U.S. patent application number 12/137445 was filed with the patent office on 2009-01-08 for biodegradable polyphosphoramidates for controlled release of bioactive substances.
Invention is credited to Kam Weng Leong, Hai-Quan Mao, Jun Wang.
Application Number | 20090012027 12/137445 |
Document ID | / |
Family ID | 23117736 |
Filed Date | 2009-01-08 |
United States Patent
Application |
20090012027 |
Kind Code |
A1 |
Wang; Jun ; et al. |
January 8, 2009 |
BIODEGRADABLE POLYPHOSPHORAMIDATES FOR CONTROLLED RELEASE OF
BIOACTIVE SUBSTANCES
Abstract
The present invention is directed to a series of new
polycationic biodegradable polyphosphoramidates. Process for making
the polymers, compositions containing these polymers and bioactive
ligands to enhance the cellular uptake ad intracellular
trafficking, articles and methods for delivery of drugs and genes
using these polymers are described. A gene delivery system based on
these polymers is prepared by complex coacervation of nucleic acid
(DNA or RNA) with polymers. Targeting ligands and molecules that
could facilitate gene transfer can be conjugated to polymers to
achieve selective and enhanced gene delivery. The current invention
also provides a complex composition with buffering capacity.
Inventors: |
Wang; Jun; (Baltimore,
MD) ; Mao; Hai-Quan; (Singapore, SG) ; Leong;
Kam Weng; (Ellicott City, MD) |
Correspondence
Address: |
GREENBERG TRAURIG LLP (LA)
2450 COLORADO AVENUE, SUITE 400E, INTELLECTUAL PROPERTY DEPARTMENT
SANTA MONICA
CA
90404
US
|
Family ID: |
23117736 |
Appl. No.: |
12/137445 |
Filed: |
June 11, 2008 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10500565 |
Jan 7, 2005 |
7417110 |
|
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PCT/SG02/00091 |
May 14, 2002 |
|
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12137445 |
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60290833 |
May 14, 2001 |
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Current U.S.
Class: |
514/44R ;
435/375; 435/440; 514/772; 525/538; 525/54.1; 525/54.2 |
Current CPC
Class: |
A61K 31/7088 20130101;
A61P 11/02 20180101; A61K 47/34 20130101; C08L 85/02 20130101; A61P
1/12 20180101; A61P 21/06 20180101; A61P 1/10 20180101; C08L
2203/02 20130101; A61P 11/06 20180101; A61K 47/605 20170801; C12N
2320/32 20130101; C08G 79/02 20130101; C08G 79/04 20130101; A61K
9/146 20130101; C12N 15/111 20130101; A61P 35/00 20180101; A61P
9/10 20180101; A61P 1/08 20180101 |
Class at
Publication: |
514/44 ; 525/538;
525/54.1; 525/54.2; 435/375; 435/440; 514/772 |
International
Class: |
A61K 47/48 20060101
A61K047/48; C08G 79/02 20060101 C08G079/02; C12N 5/06 20060101
C12N005/06; C12N 15/11 20060101 C12N015/11; A61K 31/7088 20060101
A61K031/7088 |
Claims
1. A positively charged biodegradable polyphosphoramidate
composition formed by complexation in aqueous solution comprising:
(a) at least one negatively charged bioactive molecule; and (b) a
water soluble and positively charged biodegradable
polyphosphoramidate that is capable of forming a complex with
negatively charged bioactive molecules in aqueous solution and
comprises the recurring monomeric unit shown in Formula I:
##STR00005## wherein R.sub.1 is a divalent aliphatic organic
moiety; R.sub.2 and R.sub.3 are each independently selected from
the group consisting of hydrogen, alkyl, aryl, heteroaryl,
heteroalicyclic, cycloalkyl, aralkyl, and cycloalkylalkyl groups;
each non-hydrogen occurrence of R.sub.2 and R.sub.3 is substituted
with one or more positively charged groups; and n is from 20 to
2,000; wherein when R.sub.1 is --(CH.sub.2).sub.a-- and one of
R.sub.2 and R.sub.3 is hydrogen, the other of R.sub.3 and R.sub.2
is selected from the group consisting of hydrogen, aryl,
heteroaryl, heteroalicylcic, cycloalkyl, aralkyl, and
cycloalkylaklyl; and wherein .alpha. ranges from 2 to 6.
2. The positively charged biodegradable polyphosphoramidate
composition of claim 1, wherein each non-hydrogen occurrence of
R.sub.2 and R.sub.3 is substituted with at least one of an amine
group and an imidazoyl group.
3. The positively charged biodegradable polyphosphoramidate
composition of claim 1, wherein one or more of R.sub.1, R.sub.2, or
R.sub.3 is substituted with one or more groups capable of
facilitating intracellular delivery of negatively charged bioactive
molecules, selected from the group consisting of a lysosomalytic
agent, an amphiphilic peptide, or a steroid derivative.
4. The positively charged biodegradable polyphosphoramidate
composition of claim 3, wherein the group capable of facilitating
intracellular delivery of the negatively charged bioactive
molecules is a cholesterol group.
5. The positively charged biodegradable polyphosphoramidate of
claim 1, wherein R.sub.1 is defined in Formula II: ##STR00006##
wherein each occurrence of R.sub.3 and R.sub.4 are independently
selected from the group consisting of hydrogen or alkyl group; and
q is 2to 4.
6. A positively charged biodegradable polyphosphoramidate
composition formed by complexation in aqueous solutions comprising:
(a) at least one negatively charged nucleic acid; and (b) a water
soluble and positively charged biodegradable polyphosphoramidate
that is capable of forming a complex with negatively charged
nucleic acid in aqueous solution and comprises the recurring
monomeric unit shown in Formula I: ##STR00007## wherein R.sub.1 is
a divalent aliphatic organic moiety; R.sub.2 and R.sub.3 are each
independently selected from the group consisting of hydrogen,
alkyl, aryl, heteroaryl, heteroalicyclic, cycloalkyl, aralkyl, and
cycloalkylalkyl groups; each non-hydrogen occurrence of R.sub.2 and
R.sub.3 is substituted with one or more positively charged groups;
and n is from 20 to 2,000.
7. The positively charged biodegradable polyphosphoramidate
composition of claim 6, wherein each non-hydrogen occurrence of
R.sub.2 and R.sub.3 is substituted with at least one of an amine
group and an imidazoyl group.
8. The positively charged biodegradable polyphosphoramidate
composition of claim 6, wherein one or more of R.sub.1, R.sub.2, or
R.sub.3 is substituted with one or more groups capable of
facilitating intracellular delivery of the negatively charged
nucleic acid, selected from the group consisting of a lysosomalytic
agent, an amphiphilic peptide, or a steroid derivative.
9. The positively charged biodegradable polyphosphoramidate
composition of claim 8, wherein the group capable of facilitating
intracellular delivery of the negatively charged nucleic acid is a
cholesterol group.
10. The positively charged biodegradable polyphosphoramidate of
claim 6, wherein R.sub.1 is defined in Formula II: ##STR00008##
wherein each occurrence of R.sub.3 and R.sub.4 are independently
selected from the group consisting of hydrogen or alkyl group; and
q is 2 to 4.
11. A method comprising: mixing a solution of a water soluble
positively charged biodegradable polymer comprising the recurring
monomeric unit shown in Formula I: ##STR00009## wherein R.sub.1 is
a divalent aliphatic organic moiety; R.sub.2 and R.sub.3 are each
independently selected from the group consisting of hydrogen,
alkyl, aryl, heteroaryl, heteroalicyclic, cycloalkyl, aralkyl, and
cycloalkylalkyl groups; each non-hydrogen occurrence of R.sub.2 and
R.sub.3 is substituted with one or more positively charged groups;
and n is from 20 to 2,000; wherein when R.sub.1 is
--(CH.sub.2).sub.a-- and one of R.sub.2 and R.sub.3 is hydrogen,
the other of R.sub.3 and R.sub.2 is selected from the group
consisting of hydrogen, aryl, heteroaryl, heteroalicylcic,
cycloalkyl, aralkyl, and cycloalkylaklyl; and wherein .alpha.
ranges from 2 to 6; with a solution of at least one negatively
charged bioactive molecule that is able to complex with the polymer
of Formula I to form complexes of the water soluble, positively
charged biodegradable polymer of Formula I and at least one
negatively charged molecule.
12. The method of claim 11, wherein the at least one negatively
charged bioactive molecule is selected from the group consisting of
DNA, RNA, proteins, and polysaccharides.
13. The method of claim 11, wherein the biodegradable polymer is
capable of complexing about 20% to about 60% by weight of the at
least one negatively charged bioactive molecule.
14. The method of claim 11, wherein the water soluble, positively
charged biodegradable polymer has between about 20 and about 200
phosphoramidate units.
15. The method of claim 11, wherein the concentration of the water
soluble, positively charged biodegradable polymer in the solution
ranges from about 1 .mu.g/ml to about 500 .mu.g/ml.
16. The method of claim 11, wherein the complexes are
nanoparticles.
17. The method of claim 11, wherein the complex is delivered to at
least one of a biological fluid, tissue, or cell.
18. The method of claim 11, wherein the complex is delivered to an
animal.
19. A method comprising: mixing a solution of a water soluble
positively charged biodegradable polymer comprising the recurring
monomeric unit shown in Formula I: ##STR00010## wherein R.sub.1 is
a divalent aliphatic organic moiety; R.sub.2 and R.sub.3 are each
independently selected from the group consisting of hydrogen,
alkyl, aryl, heteroaryl, heteroalicyclic, cycloalkyl, aralkyl, and
cycloalkylalkyl groups; each non-hydrogen occurrence of R.sub.2 and
R.sub.3 is substituted with one or more positively charged groups;
and n is from 20 to 2,000; with a solution of at least one
negatively charged nucleic acid molecule that is able to complex
with the polymer of Formula I to form complexes of the water
soluble, positively charged biodegradable polymer of Formula I and
at least one negatively charged nucleic acid molecule.
20. The method of claim 19, wherein the biodegradable polymer is
capable of complexing about 20% to about 60% by weight of the at
least one negatively charged nucleic acid.
21. The method of claim 19, wherein the water soluble, positively
charged biodegradable polymer has between about 20and about 200
phosphoramidate units.
22. The method of claim 19, wherein the concentration of the water
soluble, positively charged biodegradable polymer in the solution
ranges from about 1 .mu.g/ml to about 500 .mu.g/ml.
23. The method of claim 19, wherein the complexes are
nanoparticles.
24. The method of claim 19, wherein the complex is delivered to at
least one of a biological fluid, tissue, or cell.
25. The method of claim 19, wherein the complex is delivered to an
animal.
Description
[0001] This application is a divisional application and claims the
Paris Convention priority of U.S. Utility patent application Ser.
No. 10/500,565 filed on Jan. 7, 2005, which is incorporated by
reference.
BACKGROUND OF THE INVENTION
[0002] 1. Field of the Invention
[0003] The present invention generally relates to biodegradable
polymer compositions, in particular those containing both
phosphoester linkages in the polymer backbone and chargeable groups
linked to the backbone through a P--N bond. The polymers of the
invention are useful for drug and gene delivery, particularly as
carriers for gene therapy and for the delivery of protein
drugs.
[0004] 2. Background
[0005] Gene therapy has been progressively developed with the hope
that it will be an integral part of medical modalities in the
future. Gene delivery system is one of the key components in gene
medicine, which directs the gene expression plasmids to the
specific locations within the body. The control of gene expression
is achieved by influencing the distribution and stability of
plasmids in vivo and the access of the plasmids to the target
cells, and affecting the intracellular trafficking steps of the
plasmids (Mahato, et al., 1999, Pharmaceutical perspectives of
nonviral gene therapy, Adv. Genet. 41: 95-156). An ideal gene
delivery carrier should be bioabsorable, non-toxic,
non-immunogenic, stable during storage and after administration,
able to access target cells, and efficient in aiding gene
expression. As many studies demonstrated, the limitations of viral
vectors make synthetic vectors an attractive alternative.
Advantages of non-viral vectors include non-immunogenicity, low
acute toxicity, versatility, reproducibility and feasibility to be
produced on a large scale. Cationic liposome and cationic polymers
are the two major types of non-viral gene delivery carriers.
Cationic lipids self assemble into organized structures include
micelles, plannar bilayer sheets, and lamellar vesicles. Through
the condensation process, liposomes and cationic polymers form
complexes with DNA due to charge interaction. A large variety of
liposomal compositions have been developed for gene delivery
(Chesnoy and Huang, 2000, Structure and function of lipid-DNA
complexes for gene delivery, Annu. Rev. Biophys. Biomol. Struct.
29: 27-47). An effective liposome vector generally composed of a
positively charged lipid (e.g., cationic derivatives of cholesterol
and diacyl glycerol, quaternary ammonium detergents, lipid
derivatives of polyamines, etc.) and a neutral helper lipid (e.g.,
dioleoyl phosphatidylethanolamine (DOPE) or dioleoyl
phosphotidylcholine (DOPC)). Despite early excitement, there are
serious limitations to most cationic lipid systems. Several
observations have suggested that liposomal systems are relatively
unstable after the administration. Significant toxicity upon
repeated use has been shown to be associated to liposomal vectors,
especially the fusogenic phospholipid (neutral lipid), include the
down regulation of PKC dependent immunomodulator synthesis,
macrophage toxicity, neurotoxicity and acute pulmonary inflammation
(Filion and Phillips, 1998, Major limitations in the use of
cationic liposomes for DNA delivery, Int. J. Pharm. 162:
159-170).
[0006] Because of the limitations of viral vectors, cationic
lipids, cationic polymers as the basis of gene delivery systems
have gained increasing attention recently. A number of polycations
have been reported to effect transfection of DNA, including
poly-L-lysine, poly-L-ornithine, poly(4-hydroxy-L-proline ester),
polyiminocarbonate containing cyclodextrin,
poly[.alpha.-(4-aminobutyl)-L-glycolic acid], polyamidoamines,
polyamidoamine dendrimers, chitosan, polyethylenimine,
poly(2-dimethylaminoethyl methacrylate), etc. Significant progress
has been made in the development of polymer based systems,
especially biodegradable polymers that have lower toxicity and can
mediate gene transfection via condensing DNA into small particles
and protecting DNA from enzymatic degradation. Nevertheless,
searching for a safer and more efficient gene carrier still remains
a major challenge in the field of non-viral gene delivery.
SUMMARY OF THE INVENTION
[0007] The invention provides positively chargeable biodegradable
polymers that comprises at least one phosphoester linkage in the
polymer backbone and at least one positively chargeable group
wherein the positively chargeable group is a substitutent of a side
chain attached to the polymer backbone through a phosphoramidate
linkage, e.g., a P--N bond.
[0008] The invention further provides positively chargeable
biodegradable polymer compositions comprising:
[0009] (a) at least one biologically active substance; and
[0010] (b) a positively chargeable biodegradable polymer comprising
at least one phosphoester linkage in the polymer backbone and at
least one positively chargeable group wherein the positively
chargeable group is a substituent of a side chain attached to the
polymer backbone through a phosphoramidate linkage.
[0011] The invention additionally provides a method of preparing a
positively chargeable biodegradable polymers. The method comprising
the steps of:
[0012] polymerizing at least two monomers to form a polymer with at
least one phosphoester linkage in the polymer backbone;
[0013] reacting the polymer with a primary or secondary amine
having a positively chargeable group or a substituent that can be
functionalized to a positively chargeable group under conditions
conducive to the formation of a positively chargeable biodegradable
polymer comprising at least one phosphoester linkage in the polymer
backbone and at least one positively chargeable group wherein the
positively chargeable group is a substitutent of a side chain
attached to the polymer backbone through a phosphoramidate
linkage.
[0014] The invention provides a method of preparing a positively
chargeable biodegradable polymer composition. The method comprises
the steps of:
[0015] providing a positively chargeable biodegradable polymer
comprising at least one phosphoester linkage in the polymer
backbone and at least one positively chargeable group wherein the
positively chargeable group is a substitutent of a side chain
attached to the polymer backbone through a phosphoramidate linkage;
contacting the positively chargeable biodegradable polymer with a
biologically active substance under conditions conducive to the
formation of a complex, e.g., a composition, comprising the
positively chargeable biodegradable polymer and the biologically
active substance.
[0016] The invention also provides for the controlled release of a
biologically active substance. The method comprises the steps
of:
[0017] providing a positively chargeable biodegradable polymer
composition comprising:
[0018] (a) at least one biologically active substance; and
[0019] (b) a positively chargeable biodegradable polymer comprising
at least one phosphoester linkage in the polymer backbone and at
least one positively chargeable group wherein the positively
chargeable group is a substituent of a side chain attached to the
polymer backbone through a phosphoramidate linkage;
[0020] contacting the composition with a biological fluid, cell or
tissue under conditions conducive to the delivery of at least a
portion of the biologically active substance to the biological
fluid, cell or tissue.
[0021] The invention further provides methods for gene transfection
using the controlled release methods and the positively chargeable
biodegradable polymer composition comprising a DNA sequence, a gene
or a gene fragment, to deliver a DNA sequence, a gene or a gene
fragment to a specified tissue target in a patient. Gene
transfection methods of the invention are suitable for use in
treatment of any disease or disorder which is currently treatable
by gene therapy or is contemplated as a disease or disorder
suitable for treatment by gene therapy in the future. Gene
transfection methods of the invention comprise the steps of:
[0022] providing a positively chargeable biodegradable polymer
composition comprising:
[0023] (a) at least one DNA fragment, gene or gene fragment;
and
[0024] (b) a positively chargeable biodegradable polymer comprising
at least one phosphoester linkage in the polymer backbone and at
least one positively chargeable group wherein the positively
chargeable group is a substituent of a side chain attached to the
polymer backbone through a phosphoramidate linkage; contacting the
composition with a biological fluid, cell or tissue under
conditions conducive to the delivery of at least a portion of the
DNA sequence, gene or gene fragment to the biological fluid, cell
or tissue.
BRIEF DESCRIPTION OF THE DRAWINGS
[0025] FIG. 1. Synthesis scheme of P5-SP;
[0026] FIG. 2. Gel permeation chromatograph of P5-SP;
[0027] FIG. 3. Structures of P5-SP, P5-BA, P5-DMA, P5-DEA and
P5-TMA;
[0028] FIG. 4. Cytotoxicity of PPAs in COS-7 Cells as compared with
PEI and PLL;
[0029] FIG. 5. Gel electrophoretic analysis of the complexation of
PPAs with DNA;
[0030] FIG. 6. In vitro transfection efficiency of PPA-DNA
coacervates in HEK 293 cells;
[0031] FIG. 7. In vitro transfection efficiency of P5-SP-DNA
coacervates in HEK 293 cells at different charge ratios (+/-);
[0032] FIG. 8. Transfection of several cell lines using different
polymeric carriers and PRE-Luciferase as a model plasmid. P5-SP-DNA
coacervates are tested with different charge ratios (5 and 10 for
CaCo-2 cells, HeLa cells and HUH 7 cells; 7.5 and 10 for HEK293 HEK
293 cells, COS-7 cells and HepG2 cells); and
[0033] FIG. 9. Transfection mediated by PPA-SP/PPA-DMA mixtures at
different molar ratios in COS-7 cells and HeLa cells compared with
PPA-SP and PPA-DMA alone.
DETAILED DESCRIPTION OF THE INVENTION
[0034] This invention discloses a new class of cationic
biodegradable polymers containing phosphoester group in the
backbone and chargeable groups linked to the backbone through a
phosphoramidate linkage, e.g., a P--N bond. The biodegradable
polyphosphoramidate of the invention comprise the recurring
monomeric units shown in the Formula I:
##STR00001##
wherein:
[0035] R.sub.1 is a divalent organic moiety that is aliphatic,
aromatic or heterocyclic;
[0036] R.sub.2 and R.sub.3 are each independently selected from the
group consisting of hydrogen, alkyl, aryl, heteroaryl,
heteroalicyclic, cycloalkyl, aralkyl or cycloalkylalkyl;
[0037] each non-hydrogen occurrence of R.sub.2 and R.sub.3 is
substituted with one or more positively chargeable functional
groups (e.g., primary amino group, secondary amino group, tertiary
amino group and quaternary amino group, etc.); and n is 5 to
2,000.
[0038] According to a specific embodiment of the polymers of the
present disclosure, the biodegradable polyphosphoramidate of the
invention comprises the recurring monomeric unit shown in Formula I
where:
[0039] R.sub.1 is a divalent aliphatic organic moiety;
[0040] R.sub.2 and R.sub.3 are each independently selected from the
group consisting of hydrogen, alkyl, aryl, heteroaryl,
heteroalicyclic, cycloalkyl, aralkyl, and cycloalkylalkyl
groups;
[0041] each non-hydrogen occurrence of R.sub.2 and R.sub.3 is
substituted with one or more positively charged groups; and
[0042] n is from 20 to 2,000;
[0043] wherein when R.sub.1 is --(CH.sub.2).sub.n-- and one of
R.sub.2 and R.sub.3 is hydrogen, the other of R.sub.3 and R.sub.2
is selected from the group consisting of hydrogen, aryl,
heteroaryl, heteroalicylcic, cycloalkyl, aralkyl, and
cycloalkylaklyl; and
[0044] wherein .alpha. ranges from 2 to 6.
[0045] Particularly preferred polymers according to Formula I
include polymers of Formula II:
##STR00002##
wherein:
[0046] n, R.sub.2 and R.sub.3 are as defined in Formula I;
[0047] R.sub.4 and R.sub.5 are independently selected from the
group consisting of hydrogen, alkyl, cycloalkyl, alkoxy, aryl,
heteroaryl, heteroalicyclic, aralkyl, a steroid derivative; and
[0048] q is an integer from about 1 to about 5.
[0049] Preferred positively chargeable biodegradable polymers of
the invention are capable of forming a complex with biologically
active substances. Preferred biologically active substances include
DNA, RNA, proteins, small molecule therapeutics, and the like.
[0050] Other preferred positively chargeable biodegradable polymers
of the invention include polymers capable of complexing 20-60% by
weight of a biologically active substance such as DNA, RNA,
proteins, small molecule therapeutics, and the like.
[0051] Furthermore, preferred positively chargeable biodegradable
polymers of the invention include polymers having between about 5
and about 2,000 phosphoramidate groups, more preferably between
about 10 and about 1,500 phosphoramidate groups, and particularly
preferred are polymers having between about 20 and 1,000
phosphoramidate groups. Also preferred are polymers having a
molecular weight of between about 1,000 and 500,000, more
preferably having a molecular weight of between about 2,000 and
200,000, particularly preferable are polymers having a molecular
weight of between about 2,000 and 100,000.
[0052] In additional preferred embodiments, positively chargeable
biodegradable polymers of the invention further comprise one or
more groups that facilitate intracellular or extracellular delivery
of a biologically active substance. Preferred groups for
facilitating intracellular delivery of a biologically active
substance include a lysosomalytic agent, an amphiphilic peptide, a
steroid derivative, and the like.
[0053] In preferred embodiments, the biodegradable
polyphosphoramidate polymers of the invention, including polymers
according to Formula I or Formula II, are biocompatible before and
upon degradation.
[0054] In preferred embodiments, the biologically active substance
is negatively charged. Preferred biologically active substances
include anionic groups such as phosphate groups, carboxylate
groups, sulfate groups and other negatively charged bio-compatible
groups.
[0055] In another embodiment, the invention features a coacervate
system useful for the delivery of bioactive macromolecules
comprising the biodegradable polymer of Formula I.
[0056] In another embodiment, the invention features polymer
conjugates comprising polymers of Formula I and bioactive ligands
that could facilitate cell uptake and intracellular trafficking
steps.
[0057] In another embodiment of the invention, coacervate systems
useful for delivery of nucleic acids (DNA or RNA) and/or protein
drugs and comprise the biodegradable polymer of Formula I or the
above-described polymer conjugates are described.
[0058] In a further embodiment, the invention contemplates a
process of making polymeric coacervates for delivery of protein
drugs or nucleic acid.
[0059] This invention also describes a number of procedures for
preparing the biodegradable polymers described above.
[0060] The biodegradable polymers could be copolymers having one or
several different monomeric recurring units described in Formula
I.
[0061] A lipophilic moiety, e.g., a group bearing cholesterol
structural or lipid, could be conjugated to the carriers to enhance
the interaction between complexes and cell membrane therefore
facilitates cell uptake.
[0062] An endolysosomalytic agent, e.g., an amphiphilic peptide,
could be conjugated to the carriers to enhance the endosomal escape
after cell uptake step.
[0063] A nucleus localization signal could be conjugated to the
carriers to facilitate the nucleus translocation.
[0064] It is a discovery of the present invention that nucleic acid
molecules of various chain lengths can complex with these
biodegradable polymers of Formula I in aqueous conditions to form
coacervates or solid microparticles ranging from submicron to
microns in size. These coacervates containing nucleic acids, when
appropriately targeted, can transfect cells with phagocytic
activity.
[0065] According to the present invention, other molecules,
especially those that carry charges and have relatively higher
molecular weights could also be incorporated into the
complexes/coacervates.
[0066] In a further embodiment, the invention contemplates a
process of making polymeric coacervates for delivery of bioactive
macromolecules.
[0067] In yet another embodiment, the invention comprises articles
comprising one or several different polymers with structures shown
in Formula I and bioactive substances, e.g., nucleic acids and
other negatively charged macromolecules for sustained release of
these bioactive substances in-vivo and/or in-vitro. Additionally,
the bioactive substances can be released in a controlled, sustained
manner either intracellularly or extracellularly.
[0068] In a still further embodiment, the invention contemplates a
process for preparing biodegradable polyphosphoramidates, which
comprises a step of reacting a polymer shown in Formula III,
wherein X is a halogen and R.sub.1 is as defined in Formula I, with
a primary or secondary amine having a general structure as
R.sub.2R.sub.3NH, wherein R.sub.2 and R.sub.3 are each
independently selected from the group consisting of hydrogen,
alkyl, aryl, heteroaryl, heteroalicyclic, cycloalkyl, aralkyl or
cycloalkylalkyl wherein each non-hydrogen occurrence of R.sub.2 and
R.sub.3 is substituted with one or more positively chargeable
functional groups (e.g., primary amino group, secondary amino
group, tertiary amino group and quaternary amino group, etc.).
##STR00003##
[0069] In specific embodiments, one or more charged groups that are
present in R.sub.2 or R.sub.3 are capable of reacting with a
P-halogen bond. Preferably, such reactive positively chargeable
groups are protected using standard organic chemistry protecting
group techniques to prevent reaction of such groups with the P--X
bond. The protected primary or secondary amine, R.sub.2R.sub.3NH,
is then reacted with the polymer of Formula III where X is a
halogen. In particularly preferred embodiments, reactive positively
chargeable groups include primary or secondary amine groups which
are protected using standard amine protection methodologies.
[0070] In other preferred embodiments, phosphoramidate polymers of
the invention can be prepared by formation of a P--N linkage by
reacting a polymer of Formula II wherein X is hydrogen with a
primary or secondary amine in a polar aprotic solvent mixture such
as DMF/CCl.sub.4 to Scheme 1.
##STR00004##
[0071] The biodegradable polymeric system described in the present
invention achieves sustained and localized delivery of one or more
therapeutic agents to a designated biological tissue or site in a
patient. In particular, the polymeric system of the invention
achieve sustained and localized delivery of one or more genes in
skeletal muscles or intradermally and achieve a higher gene
transfer efficiencies than other plasmid delivery systems currently
under investigation. The biodegradable polymeric carriers described
in the present invention achieve gene transfer efficiencies in
vitro and in vivo that are superior to other polycationic carriers
currently under investigation.
[0072] The polyphosphoramidate carriers of the present invention
typically offer the following advantages over other biodegradable
carriers described in the literatures and patents:
[0073] Polyphosphoramidate polymers of the invention are
biodegradable wherein the polymers have a cleavable backbone,
either hydrolytically or enzymatically. The two most effective
polymeric carriers currently available, PEI and various dendrimeric
materials, are not biodegradable and their fate, in vivo, after
administration is still unclear.
[0074] Polyphosphoramidate polymers of the invention are
biocompatible before, during and after biodegradation.
Biodegradation breakdown products are typically non-toxic. The
polyphosphoramidate polymers of the invention are less cytotoxic
than poly-L-lysine, PEI and liposome compositions in vitro. In a
preferred embodiment, polymers of Formula I are degraded to
phosphate, 1,2-propanediol and amines R.sub.2R.sub.3NH. By prudent
selection of the side chains, the polymer potentially has minimal
toxicity before and upon degradation.
[0075] Polyphosphoramidate polymers described in the present patent
have higher molecule weight than most other biodegradable carriers
reported in the literatures whose number average molecular weights
are in the range of 3,000 to 9,000. The biodegradable polymers
described here generally have average molecular weights in the
range of 10,000 to 500,000. Higher molecular weight of the
polymeric carriers generally increases the binding capacity of the
carriers such that the polymers of the invention typically exhibit
superior uptake of DNA and protein.
[0076] The structures of polyphosphoramidate polymers are
tailorable to have variable charged groups with different pKb,
different charge density, molecular weight,
hydrophilicity/hydrophobicity balance to optimize the transfection
activity of the carriers. An endolysosomalytic agent, e.g. an
amphiphilic peptide, may be conjugated to the carriers to enhance
the endosomal escape after cell uptake step. A lipophilic moiety,
e.g. a group bearing cholesterol structural or lipid, may be
conjugated to the carriers to enhance the interaction between
complexes and cell membrane therefore facilitate cell uptake. A
nucleus localization signal could be conjugated to the carriers to
facilitate the nucleus translocation.
[0077] Polyphosphoramidates suitable for use in the invention may
be modified to comprise one or more specific ligands conjugated to
the side chain or as a side chain group to enhance the cellular
uptake of one or more bioactive molecules (nucleic acids and
proteins) dispersed in carrier polymer and/or achieve tissue/cell
specific delivery of the bioactive cargo.
[0078] Polyphosphoramidate polymers suitable for use in the methods
of the invention typically posses higher molecular weights than
polymeric carriers disclosed in the art such that
complexes/coacervates comprising the polyphosphoramidates of the
invention are more stable than other polycationic materials with
lower molecular weights.
[0079] Polyphosphoramidate polymers and compositions comprising at
least one bioactive molecule and a polyphosphoramidate polymer are
prepared by reproducible and easily scalable procedures.
[0080] An attractive coacervate delivery system requires a delicate
balance among factors such as the simplicity of preparation, cost
effectiveness, nucleic acids loading level, controlled release
ability, storage stability, and immunogenicity of the components.
The gene delivery system described here may offer advantages
compared to other particulate delivery systems, including the
liposomal system. The problems of instability, low loading level,
and controlled release ability are better resolved with these
polymeric systems. Compared to other synthetic polymeric systems,
such as the extensively studied polylactic/polyglycolic copolymers,
the mild conditions of coacervate formulation are appealing. Unlike
the solvent evaporation and hot-melt techniques used to formulate
synthetic polymeric coacervates, complex coacervation requires
neither contact with organic solvents nor heat. It is also
particularly suitable for encapsulating bio-macromolecules such as
nucleic acids and proteins not only through passive solvent
capturing but also by direct charge-charge interactions.
[0081] Targeting ligands can be directly bound to the surface of
the coacervates. Alternatively, such ligands can be conjugated to
the polymeric carriers to form molecular conjugates, which then
complex with nucleic acids and/or proteins. Targeting ligands
according to the present invention are any molecules, which bind to
specific types of cells in the body. These may be any types of
molecules for which a cellular receptor exists. Preferably the
cellular receptors are expressed on specific cell types only.
Examples of targeting ligands that may be used are hormones,
antibodies, cell-adhesion molecules, oligosaccharides, drugs, and
neurotransmitters.
[0082] The method of the present invention involves a coacervation
process described in U.S. Pat. No. 5,972,707 (Roy, et al., 1999,
Gene Delivery System) and U.S. Pat. No. 6,025,337 (Truong, et al.,
2000, Solid Microparticles for Gene Delivery). The process is
optimized in this invention to best suit the complexation of
nucleic acids and biodegradable carriers of Formula I.
[0083] It is a discovery of the present invention that different
polymers with different charged groups, e.g., different amino
groups with a wide range of acidity (pKb), could be included into
one coacervate/complex system for the intracellular delivery. Such
a system could offer buffering capacity similar to that of PEI.
[0084] Polyphosphoramidates suitable for use in the methods of the
present invention include any and all different single pure isomers
and mixtures of two or more isomers. The term isomer is intended to
include diastereoisomers, enantiomers, regioisomers, structural
isomers, rotational isomers, tautomers, and the like. For compounds
which contain one or more stereogenic centers, e.g., chiral
compounds, the methods of the invention may be carried out with a
enantiomerically enriched compound, a racemate, or a mixture of
diastereomers. Preferred enantiomerically enriched compounds have
an enantiomeric excess of 50% or more, more preferably the compound
has an enantiomeric excess of 60%, 70%, 80%, 90%, 95%, 98%, or 99%
or more.
[0085] Polyphosphoramidates suitable for use in the methods of the
present invention include any and all molecular weight distribution
profiles, i.e., polymers having a M.sub.w, or M.sub.n of between 1
and about 50, more typically a M.sub.w, or M.sub.n between about
1.2 and about 10. Moreover, polyphosphoramidates of the invention
have a polydispersity index of between about 1 and about 5.
[0086] As also discussed above, typical subjects for administration
in accordance with the invention are mammals, such as primates,
especially humans.
[0087] Biodegradable polymers differ from non-biodegradable
polymers in that they can be degraded during in vivo therapy. This
generally involves breaking down the polymer into its monomeric
subunits. In principle, the ultimate hydrolytic breakdown products
of polymers suitable for use in the methods of the present
invention should be biocompatible, non-toxic and easily excreted
from a patient's body. However, the intermediate oligomeric
products of the hydrolysis may have different properties. Thus,
toxicology of a biodegradable polymer intended for implantation or
injection, even one synthesized from apparently innocuous monomeric
structures, is typically determined after one or more toxicity
analyses.
[0088] The biodegradable polymer of the invention is preferably
sufficiently pure to be biocompatible itself and remains
biocompatible upon biodegradation. "Biocompatible" is defined to
mean that the biodegradation products and/or the polymer itself are
nontoxic and result in only minimal tissue irritation when
instilled in the bladder or transported or otherwise localized to
other tissues within a patient.
[0089] It will be appreciated that the actual preferred amounts of
therapeutic agent or other component used in a given composition
will vary according to the therapeutic agent being utilized
including the polymer system being employed, the mode of
application, the particular site of administration, etc. Optimal
administration rates for a given protocol of administration can be
readily ascertained by those skilled in the art using conventional
dosage determination tests conducted with regard to the foregoing
guidelines.
[0090] As used herein, "alkyl" is intended to include branched,
straight-chain and cyclic saturated aliphatic hydrocarbon groups
including alkylene, having the specified number of carbon atoms.
Examples of alkyl include, but are not limited to, methyl, ethyl,
n-propyl, i-propyl, n-butyl, s-butyl, t-butyl, n-pentyl, and
s-pentyl. Alkyl groups typically have 1 to about 16 carbon atoms,
more typically 1 to about 20 or 1 to about 12 carbon atoms.
Preferred alkyl groups are C.sub.1-C.sub.20 alkyl groups, more
preferred are C.sub.1-12-alkyl and C.sub.1-6-alkyl groups.
Especially preferred alkyl groups are methyl, ethyl, and
propyl.
[0091] As used herein, "heteroalkyl" is intended to include
branched, straight-chain and cyclic saturated aliphatic hydrocarbon
groups including alkylene, having the specified number of carbon
atoms and at least one heteroatom, e.g., N, O or S. Heteroalkyl
groups will typically have between about 1 and about 20 carbon
atoms and about 1 to about 8 heteroatoms, preferably about 1 to
about 12 carbon atoms and about 1 to about 4 heteroatoms. Preferred
heteroalkyl groups include the following groups. Preferred
alkylthio groups include those groups having one or more thioether
linkages and from 1 to about 12 carbon atoms, more preferably from
1 to about 8 carbon atoms, and still more preferably from 1 to
about 6 carbon atoms. Alylthio groups having 1, 2, 3, or 4 carbon
atoms are particularly preferred. Preferred alkylsulfinyl groups
include those groups having one or more sulfoxide (SO) groups and
from 1 to about 12 carbon atoms, more preferably from 1 to about 8
carbon atoms, and still more preferably from 1 to about 6 carbon
atoms. Alkylsulfinyl groups having 1, 2, 3, or 4 carbon atoms are
particularly preferred. Preferred alkylsulfonyl groups include
those groups having one or more sulfonyl (SO.sub.2) groups and from
1 to about 12 carbon atoms, more preferably from 1 to about 8
carbon atoms, and still more preferably from 1 to about 6 carbon
atoms. Alylsulfonyl groups having 1, 2, 3, or 4 carbon atoms are
particularly preferred. Preferred aminoalkyl groups include those
groups having one or more primary, secondary and/or tertiary amine
groups, and from 1 to about 12 carbon atoms, more preferably from 1
to about 8 carbon atoms, and still more preferably from 1 to about
6 carbon atoms. Aminoalkyl groups having 1, 2, 3, or 4 carbon atoms
are particularly preferred.
[0092] As used herein, "heteroalkenyl" is intended to include
branched, straight-chain and cyclic saturated aliphatic hydrocarbon
groups including alkenylene, having the specified number of carbon
atoms and at least one heteroatom, e.g., N, O or S. Heteroalkenyl
groups will typically have between about 1 and about 20 carbon
atoms and about 1 to about 8 heteroatoms, preferably about 1 to
about 12 carbon atoms and about 1 to about 4 heteroatoms. Preferred
heteroalkenyl groups include the following groups. Preferred
alkylthio groups include those groups having one or more thioether
linkages and from 1 to about 12 carbon atoms, more preferably from
1 to about 8 carbon atoms, and still more preferably from 1 to
about 6 carbon atoms. Alkenylthio groups having 1, 2, 3, or 4
carbon atoms are particularly preferred. Preferred alkenylsulfinyl
groups include those groups having one or more sulfoxide (SO)
groups and from 1 to about 12 carbon atoms, more preferably from 1
to about 8 carbon atoms, and still more preferably from 1 to about
6 carbon atoms. Alkenylsulfinyl groups having 1, 2, 3, or 4 carbon
atoms are particularly preferred. Preferred alkenylsulfonyl groups
include those groups having one or more sulfonyl (SO.sub.2) groups
and from 1 to about 12 carbon atoms, more preferably from 1 to
about 8 carbon atoms, and still more preferably from 1 to about 6
carbon atoms. Alkenylsulfonyl groups having 1, 2, 3, or 4 carbon
atoms are particularly preferred. Preferred aminoalkenyl groups
include those groups having one or more primary, secondary and/or
tertiary amine groups, and from 1 to about 12 carbon atoms, more
preferably from 1 to about 8 carbon atoms, and still more
preferably from 1 to about 6 carbon atoms. Aminoalkenyl groups
having 1, 2, 3, or 4 carbon atoms are particularly preferred.
[0093] As used herein, "heteroalkynyl" is intended to include
branched, straight-chain and cyclic saturated aliphatic hydrocarbon
groups including alkynylene, having the specified number of carbon
atoms and at least one heteroatom, e.g., N, O or S. Heteroalkynyl
groups will typically have between about 1 and about 20 carbon
atoms and about 1 to about 8 heteroatoms, preferably about 1 to
about 12 carbon atoms and about 1 to about 4 heteroatoms. Preferred
heteroalkynyl groups include the following groups. Preferred
alkynylthio groups include those groups having one or more
thioether linkages and from 1 to about 12 carbon atoms, more
preferably from 1 to about 8 carbon atoms, and still more
preferably from 1 to about 6 carbon atoms. Alkynylthio groups
having 1, 2, 3, or 4 carbon atoms are particularly preferred.
Prefered alkynylsulfinyl groups include those groups having one or
more sulfoxide (SO) groups and from 1 to about 12 carbon atoms,
more preferably from 1 to about 8 carbon atoms, and still more
preferably from 1 to about 6 carbon atoms. Alkynylsulfinyl groups
having 1, 2, 3, or 4 carbon atoms are particularly preferred.
Preferred alkynylsulfonyl groups include those groups having one or
more sulfonyl (SO.sub.2) groups and from 1 to about 12 carbon
atoms, more preferably from 1 to about 8 carbon atoms, and still
more preferably from 1 to about 6 carbon atoms. Alkynylsulfonyl
groups having 1, 2, 3, or 4 carbon atoms are particularly
preferred. Preferred aminoalkynyl groups include those groups
having one or more primary, secondary and/or tertiary amine groups,
and from 1 to about 12 carbon atoms, more preferably from 1 to
about 8 carbon atoms, and still more preferably from 1 to about 6
carbon atoms. Aminoalkynyl groups having 1, 2, 3, or 4 carbon atoms
are particularly preferred.
[0094] As used herein, "cycloalkyl" is intended to include
saturated ring groups, having the specified number of carbon atoms,
such as cyclopropyl, cyclobutyl, cyclopentyl, or cyclohexyl.
Cycloalkyl groups typically will have 3 to about 8 ring
members.
[0095] In the term "(C.sub.3-6 cycloalkyl)C.sub.1-4 alkyl", as
defined above, the point of attachment is on the alkyl group. This
term encompasses, but is not limited to, cyclopropylmethyl,
cyclohexylmethyl, cyclohexylmethyl.
[0096] As used here, "alkenyl" is intended to include hydrocarbon
chains of straight, cyclic or branched configuration, including
alkenylene, and one or more unsaturated carbon-carbon bonds which
may occur in any stable point along the chain, such as ethenyl and
propenyl. Alkenyl groups typically will have 2 to about 12 carbon
atoms, more typically 2 to about 12 carbon atoms.
[0097] As used herein, "alkynyl" is intended to include hydrocarbon
chains of straight, cyclic or branched configuration, including
alkynylene, and one or more triple carbon-carbon bonds which may
occur in any stable point along the chain, such as ethynyl and
propynyl. Alkynyl groups typically will have 2 to about 20 carbon
atoms, more typically 2 to about 12 carbon atoms.
[0098] As used herein, "haloalkyl" is intended to include both
branched and straight-chain saturated aliphatic hydrocarbon groups
having the specified number of carbon atoms, substituted with 1 or
more halogen (for example --C.sub.vF.sub.w where v=1 to 3 and w=1
to (2v+1). Examples of haloalkyl include, but are not limited to,
trifluoromethyl, trichloromethyl, pentafluoroethyl, and
pentachloroethyl. Typical haloalkyl groups will have 1 to about 16
carbon atoms, more typically 1 to about 12 carbon atoms.
[0099] As used herein, "alkoxy" represents an alkyl group as
defined above with the indicated number of carbon atoms attached
through an oxygen bridge. Examples of alkoxy include, but are not
limited to, methoxy, ethoxy, n-propoxy, i-propoxy, n-butoxy,
2-butoxy, t-butoxy, n-pentoxy, 2-pentoxy, 3-pentoxy, isopentoxy,
neopentoxy, n-hexoxy, 2-hexoxy, 3-hexoxy, and 3-methylpentoxy.
Alkoxy groups typically have 1 to about 16 carbon atoms, more
typically 1 to about 12 carbon atoms.
[0100] "Prodrugs" are intended to include any covalently bonded
carriers which release the active parent drug according to Formula
I in vivo when such prodrug is administered to a mammalian subject.
Prodrugs of a compound are prepared by modifying functional groups
present in the drug compound in such a way that the modifications
are cleaved, either in routine manipulation or in vivo, to the
parent compound.
[0101] Combinations of substituents and/or variables are
permissible only if such combinations result in stable compounds. A
stable compound or stable structure is meant to imply a compound
that is sufficiently robust to survive isolation to a useful degree
of purity from a reaction mixture, and formulation into an
effective therapeutic agent.
[0102] As used herein, the term "aliphatic" refers to a linear,
branched, cyclic alkane, alkene, or alkyne. Preferred aliphatic
groups in the poly(phosphoester-co-amide) polymer of the invention
are linear or branched and have from 1 to 20 carbon atoms.
[0103] As used herein, the term "aryl" refers to an unsaturated
cyclic carbon compound with 4n+2 electrons where n is a
non-negative integer, about 5-18 aromatic ring atoms and about 1 to
about 3 aromatic rings.
[0104] As used herein, the term "heterocyclic" refers to a
saturated or unsaturated ring compound having one or more atoms
other than carbon in the ring, for example, nitrogen, oxygen or
sulfur.
[0105] The polymers of the invention are usually characterized by a
release rate of the therapeutic agent in vivo that is controlled at
least in part as a function of hydrolysis of the phosphoester bond
of the polymer during biodegradation. Additionally, the therapeutic
agent to be released may be conjugated to the sidechain of the
phosphramidate repeat unit to form a pendant drug delivery system.
Further, other factors are also important.
[0106] The life of a biodegradable polymer in vivo also depends
upon its molecular weight, crystallinity, biostability, and the
degree of cross-linking. In general, the greater the molecular
weight, the higher the degree of crystallinity, and the greater the
biostability, the slower biodegradation will be.
[0107] The therapeutic agent of the invention can vary widely with
the purpose for the composition. The agent(s) may be described as a
single entity or a combination of entities. The delivery system is
designed to be used with therapeutic agents having high
water-solubility as well as with those having low water-solubility
to produce a delivery system that has controlled release rates. The
terms "therapeutic agent" and "biologically active substance"
include without limitation, medicaments; vitamins; mineral
supplements; substances used for the treatment, prevention,
diagnosis, cure or mitigation of disease or illness; or substances
which affect the structure or function of the body; or prodrugs,
which become biologically active or more active after they have
been placed in a predetermined physiological environment.
[0108] Non-limiting examples of useful therapeutic agents and
biologically active substances include the following expanded
therapeutic categories: anabolic agents, antacids, anti-asthmatic
agents, anti-cholesterolemic and anti-lipid agents,
anti-coagulants, anti-convulsants, anti-diarrheals, anti-emetics,
anti-infective agents, anti-inflammatory agents, anti-manic agents,
anti-nauseants, anti-neoplastic agents, anti-obesity agents,
anti-pyretic and analgesic agents, anti-spasmodic agents,
anti-thrombotic agents, anti-uricemic agents, anti-anginal agents,
antihistamines, anti[[-]]tussives, appetite suppressants,
biologicals, cerebral dilators, coronary dilators, decongestants,
diuretics, diagnostic agents, erythropoietic agents, expectorants,
gastrointestinal sedatives, hyperglycemic agents, hypnotics,
hypoglycemic agents, ion exchange resins, laxatives, mineral
supplements, mucolytic agents, neuromuscular drugs, peripheral
vasodilators, psychotropics, sedatives, stimulants, thyroid and
anti-thyroid agents, uterine relaxants, vitamins, antigenic
materials, and prodrugs.
[0109] Specific examples of useful therapeutic agents and
biologically active substances, i.e., bioactive molecules, from the
above categories include: (a) anti-neoplastics such as androgen
inhibitors, antimetabolites, cytotoxic agents, immunomodulators;
(b) anti-tussives such as dextromethorphan, dextromethorphan
hydrobromide, noscapine, carbetapentane citrate, and chlophedianol
hydrochloride; (c) antihistamines such as chlorpheniramine maleate,
phenindamine tartrate, pyrilamine maleate, doxylamine succinate,
and phenyltoloxamine citrate; (d) decongestants such as
phenylephrine hydrochloride, phenylpropanolamine hydrochloride,
pseudoephedrine hydrochloride, and ephedrine; (e) various alkaloids
such as codeine phosphate, codeine sulfate and morphine; (f)
mineral supplements such as potassium chloride, zinc chloride,
calcium carbonates, magnesium oxide, and other alkali metal and
alkaline earth metal salts; (g) ion exchange resins such as
cholestryramine; (h) anti-arrhythmics such as N-acetylprocainamide;
(i) antipyretics and analgesics such as acetaminophen, aspirin and
ibuprofen; (j) appetite suppressants such as phenyl-propanolamine
hydrochloride or caffeine; (k) expectorants such as guaifenesin;
(l) antacids such as aluminum hydroxide and magnesium hydroxide;
(m) biologicals such as peptides, polypeptides, proteins and amino
acids, hormones, interferons or cytokines and other bioactive
peptidic compounds, such as hGH, tPA, calcitonin, ANF, EPO and
insulin; (n) anti-infective agents such as anti-fungals,
anti-virals, antiseptics and antibiotics; and (o) antigenic
materials, partricularly those useful in vaccine applications.
[0110] Preferably, the therapeutic agent or biologically active
substance is selected from the group consisting of DNA,
polysaccharides, growth factors, hormones, anti-angiogenesis
factors, interferons or cytokines, and prodrugs. In a particularly
preferred embodiment, the therapeutic agent is a DNA vaccine
comprising a DNA sequence encoding an antigen, a DNA sequence
encoding a cytokine or a mixture of DNA sequences encoding an
antigen and a cytokine.
[0111] The therapeutic agents are used in amounts that are
therapeutically effective. While the effective amount of a
therapeutic agent will depend on the particular material being
used, amounts of the therapeutic agent from about 1% to about 65%
have been easily incorporated into the present delivery systems
while achieving controlled release. Lesser amounts may be used to
achieve efficacious levels of treatment for certain therapeutic
agents.
[0112] In addition, the polymer composition of the invention can
also comprise polymer blends of the polymer of the invention with
other biocompatible polymers, so long as they do not interfere
undesirably with the biodegradable characteristics of the
composition. Blends of the polymer of the invention with such other
polymers may offer even greater flexibility in designing the
precise release profile desired for targeted drug delivery or the
precise rate of biodegradability desired for structural implants
such as for orthopedic applications. Examples of such additional
biocompatible polymers include other polycarbonates; polyesters;
polyorthoesters; polyamides; polyurethanes; poly(iminocarbonates);
and polyanhydrides.
[0113] As a drug delivery device, the polymer compositions of the
invention provide a polymeric matrix capable of sequestering a
biologically active substance and provide predictable, controlled
delivery of the substance. The polymeric matrix then degrades to
non-toxic residues.
[0114] It will be understood, however, that the specific dose level
for any particular patient will depend upon a variety of factors
including the activity of the specific compound employed, the age,
body weight, general health, sex, diet, time of administration,
route of administration, and rate of excretion, drug combination
(i.e., other drugs being administered to the patient), the severity
of the particular disease undergoing therapy, and other factors,
including the judgment of the prescribing medical practitioner.
[0115] A positively chargeable biodegradable polymer composition of
the invention also may be packaged together with instructions
(i.e., written, such as a written sheet) for treatment of a
disorder as disclosed herein, e.g., instruction for treatment of a
subject that is susceptible to or suffering from a disease or
disorder which may be treated by administration of a bioactive
molecule, e.g., therapeutic agent, dispersed in the positively
chargeable biodegradable polymer composition.
[0116] A positively chargeable biodegradable polymer composition of
the invention be administered parenterally, preferably in a sterile
non-toxic, pyrogen-free medium. The drug, depending on the vehicle
and concentration used, can either be suspended or dissolved in the
vehicle. Advantageously, adjuvants such as local anesthetics,
preservatives and buffering agents can be dissolved in the vehicle.
The term parenteral as used herein includes injections and the
like, such as subcutaneous, intradermal, intravascular (e.g.,
intravenous), intramuscular, intrasternal, spinal, intrathecal, and
like injection or infusion techniques, with subcutaneous,
intramuscular and intravascular injections or infusions being
preferred.
[0117] A positively chargeable biodegradable polymer composition of
the invention also may be packaged together with instructions
(i.e., written, such as a written sheet) for treatment of a
disorder as disclosed herein, e.g. instruction for treatment of a
subject that is susceptible to or suffering from inflammation,
cellular injury disorders, or immune system disorders.
[0118] The following examples are illustrative of the invention.
All documents mentioned herein are incorporated herein by
reference.
EXAMPLES
[0119] The following examples are offered by way of illustration
and are not intended to limit the invention in any manner.
Example 1
Synthesis and Characterization of Polyphosphoramidates
1.1 Synthesis of P5-SP (Structure Shown in FIG. 3)
[0120] The synthetic scheme of P5-SP is shown in FIG. 1.
Poly(4-methyl-2-oxo-2-hydro-1,3,2-dioxaphospholane) is synthesized
according to the procedure described in the literature (Biela T.
Penczek S, and Slomhowski S. 1982, Racemic and optimal active
poly(4-methyl-2-oxo-2-hydro-1,3,2-dioxaphospholane): Synthesis and
oxidation to the polyacids. Makromol. Chem. Rapid Commun. 3:
667-671). Briefly, 2-hydroxy-4-methyl-1,3,2-dioxaphospholane (58 g,
0.475 mol) [freshly freshly prepared according to Lucas' method
(Lucas H J, Mitchell F W, Jr., and Scully C N, 1950, Cyclic
phosphites of some aliphatic glycols. J. Am. Chem. Soc. 72:
5491-5497) is polymerized in 200 ml of freshly dried CHCl.sub.3 at
room temperature for 48 hours. Polymerization is initiated with
triisobutylaluminum (1 wt %, 4 ml of 15% solution in heptane). The
polymer is obtained by precipitation into anhydrous benzene. This
polymer becomes insoluble in chloroform after precipitation, but it
is soluble in anhydrous DMF.
[0121] Poly(4-methyl-2-oxo-2-hydro-1,3,2-dioxaphospholane) (1.094
g, 8.9 mmol P--H groups) is dissolved in anhydrous DMF (10 ml). To
this solution is added 5 ml of anhydrous CCl.sub.4 and
N.sup.1,N.sup.8-bis(trifluroacetyl)spermidine trifluroacetate (10.8
mmol, 4.9 g) in 10 ml of DMF using a syringe, followed by addition
of 5 ml of anhydrous triethylamine under ice-water bath. The
reaction is performed at 0.degree. C. for 30 minutes then at room
temperature overnight. The resulted solution is concentrated and
product is obtained by precipitating in water followed by drying
under vacuum.
[0122] The resulted polymer is suspended in 30 ml of concentrated
ammonia solution and the mixture is stirred at 60.degree. C. for 16
hours. The solution is concentrated and dialyzed against water
overnight using a dialysis tubing with a MWCO of 7,500. P5-SP is
obtained after lyophilizing the dialyzed solution. The structure of
P5-SP is confirmed by proton-NMR: 8 (ppm): 1.35-1.4 (d, 3H),
1.5-1.7 (4H), 1.75-1.95 (2H), 2.65-2.95 (4H), 3.0-3.2 (4H),
3.75-4.15 (m, 2H), 4.35-4.65 (d, b, 1H). FIG. 2 shows a typical
chromatograph by gel permeation chromatography analysis of P5-SP.
It is indicated that P5-SP synthesized has a weight average
molecular weight of 4.58.times.10.sup.4, and number average
molecular weight of 3.14.times.10.sup.4 (Polydispersity=1.46).
1.2 Synthesis of P5-DMA (Structure Shown in FIG. 3)
[0123] To a solution of
poly(4-methyl-2-oxo-2-hydro-1,3,2-dioxaphospholane) in anhydrous
DMF cooled with ice-water bath, was added 5 ml of anhydrous
CCl.sub.4 and N,N-dimethylethylenediamine (20% excess to P--H)
solution in, followed by addition of large excess of triethylamine.
The reaction was performed at 0.degree. C. for 30 minutes and at
room temperature overnight. P5-DMA was obtained by dialysis against
water using a dialysis member with a MWCO of 2,000.
[0124] To the solution of P5-DMA (100 mg) in 5 ml of methanol was
added CH.sub.3I (1 ml) and the mixture was allowed to stay at room
temperature overnight. The resulted solution was concentrated and
precipitated into ether. P5-TMA was obtained as yellowish powder.
The number average molecular weight of P5-TMA was
2.62.times.10.sup.4 as measured by GPC/LS/RI method.
[0125] P5-BA, P5-DEA and P5-TMA (Structures are shown in FIG. 3)
are synthesized according to a similar procedure.
Example 2
Assay for the Cytotoxicity of Polyphosphoramidates (PPAs)
[0126] Cytotoxicity of polyphosphoramidates (P5-SP, P5-BA, P5-DMA,
P5-DEA and P5-TMA) in comparison with other potential gene carriers
[poly-L-lysine (PLL) and polyethylenimine (PEI)] is evaluated using
the WST-1 dye reduction assay. COS-7 cells are seeded in a 96 well
plate 24 hours before the assay at the density of 5.times.10.sup.4
cells/well. The cells are incubated for 4 hours with 100 .mu.l of
DMEM medium complemented with 10% fetal bovine serum (FBS)
containing various PPAs, or PLL or PEI at different concentrations
ranging from 0 to 500 .mu.g/ml. The medium in each well is replaced
with 100 .mu.l of fresh complete medium and cells are cultured for
an additional 20 hrs. Ten microliters of WST-1 reagent (Roche
Molecular Biochemicals) is added to each well and allowed reacting
for 4 hrs at 37.degree. C. The absorbance of the supernatant at 450
nm (use 655 nm as a reference wavelength) is measured using a
microplate reader (Model 550, Bio-Rad Lab. Hercules, Calif.).
[0127] The assay results (FIG. 4) indicate that
polyphosphoramidates exhibit lower cytotoxicity in culture than
widely used polycationic carrier, PLL and PEI. The LD.sub.50 of PEI
in this assay is 20 .mu.g/ml, LD.sub.50 of PLL is 42 .mu.g/ml,
LD.sub.50 of P5-SP is 85 .mu.g/ml, LD.sub.50 of P5-BA is 300
.mu.g/ml, LD.sub.50 of DMA or DEA or TMA is well beyond 500
.mu.g/ml. It is clear that PPAs have lower cytotoxicity than PLL
and PEI. PPAs with tertiary amino group and quaternary amino groups
have the lowest cytotoxicity in this assay.
Example 3
Gel Retardation Assay for the DNA Binding Capacity of PPAs
[0128] The formation of PPA-DNA coacervates is examined by their
electrophoretic mobility on an agarose gel at various charge ratios
of PPAs to plasmid DNA (FIG. 5). No migration of the plasmid DNA
occurred at charge ratio larger than 1.0 (P5-DMA, P5-DEA and
P5-TMA) or 1.5 (P5-BA) or 2.0 (P5-SP). This lack of migration is
due to neutralization of the nucleic acid by PPAs, suggesting the
polycationic nature of PPAs. PPAs with tertiary amino groups and
quternary amino groups appear to have higher DNA binding capacity
at the same charge ratio.
Example 4
Preparation of PPA-DNA Coacervates and Coacervates with Chloroquine
Sulfate
[0129] PPA is dissolved in saline at a concentration of 2-10 mg/ml.
To this solution is added plasmid DNA dissolved in saline to yield
desired N/P ratios, followed by brief vortexing, and the mixture is
allowed to stand at room temperature for 30 minutes. The
coacervates prepared according to this procedure are used directly
for transfection study unless stated otherwise. The efficiency of
complexation of DNA is close to 100% when the N/P ratio is over 1.0
as revealed by gel electrophoretic mobility analysis.
[0130] Chloroquine sulfate (CQ) has been widely proven to be an
effective reagent to disrupt lysosomes and enhance the transfection
efficiency in many polycationic gene delivery systems. CQ is
co-encapsulated into the coacervates simply by incorporating CQ
into the PPA solution and then forming coacervates according to the
same procedure. The CQ incorporated coacervates are used for in
vitro transfection without further purification since the total
amount of CQ added is still within the non-toxic concentration
range.
Example 5
Transfection Efficiency of PCEP-DNA Complex in Different Cell
Lines
[0131] In vitro transfection of HEK 293 cells with PPA-DNA
coacervates is evaluated using luciferase as a marker gene. Cells
are seeded 24 hours prior to transfection into a 24-well plate
(Becton-Dickinson, Lincoln Park, N.J.) at a density of
8.times.10.sup.4 per well with 1 ml of complete medium (DMEM
containing 10% FBS, supplemented with 2 mM L-glutamate, 50 units/ml
penicillin and 50 .mu.g/ml streptomycin). At the time of
transfection, the medium in each well is replaced with 1 ml of
serum free DMEM. PPA-DNA coacervates or PEI-DNA complexes or
PLL-DNA complexes or Transfast.TM.-DNA complexes are incubated with
the cells for 3 hours at 37.degree. C. The medium is replaced with
1 ml of fresh complete medium and cells are further incubated for
48 hours. All the transfection tests are performed in triplicate.
After the incubation, cells are permeabilized with 200 .mu.l of
cell lysis buffer (Promega Co., Madison, Wis.). The luciferase
activity in cell extracts is measured using a luciferase assay kit
(Promega Co., Madison, Wis.) on a luminometer (Lumat9605, EG&G
Wallac). The light units (LU) are normalized against protein
concentration in the cell extracts, which is measured using BCA
protein assay kit (Pierce, Rockford, Ill.).
[0132] FIG. 6 shows the transfection efficiency of PPA-DNA
coacervates prepared from five different PPAs with 100 .mu.g/ml of
CQ or without CQ, comparing with PEI, PLL and Transfast.TM. as gene
carriers. Coacervates prepared with P5-SP in the presence of CQ
result in the highest transfection efficiency, similar to the level
obtained by Transfast.TM.-DNA complexes and PEI-DNA complexes.
Other PPAs only show moderate transfection activity. It is also
evident that CQ can enhance the transfection efficiency for about 4
times at a concentration of 40 .mu.g/ml of CQ, transfection
efficiency increases with dose and peaks at a dose of 80 .mu.g/ml
of CQ (data not shown). The following experiments are performed
with 100 .mu.g/ml of CQ incorporated in the coacervates.
[0133] As the gel electrophoresis analysis shows, at a +/- charge
ratio of 1.0 (P5-DMA, P5-DEA and P5-TMA) or 1.5 (P5-BA) or 2.0
(P5-SP) and above, all the plasmid DNA added to the preparation
mixture is complexed with PPAs. Coacervates prepared at different
charge ratios are also examined for their abilities to transfect
HEK 293 cells (FIG. 7). Although complete DNA incorporation occurs
at charge ratio of 2.0 and above for P5-SP, the highest level of
gene transfection is observed when the coacervates are synthesized
at the +/- charge ratios between 7.5 and above. Transfection
efficiency slightly decreases when the charge ratio is 12.5 and
above.
[0134] The transfection efficiency is measured against five other
cell lines using PPA-DNA coacervates containing pRE-Luciferase
plasmid (FIG. 8). Like in HEK 293 cells, the highest level of
luciferase expression in CaCo-2 cells, HeLa cells, HuH-7 cells,
COS-7 cells and HepG2 cells is also found to be at a +/- ratio
between 5 and 10. The transfection efficiency in CaCo-2 cells, HeLa
cells or HuH 7 cells is about 100 to 200 times higher than PLL
mediated transfection, and 10 to 50 times lower than that obtained
with PEI-DNA complexes. Transfection efficiency in COS-7 cells or
HepG2 cells is about 100 to 300 times higher than PLL mediated
transfection and 2 folds lower than that obtained with PEI-DNA
complexes.
Example 6
Gene Transfection Mediated by PPA Mixtures
[0135] Complexes comprising plasmid DNA and PPA mixture were
prepared according to a similar procedure as described in Example
4, except that PPA-SP and PPA-DMA were pre-mixed at different
ratios before complexation with plasmid DNA. DNA-polymer complexes
were formed by adding 50 of polymer solution containing varying
amounts of polymer to 50 .mu.l of vortexing pRE-luciferase (60
.mu.g/ml, in 0.9% NaCl, pH 7.4) and vortexed for 15-30 s. Complexes
were allowed to form for 30 minutes at room temperature. The
complexes were used for transfection study without further
purification.
[0136] This is based on the hypothesis that complexes containing
various types of amino groups would increase the buffering
capacity, thus improve the intracellular delivery of the DNA to
cytosol and nucleus. Transfection of COS-7 cells using PPA-SP
(containing primary amino group), PPA-DMA (containing tertiary
amino group) or PPA-SP/PPA-DMA mixture showed that PPA-SP/PPA-DMA
mixture mediated significantly higher levels of gene expression
than either polymer alone (FIG. 9, structures see FIG. 3).
Transfection was performed with 3 .mu.g DNA per well. The charge
ratio of total positive charges in PPA to negative charges in DNA
was maintained at 9. Under optimal condition (at a PPA-SP/PPA-DMA
molar ratio of 4 to 9), transfection efficiency achieved by
PPA-SP/PPA-DMA mixture was 20 and 160 times higher than PPA-SP and
PPA-DMA mediated transfection, respectively.
[0137] This method of introducing polymeric carriers with different
charged groups into the same complexes represents a simple yet
effective approach in developing polymeric gene carriers and
understanding the mechanisms of polymer mediated gene transfer.
* * * * *