U.S. patent number 8,629,151 [Application Number 12/788,266] was granted by the patent office on 2014-01-14 for immunomodulatory agent-polymeric compounds.
This patent grant is currently assigned to Selecta Biosciences, Inc.. The grantee listed for this patent is Sam Baldwin, Fen-ni Fu, Yun Gao, Lloyd Johnston, Mark J. Keegan, Grayson B. Lipford, Charles Zepp. Invention is credited to Sam Baldwin, Fen-ni Fu, Yun Gao, Lloyd Johnston, Mark J. Keegan, Grayson B. Lipford, Charles Zepp.
United States Patent |
8,629,151 |
Zepp , et al. |
January 14, 2014 |
**Please see images for:
( Certificate of Correction ) ** |
Immunomodulatory agent-polymeric compounds
Abstract
This invention relates to compositions, and related compounds
and methods, of conjugates of immunomodulatory agents and polymers
or unit(s) thereof. The conjugates may be contained within
synthetic nanocarriers, and the immunomodulatory agents may be
released from the synthetic nanocarriers in a pH sensitive
manner.
Inventors: |
Zepp; Charles (Hardwick,
MA), Lipford; Grayson B. (Watertown, MA), Gao; Yun
(Southborough, MA), Johnston; Lloyd (Belmont, MA), Fu;
Fen-ni (Northborough, MA), Keegan; Mark J. (Groton,
MA), Baldwin; Sam (Westford, MA) |
Applicant: |
Name |
City |
State |
Country |
Type |
Zepp; Charles
Lipford; Grayson B.
Gao; Yun
Johnston; Lloyd
Fu; Fen-ni
Keegan; Mark J.
Baldwin; Sam |
Hardwick
Watertown
Southborough
Belmont
Northborough
Groton
Westford |
MA
MA
MA
MA
MA
MA
MA |
US
US
US
US
US
US
US |
|
|
Assignee: |
Selecta Biosciences, Inc.
(Watertown, MA)
|
Family
ID: |
43012672 |
Appl.
No.: |
12/788,266 |
Filed: |
May 26, 2010 |
Prior Publication Data
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Document
Identifier |
Publication Date |
|
US 20110027217 A1 |
Feb 3, 2011 |
|
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
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61217129 |
May 27, 2009 |
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61217117 |
May 27, 2009 |
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61217124 |
May 27, 2009 |
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61217116 |
May 27, 2009 |
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Current U.S.
Class: |
514/263.1;
525/450; 525/437; 525/419; 525/410; 514/263.4; 525/411; 525/415;
525/462; 525/420 |
Current CPC
Class: |
A61K
47/60 (20170801); A61K 47/58 (20170801); A61P
29/00 (20180101); A61K 47/6937 (20170801); A61P
37/02 (20180101); A61K 47/6935 (20170801); A61K
47/593 (20170801); C08G 63/08 (20130101); C08G
63/912 (20130101); A61K 39/385 (20130101); A61P
25/30 (20180101); A61P 37/04 (20180101); C08G
63/06 (20130101); A61K 39/39 (20130101); A61P
31/00 (20180101); A61K 39/0013 (20130101); A61K
47/59 (20170801); A61P 3/00 (20180101); A61P
25/28 (20180101); A61P 25/34 (20180101); C07D
471/04 (20130101); A61P 43/00 (20180101); A61K
47/64 (20170801); A61P 35/00 (20180101); B82Y
5/00 (20130101); C08G 64/42 (20130101); A61K
9/5138 (20130101); C08J 3/24 (20130101); C07D
473/34 (20130101); A61K 47/6925 (20170801); A61K
2039/6093 (20130101); A61K 2039/55561 (20130101); A61K
2039/627 (20130101); A61K 2039/55511 (20130101); C08J
2367/04 (20130101); A61K 2039/62 (20130101); A61K
2039/55544 (20130101); A61K 2039/55555 (20130101) |
Current International
Class: |
A01N
43/90 (20060101) |
Field of
Search: |
;525/410,411,415,450,437,419,420,462 ;514/263.1,263.4 |
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|
Primary Examiner: Jones, Jr.; Robert
Attorney, Agent or Firm: Wolf, Greenfield & Sacks,
P.C.
Parent Case Text
RELATED APPLICATIONS
This application claims the benefit under 35 U.S.C. .sctn.119 of
U.S. provisional applications 61/217,129, 61/217,117, 61/217,124,
and 61/217,116, each filed May 27, 2009, the contents of each of
which are incorporated herein by reference in their entirety.
Claims
What is claimed is:
1. A compound that comprises a structure as in formula (I):
##STR00064## wherein R.sub.1=H, OH, SH, NH.sub.2, or substituted or
unsubstituted alkyl, alkoxy, alkylthio, or alkylamino; R.sub.2=H,
alkyl, or substituted alkyl; Y=N or C; R.sub.3 is absent if Y=N; or
is H, alkyl, substituted alkyl, or combined with R.sub.4 to form a
carbocycle or heterocycle with the carbon atoms of the pyridine
ring to which they are connected if Y=C; R.sub.4 is H, or
substituted or unsubstituted alkyl, alkoxy, alkylthio, or
alkylamino when not combined with R.sub.3 to form a carbocycle or
heterocycle with the carbon atoms of the pyridine ring to which
they are connected; or is combined with R.sub.3 to form a
carbocycle or heterocycle with the carbon atoms of the pyridine
ring to which they are connected; and R.sub.8 is a biodegradable
polymer or unit thereof, wherein the biodegradable polymer or unit
thereof comprises a polyester, polycarbonate, or a polyamide, or
unit thereof.
2. The compound of claim 1, wherein the biodegradable polymer or
unit thereof comprises poly(lactic acid), poly(glycolic acid),
poly(lactic-co-glycolic acid), or polycaprolacton, or unit
thereof.
3. A composition comprising the compound of claim 1.
4. A synthetic nanocarrier that comprises the compound of claim
1.
5. A composition comprising the synthetic nanocarrier of claim
4.
6. A composition comprising a vaccine comprising the compound of
claim 1.
7. A method comprising: administering the compound of claim 1 to a
subject.
8. A method for making a conjugate that comprises a structure as in
formula (I): ##STR00065## comprising: activating a biodegradable
polymer or unit thereof, and exposing the activated biodegradable
polymer or unit thereof and a compound comprising a structure as in
formula (III) to a base and/or solvent: ##STR00066## wherein
R.sub.1=H, OH, SH, NH.sub.2, or substituted or unsubstituted alkyl,
alkoxy, alkylthio, or alkylamino; R.sub.2=H, alkyl, or substituted
alkyl; Y=N or C; R.sub.3 is absent if Y=N; or is H, alkyl,
substituted alkyl, or combined with R.sub.4 to form a carbocycle or
heterocycle with the carbon atoms of the pyridine ring to which
they are connected if Y=C; R.sub.4 is H, or substituted or
unsubstituted alkyl, alkoxy, alkylthio, or alkylamino when not
combined with R.sub.3 to form a carbocycle or heterocycle with the
carbon atoms of the pyridine ring to which they are connected; or
is combined with R.sub.3 to form a carbocycle or heterocycle with
the carbon atoms of the pyridine ring to which they are connected;
and R.sub.8 is a biodegradable polymer or unit thereof.
9. A method for making a conjugate that comprises a structure as in
formula (I): ##STR00067## comprising: exposing a composition
comprising a polymer or unit thereof and a compound comprising a
structure as in formula (III) to a coupling agent and base and/or
solvent: ##STR00068## wherein R.sub.1=H, OH, SH, NH.sub.2, or
substituted or unsubstituted alkyl, alkoxy, alkylthio, or
alkylamino; R.sub.2=H, alkyl, or substituted alkyl; Y=N or C;
R.sub.3 is absent if Y=N; or is H, alkyl, substituted alkyl, or
combined with R.sub.4 to form a carbocycle or heterocycle with the
carbon atoms of the pyridine ring to which they are connected if
Y=C; R.sub.4 is H, or substituted or unsubstituted alkyl, alkoxy,
alkylthio, or alkylamino when not combined with R.sub.3 to form a
carbocycle or heterocycle with the carbon atoms of the pyridine
ring to which they are connected; or is combined with R.sub.3 to
form a carbocycle or heterocycle with the carbon atoms of the
pyridine ring to which they are connected; and R.sub.8 is a polymer
or unit thereof.
10. The compound of claim 1, wherein R1 is H, R2 is isobutyl, Y is
C, and R3 and R4 are combined to form a benzene ring with the
carbon atoms of the pyridine ring to which they are connected.
11. The compound of claim 1, wherein R1 is ethoxymethyl, R2 is
hydroxyisobutyl, Y=C, and R3 and R4 are combined to form a benzene
ring with the carbon atoms of the pyridine ring to which they are
connected.
12. The compound of claim 1, wherein R1 is ethoxymethyl, R2 is
methanesulfonamidoisobutyl, Y=C, and R3 and R4 are combined to form
a benzene ring with the carbon atoms of the pyridine ring to which
they are connected.
13. The compound of claim 1, wherein R1 is OH, R2 is benzyl, Y=N,
R3 is absent, and R4 is butoxy.
14. The compound of claim 1, wherein Y is N, R1 is OH, R2 is
benzyl, R3 is absent, and R4 is butylamino.
15. The compound of claim 1, wherein Y is N, R1 is OH, R2 is
benzyl, R3 is absent, and R4 is butoxy.
16. The compound of claim 1, wherein Y is N, R1 is OH, R2 is
benzyl, R3 is absent, and R4 is benzylamino.
17. The compound of claim 1, wherein Y is N, R1 is OH, R2 is
benzyl, R3 is absent, and R4 is pentyl.
18. The compound of claim 1, wherein the polymer is insoluble in
water at pH=7.4 and at 25.degree. C.
19. The compound of claim 1, wherein the polymer has a weight
average molecular weight ranging from 800 Daltons to 10,000
Daltons, as determined using gel permeation chromatography.
Description
FIELD OF THE INVENTION
This invention relates to compositions, and related compounds and
methods, of conjugates of immunomodulatory agents and polymers or
unit(s) thereof. The conjugates may be contained within synthetic
nanocarriers, and the immunomodulatory agents may be released from
the synthetic nanocarriers in a pH sensitive manner.
BACKGROUND
Immunomodulatory agents are used to produce immune responses in
subjects. It is at times advantageous to attach such agents to
delivery vehicles. Currently, known attachment chemistries often
require certain reactive groups, utilize certain activation steps
for attachment to occur, and/or result in conjugates that do not
exhibit optimal properties. There is a need, therefore, for new
methods for the attachment of immunomodulatory agents to delivery
vehicles as well as a need for the resulting conjugates that
exhibit desired properties.
SUMMARY OF THE INVENTION
In one aspect, the present invention provides a compound that
comprises a structure as in formula (I):
##STR00001## wherein R.sub.1=H, OH, SH, NH.sub.2, or substituted or
unsubstituted alkyl, alkoxy, alkylthio, or alkylamino; R.sub.2=H,
alkyl, or substituted alkyl; Y=N or C; R.sub.3 is absent if Y=N; or
is H, alkyl, substituted alkyl, or combined with R.sub.4 to form a
carbocycle or heterocycle with the carbon atoms of the pyridine
ring to which they are connected if Y=C; R.sub.4 is H, or
substituted or unsubstituted alkyl, alkoxy, alkylthio, or
alkylamino when not combined with R.sub.3 to form a carbocycle or
heterocycle with the carbon atoms of the pyridine ring to which
they are connected; or is combined with R.sub.3 to form a
carbocycle or heterocycle with the carbon atoms of the pyridine
ring to which they are connected; and R.sub.8 is a biodegradable
polymer or unit thereof. In one embodiment, for the compound of
formula (I), the biodegradable polymer or unit thereof comprises a
polyester, polycarbonate, or a polyamide, or unit thereof. In
another embodiment, the biodegradable polymer or unit thereof
comprises poly(lactic acid), poly(glycolic acid),
poly(lactic-co-glycolic acid), or polycaprolactone, or unit
thereof.
In another aspect, the present invention provides a compound that
comprises a structure as in formula (II):
##STR00002## wherein R.sub.1=H, OH, SH, NH.sub.2, or substituted or
unsubstituted alkyl, alkoxy, alkylthio, or alkylamino; R.sub.2=H,
alkyl, or substituted alkyl; Y=N or C; R.sub.3 is absent if Y=N; or
is H, alkyl, substituted alkyl, or combined with R.sub.4 to form a
carbocycle or heterocycle with the carbon atoms of the pyridine
ring to which they are connected if Y=C; R.sub.4 is H, or
substituted or unsubstituted alkyl, alkoxy, alkylthio, or
alkylamino when not combined with R.sub.3 to form a carbocycle or
heterocycle with the carbon atoms of the pyridine ring to which
they are connected; or is combined with R.sub.3 to form a
carbocycle or heterocycle with the carbon atoms of the pyridine
ring to which they are connected; R.sub.5 is a polymer or unit
thereof; X=C, N, O, or S; R.sub.6 and R.sub.7 are each
independently absent, H, or substituted; and R.sub.9, R.sub.10,
R.sub.11, and R.sub.12 are each independently H, a halogen, OH,
thio, NH.sub.2, or substituted or unsubstituted alkyl, aryl,
heterocyclic, alkoxy, aryloxy, alkylthio, arylthio, alkylamino, or
arylamino. In a further embodiment, for the compound of formula
(II), the polymer or unit thereof comprises a polyester,
polycarbonate, polyamide, or a polyether, or unit thereof. In
another embodiment, the polymer or unit thereof comprises
poly(lactic acid), poly(glycolic acid), poly(lactic-co-glycolic
acid), polycaprolactone, or poly(ethylene glycol), or unit thereof.
In yet another embodiment, the polymer is biodegradable.
In one embodiment, for a compound of formula (I) or (II), R.sub.1
is H, R.sub.2 is isobutyl, Y is C, and R.sub.3 and R.sub.4 are
combined to form a benzene ring with the carbon atoms of the
pyridine ring to which they are connected. In another embodiment,
R.sub.1 is ethoxymethyl, R.sub.2 is hydroxyisobutyl, Y=C, and
R.sub.3 and R.sub.4 are combined to form a benzene ring with the
carbon atoms of the pyridine ring to which they are connected. In
yet another embodiment, R.sub.1 is ethoxymethyl, R.sub.2 is
methanesulfonamidoisobutyl, Y=C, and R.sub.3 and R.sub.4 are
combined to form a benzene ring with the carbon atoms of the
pyridine ring to which they are connected. In one embodiment,
R.sub.1 is OH, R.sub.2 is benzyl, Y=N, R.sub.3 is absent, and
R.sub.4 is butoxy. In another embodiment, Y is N, R.sub.1 is OH,
R.sub.2 is benzyl, R.sub.3 is absent, and R.sub.4 is butylamino. In
yet another embodiment, Y is N, R.sub.1 is OH, R.sub.2 is benzyl,
R.sub.3 is absent, and R.sub.4 is butoxy. In still yet another
embodiment, Y is N, R.sub.1 is OH, R.sub.2 is benzyl, R.sub.3 is
absent, and R.sub.4 is benzylamino. In one embodiment, Y is N,
R.sub.1 is OH, R.sub.2 is benzyl, R.sub.3 is absent, and R.sub.4 is
pentyl.
In one embodiment, for a compound of formula (I) or (II), the
polymer is insoluble in water at pH=7.4 and at 25.degree. C. In
another embodiment, for a compound of formula (I) or (II), the
polymer is insoluble in water at pH=7.4 and at 25.degree. C. but
soluble at pH=4.5 and at 25.degree. C. In one embodiment, for a
compound of formula (I) or (II), the polymer has a weight average
molecular weight ranging from 800 Daltons to 10,000 Daltons, as
determined using gel permeation chromatography. In another
embodiment, for a compound of formula (I) or (II), the polymer or
unit thereof does not comprise polyketal or unit thereof. In one
embodiment, a composition is provided comprising a compound having
a formula (I) or (II). In a further embodiment, the composition
further comprises a pharmaceutically acceptable excipient.
In one embodiment, a synthetic nanocarrier is provided that
comprises the compound having a formula (I) or (II). In a further
embodiment, the synthetic nanocarrier further comprises a B cell
antigen and/or a T cell antigen. In yet another embodiment, the
synthetic nanocarrier further comprises an antigen presenting cell
(APC) targeting feature. In a further embodiment, the synthetic
nanocarrier is a dendrimer, buckyball, nanowire, peptide or
protein-based nanoparticle, nanoparticle that comprises a
combination of nanomaterials, spheroidal nanoparticle, cubic
nanoparticle, pyramidal nanoparticle, oblong nanoparticle,
cylindrical nanoparticle, or toroidal nanoparticle. In another
embodiment, a composition is provided comprising a synthetic
nanocarrier. In yet a further embodiment, the composition further
comprises a pharmaceutically acceptable excipient.
In one embodiment, a composition comprising a vaccine comprising a
compound of formula (I) or (II) is provided. In another embodiment,
a composition comprising a vaccine comprising a composition
comprising a compound of formula (I) or (II) is provided. In yet
another embodiment, a composition comprising a vaccine comprising
the synthetic nanocarrier comprising a compound of formula (I) or
(II) is provided. In still yet another embodiment, a method
comprises a administering to a subject any of the above described
compounds, compositions, or synthetic nanocarrier is provided. In a
further embodiment, an immune response is induced or enhanced in
the subject following administering to a subject any of the above
described compounds, compositions, or synthetic nanocarrier.
In one aspect, a method for making a conjugate that comprises a
structure as in formula (I):
##STR00003## comprises: activating a biodegradable polymer or unit
thereof, and exposing the activated biodegradable polymer or unit
thereof and a compound comprising a structure as in formula (III)
to a base and/or solvent
##STR00004## wherein R.sub.1=H, OH, SH, NH.sub.2, or substituted or
unsubstituted alkyl, alkoxy, alkylthio, or alkylamino; R.sub.2=H,
alkyl, or substituted alkyl; Y=N or C; R.sub.3 is absent if Y=N; or
is H, alkyl, substituted alkyl, or combined with R.sub.4 to form a
carbocycle or heterocycle with the carbon atoms of the pyridine
ring to which they are connected if Y=C; R.sub.4 is H, or
substituted or unsubstituted alkyl, alkoxy, alkylthio, or
alkylamino when not combined with R.sub.3 to form a carbocycle or
heterocycle with the carbon atoms of the pyridine ring to which
they are connected; or is combined with R.sub.3 to form a
carbocycle or heterocycle with the carbon atoms of the pyridine
ring to which they are connected; and R.sub.8 is a biodegradable
polymer or unit thereof. In one embodiment, the biodegradable
polymer or unit thereof comprises a polyester, polycarbonate, or a
polyamide, or unit thereof. In a further embodiment, the
biodegradable polymer or unit thereof comprises poly(lactic acid),
poly(glycolic acid), poly(lactic-co-glycolic acid), or
polycaprolactone, or unit thereof.
In another aspect, a method for making a conjugate that comprises a
structure as in formula (I):
##STR00005## comprises exposing a composition comprising a polymer
or unit thereof and a compound comprising a structure as in formula
(III) to a coupling agent and base and/or solvent:
##STR00006## wherein R.sub.1=H, OH, SH, NH.sub.2, or substituted or
unsubstituted alkyl, alkoxy, alkylthio, or alkylamino; R.sub.2=H,
alkyl, or substituted alkyl; Y=N or C; R.sub.3 is absent if Y=N; or
is H, alkyl, substituted alkyl, or combined with R.sub.4 to form a
carbocycle or heterocycle with the carbon atoms of the pyridine
ring to which they are connected if Y=C; R.sub.4 is H, or
substituted or unsubstituted alkyl, alkoxy, alkylthio, or
alkylamino when not combined with R.sub.3 to form a carbocycle or
heterocycle with the carbon atoms of the pyridine ring to which
they are connected; or is combined with R.sub.3 to form a
carbocycle or heterocycle with the carbon atoms of the pyridine
ring to which they are connected; and R.sub.8 is a polymer or unit
thereof.
In another aspect, a method for making a conjugate that comprises a
structure as in formula (II):
##STR00007## comprises combining an alcohol, a catalyst, and a
compound comprising a structure as in formula (IV):
##STR00008## wherein R.sub.1=H, OH, SH, NH.sub.2, or substituted or
unsubstituted alkyl, alkoxy, alkylthio, or alkylamino; R.sub.2=H,
alkyl, or substituted alkyl; Y=N or C; R.sub.3 is absent if Y=N; or
is H, alkyl, substituted alkyl, or combined with R.sub.4 to form a
carbocycle or heterocycle with the carbon atoms of the pyridine
ring to which they are connected if Y=C; R.sub.4 is H, or
substituted or unsubstituted alkyl, alkoxy, alkylthio, or
alkylamino when not combined with R.sub.3 to form a carbocycle or
heterocycle with the carbon atoms of the pyridine ring to which
they are connected; or is combined with R.sub.3 to form a
carbocycle or heterocycle with the carbon atoms of the pyridine
ring to which they are connected; R.sub.5 is a polymer or unit
thereof; X is C, N, O, or S; R.sub.6 and R.sub.7 are each
independently H or substituted; and R.sub.9, R.sub.10, R.sub.11,
and R.sub.12 are each independently H, a halogen, OH, thio,
NH.sub.2, or substituted or unsubstituted alkyl, aryl,
heterocyclic, alkoxy, aryloxy, alkylthio, arylthio, alkylamino, or
arylamino; and heating the alcohol, catalyst, and compound. In some
embodiment, the alcohol, catalyst, and compound are heated in the
presence of a solvent.
In yet another aspect, a method for making a conjugate that
comprises a structure as in formula (II):
##STR00009## comprises combining an alcohol and a compound
comprising a structure as in formula (IV):
##STR00010## wherein R.sub.1=H, OH, SH, NH.sub.2, or substituted or
unsubstituted alkyl, alkoxy, alkylthio, or alkylamino; R.sub.2=H,
alkyl, or substituted alkyl; Y=N or C; R.sub.3 is absent if Y=N; or
is H, alkyl, substituted alkyl, or combined with R.sub.4 to form a
carbocycle or heterocycle with the carbon atoms of the pyridine
ring to which they are connected if Y=C; R.sub.4 is H, or
substituted or unsubstituted alkyl, alkoxy, alkylthio, or
alkylamino when not combined with R.sub.3 to form a carbocycle or
heterocycle with the carbon atoms of the pyridine ring to which
they are connected; or is combined with R.sub.3 to form a
carbocycle or heterocycle with the carbon atoms of the pyridine
ring to which they are connected; R.sub.5 is a polymer or unit
thereof; X is C, N, O, or S; R.sub.6 and R.sub.7 are each
independently H or substituted; and R.sub.9, R.sub.10, R.sub.11,
and R.sub.12 are each independently H, a halogen, OH, thio,
NH.sub.2, or substituted or unsubstituted alkyl, aryl,
heterocyclic, alkoxy, aryloxy, alkylthio, arylthio, alkylamino, or
arylamino; heating the alcohol and compound; and adding a catalyst.
In one embodiment, the alcohol, compound, and catalyst are heated
while and/or after the catalyst is added.
In yet another aspect, a method for making a conjugate that
comprises a structure as in formula (II):
##STR00011## comprises combining an alcohol, a catalyst, and a
compound comprising a structure as in formula (IV):
##STR00012## wherein R.sub.1=H, OH, SH, NH.sub.2, or substituted or
unsubstituted alkyl, alkoxy, alkylthio, or alkylamino; R.sub.2=H,
alkyl, or substituted alkyl; Y=N or C; R.sub.3 is absent if Y=N; or
is H, alkyl, substituted alkyl, or combined with R.sub.4 to form a
carbocycle or heterocycle with the carbon atoms of the pyridine
ring to which they are connected if Y=C; R.sub.4 is H, or
substituted or unsubstituted alkyl, alkoxy, alkylthio, or
alkylamino when not combined with R.sub.3 to form a carbocycle or
heterocycle with the carbon atoms of the pyridine ring to which
they are connected; or is combined with R.sub.3 to form a
carbocycle or heterocycle with the carbon atoms of the pyridine
ring to which they are connected; R.sub.5 is a polymer or unit
thereof; X is C, N, O, or S; R.sub.6 and R.sub.7 are each
independently H or substituted; and R.sub.9, R.sub.10, R.sub.11,
and R.sub.12 are each independently H, a halogen, OH, thio,
NH.sub.2, or substituted or unsubstituted alkyl, aryl,
heterocyclic, alkoxy, aryloxy, alkylthio, arylthio, alkylamino, or
arylamino.
In one embodiment, for a method of making a compound of formula
(II), the alcohol is a polymer or unit thereof with a terminal
hydroxyl group. In a further embodiment, the polymer or unit
thereof comprises a polyester, polycarbonate, polyamide, or a
polyether, or unit thereof. In yet another embodiment, the polymer
or unit thereof comprises, poly(lactic acid), poly(glycolic acid),
poly(lactic-co-glycolic acid), polycaprolactone, or poly(ethylene
glycol), or unit thereof.
In one embodiment, for a method of making a compound of formula
(II), the catalyst is a phosphazine base,
1,8-diazabicycloundec-7-ene, 1,4,7-triazabicyclodecene, or
N-methyl-1,4,7-triazabicyclodecene. In another embodiment, the
polymer has a weight average molecular weight ranging from 800
Daltons to 10,000 Daltons, as determined using gel permeation
chromatography. In yet another embodiment, the polymer is insoluble
in water at pH=7.4 and at 25.degree. C. In another embodiment, the
polymer is insoluble in water at pH=7.4 and at 25.degree. C. but
soluble at pH=4.5 and at 25.degree. C. In still yet another
embodiment, the polymer or unit thereof does not comprise polyketal
or unit thereof.
In one embodiment, for a method of making a compound of formula
(II), R.sub.1 is H, R.sub.2 is isobutyl, Y is C, and R.sub.3 and
R.sub.4 are combined to form a benzene ring with the carbon atoms
of the pyridine ring to which they are connected. In another
embodiment, R.sub.1 is ethoxymethyl, R.sub.2 is hydroxyisobutyl,
Y=C, and R.sub.3 and R.sub.4 are combined to form a benzene ring
with the carbon atoms of the pyridine ring to which they are
connected. In yet another embodiment, R.sub.1 is ethoxymethyl,
R.sub.2 is methanesulfonamidoisobutyl, Y=C, and R.sub.3 and R.sub.4
are combined to form a benzene ring with the carbon atoms of the
pyridine ring to which they are connected. In still yet another
embodiment, R.sub.1 is OH, R.sub.2 is benzyl, Y=N, R.sub.3 is
absent, and R.sub.4 is butoxy. In another embodiment, Y is N,
R.sub.1 is OH, R.sub.2 is benzyl, R.sub.3 is absent, and R.sub.4 is
butylamino. In one embodiment, Y is N, R.sub.1 is OH, R.sub.2 is
benzyl, R.sub.3 is absent, and R.sub.4 is butoxy. In another
embodiment, Y is N, R.sub.1 is OH, R.sub.2 is benzyl, R.sub.3 is
absent, and R.sub.4 is benzylamino. In yet another embodiment, Y is
N, R.sub.1 is OH, R.sub.2 is benzyl, R.sub.3 is absent, and R.sub.4
is pentyl.
In one aspect, the present invention provides a compound that
comprises a structure as in formula (IV):
##STR00013## wherein R.sub.1=H, OH, SH, NH.sub.2, or substituted or
unsubstituted alkyl, alkoxy, alkylthio, or alkylamino; R.sub.2=H,
alkyl, or substituted alkyl; Y=N or C; R.sub.3 is absent if Y=N; or
is H, alkyl, substituted alkyl, or combined with R.sub.4 to form a
carbocycle or heterocycle with the carbon atoms of the pyridine
ring to which they are connected if Y=C; R.sub.4 is H, or
substituted or unsubstituted alkyl, alkoxy, alkylthio, or
alkylamino when not combined with R.sub.3 to form a carbocycle or
heterocycle with the carbon atoms of the pyridine ring to which
they are connected; or is combined with R.sub.3 to form a
carbocycle or heterocycle with the carbon atoms of the pyridine
ring to which they are connected; X is C, N, O, or S; R.sub.6 and
R.sub.7 are each independently H or substituted; and R.sub.9,
R.sub.10, R.sub.11, and R.sub.12 are each independently H, a
halogen, OH, thio, NH.sub.2, or substituted or unsubstituted alkyl,
aryl, heterocyclic, alkoxy, aryloxy, alkylthio, arylthio,
alkylamino, or arylamino.
In one embodiment, for a compound of formula (IV), R.sub.1 is H,
R.sub.2 is isobutyl, Y is C, and R.sub.3 and R.sub.4 are combined
to form a benzene ring with the carbon atoms of the pyridine ring
to which they are connected. In another embodiment, R.sub.1 is
ethoxymethyl, R.sub.2 is hydroxyisobutyl, Y=C, and R.sub.3 and
R.sub.4 are combined to form a benzene ring with the carbon atoms
of the pyridine ring to which they are connected. In yet another
embodiment, R.sub.1 is ethoxymethyl, R.sub.2 is
methanesulfonamidoisobutyl, Y=C, and R.sub.3 and R.sub.4 are
combined to form a benzene ring with the carbon atoms of the
pyridine ring to which they are connected. In still yet another
embodiment, R.sub.1 is OH, R.sub.2 is benzyl, Y=N, R.sub.3 is
absent, and R.sub.4 is butoxy. In one embodiment, Y is N, R.sub.1
is OH, R.sub.2 is benzyl, R.sub.3 is absent, and R.sub.4 is
butylamino. In another embodiment, Y is N, R.sub.1 is OH, R.sub.2
is benzyl, R.sub.3 is absent, and R.sub.4 is butoxy. In yet another
embodiment, Y is N, R.sub.1 is OH, R.sub.2 is benzyl, R.sub.3 is
absent, and R.sub.4 is benzylamino. In still yet another
embodiment, Y is N, R.sub.1 is OH, R.sub.2 is benzyl, R.sub.3 is
absent, and R.sub.4 is pentyl. In one embodiment, a composition is
provided having a compound of formula (IV).
In one aspect, the present invention provides a method for making a
compound that comprises a structure as in formula (IV):
##STR00014## comprising combining, in the presence of a solvent
and/or heat, a compound that comprises a structure as in formula
(III):
##STR00015## and a compound comprising a structure as in formula
(V):
##STR00016## wherein R.sub.1=H, OH, SH, NH.sub.2, or substituted or
unsubstituted alkyl, alkoxy, alkylthio, or alkylamino; R.sub.2=H,
alkyl, or substituted alkyl; Y=N or C; R.sub.3 is absent if Y=N; or
is H, alkyl, substituted alkyl, or combined with R.sub.4 to form a
carbocycle or heterocycle with the carbon atoms of the pyridine
ring to which they are connected if Y=C; R.sub.4 is H, or
substituted or unsubstituted alkyl, alkoxy, alkylthio, or
alkylamino when not combined with R.sub.3 to form a carbocycle or
heterocycle with the carbon atoms of the pyridine ring to which
they are connected; or is combined with R.sub.3 to form a
carbocycle or heterocycle with the carbon atoms of the pyridine
ring to which they are connected; X is C, N, O, or S; R.sub.6 and
R.sub.7 are each independently H or substituted; and R.sub.9,
R.sub.10, R.sub.11, and R.sub.12 are each independently H, a
halogen, OH, thio, NH.sub.2, or substituted or unsubstituted alkyl,
aryl, heterocyclic, alkoxy, aryloxy, alkylthio, arylthio,
alkylamino, or arylamino.
In one embodiment, for a method comprising a compound of formula
(IV), R.sub.1 is H, R.sub.2 is isobutyl, Y is C, and R.sub.3 and
R.sub.4 are combined to form a benzene ring with the carbon atoms
of the pyridine ring to which they are connected. In another
embodiment, R.sub.1 is ethoxymethyl, R.sub.2 is hydroxyisobutyl,
Y=C, and R.sub.3 and R.sub.4 are combined to form a benzene ring
with the carbon atoms of the pyridine ring to which they are
connected. In yet another embodiment, R.sub.1 is ethoxymethyl,
R.sub.2 is methanesulfonamidoisobutyl, Y=C, and R.sub.3 and R.sub.4
are combined to form a benzene ring with the carbon atoms of the
pyridine ring to which they are connected. In still yet another
embodiment, R.sub.1 is OH, R.sub.2 is benzyl, Y=N, R.sub.3 is
absent, and R.sub.4 is butoxy. In one embodiment, Y is N, R.sub.1
is OH, R.sub.2 is benzyl, R.sub.3 is absent, and R.sub.4 is
butylamino. In another embodiment, Y is N, R.sub.1 is OH, R.sub.2
is benzyl, R.sub.3 is absent, and R.sub.4 is butoxy. In yet another
embodiment, Y is N, R.sub.1 is OH, R.sub.2 is benzyl, R.sub.3 is
absent, and R.sub.4 is benzylamino. In still yet another
embodiment, Y is N, R.sub.1 is OH, R.sub.2 is benzyl, R.sub.3 is
absent, and R.sub.4 is pentyl.
In one aspect, the present invention provides a method for making a
conjugate that comprises a structure as in formula (VI):
##STR00017## comprising combining a catalyst, a diol having the
formula (VII): HO-polymer-OH (VII), and a compound comprising a
structure as in formula (IV):
##STR00018## wherein R.sub.1=H, OH, SH, NH.sub.2, or substituted or
unsubstituted alkyl, alkoxy, alkylthio, or alkylamino; R.sub.2=H,
alkyl, or substituted alkyl; Y=N or C; R.sub.3 is absent if Y=N; or
is H, alkyl, substituted alkyl, or combined with R.sub.4 to form a
carbocycle or heterocycle with the carbon atoms of the pyridine
ring to which they are connected if Y=C; R.sub.4 is H, or
substituted or unsubstituted alkyl, alkoxy, alkylthio, or
alkylamino when not combined with R.sub.3 to form a carbocycle or
heterocycle with the carbon atoms of the pyridine ring to which
they are connected; or is combined with R.sub.3 to form a
carbocycle or heterocycle with the carbon atoms of the pyridine
ring to which they are connected; X is C, N, O, or S; R.sub.6 and
R.sub.7 are each independently H or substituted; and R.sub.9,
R.sub.10, R.sub.11, and R.sub.12 are each independently H, a
halogen, OH, thio, NH.sub.2, or substituted or unsubstituted alkyl,
aryl, heterocyclic, alkoxy, aryloxy, alkylthio, arylthio,
alkylamino, or arylamino; and heating the alcohol, catalyst, and
compound. In one embodiment, for a method comprising a compound of
formula (VI), the alcohol, catalyst, and compound are heated in the
presence of a solvent. In one embodiment of this aspect, the
polymer is intended to include a unit of a polymer provided
herein.
In another aspect, the present invention provides a method for
making a conjugate that comprises a structure as in formula
(VI):
##STR00019## comprising combining a diol having the formula (VII):
HO-polymer-OH (VII), and a compound comprising a structure as in
formula (IV):
##STR00020## wherein R.sub.1=H, OH, SH, NH.sub.2, or substituted or
unsubstituted alkyl, alkoxy, alkylthio, or alkylamino; R.sub.2=H,
alkyl, or substituted alkyl; Y=N or C; R.sub.3 is absent if Y=N; or
is H, alkyl, substituted alkyl, or combined with R.sub.4 to form a
carbocycle or heterocycle with the carbon atoms of the pyridine
ring to which they are connected if Y=C; R.sub.4 is H, or
substituted or unsubstituted alkyl, alkoxy, alkylthio, or
alkylamino when not combined with R.sub.3 to form a carbocycle or
heterocycle with the carbon atoms of the pyridine ring to which
they are connected; or is combined with R.sub.3 to form a
carbocycle or heterocycle with the carbon atoms of the pyridine
ring to which they are connected; X is C, N, O, or S; R.sub.6 and
R.sub.7 are each independently H or substituted; and R.sub.9,
R.sub.10, R.sub.11, and R.sub.12 are each independently H, a
halogen, OH, thio, NH.sub.2, or substituted or unsubstituted alkyl,
aryl, heterocyclic, alkoxy, aryloxy, alkylthio, arylthio,
alkylamino, or arylamino; heating the alcohol and compound; and
adding a catalyst. In a further embodiment, the alcohol, compound,
and catalyst are heated while and/or after the catalyst is added.
In one embodiment of this aspect, the polymer is intended to
include comprising a unit of a polymer provided herein.
In one aspect, the present invention provides a method for making a
conjugate that comprises a structure as in formula (VI):
##STR00021## comprising combining, a catalyst, a diol having the
formula (VII): HO-polymer-OH (VII), and a compound comprising a
structure as in formula (IV):
##STR00022## wherein R.sub.1=H, OH, SH, NH.sub.2, or substituted or
unsubstituted alkyl, alkoxy, alkylthio, or alkylamino; R.sub.2=H,
alkyl, or substituted alkyl; Y=N or C; R.sub.3 is absent if Y=N; or
is H, alkyl, substituted alkyl, or combined with R.sub.4 to form a
carbocycle or heterocycle with the carbon atoms of the pyridine
ring to which they are connected if Y=C; R.sub.4 is H, or
substituted or unsubstituted alkyl, alkoxy, alkylthio, or
alkylamino when not combined with R.sub.3 to form a carbocycle or
heterocycle with the carbon atoms of the pyridine ring to which
they are connected; or is combined with R.sub.3 to form a
carbocycle or heterocycle with the carbon atoms of the pyridine
ring to which they are connected; X is C, N, O, or S; R.sub.6 and
R.sub.7 are each independently H or substituted; and R.sub.9,
R.sub.10, R.sub.11, and R.sub.12 are each independently H, a
halogen, OH, thio, NH.sub.2, or substituted or unsubstituted alkyl,
aryl, heterocyclic, alkoxy, aryloxy, alkylthio, arylthio,
alkylamino, or arylamino.
In one embodiment, for a method of making a compound comprising the
formula (VI), the compound of formula (VII) is selected from the
group consisting polyketaldiols, poly(ethylene)glycol,
polycaprolactone diol, diblock polylactide-co-poly(ethylene)glycol,
diblock polylactide/polyglycolide-co-poly(ethylene)glycol, diblock
polyglycolide-co-poly(ethylene)glycol, poly(propylene)glycol, and
poly(hexamethylene carbonate)diol. In one embodiment, for a method
of making a compound comprising the formula (VI), the catalyst is a
phosphazine base, 1,8-diazabicycloundec-7-ene,
1,4,7-triazabicyclodecene, or N-methyl-1,4,7-triazabicyclodecene.
In a further embodiment, the polymer has a weight average molecular
weight ranging from 800 Daltons to 10,000 Daltons, as determined
using gel permeation chromatography. In another embodiment, the
polymer is insoluble in water at pH=7.4 and at 25.degree. C. In
another embodiment, the polymer is insoluble in water at pH=7.4 and
at 25.degree. C. but soluble at pH=4.5 and at 25.degree. C. In yet
another embodiment, the polymer does not comprise polyketal or unit
thereof.
In one embodiment, for a method of making a compound having the
formula (VI), R.sub.1 is H, R.sub.2 is isobutyl, Y is C, and
R.sub.3 and R.sub.4 are combined to form a benzene ring with the
carbon atoms of the pyridine ring to which they are connected. In
another embodiment, R.sub.1 is ethoxymethyl, R.sub.2 is
hydroxyisobutyl, Y=C, and R.sub.3 and R.sub.4 are combined to form
a benzene ring with the carbon atoms of the pyridine ring to which
they are connected. In yet another embodiment, R.sub.1 is
ethoxymethyl, R.sub.2 is methanesulfonamidoisobutyl, Y=C, and
R.sub.3 and R.sub.4 are combined to form a benzene ring with the
carbon atoms of the pyridine ring to which they are connected. In
still yet another embodiment, R.sub.1 is OH, R.sub.2 is benzyl,
Y=N, R.sub.3 is absent, and R.sub.4 is butoxy. In one embodiment, Y
is N, R.sub.1 is OH, R.sub.2 is benzyl, R.sub.3 is absent, and
R.sub.4 is butylamino. In another embodiment, Y is N, R.sub.1 is
OH, R.sub.2 is benzyl, R.sub.3 is absent, and R.sub.4 is butoxy. In
yet another embodiment, Y is N, R.sub.1 is OH, R.sub.2 is benzyl,
R.sub.3 is absent, and R.sub.4 is benzylamino. In still yet another
embodiment, Y is N, R.sub.1 is OH, R.sub.2 is benzyl, R.sub.3 is
absent, and R.sub.4 is pentyl.
In one aspect, a compound that comprises a structure as in formula
(VI):
##STR00023##
wherein each R.sub.1, independently, =H, OH, SH, NH.sub.2, or
substituted or unsubstituted alkyl, alkoxy, alkylthio, or
alkylamino;
each R.sub.2, independently, =H, alkyl, or substituted alkyl;
each Y, independently, =N or C;
each R.sub.3, independently, is absent if Y=N; or is H, alkyl,
substituted alkyl, or combined with R.sub.4 to form a carbocycle or
heterocycle with the carbon atoms of the pyridine ring to which
they are connected if Y=C;
each R.sub.4, independently, is H, or substituted or unsubstituted
alkyl, alkoxy, alkylthio, or alkylamino when not combined with
R.sub.3 to form a carbocycle or heterocycle with the carbon atoms
of the pyridine ring to which they are connected; or is combined
with R.sub.3 to form a carbocycle or heterocycle with the carbon
atoms of the pyridine ring to which they are connected;
each X, independently, is C, N, O, or S;
each R.sub.6 and R.sub.7, independently, are each independently H
or substituted; and
each R.sub.9, R.sub.19, R.sub.11, and R.sub.12, independently, are
each independently H, a halogen, OH, thio, NH.sub.2, or substituted
or unsubstituted alkyl, aryl, heterocyclic, alkoxy, aryloxy,
alkylthio, arylthio, alkylamino, or arylamino, is provided.
In one embodiment, the polymer is selected from the group
consisting of polyketaldiols, poly(ethylene)glycol,
polycaprolactone diol, diblock polylactide-co-poly(ethylene)glycol,
diblock polylactide/polyglycolide-co-poly(ethylene)glycol, diblock
polyglycolide-co-poly(ethylene)glycol, poly(propylene)glycol,
poly(hexamethylene carbonate)diol, and poly(tetrahydrofuran). In
another embodiment of this aspect, the polymer includes a unit of a
polymer. In another embodiment, the polymer has a weight average
molecular weight ranging from 800 Daltons to 10,000 Daltons, as
determined using gel permeation chromatography. In a further
embodiment, the polymer is insoluble in water at pH=7.4 and at
25.degree. C. In yet another embodiment, the polymer does not
comprise polyketal or unit thereof.
In one embodiment, R.sub.1 is H, R.sub.2 is isobutyl, Y is C, and
R.sub.3 and R.sub.4 are combined to form a benzene ring with the
carbon atoms of the pyridine ring to which they are connected. In
another embodiment, R.sub.1 is ethoxymethyl, R.sub.2 is
hydroxyisobutyl, Y=C, and R.sub.3 and R.sub.4 are combined to form
a benzene ring with the carbon atoms of the pyridine ring to which
they are connected. In yet another embodiment, R.sub.1 is
ethoxymethyl, R.sub.2 is methanesulfonamidoisobutyl, Y=C, and
R.sub.3 and R.sub.4 are combined to form a benzene ring with the
carbon atoms of the pyridine ring to which they are connected. In
still another embodiment, R.sub.1 is OH, R.sub.2 is benzyl, Y=N,
R.sub.3 is absent, and R.sub.4 is butoxy. In a further embodiment,
Y is N, R.sub.1 is OH, R.sub.2 is benzyl, R.sub.3 is absent, and
R.sub.4 is butylamino. In still another embodiment, Y is N, R.sub.1
is OH, R.sub.2 is benzyl, R.sub.3 is absent, and R.sub.4 is butoxy.
In a further embodiment, Y is N, R.sub.1 is OH, R.sub.2 is benzyl,
R.sub.3 is absent, and R.sub.4 is benzylamino. In yet a further
embodiment, Y is N, R.sub.1 is OH, R.sub.2 is benzyl, R.sub.3 is
absent, and R.sub.4 is pentyl.
In one embodiment, a composition comprising the above compounds is
provided. In another embodiment, the composition further comprises
a pharmaceutically acceptable excipient.
In another embodiment, a synthetic nanocarrier that comprises any
of the foregoing compounds is provided. In one embodiment, the
synthetic nanocarrier further comprises a B cell antigen and/or a T
cell antigen. In another embodiment, the synthetic nanocarrier
further comprises an antigen presenting cell (APC) targeting
feature. In still another embodiment, the synthetic nanocarrier is
a dendrimer, buckyball, nanowire, peptide or protein-based
nanoparticle, nanoparticle that comprises a combination of
nanomaterials, spheroidal nanoparticle, cubic nanoparticle,
pyramidal nanoparticle, oblong nanoparticle, cylindrical
nanoparticle, or toroidal nanoparticle.
In one embodiment, a composition comprising any of the foregoing
synthetic nanocarriers is provided. In one embodiment, the
composition further comprises a pharmaceutically acceptable
excipient.
In another embodiment, a composition comprising a vaccine
comprising any of the foregoing compounds is provided. In yet
another embodiment, a composition comprising a vaccine comprising
any of the foregoing compositions is provided. In another
embodiment, a composition comprising a vaccine comprising any of
the foregoing synthetic nanocarriers is provided.
In another embodiment, a method comprising administering any of the
foregoing compounds, compositions or synthetic nanocarriers to a
subject is provided. In one embodiment, the method is one where an
immune response is induced or enhanced in the subject.
In another aspect, a compound having a structure of any of the
compounds provided herein is provided. Compositions, synthetic
nanocarriers, and vaccines comprising any of the compounds provided
are also provided.
In a further aspect, any of the methods of making a compound
provided herein are also provided.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 shows the release of resiquimod (R848) from synthetic
nanocarrier formulations at pH 7.4, 37.degree. C.
FIG. 2 shows the release of R848 from synthetic nanocarrier
formulations at pH 4.5, 37.degree. C.
FIG. 3 shows the release of R848 from synthetic nanocarrier
formulations at pH 7.4 and pH 4.5 at 24 hours.
FIG. 4 shows the level of antibody induction by synthetic
nanocarriers with a CpG-containing immunostimulatory nucleic acid
(Groups 2 and 3) as compared to the level of antibody induction by
synthetic nanocarriers without the CpG-containing immunostimulatory
nucleic acid (Group 1).
FIG. 5 shows the level of antibody induction by synthetic
nanocarriers that release a phosphodiester, non-thioated
CpG-containing immunostimulatory nucleic acid or a thioated
CpG-containing immunostimulatory nucleic acid.
FIG. 6 shows the level of antibody induction by synthetic
nanocarriers that release R848 at different rates.
DETAILED DESCRIPTION
Before describing the present invention in detail, it is to be
understood that this invention is not limited to particularly
exemplified materials or process parameters as such may, of course,
vary. It is also to be understood that the terminology used herein
is for the purpose of describing particular embodiments of the
invention only, and is not intended to be limiting of the use of
alternative terminology to describe the present invention.
All publications, patents and patent applications cited herein,
whether supra or infra, are hereby incorporated by reference in
their entirety for all purposes.
As used in this specification and the appended claims, the singular
forms "a," "an" and "the" include plural referents unless the
content clearly dictates otherwise. For example, reference to "a
polymer" includes a mixture of two or more such molecules,
reference to "a solvent" includes a mixture of two or more such
solvents, reference to "an adhesive" includes mixtures of two or
more such materials, and the like.
INTRODUCTION
The inventors have unexpectedly and surprisingly discovered that
the problems and limitations noted above can be overcome by
practicing the invention disclosed herein. In particular, the
inventors have unexpectedly discovered that it is possible to
provide compounds, together with related compositions and methods,
that comprise:
a structure as in formula (I):
##STR00024##
wherein R.sub.1=H, OH, SH, NH.sub.2, or substituted or
unsubstituted alkyl, alkoxy, alkylthio, or alkylamino;
R.sub.2=H, alkyl, or substituted alkyl;
Y=N or C;
R.sub.3 is absent if Y=N; or is H, alkyl, substituted alkyl, or
combined with R.sub.4 to form a carbocycle or heterocycle with the
carbon atoms of the pyridine ring to which they are connected if
Y=C;
R.sub.4 is H, or substituted or unsubstituted alkyl, alkoxy,
alkylthio, or alkylamino when not combined with R.sub.3 to form a
carbocycle or heterocycle with the carbon atoms of the pyridine
ring to which they are connected; or is combined with R.sub.3 to
form a carbocycle or heterocycle with the carbon atoms of the
pyridine ring to which they are connected; and
R.sub.8 is a biodegradable polymer or unit thereof.
When using synthetic nanocarriers to produce an immune response in
a subject, it is advantageous to include with the synthetic
nanocarriers an immunomodulatory agent. Such an agent includes
agents that are immunomodulatory when uncoupled from the synthetic
nanocarrier but may not exhibit immunomodulatory properties when
coupled to the synthetic nanocarrier. It is particularly
advantageous to include the immunomodulatory agent as part of the
synthetic nanocarriers itself. To achieve this, the
immunomodulatory agent may be covalently attached to an appropriate
polymer or unit thereof. It follows that the compounds and
conjugates provided herein, in some embodiments, comprise an
immunomodulatory agent, which also is intended to include an agent
that is immunomodulatory when uncoupled from the polymer or unit
thereof but that may not exhibit immunomodulatory properties when
coupled to the polymer or unit thereof. The compounds provided
herein can be incorporated into one or more synthetic nanocarriers.
The compounds are incorporated into synthetic nanocarriers by
methods known in the art or described elsewhere herein.
In some embodiments, the polymer or unit thereof of the compounds
or conjugates provided is a biodegradable polymer or unit thereof.
The polymer or unit thereof, therefore, may comprise a polyester,
polycarbonate, or polyamide, or unit thereof. It follows that the
polymer or unit thereof may comprise poly(lactic acid),
poly(glycolic acid), poly(lactic-co-glycolic acid), or
polycaprolactone, or unit thereof. Generally, it is preferred that
if the polymer comprises a polyether, such as poly(ethylene glycol)
(PEG) or unit thereof, the polymer is a block-co-polymer of a
polyether and a biodegradable polymer such that the polymer is
biodegradable. In some embodiments, the polymer or unit thereof
does not comprise a polyether, such as poly(ethylene glycol), or
unit thereof. In other embodiments, the polymer does not solely
comprise a polyether or unit thereof, such as poly(ethylene
glycol), or unit thereof. Generally, for use as part of a synthetic
nanocarrier the polymer of the compounds or conjugates provided
herein is insoluble in water at pH=7.4 and at 25.degree. C., is
biodegradable, or both. In other embodiments, the polymer is
insoluble in water at pH=7.4 and at 25.degree. C. but soluble at
pH=4.5 and at 25.degree. C. In still other embodiments, the polymer
is insoluble in water at pH=7.4 and at 25.degree. C. but soluble at
pH=4.5 and at 25.degree. C. and biodegradable. The compounds,
conjugates, and synthetic nanocarriers provided herein are unique
in composition and are useful for the preparation of vaccines and
associated materials.
Methods for making the aforementioned compounds are also provided.
In embodiments, a method for making a conjugate that comprises a
structure as in formula (I):
##STR00025## comprises:
activating a biodegradable polymer or unit thereof, and
exposing the activated biodegradable polymer or unit thereof and a
compound comprising a structure as in formula (III) to a base
and/or solvent:
##STR00026##
wherein R.sub.1=H, OH, SH, NH.sub.2, or substituted or
unsubstituted alkyl, alkoxy, alkylthio, or alkylamino;
R.sub.2=H, alkyl, or substituted alkyl;
Y=N or C;
R.sub.3 is absent if Y=N; or is H, alkyl, substituted alkyl, or
combined with R.sub.4 to form a carbocycle or heterocycle with the
carbon atoms of the pyridine ring to which they are connected if
Y=C;
R.sub.4 is H, or substituted or unsubstituted alkyl, alkoxy,
alkylthio, or alkylamino when not combined with R.sub.3 to form a
carbocycle or heterocycle with the carbon atoms of the pyridine
ring to which they are connected; or is combined with R.sub.3 to
form a carbocycle or heterocycle with the carbon atoms of the
pyridine ring to which they are connected; and
R.sub.8 is a biodegradable polymer or unit thereof.
In other embodiments, a method for making a conjugate that
comprises a structure as in formula (I):
##STR00027## comprises:
exposing a composition comprising a polymer or unit thereof and a
compound comprising a structure as in formula (III) to a coupling
agent and base and/or solvent:
##STR00028##
wherein R.sub.1=H, OH, SH, NH.sub.2, or substituted or
unsubstituted alkyl, alkoxy, alkylthio, or alkylamino;
R.sub.2=H, alkyl, or substituted alkyl;
Y=N or C;
R.sub.3 is absent if Y=N; or is H, alkyl, substituted alkyl, or
combined with R.sub.4 to form a carbocycle or heterocycle with the
carbon atoms of the pyridine ring to which they are connected if
Y=C;
R.sub.4 is H, or substituted or unsubstituted alkyl, alkoxy,
alkylthio, or alkylamino when not combined with R.sub.3 to form a
carbocycle or heterocycle with the carbon atoms of the pyridine
ring to which they are connected; or is combined with R.sub.3 to
form a carbocycle or heterocycle with the carbon atoms of the
pyridine ring to which they are connected; and
R.sub.8 is a polymer or unit thereof.
The inventors have also unexpectedly discovered that it is possible
to provide compounds, together with related compositions and
methods, that comprise:
a structure as in formula (II):
##STR00029##
wherein R.sub.1=H, OH, SH, NH.sub.2, or substituted or
unsubstituted alkyl, alkoxy, alkylthio, or alkylamino;
R.sub.2=H, alkyl, or substituted alkyl;
Y=N or C;
R.sub.3 is absent if Y=N; or is H, alkyl, substituted alkyl, or
combined with R.sub.4 to form a carbocycle or heterocycle with the
carbon atoms of the pyridine ring to which they are connected if
Y=C;
R.sub.4 is H, or substituted or unsubstituted alkyl, alkoxy,
alkylthio, or alkylamino when not combined with R.sub.3 to form a
carbocycle or heterocycle with the carbon atoms of the pyridine
ring to which they are connected; or is combined with R.sub.3 to
form a carbocycle or heterocycle with the carbon atoms of the
pyridine ring to which they are connected;
R.sub.5 is a polymer or unit thereof;
X=C, N, O, or S;
R.sub.6 and R.sub.7 are each independently absent, H, or
substituted; and
R.sub.9, R.sub.10, R.sub.11, and R.sub.12 are each independently H,
a halogen, OH, thio, NH.sub.2, or substituted or unsubstituted
alkyl, aryl, heterocyclic, alkoxy, aryloxy, alkylthio, arylthio,
alkylamino, or arylamino.
It has been discovered that it is possible to attach agents, such
as immunomodulatory agents, comprising a structure as in formula
(III), to a polymer or unit thereof with a terminal alcohol.
Generally, terminal alcohols are less reactive, making attachment
chemistry problematic. It has been found that imides, such as those
comprising a structure as in formula (IV), will react with a
terminal alcohol using catalysts commonly used in ring opening
polymerizations. The resulting reaction product links the imide to
the alcohol via an ester bond.
Accordingly, methods for making conjugates via the aforementioned
chemistry are also provided. In some embodiments, a method for
making a conjugate that comprises a structure as in formula
(II):
##STR00030## comprises:
combining an alcohol, a catalyst, and a compound comprising a
structure as in formula (IV):
##STR00031##
wherein R.sub.1=H, OH, SH, NH.sub.2, or substituted or
unsubstituted alkyl, alkoxy, alkylthio, or alkylamino;
R.sub.2=H, alkyl, or substituted alkyl;
Y=N or C;
R.sub.3 is absent if Y=N; or is H, alkyl, substituted alkyl, or
combined with R.sub.4 to form a carbocycle or heterocycle with the
carbon atoms of the pyridine ring to which they are connected if
Y=C;
R.sub.4 is H, or substituted or unsubstituted alkyl, alkoxy,
alkylthio, or alkylamino when not combined with R.sub.3 to form a
carbocycle or heterocycle with the carbon atoms of the pyridine
ring to which they are connected; or is combined with R.sub.3 to
form a carbocycle or heterocycle with the carbon atoms of the
pyridine ring to which they are connected;
R.sub.5 is a polymer or unit thereof;
X is C, N, O, or S;
R.sub.6 and R.sub.7 are each independently H or substituted;
and
R.sub.9, R.sub.10, R.sub.11, and R.sub.12 are each independently H,
a halogen, OH, thio, NH.sub.2, or substituted or unsubstituted
alkyl, aryl, heterocyclic, alkoxy, aryloxy, alkylthio, arylthio,
alkylamino, or arylamino; and
heating the alcohol, catalyst, and compound.
In other embodiments, a method for making a conjugate that
comprises a structure as in formula (II):
##STR00032## comprises:
combining an alcohol and a compound comprising a structure as in
formula (IV):
##STR00033##
wherein R.sub.1=H, OH, SH, NH.sub.2, or substituted or
unsubstituted alkyl, alkoxy, alkylthio, or alkylamino;
R.sub.2=H, alkyl, or substituted alkyl;
Y=N or C;
R.sub.3 is absent if Y=N; or is H, alkyl, substituted alkyl, or
combined with R.sub.4 to form a carbocycle or heterocycle with the
carbon atoms of the pyridine ring to which they are connected if
Y=C;
R.sub.4 is H, or substituted or unsubstituted alkyl, alkoxy,
alkylthio, or alkylamino when not combined with R.sub.3 to form a
carbocycle or heterocycle with the carbon atoms of the pyridine
ring to which they are connected; or is combined with R.sub.3 to
form a carbocycle or heterocycle with the carbon atoms of the
pyridine ring to which they are connected;
R.sub.5 is a polymer or unit thereof;
X is C, N, O, or S;
R.sub.6 and R.sub.7 are each independently H or substituted;
and
R.sub.9, R.sub.10, R.sub.11, and R.sub.12 are each independently H,
a halogen, OH, thio, NH.sub.2, or substituted or unsubstituted
alkyl, aryl, heterocyclic, alkoxy, aryloxy, alkylthio, arylthio,
alkylamino, or arylamino;
heating the alcohol and compound; and
adding a catalyst.
In yet other embodiments, a method for making a conjugate that
comprises a structure as in formula (II):
##STR00034## comprises:
combining an alcohol, a catalyst, and a compound comprising a
structure as in formula (IV):
##STR00035##
wherein R.sub.1=H, OH, SH, NH.sub.2, or substituted or
unsubstituted alkyl, alkoxy, alkylthio, or alkylamino;
R.sub.2=H, alkyl, or substituted alkyl;
Y=N or C;
R.sub.3 is absent if Y=N; or is H, alkyl, substituted alkyl, or
combined with R.sub.4 to form a carbocycle or heterocycle with the
carbon atoms of the pyridine ring to which they are connected if
Y=C;
R.sub.4 is H, or substituted or unsubstituted alkyl, alkoxy,
alkylthio, or alkylamino when not combined with R.sub.3 to form a
carbocycle or heterocycle with the carbon atoms of the pyridine
ring to which they are connected; or is combined with R.sub.3 to
form a carbocycle or heterocycle with the carbon atoms of the
pyridine ring to which they are connected;
R.sub.5 is a polymer or unit thereof;
X is C, N, O, or S;
R.sub.6 and R.sub.7 are each independently H or substituted;
and
R.sub.9, R.sub.10, R.sub.11, and R.sub.12 are each independently H,
a halogen, OH, thio, NH.sub.2, or substituted or unsubstituted
alkyl, aryl, heterocyclic, alkoxy, aryloxy, alkylthio, arylthio,
alkylamino, or arylamino.
Imides that may be used in the aforementioned reactions are also
provided herein. In one embodiment, the imide compound comprises a
structure as in formula (IV):
##STR00036##
wherein R.sub.1=H, OH, SH, NH.sub.2, or substituted or
unsubstituted alkyl, alkoxy, alkylthio, or alkylamino;
R.sub.2=H, alkyl, or substituted alkyl;
Y=N or C;
R.sub.3 is absent if Y=N; or is H, alkyl, substituted alkyl, or
combined with R.sub.4 to form a carbocycle or heterocycle with the
carbon atoms of the pyridine ring to which they are connected if
Y=C;
R.sub.4 is H, or substituted or unsubstituted alkyl, alkoxy,
alkylthio, or alkylamino when not combined with R.sub.3 to form a
carbocycle or heterocycle with the carbon atoms of the pyridine
ring to which they are connected; or is combined with R.sub.3 to
form a carbocycle or heterocycle with the carbon atoms of the
pyridine ring to which they are connected;
X is C, N, O, or S;
R.sub.6 and R.sub.7 are each independently H or substituted;
and
R.sub.9, R.sub.10, R.sub.11, and R.sub.12 are each independently H,
a halogen, OH, thio, NH.sub.2, or substituted or unsubstituted
alkyl, aryl, heterocyclic, alkoxy, aryloxy, alkylthio, arylthio,
alkylamino, or arylamino.
Such a compound can be made by methods that comprise combining, in
the presence of a solvent and/or heat, with or without a
dehydrating agent, such as a carboxylic acid anhydride or acetic
anhydride, and a base, such as pyridine compound, a compound that
comprises a structure as in formula (III):
##STR00037## and
a compound comprising a structure as in formula (V):
##STR00038##
wherein R.sub.1=H, OH, SH, NH.sub.2, or substituted or
unsubstituted alkyl, alkoxy, alkylthio, or alkylamino;
R.sub.2=H, alkyl, or substituted alkyl;
Y=N or C;
R.sub.3 is absent if Y=N; or is H, alkyl, substituted alkyl, or
combined with R.sub.4 to form a carbocycle or heterocycle with the
carbon atoms of the pyridine ring to which they are connected if
Y=C;
R.sub.4 is H, or substituted or unsubstituted alkyl, alkoxy,
alkylthio, or alkylamino when not combined with R.sub.3 to form a
carbocycle or heterocycle with the carbon atoms of the pyridine
ring to which they are connected; or is combined with R.sub.3 to
form a carbocycle or heterocycle with the carbon atoms of the
pyridine ring to which they are connected;
X is C, N, O, or S;
R.sub.6 and R.sub.7 are each independently H or substituted;
and
R.sub.9, R.sub.10, R.sub.11, and R.sub.12 are each independently H,
a halogen, OH, thio, NH.sub.2, or substituted or unsubstituted
alkyl, aryl, heterocyclic, alkoxy, aryloxy, alkylthio, arylthio,
alkylamino, or arylamino.
The inventors have also unexpectedly and surprisingly discovered
that it is possible to make polymeric synthetic nanocarriers using
polymers that have a weight average molecular weight ranging from
about 800 Daltons to about 10,000 Daltons, as determined using gel
permeation chromatography. In the formulation of polymeric
synthetic nanocarriers, it has been generally believed that the
molecular weight of polymers should be or exceed 10,000 Daltons. At
times, it is advantageous to append to the polymers an
immunomodulatory compound that can be released from the synthetic
nanocarrier by a nonspecific degradation step within the body. If
the synthetic nanocarriers are to be used to target the
endosomal/lysosomal compartment, then it is particularly
advantageous to have this degradation step occur preferentially at
an acidic pH. One drawback to appending the immunomodulatory agent
to the polymer is that the loading is diminished as the molecular
weight of the polymer increases. In addition, as the molecular
weight of the polymer increases so does the hydrophobicity of the
polymer with the results that the degradation rate at a given pH
can decrease. This leads to an undesirably decreased release rate
of the immunomodulatory agent. Surprisingly, it has been found that
low molecular weight polymers with a weight average molecular
weight ranging from about 800 Daltons to about 10,000 Daltons form
stable synthetic nanocarriers and that the rate of release of
immunomodulatory agent from the synthetic nanocarrier is increased
as the molecular weight decreases. The polymer of the compounds
provided herein, therefore, in embodiments, have a weight average
molecular weight ranging from about 800 Daltons to about 10,000
Daltons, and such compounds may be used to produce synthetic
nanocarriers.
The compounds provided herein or the synthetic nanocarriers that
comprise the compounds may also be pH sensitive (i.e., exhibit
increased release of the immunomodulatory agent at or about a pH of
4.5 as compared to the release of the immunomodulatory agent at or
about physiological pH (i.e., pH or 7.4). The property of having
relatively low release of immunomodulatory agents at or about
physiological pH but increased release at or about a pH of 4.5 is
desirable for it targets the immunomodulatory agents to the
endosomal/lysosomal compartment of, for example, antigen presenting
cells (APCs) which tend to possess a pH that is at or about 4.5.
This low pH level is found primarily in the upper gastrointestinal
tract and endosome/lysosomes. Accordingly, unless the inventive
compounds and compositions are administered via an oral route of
administration, accelerated release at pH at or about 4.5 provides
for an enhanced concentration of the immunomodulatory agent in the
target compartment. Under these conditions, the immunomodulatory
agent exhibits a pH sensitive dissociation and is then free to
interact with receptors within the endosome/lysosome and stimulate
a desired immune response. Additionally, because the coupling of
the polymer may occur at a position on the immunomodulatory agent
or compound of interest that, generally, substantially reduces or
eliminates the biological activity of the immunomodulatory agent or
compound of interest, the coupling can effectively produce a
"pro-drug" like effect. This effect, in combination with
accelerated release in conditions present in the endosome/lysosome,
means that off-target effects (e.g., adverse events) are reduced
and safety margins increased for compositions and vaccines that
comprise the inventive compounds and compositions.
The present invention will now be described in more detail.
DEFINITIONS
"Administering" or "administration" means providing a compound,
conjugate, synthetic nanocarrier, or composition provided herein to
a patient in a manner that is pharmacologically useful.
"APC targeting feature" means one or more portions of which the
inventive synthetic nanocarriers are comprised that target the
synthetic nanocarriers to professional antigen presenting cells
("APCs"), such as but not limited to dendritic cells, SCS
macrophages, follicular dendritic cells, and B cells. In
embodiments, APC targeting features may comprise immunofeature
surface(s) and/or targeting moieties that bind known targets on
APCs. In embodiments, APC targeting features may comprise one or
more B cell antigens present on a surface of synthetic
nanocarriers. In embodiments, APC targeting features may also
comprise one or more dimensions of the synthetic nanoparticles that
is selected to promote uptake by APCs.
In embodiments, targeting moieties for known targets on macrophages
("Mphs") comprise any targeting moiety that specifically binds to
any entity (e.g., protein, lipid, carbohydrate, small molecule,
etc.) that is prominently expressed and/or present on macrophages
(i.e., subcapsular sinus-Mph markers). Exemplary SCS-Mph markers
include, but are not limited to, CD4 (L3T4, W3/25, T4); CD9 (p24,
DRAP-1, MRP-1); CD11a (LFA-1.alpha., .alpha. L Integrin chain);
CD11b (.alpha.M Integrin chain, CR3, Mo1, C3niR, Mac-1); CD11c
(.alpha.X Integrin, p150, 95, AXb2); CDw12 (p90-120); CD13 (APN,
gp150, EC 3.4.11.2); CD14 (LPS-R); CD15 (X-Hapten, Lewis, X,
SSEA-1,3-FAL); CD15s (Sialyl Lewis X); CD15u (3' sulpho Lewis X);
CD15su (6 sulpho-sialyl Lewis X); CD16a (FCRIIIA); CD16b
(FcgRIIIb); CDw17 (Lactosylceramide, LacCer); CD18 (Integrin
.beta.2, CD11a,b,c .beta.-subunit); CD26 (DPP IV ectoeneyme, ADA
binding protein); CD29 (Platelet GPIIa, .beta.-1 integrin, GP);
CD31 (PECAM-1, Endocam); CD32 (FC.gamma.RII); CD33 (gp67); CD35
(CR1, C3b/C4b receptor); CD36 (GpIIIb, GPIV, PASIV); CD37
(gp52-40); CD38 (ADP-ribosyl cyclase, T10); CD39 (ATPdehydrogenase,
NTPdehydrogenase-1); CD40 (Bp50); CD43 (Sialophorin, Leukosialin);
CD44 (EMCRII, H-CAM, Pgp-1); CD45 (LCA, T200, B220, Ly5); CD45RA;
CD45RB; CD45RC; CD45RO (UCHL-1); CD46 (MCP); CD47 (gp42, IAP, OA3,
Neurophillin); CD47R (MEM-133); CD48 (Blast-1, Hulym3, BCM-1,
OX-45); CD49a (VLA-1.alpha., .alpha.1 Integrin); CD49b
(VLA-2.alpha., gp1a, .alpha.2 Integrin); CD49c (VLA-3.alpha.,
.alpha.3 Integrin); CD49e (VLA-5.alpha., .alpha.5 Integrin); CD49f
(VLA-6.alpha., .alpha.6 Integrin, gp1c); CD50 (ICAM-3); CD51
(Integrin .alpha., VNR-.alpha., Vitronectin-R.alpha.); CD52
(CAMPATH-1, HE5); CD53 (OX-44); CD54 (ICAM-1); CD55 (DAF); CD58
(LFA-3); CD59 (1F5Ag, H19, Protectin, MACIF, MIRL, P-18); CD60a
(GD3); CD60b (9-O-acetyl GD3); CD61 (GP IIIa, .beta.3 Integrin);
CD62L (L-selectin, LAM-1, LECAM-1, MEL-14, Leu8, TQ1); CD63 (LIMP,
MLA1, gp55, NGA, LAMP-3, ME491); CD64 (Fc.gamma.RI); CD65
(Ceramide, VIM-2); CD65s (Sialylated-CD65, VIM2); CD72 (Ly-19.2,
Ly-32.2, Lyb-2); CD74 (Ii, invariant chain); CD75 (sialo-masked
Lactosamine); CD75S (.alpha.-2,6 sialylated Lactosamine); CD80 (B7,
B7-1, BB1); CD81 (TAPA-1); CD82 (4F9, C33, IA4, KAI1, R2); CD84
(p75, GR6); CD85a (ILT5, LIR2, HL9); CD85d (ILT4, LIR2, MIR10);
CD85j (ILT2, LIR1, MIR7); CD85k (ILT3, LIR5, HM18); CD86
(B7-2/B70); CD87 (uPAR); CD88 (C5aR); CD89 (IgA Fc receptor,
Fc.alpha.R); CD91 (.alpha.2M-R, LRP); CDw92 (p70); CDw93 (GR11);
CD95 (APO-1, FAS, TNFRSF6); CD97 (BL-KDD/F12); CD98 (4F2, FRP-1,
RL-388); CD99 (MIC2, E2); CD99R (CD99 Mab restricted); CD100
(SEMA4D); CD101 (IGSF2, P126, V7); CD102 (ICAM-2); CD111 (PVRL1,
HveC, PRR1, Nectin 1, HIgR); CD112 (HveB, PRR2, PVRL2, Nectin2);
CD114 (CSF3R, G-CSRF, HG-CSFR); CD115 (c-fms, CSF-1R, M-CSFR);
CD116 (GMCSFR.alpha.); CDw119 (IFN.gamma.R, IFN.gamma.RA); CD120a
(TNFR1, p55); CD120b (TNFRII, p75, TNFR p80); CD121b (Type 2
IL-1R); CD122 (IL2R.beta.); CD123 (IL-3R.alpha.); CD124
(IL-4R.alpha.); CD127 (p90, IL-7R, IL-7R.alpha.); CD128a (IL-8Ra,
CXCR1, (Tentatively renamed as CD181)); CD128b (IL-8Rb, CSCR2,
(Tentatively renamed as CD182)); CD130 (gp130); CD131 (Common (3
subunit); CD132 (Common .gamma. chain, IL-2R.gamma.); CDw136
(MSP-R, RON, p158-ron); CDw137 (4-1BB, ILA); CD139; CD141
(Thrombomodulin, Fetomodulin); CD147 (Basigin, EMMPRIN, M6, OX47);
CD148 (HPTP-.eta., p260, DEP-1); CD155 (PVR); CD156a (CD156, ADAMS,
MS2); CD156b (TACE, ADAM17, cSVP); CDw156C (ADAM10); CD157 (Mo5,
BST-1); CD162 (PSGL-1); CD164 (MGC-24, MUC-24); CD165 (AD2, gp37);
CD168 (RHAMM, IHABP, HMMR); CD169 (Sialoadhesin, Siglec-1); CD170
(Siglec 5); CD171 (L1CAM, NILE); CD172 (SIRP-1.alpha., MyD-1);
CD172b (SIRP.beta.); CD180 (RP105, Bgp95, Ly64); CD181 (CXCR1,
(Formerly known as CD128a)); CD182 (CXCR2, (Formerly known as
CD128b)); CD184 (CXCR4, NPY3R); CD191 (CCR1); CD192 (CCR2); CD195
(CCR5); CDw197 (CCR7 (was CDw197)); CDw198 (CCR8); CD204 (MSR);
CD205 (DEC-25); CD206 (MMR); CD207 (Langerin); CDw210 (CK); CD213a
(CK); CDw217 (CK); CD220 (Insulin R); CD221 (IGF1 R); CD222 (M6P-R,
IGFII-R); CD224 (GGT); CD226 (DNAM-1, PTA1); CD230 (Prion Protein
(PrP)); CD232 (VESP-R); CD244 (2B4, P38, NAIL); CD245 (p220/240);
CD256 (APRIL, TALL2, TNF (ligand) superfamily, member 13); CD257
(BLYS, TALL1, TNF (ligand) superfamily, member 13b); CD261
(TRAIL-R1, TNF-R superfamily, member 10a); CD262 (TRAIL-R2, TNF-R
superfamily, member 10b); CD263 (TRAIL-R3, TNBF-R superfamily,
member 10c); CD264 (TRAIL-R4, TNF-R superfamily, member 10d); CD265
(TRANCE-R, TNF-R superfamily, member 11a); CD277 (BT3.1, B7 family:
Butyrophilin 3); CD280 (TEM22, ENDO180); CD281 (TLR1, TOLL-like
receptor 1); CD282 (TLR2, TOLL-like receptor 2); CD284 (TLR4,
TOLL-like receptor 4); CD295 (LEPR); CD298 (ATP1B3, Na K ATPase,
.beta.3 subunit); CD300a (CMRF-35H); CD300c (CMRF-35A); CD300e
(CMRF-35L1); CD302 (DCL1); CD305 (LAIR1); CD312 (EMR2); CD315
(CD9P1); CD317 (BST2); CD321 (JAM1); CD322 (JAM2); CDw328
(Siglec7); CDw329 (Siglec9); CD68 (gp 110, Macrosialin); and/or
mannose receptor; wherein the names listed in parentheses represent
alternative names.
In embodiments, targeting moieties for known targets on dendritic
cells ("DCs") comprise any targeting moiety that specifically binds
to any entity (e.g., protein, lipid, carbohydrate, small molecule,
etc.) that is prominently expressed and/or present on DCs (i.e., a
DC marker). Exemplary DC markers include, but are not limited to,
CD1a (R4, T6, HTA-1); CD1b (R1); CD1c (M241, R7); CD1d (R3); CD1e
(R2); CD11b (.alpha.M Integrin chain, CR3, Mo1, C3niR, Mac-1);
CD11c (.alpha.X Integrin, p150, 95, AXb2); CDw117
(Lactosylceramide, LacCer); CD19 (B4); CD33 (gp67); CD 35 (CR1,
C3b/C4b receptor); CD 36 (GpIIIb, GPIV, PASIV); CD39
(ATPdehydrogenase, NTPdehydrogenase-1); CD40 (Bp50); CD45 (LCA,
T200, B220, Ly5); CD45RA; CD45RB; CD45RC; CD45RO (UCHL-1); CD49d
(VLA-4.alpha., .alpha.4 Integrin); CD49e (VLA-5.alpha., .alpha.5
Integrin); CD58 (LFA-3); CD64 (Fc.gamma.RI); CD72 (Ly-19.2,
Ly-32.2, Lyb-2); CD73 (Ecto-5' nucloticlase); CD74 (Ii, invariant
chain); CD80 (B7, B7-1, BB1); CD81 (TAPA-1); CD83 (HB15); CD85a
(ILT5, LIR3, HL9); CD85d (ILT4, LIR2, MIR10); CD85j (ILT2, LIR1,
MIR7); CD85k (ILT3, LIR5, HM18); CD86 (B7-2/B70); CD88 (C5aB); CD97
(BL-KDD/F12); CD101 (IGSF2, P126, V7); CD116 (GM-CSFR.alpha.);
CD120a (TMFRI, p55); CD120b (TNFRII, p75, TNFR p80); CD123
(IL-3R.alpha.); CD139; CD148 (HPTP-.eta., DEP-1); CD150 (SLAM,
IPO-3); CD156b (TACE, ADAM17, cSVP); CD157 (Mo5, BST-1); CD167a
(DDR1, trkE, cak); CD168 (RHAMM, IHABP, HMMR); CD169 (Sialoadhesin,
Siglec-1); CD170 (Siglec-5); CD171 (L1CAM, NILE); CD172
(SIRP-1.alpha., MyD-1); CD172b (SIRP.beta.); CD180 (RP105, Bgp95,
Ly64); CD184 (CXCR4, NPY3R); CD193 (CCR3); CD196 (CCR6); CD197
(CCR7 (ws CDw197)); CDw197 (CCR7, EBI1, BLR2); CD200 (OX2); CD205
(DEC-205); CD206 (MMR); CD207 (Langerin); CD208 (DC-LAMP); CD209
(DCSIGN); CDw218a (IL18R.alpha.); CDw218b (IL8R.beta.); CD227
(MUC1, PUM, PEM, EMA); CD230 (Prion Protein (PrP)); CD252 (OX40L,
TNF (ligand) superfamily, member 4); CD258 (LIGHT, TNF (ligand)
superfamily, member 14); CD265 (TRANCE-R, TNF-R superfamily, member
11a); CD271 (NGFR, p75, TNFR superfamily, member 16); CD273 (B7DC,
PDL2); CD274 (B7H1, PDL1); CD275 (B7H2, ICOSL); CD276 (B7H3); CD277
(BT3.1, B7 family: Butyrophilin 3); CD283 (TLR3, TOLL-like receptor
3); CD289 (TLR9, TOLL-like receptor 9); CD295 (LEPR); CD298
(ATP1B3, Na K ATPase .beta.3 submit); CD300a (CMRF-35H); CD300c
(CMRF-35A); CD301 (MGL1, CLECSF14); CD302 (DCL1); CD303 (BDCA2);
CD304 (BDCA4); CD312 (EMR2); CD317 (BST2); CD319 (CRACC, SLAMF7);
CD320 (8D6); and CD68 (gp110, Macrosialin); class II MHC; BDCA-1;
Siglec-H; wherein the names listed in parentheses represent
alternative names.
In embodiments, targeting can be accomplished by any targeting
moiety that specifically binds to any entity (e.g., protein, lipid,
carbohydrate, small molecule, etc.) that is prominently expressed
and/or present on B cells (i.e., B cell marker). Exemplary B cell
markers include, but are not limited to, CD1c (M241, R7); CD1d
(R3); CD2 (E-rosette R, T11, LFA-2); CD5 (T1, Tp67, Leu-1, Ly-1);
CD6 (T12); CD9 (p24, DRAP-1, MRP-1); CD11a (LFA-1.alpha., .alpha.L
Integrin chain); CD11b (.alpha.M Integrin chain, CR3, Mo1, C3niR,
Mac-1); CD11c (.alpha.X Integrin, P150, 95, AXb2); CDw17
(Lactosylceramide, LacCer); CD18 (Integrin .beta.2, CD11a, b, c
.beta.-subunit); CD19 (B4); CD20 (B1, Bp35); CD21 (CR2, EBV-R,
C3dR); CD22 (BL-CAM, Lyb8, Siglec-2); CD23 (FceRII, B6, BLAST-2,
Leu-20); CD24 (BBA-1, HSA); CD25 (Tac antigen, IL-2R.alpha., p55);
CD26 (DPP IV ectoeneyme, ADA binding protein); CD27 (T14, S152);
CD29 (Platelet GPIIa, .beta.-1 integrin, GP); CD31 (PECAM-1,
Endocam); CD32 (FC.gamma.RII); CD35 (CR1, C3b/C4b receptor); CD37
(gp52-40); CD38 (ADPribosyl cyclase, T10); CD39 (ATPdehydrogenase,
NTPdehydrogenase-1); CD40 (Bp50); CD44 (ECMRII, H-CAM, Pgp-1); CD45
(LCA, T200, B220, Ly5); CD45RA; CD45RB; CD45RC; CD45RO (UCHL-1);
CD46 (MCP); CD47 (gp42, IAP, OA3, Neurophilin); CD47R (MEM-133);
CD48 (Blast-1, Hulym3, BCM-1, OX-45); CD49b (VLA-2.alpha., gp1a,
.alpha.2 Integrin); CD49c (VLA-3.alpha., .alpha.3 Integrin); CD49d
(VLA-4.alpha., .alpha.4 Integrin); CD50 (ICAM-3); CD52 (CAMPATH-1,
HES); CD53 (OX-44); CD54 (ICAM-1); CD55 (DAF); CD58 (LFA-3); CD60a
(GD3); CD62L (L-selectin, LAM-1, LECAM-1, MEL-14, Leu8, TQ1); CD72
(Ly-19.2, Ly-32.2, Lyb-2); CD73 (Ecto-5'-nuciotidase); CD74 (Ii,
invariant chain); CD75 (sialo-masked Lactosamine); CD75S (.alpha.2,
6 sialytated Lactosamine); CD77 (Pk antigen, BLA, CTH/Gb3); CD79a
(Ig.alpha., MB1); CD79b (Ig.beta., B29); CD80; CD81 (TAPA-1); CD82
(4F9, C33, IA4, KAI1, R2); CD83 (HB15); CD84 (P75, GR6); CD85j
(ILT2, LIR1, MIR7); CDw92 (p70); CD95 (APO-1, FAS, TNFRSF6); CD98
(4F2, FRP-1, RL-388); CD99 (MIC2, E2); CD100 (SEMA4D); CD102
(ICAM-2); CD108 (SEMA7A, JMH blood group antigen); CDw119
(IFN.gamma.R, IFN.gamma.Ra); CD120a (TNFRI, p55); CD120b (TNFRII,
p75, TNFR p80); CD121b (Type 2 IL-1R); CD122 (IL2R.beta.); CD124
(IL-4R.alpha.); CD130 (gp130); CD132 (Common .gamma. chain,
IL-2R.gamma.); CDw137 (4-1BB, ILA); CD139; CD147 (Basigin, EMMPRIN,
M6, OX47); CD150 (SLAM, IPO-3); CD162 (PSGL-1); CD164 (MGC-24,
MUC-24); CD166 (ALCAM, KG-CAM, SC-1, BEN, DM-GRASP); CD167a (DDR1,
trkE, cak); CD171 (L1CMA, NILE); CD175s (Sialyl-Tn (S-Tn)); CD180
(RP105, Bgp95, Ly64); CD184 (CXCR4, NPY3R); CD185 (CXCR5); CD192
(CCR2); CD196 (CCR6); CD197 (CCR7 (was CDw197)); CDw197 (CCR7,
EBI1, BLR2); CD200 (OX2); CD205 (DEC-205); CDw210 (CK); CD213a
(CK); CDw217 (CK); CDw218a (IL18R.alpha.); CDw218b (IL18R.beta.);
CD220 (Insulin R); CD221 (IGF1 R); CD222 (M6P-R, IGFII-R); CD224
(GGT); CD225 (Leu13); CD226 (DNAM-1, PTA1); CD227 (MUC1, PUM, PEM,
EMA); CD229 (Ly9); CD230 (Prion Protein (Prp)); CD232 (VESP-R);
CD245 (p220/240); CD247 (CD3 Zeta Chain); CD261 (TRAIL-R1, TNF-R
superfamily, member 10a); CD262 (TRAIL-R2, TNF-R superfamily,
member 10b); CD263 (TRAIL-R3, TNF-R superfamily, member 10c); CD264
(TRAIL-R4, TNF-R superfamily, member 10d); CD265 (TRANCE-R, TNF-R
superfamily, member 11a); CD267 (TACI, TNF-R superfamily, member
13B); CD268 (BAFFR, TNF-R superfamily, member 13C); CD269 (BCMA,
TNF-R superfamily, member 16); CD275 (B7H2, ICOSL); CD277 (BT3.1.B7
family: Butyrophilin 3); CD295 (LEPR); CD298 (ATP1B3 Na K ATPase
.beta.3 subunit); CD300a (CMRF-35H); CD300c (CMRF-35A); CD305
(LAIR1); CD307 (IRTA2); CD315 (CD9P1); CD316 (EW12); CD317 (BST2);
CD319 (CRACC, SLAMF7); CD321 (JAM1); CD322 (JAM2); CDw327 (Siglec6,
CD33L); CD68 (gp 100, Macrosialin); CXCR5; VLA-4; class II MHC;
surface IgM; surface IgD; APRL; and/or BAFF-R; wherein the names
listed in parentheses represent alternative names. Examples of
markers include those provided elsewhere herein.
In some embodiments, B cell targeting can be accomplished by any
targeting moiety that specifically binds to any entity (e.g.,
protein, lipid, carbohydrate, small molecule, etc.) that is
prominently expressed and/or present on B cells upon activation
(i.e., activated B cell marker). Exemplary activated B cell markers
include, but are not limited to, CD 1a (R4, T6, HTA-1); CD1b (R1);
CD15s (Sialyl Lewis X); CD15u (3' sulpho Lewis X); CD15su (6
sulpho-sialyl Lewis X); CD30 (Ber-H2, Ki-1); CD69 (AIM, EA 1, MLR3,
gp34/28, VEA); CD70 (Ki-24, CD27 ligand); CD80 (B7, B7-1, BB1);
CD86 (B7-2/B70); CD97 (BLKDD/F12); CD125 (IL-5R.alpha.); CD126
(IL-6R.alpha.); CD138 (Syndecan-1, Heparan sulfate proteoglycan);
CD152 (CTLA-4); CD252 (OX40L, TNF(ligand) superfamily, member 4);
CD253 (TRAIL, TNF(ligand) superfamily, member 10); CD279 (PD1);
CD289 (TLR9, TOLL-like receptor 9); and CD312 (EMR2); wherein the
names listed in parentheses represent alternative names. Examples
of markers include those provided elsewhere herein.
"B cell antigen" means any antigen that naturally is or could be
engineered to be recognized by a B cell, and triggers (naturally or
being engineered as known in the art) an immune response in a B
cell (e.g., an antigen that is specifically recognized by a B cell
receptor on a B cell). In some embodiments, an antigen that is a T
cell antigen is also a B cell antigen. In other embodiments, the T
cell antigen is not also a B cell antigen. B cell antigens include,
but are not limited to proteins, peptides, small molecules, and
carbohydrates. In some embodiments, the B cell antigen is a
non-protein antigen (i.e., not a protein or peptide antigen). In
some embodiments, the B cell antigen is a carbohydrate associated
with an infectious agent. In some embodiments, the B cell antigen
is a glycoprotein or glycopeptide associated with an infectious
agent. The infectious agent can be a bacterium, virus, fungus,
protozoan, parasite or prion. In some embodiments, the B cell
antigen is a poorly immunogenic antigen. In some embodiments, the B
cell antigen is an abused substance or a portion thereof. In some
embodiments, the B cell antigen is an addictive substance or a
portion thereof. Addictive substances include, but are not limited
to, nicotine, a narcotic, a cough suppressant, a tranquilizer, and
a sedative. In some embodiments, the B cell antigen is a toxin,
such as a toxin from a chemical weapon or natural sources, or a
pollutant. The B cell antigen may also be a hazardous environmental
agent. In other embodiments, the B cell antigen is an alloantigen,
an allergen, a contact sensitizer, a degenerative disease antigen,
a hapten, an infectious disease antigen, a cancer antigen, an
atopic disease antigen, an addictive substance, a xenoantigen, or a
metabolic disease enzyme or enzymatic product thereof.
"Biodegradable polymer" means a polymer that degrades over time
when introduced into the body of a subject. Biodegradable polymers,
include but are not limited to, polyesters, polycarbonates,
polyketals, or polyamides. Such polymers may comprise poly(lactic
acid), poly(glycolic acid), poly(lactic-co-glycolic acid), or
polycaprolactone. In some embodiments, the biodegradable polymer
comprises a block-co-polymer of a polyether, such as poly(ethylene
glycol), and a polyester, polycarbonate, or polyamide, or other
biodegradable polymer. In embodiments, the biodegradable polymer
comprises a block-co-polymer of poly(ethylene glycol) and
poly(lactic acid), poly(glycolic acid), poly(lactic-co-glycolic
acid), or polycaprolactone. In some embodiments, however, the
biodegradable polymer does not comprise a polyether, such as
poly(ethylene glycol), or consist solely of the polyether.
Generally, for use as part of a synthetic nanocarrier the
biodegradable polymer is insoluble in water at pH=7.4 and at
25.degree. C. The biodegradable polymer, in embodiments, have a
weight average molecular weight ranging from about 800 to about
50,000 Daltons, as determined using gel permeation chromatography.
In some embodiments, the weight average molecular weight is from
about 800 Daltons to about 10,000 Daltons, preferably from 800
Daltons to 10,000 Daltons, as determined using gel permeation
chromatography. In other embodiments, the weight average molecular
weight is from 1000 Daltons to 10,000 Daltons, as determined by gel
permeation chromatography. In an embodiment, the biodegradable
polymer does not comprise polyketal or a unit thereof.
"Couple" or "Coupled" or "Couples" (and the like) means attached to
a polymer or unit thereof or attached to or contained within the
synthetic nanocarrier. In some embodiments, the covalent coupling
is mediated by one or more linkers. In some embodiments, the
coupling is non-covalent. In some embodiments, the non-covalent
coupling is mediated by charge interactions, affinity interactions,
metal coordination, physical adsorption, hostguest interactions,
hydrophobic interactions, TT stacking interactions, hydrogen
bonding interactions, van der Waals interactions, magnetic
interactions, electrostatic interactions, dipole-dipole
interactions, and/or combinations thereof. In embodiments, the
coupling may arise in the context of encapsulation within the
synthetic nanocarriers, using conventional techniques. Any of the
aforementioned couplings may be arranged to be on a surface or
within an inventive synthetic nanocarrier.
"Dosage form" means a compound, conjugate, synthetic nanocarrier,
or composition provided herein in a medium, carrier, vehicle, or
device suitable for administration to a subject.
"Encapsulate" means to enclose within a synthetic nanocarrier,
preferably enclose completely within a synthetic nanocarrier. Most
or all of a substance that is encapsulated is not exposed to the
local environment external to the synthetic nanocarrier.
Encapsulation is distinct from absorption, which places most or all
of a substance on a surface of a synthetic nanocarrier, and leaves
the substance exposed to the local environment external to the
synthetic nanocarrier. In embodiments, the immunomodulatory agent
or B cell and/or T cell antigen is encapsulated within the
synthetic nanocarrier.
"Immunomodulatory agent" means an agent that modulates an immune
response. "Modulate", as used herein, refers to inducing,
enhancing, stimulating, or directing an immune response. Such
agents include immunostimulatory agents that stimulate (or boost)
an immune response to an antigen but is not an antigen or derived
from an antigen. In some embodiments, the immunomodulatory agent is
on the surface of the synthetic nanocarrier and/or is incorporated
within the synthetic nanocarrier. In embodiments, the
immunomodulatory agent is coupled to the synthetic nanocarrier via
the polymer or unit thereof of the compounds or conjugates
provided.
In some embodiments, all of the immunomodulatory agents of a
synthetic nanocarrier are identical to one another. In some
embodiments, a synthetic nanocarrier comprises a number of
different types of immunomodulatory agents. In some embodiments, a
synthetic nanocarrier comprises multiple individual
immunomodulatory agents, all of which are identical to one another.
In some embodiments, a synthetic nanocarrier comprises exactly one
type of immunomodulatory agent. In some embodiments, a synthetic
nanocarrier comprises exactly two distinct types of
immunomodulatory agents. In some embodiments, a synthetic
nanocarrier comprises greater than two distinct types of
immunomodulatory agents.
"Maximum dimension of a synthetic nanocarrier" means the largest
dimension of a nanocarrier measured along any axis of the synthetic
nanocarrier. "Minimum dimension of a synthetic nanocarrier" means
the smallest dimension of a synthetic nanocarrier measured along
any axis of the synthetic nanocarrier. For example, for a
spheroidal synthetic nanocarrier, the maximum and minimum dimension
of a synthetic nanocarrier would be substantially identical, and
would be the size of its diameter. Similarly, for a cubic synthetic
nanocarrier, the minimum dimension of a synthetic nanocarrier would
be the smallest of its height, width or length, while the maximum
dimension of a synthetic nanocarrier would be the largest of its
height, width or length. In an embodiment, a minimum dimension of
at least 75%, preferably at least 80%, more preferably at least
90%, of the synthetic nanocarriers in a sample, based on the total
number of synthetic nanocarriers in the sample, is greater than 100
nm. In an embodiment, a maximum dimension of at least 75%,
preferably at least 80%, more preferably at least 90%, of the
synthetic nanocarriers in a sample, based on the total number of
synthetic nanocarriers in the sample, is equal to or less than 5
.mu.m. Preferably, a minimum dimension of at least 75%, preferably
at least 80%, more preferably at least 90%, of the synthetic
nanocarriers in a sample, based on the total number of synthetic
nanocarriers in the sample, is equal to or greater than 110 nm,
more preferably equal to or greater than 120 nm, more preferably
equal to or greater than 130 nm, and more preferably still equal to
or greater than 150 nm Preferably, a maximum dimension of at least
75%, preferably at least 80%, more preferably at least 90%, of the
synthetic nanocarriers in a sample, based on the total number of
synthetic nanocarriers in the sample is equal to or less than 3
.mu.m, more preferably equal to or less than 2 .mu.m, more
preferably equal to or less than 1 .mu.m, more preferably equal to
or less than 800 nm, more preferably equal to or less than 600 nm,
and more preferably still equal to or less than 500 nm. In
preferred embodiments, a maximum dimension of at least 75%,
preferably at least 80%, more preferably at least 90%, of the
synthetic nanocarriers in a sample, based on the total number of
synthetic nanocarriers in the sample, is equal to or greater than
100 nm, more preferably equal to or greater than 120 nm, more
preferably equal to or greater than 130 nm, more preferably equal
to or greater than 140 nm, and more preferably still equal to or
greater than 150 nm. Measurement of synthetic nanocarrier sizes is
obtained by suspending the synthetic nanocarriers in a liquid
(usually aqueous) media and using dynamic light scattering (e.g.
using a Brookhaven ZetaPALS instrument).
"Pharmaceutically acceptable excipient" means a pharmacologically
inactive substance added to an inventive compound, conjugate,
synthetic nanocarrier or composition to further facilitate its
administration. Examples, without limitation, of pharmaceutically
acceptable excipients include calcium carbonate, calcium phosphate,
various diluents, various sugars and types of starch, cellulose
derivatives, gelatin, vegetable oils, and polyethylene glycols.
"Release Rate" means the rate that an entrapped immunomodulatory
agent flows from a composition, such as a synthetic nanocarrier,
into a surrounding media in an in vitro release test. First, the
synthetic nanocarrier is prepared for the release testing by
placing into the appropriate in vitro release media. This is
generally done by exchanging the buffer after centrifugation to
pellet the synthetic nanocarrier and reconstitution of the
synthetic nanocarriers using a mild condition. The assay is started
by placing the sample at 37.degree. C. in an appropriate
temperature-controlled apparatus. A sample is removed at various
time points.
The synthetic nanocarriers are separated from the release media by
centrifugation to pellet the synthetic nanocarriers. The release
media is assayed for the immunomodulatory agent that has dispersed
from the synthetic nanocarriers. The immunomodulatory agent is
measured using HPLC to determine the content and quality of the
immunomodulatory agent. The pellet containing the remaining
entrapped immunomodulatory agent is dissolved in solvents or
hydrolyzed by base to free the entrapped immunomodulatory agent
from the synthetic nanocarriers. The pellet-containing
immunomodulatory agent is then also measured by HPLC to determine
the content and quality of the immunomodulatory agent that has not
been released at a given time point.
The mass balance is closed between immunomodulatory agent that has
been released into the release media and what remains in the
synthetic nanocarriers. Data are presented as the fraction released
or as the net release presented as micrograms released over
time.
"Subject" means an animal, including mammals such as humans and
primates; avians; domestic household or farm animals such as cats,
dogs, sheep, goats, cattle, horses and pigs; laboratory animals
such as mice, rats and guinea pigs; fish; and the like.
"Synthetic nanocarrier(s)" means a discrete object that is not
found in nature, and that possesses at least one dimension that is
less than or equal to 5 microns in size. Albumin nanoparticles are
expressly included as synthetic nanocarriers.
Synthetic nanocarriers include the compounds and compositions
provided herein and, therefore, can be polymeric nanoparticles. In
some embodiments, synthetic nanocarriers can comprise one or more
polymeric matrices. The synthetic nanocarriers, however, can also
include other nanomaterials and may be, for example, lipid-polymer
nanoparticles. In some embodiments, a polymeric matrix can be
surrounded by a coating layer (e.g., liposome, lipid monolayer,
micelle, etc.). In some embodiments, the synthetic nanocarrier is
not a micelle. In some embodiments, a synthetic nanocarrier may
comprise a core comprising a polymeric matrix surrounded by a lipid
layer (e.g., lipid bilayer, lipid monolayer, etc.). In some
embodiments, the various elements of the synthetic nanocarriers can
be coupled with the polymeric matrix.
The synthetic nanocarriers may comprise one or more lipids. In some
embodiments, a synthetic nanocarrier may comprise a liposome. In
some embodiments, a synthetic nanocarrier may comprise a lipid
bilayer. In some embodiments, a synthetic nanocarrier may comprise
a lipid monolayer. In some embodiments, a synthetic nanocarrier may
comprise a micelle. In some embodiments, a synthetic nanocarrier
may comprise a non-polymeric core (e.g., metal particle, quantum
dot, ceramic particle, bone particle, viral particle, proteins,
nucleic acids, carbohydrates, etc.) surrounded by a lipid layer
(e.g., lipid bilayer, lipid monolayer, etc.).
The synthetic nanocarriers may comprise lipid-based nanoparticles,
metallic nanoparticles, surfactant-based emulsions, dendrimers,
buckyballs, nanowires, virus-like particles, peptide or
protein-based particles (such as albumin nanoparticles). Synthetic
nanocarriers may be a variety of different shapes, including but
not limited to spheroidal, cubic, pyramidal, oblong, cylindrical,
toroidal, and the like. Synthetic nanocarriers according to the
invention comprise one or more surfaces. Exemplary synthetic
nanocarriers that can be adapted for use in the practice of the
present invention comprise: (1) the biodegradable nanoparticles
disclosed in U.S. Pat. No. 5,543,158 to Gref et al., (2) the
polymeric nanoparticles of Published U.S. Patent Application
20060002852 to Saltzman et al., (3) the lithographically
constructed nanoparticles of Published U.S. Patent Application
20090028910 to DeSimone et al., (4) the disclosure of WO
2009/051837 to von Andrian et al., or (5) the nanoparticles
disclosed in Published U.S. Patent Application 2008/0145441 to
Penades et al.
Synthetic nanocarriers according to the invention that have a
minimum dimension of equal to or less than about 100 nm, preferably
equal to or less than 100 nm, do not comprise a surface with
hydroxyl groups that activate complement or alternatively comprise
a surface that consists essentially of moieties that are not
hydroxyl groups that activate complement. In a preferred
embodiment, synthetic nanocarriers according to the invention that
have a minimum dimension of equal to or less than about 100 nm,
preferably equal to or less than 100 nm, do not comprise a surface
that substantially activates complement or alternatively comprise a
surface that consists essentially of moieties that do not
substantially activate complement. In a more preferred embodiment,
synthetic nanocarriers according to the invention that have a
minimum dimension of equal to or less than about 100 nm, preferably
equal to or less than 100 nm, do not comprise a surface that
activates complement or alternatively comprise a surface that
consists essentially of moieties that do not activate complement.
In embodiments, synthetic nanocarriers may possess an aspect ratio
greater than 1:1, 1:1.2, 1:1.5, 1:2, 1:3, 1:5, 1:7, or greater than
1:10.
In some embodiments, synthetic nanocarriers are spheres or
spheroids. In some embodiments, synthetic nanocarriers are flat or
plate-shaped. In some embodiments, synthetic nanocarriers are cubes
or cubic. In some embodiments, synthetic nanocarriers are ovals or
ellipses. In some embodiments, synthetic nanocarriers are
cylinders, cones, or pyramids.
It is often desirable to use a population of synthetic nanocarriers
that is relatively uniform in terms of size, shape, and/or
composition so that each synthetic nanocarrier has similar
properties. For example, at least 80%, at least 90%, or at least
95% of the synthetic nanocarriers may have a minimum dimension or
maximum dimension that falls within 5%, 10%, or 20% of the average
diameter or average dimension. In some embodiments, a population of
synthetic nanocarriers may be heterogeneous with respect to size,
shape, and/or composition.
Synthetic nanocarriers can be solid or hollow and can comprise one
or more layers. In some embodiments, each layer has a unique
composition and unique properties relative to the other layer(s).
To give but one example, synthetic nanocarriers may have a
core/shell structure, wherein the core is one layer (e.g., a
polymeric core) and the shell is a second layer (e.g., a lipid
bilayer or monolayer). Synthetic nanocarriers may comprise a
plurality of different layers.
"T cell antigen" means any antigen that is recognized by and
triggers an immune response in a T cell (e.g., an antigen that is
specifically recognized by a T cell receptor on a T cell or an NKT
cell via presentation of the antigen or portion thereof bound to a
Class I or Class II major histocompatability complex molecule
(MHC), or bound to a CD1 complex). In some embodiments, an antigen
that is a T cell antigen is also a B cell antigen. In other
embodiments, the T cell antigen is not also a B cell antigen. T
cell antigens generally are proteins or peptides. T cell antigens
may be an antigen that stimulates a CD8+ T cell response, a CD4+ T
cell response, or both. The T cell antigens, therefore, in some
embodiments can effectively stimulate both types of responses.
In some embodiments the T cell antigen is a T-helper antigen, which
is a T cell antigen that can generate an augmented response to an
unrelated B cell antigen through stimulation of T cell help. In
embodiments, a T-helper antigen may comprise one or more peptides
derived from tetanus toxoid, Epstein-Barr virus, influenza virus,
respiratory syncytial virus, measles virus, mumps virus, rubella
virus, cytomegalovirus, adenovirus, diphtheria toxoid, or a PADRE
peptide. In other embodiments, a T-helper antigen may comprise one
or more lipids, or glycolipids, including but not limited to:
.alpha.-galactosylceramide (.alpha.-GalCer), .alpha.-linked
glycosphingolipids (from Sphingomonas spp.), galactosyl
diacylglycerols (from Borrelia burgdorferi), lypophosphoglycan
(from Leishmania donovani), and phosphatidylinositol tetramannoside
(PIM4) (from Mycobacterium leprae). For additional lipids and/or
glycolipids useful as T-helper antigens, see V. Cerundolo et al.,
"Harnessing invariant NKT cells in vaccination strategies." Nature
Rev Immun, 9:28-38 (2009). In embodiments, CD4+ T-cell antigens may
be derivatives of a CD4+ T-cell antigen that is obtained from a
source, such as a natural source. In such embodiments, CD4+ T-cell
antigen sequences, such as those peptides that bind to MHC II, may
have at least 70%, 80%, 90%, or 95% identity to the antigen
obtained from the source. In embodiments, the T cell antigen,
preferably a T-helper antigen, may be coupled to, or uncoupled
from, a synthetic nanocarrier.
"Unit thereof" refers to a monomeric unit of a polymer, the polymer
generally being made up of a series of linked monomers.
"Vaccine" means a composition of matter that improves the immune
response to a particular pathogen or disease. A vaccine typically
contains factors that stimulate a subject's immune system to
recognize a specific antigen as foreign and eliminate it from the
subject's body. A vaccine also establishes an immunologic `memory`
so the antigen will be quickly recognized and responded to if a
person is re-challenged. Vaccines can be prophylactic (for example
to prevent future infection by any pathogen), or therapeutic (for
example a vaccine against a tumor specific antigen for the
treatment of cancer). Vaccines according to the invention may
comprise one or more of the compounds, conjugates, synthetic
nanocarriers, or compositions provided herein.
Methods of Making the Inventive Compounds, Conjugates, or Synthetic
Nanocarriers
The immunomodulatory agent and polymers or unit thereof are coupled
covalently via an amide or ester bond. In some embodiments, these
conjugates form part of a synthetic nanocarrier. In general, a
polymer, such as polylactide (PLA) or polylactide-co-glycolide
(PLGA), can be conjugated with an immunostimulatory agent, such as
resiquimod (also known as R848), in several ways. Methods for
coupling are provided below and in the EXAMPLES.
The following methods or any step of the methods provided are
exemplary and may be carried out under any suitable conditions. In
some cases, the reaction or any step of the methods provided may be
carried out in the presence of a solvent or a mixture of solvents.
Non-limiting examples of solvents that may be suitable for use in
the invention include, but are not limited to, p-cresol, toluene,
xylene, mesitylene, diethyl ether, glycol, petroleum ether, hexane,
cyclohexane, pentane, dichloromethane (or methylene chloride),
chloroform, dioxane, tetrahydrofuran (THF), dimethyl sulfoxide
(DMSO), dimethylformamide (DMF), ethyl acetate (EtOAc),
triethylamine, acetonitrile, methyl-t-butyl ether (MTBE),
N-methylpyrrolidone (NMP), dimethylacetamide (DMAC), isopropanol
(IPA), mixtures thereof, or the like. In some cases, the solvent is
selected from the group consisting of ethyl acetate, methylene
chloride, THF, DMF, NMP, DMAC, DMSO, and toluene, or a mixture
thereof.
A reaction or any step of the methods provided may be carried out
at any suitable temperature. In some cases, a reaction or any step
of the methods provided is carried out at about room temperature
(e.g., about 25.degree. C., about 20.degree. C., between about
20.degree. C. and about 25.degree. C., or the like). In some cases,
however, the reaction or any step of the methods provided may be
carried out at a temperature below or above room temperature, for
example, at about -20.degree. C., at about -10.degree. C., at about
0.degree. C., at about 10.degree. C., at about 30.degree. C., about
40.degree. C., about 50.degree. C., about 60.degree. C., about
70.degree. C., about 80.degree. C., about 90.degree. C., about
100.degree. C., about 120.degree. C., about 140.degree. C., about
150.degree. C. or greater. In particular embodiments, the reaction
or any step of the methods provided is conducted at temperatures
between 0.degree. C. and 120.degree. C. In some embodiments, the
reaction or any step of the methods provided may be carried out at
more than one temperature (e.g., reactants added at a first
temperature and the reaction mixture agitated at a second wherein
the transition from a first temperature to a second temperature may
be gradual or rapid).
The reaction or any step of the methods provided may be allowed to
proceed for any suitable period of time. In some cases, the
reaction or any step of the methods provided is allowed to proceed
for about 10 minutes, about 20 minutes, about 30 minutes, about 40
minutes, about 50 minutes, about 1 hour, about 2 hours, about 4
hours, about 8 hours, about 12 hours, about 16 hours, about 24
hours, about 2 days, about 3 days, about 4 days, or more. In some
cases, aliquots of the reaction mixture may be removed and analyzed
at an intermediate time to determine the progress of the reaction
or any step of the methods provided. In some embodiments, a
reaction or any step of the methods provided may be carried out
under an inert atmosphere in anhydrous conditions (e.g., under an
atmosphere of nitrogen or argon, anhydrous solvents, etc.)
The reaction products and/or intermediates may be isolated (e.g.,
via distillation, column chromatography, extraction, precipitation,
etc.) and/or analyzed (e.g., gas liquid chromatography, high
performance liquid chromatography, nuclear magnetic resonance
spectroscopy, etc.) using commonly known techniques. In some cases,
a conjugate or synthetic nanocarrier that includes the conjugated
may be analyzed to determine the loading of immunomodulatory agent,
for example, using reverse phase HPLC.
The polymers may have any suitable molecular weight. For example,
the polymers may have a low or high molecular weight. Non-limiting
molecular weight values include 100 Da, 200 Da, 300 Da, 500 Da, 750
Da, 1000 Da, 2000 Da, 3000 Da, 4000 Da, 5000 Da, 6000 Da, 7000 Da,
8000 Da, 9000 Da, 10,000 Da, or greater. In some embodiments, the
polymers have a weight average molecular weight of about 800 Da to
about 10,000 Da. The molecular weight of a polymer may be
determined using gel permeation chromatography.
Provided below are exemplary conjugation reactions that are not
intended to be limiting.
Method 1
A polymer (e.g., PLA, PLGA) or unit thereof with at least one acid
end groups is converted to a reactive acylating agent such as an
acyl halide, acylimidazole, active ester, etc. using an activating
reagent commonly used in amide synthesis.
In this two-step method, the resulting activated polymer or unit
thereof (e.g., PLA, PLGA) is isolated and then reacted with an
immunomodulatory agent (e.g., R848) in the presence of a base to
give the desired conjugate (e.g., PLA-R848), for example, as shown
in the following scheme:
##STR00039##
Activating reagents that can be used to convert polymers or units
thereof, such as PLA or PLGA, to an activated acylating form
include, but are not limited to cyanuric fluoride,
N,N-tetramethylfluoroformamidinium hexafluorophosphate (TFFH);
Acylimidazoles, such as carbonyl diimidazole (CDI),
N,N'-carbonylbis(3-methylimidazolium)triflate (CBMIT); and Active
esters, such as N-hydroxylsuccinimide (NHS or HOSu) in the presence
of a carbodiimide such as N,N'-dicyclohexylcarbodiimide (DCC),
N-ethyl-N'-(3-(dimethylamino)propyl)carbodiimide hydrochloride
(EDC) or N,N'-diisopropylcarbodiimide (DIC); N,N'-disuccinimidyl
carbonate (DSC); pentafluorophenol in the presence of DCC or EDC or
DIC; pentafluorophenyl trifluoroacetate.
The activated polymer or unit thereof may be isolated (e.g., via
precipitation, extraction, etc.) and/or stored under suitable
conditions (e.g., at low temperature, under argon) following
activation, or may be used immediately. The activated polymer or
unit thereof may be reacted with an immunostimulatory agent under
any suitable conditions. In some cases, the reaction is carried out
in the presence of a base and/or catalyst. Non-limiting examples of
bases/catalysts include diisopropylethylamine (DIPEA) and
4-dimethylaminopyridine (DMAP).
Method 2
A polymer or unit thereof (e.g., PLA, PLGA having any suitable
molecular weight) with an acid end group reacts with an
immunomodulatory agent (e.g., R848) in the presence of an
activating or coupling reagent, which converts the polymer or unit
thereof (e.g., PLA, PLGA) to a reactive acylating agent in situ, to
give the desired conjugate (e.g., PLA-R848, PLGA-R848).
##STR00040##
Coupling or activating agents include but are not limited to:
activating agents used in the presence of an carbodiimide such as
EDC or DCC or DIC, such as 1-Hydroxybenzotriazole (HOBt),
1-Hydroxy-7-azabenzotriazole (HOAt),
3,4-Dihydro-3-hydroxy-4-oxo-1,2,3-benzotriazine (HO-Dhbt),
N-Hydroxysuccinimide (NHS or HOSu), Pentafluorophenol (PFP);
Activating agents without carbodiimide: Phosphonium salts, such as
O-Benzotriazol-1-yloxytris(dimethylamino)phosphonium
hexafluorophosphate (BOP),
O-Benzotriazol-1-yloxytris(pyrrolidino)phosphonium
hexafluorophosphate (PyBOP),
7-Azabenzotriazol-1-yloxytris(pyrrolidino)phosphonium
hexafluorophosphate (PyAOP); uronium salts such as
O-Benzotriazol-1-yloxytris-1,1,3,3-tetramethyluronium
tetrafluoroborate (TBTU) and hexafluorophosphate (HBTU),
O-(7-Azabenzotriazol-1-yl)-1,1,3,3-tetramethyluronium
hexafluorophosphate (HATU),
O-(1,2-dihydro-2-oxo-1-pyridyl)-1,1,3,3-tetramethyl-uronium
tetrafluoroborate (TPTU); Halouronium and halophosphonium salts
such as bis(tetramethylene)fluoroformamidinium hexafluorophosphate
(BTFFH), bromotris(dimethylamino)phosphonium hexafluoro-phosphate
(BroP), bromotripyrrolidino phosphonium hexafluorophosphate
(PyBroP) and chlorotripyrrolidino phosphonium hexafluorophosphate
(PyClop); Benzotriazine derivatives such as
O-(3,4-Dihydro-4-oxo-1,2,3-benzotriazine-3-yl)-N,N,N',N'-tetramethyluroni-
um tetrafluoroborate (TDBTU) and
3-(diethyloxyphosphoryloxy)-1,2,3-benzotriazin-4(3H)-one (DEPBT).
Non-limiting examples of suitable solvents include DMF, DCM,
toluene, ethyl acetate, etc., as described herein.
Method 3
Immunomodulatory agents, such as R848, can also be coupled to
polymers or units thereof that are terminated in a hydroxyl group.
Such polymers or units thereof include polyethylene glycol,
polylactide, polylactide-co-glycolide, polycaprolactone, and other
like polyesters, or units thereof. In general, the reaction
proceeds as follows where an imide of the general structure (IV)
will react with the terminal hydroxyl of the aforementioned
polymers or units thereof using a catalyst used in lactone ring
opening polymerizations. The resulting reaction product (II) links
the amide of the agent to the polymer or unit thereof via an ester
bond. The compounds of formula (IV) and (II) are as follows:
##STR00041## wherein R.sub.1=H, OH, SH, NH.sub.2, or substituted or
unsubstituted alkyl, alkoxy, alkylthio, or alkylamino; R.sub.2=H,
alkyl, or substituted alkyl; Y=N or C; R.sub.3 is absent if Y=N; or
is H, alkyl, substituted alkyl, or combined with R.sub.4 to form a
carbocycle or heterocycle with the carbon atoms of the pyridine
ring to which they are connected if Y=C; R.sub.4 is H, or
substituted or unsubstituted alkyl, alkoxy, alkylthio, or
alkylamino when not combined with R.sub.3 to form a carbocycle or
heterocycle with the carbon atoms of the pyridine ring to which
they are connected; or is combined with R.sub.3 to form a
carbocycle or heterocycle with the carbon atoms of the pyridine
ring to which they are connected; R.sub.5 is a polymer or unit
thereof; X is C, N, O, or S; R.sub.6 and R.sub.7 are each
independently H or substituted; and R.sub.9, R.sub.10, R.sub.11,
and R.sub.12 are each independently H, a halogen, OH, thio,
NH.sub.2, or substituted or unsubstituted alkyl, aryl,
heterocyclic, alkoxy, aryloxy, alkylthio, arylthio, alkylamino, or
arylamino.
Catalysts include, but are not limited to, phosphazine bases,
1,8-diazabicycloundec-7-ene (DBU), 1,4,7-triazabicyclodecene (TBD),
and N-methyl-1,4,7-triazabicyclodecene (MTDB). Other catalysts are
known in the art and provided, for example, in Kamber et al.,
Organocatalytic Ring-Opening Polymerization, Chem. Rev. 2007, 107,
58-13-5840. Non-limiting examples of suitable solvents include
methylene chloride, chloroform, and THF.
A specific example of a reaction completed by such a method is
shown here:
##STR00042## wherein R.sub.5--OH contains two hydroxyl groups
(e.g., a diol, HO--R.sub.5--OH), each of which are functionalized
by reaction with an imide associated with R848. In some cases,
HO--R.sub.5--OH is a poly-diol such as poly(hexamethyl
carbonate)diol or polycaprolactone diol. For example, the reaction
may be carried out as follows:
##STR00043## wherein the R groups are as described herein.
Non-limiting examples of suitable polymers include polyketaldiols,
poly(ethylene)glycol, polycaprolactone diol, diblock
polylactide-co-poly(ethylene)glycol, diblock
polylactide/polyglycolide-co-poly(ethylene)glycol, diblock
polyglycolide-co-poly(ethylene)glycol, poly(propylene)glycol,
poly(hexamethylene carbonate)diol, and poly(tetrahydrofuran).
In embodiments where a poly-diol is employed, one of the diol
groups may be protected with a protecting group (e.g.,
t-butyloxycarbonyl), thus the poly-diol would be a compound of
formula HO--R.sub.5--OP, wherein P is a protecting group. Following
reaction with an immunomodulatory agent to form a immunomodulatory
agent-R.sub.5--OP conjugate, the protecting group may be removed
and the second diol group may be reacted with any suitable reagent
(e.g., PLGA, PLA).
Method 4
A conjugate (e.g., R848-PLA) can be formed via a one-pot
ring-opening polymerization of an immunomodulatory agent (e.g.,
R848) with a polymer or unit thereof (e.g., D/L-lactide) in the
presence of a catalyst, for example, as shown in the following
scheme:
##STR00044##
In a one-step procedure, the immunomodulatory agent and the polymer
or unit thereof may be combined into a single reaction mixture
comprising a catalyst. The reaction may proceed at a suitable
temperature (e.g., at about 150.degree. C.) and the resulting
conjugate may be isolated using commonly known techniques.
Non-limiting examples of suitable catalysts include DMAP and tin
ethylhexanoate.
Method 5
A conjugate can be formed via two-step ring opening polymerization
of an immunomodulatory agent (e.g., R848) with one or more polymers
or units thereof (e.g., D/L-lactide and glycolide) in the presence
of a catalyst, for example, as shown in the following scheme:
##STR00045##
The polymers or units thereof may be first combined, and in some
cases, heated (e.g., to 135.degree. C.) to form a solution. The
immunomodulatory agent may be added to a solution comprising the
polymers or units thereof, followed by addition of a catalyst
(e.g., tin ethylhexanoate). The resulting conjugate may be isolated
using commonly known techniques. Non-limiting examples of suitable
catalysts include DMAP and tin ethylhexanoate.
In some embodiments, a compound or conjugate provided herein,
another immunomodulatory agent, antigen, and/or targeting moiety
can be covalently associated with a polymeric matrix. In some
embodiments, covalent association is mediated by a linker. In some
embodiments, a compound or conjugate provided herein, another
immunomodulatory agent, antigen, and/or targeting moiety can be
noncovalently associated with a polymeric matrix. For example, in
some embodiments, a compound or conjugate provided herein, another
immunomodulatory agent, antigen, and/or targeting moiety can be
encapsulated within, surrounded by, and/or dispersed throughout a
polymeric matrix. Alternatively or additionally, a compound or
conjugate provided herein, another immunomodulatory agent, antigen,
and/or targeting moiety can be associated with a polymeric matrix
by hydrophobic interactions, charge interactions, van der Waals
forces, etc.
A wide variety of polymers and methods for forming polymeric
matrices therefrom are known conventially. In general, a polymeric
matrix comprises one or more polymers. Polymers may be natural or
unnatural (synthetic) polymers. Polymers may be homopolymers or
copolymers comprising two or more monomers. In terms of sequence,
copolymers may be random, block, or comprise a combination of
random and block sequences. Typically, polymers in accordance with
the present invention are organic polymers.
Examples of polymers suitable for use in the present invention
include, but are not limited to polyethylenes, polycarbonates
(e.g., poly(1,3-dioxan-2one)), polyanhydrides (e.g., poly(sebacic
anhydride)), polyhydroxyacids (e.g.,
poly(.beta.-hydroxyalkanoate)), polypropylfumerates,
polycaprolactones, polyamides (e.g., polycaprolactam), polyacetals,
polyethers, polyesters (e.g., polylactide, polyglycolide),
poly(orthoesters), polycyanoacrylates, polyvinyl alcohols,
polyurethanes, polyphosphazenes, polyacrylates, polymethacrylates,
polyureas, polystyrenes, polyamines, and polysaccharides (e.g.,
chitosan).
In some embodiments, polymers in accordance with the present
invention include polymers which have been approved for use in
humans by the U.S. Food and Drug Administration (FDA) under 21
C.F.R. .sctn.177.2600, including but not limited to polyesters
(e.g., polylactic acid, poly(lactic-co-glycolic acid),
polycaprolactone, polyvalerolactone, poly(1,3-dioxan-2one));
polyanhydrides (e.g., poly(sebacic anhydride)); polyethers (e.g.,
polyethylene glycol); polyurethanes; polymethacrylates;
polyacrylates; and polycyanoacrylates.
In some embodiments, polymers can be hydrophilic. For example,
polymers may comprise anionic groups (e.g., phosphate group,
sulphate group, carboxylate group); cationic groups (e.g.,
quaternary amine group); or polar groups (e.g., hydroxyl group,
thiol group, amine group). In some embodiments, a synthetic
nanocarrier comprising a hydrophilic polymeric matrix generates a
hydrophilic environment within the synthetic nanocarrier. In some
embodiments, polymers can be hydrophobic. In some embodiments, a
synthetic nanocarrier comprising a hydrophobic polymeric matrix
generates a hydrophobic environment within the synthetic
nanocarrier. Selection of the hydrophilicity or hydrophobicity of
the polymer may have an impact on the nature of materials that are
incorporated (e.g., coupled) within the synthetic nanocarrier.
In some embodiments, polymers may be modified with one or more
moieties and/or functional groups. A variety of moieties or
functional groups can be used in accordance with the present
invention. In some embodiments, polymers may be modified with PEG,
with a carbohydrate, and/or with acyclic polyacetals derived from
polysaccharides (Papisov, 2001, ACS Symposium Series, 786:301).
In some embodiments, polymers may be modified with a lipid or fatty
acid group. In some embodiments, a fatty acid group may be one or
more of butyric, caproic, caprylic, capric, lauric, myristic,
palmitic, stearic, arachidic, behenic, or lignoceric acid. In some
embodiments, a fatty acid group may be one or more of palmitoleic,
oleic, vaccenic, linoleic, alpha-linoleic, gamma-linoleic,
arachidonic, gadoleic, arachidonic, eicosapentaenoic,
docosahexaenoic, or erucic acid.
In some embodiments, polymers may be polyesters, including
copolymers comprising lactic acid and glycolic acid units, such as
poly(lactic acid-co-glycolic acid) and poly(lactide-co-glycolide),
collectively referred to herein as "PLGA"; and homopolymers
comprising glycolic acid units, referred to herein as "PGA," and
lactic acid units, such as poly-L-lactic acid, poly-D-lactic acid,
poly-D,L-lactic acid, poly-L-lactide, poly-D-lactide, and
poly-D,L-lactide, collectively referred to herein as "PLA." In some
embodiments, exemplary polyesters include, for example,
polyhydroxyacids; PEG copolymers and copolymers of lactide and
glycolide (e.g., PLA-PEG copolymers, PGA-PEG copolymers, PLGA-PEG
copolymers, and derivatives thereof. In some embodiments,
polyesters include, for example, polyanhydrides, poly(ortho ester),
poly(ortho ester)-PEG copolymers, poly(caprolactone),
poly(caprolactone)-PEG copolymers, polylysine, polylysine-PEG
copolymers, poly(ethyleneimine), poly(ethylene imine)-PEG
copolymers, poly(L-lactide-co-L-lysine), poly(serine ester),
poly(4-hydroxy-L-proline ester),
poly[.alpha.-(4-aminobutyl)-L-glycolic acid], and derivatives
thereof.
In some embodiments, a polymer may be PLGA. PLGA is a biocompatible
and biodegradable co-polymer of lactic acid and glycolic acid, and
various forms of PLGA are characterized by the ratio of lactic
acid:glycolic acid. Lactic acid can be L-lactic acid, D-lactic
acid, or D,L-lactic acid. The degradation rate of PLGA can be
adjusted by altering the lactic acid:glycolic acid ratio. In some
embodiments, PLGA to be used in accordance with the present
invention is characterized by a lactic acid:glycolic acid ratio of
approximately 85:15, approximately 75:25, approximately 60:40,
approximately 50:50, approximately 40:60, approximately 25:75, or
approximately 15:85.
In some embodiments, polymers may be one or more acrylic polymers.
In certain embodiments, acrylic polymers include, for example,
acrylic acid and methacrylic acid copolymers, methyl methacrylate
copolymers, ethoxyethyl methacrylates, cyanoethyl methacrylate,
aminoalkyl methacrylate copolymer, poly(acrylic acid),
poly(methacrylic acid), methacrylic acid alkylamide copolymer,
poly(methyl methacrylate), poly(methacrylic acid anhydride), methyl
methacrylate, polymethacrylate, poly(methyl methacrylate)
copolymer, polyacrylamide, aminoalkyl methacrylate copolymer,
glycidyl methacrylate copolymers, polycyanoacrylates, and
combinations comprising one or more of the foregoing polymers. The
acrylic polymer may comprise fully-polymerized copolymers of
acrylic and methacrylic acid esters with a low content of
quaternary ammonium groups.
In some embodiments, polymers can be cationic polymers. In general,
cationic polymers are able to condense and/or protect negatively
charged strands of nucleic acids (e.g., DNA, RNA, or derivatives
thereof). Amine-containing polymers such as poly(lysine) (Zauner et
al., 1998, Adv. Drug Del. Rev., 30:97; and Kabanov et al., 1995,
Bioconjugate Chem., 6:7), poly(ethylene imine) (PEI; Boussif et
al., 1995, Proc. Natl. Acad. Sci., USA, 1995, 92:7297), and
poly(amidoamine) dendrimers (Kukowska-Latallo et al., 1996, Proc.
Natl. Acad. Sci., USA, 93:4897; Tang et al., 1996, Bioconjugate
Chem., 7:703; and Haensler et al., 1993, Bioconjugate Chem., 4:372)
are positively-charged at physiological pH, form ion pairs with
nucleic acids, and mediate transfection in a variety of cell
lines.
In some embodiments, polymers can be degradable polyesters bearing
cationic side chains (Putnam et al., 1999, Macromolecules, 32:3658;
Barrera et al., 1993, J. Am. Chem. Soc., 115:11010; Kwon et al.,
1989, Macromolecules, 22:3250; Lim et al., 1999, J. Am. Chem. Soc.,
121:5633; and Zhou et al., 1990, Macromolecules, 23:3399). Examples
of these polyesters include poly(L-lactide-co-L-lysine) (Barrera et
al., 1993, J. Am. Chem. Soc., 115:11010), poly(serine ester) (Zhou
et al., 1990, Macromolecules, 23:3399), poly(4-hydroxy-L-proline
ester) (Putnam et al., 1999, Macromolecules, 32:3658; and Lim et
al., 1999, J. Am. Chem. Soc., 121:5633), and
poly(4-hydroxy-L-proline ester) (Putnam et al., 1999,
Macromolecules, 32:3658; and Lim et al., 1999, J. Am. Chem. Soc.,
121:5633).
The properties of these and other polymers and methods for
preparing them are well known in the art (see, for example, U.S.
Pat. Nos. 6,123,727; 5,804,178; 5,770,417; 5,736,372; 5,716,404;
6,095,148; 5,837,752; 5,902,599; 5,696,175; 5,514,378; 5,512,600;
5,399,665; 5,019,379; 5,010,167; 4,806,621; 4,638,045; and
4,946,929; Wang et al., 2001, J. Am. Chem. Soc., 123:9480; Lim et
al., 2001, J. Am. Chem. Soc., 123:2460; Langer, 2000, Acc. Chem.
Res., 33:94; Langer, 1999, J. Control. Release, 62:7; and Uhrich et
al., 1999, Chem. Rev., 99:3181). More generally, a variety of
methods for synthesizing certain suitable polymers are described in
Concise Encyclopedia of Polymer Science and Polymeric Amines and
Ammonium Salts, Ed. by Goethals, Pergamon Press, 1980; Principles
of Polymerization by Odian, John Wiley & Sons, Fourth Edition,
2004; Contemporary Polymer Chemistry by Allcock et al.,
Prentice-Hall, 1981; Deming et al., 1997, Nature, 390:386; and in
U.S. Pat. Nos. 6,506,577, 6,632,922, 6,686,446, and 6,818,732.
In some embodiments, polymers can be linear or branched polymers.
In some embodiments, polymers can be dendrimers. In some
embodiments, polymers can be substantially cross-linked to one
another. In some embodiments, polymers can be substantially free of
cross-links. In some embodiments, polymers can be used in
accordance with the present invention without undergoing a
cross-linking step. It is further to be understood that inventive
compounds and synthetic nanocarriers may comprise block copolymers,
graft copolymers, blends, mixtures, and/or adducts of any of the
foregoing and other polymers. Those skilled in the art will
recognize that the polymers listed herein represent an exemplary,
not comprehensive, list of polymers that can be of use in
accordance with the present invention.
In some embodiments, synthetic nanocarriers may comprise metal
particles, quantum dots, ceramic particles, etc.
In some embodiments, synthetic nanocarriers may optionally comprise
one or more amphiphilic entities. In some embodiments, an
amphiphilic entity can promote the production of synthetic
nanocarriers with increased stability, improved uniformity, or
increased viscosity. In some embodiments, amphiphilic entities can
be associated with the interior surface of a lipid membrane (e.g.,
lipid bilayer, lipid monolayer, etc.). Many amphiphilic entities
known in the art are suitable for use in making synthetic
nanocarriers in accordance with the present invention. Such
amphiphilic entities include, but are not limited to,
phosphoglycerides; phosphatidylcholines; dipalmitoyl
phosphatidylcholine (DPPC); dioleylphosphatidyl ethanolamine
(DOPE); dioleyloxypropyltriethylammonium (DOTMA);
dioleoylphosphatidylcholine; cholesterol; cholesterol ester;
diacylglycerol; diacylglycerolsuccinate; diphosphatidyl glycerol
(DPPG); hexanedecanol; fatty alcohols such as polyethylene glycol
(PEG); polyoxyethylene-9-lauryl ether; a surface active fatty acid,
such as palmitic acid or oleic acid; fatty acids; fatty acid
monoglycerides; fatty acid diglycerides; fatty acid amides;
sorbitan trioleate (Span.RTM.85) glycocholate; sorbitan monolaurate
(Span.RTM.20); polysorbate 20 (Tween.RTM.20); polysorbate 60
(Tween.RTM.60); polysorbate 65 (Tween.RTM.65); polysorbate 80
(Tween.RTM.80); polysorbate 85 (Tween.RTM.85); polyoxyethylene
monostearate; surfactin; a poloxomer; a sorbitan fatty acid ester
such as sorbitan trioleate; lecithin; lysolecithin;
phosphatidylserine; phosphatidylinositol; sphingomyelin;
phosphatidylethanolamine (cephalin); cardiolipin; phosphatidic
acid; cerebrosides; dicetylphosphate;
dipalmitoylphosphatidylglycerol; stearylamine; dodecylamine;
hexadecyl-amine; acetyl palmitate; glycerol ricinoleate; hexadecyl
sterate; isopropyl myristate; tyloxapol; poly(ethylene
glycol)5000-phosphatidylethanolamine; poly(ethylene
glycol)400-monostearate; phospholipids; synthetic and/or natural
detergents having high surfactant properties; deoxycholates;
cyclodextrins; chaotropic salts; ion pairing agents; and
combinations thereof. An amphiphilic entity component may be a
mixture of different amphiphilic entities. Those skilled in the art
will recognize that this is an exemplary, not comprehensive, list
of substances with surfactant activity. Any amphiphilic entity may
be used in the production of synthetic nanocarriers to be used in
accordance with the present invention.
In some embodiments, synthetic nanocarriers may optionally comprise
one or more carbohydrates. Carbohydrates may be natural or
synthetic. A carbohydrate may be a derivatized natural
carbohydrate. In certain embodiments, a carbohydrate comprises
monosaccharide or disaccharide, including but not limited to
glucose, fructose, galactose, ribose, lactose, sucrose, maltose,
trehalose, cellulose, mannose, xylose, arabinose, glucoronic acid,
galactoronic acid, mannuronic acid, glucosamine, galatosamine, and
neuramic acid. In certain embodiments, a carbohydrate is a
polysaccharide, including but not limited to pullulan, cellulose,
microcrystalline cellulose, hydroxypropyl methylcellulose (HPMC),
hydroxycellulose (HC), methylcellulose (MC), dextran, cyclodextran,
glycogen, starch, hydroxyethylstarch, carageenan, glycon, amylose,
chitosan, N,O-carboxylmethylchitosan, algin and alginic acid,
starch, chitin, heparin, konjac, glucommannan, pustulan, heparin,
hyaluronic acid, curdlan, and xanthan. In certain embodiments, the
carbohydrate is a sugar alcohol, including but not limited to
mannitol, sorbitol, xylitol, erythritol, maltitol, and
lactitol.
Synthetic nanocarriers may be prepared using a wide variety of
methods known in the art. For example, synthetic nanocarriers can
be formed by methods as nanoprecipitation, flow focusing fluidic
channels, spray drying, single and double emulsion solvent
evaporation, solvent extraction, phase separation, milling,
microemulsion procedures, microfabrication, nanofabrication,
sacrificial layers, simple and complex coacervation, and other
methods well known to those of ordinary skill in the art.
Alternatively or additionally, aqueous and organic solvent
syntheses for monodisperse semiconductor, conductive, magnetic,
organic, and other nanomaterials have been described (Pellegrino et
al., 2005, Small, 1:48; Murray et al., 2000, Ann. Rev. Mat. Sci.,
30:545; and Trindade et al., 2001, Chem. Mat., 13:3843). Additional
methods have been described in the literature (see, e.g., Doubrow,
Ed., "Microcapsules and Nanoparticles in Medicine and Pharmacy,"
CRC Press, Boca Raton, 1992; Mathiowitz et al., 1987, J. Control.
Release, 5:13; Mathiowitz et al., 1987, Reactive Polymers, 6:275;
and Mathiowitz et al., 1988, J. Appl. Polymer Sci., 35:755, and
also U.S. Pat. Nos. 5,578,325 and 6,007,845).
In certain embodiments, synthetic nanocarriers are prepared by a
nanoprecipitation process or spray drying. Conditions used in
preparing synthetic nanocarriers may be altered to yield particles
of a desired size or property (e.g., hydrophobicity,
hydrophilicity, external morphology, "stickiness," shape, etc.).
The method of preparing the synthetic nanocarriers and the
conditions (e.g., solvent, temperature, concentration, air flow
rate, etc.) used may depend on the materials to be coupled to the
synthetic nanocarriers and/or the composition of the polymer
matrix.
If particles prepared by any of the above methods have a size range
outside of the desired range, particles can be sized, for example,
using a sieve.
Coupling can be achieved in a variety of different ways, and can be
covalent or non-covalent. Such couplings may be arranged to be on a
surface or within an inventive synthetic nanocarrier. Elements of
the inventive synthetic nanocarriers (such as moieties of which an
immunofeature surface is comprised, targeting moieties, polymeric
matrices, and the like) may be directly coupled with one another,
e.g., by one or more covalent bonds, or may be coupled by means of
one or more linkers. Additional methods of functionalizing
synthetic nanocarriers may be adapted from Published US Patent
Application 2006/0002852 to Saltzman et al., Published US Patent
Application 2009/0028910 to DeSimone et al., or Published
International Patent Application WO/2008/127532 A1 to Murthy et
al.
Any suitable linker can be used in accordance with the present
invention. Linkers may be used to form amide linkages, ester
linkages, disulfide linkages, etc. Linkers may contain carbon atoms
or heteroatoms (e.g., nitrogen, oxygen, sulfur, etc.). In some
embodiments, a linker is an aliphatic or heteroaliphatic linker. In
some embodiments, the linker is a polyalkyl linker. In certain
embodiments, the linker is a polyether linker. In certain
embodiments, the linker is a polyethylene linker. In certain
specific embodiments, the linker is a polyethylene glycol (PEG)
linker.
In some embodiments, the linker is a cleavable linker. To give but
a few examples, cleavable linkers include protease cleavable
peptide linkers, nuclease sensitive nucleic acid linkers, lipase
sensitive lipid linkers, glycosidase sensitive carbohydrate
linkers, pH sensitive linkers, hypoxia sensitive linkers,
photo-cleavable linkers, heat-labile linkers, enzyme cleavable
linkers (e.g., esterase cleavable linker), ultrasound-sensitive
linkers, x-ray cleavable linkers, etc. In some embodiments, the
linker is not a cleavable linker.
A variety of methods can be used to couple a linker or other
element of a synthetic nanocarrier with the synthetic nanocarrier.
General strategies include passive adsorption (e.g., via
electrostatic interactions), multivalent chelation, high affinity
non-covalent binding between members of a specific binding pair,
covalent bond formation, etc. (Gao et al., 2005, Curr. Op.
Biotechnol., 16:63). In some embodiments, click chemistry can be
used to associate a material with a synthetic nanocarrier.
Non-covalent specific binding interactions can be employed. For
example, either a particle or a biomolecule can be functionalized
with biotin with the other being functionalized with streptavidin.
These two moieties specifically bind to each other noncovalently
and with a high affinity, thereby associating the particle and the
biomolecule. Other specific binding pairs could be similarly used.
Alternately, histidine-tagged biomolecules can be associated with
particles conjugated to nickel-nitrolotriaceteic acid (Ni-NTA).
For additional general information on coupling, see the journal
Bioconjugate Chemistry, published by the American Chemical Society,
Columbus Ohio, PO Box 3337, Columbus, Ohio, 43210; "Cross-Linking,"
Pierce Chemical Technical Library, available at the Pierce web site
and originally published in the 1994-95 Pierce Catalog, and
references cited therein; Wong S S, Chemistry of Protein
Conjugation and Cross-linking, CRC Press Publishers, Boca Raton,
1991; and Hermanson, G. T., Bioconjugate Techniques, Academic
Press, Inc., San Diego, 1996.
It is to be understood that the compositions of the invention can
be made in any suitable manner, and the invention is in no way
limited to compositions that can be produced using the methods
described herein. Selection of an appropriate method may require
attention to the properties of the particular moieties being
associated.
Pharmaceutical Compositions and Methods of Use
Compositions according to the invention comprise inventive
compounds, conjugates, or synthetic nanocarriers, optionally, in
combination with pharmaceutically acceptable excipients. The
compositions may be made using conventional pharmaceutical
manufacturing and compounding techniques to arrive at useful dosage
forms. In an embodiment, inventive compounds, conjugates, synthetic
nanocarriers, or compositions are suspended in sterile saline
solution for injection together with a preservative.
In some embodiments, inventive compounds, conjugates, synthetic
nanocarriers, or compositions are manufactured under sterile
conditions or are terminally sterilized. This can ensure that
resulting compositions are sterile and non-infectious, thus
improving safety when compared to non-sterile compositions. This
provides a valuable safety measure, especially when subjects
receiving inventive compounds, conjugates, synthetic nanocarriers,
or compositions have immune defects, are suffering from infection,
and/or are susceptible to infection. In some embodiments, inventive
compounds, conjugates, synthetic nanocarriers, or compositions may
be lyophilized and stored in suspension or as lyophilized powder
depending on the formulation strategy for extended periods without
losing activity.
The inventive compounds, conjugates, synthetic nanocarriers, or
compositions may be administered by a variety of routes of
administration, including but not limited to parenteral,
subcutaneous, intramuscular, intradermal, oral, intranasal,
transmucosal, rectal; ophthalmic, transdermal, transcutaneous or by
a combination of these routes.
The inventive compounds, conjugates, synthetic nanocarriers, or
compositions and methods described herein can be used to induce,
enhance, stimulate, modulate, or direct an immune response. The
inventive compounds, conjugates, synthetic nanocarriers, or
compositions and methods described herein can be used in the
diagnosis, prophylaxis and/or treatment of conditions such as
cancers, infectious diseases, metabolic diseases, degenerative
diseases, inflammatory diseases, immunological diseases, or other
disorders and/or conditions. The inventive compounds, conjugates,
synthetic nanocarriers, or compositions and methods described
herein can also be used for the prophylaxis or treatment of an
addiction, such as an addiction to nicotine or a narcotic. The
inventive compounds, conjugates, synthetic nanocarriers, or
compositions and methods described herein can also be used for the
prophylaxis and/or treatment of a condition resulting from the
exposure to a toxin, hazardous substance, environmental toxin, or
other harmful agent.
EXAMPLES
Example 1
One-Pot Ring-Opening Polymerization of R848 with D/L-Lactide in the
Presence of a Catalyst
##STR00046##
A mixture of R848 (0.2 mmol, 63 mg), D/L-lactide (40 mmol, 5.8 g),
and 4-dimethylaminopyridine (DMAP) (50 mg, 0.4 mmol) in 2 mL of
anhydrous toluene was heated slowly to 150.degree. C. (oil bath
temperature) and maintained at this temperature for 18 h (after 3
hr, no R848 was left). The mixture was cooled to ambient
temperature and the resulting mixture was quenched with water (50
mL) to precipitate out the resulting polymer, R848-PLA. The polymer
was then washed sequentially with 45 mL each of MeOH, iPrOH, and
ethyl ether. The polymer was dried under vacuum at 30.degree. C. to
give an off-white puffy solid (5.0 g). Polymeric structure was
confirmed by .sup.1H NMR in CDCl.sub.3. A small sample of the
polymer was treated with 2 N NaOH aq in THF/MeOH to determine the
loading of R848 on the polymer by reverse phase HPLC. The loading
of R848 is 3 mg per gram of polymer (0.3% loading-27.5% of
theory).
Example 2
Two Step Ring Opening Polymerization of R848 with D/L-Lactide and
Glycolide
##STR00047##
A mixture of D/L-lactide (10.8 g, 0.075 moles) and glycolide (2.9
g, 0.025 moles) was heated to 135.degree. C. under argon. Once all
of the materials had melted and a clear solution had resulted, R848
(1.08 g, 3.43.times.10.sup.-3 moles) was added. This solution was
stirred at 135.degree. C. under a slow stream of argon for one
hour. Tin ethylhexanoate (150 .mu.L) was added and heating was
continued for 4 hours. After cooling, the solid pale brown mass was
dissolved in methylene chloride (250 mL) and the solution was
washed with 5% tartaric acid solution (2.times.200 mL). The
methylene chloride solution was dried over magnesium sulfate,
filtered, and then concentrated under vacuum. The residue was
dissolved in methylene chloride (20 mL) and 2-propanol (250 mL) was
added with stirring. The polymer that separated was isolated by
decantation of the 2-propanol and was dried under high vacuum. NMR
showed that the polymer was 71.4% lactide and 28.6% glycolide with
a molecular weight of 4000. The loading of R848 was close to
theoretical by NMR.
Example 3
Preparation of PLGA-R848 Conjugate
##STR00048##
A mixture of PLGA (Lakeshores Polymers, MW .about.5000, 7525DLG1A,
acid number 0.7 mmol/g, 10 g, 7.0 mmol) and HBTU (5.3 g, 14 mmol)
in anhydrous EtOAc (160 mL) was stirred at room temperature under
argon for 50 minutes. Compound R848 (2.2 g, 7 mmol) was added,
followed by diisopropylethylamine (DIPEA) (5 mL, 28 mmol). The
mixture was stirred at room temperature for 6 h and then at
50-55.degree. C. overnight (about 16 h). After cooling, the mixture
was diluted with EtOAc (200 mL) and washed with saturated
NH.sub.4Cl solution (2.times.40 mL), water (40 mL) and brine
solution (40 mL). The solution was dried over Na.sub.2SO.sub.4 (20
g) and concentrated to a gel-like residue. Isopropyl alcohol (IPA)
(300 mL) was then added and the polymer conjugate precipitated out
of solution. The polymer was then washed with IPA (4.times.50 mL)
to remove residual reagents and dried under vacuum at 35-40.degree.
C. for 3 days as a white powder (10.26 g, MW by GPC is 5200, R848
loading is 12% by HPLC).
Example 4
Preparation of PLGA-854A Conjugate
##STR00049##
A mixture of PLGA (Lakeshores Polymers, MW .about.5000, 7525DLG1A,
acid number 0.7 mmol/g, 1.0 g, 7.0 mmol) and HBTU (0.8 g, 2.1 mmol)
in anhydrous EtOAc (20 mL) was stirred at room temperature under
argon for 45 minutes. Compound 845A (0.29 g, 0.7 mmol) was added,
followed by diisopropylethylamine (DIPEA) (0.73 mL, 4.2 mmol). The
mixture was stirred at room temperature for 6 h and then at
50-55.degree. C. overnight (about 15 h). After cooling, the mixture
was diluted with EtOAc (100 mL) and washed with saturated NH4Cl
solution (2.times.20 mL), water (20 mL) and brine solution (20 mL).
The solution was dried over Na.sub.2SO.sub.4 (10 g) and
concentrated to a gel-like residue. Isopropyl alcohol (IPA) (40 mL)
was then added and the polymer conjugate precipitated out of
solution. The polymer was then washed with IPA (4.times.25 mL) to
remove residual reagents and dried under vacuum at 35-40.degree. C.
for 2 days as a white powder (1.21 g, MW by GPC is 4900, 854A
loading is 14% by HPLC).
Example 5
Preparation of PLGA-BBHA Conjugate
##STR00050##
A mixture of PLGA (Lakeshores Polymers, MW .about.5000, 7525DLG1A,
acid number 0.7 mmol/g, 1.0 g, 7.0 mmol) and HBTU (0.8 g, 2.1 mmol)
in anhydrous EtOAc (30 mL) was stirred at room temperature under
argon for 30 minutes. Compound BBHA (0.22 g, 0.7 mmol) in 2 mL of
dry DMSO was added, followed by diisopropylethylamine (DIPEA) (0.73
mL, 4.2 mmol). The mixture was stirred at room temperature for 20
h. Additional amounts of HBTU (0.53 g, 1.4 mmol) and DIPEA (0.5 mL,
2.8 mmol) were added and the mixture was heated at 50-55.degree. C.
for 4 h. After cooling, the mixture was diluted with EtOAc (100 mL)
and washed with saturated NH4Cl solution 20 mL), water (2.times.20
mL) and brine solution (20 mL). The solution was dried over
Na.sub.2SO.sub.4 (10 g) and concentrated to a gel-like residue.
Isopropyl alcohol (IPA) (35 mL) was then added and the brownish
polymer conjugate precipitated out of solution. The polymer was
then washed with IPA (2.times.20 mL) to remove residual reagents
and dried under vacuum at 35-40.degree. C. for 2 days as a brownish
powder (1.1 g).
Example 6
Preparation of Low MW PLA-R848 Conjugate
##STR00051##
A solution of PLA-CO2H (average MW: 950, DPI: 1.32; 5.0 g, 5.26
mmol) and HBTU (4.0 g, 10.5 mmol) in EtOAc (120 mL) was stirred at
room temperature under argon for 45 min Compound R848 (1.65 g, 5.26
mmol) was added, followed by DIPEA (5.5 mL, 31.6 mmol). The mixture
was stirred at room temperature for 6 h and then at 50-55.degree.
C. for 15 h. After cooling, the mixture was diluted with EtOAc (150
mL) and washed with 1% citric acid solution (2.times.40 mL), water
(40 mL) and brine solution (40 mL). The solution was dried over
Na.sub.2SO.sub.4 (10 g) and concentrated to a gel-like residue.
Methyl t-butyl ether (MTBE) (150 mL) was then added and the polymer
conjugate precipitated out of solution. The polymer was then washed
with MTBE (50 mL) and dried under vacuum at room temperature for 2
days as a white foam (5.3 g, average MW by GPC is 1200, PDI: 1.29;
R848 loading is 20% by HPLC).
Example 7
Preparation of Low MW PLA-R848 Conjugate
##STR00052##
A solution of PLA-CO2H (average MW: 1800, DPI:1.44; 9.5 g, 5.26
mmol) and HBTU (4.0 g, 10.5 mmol) in EtOAc (120 mL) was stirred at
room temperature under argon for 45 min Compound R848 (1.65 g, 5.26
mmol) was added, followed by DIPEA (5.5 mL, 31.6 mmol). The mixture
was stirred at room temperature for 6 h and then at 50-55.degree.
C. for 15 h. After cooling, the mixture was diluted with EtOAc (150
mL) and washed with 1% citric acid solution (2.times.40 mL), water
(40 mL) and brine solution (40 mL). The solution was dried over
Na.sub.2SO.sub.4 (10 g) and concentrated to a gel-like residue.
Methyl t-butyl ether (MTBE) (150 mL) was then added and the polymer
conjugate precipitated out of solution. The polymer was then washed
with MTBE (50 mL) and dried under vacuum at room temperature for 2
days as a white foam (9.5 g, average MW by GPC is 1900, PDI: 1.53;
R848 loading is 17% by HPLC).
Example 8
Conjugation of R848 to PCADK Via Imide Ring Opening
The following examples describes the synthesis of a polyketal,
PCADK, according to a method provided in Pulendran et al, WO
2008/127532, illustrated in step 1 below.
PCADK is synthesized in a 50 mL two-necked flask, connected to a
short-path distilling head. First, 5.5 mg of re-crystallized
p-toluenesulfonic acid (0.029 mmol, Aldrich, St. Louis, Mo.), is
dissolved in 6.82 mL of ethyl acetate, and added to a 30 mL benzene
solution (kept at 100.degree. C.), which contains
1,4-cyclohexanedimethanol (12.98 g, 90.0 mmol, Aldrich). The ethyl
acetate is allowed to boil off, and distilled 2,2-dimethoxypropane
(10.94 mL, 90.0 mmol, Aldrich) is added to the benzene solution,
initiating the polymerization reaction. Additional doses of
2,2-dimethoxypropane (5 mL) and benzene (25 mL) are subsequently
added to the reaction every hour for 6 hours via a metering funnel
to compensate for 2,2-dimethoxypropane and benzene that is
distilled off. After 8 hours, the reaction is stopped by addition
of 500 .mu.L of triethylamine. The polymer is isolated by
precipitation in cold hexane (stored at -20.degree. C.) followed by
vacuum filtration. The molecular weight of PCADK is determined by
gel permeation chromatography (GPC) (Shimadzu, Kyoto, Japan)
equipped with a UV detector. THF is used as the mobile phase at a
flow rate of 1 ml/min Polystyrene standards from Polymer
Laboratories (Amherst, Mass.) are used to establish a molecular
weight calibration curve. This compound is used to generate the
PCADK particles in all subsequent experiments.
R848 may be conjugated to the terminal alcohol groups of the PCADK
having molecular weight 6000 via imide ring opening, according to
the step 2 shown below.
##STR00053##
In step 2, the polymer from step 1 (12 g, 2.0.times.10.sup.-3
moles) is dissolved in methylene chloride 100 mL, and the lactam of
R848 (3.3 g, 8.0.times.10.sup.-3 moles) is added. This slurry is
stirred as 1,5,7-triazabicyclo-[4,4,0]dec-5-ene (TBD, 0.835 g,
6.times.10.sup.-3 moles) is added in a single portion. After
stirring at room temperature overnight, a clear solution forms. The
solution is diluted with methylene chloride (100 mL) and the
solution is washed with 5% citric acid. This solution is dried over
sodium sulfate after which it is filtered and evaporated under
vacuum. After drying under high vacuum there is obtained 11.3 grams
(81%) of polymer. A portion is hydrolyzed in acid and the R848
content is determined to be 9% by weight.
Example 9
Conjugation of R848 to Poly-Caprolactonediol Via Imide Ring
Opening
Imide ring opening is used to attach R854 to the terminal alcohol
groups of poly-caprolactonediol of molecular weight 2000. The
polycaprolactone diol is purchased from Aldrich Chemical Company,
Cat. #189421, and has the following structure:
##STR00054##
The polycaprolactone diol-R854 conjugate has the following
structure:
##STR00055##
The polymer (5 g, 2.5.times.10.sup.-3 moles) is dissolved in
methylene chloride 25 mL and the lactam of R854 (2.4 g,
5.0.times.10.sup.-3 moles) is added. This slurry is stirred as
1,5,7-triazabicyclo-[4,4,0]dec-5-ene (TBD, 0.557 g,
4.times.10.sup.-3 moles) is added in a single portion. After
stirring at room temperature for 15 minutes, a clear pale yellow
solution forms. The solution is diluted with methylene chloride
(100 mL) and the solution is washed with 5% citric acid. This
solution is dried over sodium sulfate after which it is filtered
and evaporated under vacuum. After drying under high vacuum there
is obtained 5.2 grams (70%) of polymer. A portion is hydrolyzed in
acid and the R848 content is determined to be 18.5% by weight.
Example 10
Conjugation of R848 to Poly-(Hexamethylene Carbonate)Diol Via Imide
Ring Opening
Imide ring opening is used to attach R848 to the terminal alcohol
groups of poly-(hexamethylene carbonate)diol of molecular weight
2000. The poly(hexamethylene carbonate)diol is purchased from
Aldrich Chemical Company, Cat #461164, and has the following
structure:
HO--[CH.sub.2(CH.sub.2).sub.4CH.sub.2OCO.sub.2]nCH.sub.2(CH.sub.2).sub.4C-
H.sub.2--OH.
The poly(hexamethylene carbonate)diol-R848 conjugate has the
following structure:
##STR00056##
The polymer (5 g, 2.5.times.10.sup.-3 moles) is dissolved in
methylene chloride 25 mL and the lactam of R848 (2.06 g,
5.0.times.10.sup.-3 moles) is added. This slurry is stirred as
1,5,7-triazabicyclo-[4,4,0]dec-5-ene (TBD, 0.557 g,
4.times.10.sup.-3 moles) is added in a single portion. After
stirring at room temperature overnight a clear pale yellow solution
forms. The solution is diluted with methylene chloride (100 mL) and
the solution is washed with 5% citric acid. This solution is dried
over sodium sulfate after which it is filtered and evaporated under
vacuum. After drying under high vacuum there is obtained 5.9 grams
(84%) of polymer. NMR is used to determine the R848 content which
is determined to be 21%.
Example 11
Polylactic Acid Conjugates of an Imidazoquinoline Using a Tin
Ethylhexanoate Catalyst
##STR00057##
To a two necked round bottom flask equipped with a stir bar and
condenser was added the imidazoquinoline resiquimod (R-848, 100 mg,
3.18.times.10.sup.-4 moles), D/L lactide (5.6 g,
3.89.times.10.sup.-2 moles) and anhydrous sodium sulfate (4.0 g).
The flask and contents were dried under vacuum at 50.degree. C. for
8 hours. The flask was then flushed with argon and toluene (100 mL)
was added. The reaction was stirred in an oil bath set at
120.degree. C. until all of the lactide had dissolved and then tin
ethylhexanoate (75 mg, 60 .mu.L) was added via pipette. Heating was
continued under argon for 16 hours. After cooling, water (20 mL)
was added and stirring was continued for 30 minutes. The reaction
was diluted with additional toluene (200 mL) and was then washed
with water (200 mL). The toluene solution was then washed in turn
with 10% sodium chloride solution containing 5% conc. Hydrochloric
acid (200 mL) followed by saturated sodium bicarbonate (200 mL).
TLC (silica, 10% methanol in methylene chloride) showed that the
solution contained no free R-848. The solution was dried over
magnesium sulfate, filtered and evaporated under vacuum to give
3.59 grams of polylactic acid-R-848 conjugate. A portion of the
polymer was hydrolyzed in base and examined by HPLC for R-848
content. By comparison to a standard curve of R-848 concentration
vs. HPLC response, it was determined that the polymer contained
4.51 mg of R-848 per gram of polymer. The molecular weight of the
polymer was determined by GPC to be about 19,000.
Example 12
Low Molecular Weight Polylactic Acid Conjugates of an
Imidazoquinoline
##STR00058##
To a round bottom flask equipped with a stir bar and condenser was
added the imidazoquinoline, resiquimod (R-848, 218 mg,
6.93.times.10.sup.-4 moles), D/L lactide (1.0 g,
6.93.times.10.sup.-3 moles) and anhydrous sodium sulfate (800 mg).
The flask and contents were dried under vacuum at 55.degree. C. for
8 hours. After cooling, the flask was then flushed with argon and
toluene (50 mL) was added. The reaction was stirred in an oil bath
set at 120.degree. C. until all of the lactide had dissolved and
then tin ethylhexanoate (19 mg, 15 .mu.L) was added via pipette.
Heating was continued under argon for 16 hours. After cooling, the
reaction was diluted with ether (200 mL) and the solution was
washed with water (200 mL). The solution was dried over magnesium
sulfate, filtered and evaporated under vacuum to give 880 mg. of
crude polylactic acid-R-848 conjugate. The crude polymer was
chromatographed on silica using 10% methanol in methylene chloride
as eluent. The fractions containing the conjugate were pooled and
evaporated to give the purified conjugate. This was dried under
high vacuum to provide the conjugate as a solid foam in a yield of
702 mg (57.6%). By integrating the NMR signals for the aromatic
protons of the quinoline and comparing this to the integrated
intensity of the lactic acid CH proton it was determined that the
molecular weight of the conjugate was approximately 2 KD. GPC
showed that the conjugate contained less than 5% of free R848.
Example 13
Low Molecular Weight Polylactic Acid Co-Glycolic Acid Conjugates of
an Imidazoquinoline
##STR00059##
To a round bottom flask equipped with a stir bar and condenser was
added the imidazoquinoline, resiquimod (R-848, 436 mg,
1.39.times.10.sup.-3 moles), glycolide (402 mg,
3.46.times.10.sup.-3 moles), D/L lactide (2.0 g,
1.39.times.10.sup.-2 moles) and anhydrous sodium sulfate (1.6 g).
The flask and contents were dried under vacuum at 55.degree. C. for
8 hours. After cooling, the flask was then flushed with argon and
toluene (60 mL) was added. The reaction was stirred in an oil bath
set at 120.degree. C. until all of the R848, glycolide and lactide
had dissolved and then tin ethylhexanoate (50 mg, 39 .mu.L) was
added via pipette. Heating was continued under argon for 16 hours.
After cooling, the reaction was diluted with ethyl acetate (200 mL)
and the solution was washed with water (200 mL). The solution was
dried over magnesium sulfate, filtered and evaporated under vacuum
to give crude PLGA-R-848 conjugate. The crude polymer was
chromatographed on silica using 10% methanol in methylene chloride
as eluent. The fractions containing the conjugate were pooled and
evaporated to give the purified conjugate. This was dried under
high vacuum to provide the conjugate as a solid foam in a yield of
1.55 g (54.6%). By integrating the NMR signals for the aromatic
protons of the quinoline and comparing this to the integrated
intensity of the lactic acid CH proton it was determined that the
molecular weight of the conjugate was approximately 2 KD. GPC
showed that the conjugate contained no detectable free R848.
Example 14
Polylactic Acid Conjugates of an Imidazoquinoline Using a Lithium
Diisopropylamide Catalysis
The imidazoquinoline (R-848), D/L lactide, and associated glassware
were all dried under vacuum at 50.degree. C. for 8 hours prior to
use. To a round bottom flask equipped with a stir bar and condenser
was added the R-848 (33 mg, 1.05.times.10.sup.-4 moles), and dry
toluene (5 mL). This was heated to reflux to dissolve all of the
R-848. The solution was stirred under nitrogen and cooled to room
temperature to provide a suspension of finely divided R-848. To
this suspension was added a solution of lithium diisopropyl amide
(2.0 M in THF, 50 .mu.L, 1.0.times.10.sup.-4 moles) after which
stiffing was continued at room temperature for 5 minutes. The pale
yellow solution that had formed was added via syringe to a hot
(120.degree. C.) solution of D/L lactide (1.87 g,
1.3.times.10.sup.-2 moles) under nitrogen. The heat was removed and
the pale yellow solution was stirred at room temperature for one
hour. The solution was diluted with methylene chloride (200 mL) and
this was then washed with 1% hydrochloric acid (2.times.50 mL)
followed by saturated sodium bicarbonate solution (50 mL). The
solution was dried over magnesium sulfate, filtered and evaporated
under vacuum to give the polylactic acid-R-848 conjugate. TLC
(silica, 10% methanol in methylene chloride) showed that the
solution contained no free R-848. The polymer was dissolved in
methylene chloride (10 mL) and the solution was dripped into
stirred hexane (200 mL). The precipitated polymer was isolated by
decantation and was dried under vacuum to give 1.47 grams of the
polylactic acid--R-848 conjugate as a white solid. A portion of the
polymer was hydrolyzed in base and examined by HPLC for R-848
content. By comparison to a standard curve of R-848 concentration
vs. HPLC response, it was determined that the polymer contained
10.96 mg of R-848 per gram of polymer.
Example 15
Polylactic Acid Activation
PLA (D/L-polylactide) (Resomer R202H from Boehringer-Ingelheim, KOH
equivalent acid number of 0.21 mmol/g, intrinsic viscosity (iv):
0.21 dl/g) (10 g, 2.1 mmol, 1.0 eq) was dissolved in
dichloromethane (DCM) (35 mL). EDC (2.0 g, 10.5 mmol, 5 eq) and NHS
(1.2 g, 10.5 mmol, 5 eq) were added. The solids were dissolved with
the aid of sonication. The resulting solution was stirred at room
temperature for 6 days. The solution was concentrated to remove
most of DCM and the residue was added to a solution of 250 mL of
diethyl ether and 5 mL of MeOH to precipitate out the activated
PLA-NHS ester. The solvents were removed and the polymer was washed
twice with ether (2.times.200 mL) and dried under vacuum to give
PLA-NHS activated ester as a white foamy solid (.about.8 g
recovered, .sup.1H NMR confirmed the presence of NHS ester). The
PLA-NHS ester is stored under argon in a below -10.degree. C.
freezer before use.
Alternatively, the reaction can be performed in DMF, THF, dioxane,
or CHCl.sub.3 instead of DCM. DCC can be used instead of EDC
(resulting DCC-urea is filtered off before precipitation of the
PLA-NHS ester from ether). The amount of EDC or DCC and NHS can be
in the range of 2-10 eq of the PLA.
Example 16
PLA Activation
PLA (D/L-polylactide) with MW of 5000 (10.5 g, 2.1 mmol, 1.0 eq) is
dissolved in dichloromethane (DCM) (35 mL). EDC (2.0 g, 10.5 mmol,
5 eq) and NHS (1.2 g, 10.5 mmol, 5 eq) are added. The resulting
solution is stirred at room temperature for 3 days. The solution is
concentrated to remove most of DCM and the residue is added to a
solution of 250 mL of diethyl ether and 5 mL of MeOH to precipitate
out the activated PLA-NHS ester. The solvents are removed and the
polymer is washed twice with ether (2.times.200 mL) and dried under
vacuum to give PLA-NHS activated ester as a white foamy solid
(.about.8 g recovered, .sup.1H NMR can be used to confirm the
presence of NHS ester). The PLA-NHS ester is stored under argon in
a below -10.degree. C. freezer before use.
Alternatively, the reaction can be performed in DMF, THF, dioxane,
or CHCl.sub.3 instead of DCM. DCC can be used instead of EDC
(resulting DCC-urea is filtered off before precipitation of the
PLA-NHS ester from ether). The amount of EDC or DCC and NHS can be
in the range of 2-10 eq of the PLA.
Example 17
Low MW PLGA Activation
In the same manner as provided above for polymer activation, low MW
PLGA with 50% to 75% glycolide is converted to the corresponding
PLGA-NHS activated ester and is stored under argon in a below
-10.degree. C. freezer before use.
Example 18
Polylactic Acid Activation
PLA (R202H, acid number of 0.21 mmol/g) (2.0 g, 0.42 mmol, 1.0 eq)
was dissolved in 10 mL of dry acetonitrile. N,N'-disuccinimidyl
carbonate (DSC) (215 mg, 1.26 mmol, 3.0 eq) and catalytic amount of
4-(N,N-dimethylamino)pyridine (DMAP) were added. The resulting
mixture was stirred under argon for 1 day. The resulting solution
was concentrated to almost dryness. The residue was then added to
40 mL of ether to precipitate out the polymer which was washed
twice with ether (2.times.30 mL) and dried under vacuum to give
PLA-NHS activated ester (1H NMR showed the amount of NHS ester at
about 80%).
Example 19
Polylactic Acid Activation
PLA (R202H) (5.0 g, 1.05 mmol) was dissolved in 25 mL of anhydrous
DCM and 2.5 mL of anhydrous DMF. DCC (650 mg, 3.15 mmol, 5.0 eq)
and pentafluorophenol (PFP) (580 mg, 3.15 mmol, 5.0 eq) were added.
The resulting solution was stirred at room temperature for 6 days
and then concentrated to remove DCM. The resulting residue was
added to 250 mL of ether to precipitate out the activated PLA
polymer which was washed with ether (2.times.100 mL) and dried
under vacuum to give PLA-PFP activated ester as a white foamy solid
(4.0 g).
Example 20
Polylactic Acid or PLGA Conjugates of an Imidazoquinoline
PLA-NHS (1.0 g), R848 (132 mg, 0.42 mmol), and
diisopropylethylamine (DIPEA) (0.073 mL, 0.42 mmol) were dissolved
in 2 mL of dry DMF under argon. The resulting solution was heated
at 50-60.degree. C. for 2 days. The solution was cooled to room
temperature and added to 40 mL of de-ionized (DI) water to
precipitate out the polymer product. The polymer was then washed
with DI water (40 mL) and ether (2.times.40 mL) and dried at
30.degree. C. under vacuum to give R848-PLA conjugate as a white
foamy solid (0.8 g, .sup.1H NMR showed the conjugation of R848 to
PLA via the amide bond). The degree of conjugation (loading) of
R848 on the polymer was confirmed by HPLC analysis as follows: a
weighed amount of polymer was dissolved in THF/MeOH and treated
with 15% NaOH. The resulting hydrolyzed polymer products were
analyzed for the amount of R848 by HPLC in comparison with a
standard curve.
Example 21
Polylactic Acid or PLGA Conjugates of an Imidazoquinoline
PLA-NHS (1.0 g, 0.21 mmol, 1.0 eq), R848 (132 mg, 0.42 mmol, 2.0
eq), DIPEA (0.15 mL, 0.84 mmol, 4.0 eq) and DMAP (25 mg, 0.21 mmol,
1.0 eq) were dissolved in 2 mL of dry DMF under argon. The
resulting solution was heated at 50-60.degree. C. for 2 days. The
solution was cooled to room temperature and added to 40 mL of
de-ionized (DI) water to precipitate out the polymer product. The
polymer was then washed with DI water (40 mL) and ether (2.times.40
mL) and dried at 30.degree. C. under vacuum to give PLA-R848
conjugate as a white foamy solid (0.7 g, 20 mg of the polymer was
hydrolyzed in solution of 0.2 mL of THF, 0.1 mL of MeOH and 0.1 mL
of 15% NaOH. The amount of R848 on the polymer was determined to be
about 35 mg/g by reverse phase HPLC analysis (C18 column, mobile
phase A: 0.1% TFA in water, mobile phase B: 0.1% TFA in CH3CN,
gradient).
Example 22
Polylactic Acid Conjugates of an Imidazoquinoline
PLA (R202H) (2.0 g, 0.42 mmol, 1.0 eq), DCC (260 mg, 1.26 mmol, 3.0
eq), NHS (145 mg, 1.26 mmol, 3.0 eq), R848 (200 mg, 0.63 mmol, 1.5
eq), DMAP (77 mg, 0.63 mmol, 1.5 eq) and DIPEA (0.223 mL, 1.26
mmol, 3.0 eq) were dissolved in 4 mL of dry DMF. The mixture was
heated at 50-55.degree. C. for 3 days. The mixture was cooled to
room temperature and diluted with DCM. The DCC-urea was filtered
off and the filtrate was concentrated to remove DCM. The resulting
residue in DMF was added to water (40 mL) to precipitate out the
polymer product which was washed with water (40 mL), ether/DCM (40
mL/4 mL) and ether (40 mL). After drying under vacuum at 30.degree.
C., the desired PLA-R848 conjugate was obtained as a white foamy
solid (1.5 g).
Example 23
Polylactic Acid Conjugates of an Imidazoquinoline
PLA (R202H) (2.0 g, 0.42 mmol, 1.0 eq), EDC (242 mg, 1.26 mmol, 3.0
eq), HOAt (171 mg, 1.26 mmol, 3.0 eq), R848 (200 mg, 0.63 mmol, 1.5
eq), and DIPEA (0.223 mL, 1.26 mmol, 3.0 eq) were dissolved in 4 mL
of dry DMF. The mixture was heated at 50-55.degree. C. for 2 days.
The solution was cooled to room temperature and added to water (40
mL) to precipitate out the polymer product which was washed with
water (40 mL), ether/MeOH (40 mL/2 mL) and ether (40 mL). The
orange colored polymer was dissolved in 4 mL of DCM and the
resulting solution was added to 40 mL of ether to precipitate out
the polymer without much of the orange color. The light colored
polymer was washed with ether (40 mL). After drying under vacuum at
30.degree. C., the desired PLA-R848 conjugate was obtained as a
light brown foamy solid (1.5 g).
Example 24
Polylactic Acid or PLGA Conjugates of an Imidazoquinoline
PLA (R202H) (1.0 g, 0.21 mmol, 1.0 eq), EDC (161 mg, 0.84 mmol, 4.0
eq), HOBt.H2O (65 mg, 0.42 mmol, 2.0 eq), R848 (132 mg, 0.42 mmol,
2.0 eq), and DIPEA (0.150 mL, 0.84 mmol, 4.0 eq) were dissolved in
2 mL of dry DMF. The mixture was heated at 50-55.degree. C. for 2
days. The solution was cooled to room temperature and added to
water (40 mL) to precipitate out the polymer product. The orange
colored polymer was dissolved in 2 mL of DCM and the resulting
solution was added to 40 mL of ether to precipitate out the polymer
which was washed with water/acetone (40 mL/2 mL) and ether (40 mL).
After drying under vacuum at 30.degree. C., the desired PLA-R848
conjugate was obtained as an off-white foamy solid (1.0 g, loading
of R848 on polymer was about 45 mg/g based on HPLC analysis and
confirmed by .sup.1H NMR). In the same manner, PLGA (75%
Lactide)-R848 and PLGA (50% lactide)-R848 were prepared.
Example 25
Conjugation of R848 to Polyglycine, A Polyamide
##STR00060##
The t-butyloxycarbonyl (tBOC) protected polyglycine carboxylic acid
(I) is prepared by ring opening polymerization of glycine
N-carboxyanhydride (Aldrich cat #369772) using 6-aminohexanoic acid
benzyl ester (Aldrich cat #S33465) by the method of Aliferis et al.
(Biomacromolecules, 5, 1653, (2004)). Protection of the end amino
group as the t-BOC carbamate followed by hydrogenation over
palladium on carbon to remove the benzyl ester completes the
synthesis of BOC protected polyglycine carboxylic acid (I).
A mixture of BOC-protected polyglycine carboxylic acid (5 gm,
MW=2000, 2.5.times.10.sup.-3 moles) and HBTU (3.79 gm,
1.0.times.10.sup.-2 moles) in anhydrous DMF (100 mL) is stirred at
room temperature under argon for 50 minutes. Then R848 (1.6 gm,
5.0.times.10.sup.-3 moles) is added, followed by
diisopropylethylamine (4 mL, 2.2.times.10.sup.-2 moles). The
mixture is stirred at RT for 6 h and then at 50-55.degree. C.
overnight (16 h). After cooling, the DMF is evaporated under vacuum
and the residue is triturated in EtOAc (100 mL). The polymer is
isolated by filtration and the polymer is then washed with
2-propanol (4.times.25 mL) to remove residual reagents and dried
under vacuum at 35-40.degree. C. for 3 days. The polymer is
isolated as an off white solid in a yield of 5.1 g (88%). The R848
loading that can be determined by NMR is 10.1%.
The t-BOC protecting group is removed using trifluoroacetic acid
and the resulting polymer is grafted to PLA with carboxyl end
groups by conventional methods.
Example 26
Preparation of a PLGA Conjugate of the Polyglycine/R848 Polymer
Step 1: A t-BOC protected polyglycine/R848 conjugate (5 g) is
dissolved in trifluoroacetic acid (25 mL) and this solution is
warmed at 50.degree. C. for one hour. After cooling, the
trifluoroacetic acid is removed under vacuum and the residue is
triturated in ethyl acetate (25 mL). The polymer is isolated by
filtration and is washed well with 2-propanol. After drying under
vacuum there is obtained 4.5 grams of polymer as an off white
solid.
Step 2: A mixture of PLGA (Lakeshores Polymers, MW .about.5000,
7525DLG1A, acid number 0.7 mmol/g, 10 g, 7.0 mmol) and HBTU (5.3 g,
14 mmol) in anhydrous DMF (100 mL) is stirred at RT under argon for
50 minutes. The polymer from above (1.4 g, 7 mmol) dissolved in dry
DMF (20 mL) is added, followed by diisopropylethylamine (DIPEA) (5
mL, 28 mmol). The mixture is stirred at RT for 6 h and then at
50-55.degree. C. overnight (16 h). After cooling, the DMF is
evaporated under vacuum, and the residue is dissolved in methylene
chloride (50 mL). The polymer is precipitated by the addition of
2-propanol (200 mL). The polymer is isolated by decantation and is
washed with 2-propanol (4.times.50 mL) to remove residual reagents
and then dried under vacuum at 35-40 C overnight. There is obtained
9.8 g (86%) of the block copolymer.
Example 27
Preparation of PLGA-2-Butoxy-8-Hydroxy-9-Benzyl Adenine
Conjugate
##STR00061##
A mixture of PLGA (Lakeshores Polymers, MW .about.5000, 7525DLG1A,
acid number 0.7 mmol/g, 1.0 g, 7.0 mmol) and HBTU (0.8 g, 2.1 mmol)
in anhydrous EtOAc (30 mL) is stirred at RT under argon for 30
minutes. Compound (I) (0.22 g, 0.7 mmol) in 2 mL of dry DMSO is
added, followed by diisopropylethylamine (DIPEA) (0.73 mL, 4.2
mmol). The mixture is stirred at room temperature for 20 h.
Additional amounts of HBTU (0.53 g, 1.4 mmol) and DIPEA (0.5 mL,
2.8 mmol) are added and the mixture is heated at 50-55.degree. C.
for 4 h. After cooling, the mixture is diluted with EtOAc (100 mL)
and washed with saturated NH.sub.4Cl solution 20 mL), water
(2.times.20 mL) and brine solution (20 mL). The solution is dried
over Na.sub.2SO.sub.4 (10 g) and concentrated to a gel-like
residue. Isopropyl alcohol (IPA) (35 mL) is then added and the
brownish polymer conjugate precipitates out of solution. The
polymer is then washed with IPA (2.times.20 mL) to remove residual
reagents and dried under vacuum at 35-40.degree. C. for 2 days as a
brownish powder (1.0 g).
Example 28
Preparation of PLGA-2,9-Dibenzyl-8-Hydroxyadenine Conjugate
##STR00062##
A mixture of PLGA (Lakeshores Polymers, MW .about.5000, 7525DLG1A,
acid number 0.7 mmol/g, 1.0 g, 7.0 mmol) and HBTU (0.8 g, 2.1 mmol)
in anhydrous EtOAc (30 mL) is stirred at RT under argon for 30
minutes. Compound (II) (0.24 g, 0.7 mmol) in 2 mL of dry DMSO is
added, followed by diisopropylethylamine (DIPEA) (0.73 mL, 4.2
mmol). The mixture is stirred at RT for 20 h. Additional amounts of
HBTU (0.53 g, 1.4 mmol) and DIPEA (0.5 mL, 2.8 mmol) are added and
the mixture is heated at 50-55.degree. C. for 4 h. After cooling,
the mixture is diluted with EtOAc (100 mL) and washed with
saturated NH.sub.4Cl solution 20 mL), water (2.times.20 mL) and
brine solution (20 mL). The solution is dried over Na.sub.2SO.sub.4
(10 g) and concentrated to a gel-like residue. Isopropyl alcohol
(IPA) (35 mL) is then added and the brownish polymer conjugate
precipitated out of solution. The polymer is then washed with IPA
(2.times.20 mL) to remove residual reagents and dried under vacuum
at 35-40.degree. C. for 2 days as a brownish powder (1.2 g).
Example 29
Imide Ring Opening Used to Attach
2-Pentyl-8-Hydroxy-9-Benzyladenine to the Terminal Alcohol Groups
of Poly-Hexamethylene Carbonate)Diol of Molecular Weight 2000
The poly(hexamethylene carbonate)diol is purchased from Aldrich
Chemical Company, Cat #461164.
Poly(hexamethylene carbonate)diol:
HO--[CH.sub.2(CH.sub.2).sub.4CH.sub.2OCO.sub.2]nCH.sub.2(CH.sub.2).sub.4C-
H.sub.2--OH
Poly(hexamethylene carbonate)diol-8-oxoadenine conjugate:
##STR00063##
The polymer (5 g, 2.5.times.10.sup.-3 moles) is dissolved in
methylene chloride 25 mL and the lactam of
2-pentyl-8-hydroxy-9-benzyladenine (2.05 g, 5.0.times.10.sup.-3
moles) is added. This slurry is stirred as
1,5,7-triazabicyclo-[4,4,0]dec-5-ene (TBD, 0.557 g,
4.times.10.sup.-3 moles) is added in a single portion. After
stirring at room temperature overnight a clear pale yellow solution
forms. The solution is diluted with methylene chloride (100 mL),
and the solution is washed with 5% citric acid. This solution is
dried over sodium sulfate after which it is filtered and evaporated
under vacuum. After drying under high vacuum there is obtained 5.5
grams (78%) of polymer. NMR is used to determine the benzyladenine
content which is 18%.
Example 30
Nicotine-Peg-Pla Conjugates
A 3-nicotine-PEG-PLA polymer was synthesized as follows:
First, monoamino poly(ethylene glycol) from JenKem.RTM. with a
molecular weight of 3.5 KD (0.20 g, 5.7.times.10.sup.-5 moles) and
an excess of 4-carboxycotinine (0.126 g, 5.7.times.10.sup.-4 moles)
were dissolved in dimethylformamide (5.0 mL). The solution was
stirred and dicyclohexylcarbodiimide (0.124 g, 6.0.times.10.sup.-4
moles) was added. This solution was stirred overnight at room
temperature. Water (0.10 mL) was added and stirring was continued
for an additional 15 minutes. The precipitate of dicyclohexyl urea
was removed by filtration and the filtrates were evaporated under
vacuum. The residue was dissolved in methylene chloride (4.0 mL)
and this solution was added to diethyl ether (100 mL). The solution
was cooled in the refrigerator for 2 hours and the precipitated
polymer was isolated by filtration. After washing with diethyl
ether, the solid white polymer was dried under high vacuum. The
yield was 0.188 g. This polymer was used without further
purification for the next step.
The nicotine/PEG polymer (0.20 g, 5.7.times.10.sup.-5 moles) was
dissolved in dry tetrahydrofuran (10 mL) under nitrogen and the
solution was stirred as a solution of lithium aluminum hydride in
tetrahydrofuran (1.43 mL of 2.0 M, 2.85.times.10.sup.-3 moles) was
added. The addition of the lithium aluminum hydride caused the
polymer to precipitate as a gelatinous mass. The reaction was
heated to 80.degree. C. under a slow stream of nitrogen and the
tetrahydrofuran was allowed to evaporate. The residue was then
heated at 80.degree. C. for 2 hours. After cooling, water (0.5 mL)
was cautiously added. Once the hydrogen evolution had stopped, 10%
methanol in methylene chloride (50 mL) was added and the reaction
mixture was stirred until the polymer had dissolved. This mixture
was filtered through Celite.RTM. brand diatomaceous earth
(available from EMD Inc. as Celite.RTM. 545, part #CX0574-3) and
the filtrates were evaporated to dryness under vacuum. The residue
was dissolved in methylene chloride (4.0 mL) and this solution was
slowly added to diethyl ether (100 mL). The polymer separated as a
white flocculent solid and was isolated by centrifugation. After
washing with diethyl ether, the solid was dried under vacuum. The
yield was 0.129 g.
Next, a 100 mL round bottom flask, equipped with a stir bar and
reflux condenser was charged with the PEG/nicotine polymer (0.081
g, 2.2.times.10.sup.-5 moles), D/L lactide (0.410 g,
2.85.times.10.sup.-3 moles) and anhydrous sodium sulfate (0.380 g).
This was dried under vacuum at 55.degree. C. for 8 hours. The flask
was cooled and flushed with argon and then dry toluene (10 mL) was
added. The flask was placed in an oil bath set at 120.degree. C.,
and once the lactide had dissolved, tin ethylhexanoate (5.5 mg,
1.36.times.10.sup.-5 moles) was added. The reaction was allowed to
proceed at 120.degree. C. for 16 hours. After cooling to room
temperature, water (15 mL) was added and stirring was continued for
30 minutes. Methylene chloride (200 mL) was added, and after
agitation in a separatory funnel, the phases were allowed to
settle. The methylene chloride layer was isolated and dried over
anhydrous magnesium sulfate. After filtration to remove the drying
agent, the filtrates were evaporated under vacuum to give the
polymer as a colorless foam. The polymer was dissolved in
tetrahydrofuran (10 mL) and this solution was slowly added to water
(150 mL) with stirring. The precipitated polymer was isolated by
centrifugation and the solid was dissolved in methylene chloride
(10 mL). The methylene chloride was removed under vacuum and the
residue was dried under vacuum. 3-nicotine-PEG-PLA polymer yield
was 0.38 g
Example 31
Synthetic Nanocarrier Formulation
For encapsulated adjuvant formulations, Resiquimod (aka R848) was
synthesized according to the synthesis provided in Example 99 of
U.S. Pat. No. 5,389,640 to Gerster et al.
R848 was conjugated to PLA by a method provided above, and the PLA
structure was confirmed by NMR.
PLA-PEG-nicotine conjugate was prepared according to Example
30.
PLA was purchased (Boehringer Ingelheim Chemicals, Inc., 2820 North
Normandy Drive, Petersburg, Va. 23805). The polyvinyl alcohol
(Mw=11 KD-31 KD, 85-89% hydrolyzed) was purchased from VWR
scientific. Ovalbumin peptide 323-339 was obtained from Bachem
Americas Inc. (3132 Kashiwa Street, Torrance Calif. 90505. Part
#4064565).
The above materials were used to prepare the following solutions:
1. Resiquimod (R848) @ 10 mg/mL and PLA @ 100 mg/mL in methylene
chloride or PLA-R848 conjugate @ 100 mg/mL in methylene chloride 2.
PLA-PEG-nicotine in methylene chloride @ 100 mg/mL 3. PLA in
methylene chloride @ 100 mg/mL 4. Ovalbumin peptide 323-339 in
water @ 10 or 69 mg/mL 5. Polyvinyl alcohol in water @50 mg/mL.
Solution #1 (0.25 to 0.75 mL), solution #2 (0.25 mL), solution #3
(0.25 to 0.5 mL) and solution #4 (0.1 mL) were combined in a small
vial and the mixture was sonicated at 50% amplitude for 40 seconds
using a Branson Digital Sonifier 250. To this emulsion was added
solution #5 (2.0 mL) and sonication at 35% amplitude for 40 seconds
using the Branson Digital Sonifier 250 forms the second emulsion.
This was added to a beaker containing phosphate buffer solution (30
mL) and this mixture was stirred at room temperature for 2 hours to
form the nanoparticles.
To wash the particles a portion of the nanoparticle dispersion (7.4
mL) was transferred to a centrifuge tube and spun at 5,300 g for
one hour, supernatant was removed, and the pellet was re-suspended
in 7.4 mL of phosphate buffered saline. The centrifuge procedure
was repeated and the pellet was re-suspended in 2.2 mL of phosphate
buffered saline for a final nanoparticle dispersion of about 10
mg/mL.
Example 32
Double Emulsion with Multiple Primary Emulsions
Materials
Ovalbumin peptide 323-339, a 17 amino acid peptide known to be a T
cell epitope of Ovalbumin protein, was purchased from Bachem
Americas Inc. (3132 Kashiwa Street, Torrance Calif. 90505.)
Resiquimod (aka R848) was synthesized according to a method
provided in U.S. Pat. No. 6,608,201.
PLA-R848, resiquimod, was conjugated to PLA with a molecular weight
of approximately 2,500 Da according to a method provided above.
PLGA-R848, resiquimod, was conjugated to PLGA with a molecular
weight of approximately 4,100 Da according to a method provided
above.
PS-1826 DNA oligonucleotide with fully phosphorothioated backbone
having nucleotide sequence 5'-TCC ATG ACG TTC CTG ACG TT-3' (SEQ ID
NO: 1) with a sodium counter-ion was purchased from Oligos Etc
(9775 SW Commerce Circle C-6, Wilsonville, Oreg. 97070.)
PO-1826 DNA oligonucleotide with phosphodiester backbone having
nucleotide sequence 5'-TCC ATG ACG TTC CTG ACG TT-3' (SEQ ID NO: 2)
with a sodium counter-ion was purchased from Oligos Etc. (9775 SW
Commerce Circle C-6, Wilsonville, Oreg. 97070.)
PLA with an inherent viscosity of 0.21 dL/g was purchased from
SurModics Pharmaceuticals (756 Tom Martin Drive, Birmingham, Ala.
35211. Product Code 100 DL 2A.)
PLA with an inherent viscosity of 0.71 dL/g was purchased from
SurModics Pharmaceuticals (756 Tom Martin Drive, Birmingham, Ala.
35211. Product Code 100 DL 7A.)
PLA with an inherent viscosity of 0.19 dL/g was purchased from
Boehringer Ingelheim Chemicals, Inc. (Petersburg, Va. Product Code
R202H.)
PLA-PEG-nicotine with a molecular weight of approximately 18,500 to
22,000 Da was prepared according to a method provided above.
PLA-PEG-R848 with a molecular weight of approximately 15,000 Da was
synthesized was prepared according to a method provided above.
Polyvinyl alcohol (Mw=11,000-31,000, 87-89% hydrolyzed) was
purchased from J. T. Baker (Part Number U232-08).
Batches were produced using a double emulsion process with multiple
primary emulsions. The table below references the solution suffix
(e.g., B in Solution #1 column indicates Solution #1B was used) and
volume of solution used.
TABLE-US-00001 Sample Solution #1 Solution #2 Solution #3 Solution
#4 Solution #5 Number (Volume) (Volume) (Volume) (Volume) (Volume)
1 B (0.1 ml) C (1.0 ml) A (0.1 ml) C (1 0 ml) A (2.0 ml) 2 A (0.2
ml) A (1.0 ml) A (0.1 ml) A (1.0 ml) A (3.0 ml) 3 A (0.2 ml) B (1.0
ml) A (0.1 ml) B (1.0 ml) A (3.0 ml) 4 A (0.2 ml) B (1.0 ml) A (0.1
ml) B (1.0 ml) A (3.0 ml)
Solution 1A: Ovalbumin peptide 323-339 @ 35 mg/mL in dilute
hydrochloric acid aqueous solution. The solution was prepared by
dissolving ovalbumin peptide in 0.13N hydrochloric acid solution at
room temperature.
Solution 1B: Ovalbumin peptide 323-339 @ 70 mg/mL in dilute
hydrochloric acid aqueous solution. The solution was prepared by
dissolving ovalbumin peptide in 0.13N hydrochloric acid solution at
room temperature.
Solution 2A: 0.21-IV PLA @ 75 mg/mL and PLA-PEG-nicotine @ 25 mg/ml
in methylene chloride. The solution was prepared by first preparing
two separate solutions at room temperature: 0.21-IV PLA @ 100 mg/mL
in pure methylene chloride and PLA-PEG-nicotine @ 100 mg/mL in pure
methylene chloride. The final solution was prepared by adding 3
parts PLA solution for each part of PLA-PEG-nicotine solution.
Solution 2B: 0.71-IV PLA @ 75 mg/mL and PLA-PEG-nicotine @ 25 mg/ml
in methylene chloride. The solution was prepared by first preparing
two separate solutions at room temperature: 0.71-IV PLA @ 100 mg/mL
in pure methylene chloride and PLA-PEG-nicotine @ 100 mg/mL in pure
methylene chloride. The final solution was prepared by adding 3
parts PLA solution for each part of PLA-PEG-nicotine solution.
Solution 2C, 0.19-IV PLA @ 75 mg/mL and PLA-PEG-nicotine @ 25 mg/ml
in methylene chloride. The solution was prepared by first preparing
two separate solutions at room temperature: 0.19-IV PLA @ 100 mg/mL
in pure methylene chloride and PLA-PEG-nicotine @ 100 mg/mL in pure
methylene chloride. The final solution was prepared by adding 3
parts PLA solution for each part of PLA-PEG-nicotine solution.
Solution 3A: Oligonucleotide (either PS-1826 or PO-1826) @ 200
mg/ml in purified water. The solution was prepared by dissolving
oligonucleotide in purified water at room temperature.
Solution 4A: Same as Solution #2A.
Solution 4B: Same as Solution #2B.
Solution 4C: Same as Solution #2C.
Solution 5A: Polyvinyl alcohol @ 50 mg/mL in 100 mM pH 8 phosphate
buffer.
Two separate primary water in oil emulsions were prepared. W1/O2
was prepared by combining solution 1 and solution 2 in a small
pressure tube and sonicating at 50% amplitude for 40 seconds using
a Branson Digital Sonifier 250. W3/O4 was prepared by combining
solution 3 and solution 4 in a small pressure tube and sonicating
at 50% amplitude for 40 seconds using a Branson Digital Sonifier
250. A third emulsion with two inner emulsion ([W1/O2, W3/O4]/W5)
emulsion was prepared by combining 0.5 ml of each primary emulsion
(W1/O2 and W3/O4) and solution 5 and sonicating at 30% amplitude
for 40 to 60 seconds using the Branson Digital Sonifier 250.
The third emulsion was added to a beaker containing 70 mM phosphate
buffer solution (30 mL) and stirred at room temperature for 2 hours
to allow for the methylene chloride to evaporate and for the
nanocarriers to form. A portion of the nanocarriers were washed by
transferring the nanocarrier suspension to a centrifuge tube and
spinning at 13,823 g for one hour, removing the supernatant, and
re-suspending the pellet in phosphate buffered saline. The washing
procedure was repeated and the pellet was re-suspended in phosphate
buffered saline for a final nanocarrier dispersion of about 10
mg/mL.
The amounts of oligonucleotide and peptide in the nanocarrier were
determined by HPLC analysis.
Example 33
Standard Double Emulsion
Materials
As provided in Example 32 above.
Batches were produced using a standard double emulsion process. The
table below references the solution suffix (e.g., B in Solution #1
column indicates Solution #1B was used) and volume of solution
used.
TABLE-US-00002 Sample Solution #1 Solution #2 Solution #3 Solution
#4 Solution #5 Number (Volume) (Volume) (Volume) (Volume) (Volume)
1 A (0.1 ml) A (0.75 ml) A (0.25 ml) None A (2.0 ml) 2 A (0.1 ml)
None A (0.25 ml) A (0.75 ml) A (2.0 ml) 3 A (0.1 ml) B (0.75 ml) A
(0.25 ml) None A (2.0 ml) 4 B (0.1 ml) C (0.75 ml) A (0.25 ml) None
B (2.0 ml) 5 B (0.1 ml) D (0.25 ml) A (0.25 ml) A (0.50 ml) B (2.0
ml) 6 C (0.2 ml) None A (0.25 ml) A (0.75 ml) B (2.0 ml) 7 D (0.1
ml) None A (0.25 ml) A (0.75 ml) B (2.0 ml)
Solution 1A: Ovalbumin peptide 323-339 @ 69 mg/mL in de-ionized
water. The solution was prepared by slowly adding ovalbumin peptide
to the water while mixing at room temperature.
Solution 1B: Ovalbumin peptide 323-339 @ 70 mg/mL in dilute
hydrochloric acid aqueous solution. The solution was prepared by
dissolving ovalbumin peptide in 0.13N hydrochloric acid solution at
room temperature.
Solution 1C: Oligonucleotide (PS-1826) @ 50 mg/ml in purified
water. The solution was prepared by dissolving oligonucleotide in
purified water at room temperature.
Solution 1D: Ovalbumin peptide 323-339 @ 17.5 mg/mL in dilute
hydrochloric acid aqueous solution. The solution was prepared by
dissolving ovalbumin peptide @ 70 mg/ml in 0.13N hydrochloric acid
solution at room temperature and then diluting the solution with 3
parts purified water per one part of starting solution.
Solution 2A: R848 @ 10 mg/ml and 0.19-IV PLA @ 100 mg/mL in pure
methylene chloride prepared at room temperature.
Solution 2B: PLA-R848 @ 100 mg/ml in pure methylene chloride
prepared at room temperature.
Solution 2C: PLGA-R848 @ 100 mg/ml in pure methylene chloride
prepared at room temperature.
Solution 2D: PLA-PEG-R848 @ 100 mg/ml in pure methylene chloride
prepared at room temperature.
Solution 3A: PLA-PEG-nicotine @ 100 mg/ml in pure methylene
chloride prepared at room temperature.
Solution 4A: 0.19-IV PLA @ 100 mg/mL in pure methylene chloride
prepared at room temperature.
Solution 5A: Polyvinyl alcohol @ 50 mg/mL in de-ionized water.
Solution 5B: Polyvinyl alcohol @ 50 mg/mL in 100 mM pH 8 phosphate
buffer.
The water in oil (W/O) primary emulsion was prepared by combining
solution 1 and solution 2, solution 3, and solution 4 in a small
pressure tube and sonicating at 50% amplitude for 40 seconds using
a Branson Digital Sonifier 250. The water/oil/water (W/O/W) double
emulsion was prepared by adding solution 5 to the primary emulsion
and sonicating at 30% to 35% amplitude for 40 seconds using the
Branson Digital Sonifier 250.
The double emulsion was added to a beaker containing phosphate
buffer solution (30 mL) and stirred at room temperature for 2 hours
to allow for the methylene chloride to evaporate and for the
nanocarriers to form. A portion of the nanocarriers were washed by
transferring the nanocarrier suspension to a centrifuge tube and
spinning at 5,000 to 9,500 RPM for one hour, removing the
supernatant, and re-suspending the pellet in phosphate buffered
saline. The washing procedure was repeated and the pellet was
re-suspended in phosphate buffered saline for a final nanocarrier
dispersion of about 10 mg/mL.
Example 34
Determination of Amount of Agents
Method for R848 and Peptides (e.g., Ova Peptide, Human Peptide,
TT2pDT5T)
The amount of R848 (immunostimulatory agent) and ova peptide (T
cell antigen) was measured using reverse phase HPLC on an Agilent
1100 system at appropriate wavelengths (.lamda.=254 nm for R848 and
215 nm for ova peptide) equipped with an Agilent Zorbax SB-C18
column (3.5 .mu.m. 75.times.4.6 mm Column Temp=40.degree. C. (part
no. 866953-902)) using Mobile Phase A (MPA) of 95% water/5%
acetonitrile/0.1% TFA and Mobile Phase B (MPB) of 90%
acetonitrile/10% water/0.09% TFA (Gradient: B=5 to 45% in 7
minutes; ramp to 95% B to 9 min; decrease back to 5% B to 9.5 min
and kept equilibrating to end. Total run time was 13 minute with
flow rate of 1 mL/min).
Method for CpG
The amount of CpG (immunostimulatory agent) was measured using
reverse phase HPLC on Agilent 1100 system at 260 nm equipped with
Waters XBridge C-18 (2.5 micron particle, 50.times.4.6 mm ID (part
No. 186003090), column temp. 600 C) using mobile phase A of 2%
acetonitrile in 100 mM TEA-acetic acid buffer, pH about 8.0 and
mobile B as 90% acetonitrile, 10% water (column equilibrated at 5%
B, increased to 55% B in 8.5 min, then ramped to 90% B to 12
minutes. Strength of B was rapidly decreased to 5% in one minute
and equilibrated until stop time, 16 minutes. The flow rate was 1
mL/min until end of the method, 16 minutes).
Method for Nicotine Analog
Nicotine analog was measured using reverse phase HPLC on Agilent
1100 system at 254 nm equipped with Waters X-Bridge C-18 (5 micron
particle, 100.times.4.6 mm ID, column temp at 40.degree. C.) using
Mobile Phase A (MPA) of 95% water/5% acetonitrile/0.1% TFA and
Mobile Phase B (MPB) of 90% acetonitrile/10% water/0.09% TFA
(gradient: column was equilibrated at 5% B increased to 45% B in 14
minutes. Then ramped up to 95% B from 14 to 20 minutes. Mobile B
strength was quickly decreased back to 5% and requilibrated until
the end of the method. The flow rate of the method was maintained
at 0.5 ml/min with total run time of 25 minutes. The NC suspension
was centrifuged @14000 rpm for about 15-30 minutes depending on
particle size. The collected pellets were treated with 200 uL of
conc. NH.sub.4OH (8 M) for 2 h with agitation until the solution
turns clear. A 200 uL of 1% TFA was added to neutralize the mixture
solution, which brought the total volume of the pellet solution to
200 uL. An aliquot of 50 uL of the solution was diluted with MPA
(or water) to 200 uL and analyzed on HPLC as above to determine the
amount present in the pellets.
Encapsulated Free R848 in Nanocarrier
0.5 mL of the NC suspension was centrifuged @14000 rpm for about 15
minutes. The collected pellet was dissolved with 0.3 mL of
acetonitrile and centrifuged briefly @14000 rpm to remove any
residual insolubles. The clear solution was further diluted with 4
times equivalent volume of MPA and assayed on reverse phase HPLC
described above.
Encapsulated CpG in Nanocarrier
330 uL of NC suspension from the manufacture (about 10 mg/mL
suspension in PBS) was spun down at 14000 rpm for 15 to 30 minutes
depending on particle size. The collected pellets were re-suspended
with 500 uL of water and sonicated for 30 minutes to fully disperse
the particles. The NC was then heated at 600.degree. C. for 10
minutes. Additional 200 uL of 1 N NaOH was added to the mixture,
heated for another 5 minutes where the mixture becomes clear. The
hydrolyzed NC solution was centrifuged briefly at 14000 rpm. A
final 2.times. dilution of the clear solution using water was then
made and assayed on the reverse HPLC described above.
Encapsulated T Cell Antigens (e.g., Ova Peptide, or Human Peptide,
TT2pDT5T)
330 uL of NC suspension from the manufacture (about 10 mg/mL
suspension in PBS) was spun down at 14000 rpm for 15 to 30 minutes.
100 uL of acetonitrile was added to the pellets to dissolve the
polymer components of the NC. The mixture was vortexed and
sonicated for 1 to 5 minutes. 100 uL 0.2% TFA was added to the
mixture to extract the peptides and sonicated for another 5 minutes
to ensure the break down of the aggregates. The mixture was
centrifuged at 14000 rpm for 15 minutes to separate any insoluble
materials (e.g., polymers). A 50 uL aliquot of the supernatant
diluted with 150 uL of MPA (or water) was taken and assayed on the
reverse phase HPLC as described above.
Amount of Conjugated Nicotine Analog (B Cell Antigen) in
Nanocarriers
1.5 mL of NC suspension was spun down @ 14000 rpm for about 15
minutes, the pellets were hydrolyzed using 150 uL of concentrated
NH.sub.4OH (8M) for about 2-3 h until the solution turns clear. A
150 uL of 2% TFA(aq) solution was added to the pellet mixture to
neutralize the solution. A 100 uL aliquot of the mixture was
diluted with 200 uL of water and assayed on reverse phase HPLC
described above and quantified based on the standard curve
established using the precursor (PEG-nicotine) of the
PLA-PEG-nicotine used in the manufacture.
Example 35
Release Rate of Immunomodulatory Agent from Synthetic
Nanocarriers
The following data show the rates of release of R-848 from
nanoparticles made from the low molecular weight polylactic
acid-R-848 conjugate shown above. Table 1 provides relevant
formulation information for the experiments.
The release of T-cell antigen, ova peptide and adjuvant, R848 from
the synthetic nanocarrier (nanoparticles) in PBS (100 mM, pH=7.4)
and Citrate buffer (100 mM, pH=4.5) at 37.degree. C. were performed
as follows:
Analytical Method: The amount of R848 and ova peptide released is
measured using reverse phase HPLC on a Agilent 1100 system at
.lamda.=215 nm equipped with an Agilent Zorbax SB-C18 column (3.5
.mu.m. 75.times.4.6 mm Column Temp=40.degree. C. (part no.
866953-902)) using Mobile Phase A (MPA) of 98% water/2%
acetonitrile/0.1% TFA and Mobile Phase B (MPB) of 90%
acetonitrile/10% water/0.09% TFA with Gradient: B=5 to 45% in 7
minutes; ramp to 95% B to 9 min; re-EQ to end. 13 minute run time.
Flow=1 mL/min.
The total amount of R848 and ova peptide present in the
nanoparticles was as shown in Table 1. An aqueous suspension of the
tested synthetic nanocarriers was then diluted to a final stock
volume of 4.4 mL with PBS.
(A) In Vitro Release Rate Measurement in PBS (pH=7.4):
For TO sample, a 200 .mu.L aliquot was immediately removed from
each of the NP sample and centrifuged @ 14000 rpm in a
microcentrifuge tubes using a Microcentrifuge (Model: Galaxy 16).
100 .mu.L of supernatant was removed and diluted to 200 .mu.L in
HPLC Mobile Phase A (MPA) and assayed for the amount of R848 and
ova peptide released on the reverse phase HPLC.
For time point measurements: 9.times.200 .mu.L of each of the
samples were added to microcentrifuge tubes (3.times.200 for
unconjugated) and 300 .mu.L of 37 C PBS was added to each above
aliquot and the samples were placed immediately in 37.degree. C.
oven. At the following time points: 24 hr, 48 hr, 96 hr and 144 hr
(for conjugated R848) or 2 h, 16 h and 24 h (for unconjugated
(encapsulated) R848), the samples were centrifuged and assayed for
the amount of R848 and ova peptide released as above for T0
sample.
(B) In Vitro Release Rate Measurement in Citrate Buffer
(pH=4.5):
For T0 sample, a 200 .mu.L aliquot was removed from each of the
samples and centrifuged @ 6000 rpm for 20 minutes and the
supernatant was removed. The residue nanoparticles was resuspended
in 200 uL of citrate buffer and centrifuged @ 14000 rpm for 15
minutes. 100 uL of the supernatant was removed and diluted to 200
uL with MPA and assayed for R848 and peptide as above.
For time point measurements: 9.times.200 uL of each of the samples
were added to microcentrifuge tubes (3.times.200 for unconjugated)
and centrifuged for 20 minutes @ 6000 rpm and the supernatants were
removed. The residue NPs were then resuspended in 500 uL of citrate
buffer and placed in 37.degree. C. oven. At the following time
points: 24 hr, 48 hr, 96 hr and 144 hr (for conjugated R848) or 2
h, 16 h and 24 h (for unconjugated (encapsulated) R848), the
samples were centrifuged and assayed for the amount of R848 and ova
peptide released as above for T0 sample.
In order to complete the mass balance from above measurements in
PBS and Citrate buffer, the remaining pellets (conjugated R848
samples only) from each sample was treated with 200 uL of conc.
NH4OH (8 M) for 3 h with mixing. After the mixture was settled, 200
uL of 1% TFA was added to bring total volume of the pellet to 400
uL. An aliquot of 50 uL of the solution was diluted with MPA to 200
uL and analyzed on HPLC as above to determine the amount of R848
and ova peptide that remained in the pellet after in vitro release
to close the mass balance. For unconjugated samples, the sample was
diluted with TFA in acetonitrile and assayed as above for R848 and
peptide.
The results are summarized in FIGS. 1-3.
TABLE-US-00003 TABLE 1 Formulation Targets With A Covalent R848 Ova
PLA- PLA-R848 R848 peptide PEG- conjugate PLA (15-20K, Formulation
load* load NIC type** BI R202H) Chemistry 1 E1.5% 1.1-2.2% 25% 75%
2 E1.5%++ 1.1-2.2% 25% 75% 3 C75% 0.15- 25% Method 1 Amine 0.31% 4
C75% 0.15- 25% Method 1 Amine 0.31% 5 C75% 0.15- 25% Method 5
ROP-hi 0.31% MW 6 C75% 0.15- 25% Method 5 ROP-lo MW 0.31% 7 C50%
0.15- 25% Method 5 25% ROP-lo MW 0.31% 8 C25% 0.15- 25% Method 5
50% ROP-lo MW 0.31% *C = covalent R848; E = encapsulation of
R848
Materials and Method-- HPLC--Agilent 1100. .lamda.=215 nm Column
Temp=40.degree. C. Column--Agilent Zorbax SB-C18, 3.5 .mu.m.
75.times.4.6 mm (part no. 866953-902) C18 guard column Mobile Phase
A (MPA)--98% water/2% acetonitrile/0.1% TFA Mobile Phase B
(MPB)--90% acetonitrile/10% water/0.09% TFA Gradient: B=5 to 45% in
7 minutes; ramp to 95% B to 9 min; re-EQ to end. 13 minute run
time. Flow=1 mL/min. PBS--100 mM, pH=7.4. Citrate Buffer--100 mM,
pH=4.5. Oven-- Microcentrifuge--Galaxy 16 Microcentrifuge tubes
Sonicator Pipets--20, 200, 1000 .mu.L adjustable HPLC grade
water--EMD-#WX0008-1. NH.sub.4OH--.about.8M. Mallinkcrodt. TFA,
0.2%. Prep Apr. 27, 2009. TFA, 1%. Prep May 13, 2009. Thermometer
Samples
"6-1" and "6-2" have entrapped R848. All of the rest have
conjugated R848.
The estimated values are based on the loading results from the "62"
series.
TABLE-US-00004 TABLE 2 Estimated R848 and Ova peptide in synthetic
nanocarriers: Estimated R848 in Estimated Ova in Sample ID NPs
(.mu.g/mL) NPs (.mu.g/mL) 1 54 146 2 166 184 3 119 32 4 114 34 5
465 37 6 315 34 7 116 40
Sample volumes were slightly below what was planned. To ensure
enough material is available for all time points, the following
volumes of PBS were added to the samples to bring them all to 4.4
mL.
TABLE-US-00005 TABLE 3 Sample Volume PBS added Sample ID Volume
(mL) (mL) 1 4.35 0.05 2 4.23 0.17 3 4.21 0.19 4 4.20 0.20 5 4.21
0.19 6 4.19 0.21 7 4.20 0.20
Procedure-- 1) T=0 Sample Prep a. PBS i. Remove a 200 .mu.L aliquot
from each of the samples. Microcentrifuge @ 14000 rpm. Remove
supernatant. ii. Dilute supernatant 100 .mu.L>200 .mu.L in MPA.
(DF=2). iii. Assay for peptide and R848. b. Citrate i. Remove a 200
.mu.L aliquot from each of the samples. Microcentrifuge @ 6000 rpm
for 20 minutes. Remove supernatant. ii. Add 200 uL of citrate
buffer and thoroughly resuspend. iii. Microcentrifuge @ 14000 rpm
for 15 minutes. Remove supernatant. iv. Dilute supernatant 100
.mu.L>200 .mu.L in MPA. (DF=2) v. Assay for peptide and R848. 2)
PBS IVR a. Add 9.times.200 .mu.L of each of the samples to
microcentrifuge tubes. (3.times.200 for unconjugated) b. To each
aliquot add 300 .mu.L of 37 C PBS. c. Immediately place samples in
37 C oven. 3) Citrate IVR a. Add 9.times.200 uL of each of the
samples to microcentrifuge tubes. (3.times.200 for unconjugated) b.
Centrifuge for 20 minutes @ 6000 rpm. c. Remove the supernatants.
d. To each tube, add 500 .mu.L of citrate buffer and resuspend
thoroughly. e. Place samples in 37 C oven 4) For lots 1-4 and 8,
remove the samples (see step 6) at the following time points: a.
Conjugated i. 24 hr ii. 48 hr (2 days) iii. 96 hr (4 days) iv. 144
hr (6 days) v. Further time points TBD based on the above data. b.
Non conjugated i. 2 hr ii. 16 hr iii. 24 hr 5) For lots 6 and 7,
remove samples at the following time points: a. PBS i. 24 hr ii. 48
hr (2 days) iii. 96 hr (4 days) iv. 144 hr (6 days) v. Further time
points TBD based on the above data. b. Citrate i. 2 hr ii. 16 hr
iii. 24 hr iv. 48 hr (2 days) v. 72 hr (3 days) vi. 96 hr (4 days)
vii. 120 hr (5 days) viii. Further time points TBD based on the
above data. 6) Sample as follows: a. Microcentrifuge @ 14000 rpm
for 15 minutes. b. Remove supernatant. c. Dilute 100 .mu.L to 200
.mu.L in MPA. (DF=2) 7) Assay for peptide and R848. This will
provide the amount released at each time point.
To Complete Mass Balance, Perform the Following: 8) To the
remaining pellets (conjugated only) add 200 uL NH.sub.4OH. 9)
Vortex briefly and sonicate to disperse. 10) Add stir bar. Allow to
sit until clear (at least 3 hours). 11) Add 200 uL of 1% TFA (total
pellet volume=400 .mu.L). 12) Dilute 50 .mu.L to 200 .mu.L in MPA.
Analyze by HPLC to determine peptide and R848 remaining in the
pellet. (DF=4). 13) For unconjugated lots, assay for peptide and
R848 with typical AcN/TFA method.
Example 36
Release Rate Testing
The release of antigen (e.g., ova peptide, T cell antigen) and
immunostimulatory agents (e.g., R848, CpG) from synthetic
nanocarriers in phosphate buffered saline solution (PBS) (100 mM,
pH=7.4) and citrate buffer (100 mM, pH=4.5) at 37.degree. C. was
determined as follows:
The release of R848 from the nanocarrier composed of conjugated
R848 and the ova peptide was achieved by exchanging desired amount
of the aqueous suspension of the tested synthetic nanocarriers
obtained from the manufacture (e.g., about 10 mg/mL in PBS) into
the same volume of the appropriate release media (Citrate buffer
100 mM) via centrifugation and re-suspension.
In Vitro Release Rate Measurement in PBS (pH=7.4)
1 mL of the PBS suspension NC was centrifuged @ 14000 rpm in
microcentrifuge tubes generally from 15-30 minutes depending on
particle size. The collected supernatant was then diluted with
equal volume of the mobile phase A (MPA) or water and assayed on
reverse phase HPLC for the amount of the R848 release during the
storage. The remaining pellet was re-suspended to homogeneous
suspension in 1 mL of PBS and placed to 37.degree. C. thermal
chamber with constant gentle agitation
For T0 sample, a 150 .mu.L aliquot was immediately removed from NC
suspension prior placing the NC suspension to 37.degree. C. thermal
chamber and centrifuged @ 14000 rpm in microcentrifuge tubes using
a microcentrifuge (Model: Galaxy 16). 100 .mu.L of the supernatant
was removed and diluted to 200 .mu.L with HPLC Mobile Phase A (MPA)
or water and assayed for the amount of R848 and ova peptide
released on the reverse phase HPLC.
For time point measurements, 150 .mu.L aliquot was removed from the
37.degree. C. NC sample suspension, and the samples were
centrifuged and assayed for the amount of R848 and ova peptide
released in the same manner as for TO sample. The R848 and ova
peptide released was tested at 6 h, 24 h for routine monitoring
with additional 2 h, 48 h, 96 h and 144 h for complete release
profile establishment.
In Vitro Release Rate Measurement in Citrate Buffer (pH=4.5)
A 100 mM sodium citrate buffer (pH=4.5) was applied to exchange the
original NC storage solution (e.g., PBS) instead of the PBS buffer,
pH=7.4. In order to complete the mass balance from above
measurements in PBS and Citrate buffer, the remaining pellets from
each time point were treated with 100 uL of NH.sub.4OH (8 M) for 2
h (or more) with agitation until solution turn clear. A 100 uL of
1% TFA was added to neutralize the mixture, which brought the total
volume of the pellet solution to 200 uL. An aliquot of 50 uL of the
mixture was diluted with MPA (or water) to 200 uL and analyzed on
HPLC as above to determine the amount of unreleased R848 remaining
in the pellets after in vitro release to close the mass balance.
For unconjugated samples, the sample was diluted with TFA in
acetonitrile and assayed as above for R848.
The release of CpG was determined similar to the measurement of
R848 and ova peptide in terms of sample preparation and monitored
time points. However, the amount of the CpG in the release media
was assayed by the reverse phase HPLC method described above.
Example 37
Immunization with NC-Nic Carrying CpG Adjuvant
Groups of five mice were immunized three times (subcutaneously,
hind limbs) at 2-week intervals (days 0, 14 and 28) with 100 .mu.g
of NC-Nic. NC-Nic was a composition of nanocarriers exhibiting
nicotine on the outer surface and, for all groups of mice except
for Group 1, carrying CpG-1826 (thioated) adjuvant, which was
released from the nanocarriers at different rates. The nanocarriers
were prepared according to a method provided above. Serum
anti-nicotine antibodies were then measured on days 26 and 40.
EC.sub.50 for anti-nicotine antibodies as measured in standard
ELISA against polylysine-nicotine are shown in FIG. 4.
The Group 1 mice were administered NC-Nic w/o CpG-1826 containing
Ova peptide and polymers, 75% of which were PLA and 25% were
PLA-PEG-Nic. The Group 2 mice were administered NC-Nic containing
ova peptide, polymers, 75% of which were PLA and 25% were
PLA-PEG-Nic, and 3.2% CpG-1826; release rate at 24 hours: 4.2 .mu.g
CpG per mg of NC. The Group 3 mice were administered NC-Nic
containing polymers, 75% of which were PLA and 25% were
PLA-PEG-Nic, and 3.1% CpG-1826; release rate at 24 hours: 15 .mu.g
CpG per mg of NC. Release was determined at a pH of 4.5.
The results shown in FIG. 4 demonstrate that entrapment of adjuvant
into nanocarriers is beneficial for the immune response against
NC-associated antigen, and, furthermore, that the higher release
rate of entrapped CpG adjuvant from within the nanocarriers (NC) at
24 hours produced an immune response, which was elevated compared
to one induced by NC with a slower release rate of CpG adjuvant (a
TLR9 agonist).
Example 38
Immunization with NC-Nic Carrying Two Forms of CpG Adjuvant
Groups of five mice were immunized two times (subcutaneously, hind
limbs) at 4-week intervals (days 0, and 28) with 100 .mu.g of
NC-Nic and serum anti-nicotine antibodies were then measured on
days 12, 24 and 40. NC-Nic was a composition of nanocarriers
exhibiting nicotine on the outer surface and carrying one of two
forms of CpG-1826 adjuvant. The nanocarriers were prepared
according to a method provided above. EC.sub.50 for anti-nicotine
antibodies as measured in standard ELISA against
polylysine-nicotine are shown in FIG. 5.
The Group 1 mice were administered NC-Nic containing ova peptide,
polymers, 75% of which were PLA and 25% were PLA-PEG-Nic, and 6.2%
CpG-1826 (thioated); release rate at 24 hours: 16.6 .mu.g CpG per
mg of NC. The Group 2 mice were administered NC-Nic containing ova
peptide, polymers, 75% of which were PLA and 25% were PLA-PEG-Nic,
and 7.2% CpG-1826 (thioated); release rate at 24 hours: 13.2 .mu.g
CpG per mg of NC. The Group 3 mice were administered NC-Nic
containing ova peptide, polymers, 75% of which were PLA and 25%
were PLA-PEG-Nic, and 7.9% CpG-1826 (phosphodiester or PO,
non-thioated); release rate at 24 hours: 19.6 .mu.g CpG per mg of
NC. The Group 4 mice were administered NC-Nic containing ova
peptide, polymers, 75% of which were PLA and 25% were PLA-PEG-Nic,
and 8.5% CpG-1826 (PO, non-thioated); release rate at 24 hours: 9.3
.mu.g CpG per mg of NC. Release was determined at a pH of 4.5.
The results shown in FIG. 5 demonstrate that the rate of release of
entrapped adjuvant (CpG, TLR9 agonist) from nanocarriers influenced
production of an antibody to NC-bound antigen (nicotine) with the
nanocarrier exhibiting higher release rate at 24 hours induced
stronger humoral immune response (group 1>group 2 and group
3>group 4). This was true irrespective of CpG form used (more
stable, thioated or less stable non-thioated).
Example 39
Immunization with NC-Nic Carrying R848
Groups of five mice were immunized three times (subcutaneously,
hind limbs) at 2-week intervals (days 0, 14 and 28) with 100 .mu.g
of NC-Nic and serum anti-nicotine antibodies were then measured on
days 26, 40 and 54. The nanocarriers were prepared according to a
method provided above. EC.sub.50 for anti-nicotine antibodies as
measured in standard ELISA against polylysine-nicotine are shown in
FIG. 6.
The Group 1 mice were administered NC-Nic containing ova peptide
and polymers, 75% of which were PLA and 25% were PLA-PEG-Nic, but
without adjuvant. The Group 2 mice were administered NC-Nic
containing ova peptide, polymers, 75% of which were PLA and 25%
were PLA-PEG-Nic, and 1.0% R848; of which 92% is released at 2
hours and more than 96% is released at 6 hours. The Group 3 mice
were administered NC-Nic containing ova peptide, polymers, 75% of
which were PLA-R848 and 25% were PLA-PEG-Nic, and 1.3% R848, of
which 29.4% is released at 6 hours and 67.8% is released at 24
hours. The Group 4 mice were administered NC-Nic containing ova
peptide, polymers, 75% of which were PLA-R848 and 25% were
PLA-PEG-Nic, and 1.4% of R848, of which 20.4% is released at 6
hours and 41.5% is released at 24 hours. The Group 5 mice were
administered NC-Nic containing ova peptide, polymers, 25% of which
were PLA-PEG-R848, 50% PLA, and 25% were PLA-PEG-Nic, and 0.7% of
R848; of which less than 1% is released at 24 hours. Release was
determined at a pH of 4.5.
The results shown in FIG. 6 demonstrate that R848 adjuvant (a TLR
7/8 agonist) contained in the NC augments humoral immune response
against NC-associated antigen (groups 2-5>>group 1).
Furthermore, neither fast (group 2), nor slow (group 5) release of
R848 was elevated an immune response to the same level as NC
releasing R848 at intermediate rate (group 3.apprxeq.group
4>group 2.apprxeq.group 5).
SEQUENCE LISTINGS
1
2120DNAArtificial Sequencesynthetic oligonucleotide 1tccatgacgt
tcctgacgtt 20220DNAArtificial Sequencesynthetic oligonucleotide
2tccatgacgt tcctgacgtt 20
* * * * *
References