mu.M.
[0095] As shown in the figures, fluorescently labeled peptide
polymers composed of 6 or more arginine residues entered cells more
efficiently than the tat sequence 49-57. In particular, uptake was
enhanced to at least about twice the uptake level of tat 49-57, and
as much as about 6-7 times the uptake level of tat 49-57. Uptake of
fluorescein alone was negligible. Also, the lysine nonamer (K9)
showed very little uptake, indicating that short lysine polymers
are ineffective as trans-membrane transports, in contrast to
comparable-length guanidinium-containing polymers.
[0096] With reference to FIG. 1B, homopolymers of D-arginine
exhibited even greater transport activity than the L-counterparts.
However, the order of uptake levels was about the same. For the
D-homopolymers, the peptides with 7 to 9 arginines exhibited
roughly equal activity. The hexamer (R6 or r6) was somewhat less
effective, but still exhibited at least about 2 to 3-fold higher
transport activity than tat(49-57).
[0097] The ability of the D- and L-arginine polymers to enhance
trans-membrane transport was confirmed by confocal microscopy
(FIGS. 2A-2F and Example 4). Consistent with the FACS data
described above, the cytosol of cells incubated with either R9
(FIGS. 2B and 2E) or r9 (FIGS. 2C and 2F) was brightly fluorescent,
indicating high levels of conjugate transport into the cells. In
contrast, tat(49-57) at the same concentration showed only weak
straining (FIGS. 2A and 2D). The confocal micrographs also
emphasize the point that the D-arginine polymer (FIG. 2C) was more
effective at entering cells than the polymer composed of L-arginine
(FIG. 2F).
[0098] From the foregoing, it is apparent that transport polymers
of the invention are significantly more effective than HIV tat
peptide 47-59 in transporting drugs across the plasma membranes of
cells. Moreover, the poly-Lys nonamer was ineffective as a
transporter.
[0099] To determine whether there was an optimal length for
contiguous guanidinium-containing homopolymers, a set of longer
arginine homopolymer conjugates (R15, R20, R25, and R30) were
examined. To examine the effect of substantially longer polymers, a
mixture of L-arginine polymers with an average molecular weight of
.about.12,000 daltons (.about.100 amino acids) was also tested
(Example 5). However, to avoid precipitation problems, the level of
serum in the assay had to be reduced for testing conjugates with
the .about.12,000 MW polymer material. Cell uptake was analyzed by
FACS as above, and the mean fluorescence of live cells was
measured. Cytotoxicity of each conjugate was also measured.
[0100] With reference to FIG. 3, uptake of L-arginine homopolymer
conjugates with 15 or more arginines exhibited patterns of cellular
uptake distinctly different from polymers containing nine arginines
or less. The curves of the longer conjugates were flatter, crossing
those of the R9 and r9 conjugates. At higher concentrations (>3
.mu.M), uptake of R9 and r9 was significantly better than for the
longer polymers. However, at lower concentrations, cells incubated
with the longer peptides exhibited greater fluorescence.
[0101] Based on this data, it appears that r9 and R9 enter the
cells at higher rates than polymers containing 15 or more
contiguous arginines. However, the biological half-life of R9
(L-peptide) was shorter than for the longer conjugates, presumably
because proteolysis of the longer peptides (due to serum enzymes)
produces fragments that retain transport activity. In contrast, the
D-isomer (r9) did not show evidence of proteolytic degradation,
consistent with the high specificity of serum proteases for
L-polypeptides.
[0102] Thus, overall transport efficacy of a transport polymer
appears to depend on a combination of (i) rate of trans-membrane
uptake (polymer with less than about 15 continuous arginines are
better) versus susceptibility to proteolytic inactivation (longer
polymers are better). Accordingly, polymers containing 7 to 20
contiguous guanidinium residues, and preferably 7 to 15, are
preferred.
[0103] Notably, the high molecular weight polyarginine conjugate
(12,000 MW) did not exhibit detectable uptake. This result is
consistent with the observations of Barsoum et al. (PCT Pub. No. WO
94/04686), and suggests that arginine polymers have transport
properties that are significantly different from those that may be
exhibited by lysine polymers. Furthermore, the 12,000 MW
polyarginine conjugate was found to be highly toxic (Example 5). In
general, toxicity of the polymers increased with length, though
only the 12,000 MW conjugate showed high toxicity at all
concentrations tested.
[0104] When cellular uptake of polymers of D- and L-arginine were
analyzed by Michaelis-Menten kinetics (Example 6), the rate of
uptake by Jurkat cells was so efficient that precise K.sub.m values
could only be obtained when the assays were carried out at
3.degree. C. (on ice). Both the maximal rate of transport
(V.sub.max) and the apparent affinity of the peptides for the
putative receptor of the Michaelis constant (K.sub.m) were derived
from Lineweaver-Burk plots of the observed fluorescence of Jurkat
cells after incubation with varying concentrations of nonamers of
D- and L-arginine for 30, 60, 120, and 240 seconds.
[0105] Kinetic analysis also reveals that polymers rich in arginine
exhibit a better ability to bind to and traverse a putative
cellular transport site than, for example, the tat(49-57) peptide,
since the K.sub.m for transport of the nonameric poly-L-arginine
(44 .mu.M) was substantially lower than the K.sub.m of the tat
peptide (722 .mu.M). Moreover, the nonamer of D-arginine exhibited
the lowest K.sub.m (7 .mu.M) of the polymers tested in this assay
(Table 1), i.e., an approximately 100-fold greater apparent
affinity.
[0106] According to a preferred embodiment of the invention, the
transport polymer of the invention has an apparent affinity (Km)
that is at least 10-fold greater, and preferably at least 100-fold
greater, than the affinity measured for tat by the procedure of
Example 6 when measured at room temperature (23.degree. C.) or
37.degree. C.
1 TABLE 1 V.sub.MAX K.sub.M (.mu.M) (.mu.M/sec)
H.sub.3N-RRRRRRRRR-COO.sup.31 (SEQ ID NO: 8) 44.43 0.35
H3N-rrrrrrrrr-COO (SEQ ID NO: 17) 7.21 0.39 tat 49-57 (SEQ ID NO:
1) 722 0.38
[0107] Experiments carried out in support of the present invention
indicate that polymer-facilitated transport is dependent upon
metabolic integrity of cells. Addition of a toxic amount of sodium
azide (0.5% w/v) to cells resulted in inhibition of uptake of
conjugates by about 9% (Example 7). The results shown in FIG. 4
demonstrate (i) sodium azide sensitivity of transmembrane
transport, suggesting energy-dependence (cellular uptake), and (ii)
the superiority of poly-guanidinium polymers of the invention (R9,
R8, R7) relative to HIV tat (49-57).
[0108] Without ascribing to any particular theory, the data suggest
that the transport process is an energy-dependent process mediated
by specific recognition of guanidinium or amidinium-containing
polymers by a molecular transporter present in cellular plasma
membranes.
[0109] Other experiments in support of the invention have shown
that the conjugates of the invention are effective to transport
biologically active agents across membranes of a variety of cell
types, including human T cells (Jurkat), B cells (murine CH27),
lymphoma T cells (murine EL-4), mastocytoma cells (murine P388),
several murine T cell hybridomas, neuronal cells (PC-12),
fibroblasts (murine RT), kidney cells (murine HELA), myeloblastoma
(murine K562); and primary tissue cells, including all human blood
cells (except red blood cells), such as T and B lymphocytes,
macrophages, dendritic cells, and eosinophils; basophiles, mast
cells, endothelial cells, cardiac tissue cells, liver cells, spleen
cells, lymph node cells, and keratinocytes.
[0110] The conjugates are also effective to traverse both gram
negative and gram positive bacterial cells, as disclosed in Example
8 and FIGS. 5A-5C. In general, polymers of D-arginine subunits were
found to enter both gram-positive and gram-negative bacteria at
rates significantly faster than the transport rates observed for
polymers of L-arginine. This is illustrated by FIG. 5A, which shows
much higher uptake levels for r9 conjugate (D-arginines), than for
the R9 conjugate (L-arginines), when incubated with E. coli IIB 101
(prokaryotic) cells. This observation may be attributable to
proteolytic degradation of the L-polymers by bacterial enzymes.
[0111] FIG. 5B shows uptake levels for D-arginine conjugates as a
function of length (r4 to r9) in comparison to a poly-L-lysine
conjugate (K9), when incubated with E. coli HB 101 cells. As can be
seen, the polyarginine conjugates showed a trend similar to that in
FIG. 2B observed with eukaryotic cells, such that polymers shorter
than r6 showed low uptake levels, with uptake levels increasing as
a function of length.
[0112] Gram-positive bacteria, as exemplified by Strep. bovis, were
also stained efficiently with polymers of arginine, but not lysine,
as shown in FIG. 5C.
[0113] More generally, maximum uptake levels by the bacteria were
observed at 37.degree. C. However, significant staining was
observed when incubation was performed either at room temperature
or at 3.degree. C. Confocal microscopy revealed that pretreatment
of the bacteria with 0.5% sodium azide inhibited transport across
the inner plasma membranes of both gram-positive and gram-negative
bacteria, but not transport across the cell wall (gram-positive
bacteria) into the periplasmic space.
[0114] Thus, the invention includes conjugates that contain
antimicrobial agents, such as antibacterial and antifungal
compounds, for use in preventing or inhibiting microbial
proliferation or infection, and for disinfecting surfaces to
improve medical safety. In addition, the invention can be used for
transport into plant cells, particularly in green leafy plants.
[0115] Additional studies in support of the invention have shown
that translocation across bacterial membranes is both energy- and
temperature-dependent, consistent with observations noted earlier
for other cell-types.
[0116] V. Therapeutic Compositions
[0117] A. Small Organic Molecules
[0118] Small organic molecule therapeutic agents may be
advantageously attached to linear transport polymers as described
herein, to facilitate or enhance transport across biological
membranes. For example, delivery of highly charged agents, such as
levodopa (L-3,4-dihydroxy-phenylalanine- ; L-DOPA) may benefit by
linkage to polymeric transport molecules as described herein.
Peptoid and peptidomimetic agents are also contemplated (e.g.,
Langston (1997) DDT2:255; 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.
[0119] 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 transport
polymer and linker moiety (if any) for the small molecule agent to
exert biological activity after crossing the target biological
membrane. For such situations, the conjugate preferably includes a
cleavable linker for releasing free drug after passing through a
biological membrane.
[0120] In one approach, the conjugate can include a disulfide
linkage, as illustrated in FIG. 6A, 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.
[0121] A method for synthesizing a disulfide-containing conjugate
is provided in Example 9A. The product 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 include 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.
[0122] In another approach, the conjugate includes a photocleavable
linker that is cleaved upon exposure to electromagnetic radiation.
An exemplary linkage is illustrated in FIG. 6B, 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
(Example 9B). 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.
[0123] Preferably, the cleavable linker contains first and second
cleavable groups that can cooperate to cleave the polymer 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 polymer.
[0124] FIG. 6C shows a conjugate (III) containing a transport
polymer T linked to the anticancer agent, 5-fluorouracil (5FU).
Here, the linkage is provided by a modified lysyl residue. The
transport polymer is linked to the a-amino group, and the
5-fluorouracil is linked via the .alpha.-carbonyl. The lysyl
.epsilon.-amino group has been modified to a carbamate ester of
o-hydroxymethyl nitrobenzene, which comprises a first, photolabile
cleavable group in the conjugate. Photo-illumination severs the
nitrobenzene moiety from the conjugate, leaving a carbamate that
also rapidly decomposes to give the free E-amino group, an
effective nucleophile. Intramolecular reaction of the
.epsilon.-amino group with the amide linkage to the 5-fluorouracil
group leads to cyclization with release of the 5-fluorouracil
group.
[0125] FIG. 6D illustrates a conjugate (IV) containing a transport
polymer 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 transport polymer, (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, for binding to its biological target. Example 9C
describes a synthetic protocol for preparing this type of
conjugate.
[0126] FIG. 6E illustrates yet another approach wherein a transport
polymer 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. 6E, the
linker amino group is joined to the transport polymer by an amide
linkage, and is joined to the paclitaxel moiety by an ester
linkage. Enzymatic cleavage of the amide linkage releases the
polymer 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.
[0127] FIGS. 6D and 6E are illustrative of another aspect of the
invention, comprising taxane- and taxoid anticancer conjugates
which have enhanced trans-membrane 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.
[0128] The term "taxane" refers to paclitaxel (FIG. 6F, 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. 6G. FIG. 6F 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. 6H. Substantial synthetic and
biological information is available on syntheses and activities of
a variety of taxane and taxoid compounds, as reviewed in Suffiness
Suffness, M., Ed., Taxol: Science and Applications, CRC Press, New
York, N.Y., pp. 237-239 (1995), 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.
[0129] The transport polymer is conjugated to the taxane or taxoid
moiety via any suitable site of attachment in the taxane or taxoid.
Conveniently, the transport polymer is linked via a C2'-oxygen
atom, C7-oxygen atom or, 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.
[0130] It will be appreciated that the taxane and taxoid conjugates
of the invention have improved water solubility relative to taxol
(.about.0.25 .mu.g/mL) and taxotere (6-7 .mu.g/mL). Therefore,
large amounts of solubilizing agents such as "CREMOPHOR.RTM. EL"
(polyoxyethylated castor oil), polysorbate 80 (polyoxyethylene
sorbitan monooleate, also known as "TWEEN.RTM. 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.
[0131] B. Metals
[0132] Metals can be transported into eukaryotic and prokaryotic
cells using chelating agents such as texaphyrin or diethylene
triamine pentaacetic acid (DTPA), conjugated to a transport
membrane of the invention, as illustrated by Example 10. 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.
[0133] C. Macromolecules The enhanced transport method of the
invention is particularly suited for enhancing transport across
biological membranes 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.
[0134] By way of example, protein nucleic acids (PNA) are analogs
of DNA in which the backbone is structurally homomorphous with a
deoxyribose backbone. It 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 are true DNA mimics 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.
[0135] Examples of anti-sense oligonucleotides whose transport into
cells may 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.
[0136] FIG. 7 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.
[0137] Another class of macromolecules that can be transported
across biological membranes 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.
[0138] 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.
[0139] 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.
[0140] Example 14 provides details of experiments carried out in
support of the present invention in which an exemplary protein
antigen, ovalbumin, was delivered to APCs after conjugation to an
R7 polymer. Subsequent addition of the APCs to cytotoxic T
lymphocytes (CTLs) resulted in CD8+albumin-specific cytotoxic T
cells (stimulated CTLs). In contrast, APCs that had been exposed to
unmodified ovalbumin failed to stimulate the CTLs.
[0141] In parallel experiments, histocompatible dendritic cells (a
specific type of APC) were exposed to ovalbumin-R7 conjugates, then
injected into mice. Subsequent analysis of blood from these mice
revealed the presence of albumin-specific CTLs. Control mice were
given dendritic cells that had been exposed to unmodified albumin.
The control mice did not exhibit the albumin-specific CTL response.
These experiments exemplify one of the specific utilities
associated with delivery of macromolecules in general, and proteins
in particular, into cells.
[0142] 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. Virology 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. By
attaching transport polymers to scFv fragments, however, the degree
of cellular uptake can be increased, allowing the immunospecific
fragments to bind and disable important microbial components, such
as HIV Rev, HIV reverse transcriptase, and integrase proteins.
[0143] D. Peptides
[0144] 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
intracellular signals. Examples of such proteins include, but are
not limited to, protein kinase C, RAF-1, p21Ras, NF-.kappa.B,
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.
[0145] When the transport enhancing molecule 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.
[0146] In experiments carried out in support of the present
invention (Example 15) a 10-amino acid segment of the cytoplasmic
tail region of the transmembrane protein V7 (also known as CD101)
was synthesized with an R7 polymer sequence at its C terminus. This
tail region is known to physically associate with and mediate the
inactivation of RAF-1 kinase, a critical enzyme in the MAP kinase
pathway of cellular activation. The V7-R7 conjugate was added to T
cells, which were subsequently lysed with detergent. The soluble
fraction was tested for immunoprecipitation by anti-V7 murine
antibody in conjunction with goat anti-mouse IgG.
[0147] In the absence of peptide treatment, RAF-1, a kinase known
to associate with and be inactivated by association with V7,
co-precipitated with V7. In peptide treated cells, RAF-1 protein
was eliminated from the V7 immuno-complex. The same peptide
treatment did not disrupt a complex consisting of RAF-1 and p21
Ras, ruling out any non-specific modification of RAF-1 by the V7
peptides. These results showed that a cytoplasmic tail region V7
peptide, when conjugated to a membrane transport enhancing peptide
of the present invention, enters a target cell and specifically
associates with a physiological effector molecule, RAF-1. Such
association can be used to disrupt intracellular processes.
[0148] In a second set of studies, the V7 portion of the conjugate
was phosphorylated in vitro using protein kinase C. Anti-RAF-1
precipitates of T cells that had been exposed to the phosphorylated
V7 tail peptides, but not the unphosphorylated V7 tail peptide,
demonstrated potent inhibition of RAF-kinase activity. These
studies demonstrate two principles. First, the transport polymers
of the invention can effect transport of a highly charged
(phosphorylated) molecule across the cell membrane. Second, while
both phosphorylated and unphosphorylated V7 tail peptides can bind
to RAF-1, only the phosphorylated peptide modified RAF-1 kinase
activity.
[0149] VI. Screening Assay Method and Library
[0150] In another embodiment, the invention can be used to screen
one or more conjugates for a selected biological activity, wherein
the conjugate(s) are formed from one or more candidate agents.
Conjugate(s) are contacted with a cell that exhibits a detectable
signal upon uptake of the conjugate into the cell, such that the
magnitude of the signal is indicative of the efficacy of the
conjugate with respect to the selected biological activity.
[0151] One advantage of this embodiment is that it is particularly
useful for testing the activities of agents that by themselves are
unable, or poorly able, to enter cells to manifest biological
activity. Thus, the invention provides a particularly efficient way
of identifying active agents that might not otherwise be accessible
through large-scale screening programs, for lack of an effective
and convenient way of transporting the agents into the cell or
organelle.
[0152] Preferably, the one or more candidate agents are provided as
a combinatorial library of conjugates which are prepared using any
of a number of combinatorial synthetic methods known in the art.
For example, Thompson and Ellman (1996) recognized at least five
different general approaches for preparing combinatorial libraries
on solid supports, namely (1) synthesis of discrete compounds, (2)
split synthesis (split and pool), (3) soluble library
deconvolution, (4) structural determination by analytical methods,
and (5) encoding strategies in which the chemical compositions of
active candidates are determined by unique labels, after testing
positive for biological activity in the assay. Synthesis of
libraries in solution includes at least (1) spatially separate
syntheses and (2) synthesis of pools (Thompson and Ellman (1996)
Chem. Rev. 96:555). Further description of combinatorial synthetic
methods can be found in Lam et al. (1997) Chem. Rev. 97:411, which
particularly describes the one-bead-one-compound approach.
[0153] These approaches are readily adapted to prepare conjugates
in accordance with the present invention, including suitable
protection schemes as necessary. For example, for a library that is
constructed on one or more solid supports, a transport peptide
moiety can be attached to the support(s) first, followed by
building or appending candidate agents combinatorially onto the
polymers via suitable reactive functionalities. In an alternative
example, a combinatorial library of agents is first formed on one
or more solid supports, followed by appending a transport polymer
to each immobilized candidate agent. Similar or different
approaches can be used for solution phase syntheses. Libraries
formed on a solid support are preferably severed from the support
via a cleavable linking group by known methods (Thompson and Ellman
(1996), supra, and Lam et al. (1997), supra).
[0154] The one or more conjugate candidates can be tested with any
of a number of cell-based assays that elicit detectable signals in
proportion to the efficacy of the conjugate. Conveniently, the
candidates are incubated with cells in multiwell plates, and the
biological effects are measured via a signal (e.g., fluorescence,
reflectance, absorption, or chemiluminescence) that can be
quantitated using a plate reader. Alternatively, the incubation
mixtures can be removed from the wells for further processing
and/or analysis. The structures of active and optionally inactive
compounds, if not already known, are then determined, and this
information can be used to identify lead compounds and to focus
further synthesis and screening efforts.
[0155] For example, the .gamma.-interferon secretion assay detailed
in Example 11 is readily adapted to a multiwell format, such that
active secretion inhibitors can be detected by europium-based
fluorescence detection using a plate reader. Anticancer agents can
be screened using established cancer cell lines (e.g., provided by
the National Institutes of Health (NIH) and the National Cancer
Institute (NCI). Cytotoxic effects of anticancer agents can be
determined by trypan dye exclusion, for example.
[0156] Other examples include assays directed to inhibiting cell
signaling, such as IL-4 receptor inhibition; assays for blocking
cellular proliferation, and gene expression assays. In a typical
gene expression assay, a gene of interest is placed under the
control of a suitable promoter and is followed downstream by a gene
for producing a reporter species such as .beta.-galactosidase or
firefly luciferase. An inhibitory effect can be detected based on a
decrease in reporter signal.
[0157] The invention also includes a conjugate library that is
useful for screening in the above method. The library includes a
plurality of candidate agents for one or more selected biological
activities, each of which is conjugated to at least one transport
polymer in accordance with the invention. Preferably, the conjugate
library is a combinatorial library. In another embodiment, the
invention includes a regular array of distinct polymer-agent
conjugates distributed in an indexed or indexable plurality of
sample wells, for testing and identifying active agents of
interest.
[0158] VI. Utility
[0159] 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.
[0160] 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. The
formulations may take the form of solid, semi-solid, lyophilized
powder, or liquid dosage forms, such as, for example, tablets,
pills, capsules, powders, solutions, suspensions, emulsions,
suppositories, retention enemas, creams, ointments, lotions,
aerosols or the like, preferably in unit dosage forms suitable for
simple administration of precise dosages.
[0161] 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., (Gennaro,
Gennaro, Ed., Remington's Pharmaceutical Sciences, 18.sup.th Ed.,
Mack Publishing Co., Easton, Pa. (1990)).
[0162] 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.
[0163] 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.
[0164] The active compounds of the formulas may be formulated into
a suppository comprising, for example, about 0.5% to about 50% of a
compound of the invention, disposed in a polyethylene glycol (PEG)
carrier (e.g., PEG 1000 [96%] and PEG 4000 [4%]).
[0165] 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.
[0166] 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.
[0167] 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.
[0168] 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 (1980). 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.
[0169] 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.
[0170] Stability of the conjugate can be further controlled by the
composition and stereochemistry of the backbone and sidechains of
the polymer. For polypeptide polymers, 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. Homo-D-polymers are generally
preferred.
[0171] The following examples are intended to illustrate but not
limit the present invention.
EXAMPLE 1
Peptide Synthesis
[0172] Peptides were synthesized using solid phase techniques on an
Applied Biosystems Peptide synthesizer using FastMOC.TM. chemistry
and commercially available Wang resins and Fmoc protected amino
acids, according to methods well known in the art (Bonifaci et al.,
Aids 9:995-1000). Peptides were purified using C4 or C18 reverse
phase HPLC columns, and their structures were confirmed using amino
acid analysis and mass spectrometry.
EXAMPLE 2
Fluorescence Assays
[0173] Fluorescent peptides were synthesized by modification of the
amino terminus of the peptide with aminocaproic acid followed by
reaction with fluorescein isothiocyanate in the presence of
(2-1H-benzotriazol-1-yl)-1,- 1,3,3-tetramethyl uronium
hexafluorophosphate/N-hydroxy benzotriazole dissolved in N-methyl
pyrrolidone. The products were purified by gel filtration.
[0174] Suspension cells (10.sup.6/mL) were incubated for varying
times, at 37.degree. C., 23.degree. C., or 4.degree. C., with a
range of concentration of peptides or conjugates in PBS pH 7.2
containing 2% fetal calf serum (PBS/FCS) in 96 well plates. After a
15 minute incubation, the cells were pelleted by centrifugation,
washed three times with PBS/FCS containing 1% sodium azide,
incubated with trypsin/EDTA (Gibco) at 37.degree. C. for five
minutes, then washed twice more with PBS/FCS/NaN.sub.3. The
pelleted cells were resuspended in PBS containing 2% FCS and 0.1%
propidium iodide and analyzed on a FACScan (Becton Dickinson,
Mountain View, Calif.). Cells positive for propidium iodide were
excluded from the analysis. For analysis of polymers of arginine,
the voltage of the photomultiplier was reduced by an order of
magnitude to allow a more accurate measurement.
EXAMPLE 3
Tat Basic Peptide Versus Poly-Arg Peptides
[0175] Uptake levels of the following polypeptides were measured by
the method in Example 2: (1) a polypeptide comprising HIV tat
residues 49-57 (Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-Arg=SEQ ID NO:1),
(2) a nonamer of L-Lys residues (K9, SEQ ID NO:2), and (3) homo-L
or homo-D-polypeptides containing four to nine Arg residues (SEQ ID
NO:3-8 and 12-17). Results a re shown in FIGS. 1A-1C.
EXAMPLE 4
Confocal Cell Microscopy
[0176] Cells incubated with fluorescent polyarginine peptides were
prepared as described above for binding assays and analyzed at the
Cell Sciences Imaging Facility (Stanford University, Stanford,
Calif.) using a scanning, single beam laser confocal microscope,
with an excitation wavelength of 488 nm (argon-ion laser) and an
emission band-width of 510-550 using a band-pass filter. Conjugates
(6.25 .mu.M) containing tat(49-57), R7, or r7 coupled to
fluorescein were incubated with Jurkat cells for 37.degree. C. for
10 minutes. FIGS. 2A-2F show result s for emitted fluorescence
(FIGS. 2A-2C) and transmitted light (2D-2F) for tat(49-57) (FIGS.
2A and 2C), R7 (FIGS. 2B and 2E), and r7 (FIGS. 2C and 2F).
EXAMPLE 5
Length Range Studies
[0177] The following homopolymers of polyarginine were tested by
the fluorescence assay in Example 2, with incubation at 37.degree.
C. for 15 minutes prior to cell pelleting: r9, R9, R15, R20, R25,
and R30. In addition, a mixture of L-arginine polymers having an
average molecular weight of 12,000 daltons (approximately 100 amino
acids) was also tested (Sigma Chem. Co.) after being labeled with
fluorescein isothiocyanate and purified by gel filtration
("SEPHADEX" G-25). The cells were analyzed by FACS, and the mean
fluorescence of the live cells was measured. Cytotoxicity of each
conjugate was also measured by calculating the percentage of cells
that stained with propidium iodide, which is characteristic of cell
death. Uptake results for the r9, R9, R15, R20, and R25 conjugates
are shown in FIG. 3.
[0178] The commercially available polyarginine (12,000 MW)
precipitated proteins in serum, most likely al-acid glycoprotein.
Therefore, the level of fetal calf serum was reduced 10-fold in the
assay for conjugates prepared from this material.
[0179] The 12,000 MW poly-Arg composition was toxic at
concentrations from 800 nM to 50 .mu.M and is excluded from FIG. 3.
Poly-L-Arg conjugates containing 20 arginine residues or more were
toxic at concentrations greater than 12 .mu.M, such that toxicity
increased with length.
EXAMPLE 6
Kinetics of Uptake
[0180] To measure Vmax and Km parameters of cellular uptake, the
assay method of Example 2 was used with the following
modifications. Peptides were incubated with cells for 0.5, 1, 2,
and 4 minutes at 4.degree. C. in triplicate, in 50 .mu.L of PBS/FCS
in 96-well plates. At the end of incubation, the reaction was
quenched by diluting the samples in 5 mL of PBS/FCS, centrifuging
and washing once with PBS/FCS, trypsin/EDTA, and finally again with
PBS/FCS, and taking up the pellets in PBS/FCS containing propidium
iodide for analysis on a FACScan. FACS data were fitted to the
Line-weaver-Burk equation for Michaelis-Menten kinetics. Kinetic
data for fluorescent conjugates of tat(49-57), R9, and r9 are shown
in Table I above.
EXAMPLE 7
Metabolic Inhibitor Effects on Transport
[0181] Suspension cells (10.sup.6/mL) were incubated for 30 minutes
with 0.5% sodium azide in PBS containing 2% FCS. At the end of
incubation, fluorescent peptides (tat(49-57)), R7, R8, or R9) were
added to a final concentration of 12.5 .mu.M. After incubation for
30 minutes, the cells were washed as in Example 2, except that all
wash buffers contained 0.1% sodium azide. The results are shown in
FIG. 4.
EXAMPLE 8
Transport into Bacterial Cells
[0182] Gram-negative bacteria (E. coli strain HB101) and
gram-positive bacteria (Strep. bovis) were grown in appropriate
media in logarithmic phase. Cell cultures (4.times.10.sup.8 per mL)
were incubated for 30 minutes at 37.degree. C. with varying
concentrations of fluorescent conjugates containing linear polymers
of L-arginine (R4 through R9), D-arginine (r4 through r9), or
L-lysine (K9) at conjugate concentrations of 3 to 50 .mu.M. The
cells were washed and taken up in PBS-containing propidium iodide
(to distinguish dead cells) and analyzed by FACS and fluorescent
microscopy. Results are shown in FIGS. 5A-5C as discussed
above.
EXAMPLE 9
Conjugates with Exemplary Cleavable Linkers
[0183] A. 6-Mercaptopurine Cysteine Disulfide Conjugate
[0184] 1. Thiol Activation.
N-acetyl-Cys(SH)-Ala-Ala-Ala-(Arg).sub.7-CO2H (12.2 mg, 0.0083
mmol) (SEQ ID NO: 20) was dissolved in 3 mL of 3:1 AcOH:H.sub.2O
with stirring at ambient temperature. To this solution was added
dithio-bis (5-nitropyridine) (DTNP) (12.9 mg, 0.0415 mmol, 5 eq).
The solution was permitted to stir for 24 h at ambient temperature,
after which the mixture took on a bright yellow color. Solvent was
removed in vacuo, and the residue was redissolved in 5 mL of H20
and extracted 3 times with ethyl acetate to remove excess DTNP. The
aqueous layer was lyophilized, and the product was used without
further purification.
[0185] 2. Attachment of Drug.
N-acetyl-Cys(SH)-Ala-Ala-Ala-(Arg).sub.7-CO2- H (0.0083 mmol) (SEQ
ID NO: 20)was dissolved in 1 mL of degassed H20 (pH=5) under argon
at room temperature, with stirring. 6-Mercaptopurine (1.42 mg,
0.0083 mmol, 1 eq) in 0.5 mL DMF was added to the mixture. The
reaction was permitted to stir for 18 h under inert atmosphere at
ambient temperature. After 18 h, a bright yellow color developed,
indicating the presence of free 5-nitro-2-thiopyridine. Solvent was
removed under reduced pressure, and the residue was purified by
HPLC, providing the desired product (I, FIG. 6A) in 50% overall
yield.
[0186] B. Photocleavable Taxol Conjugate
3-Nitro-4-(bromomethyl)benzoic acid (100 mg, 0.384 mmol) is
dissolved in anhydrous methanol (5 mL) under an atmosphere of
nitrogen. To this solution is added sodium methoxide (88 .mu.L, 25%
(w/w) in methanol, 0.384 mmol, 1 eq) followed by addition of
6-mercaptopurine (58.2 mg, 0.384 mmol, 1 eq). The mixture is warmed
to reflux and permitted to stir for 3 h. The reaction mixture is
then cooled, filtered, and quenched by acidification with 6N HCl.
The reaction volume is then reduced to one-half at which point the
product precipitates and is collected by filtration. The residue is
redissolved in methanol, filtered (if necessary) and concentrated
under reduced pressure to provide desired sulfide (11, FIG. 6B) in
50% yield as a yellow powdery solid.
[0187] C. Phosphate-Cleavable Taxol Conjugate
[0188] 1. To a suspension of o-hydroxy phenylacetic acid (15.0 g,
0.099 mol) in H20 (39 mL) at 0.degree. C. was added a solution of
nitric acid (12 mL of 65% in 8 mL H20) slowly via pipette. The
solution was stirred for an additional 1.5 h at 0.degree. C. The
mixture was then warmed to ambient temperature and allowed to stir
for an additional 0.5 h. The heterogeneous solution was poured over
ice (10 g) and filtered to remove the insoluble ortho-nitro isomer.
The reddish solution was concentrated under reduced pressure, and
the thick residue was redissolved in 6N HCI and filtered through
celite. The solvent was again removed under reduced pressure to
provide the desired 2-hydroxy-4-nitro-phenylacetic acid as a light,
brownish-red solid (40% yield). The product (IV-a) was used in the
next step without further purification.
[0189] 2. Product IV-a (765 mg, 3.88 mmol) was dissolved in freshly
distilled THF (5 mL) under argon atmosphere. The solution was
cooled to 0.degree. C., and borane-THF (1.0 M in THF, 9.7 mL, 9.7
mmol, 2.5 eq) was added dropwise via syringe with apparent
evolution of hydrogen. The reaction was permitted to stir for an
additional 16 h, slowly warming to room temperature. The reaction
was quenched by slow addition of 1M HCl (with furious bubbling) and
10 mL of ethyl acetate. The layers were separated and the aqueous
layer extracted five times with ethyl acetate. The combined organic
layers were washed with brine and dried over magnesium sulfate. The
solvent was evaporated in vacuo and the residue purified by rapid
column chromatography (1:1 hexane:ethyl acetate) to provide the
desired nitro-alcohol (IV-b) as a light yellow solid (85w
yield).
[0190] 3. Nitro-alcohol (IV-b) (150 mg, 0.819 mmol) was dissolved
in dry DMF (5 mL) containing di-t-butyl-di-carbonate (190 mg, 1.05
eq) and 10% Pd-C (10 mg). The mixture was placed in a Parr
apparatus and pressurized/purged five times. The solution was then
pressurized to 47 psi and allowed to shake for 24 h. The reaction
was quenched by filtration through celite, and the solvent was
removed under reduced pressure. The residue was purified by column
chromatography (1:1 hexane:ethyl acetate) to provide the protected
aniline product (IV-c) as a tan crystalline solid in 70% yield.
[0191] 4. TBDMS-Cl (48 mg, 0.316 mmol) was dissolved in freshly
distilled dichloromethane (4 mL) under an argon atmosphere. To this
solution was added imidazole (24 mg, 0.347 mmol, 1.1 eq) and
immediately a white precipitate formed. The solution was stirred
for 30 min at room temperature, at which point product IV-c (80 mg,
0.316 mmol, 1.0 eq) was added rapidly as a solution in
dichloromethane/THF (1.0 mL). The resulting mixture was permitted
to stir for an additional 18 h at ambient temperature. Reaction was
quenched by addition of saturated aqueous ammonium chloride. The
layers were separated and the aqueous phase extracted 3 times with
ethyl acetate and the combined organic layers washed with brine and
dried over sodium sulfate. The organic phase was concentrated to
provide silyl ether-phenol product (IV-d) as a light yellow oil
(90% yield).
[0192] 5. Silyl ether-phenol IV-d (150 mg, 0.408 mmol) was
dissolved in freshly distilled THF (7 mL) under argon and the
solution cooled to 0.degree. C. n-BuLi (2.3 M in hexane, 214 .mu.L)
was then added dropwise via syringe. A color change from light
yellow to deep red was noticed immediately. After 5 min,
tetrabenzyl pyrophosphate (242 mg, 0.45 mmol, 1.1 eq) was added
rapidly to the stirring solution under argon. The solution was
stirred for an additional 18 h under inert atmosphere, slowly
warming to room temperature, during which time a white precipitate
forms. The reaction was quenched by addition of saturated aqueous
ammonium chloride and 10 mL of ethyl acetate. The layers were
separated, and the aqueous layer was extracted 5 times with ethyl
acetate. The combined organic phases were washed with brine and
dried over magnesium sulfate. The solvent was removed by
evaporation and the residue purified by rapid column chromatography
(1:1 hexane:ethyl acetate) to provide the desired phosphate-silyl
ether (IV-e) as a light orange oil (90% yield).
[0193] 6. Phosphate-silyl ether (IV-e) (10 mg, 0.0159 mmol) was
dissolved in 2 mL of dry ethanol at room temperature. To the
stirring solution was added 20 .mu.L of conc. HCl (1% v:v
solution), and the mixture was permitted to stir until TLC analysis
indicated the reaction was complete. Solid potassium carbonate was
added to quench the reaction, and the mixture was rapidly filtered
through silica gel and concentrated to give crude alcohol-dibenzyl
phosphate product (IV-f) as a light yellow oil (100% yield).
[0194] 7. Alcohol IV-f (78 mg, 0.152 mmol) was dissolved in freshly
distilled dichloromethane (10 mL) under an argon atmosphere. To the
solution was added Dess-Martin periodinane (90 mg, 0.213 mmol, 1.4
eq). The solution was permitted to stir, and the progress of the
reaction was monitored by TLC analysis. Once TLC indicated
completion, reaction was quencned by addition of 1:1 saturated
aqueous sodium bicarbonate:saturated aqueous sodium thiosulfite.
The biphasic mixture was permitted to stir for 1 h at ambient
temperature. The layers were separated, and the aqueous phase was
extracted 3 times with ethyl acetate. The combined organic layers
were washed with brine and dried over sodium sulfate. Solvent was
removed under reduced pressure to provide an aldehyde product
(IV-g) as a light tan oil (100% yield).
[0195] 8. Aldehyde IV-g (78 mg, 0.152 mmol) was dissolved in
t-butanol/water (3.5 mL) under inert atmosphere. To the rapidly
stirring solution was added 2-methyl-2-butene (1.0 M in THF, 1.5
mL), sodium phosphate-monobasic (105 mg, 0.76 mmol, 5 eq) and
sodium chlorite (69 mg, 0.76 mmol, 5 eq). The solution was
permitted to stir for 8 additional hours at room temperature. The
solution was concentrated, and the residue was acidified and
extracted with ethyl acetate 3 times. The combined organic phases
were dried over magnesium sulfate. The solution was again
concentrated under reduced pressure and the residue was purified
via column chromatography (2:1 ethyl acetate:hexane) to give the
desired carboxylic acid-dibenzylphosphate (IV-h) as a light yellow
oil (65% yield).
[0196] 9. Acid IV-h (8.0 mg, 0.0152 mmol, 1.1 eq) was dissolved in
freshly distilled dichloromethane (2 mL) under argon at ambient
temperature. To this mixture was added paclitaxel (12 mg, 0.0138
mmol, 1 eq) followed by DMAP (2 mg, 0.0138 mmol, 1 eq) and DCC (3.2
mg, 0.0152, 1.1 eq). The mixture was allowed to stir at room
temperature for an additional 4 h, during which a light precipitate
formed. Once TLC analysis indicated that the reaction was complete,
solvent was removed under reduced pressure, and the residue was
purified by rapid column chromatography (1:1 hexane:ethyl acetate)
to provide paclitaxel-C2'-carboxylate ester (IV-i) as a white,
crystalline solid (65% yield).
[0197] 10. Ester IV-i (5.0 mg) was dissolved in neat formic acid
(1.0 mL) under an argon atmosphere at room temperature and
permitted to stir for 30 min. Once TLC indicated that the reaction
was complete, the solution was concentrated under reduced pressure
and the residue purified by rapid filtration through silica gel to
give the desired aniline-taxol compound (IV-j) in 50% yield as a
white powder.
[0198] 11. To a solution of (poly di-CBZ)-protected
AcHN-RRRRRRR-CO2H (1.2 eq, 0.1 to 1.0 M) (SEQ ID NO: 6) in dry DMF
was added O-benzotriazolyloxy tetramethyluronium
hexafluorophosphate (HATU, 1.0 eq) and a catalytic amount of DMAP
(0.2 eq). The solution stirred under inert atmosphere for 5 min at
ambient temperature. To this mixture was then added Taxol-aniline
derivative (IV-j) as a solution in dry DMF (minimal volume to
dissolve). The resulting solution was stirred for an additional 5 h
at room temperature. Reaction was terminated by concentrating the
reaction mixture under reduced pressure. The crude reaction mixture
was then purified by HPLC to provide the desired material (IV, FIG.
6D).
EXAMPLE 10
Transport of Metal Ions
[0199] 3.93 g of DTPA is dissolved in 100 mL of HEPES buffer and
1.52 ml of europium chloride atomic standard solution (Aldrich)
dissolved in 8 ml of HEPES buffer is added and stirred for 30
minutes at room temperature. Chromatographic separation and
lyophilization affords an Eu-DTPA chelate complex. This complex is
then conjugated to the amino terminus of a polypeptide by solid
phase peptide chemistry. The cellular uptake of europium ion can be
monitored by time resolved fluorescence.
EXAMPLE 11
Uptake of PNA-Peptide Conjugates
[0200] PNA peptide conjugates were synthesized using solid phase
chemistry with commercially available Fmoc reagents (PerSeptive
Biosystems, Cambridge, Mass.) on either an Applied Biosystems 433A
peptide synthesizer or a Millipore Expedite nucleic acid synthesis
system. Polymers of D or L-arginine were attached to the amino or
carboxyl termini of the PNAs, which are analogous to the 5' and 3'
ends of the nucleic acids, respectively. The conjugates were also
modified to include fluorescein or biotin by adding an aminocaproic
acid spacer to the amino terminus of the conjugate and then
attaching biotin or fluorescein. The PNA-peptide conjugates were
cleaved from the solid phase resin using 95% TFA, 2.5% triisopropyl
silane, and 2.5% aqueous phenol. The resin was removed by
filtration, and residual acid was removed by evaporation. The
product was purified by HPLC using a C-18 reverse phase column, and
the product was lyophilized. The desired PNA-polymer conjugates
were identified using laser desorption mass spectrometry.
[0201] A. Inhibition of Cellular Secretion of Gamma-IFN
[0202] 1. PNA-Peptide Conjugates. The following sense and antisense
PNA-peptide conjugates were prepared for inhibiting gamma-IFN
production, where r=D-arginine, and R=L-arginine:
[0203] Sense:
[0204] NH.sub.2-rrrrrrr-AACGCTACAC-COOH (SEQ ID NO: 6 conjugated to
SEQ ID NO: 18)
[0205] Antisense:
[0206] NH.sub.2-rrrrrrr-GTGTAGCGTT-COOH (SEQ ID NO: 6 conjugated to
SEQ ID NO: 19)
[0207] Fluorescent antisense:
[0208] X-rrrrrrr-GTGTAGCGTT-COOH (X-SEQ ID NO: 6 conjugated to SEQ
ID NO:19) where X=fluorescein-aminocaproate
[0209] Biotinylated antisense:
[0210] Z-rrrrrrr-GTGTAGCGTT-COOH (Z-SEQ ID NO: 6 conjugated to SEQ
ID NO:19) where Z=biotin-aminocaproate
[0211] 2. Uptake by T Cells. To show that PNA-polyarginine
conjugates enter cells effectively, the fluorescent antisense
conjugate above (X-SEQ ID NO: 19) was synthesized by conjugating
fluorescein isothiocyanate to the amino terminus of r7 the above
conjugated with SEQ ID NO: 18 using an aminocaproic acid
spacer.
[0212] Cellular uptake was assayed by incubating the Jurkat human T
cell line (5.times.10.sup.5 cells/well) either pretreated for 30
minutes with 0.5% sodium azide or phosphate buffered saline, with
varying amounts (100 nM to 50 .mu.M) of the fluorescein-labeled
sense and antisense PNA-r7 conjugate, as well as the antisense PNA
alone (without r7 segment). The amount of antisense PNA that
entered the cells was analyzed by confocal microscopy and FACS. In
both cases, fluorescent signals were present only in cells not
exposed to azide, and the fluorescent signal was dependent on the
dose of the fluorescent conjugate and on the temperature and
duration of incubation.
[0213] 3. Gamma-IFN Assay. The amount of gamma interferon secreted
by a murine T cell line (clone 11.3) was measured by incubating
10.sup.5 T cells with varying amounts of antigen (peptide
consisting of residues 110-121 of sperm whale myoglobin) and
histocompatible spleen cells from DBA/2 mice (H-2d,
5.times.10.sup.5), which act as antigen-presenting cells (APCs), in
96 well plates. After incubation for 24 hours at 37.degree. C., 100
.mu.L of the supernatants were transferred to microtiter plates
coated with commercially available anti-gamma-IFN monoclonal
antibodies (Mab) (Pharmingen, San Diego, Calif.). After incubation
for an hour at room temperature, the plates were washed with PBS
containing 1% fetal calf serum and 0.1% Tween.RTM. 20, after which
a second, biotinylated gamma-IFN Mab was added. After a second hour
of incubation, the plates were washed as before, and europium
(Eu)-streptavidin (Delphia-Pharmacia) was added. Again, after an
hour of incubation, an acidic buffer was added to release Eu, which
was measured by time-resolved fluorometry on a Delphia plate
reader. The amount of fluorescence was proportional to the amount
of gamma-IFN that had been produced and could be quantified
precisely using known amounts of gamma-IFN to create a standard
curve.
[0214] 4. Inhibition of Gamma-IFN Production by Conjugates. The
ability of PNA-polyarginine conjugates to inhibit secretion of
gamma-IFN was assayed by adding various concentrations of the above
gamma-IFN conjugates with suboptimal doses of peptide antigen (0.5
.mu.M), to a mixture of clone 11.3 T cells and histocompatible
spleen cells. PNA sequences lacking polyarginine moieties, and
non-conjugated D-arginine heptamer, were also tested.
[0215] After 24 hours, aliquots of the cultured supernatants were
taken, and the amount of gamma-IFN was measured using the
fluorescent binding assay described in section 3 above. Treatment
of cells with the antisense PNA-r7 conjugate resulted in an over
70% reduction in IFN secretion, whereas equivalent molar amounts of
the sense PNA-r7, antisense PNA lacking r7, or r7 alone all showed
no inhibition (FIG. 7).
EXAMPLE 12
Transport of Large Protein Antigen Into APCs
[0216] A conjugate of ovalbumin coupled to a poly-L-arginine
heptamer was formed by reacting a cysteine-containing polypeptide
polymer (Cys-Ala-Ala-Ala-Arg.sub.7, SEQ ID NO:20) with ovalbumin
(45 kDa) in the presence of sulfo-MBS, a heterobifunctional
crosslinker (Pierce Chemical Co., Rockford, Ill.). The molar ratio
of peptide conjugated to ovalbumin was quantified by amino acid
analysis. The conjugate product was designated OV-R7. The conjugate
was added (final concentration .apprxeq.10 .mu.M) to B-cells, also
referred to as antigen-presenting cells (APCs), which were isolated
according to standard methods. The APCs were incubated with OV-R7,
and then were added to a preparation of cytotoxic T-lymphocytes
isolated by standard methods. Exposure of CTLs to APCs that had
been incubated with OV-R7 produced CD8+albumin-specific CTLs. In
contrast, APCs that had been exposed to unmodified ovalbumin failed
to stimulate the CTLs.
[0217] In another experiment, histocompatible dendritic cells (a
specific type of APC) were exposed to albumin-R7 conjugates and
were then injected into mice. Subsequent analysis of blood from
these mice revealed the presence of albumin-specific CTLs. Control
mice were given dendritic cells that had been exposed to unmodified
albumin. The control mice did not exhibit the albumin-specific CTL
response.
EXAMPLE 13
Enhanced Uptake of V7-Derived Peptide
[0218] A conjugate consisting of a portion of the C-terminal
cytoplasmic tail region of V7 (a leukocyte surface protein) having
the sequence KLSTLRSNT (SEQ ID NO:21; Ruegg et al. (1995) J.
Immunol. 154:4434-43) was synthesized with 7 arginine residues
attached to its C-terminus according to standard methods using a
peptide synthesizer (Applied Biosystems Model 433). The conjugate
was added (final concentration .apprxeq.10 .mu.M) to T cells that
had been isolated by standard methods, and was incubated at
37.degree. C. for several hours to overnight. Cells were lysed
using detergent (1% Triton X-100). DNA was removed, and the soluble
(protein-containing) fraction was subjected to immunoprecipitation
with an anti-V7 murine monoclonal antibody in combination with goat
anti-mouse IgG. RAF-1 is a kinase that associates with, and is
inactivated by association with, V7.
[0219] In the absence of peptide treatment, RAF-1 protein
co-precipitated with V7. In peptide-treated cells, RAF-1 protein
was eliminated from the V7 immunocomplex. The same peptides were
unable to disrupt a complex consisting of RAF-1 and p21 Ras, ruling
out non-specific modification of RAF-1 by the V7 peptide.
[0220] In a second study, the V7 peptide portion of the
V7-poly-arginine conjugate was phosphorylated in vitro using
protein kinase C. Anti-RAF-1 precipitates of T cells that had been
exposed to the phosphorylated V7 tail peptides, but not the
unphosphorylated V7 tail peptide, demonstrated potent inhibition of
RAF-kinase activity.
[0221] While the invention has been described with reference to
specific methods and embodiments, it will be appreciated that
various modifications and changes may be made without departing
from the spirit of the invention.
* * * * *