U.S. patent application number 13/484615 was filed with the patent office on 2012-10-18 for photosensitizers for targeted photodynamic therapy.
This patent application is currently assigned to Lynntech, Inc.. Invention is credited to Hariprasad Gali, Michael R. Hamblin, Tim Wharton.
Application Number | 20120264802 13/484615 |
Document ID | / |
Family ID | 36941699 |
Filed Date | 2012-10-18 |
United States Patent
Application |
20120264802 |
Kind Code |
A1 |
Wharton; Tim ; et
al. |
October 18, 2012 |
PHOTOSENSITIZERS FOR TARGETED PHOTODYNAMIC THERAPY
Abstract
The present invention relates to photosensitizer compounds based
on functionalized fullerenes useful in targeted photodynamic
therapy (PDT), and methods of use thereof.
Inventors: |
Wharton; Tim; (Bryan,
TX) ; Gali; Hariprasad; (College Station, TX)
; Hamblin; Michael R.; (Revere, MA) |
Assignee: |
Lynntech, Inc.
College Station
TX
The General Hospital Corporation
Boston
MA
|
Family ID: |
36941699 |
Appl. No.: |
13/484615 |
Filed: |
May 31, 2012 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11885241 |
Jul 23, 2008 |
8207211 |
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PCT/US2006/006894 |
Feb 28, 2006 |
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13484615 |
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60657181 |
Feb 28, 2005 |
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Current U.S.
Class: |
514/410 ; 422/22;
422/28; 514/183; 514/616; 540/460; 548/417; 564/152; 977/734;
977/915 |
Current CPC
Class: |
A61P 31/10 20180101;
Y10S 977/904 20130101; C07C 233/60 20130101; B82Y 5/00 20130101;
A61K 41/0057 20130101; A61K 47/6949 20170801; C07D 209/70 20130101;
C07D 257/10 20130101; C07C 233/62 20130101; C07C 2604/00 20170501;
A61P 31/04 20180101; Y10S 977/734 20130101; A61P 31/00
20180101 |
Class at
Publication: |
514/410 ;
564/152; 514/616; 548/417; 540/460; 514/183; 422/28; 422/22;
977/734; 977/915 |
International
Class: |
A61K 31/164 20060101
A61K031/164; C07D 209/70 20060101 C07D209/70; A61K 31/403 20060101
A61K031/403; C07D 245/04 20060101 C07D245/04; A61P 31/00 20060101
A61P031/00; A61P 31/04 20060101 A61P031/04; A61P 31/10 20060101
A61P031/10; A61L 2/16 20060101 A61L002/16; A61L 2/02 20060101
A61L002/02; C07C 233/60 20060101 C07C233/60; A61K 31/33 20060101
A61K031/33 |
Goverment Interests
STATEMENT OF U.S. GOVERNMENT INTEREST
[0003] Funding for the present invention was provided in part by
the Government of the United States under Grant Nos. R43 CA103268,
and R01 A1050875 from the National Institutes of Health.
Accordingly, the Government of the United States has certain rights
in and to the invention.
Claims
1. A functionized fullerene compound of the formula: ##STR00015##
wherein Z is carbon, nitrogen or phosphorus; R.sub.1 and R.sub.2
are independently selected from the group consisting of
C.sub.1-C.sub.12alkyl, C.sub.2-C.sub.12alkenyl,
C.sub.2-C.sub.12alkynyl, C.sub.3-C.sub.8cycloalkyl,
(aryl)C.sub.0-C.sub.4alkyl, (heteroaryl)C.sub.0-C.sub.4alkyl, or a
group of the formula C(O)--N(R.sub.4)(R.sub.5)(R.sub.6); or
ZR.sub.1R.sub.2 taken in combination foil a 3-20 member
heterocyclic ring having 1-6 ring heteroatoms selected from
nitrogen and phosphorus and having at least one quaternary ammonium
cation or quaternary phosphonium cation; R.sub.4 and R.sub.5 are
independently selected from hydrogen or a group selected from
C.sub.1-C.sub.12alkyl, C.sub.2-C.sub.12alkenyl,
C.sub.2-C.sub.12alkynyl, C.sub.3-C.sub.7cycloalkyl, C.sub.3-C.sub.8
(aryl)C.sub.0-C.sub.4alkyl, and (heteroaryl)C.sub.0-C.sub.4alkyl
each of which groups is substituted with 0-3 substituents selected
from hydroxy, amino, mono-, di-, or
tri-(C.sub.1-C.sub.4alkyl)amino, halogen, quaternary ammonium
cations, quaternary phosphonium cations; R.sub.6 is absent,
hydrogen or a group selected from C.sub.1-C.sub.12alkyl,
C.sub.2-C.sub.12alkenyl, C.sub.2-C.sub.12alkynyl,
C.sub.3-C.sub.7cycloalkyl, C.sub.3-C.sub.8cycloalkyl,
(aryl)C.sub.0-C.sub.4alkyl, and (heteroaryl)C.sub.0-C.sub.4alkyl
each of which groups is substituted with 0-3 substituents selected
from hydroxy, amino, mono-, di-, or
tri-(C.sub.1-C.sub.4alkyl)amino, halogen, quaternary ammonium
cations, quaternary phosphonium cations; X.sub.1 and X.sub.2 are
independently selected at each occurrence from the group consisting
of CH.sub.2 and CHR.sub.3, wherein R.sub.3 is a
C.sub.1-C.sub.6alkyl which is independently selected at each
occurrence of R.sub.3; r is 1, 2, 3, or 4; p and q are
independently selected from 0, 1, 2, or 3 such that
0.ltoreq.(p+q).ltoreq.4; ANION is at least one organic or inorganic
anion; m is a negative integer corresponding to the net negative
charge of each ANION equivalent; n is a positive integer
corresponding to the net positive charge of the substituted
buckminsterfullerene cation; and k is the quotient of n/m.
2. The compound of claim 1, wherein Z is nitrogen or phosphorus;
X.sub.1 and X.sub.2 are methylene; p=q=1; R.sub.1 and R.sub.2 are
independently selected C.sub.1-C.sub.6alkyl,
(aryl)C.sub.0-C.sub.1alkyl, or (heteroaryl)C.sub.0-C.sub.1alkyl; r
is 2, 3, or 4; and n.gtoreq.r.
3. The compound of claim 1, wherein Z is nitrogen or phosphorus;
X.sub.1 and X.sub.2 are methylene; p=q=1; R.sub.1 is
C.sub.1-C.sub.6alkyl, (aryl)C.sub.0-C.sub.1 alkyl, or
(heteroaryl)C.sub.0-C.sub.1alkyl; R.sub.2 is
(aryl)C.sub.0-C.sub.1alkyl, or (heteroaryl)C.sub.0-C.sub.1alkyl; r
is 1, 2, 3, or 4; and n.gtoreq.r.
4. The compound of claim 1, wherein Z is nitrogen; X.sub.1 and
X.sub.2 are methylene; p=q=1; R.sub.1 and R.sub.2 are independently
selected from methyl, ethyl, propyl or isopropyl; r is 2, 3, or 4;
and n.gtoreq.r.
5. The compound of claim 1, wherein Z is carbon; p=q=0; R.sub.1 and
R.sub.2 are independently selected groups of the formula
C(O)--N(R.sub.4)(R.sub.5)(R.sub.6); or ZR.sub.1R.sub.2 taken in
combination form a 6-20 member heterocyclic ring having 1-6 ring
heteroatoms selected from nitrogen and phosphorus and having at
least one quaternary ammonium cation or quaternary phosphonium
cation; R.sub.4 and R.sub.5 are independently selected from
hydrogen or a group selected from C.sub.1-C.sub.12alkyl,
C.sub.2-C.sub.12alkenyl, C.sub.2-C.sub.12alkynyl,
C.sub.3-C.sub.2cycloalkyl, C.sub.3-C.sub.8
(aryl)C.sub.0-C.sub.4alkyl, and (heteroaryl)C.sub.0-C.sub.4alkyl
each of which groups is substituted with 0-3 substituents selected
from hydroxy, amino, di-, or tri-(C.sub.1-C.sub.2alkyl)amino,
halogen, quaternary ammonium cations, quaternary phosphonium
cations; and R.sub.6 is absent, hydrogen or a group selected from
C.sub.1-C.sub.12alkyl, C.sub.2-C.sub.12alkenyl,
C.sub.2-C.sub.12alkynyl, C.sub.3-C.sub.7cycloalkyl,
C.sub.3-C.sub.8cycloalkyl, (aryl)C.sub.0-C.sub.4alkyl, and
(heteroaryl)C.sub.0-C.sub.4alkyl each of which groups is
substituted with 0-3 substituents selected from hydroxy, amino,
mono-, di-, or tri-(C.sub.1-C.sub.2alkyl)amino, halogen, quaternary
ammonium cations, quaternary phosphonium cations.
6. The compound of claim 5, wherein R.sub.1 and R.sub.2 are
independently selected groups of the formula
C(O)--N(R.sub.4)(R.sub.5)(R.sub.6); R.sub.4 is C.sub.2-C.sub.6alkyl
substituted with 1-3 substitutents selected from hydroxy, amino,
di-, or tri-(C.sub.1-C.sub.2alkyl)amino, and quaternary ammonium
cations; R.sub.5 is hydrogen, C.sub.1-C.sub.6alkyl substituted with
0-3 substitutents selected from hydroxy, amino, and quaternary
ammonium cations; and R.sub.6 is absent, hydrogen, or
C.sub.1-C.sub.6alkyl substituted with 0-3 substitutents selected
from hydroxy, amino, di-, or tri-(C.sub.1-C.sub.2alkyl)amino, and
quaternary ammonium cations.
7. The compound of claim 5, wherein R.sub.1 and R.sub.2 are the
same and are selected from the group consisting of: ##STR00016##
wherein R.sub.4 is methyl, ethyl or propyl or isopropyl; R.sub.5
and R.sub.6 are independently selected from methyl, ethyl,
2-(N,N-dimethylamino)ethyl, 3-(N,N-dimethylamino)propyl,
2-(N,N,N-trimethylammonium)ethyl, or
3-(N,N,N-trimethylammonium)propyl.
8. The compound of claim 7, wherein r is 1.
9. The compound of claim 7, wherein r is 2.
10. The compound of claim 7, wherein r is 3.
11. The compound of claim 1, wherein p=q=0; and ZR.sub.1R.sub.2,
taken in combination, form a 7-20 member heterocyclic ring having 2
to 6 nitrogen atoms wherein at least one of the nitrogen atoms is a
quaternary ammonium cation.
12. The compound of claim 11, wherein ZR.sub.1R.sub.2 is a
heterocyclic ring of the formula: ##STR00017## wherein w is
independently selected at each occurrence from 1, 2 or 3; v is 0,
1, 2, or 3; R.sub.7 is independently selected at each occurrence
from hydrogen, C.sub.1-C.sub.6alkyl substituted with 0-3
substitutents selected from hydroxy, amino, and quaternary ammonium
cations; and R.sub.8 is independently selected at each occurrence
from absent, hydrogen, or C.sub.1-C.sub.6alkyl substituted with 0-3
substitutents selected from hydroxy, amino, di-, or
tri-(C.sub.1-C.sub.2alkyl)amino, and quaternary ammonium cations;
and wherein at least one NR.sub.7R.sub.8 is a quaternary ammonium
cation or is substituted by a quaternary ammonium cation.
13. The compound of claim 12, wherein v is 1, 2 or 3; w is 2;
R.sub.7 is independently selected from the group of methyl, ethyl
or propyl or isopropyl; R.sub.8 are independently selected from
methyl, ethyl, 2-(N,N-dimethylamino)ethyl,
3-(N,N-dimethylamino)propyl, 2-(N,N,N-trimethylammonium)ethyl, or
3-(N,N,N-trimethylammonium)propyl.
14. A method for providing antimicrobial therapy, comprising:
administering an effective amount of a composition comprising a
functionalized fullerene compound of claim 1 to a subject in need
thereof; directing light onto the administered fullerene compound
to produce a cytotoxic species; and killing microbes associated
with or proximal to the fullerene compound by reaction with the
cytotoxic species, thereby providing antimicrobial therapy.
15. The method of claim 14, wherein the fullerene is functionalized
with a cationic organic moiety.
16. The method of claim 14, wherein the fullerene is functionalized
with a nonionic organic moiety.
17. The method of claim 14, further comprising: washing away excess
fullerenes that are not associated with the microbial cells prior
to the step of directing light onto the associated fullerene
compound.
18. The method of claim 14, wherein the composition is applied as a
solution having a fullerene concentration of between 1 and 100
micromolar.
19. The method of claim 14, wherein the light is visible light is
provided at an intensity of 0.5 and 160 Joules per square
centimeter.
20. The method of claim 14, wherein visible light is provided at an
intensity between 0.5 and 20 Joules per square centimeter.
21. The method of claim 14, wherein the functionalized fullerene is
water soluble.
22. The method of claim 14, wherein the killing is selective for
the microbe and infected cells of the subject.
23. The method of claim 14, wherein the microbe is selected from
the group consisting of bacteria, yeast and fungi.
24. A pharmaceutical composition, comprising: a biocompatible
carrier and an effective amount of a functionalized fullerene
compound of claim 1.
25. The pharmaceutical composition of claim 24, wherein the
composition is a solution having a fullerene concentration of
between 1 and 100 micromolar.
26. The composition of claim 24, further comprising a
hyperosmotically active chemical species.
27. The composition of claim 24, wherein the fullerene is
functionalized with a cationic organic moiety.
28. The composition of claim 24, wherein the fullerene is
functionalized with a nonionic organic moiety.
29. A method for killing a microbe, comprising: providing a
composition comprising a functionalized fullerene compound of claim
1 to a microbe; directing light onto the fullerene compound to
produce a cytotoxic species; and killing the microbe associated
with or proximal to the fullerene compound by reaction with the
cytotoxic species.
30. The method of claim 29, wherein the composition is applied as a
solution having a fullerene concentration of between 1 and 100
micromolar.
31. The method of claim 29, wherein the light is visible light is
provided at an intensity of 0.5 and 160 Joules per square
centimeter.
32. The method of claim 29, wherein visible light is provided at an
intensity between 0.5 and 20 Joules per square centimeter.
33. The method of claim 29, wherein the microbe is selected from
the group consisting of bacteria, yeast and fungi.
34. The method of claim 29, wherein the microbe is associated with
an inaminate object.
35. The method according to claim 29 or claim 14, further
comprising obtaining the functionalized fullerene compound.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS/PATENTS & INCORPORATION
BY REFERENCE
[0001] This application is a continuation of U.S. application Ser.
No. 11/885,241, filed Jul. 23, 2008, which is the U.S. national
phase application, pursuant to 35 U.S.C. .sctn.371, of PCT
international application Ser. No. PCT/US06/006894 filed Feb. 28,
2006, designating the United States and published in English on
Sep. 8, 2006 as publication WO 2006/093891 A2, which claims
priority to U.S. provisional application Ser. No. 60/657,181, filed
Feb. 28, 2005. The entire contents of the aforementioned patent
applications are incorporated herein by this reference.
[0002] Each of the applications and patents cited in this text, as
well as each document or reference cited in each of the
applications and patents (including during the prosecution of each
issued patent; "application cited documents"), and each of the PCT
and foreign applications or patents corresponding to and/or
paragraphing priority from any of these applications and patents,
and each of the documents cited or referenced in each of the
application cited documents, are hereby expressly incorporated
herein by reference. More generally, documents or references are
cited in this text, either in a Reference List, or in the text
itself; and, each of these documents or references ("herein-cited
references"), as well as each document or reference cited in each
of the herein-cited references (including any manufacturer's
specifications, instructions, etc.), is hereby expressly
incorporated herein by reference.
BACKGROUND OF THE INVENTION
[0004] Photodynamic therapy (PDT) refers to the use of
photosensitizing drugs in combination with light for treating
medical conditions. The PDT technique has shown promise as a cancer
therapy (Dolmans, D. E., Fukumura, D., and Jain, R. K. (2003).
Photodynamic therapy for cancer. Nat. Rev. Cancer 3, 380-387) and
recently has achieved success as a treatment for age-related
macular degeneration (Brown, S. B., and Mellish, K. J. (2001).
Verteporfin: a milestone in opthalmology and photodynamic therapy.
Expert Opin. Pharmacother. 2, 351-361). The PDT method uses a
compound known as a photosensitizer (PS) which is administered
directly (e.g., endoscopically or topically) to an accessible
treatment site, or alternatively, is administered systemically and
concentrates in a target tissue site within the body of a subject.
Subsequent irradiation of the target site with visible light of
suitable wavelength generates singlet oxygen, .sup.1O2, within or
on the surface of the cells of the treatment site, ultimately
leading to cell death. The singlet oxygen is catalytically
generated by energy transfer from the PS to oxygen from dissolved
O.sub.2, which is ubiquitous in the body's tissues. Photodynamic
therapy is advantageous compared with other therapies due to its
dual selectivity: not only is the PS targeted to the tumor or other
lesion, but the light can also be accurately delivered to the
affected tissue.
[0005] The potential use of a photosensitizer as an effective means
of killing microorganisms was first recognized over 100 years ago
(Moan, J., and Peng, Q. (2003). An outline of the hundred-year
history of PDT. Anticancer Res. 23, 3591-3600); however, the
possible use of PDT as a treatment for microbial infections is just
beginning to be appreciated (Wainwright, M. (1998). Photodynamic
antimicrobial chemotherapy (PACT). J. Antimicrob. Chemother. 42,
13-28, Maisch, T., Szeimies, R. M., Joni, G., and Abels, C. (2004).
Antibacterial photodynamic therapy in dermatology. Photochem.
Photobiol. Sci. 3, 907-917 and 30. Hamblin, M. R., and Hasan, T.
(2004). Photodynamic therapy: a new antimicrobial approach to
infectious disease? Photo-chem. Photobiol. Sci. 3, 436-450). For
decades, antibiotics have been the first line of defense against
microorganisms. Of great concern in current medical practice is the
proliferation of infectious microbes that display multiple
antibiotic resistance, and hence are not killed by existing
antibiotics alone or in combination. Accordingly, there is a great
unmet need to develop new antimicrobial agents to which microbes
are not easily able to develop resistance. In this regard, it is
envisioned that treatment of infections with PDT holds great
promise as an alternative or adjunct to traditional antibiotic
therapy because organisms are unlikely to develop resistance to a
killing mechanism based on bombardment of the pathogens with
reactive oxygen species.
[0006] Given the urgent need for new antimicrobial agents, it would
be desirable to develop PS compounds that are effective at killing
a broad range of microbes such as bacteria, fungi and yeast but are
not harmful to the cells of a mammalian subject receiving
antimicrobial PS therapy.
SUMMARY OF THE INVENTION
[0007] The invention relates to the development and use of a new
class of photosensitizing molecules for PDT. It has now been
demonstrated that cationic fullerene embodiments functionalized
with one, two, or three pyrrolidinium groups, after a short
incubation followed by illumination with white light, have a
broad-spectrum antimicrobial activity and can rapidly kill more
than 99.99% of bacterial and fungal cells.
[0008] In this invention, fullerene molecules, e.g., C.sub.60,
C.sub.70, C.sub.74, C.sub.76, C.sub.78, C.sub.80, C.sub.82,
C.sub.84, higher fullerenes and their functionalized derivatives,
have been modified to include a variety of properties needed for
application of PDT to microorganisms. This was achieved by
controlling hydrophobicity, molecular charge, and water solubility
of the carbon nanomaterial specifically to target microbial species
preferentially over other types of cells for PDT. A positive charge
on some embodiments allows the fullerenes to bind to cells and
overcome microbial permeability barriers. Cationic fullerenes in
particular perform better as antimicrobial photosensitizers than
the widely employed antimicrobial photosensitizer toluidine blue O.
Accordingly, cationic fullerene-mediated photodynamic therapy may
find significant application in the treatment of a wide variety of
conditions, such as for example, localized infections in wounds,
burns, and mucus membranes.
[0009] More particularly, in one embodiment the present invention
relates to compositions comprising a functionalized fullerene,
wherein the wherein the functionalized fullerene comprises a
fullerene core (C.sub.n) where n is an even integer greater than or
equal to 60, and at least one functional group bonded to at least
one carbon atom of the fullerene core.
[0010] Some embodiments are based on hydrophilic cationic fullerene
derivatives. Other embodiments are hydrophilic neutral fullerene
derivatives.
[0011] Fullerene derivatives of the invention are suitable for the
treatment of a variety of bacterial, viral, and fungal infections.
Accordingly, in another embodiment, the invention relates to a
method for providing antimicrobial therapy, which includes
administering an effective amount of a functionalized fullerene
species to a subject in need thereof. The fullerene species can be
any one of the compounds described herein. The method includes
directing light onto the administered fullerene species to produce
a cytotoxic species; and killing microbial cells associated with or
proximal to the fullerene species by reaction with the cytotoxic
species.
BRIEF DESCRIPTION OF THE DRAWINGS
[0012] The following Detailed Description, given by way of example,
but not intended to limit the invention to specific embodiments
described, may be understood in conjunction with the accompanying
drawings, incorporated herein by reference. Various preferred
features and embodiments of the present invention will now be
described by way of non-limiting examples and with reference to the
accompanying drawings in which:
[0013] FIG. 1 is a graph showing UV-Visible absorption spectra of
common photosensitizer toluidine blue O (TBO) and of derivatized
fullerenes (CI1-3) prepared in accordance with the invention.
[0014] FIG. 2 is a graph illustrating photodynamic inactivation
(PDI) of S. aureus bacteria by functionized fullerenes NI1-3 and
CI1-3 prepared in accordance with the invention, following exposure
to 405 nm light.
[0015] FIG. 3 is a dose-response curve illustrating PDI of S.
aureus by varying concentrations of cationic fullerene CU, in
accordance with an embodiment of the invention.
[0016] FIG. 4 is a graph showing PDI of several microbial species
(bacteria--S. aureus, E. coli, P. aeruginosa; and yeast--C.
albicans) by cationic fullerene CU, in accordance with an
embodiment of the invention.
[0017] FIG. 5 is a graph showing effect of a wash procedure on PDI
of P. aeruginosa by cationic fullerene CU, in accordance with an
embodiment of the invention.
[0018] FIG. 6 is a graph showing PDI of bacterial strains S.
aureus, E. coli, and P. aeruginosa and yeast strain C. albicans by
cationic fullerene CI1 following exposure to low levels of white
light.
[0019] FIG. 7 is a graph showing PDI of bacterial strains S.
aureus, E. coli, and P. aeruginosa and yeast strain C. albicans by
cationic fullerene CI2, following exposure to low levels of white
light.
[0020] FIG. 8 is a graph showing PDI of bacterial strains S.
aureus, E. coli, and P. aeruginosa and yeast strain C. albicans by
cationic fullerene CI3, following exposure to low levels of white
light.
[0021] FIG. 9 is a graph showing PDI of bacterial strains S.
aureus, E. coli, and P. aeruginosa, yeast strain C. albicans, and
mammalian fibroblast cell line L929 by cationic fullerene CI1,
following exposure to higher levels of white light than provided in
FIGS. 6-8.
[0022] FIG. 10 is a graph showing PDI of bacterial strains S.
aureus, E. coli, and P. aeruginosa, yeast strain C. albicans, and
mammalian fibroblasts by cationic fullerene CI2, following exposure
to white light under conditions as described for FIG. 9.
[0023] FIG. 11 is a graph showing PDI of bacterial strains S.
aureus, E. coli, and P. aeruginosa, yeast strain C. albicans, and
mammalian fibroblasts by cationic fullerene CI3, following exposure
to white light under conditions as described for FIG. 9.
[0024] FIG. 12 is a graph showing PDI of bacterial strain S. aureus
by cationic fullerenes CI1-3 in accordance with the invention,
relative to that of known antimicrobial photosensitizer toluidine
blue O (TBO).
[0025] FIG. 13 is a graph showing PDI of bacterial strain E. coli
by cationic fullerenes CI1-3 in accordance with the invention,
relative to that of TBO.
[0026] FIG. 14 is a graph showing PDI of bacterial strain P.
aeruginosa by cationic fullerenes CI1-3 in accordance with the
invention, relative to that of TBO.
[0027] FIG. 15 is a graph showing PDI of yeast strain C. albicans
by cationic fullerenes CI1-3, in accordance with the invention,
relative to that of TBO.
[0028] FIG. 16 is a graph showing PDI at high light levels of
mammalian fibroblasts by cationic fullerene CI2 in accordance with
the invention, relative to TBO.
[0029] FIGS. 17A and B are two graphs showing PDI of S. aureus (A)
and E. coli (B) bacteria by nonionic fullerenes NI1-3 in accordance
with the invention, under conditions with or without washing to
remove fullerenes prior to illumination.
[0030] FIG. 18 is a graph showing PDI of S. aureus with cationic
fullerene CI1 at specified concentrations, in accordance with an
embodiment of the invention.
[0031] FIGS. 19A-B are two graphs showing PDI of S. aureus (A) and
E. coli (B) with cationic fullerenes CI1-3, in accordance with an
embodiment of the invention.
[0032] FIGS. 20A-B are two graphs showing PDI of C. albicans (A)
and P. aeruginosa (B) with cationic fullerenes CI1-3, in accordance
with an embodiment of the invention.
[0033] FIGS. 21A-B are two graphs showing PDI of E. coli (A) and
mammalian fibroblasts (B) under the same conditions with cationic
fullerenes CI1-3, in accordance with an embodiment of the
invention.
DETAILED DESCRIPTION OF THE INVENTION
I. Definitions
[0034] In order that the invention may be more readily understood,
certain terms are first defined and collected here for convenience.
Other definitions appear in context throughout the application.
[0035] The term "alkyl" refers to the radical of saturated
aliphatic groups, including straight-chain alkyl groups,
branched-chain alkyl groups, cycloalkyl (alicyclic) groups, alkyl
substituted cycloalkyl groups, and cycloalkyl substituted alkyl
groups. The term alkyl further includes alkyl groups, which can
further include oxygen, nitrogen, sulfur or phosphorous atoms
replacing one or more carbons of the hydrocarbon backbone. In
certain embodiments, a straight chain or branched chain alkyl has
30 or fewer carbon atoms in its backbone (e.g., C.sub.1-C.sub.30
for straight chain, C.sub.3-C.sub.30 for branched chain),
preferably 26 or fewer, and more preferably 20 or fewer. Likewise,
certain cycloalkyls have from 3-10 carbon atoms in their ring
structure, and more preferably have 3, 4, 5, 6 or 7 carbons in the
ring structure.
[0036] Moreover, the term alkyl as used throughout the
specification and claims is intended to include both "unsubstituted
alkyls" and "substituted alkyls," the latter of which refers to
alkyl moieties having substituents replacing a hydrogen on one or
more carbons of the hydrocarbon backbone. Such substituents can
include, for example, halogen, hydroxyl, alkylcarbonyloxy,
arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy,
carboxylate, alkylcarbonyl, alkoxycarbonyl, aminocarbonyl,
alkylthiocarbonyl, alkoxyl, phosphate, phosphonato, phosphinato,
cyano, amino (including alkyl amino, dialkylamino, arylamino,
diarylamino, and alkylarylamino), acylamino (including
alkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido),
amidino, imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate,
sulfates, sulfonato, sulfamoyl, sulfonamido, nitro,
trifluoromethyl, cyano, azido, heterocyclyl, alkylaryl, or an
aromatic or heteroaromatic moiety. It will be understood by those
skilled in the art that the moieties substituted on the hydrocarbon
chain can themselves be substituted, if appropriate. Cycloalkyls
can be further substituted, e.g., with the substituents described
above. An "alkylaryl" moiety is an alkyl substituted with an aryl
(e.g., phenylmethyl (benzyl)). The term "alkyl" also includes
unsaturated aliphatic groups analogous in length and possible
substitution to the alkyls described above, but that contain at
least one double or triple bond respectively.
[0037] Unless the number of carbons is otherwise specified, "lower
alkyl" as used herein means an alkyl group, as defined above, but
having from one to ten carbons, more preferably from one to six,
and most preferably from one to four carbon atoms in its backbone
structure, which may be straight or branched-chain.
[0038] The terms "alkoxyalkyl," "polyaminoalkyl" and
"thioalkoxyalkyl" refer to alkyl groups, as described above, which
further include oxygen, nitrogen or sulfur atoms replacing one or
more carbons of the hydrocarbon backbone, e.g., oxygen, nitrogen or
sulfur atoms.
[0039] The terms "alkenyl" and "alkynyl" refer to unsaturated
aliphatic groups analogous in length and possible substitution to
the alkyls described above, but that contain at least one double or
triple bond, respectively.
[0040] The term "aryl" as used herein, refers to the radical of
aryl groups, including 5- and 6-membered single-ring aromatic
groups that may include from zero to four heteroatoms, for example,
benzene, pyrrole, furan, thiophene, imidazole, benzoxazole,
benzothiazole, triazole, tetrazole, pyrazole, pyridine, pyrazine,
pyridazine and pyrimidine, and the like. Aryl groups also include
polycyclic fused aromatic groups such as naphthyl, quinolyl,
indolyl, and the like.
[0041] Those aryl groups having heteroatoms in the ring structure
may also be referred to as "aryl heterocycles," "heteroaryls" or
"heteroaromatics." The aromatic ring can be substituted at one or
more ring positions with such substituents as described above, as
for example, halogen, hydroxyl, alkoxy, alkylcarbonyloxy,
arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy,
carboxylate, alkylcarbonyl, alkoxycarbonyl, aminocarbonyl,
alkylthiocarbonyl, phosphate, phosphonato, phosphinato, cyano,
amino (including alkyl amino, dialkcylamino, arylamino,
diarylamino, and alkylarylamino), acylamino (including
alkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido),
amidino, imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate,
sulfates, sulfonato, sulfamoyl, sulfonamido, nitro,
trifluoromethyl, cyano, azido, heterocyclyl, alkylaryl, or an
aromatic or heteroaromatic moiety. Aryl groups can also be fused or
bridged with alicyclic or heterocyclic rings which are not aromatic
so as to form a polycycle (e.g., tetralin).
[0042] The term "chiral" refers to molecules which have the
property of non-superimposability of the mirror image partner,
while the team "achiral" refers to molecules which are
superimposable on their mirror image partner.
[0043] The term "enantiomers" refers to two stereoisomers of a
compound which are non-superimposable mirror images of one another.
An equimolar mixture of two enantiomers is called a "racemic
mixture" or a "racemate."
[0044] The term "halogen" designates --F, --Cl, --Br or --I.
[0045] The term "haloalkyl" is intended to include alkyl groups as
defined above that are mono-, di- or polysubstituted by halogen,
e.g., fluoromethyl and trifluoromethyl.
[0046] The term "hydroxyl" means --OH.
[0047] The term "heteroatom" as used herein means an atom of any
element other than carbon or hydrogen. Preferred heteroatoms are
nitrogen, oxygen, sulfur and phosphorus.
[0048] The term "isomers" or "stereoisomers" refers to compounds
which have identical chemical constitution, but differ with regard
to the arrangement of the atoms or groups in space. Furthermore the
indication of stereochemistry across a carbon-carbon double bond is
also opposite from the general chemical field in that "Z" refers to
what is often referred to as a "cis" (same side) conformation
whereas "E" refers to what is often referred to as a "trans"
(opposite side) conformation. With respect to the nomenclature of a
chiral center, the terms "d" and "1" configuration are as defined
by the IUPAC Recommendations. As to the use of the terms,
diastereomer, racemate, epimer and enantiomer, these will be used
in their normal context to describe the stereochemistry of
preparations.
[0049] The term "obtaining" as in "obtaining the fullerene
derivative" is intended to include purchasing, synthesizing or
otherwise acquiring the fullerene derivative (or indicated
substance or material).
[0050] A "photosensitizer" or "photosensitive material" is defined
herein as a material, element, chemical, solution, compound,
matter, or substance which is sensitive, reactive, receptive, or
responsive to light energy. The term can refer to a
photoactivatable fullerene compound, or a precursor thereof, that
produces a reactive species (e.g., oxygen) having a phototoxic
effect on a microbe or infected cell.
[0051] The terms "polycyclyl" or "polycyclic radical" refer to the
radical of two or more cyclic rings (e.g., cycloalkyls,
cycloalkenyls, cycloalkynyls, aryls and/or heterocyclyls) in which
two or more carbons are common to two adjoining rings, e.g., the
rings are "fused rings". Rings that are joined through non-adjacent
atoms are termed "bridged" rings. Each of the rings of the
polycycle can be substituted with such substituents as described
above, as for example, halogen, hydroxyl, alkylcarbonyloxy,
arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy,
carboxylate, alkylcarbonyl, alkoxycarbonyl, aminocarbonyl,
alkylthiocarbonyl, alkoxyl, phosphate, phosphonato, phosphinato,
cyano, amino (including alkyl amino, dialkylamino, arylamino,
diarylamino, and alkylarylamino), acylamino (including
alkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido),
amidino, imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate,
sulfates, sulfonato, sulfamoyl, sulfonamido, nitro,
trifluoromethyl, cyano, azido, heterocyclyl, alkyl, alkylaryl, or
an aromatic or heteroaromatic moiety.
[0052] The term "sulfhydryl" or "thiol" means --SH. The term
"subject" refers to animals such as mammals, including, but not
limited to, primates (e.g., humans), cows, sheep, goats, horses,
dogs, cats, rabbits, rats, mice and the like. In certain
embodiments, the subject is a human.
[0053] In this disclosure, "comprises," "comprising," "containing"
and "having" and the like can have the meaning ascribed to them in
U.S. patent law and can mean "includes," "including," and the like;
"consisting essentially of or "consists essentially" likewise has
the meaning ascribed in U.S. patent law and the term is open-ended,
allowing for the presence of more than that which is recited so
long as basic or novel characteristics of that which is recited are
not changed by the presence of more than that which is recited, but
excludes prior art embodiments.
[0054] Unless otherwise defined, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention belongs. In case
of conflict, the present application, including definitions will
control.
II. Compositions of the Invention
[0055] The present invention provides photodynamic compositions for
PDT. PDT employs photoactivatable compounds known as
photosensitizers to selectively target and destroy cells. Therapy
involves delivering visible light of the appropriate wavelength to
excite the photosensitizer molecule to the excited singlet state.
This excited state can then undergo intersystem crossing to the
slightly lower energy triplet state, which can then react further
by one or both of two pathways, known as Type I and Type II
photoprocesses (Ochsner (1997) J Photochem Photobiol B 39:1-18).
The Type I pathway involves electron transfer reactions from the
photosensitizer triplet to produce radical ions that can then react
with oxygen to produce cytotoxic species such as superoxide,
hydroxyl and lipid derived radicals. The Type II pathway involves
energy transfer from the photosensitizer triplet to ground state
molecular oxygen (triplet) to produce the excited state singlet
oxygen, which can then oxidize many biological molecules such as
proteins, nucleic acids and lipids, and lead to cytotoxicity.
[0056] Functionalized Fullerenes as Photosensitizers
[0057] The therapeutic compositions of the invention comprise novel
photosensitizer compounds for PDT based on functionalized fullerene
molecules. Without being bound by theory, it is believed that the
functionalized fullerene molecules of the invention function
through the Type I pathway described herein above.
[0058] More particularly, the invention relates to fullerenes,
e.g., C.sub.60, C.sub.70, C.sub.74, C.sub.76, C.sub.78, C.sub.80,
C.sub.82, C.sub.84, higher fullerenes and their functionalized
derivatives. Buckminsterfullerenes, also known as fullerenes or,
more colloquially, "buckyballs," are cage-like molecules consisting
essentially of sp.sup.2-hybridized carbons. Fullerenes were first
reported by Kroto et al., Nature (1985) 318:162. Fullerenes are the
third form of pure carbon, in addition to diamond and graphite.
Typically, fullerenes are arranged in hexagons, pentagons, or both.
Most known fullerenes have 12 pentagons and varying numbers of
hexagons depending on the size of the molecule. Common fullerenes
include C.sub.60 and C.sub.70, although fullerenes comprising up to
about 400 carbon atoms are also known.
[0059] C.sub.60 has 30 carbon-carbon double bonds, and has been
reported to readily react with oxygen radicals (Krusic et al.,
Science, 1991, 254:1183-1185). Other fullerenes have comparable
numbers of carbon-carbon double bonds and would be expected to be
similarly reactive with oxygen radicals. Native fullerenes are
generally only soluble in apolar organic solvents, such as toluene
or benzene. To render fullerenes water-soluble, as well as to
impart other properties to fullerene-based molecules, a number of
fullerene substituents have been developed.
[0060] Methods of substituting fullerenes with various substituents
are known in the art. Methods include 1,3-dipolar additions
(Sijbesma et al., J. Am. Chem. Soc. (1993) 115:6510-6512; Suzuki,
J. Am. Chem. Soc. (1992) 114:7301-7302; Suzuki et al., Science
(1991) 254:1186-1188; Prato et al., J. Org. Chem. (1993)
58:5578-5580; Vasella et al., Angew. Chem. Int. Ed. Engl. (1992)
31:1388-1390; Prato et al., J. Am. Chem. Soc. (1993) 115:1148-1150;
Maggini et al., Tetrahedron Lett. (1994) 35:2985-2988; Maggini et
al., J. Am. Chem. Soc. (1993) 115:9798-9799; and Meier et al., J.
Am. Chem. Soc. (1994) 116:7044-7048), Diels-Alder reactions (lyoda
et al., J. Chem. Soc. Chem. Commun. (1994) 1929-1930; Belik et al.,
Angew. Chem. Int. Ed. Engl. (1993) 32:78-80; Bidell et al., J.
Chem. Soc. Chem. Commun. (1994) 1641-1642; and Meidine et al., J.
Chem. Soc. Chem. Commun. (1993) 1342-1344), other cycloaddition
processes (Saunders et al., Tetrahedron Lett. (1994) 35:3869-3872;
Tadeshita et al., J. Chem. Soc. Perkin. Trans. (1994) 1433-1437;
Beeretal., Angew. Chem. Int. Ed. Engl. (1994)33:1087-1088; Kusukawa
et al., Organometallics (1994) 13:4186-4188; Averdung et al., Chem.
Ber. (1994) 127:787-789; Akasaka et al., J. Am. Chem. Soc. (1994)
116:2627-2628; Wu et al., Tetrahedron Lett. (1994) 35:919-922; and
Wilson, J. Org. Chem. (1993) 58:6548-6549); cyclopropanation by
addition/elimination (Hirsch et al., Agnew. Chem. Int. Ed. Engl.
(1994) 33:437-438 and Bestmann et al., C. Tetra. Lett. (1994)
35:9017-9020); and addition of carbanions/alkyl lithiums/Grignard
reagents (Nagashima et al., J. Org. Chem. (1994) 59:1246-1248;
Fagan et al., J. Am. Chem. Soc. (1994) 114:9697-9699; Hirsch et
al., Agnew. Chem. Int. Ed. Engl. (1992) 31:766-768; and Komatsu et
al., J. Org. Chem. (1994) 59:6101-6102); among others. The
synthesis of substituted fullerenes is reviewed by Murphy et al.,
U.S. Pat. No. 6,162,926.
[0061] The discovery of the fullerenes in 1985, and the subsequent
development of synthetic methods for the preparation of large-scale
quantities of the allotropes of carbon has generated considerable
interest and opened a whole new field of carbon chemistry.
Fullerenes are defined as closed-cage polyhedrons made up entirely
of sp.sup.2-hybridized carbon atoms that contain exactly 12
pentagonal faces (known as Euler's theorem) and (n/2-10) hexagonal
faces where n is the number of carbon atoms (n must be even and
greater than twenty). The soccer ball-shaped fullerene C.sub.60 has
the highest theoretically possible symmetry, icosahedral (I.sub.h).
It is the most abundant fullerene that is produced during the
graphite combustion production of the materials, followed by
C.sub.70.
[0062] C.sub.60 can be functionalized by well known methods of
synthetic organic chemistry. The formation of C.sub.60 derivatives
(i.e., covalently modified C.sub.60) nearly always involves the
addition of a functional group (addend) across one or more of its
30 double bonds. When only one addend is attached, the fullerene
derivative is termed a "monoadduct," with two, a "bisadduct,"
etc.
[0063] Another advantage of the spherical C.sub.60 molecule for PDT
is its large surface area of .about.200 .ANG..sup.2 compared to
.ltoreq.100 .ANG..sup.2 for other "flat" rigid PS, maximizing
exposure to O.sub.2. Additionally, the versatility of the C.sub.60
scaffolding allows a tailoring of the hydrophobicity/hydrophilicity
by simple synthetic methods, providing, as a nonlimiting example,
any of a number of structures expected to be absorbed through the
skin. Advantageously, C.sub.60 and its derivatives are also
thermally and photochemically stable (minimal photobleaching).
[0064] The present invention relates in one aspect to compositions
comprising a functionalized (substituted, derivatized) fullerene
comprising a fullerene core (C.sub.n) where n is an even integer
greater than or equal to 60, and at least one functional group
bonded to at least one carbon atom of the fullerene core.
[0065] In one embodiment, the functionalized fullerene is a
compound of the generic formula I:
##STR00001##
[0066] wherein
[0067] Z is carbon, nitrogen or phosphorus;
[0068] R.sub.1 and R.sub.2 are independently selected from the
group consisting of C.sub.1-C.sub.12alkyl, C.sub.2-C.sub.12alkenyl,
C.sub.2-C.sub.12alkynyl, C.sub.3-C.sub.8cycloalkyl,
(aryl)C.sub.0-C.sub.4alkyl, (heteroaryl)C.sub.0-C.sub.4alkyl, or a
group of the formula C(O)--N(R.sub.4)(R.sub.5)(R.sub.6); or
[0069] ZR.sub.1R.sub.2 taken in combination form a 3-20 member
heterocyclic ring having 1-6 ring heteroatoms selected from
nitrogen and phosphorus, and having at least one quaternary
ammonium cation or quaternary phosphonium cation;
[0070] R.sub.4 and R.sub.5 are independently selected from hydrogen
or a group selected from C.sub.1-C.sub.12alkyl,
C.sub.2-C.sub.12alkenyl, C.sub.2-C.sub.12alkynyl,
C.sub.3-C.sub.7cycloalkyl, C.sub.3-C.sub.8
(aryl)C.sub.0-C.sub.4alkyl, and (heteroaryl)C.sub.0-C.sub.4alkyl
each of which groups is substituted with 0-3 substituents selected
from hydroxy, amino, mono-, di-, or
tri-(C.sub.1-C.sub.4alkyl)amino, halogen, quaternary ammonium
cations, quaternary phosphonium cations;
[0071] R.sub.6 is absent, hydrogen or a group selected from
C.sub.1-C.sub.12alkyl, C.sub.2-C.sub.12alkenyl,
C.sub.2-C.sub.12alkynyl, C.sub.3-C.sub.7cycloalkyl,
C.sub.3-C.sub.8cycloalkyl, (aryl)C.sub.0-C.sub.4alkyl, and
(heteroaryl)C.sub.0-C.sub.4alkyl each of which groups is
substituted with 0-3 substituents selected from hydroxy, amino,
mono-, di-, or tri-(C.sub.1-C.sub.4alkyl)amino, halogen, quaternary
ammonium cations, quaternary phosphonium cations;
[0072] X.sub.1 and X.sub.2 are independently selected at each
occurrence from the group consisting of CH.sub.2 and CHR.sub.3,
wherein R.sub.3 is a C.sub.1-C.sub.6alkyl which is independently
selected at each occurrence of R.sub.3;
[0073] r is 1, 2, 3, or 4;
[0074] p and q are independently selected from 0, 1, 2, or 3 such
that 0.ltoreq.(p+q).ltoreq.4;
[0075] ANION is at least one organic or inorganic anion;
[0076] m is a negative integer corresponding to the net negative
charge of each ANION equivalent;
[0077] n is a positive integer corresponding to the net positive
charge of the substituted buckminsterfullerene cation; and
[0078] k is the quotient of n/m.
[0079] Certain other compounds of formula I include those compounds
in which the C60-fullerene is substituted by a Cn-fullerene wherein
n is an integer of between 50 and about 84.
[0080] Another embodiment is a compound according to formula I,
wherein
[0081] Z is nitrogen or phosphorus;
[0082] X.sub.1 and X.sub.2 are methylene;
[0083] p=q=1;
[0084] R.sub.1 and R.sub.2 are independently selected
C.sub.1-C.sub.6alkyl, (aryl)C.sub.0-C.sub.1 alkyl, or
(heteroaryl)C.sub.0-C.sub.1alkyl;
[0085] r is 2, 3, or 4; and
[0086] n.gtoreq.r.
[0087] Another embodiment is a compound according to formula I,
referred to herein as compounds of formula II, wherein
[0088] Z is nitrogen or phosphorus;
[0089] X.sub.1 and X.sub.2 are methylene;
[0090] p=q=1;
[0091] R.sub.1 is C.sub.1-C.sub.6alkyl, (aryl)C.sub.0-C.sub.1alkyl,
or (heteroaryl)C.sub.0-C.sub.1 alkyl;
[0092] R.sub.2 is (aryl)C.sub.0-C.sub.1alkyl, or
(heteroaryl)C.sub.0-C.sub.1alkyl;
[0093] r is 1, 2, 3, or 4; and
[0094] n.gtoreq.r.
[0095] Another embodiment is a compound according to formula II,
wherein
[0096] Z is nitrogen;
[0097] X.sub.1 and X.sub.2 are methylene;
[0098] p=q=1;
[0099] R.sub.1 and R.sub.2 are independently selected from methyl,
ethyl, propyl or isopropyl;
[0100] r is 2, 3, or 4; and
[0101] n.gtoreq.r
[0102] Another embodiment is the compound according to formula I,
wherein
[0103] Z is carbon;
[0104] p=q=0;
[0105] R.sub.1 and R.sub.2 are independently selected groups of the
formula C(O)--N(R.sub.4)(R.sub.5)(R.sub.6); or
[0106] ZR.sub.1R.sub.2 taken in combination foam a 6-20 member
heterocyclic ring having 1-6 ring heteroatoms selected from
nitrogen and phosphorus and having at least one quaternary ammonium
cation or quaternary phosphonium cation;
[0107] R.sub.4 and R.sub.5 are independently selected from hydrogen
or a group selected from C.sub.1-C.sub.12alkyl,
C.sub.2-C.sub.12alkenyl, C.sub.2-C.sub.12alkynyl,
C.sub.3-C.sub.7cycloalkyl, C.sub.3-C.sub.8
(aryl)C.sub.0-C.sub.4alkyl, and (heteroaryl)C.sub.0-C.sub.4alkyl
each of which groups is substituted with 0-3 substituents selected
from hydroxy, amino, di-, or tri-(C.sub.1-C.sub.2alkyl)amino,
halogen, quaternary ammonium cations, quaternary phosphonium
cations; and
[0108] R.sub.6 is absent, hydrogen or a group selected from
C.sub.1-C.sub.12alkyl, C.sub.2-C.sub.12alkenyl,
C.sub.2-C.sub.12alkynyl, C.sub.3-C.sub.7cycloalkyl,
C.sub.3-C.sub.8cycloalkyl, (aryl)C.sub.0-C.sub.4alkyl, and
(heteroaryl)C.sub.0-C.sub.4alkyl each of which groups is
substituted with 0-3 substituents selected from hydroxy, amino,
mono-, di-, or tri-(C.sub.1-C.sub.2alkyl)amino, halogen, quaternary
ammonium cations, quaternary phosphonium cations.
[0109] Another embodiment is a compound according to formula
referred to herein as formula III, wherein
[0110] R.sub.1 and R.sub.2 are independently selected groups of the
formula C(O)--N(R.sub.4)(R.sub.5)(R.sub.6);
[0111] R.sub.4 is C.sub.2-C.sub.6alkyl substituted with 1-3
substitutents selected from hydroxy, amino, di-, or
tri-(C.sub.1-C.sub.2alkyl)amino, and quaternary ammonium
cations;
[0112] R.sub.5 is hydrogen, C.sub.1-C.sub.6alkyl substituted with
0-3 substitutents selected from hydroxy, amino, and quaternary
ammonium cations; and
[0113] R.sub.6 is absent, hydrogen, or C.sub.1-C.sub.6alkyl
substituted with 0-3 substitutents selected from hydroxy, amino,
di-, or tri-(C.sub.1-C.sub.2alkyl)amino, and quaternary ammonium
cations.
[0114] Another embodiment is a compound according to formula III,
referred to herein as formula IV, wherein
wherein R.sub.1 and R.sub.2 are the same and are selected from the
group consisting of:
##STR00002##
[0115] wherein R.sub.4 is methyl, ethyl or propyl or isopropyl;
[0116] R.sub.5 and R.sub.6 are independently selected from methyl,
ethyl, 2-(N,N-dimethylamino)ethyl, 3-(N,N-dimethylamino)propyl,
2-(N,N,N-trimethylammonium)ethyl, or
3-(N,N,N-trimethylammonium)propyl.
[0117] Another embodiment is a compound according to formula IV,
wherein r is 1.
[0118] Another embodiment is a compound according to formula IV,
wherein r is 2.
[0119] Another embodiment is a compound according to formula IV,
wherein r is 3.
[0120] Another embodiment is a compound according to formula I,
wherein
[0121] p=q=0; and
[0122] ZR.sub.1R.sub.2, taken in combination, form a 7-20 member
heterocyclic ring having 2 to 6 nitrogen atoms wherein at least one
of the nitrogen atoms is a quaternary ammonium cation. (Formula
V).
[0123] Another embodiment is a compound according to formula V,
referred to herein as formula VI wherein ZR.sub.1R.sub.2 is a
heterocyclic ring of the formula:
##STR00003##
[0124] wherein
[0125] w is independently selected at each occurrence from 1, 2 or
3;
[0126] v is 0, 1, 2, or 3;
[0127] R.sub.7 is independently selected at each occurrence from
hydrogen, C.sub.1-C.sub.6alkyl substituted with 0-3 substitutents
selected from hydroxy, amino, and quaternary ammonium cations;
and
[0128] R.sub.8 is independently selected at each occurrence from
absent, hydrogen, or C.sub.1-C.sub.6alkyl substituted with 0-3
substitutents selected from hydroxy, amino, di-, or
tri-(C.sub.1-C.sub.2alkyl)amino, and quaternary ammonium cations;
and wherein at least one NR.sub.7R.sub.8 is a quaternary ammonium
cation or is substituted by a quaternary ammonium cation.
[0129] Another embodiment is a compound according to formula VI,
wherein
[0130] v is 1, 2 or 3;
[0131] w is 2;
[0132] R.sub.7 is independently selected from the group of methyl,
ethyl or propyl or isopropyl;
[0133] R.sub.8 are independently selected from methyl, ethyl,
2-(N,N-dimethylamino)ethyl, 3-(N,N-dimethylamino)propyl,
2-(N,N,N-trimethylammonium)ethyl, or
3-(N,N,N-trimethylammonium)propyl.
[0134] The chemical structures of certain preferred embodiments of
the fullerene-based photosensitizer compounds of the invention are
shown in Table 2.
TABLE-US-00001 TABLE 1 Chemical structures of the fullerence
derivatives. ##STR00004## NI1 ##STR00005## NI2 ##STR00006## NI3
##STR00007## CI1 ##STR00008## CI2 ##STR00009## CI3 ##STR00010## N1
NI = non-ionic, CI = catonic, N = nitrogenous base.
[0135] Synthetic schemes for particular functionalized fullerene PS
are further described in Examples 1-4, infra.
[0136] A pharmaceutical composition in accordance with the
invention can contain a suitable concentration of an active agent
(i.e., a functionalized fullerene compound) and may also comprise
certain other components. For example, in some embodiments,
pharmaceutical compositions of the present invention are formulated
with pharmaceutically acceptable carriers or excipients, such as
water, saline, aqueous dextrose, glycerol, or ethanol, and may also
contain auxiliary substances such as wetting or emulsifying agents,
and pH buffering agents in addition to the active agent.
[0137] The pharmaceutical composition can also comprise, or can be
applied in combination with, an optical clearing agent to enhance
the photoactive effectiveness of the functionalized fullerene
compound by allowing more effective penetration of light through
tissue. At visible and near infrared wavelengths, optical
scattering dominates over absorption and is much more significant
in reducing light penetration into biological tissues. Optical
clearing is a method for inducing a transient reduction in optical
scattering by biological tissue. Studies have demonstrated
increased light penetration depth using hyperosmotically active
chemical agents such as glycerol, propylene glycol, ethylene
glycol, DMSO, glucose or dextrose, oleic acid, linoleic acid, etc.,
which are applied to the skin or tissue. Various mechanisms for
optical clearing have been proposed. Although the mechanism of
optical clearing is still not entirely understood, it has been
inferred that hyperosmotic agents reduce random scattering
primarily by better refractive index matching, dehydration, and
collagen dissociation.
[0138] One or more optical clearing agents can be applied to tissue
simultaneously with the pharmaceutical composition, as a combined
formulation. Alternatively, one or more optical clearing agents can
be applied some time prior to the application of the pharmaceutical
composition, as a separate formulation. One or more optical
clearing agents can be applied to tissue simultaneously with the
application of light or can be applied some time prior to the
application of light.
[0139] The pharmaceutical composition can further comprise or be
used in combination with a permeation enhancer (also termed an
"absorption enhancer"), which promotes the distribution and
penetration of the functionalized fullerene compound in the tissue
being treated by PDT. Examples include but are not be limited to:
DMSO, polyethylene glycol, nonionic surfactants, ionic surfactants,
vitamin A, and steroids.
[0140] Kits
[0141] The invention also includes kits for killing microbes and/or
treating microbial infections in a subject comprising a
functionalized fullerene compound and instructions for using the
functionalized fullerene compound to kill the microbe or to treat
the infection in accordance with the methods described herein.
[0142] The kits of the invention have many applications. For
example, the kits can be used to provide reagents and therapeutics
for killing microbes in a subject or associated with inanimate
objects. The kits of the invention include instructions for the
reagents, equipment (test tubes, reaction vessels, needles,
syringes, etc.), standards for calibrating the administration,
and/or equipment provided or used to conduct the treatment. The
standard or control information can be compared to a test sample to
determine, for example, if the dosage is correct.
III. Methods of the Invention
[0143] Photodynamic therapy according to the present invention may
be utilized in the eradication of microcellular organisms, such as
bacteria, acellular organisms, and cells infected with
microcellular and acellular organisms. Acellular organisms are
defined broadly to include organisms not composed of cells, e.g.,
bodies of protoplasm made discrete by an enveloping membrane (also
referred to as a capsule, envelope, or capsid). Examples of
acellular organisms include, but are not limited to, viruses,
spores, fungi, and other virus-like agents such as viroids,
plasmids, prions, and virinos, and other infectious particles.
Acellular and microcellular organisms are collectively referred to
herein as microbes.
[0144] Structures of cellular and acellular organisms are described
as follows. Procaryotic cells are cellular organisms, including
bacteria. The component structures of procaryotic cells include
appendages, cell envelope, and protoplasm. The cell envelope
further includes the glycocalyx (capsules, slime layers), cell
wall, and cell membrane. All bacterial cells invariably have a cell
envelope, glycocalyx, cell membrane, ribosomes, and a nucleoid; the
majority have a cell wall. Although they are common to many
species, flagella, pili, fimbriae, capsules, slime layers, and
granules are not universal components of all bacteria. Organisms of
the genera Chlamydia, Rickettsia, and Ehrlichea, referred to as
obligate intracellular bacteria, are prokaryotes that differ from
most other bacteria with respect to their very small size and
obligate intracellular parasitism.
[0145] Eucaryotic cells are typical of certain microbial groups
(fungi, algae, protozoans, and helminth worms), as well as all
animal and plants. Eucaryotic cells have component structures
including appendages, surface structures, cytoplasmic membrane,
nucleus, cytoplasm, cytoskeleton, and ribosomes. The surface
structures may include glycocalyx, capsules, and slimes.
[0146] Virus particles are not cells and they neither possess
procaryotic nor eucaryotic structural qualities. Instead, they are
large, complex macromolecules, with parts made up of repeating
molecular subunits. Virus particles include component structures of
a covering and a central core. The covering includes a capsid and
in some viruses, an envelope. All viruses have a protein capsid or
shell that surrounds the nucleic acid strand. Members of 12 of the
17 families of animal viruses possess an additional covering
external to the capsid called an envelope, which is actually a
modified piece of the host's cell membrane. Viruses that lack this
envelope are considered naked nucleocapsids. Special virus-like
infectious agents include the prion (proteinacious infectious
particles) and viroids.
[0147] Photodynamic compositions of the present invention can be
utilized in the eradication of microcellular organisms, acellular
organisms, and cells infected with microcellular and acellular
organisms. Particularly preferred photodynamic compositions are
based on functionalized fullerenes as discussed in further detail
infra. Photodynamic compositions of the invention may be provided
in a liquid, gaseous, or solid form, including but not limited to
liquids, solutions, topical ointments, or powders.
[0148] In one embodiment, the present invention is directed to a
method for providing antimicrobial therapy, comprising:
[0149] administering to a subject in need thereof an effective
amount of a composition comprising a functionalized fullerene
compound, wherein the functionalized fullerene compound is any one
of the compounds as described above;
[0150] directing light onto the administered functionalized
fullerene compound to produce a cytotoxic species; and
[0151] killing microbial cells associated with or proximal to the
functionalized fullerene compound, thereby providing antimicrobial
therapy.
[0152] In another embodiment, the present invention is directed to
a method for killing a microbe, comprising:
[0153] providing a composition comprising a functionalized
fullerene compound, wherein the functionalized fullerene is any one
of the compounds as described above;
[0154] directing light onto the functionalized fullerene compound
to produce a cytotoxic species; and
[0155] killing the microbe associated with or proximal to the
functionalized fullerene compound by reaction with the cytotoxic
species. Methods of the invention permit but do not require direct
contact with the microbe of interest. Typically, production of a
cytotoxic species proximal to the microbe is sufficient to kill the
microbe.
[0156] Administration
[0157] An "effective amount" of a functionalized fullerene compound
is an amount sufficient to effect a beneficial or desired clinical
result (e.g., a photodynamic effect). An effective amount can be
administered in one or more doses. In terms of treatment, an
effective amount is an amount that is sufficient to palliate,
ameliorate, stabilize, reverse or slow the progression of a
condition caused by infection. The effective amount is generally
determined by the physician on a case-by-case basis and is within
the skill of one in the art. In accordance with certain preferred
aspects of the invention, "an effective amount of a functionalized
fullerene compound" of the invention is defined as an amount
sufficient to yield an acceptable antimicrobial effect, i.e., to
kill pathogens such as bacteria, yeast, fungus etc. with minimal
adverse effect on the cells of the mammalian subject of the PDT
treatment.
[0158] As a rule, the dosage for in vivo therapeutics will vary.
Several factors are typically taken into account when determining
an appropriate dosage. These factors include age, sex and weight of
the patient, the condition being treated, and the severity of the
condition.
[0159] Suitable dosages and formulations of functionalized
fullerene compound can be empirically determined by the
administering physician. Standard texts, such as Remington: The
Science and Practice of Pharmacy, 17th edition, Mack Publishing
Company, and the Physician's Desk Reference, each of which is
incorporated herein by reference, can be consulted to prepare
suitable compositions and doses for administration. A determination
of the appropriate dosage is within the skill of one in the art
given the parameters for use described herein.
[0160] Administration can be in any order. Typically the
functionalized fullerene compound is administered, followed by
application of light. A light source is utilized to practice
embodiments of the present invention. The light source may be laser
light source, a high intensity flash lamp, a light-emitting diode
(LED) or other illumination source as appreciated by those skilled
in the relevant arts. A broad-spectrum light source may be
utilized; however a narrow spectrum light source is one preferred
light source. The light source may be selected with reference to
the specific photosensitive material, as photosensitive materials
may have an associated range of photoactivation. In some instances
a laser light source may be used to practice the present invention.
A variety of laser light sources is currently available, and the
selection of a particular laser light source for implementing the
PDT would readily be appreciated by those skilled in the relevant
arts. A laser source may be selected with regard to the choice of
wavelength, beam diameter, exposure time and sensitivity of the
cellular and/or acellular organisms.
[0161] In preferred embodiments, the light source is utilized for a
period of time necessary to effect a photodynamic response. The
period of time for photodynamic activation of the photosensitive
material is preferably between 5 seconds and 1 hour. Even more
preferably, the period of time for light illumination is between 2
and 20 minutes.
[0162] A variety of light delivery devices may be utilized to
practice the present invention. A hand manipulable light wand or
fiber optic device may be used to illuminate tissue within a living
body. Such fiber optic devices may include a disposable fiber optic
guide provided in kit form with a solution containing a
photosensitive material. Other potential light devices for use in
accordance with the present invention include the devices disclosed
in U.S. Pat. No. 6,159,236, entitled Expandable treatment device
for photodynamic therapy and method of using same, and U.S. Pat.
No. 6,048,359, entitled Spatial orientation and light sources and
method of using same for medical diagnosis and photodynamic
therapy, both incorporated by reference in their entireties
herein.
[0163] Repeat administrations of a treatment protocol may also be
necessary or desired, including repeat administrations of
photosensitive functionalized fullerenes and light activation. The
repeat administrations may include different photosensitive
materials and/or different compounds than earlier administered.
Repeat administrations of the treatment protocol may continue for a
period of time.
[0164] In general, an effective amount of a functionalized
fullerene compound will be in the range of from about 0.1 to about
10 mg by injection or from about 5 to about 100 mg orally. Such
dosages may vary, for example, depending on whether multiple
administrations are given, tissue type and route of administration,
the condition of the individual, the desired objective and other
factors known to those of skill in the art.
[0165] Compositions of the present invention are administered by a
mode appropriate for the form of composition. Available routes of
administration include subcutaneous, intramuscular,
intraperitoneal, intradermal, oral, intranasal, intrapulmonary
(i.e., by aerosol), intravenously, intramuscularly, subcutaneously,
intracavity, intrathecally or transdermally, alone or in
combination with other pharmaceutical agents. Therapeutic
compositions of photosensitizers are often administered by
injection or by gradual perfusion, or by topical application to the
skin or mucous membrane in need of treatment.
[0166] Compositions for oral, intranasal, or topical administration
can be supplied in solid, semi-solid or liquid forms, including
tablets, capsules, powders, liquids, and suspensions. Compositions
for injection can be supplied as liquid solutions or suspensions,
as emulsions, or as solid forms suitable for dissolution or
suspension in liquid prior to injection. For administration via the
respiratory tract, a preferred composition is one that provides a
solid, powder, or liquid aerosol when used with an appropriate
aerosolizer device. Although not required, compositions are
preferably supplied in unit dosage form suitable for administration
of a precise amount. Also contemplated by this invention are
slow-release or sustained release forms, whereby a relatively
consistent level of the active compound are provided over an
extended period.
[0167] Another method of administration is intravascular, for
instance by direct injection into the blood vessels of the infected
tissue or surrounding area.
[0168] Further, it may be desirable to administer the compositions
locally to the area in need of treatment. This can be achieved, for
example, by local infusion during surgery, by injection, by means
of a catheter, or by means of an implant, said implant being of a
porous, non-porous, or gelatinous material, including membranes,
such as silastic membranes, or fibers. A suitable such membrane is
Gliadel.RTM. provided by Guilford Pharmaceuticals Inc.
[0169] In alternative embodiments, photodynamic compositions of the
invention can be used to sterilize inaminate objects which harbor
microbes, such as surfaces, liquids (e.g., bood products, bodily
fluids), surgical equipment, textile products and the like.
[0170] Microbial Infections and Associated Disorders
[0171] Infectious diseases and conditions affect a wide range of
tissues of one or more organs or organ systems of the body
including, but are not limited to, the peripheral nervous system,
hematological system, bone marrow, the central nervous system,
skin, appendix, gastrointestinal tract (including but not limited
to esophagus, duodenum, and colon), respiratory/pulmonary system
(including but not limited to lung, nose, pharynx, larynx), eye,
genito-reproductive system, gums, liver/biliary ductal system,
renal system (including but not limited to kidneys, urinary tract,
bladder), connective tissue (including but not limited to joints,
cartilage), cardiovascular system, muscle, heart, spleen, breast,
lymphatic system, ear, endocrine/exocrine system (including but not
limited to lacrimal glands, salivary glands, thyroid gland,
pancreas), and bone/skeletal system.
[0172] Both gram negative and gram positive bacteria can be killed
by the methods of the invention. Such gram positive bacteria
include, but are not limited to, Pasteurella species, Staphylococci
species, and Streptococcus species, including S. aureus. Gram
negative bacteria include, but are not limited to, Escherichia
coli, Pseudomonas species, and Salmonella species.
[0173] Specific examples of infectious bacteria susceptible to
killing by the PDT methods and compositions of the invention
include but are not limited to: Helicobacter pyloris, Borelia
burgdorferi, Legionella pneumophilia, Mycobacteria sps (e.g. M.
tuberculosis, M. avium, M. intracellulare, M. kansaii, M.
gordonae), Staphylococcus aureus, Neisseria gonorrhoeae, Neisseria
meningitidis, Listeria monocytogenes, Streptococcus pyogenes (Group
A Streptococcus), Streptococcus agalactiae (Group B Streptococcus),
Streptococcus (viridans group), Streptococcus faecalis,
Streptococcus bovis, Streptococcus (anaerobic sps.), Streptococcus
pneumoniae, pathogenic Campylobacter sp., Enterococcus sp.,
Haemophilus influenzae, Bacillus anthracis, corynebacterium
diphtheriae, corynebacterium sp., Erysipelothrix rhusiopathiae,
Clostridium perfringens, Clostridium tetani, Enterobacter
aerogenes, Klebsiella pneumoniae, Pasturella multocida, Bacteroides
sp., Fusobacterium nucleatum, Streptobacillus moniliformis,
Treponema pallidium, Treponema pertenue, Leptospira, Rickettsia,
and Actinomyces israelli.
[0174] Fungi can also be killed by antimicrobial PDT in accordance
with the invention. Examples of fungi include Cryptococcus
neoformans, Histoplasma capsulatum, Coccidioides immitis,
Blastomyces dermatitidis, Chlamydia trachomatis, and Candida
albicans.
[0175] Other infectious organisms that can be targeted (e.g.,
protists) include Plasmodium spp. such as Plasmodium falciparum,
Plasmodium malariae, Plasmodium ovale, and Plasmodium vivax and
Toxoplasma gondii. Blood-borne and/or tissues parasites include
Plasmodium spp., Babesia microti, Babesia divergens, Leishmania
tropica, Leishmania spp., Leishmania braziliensis, Leishmania
donovani, Trypanosoma gambiense and Trypanosoma rhodesiense
(African sleeping sickness), Trypanosoma cruzi (Chagas' disease),
and Toxoplasma gondii.
[0176] Other medically relevant microorganisms have been described
extensively in the literature, e.g., see C. G. A Thomas, Medical
Microbiology, Bailliere Tindall, Great Britain 1983, the entire
contents of which is hereby incorporated by reference.
[0177] The invention will be more fully understood by reference to
the following examples. These examples, however, are merely
intended to illustrate the embodiments of the invention and are not
to be construed to limit the scope of the invention.
EXAMPLES
Example 1
Synthesis of Nonionic Fullerene Derivatives
[0178] This Example describes the synthesis of a series of
functionalized nonionic C.sub.60 fullerene derivatives with one,
two, or three polar diserinol groups (e.g., NI1, NI2, NI3, as shown
in Table 2, supra).
[0179] This synthesis was carried out as described below and shown
in Scheme 1.
##STR00011##
[0180] Serinol (2.05 equivalents) and diethylmalonate (1
equivalent) were reacted at 200.degree. C. for 45 minutes in an
open tube. Then acetic anhydride (4.1 equivalents) and pyridine
(4.1 equivalents) were added and stirred for 18 hours at room
temperature. The product termed MSA thus obtained was
recrystallized using a mixture of hexane and ethyl acetate.
[0181] Purified C.sub.60 (200 mg, 0.28 mmol) was dissolved in
toluene (250 ml) by sonicating for 10 minutes and nitrogen was
purged for 30 minutes. Then CB.sub.4 (46.1 mg, 0.14 mmol) as a
solid directly, MSA (58.2 mg. 0.14 mmol) in acetone (3 ml), and
1,8-Diazabicyclo[5.4.0]undec-7-ene (31.7 mg, 0.21 mmol) in toluene
(5 ml) were added. The reaction mixture as stirred at room
temperature for 4.5 hours under nitrogen atmosphere. Solvents were
removed on a rotavap under vacuum. The product was dissolved in a
minimum amount of chloroform and loaded onto a silica gel column (1
in.times.9 in) and eluted with dichloromethane containing 0-2%
methanol to collect pure N11, N12 and N13. The compounds were
characterized by matrix assisted laser desorption ionization mass
spectrometry (MALDI-MS) as follows: N11--calculated mass 1137.02
and observed mass 1137.56; N12--calculated mass 1553.40 and
observed mass 1153.77; N13--calculated mass 1969.78 and observed
mass 1970.26.
[0182] NMR data were obtained for C.sub.60(MSA)-protected NI1:
.sup.1H NMR. (400 MHz, CDCl.sub.3, TMS ref.) .delta. (ppm) 2.10 (s,
12H, CH.sub.3), 4.34-4.41 (m, 8H, CH.sub.2), 4.68-4.72 (m, 2H, CH),
7.37 (br d, J=56.4 Hz, 2H, NH).
[0183] Deprotection of --OH groups was achieved by treating NI1-3
with an excess of potassium carbonate in methanol and deionized
water at room temperature for 90 minutes. Potassium ions were
removed by adding strong cation exchange resin (Biorad AG MP-50W,
treated with 1M HCl) to the reaction mixture until the pH reached
7. The solution was filtered and solvents were removed on a rotavap
to obtain pure NI1, NI2, and NI3.
Example 2
Synthesis of Cationic Fullerene Derivatives
[0184] This Example describes a scheme for synthesis of cationic
fullerene derivatives (e.g., CI1, CI2, and CI3, as illustrated in
Table 2, supra).
[0185] The synthesis of compounds CI1-3 was carried out using
published procedures (Wharton, T., Kini, V. U., Mortis, R. A., and
Wilson, L. J. (2001). New non-ionic, highly water-soluble
derivatives of C60 designed for biological compatibility.
Tetrahedron Lett. 42, 5159-5162, Wharton, T., and Wilson, L. J.
(2002). Highly-iodinated fullerene as a contrast agent for X-ray
imaging. Bioorg. Med. Chem. 10, 3545-3554, Maggini, M., Scorrano,
G., and Prato, M. (1993). Addition of azomethine ylides to C60:
synthesis, characterization, and functionalization of fullerene
pyrrolidines. J. Am. Chem. Soc. 115, 9798-9799 and Cassell, A. M.,
Scrivens, W. A., and Tour, J. M. (1998). Assembly of DNA/fullerene
hybrid materials. Angew. Chem. Int. Ed. Engl. 37, 1528-1530.) with
modifications as described below, and illustrated in Scheme 2.
##STR00012##
[0186] Purified C.sub.60 (200 mg, 0.28 mmol) was dissolved in
toluene (260 ml) by sonicating for 5 minutes. To this solution were
added sarcosine (50.8 mg, 0.57 mmol) and paraformaldehyde (40.9 mg,
1.36 mmol) for CI1; sarcosine (63.5 mg, 0.71 mmol) and
paraformaldehyde (35.79 mg, 1.19 mmol) for CU; or sarcosine (88.9
mg, 1.0 mmol) and paraformaldehyde (46.0 mg, 1.53 mmol) for CI3, as
solids directly. The reaction mixture was refluxed for 2 hours for
CI1; overnight for CU; and 3 hours for CI3. Solvents were removed
on a rotavap under vacuum.
[0187] The product was dissolved in a minimum amount of toluene and
loaded onto a silica gel column (1 in.times.9 in) packed with
toluene and eluted with toluene containing 0-5% acetone to collect
pure CI1, CI2, or CI3, with yields of 30-40% purity. The purity of
the compounds in terms of mono-, bis-, and tris-substitutions was
confirmed by thin layer chromatography (TLC).
[0188] Methylation of CI1, CI2, or CI3 was carried out by
dissolving the compounds in a large excess of methyl iodide (1 ml
per 20 mg CI1-3) and stirring for 48-72 hours at room temperature
(or 7 days in the case of CI3). Pure methylated products CI1, CI2,
or CI3 were precipitated by adding hexanes, and the precipitates
were collected, washed with toluene and dichloromethane, and dried.
The compounds were characterized by electrospray mass spectrometry
(ES-MS) as follows: CH-- calculated mass 792.08 and observed mass
792.04; CI2-calculated mass 864.16 and observed mass 432.05
(M.sup.2+); and CI3-calculated mass 936.24 and observed mass 312.08
(M.sup.3+).
[0189] NMR data were obtained for CH as follows: .sup.1H NMR (400
MHz, 2:3 CDCl.sub.3:DMSO-d.sub.6, TMS ref.) .delta. (ppm) 4.08 (s,
6H, CH.sub.3), 5.72 (s, 4H, CH.sub.2). Referring to FIG. 1,
UV-visible absorption spectra of the compounds were recorded in 1:9
DMSO:water at a concentration of 10 mM. More particularly, FIG. 1
shows UV-Visible absorption spectra of CI1-3 and toluidine blue O
(TBO) at 10 .mu.M concentration in 1:9 DMSO:water.
Example 3
Synthesis of Nitrogenous Fullerene Derivatives
[0190] This Example describes a scheme for synthesis of nitrogenous
fullerene derivatives (e.g., N1 as illustrated in Table 2,
supra).
[0191] The synthesis of compound N1 was carried out as described
below and illustrated in Scheme 3.
##STR00013##
[0192] Purified C.sub.60 (360 mg, 0.5 mmol) was dissolved in
toluene (180 ml) by sonicating for 30 minutes and nitrogen was
purged for 15 minutes. Then CBr.sub.4 (83 mg, 0.25 mmol) as solid
directly, 1,4,8,11-tetraazacyclotetradecane-5,7-dione (57 mg, 0.25
mmol) in methanol (1 ml) and toluene (9 ml), and DBU (57 mg, 0.375
mmol) in toluene (10 ml) were added. The reaction mixture was
stirred at room temperature for 24 hours under nitrogen atmosphere.
The product N1 was precipitated and filtered, washed with toluene
and dried.
[0193] Methylation of N1 was carried out by suspending in a large
excess of methyl iodide and stirring for 72 hours at room
temperature. The methylated product N1 was precipitated and which
was collected and washed with toluene and dichloromethane, and
dried.
Example 4
Synthesis of Cationic CI4 and CI5 Fullerene Derivatives
[0194] The synthesis of cationic compounds CI4 and CI5 was carried
out as described below, and illustrated in Scheme 4.
##STR00014##
[0195] For synthesis of diquat-21,
(CH.sub.3).sub.2N(CH.sub.2).sub.2NH.sub.2 (2.05 equivalents) and
dimethylmalonate (1 equivalent) were dissolved in toluene and
reacted at 100.degree. C. for 2 hours. The solvents were removed on
a rotavap and added hexanes. The product was cooled in a
refrigerator overnight and filtered. The product obtained as a pink
waxy solid.
[0196] For synthesis of diquat-31,
(CH.sub.3).sub.2N(CH.sub.2).sub.2NH.sub.2 (2.05 equivalents) and
dimethylmalonate (1 equivalent) were reacted at 120.degree. C. for
2 hours. The solvents were removed on a rotavap. The product was
obtained as a high viscous pale yellow liquid after vacuum drying
for 60 hours at 20.degree. C.
[0197] For synthesis of CI4 and CI5, purified C.sub.60 (360 mg, 0.5
mmol) was dissolved in toluene (180 ml) by sonicating for 15
minutes and nitrogen was purged for 15 minutes. Then CBr.sub.4 (83
mg, 0.25 mmol) as a solid directly, diquat (0.25 mmol) in toluene
(5 ml), toluene (9 ml), and DBU (57 mg, 0.375 mmol) in toluene (10
ml) were added. The reaction mixture was stirred at room
temperature for 4 hours under nitrogen atmosphere. The product
C.sub.60-diquat was precipitated and filtered, washed with toluene,
and dried.
[0198] Methylation of C.sub.60-diquat was carried out by dissolving
the compounds in a large excess of methyl iodide and stirring for
72 hours at room temperature. The methylated product was
precipitated and collected, washed with toluene and
dichloromethane, and dried. The compounds were characterized by
electrospray mass spectrometry (ES-MS) as follows. CI4-calculated
mass 993.03 and observed mass 496.09 (M); CI5-calculated mass
1021.08 and observed mass 510.11 (M.sup.2+).
Example 5
Absorption Spectra of Derivatized Fullerenes
[0199] This Example describes one aspect of the characterization
(determination of absorption spectra) of functionalized fullerenes
NI1-3 and CI1-3 of the invention.
[0200] Functionalized fullerenes NI1-3 and CI1-3 were prepared as
described above. There are eight possible regioisomers of the
bis-substituted fullerenes and 46 possible regioisomers of the
tris-substituted fullerenes. It was not practical to separate these
mixtures of regioisomers into individual pure compounds; therefore,
NI2 and NI3, and CI2 and CI3 were studied as mixtures of
regioisomers. The identity of the compounds, however, was confirmed
by mass spectrometry, giving molecular ions identical to those
calculated. The proton and C13 NMR spectra of the immediate
precursors of BF1 and BF4 have been reported (Wharton, T., Kini, V.
U., Mortis, R. A., and Wilson, L. J. (2001). New non-ionic, highly
water-soluble derivatives of C60 designed for biological
compatibility. Tetrahedron Lett. 42, 5159-5162, Maggini, M.,
Scorrano, G., and Prato, M. (1993). Addition of azomethine ylides
to C60: synthesis, characterization, and functionalization of
fullerene pyrrolidines. J. Am. Chem. Soc. 115, 9798-9799).
[0201] The absorption spectra of CI1-3 and TBO, all at the same
concentration of 10 .mu.M in DMSO:water (i.e., 1:9), are shown in
FIG. 1. The overall extinction coefficients of the fullerenes were
in the following order: CI1>CI2>CI3. The shoulder in the UVA
range moved from 340 nm for CI1 to 310 nm for CI2 and disappeared
altogether for CI3 (FIG. 1).
Example 6
Distribution Coefficients of Derivatized Fullerenes
[0202] This Example describes studies performed to determine the
distribution coefficients of fullerenes NI1-3 and CI1-3 of the
invention.
[0203] Each compound was dissolved in a minimum amount of DMSO: CI1
(0.9 mg in 200 .mu.l), CI2 (5.3 mg in 200 .mu.l), CI3 (5.4 mg in
200 .mu.l). Ten ml of DI water and 10 ml of 1-octanol were added in
each compound and vigorously shaken for 2 min. and the vials of the
compounds were settled down overnight. The phases were separated
and UV-spectra of each phase were analyzed. Distribution
coefficient of each compound was determined using absorbance of
aqueous phases and organic phases at 330 nm.
[0204] The results of these determinations are presented in Table
2. Referring to Table 2, it will be appreciated that the
hydrophilic character of fullerene derivatives increases with
increasing number of cationic functional groups, whereas
hydrophilicity decreases with increasing number of serinol
groups.
TABLE-US-00002 TABLE 2 Octanol-water partition constants (K.sub.ow)
of Fullerene Derivatives NI1-3 and CI1-3. Compound NI1 NI2 NI3 CI1
CI2 CI3 K.sub.ow 0.025 0.032 0.078 140.80 1.28 0.37 LogK.sub.ow
-1.61 -1.49 -1.11 2.15 0.11 -0.43
Example 7
Determining Antimicrobial Properties of Derivatized Fullerenes
[0205] This Example describes exemplary materials and methods
useful for testing antimicrobial activity of derivatized fullerenes
prepared in accordance with the invention.
[0206] 1. Microbial Strains and Culture Conditions
[0207] Staphylococcus aureus (ATCC #35556), Escherichia coli (ATCC
#25922), and Pseudomonas aeruginosa (ATCC #BAA-47; PAO1) were
cultured in brain-heart infusion (BHI) broth (Difco, BD Diagnostic
Systems, Sparks, Md.) at 37.degree. C. in aerobic conditions in a
shaker at 150 rpm. Candida albicans (ATCC #18804) is grown in YM
broth (Difco). Exponential cultures obtained by reculturing
stationary overnight precultures were used for experiments.
[0208] E. coli, P. aeruginosa, and S. aureus are grown in fresh
medium for approximately 1 hr to a density of about 10.sup.8
cells/ml; the OD values at 650 nm are 0.6, 0.8, and 0.8,
respectively. C. albicans is grown for approximately 4 hr to an
approximate density of 10.sup.8 cells/ml, corresponding to an OD of
6 at 650 nm (measured at 10-fold dilution). Cells were used for
experiments in the mid-log growth phase.
[0209] 2. Photosensitizers and Light Sources
[0210] Toluidine blue O (TBO), a common PS, is available from
commercial sources, e.g., from Sigma (St. Louis, Mo.) and was
dissolved in water to give a 1 mM stock solution that is stored in
the dark at 4.degree. C. for a maximum of 2 weeks.
[0211] A noncoherent lamp with filtered liquid light guides, e.g.,
a LumaCare.TM. LC122 lamp (MBG Technologies, Inc., Newport Beach,
Calif.) was used to provide illumination of PS. More specifically,
for illumination of fullerenes, a broad-band white light band pass
filter (400-700 nm) was used, whereas for TBO, a band pass filter
at 620-650 nm was used. The lamp was adjusted to give a uniform
spot of about 4 cm diameter with an irradiance of 200 mW/cm.sup.2,
as measured with a power meter, e.g., a model DMM 199 meter with
201 Standard head (Coherent, Santa Clara, Calif.).
[0212] 3. Photodynamic Inactivation Studies
[0213] Typically, derivatized fullerenes prepared as described
above are dissolved in DMSO to provide stock solutions having final
concentrations of about 5 mM. Compound CI1 is poorly soluble;
accordingly a concentration of 2.7 mM can be used. All stock
solutions were stored in the dark at room temperature.
[0214] In some experiments, for example in studies described in
Examples below, suspensions of S. aureus cells (10.sup.8 per ml)
were incubated with derivatized fullerenes such as NI-1-3 and CI1-3
at a concentration of 100 .mu.M in PBS at room temperature for 10
min. In other experiments, the bacterial suspension was centrifuged
(4000.times.g for 10 min) after incubation and resuspended in fresh
PBS before illumination; the latter procedure was referred to
herein as a "wash." K coli, P. aeruginosa, and C. albicans were
used at concentrations of about 10.sup.8 cells per ml.
[0215] Illumination was carried out from above on microbial cell
suspensions in wells of a 24-well plate. Aliquots were removed at
times corresponding to the delivery of calculated fluences of
light, and were serially diluted in PBS and streaked on square BHI
or YM agar plates according to the method of Jett et al. (Jett, B.
D., Hatter, K. L., Huycke, M. M., and Gilmore, M. S. (1997).
Simplified agar plate method for quantifying viable bacteria.
Biotechniques 23, 648-650). Survival fractions were calculated with
reference to cells incubated in PBS alone. Values on killing curves
at 0 J/cm.sup.2 represent the dark toxicity of the fullerenes or
other PS tested. Control cells were treated with light and no PS
and also analyzed for viability.
[0216] 4. Mammalian Cell Culture Experiments
[0217] Experiments to test the effect of functionalized fullerenes
on mammalian cells can be performed in a suitable mammalian cell
line. One such cell line is the L929 murine fibroblast line (ATCC
#CCL1), a spontaneously transformed immortalized cell line
established from the normal subcutaneous areolar and adipose tissue
of a male C3H/An mouse (Earle, W. R., Schilling, E. L., Stark, T.
H., Straus, N. P., Brown, M. F., and Shelton, E. (1943). Production
of malignancy in vitro. IV. The mouse fibroblast cultures and
changes seen in the living cells. J. Natl. Cancer Inst. 4,
165-212). In a suitable assay, these cells were cultured in
Dulbecco's modified Eagle's medium (Sigma) at 37.degree. C. in a
humidified atmosphere containing 5% CO2. The medium is preferably
modified by using 4 mM L-glutamine (containing 1.5 g/l sodium
bicarbonate and 4.5 g/l glucose), 10% fetal bovine serum (FBS), 100
U/ml penicillin, and 100 mg/ml streptomycin. Cells were plated in
96-well cell culture plates, at a density of about 300 cells/well,
and are allowed to attach for 24 hr.
[0218] Fullerenes and other PS, e.g., TBO, were added at a
concentration of about 10 .mu.M in 200 .mu.l complete medium per
well. After 10 min, fresh medium is added, and the cultures were
illuminated with white light (for fullerenes) or with red light
(for TBO). At the completion of the illumination period, cells were
returned to the incubator for 24 hr. Cell viability can be
determined, for example, by using the MTT-microculture tetrazolium
assay, a method of assessing cellular response to PDT (Merlin, J.
L., Azzi, S., Lignon, D., Ramacci, C., Zeghari, N., and Guillemin,
F. (1992). MIT assays allow quick and reliable measurement of the
response of human tumour cells to photodynamic therapy. Eur. J.
Cancer 28A, 1452-1458. This assay involves the reduction of a
colorless substrate, i.e.,
3-[4,5-Dimethylthiazol-2-yl]-diphenyltetrazolium bromide (Sigma,
St. Louis, Mich.) to an insoluble dark-blue formazan product which
is formed in proportion to the amount of succinate dehydrogenase
activity in the mitochondria of living cells. After incubation with
MTT for periods ranging from 4 to 8 hr, the medium was aspirated
from each well, and 100 ml DMSO is added. The absorbance at 570 nm
was read by a microplate reader, e.g., a Spectra Max.TM. 340 PC
(Molecular Devices, Sunnyvale, Calif.). The fraction of cells
surviving was calculated by dividing the mean absorbances of
formazan produced from PDT-treated cells by the mean absorbances
from dark controls incubated with PS and kept at room temperature
for periods of time equal to the irradiation times.
[0219] 5. Statistics
[0220] Preferably values are calculated and expressed as means and
standard errors of at least six independent wells. Differences
between killing curves were tested for significance at the highest
comparable fluence by an unpaired two-tailed Student's t test,
assuming equal or unequal variation in the standard deviations, as
appropriate. P values of less than 0.05 were considered
significant.
Example 8
Studies of Derivatized Fullerenes as Antimicrobial
Photosensitizers
[0221] This Example describes a series of experiments performed to
test the ability of certain embodiments of derivatized fullerenes,
prepared as described above, to act as effective antimicrobial PS
against a range of bacterial strains and yeast.
[0222] Four microbial species were used in this study. Briefly,
Escherichia coli was purchased from ATCC (ATCC #25922), Pseudomonas
aeruginosa, two clinical isolates, were obtained; UCBP PA 14 from
L. Rahme (Massachusetts General Hospital) and PA 767K from Kim
Lewis (Tufts University), slime deficient mutant of Staphylococcus
aureus was obtained from Gerald B Pier (Charming Laboratories).
Yeast strain Candida albicans was purchased from ATCC (ATCC
#18804). Exponential cultures obtained by reculturing stationary
overnight cultures were used for all experiments. Bacteria were
grown at 37.degree. C. in BHI broth to a cell density of 10.sup.8
cells per mL. C. albicans was grown 37.degree. C. in YM medium to
10 cells/mL.
[0223] Six fullerene derivatives (NI1-3 and CI1-3) were evaluated.
Five of them were dissolved in DMSO to obtain a stock solutions of
5 .mu.M, and one (CI1) was poorly soluble and therefore the
concentration of the stock solution was 2.7 .mu.M. Concentration of
fullerene derivatives used in the experiments varied between 1 and
100 .mu.M.
[0224] Fullerene derivatives were mixed with microbial suspensions
and incubated in the dark at room temperature for 10 minutes.
Excess fullerenes were either washed out of, or left in, the
suspensions before illumination. Aliquots of 100 .mu.L were placed
on three well hanging drop slides and illuminated with either a
405-nm laser (Nichia Chemical Industries) at an irradiance of 100
mW/cm.sup.2, or with white light at room temperature. White light
was 400-700 nm Gaussian distribution from LumaCare.TM. lamp with
filtered liquid light guide, as described above.
[0225] During illumination, aliquots of 20 .mu.l, were taken to
determine the colony-forming units. The contents of the wells were
mixed before sampling. The aliquots were serially diluted 10-fold
in PBS without Ca.sup.++/Mg.sup.++ to give dilutions of
10.sup.-1-10.sup.-6 times the original concentrations, and were
streaked horizontally on square BHI agar plates. Plates were
incubated at 37.degree. C. overnight. Colonies were counted and
survival fraction was determined as percent of survival compared to
control. Microbial suspensions incubated with fullerene derivatives
in DMSO in the absence of light and bacteria illuminated in the
absence of fullerene derivatives were used as controls. Fullerene
derivatives and DMSO were not toxic for microorganisms in the dark
and light alone did not cause cell destruction.
[0226] The ability of fullerenes NI1-3 and CI1-3 to mediate
photodynamic inhibition (PDI) against the gram positive bacterium
S. aureus was initially tested at 100 .mu.M concentration for 10
minutes at light intensities (405 nm light) ranging from 0-200
J/cm.sup.2. Referring now to FIG. 2, it is seen that all fullerene
derivatives at this concentration have significant activity in
mediating PDI of S. aureus; however CI2 is the most potent.
[0227] Next, both the drug dose and the light dose were reduced in
an assay using CI2 to determine the lowest effective PDT parameters
for killing S. aureus. As can be seen from FIG. 3, substantial
killing (6 logs) was achieved at 1-.mu.M concentration of the
functionalized fullerene exposed to light intensity of 8 J/cm.sup.2
making CU approximately 1000 times more potent than the other
fullerene derivatives.
[0228] The Gram-positive slime deficient S. aureus bacterium is
very susceptible to PDT. The antimicrobial activity of CI2 was
further defined by exploring its photokilling abilities with other
pathogenic microorganisms including the Gram-negative bacterium E.
coli, and the challenging P. aeruginosa, as well as the eukaryotic
fungus C. albicans. Referring now to FIG. 4, it is seen that 10-20
.mu.M concentration and 16 J/cm.sup.2 of 405-nm light is sufficient
to achieve high levels of killing of each of these pathogens. A
comparison of the killing obtained by illuminating with and without
a wash of the cells revealed more killing after a wash, as shown
for P. aeruginosa in FIG. 5. This implies that the CI2 molecule is
able to bind and penetrate the cell walls of these pathogens,
rather than merely generating cytotoxic species outside of the
cells.
Example 9
Selectivity of Cationic Fullerenes for Broad Spectrum Antimicrobial
Photoinactivation While Sparing Mammalian Cells
[0229] This Example describes PDI assays demonstrating that
functionalized fullerenes in accordance with the present invention
can effectively kill bacteria and yeast strains while sparing
mammalian cells.
[0230] Mammalian cells used in these studies are a transformed
mouse fibroblast cell line (L929) described above. Microbes studied
included bacterial strains S. aureus, E. coli, and P. aeruginosa,
and yeast strain C. albicans. All microbes and cells were cultured
as described in Example 7 above. The concentration of fullerene
derivatives was 10 .mu.M for all cells and microbes except S
aureus, for which the concentration was reduced to 1 .mu.M.
Incubation time was 10 minutes in all cases. The survival fraction
at 0 J/cm.sup.2 gives "dark" toxicity of fullerene. It should be
noted that MTT assay used for mammalian cells does not have as big
a dynamic range as CPU assay used for microbes.
[0231] FIGS. 6 to 8 show the PDT activity of CI1, CI2, and CI3
cationic fullerenes against various microorganisms at fluences of
0-15 J/Cm.sup.2 of white light. FIGS. 9 to 11 illustrate results
with CI1, CI2, and CI3 at fluences uo to 50 J/Cm.sup.2 of white
light. As can be seen in these data graphs, mammalian cells are not
killed by this regimen whereas the microbes are effectively killed.
Further studies demonstrating selectivity of derivatized fullerenes
in accordance with the invention for killing microbial, as opposed
to mammalian, cells, are described in Examples 11 and 15 below.
Example 10
Comparison of Cationic Fullerenes with Toluidine blue O (TBO) for
Broad Spectrum Antimicrobial Photoinactivation
[0232] This Example describes studies undertaken to compare the
killing efficiency of cationic fullerenes of the present invention
with that of toluidine blue O (TBO), a well-known antimicrobial
photosensitizing agent.
[0233] TBO is used extensively for photodynamic inhibition (PDI),
as discussed in many reported studies. For example, TBO has been
investigated for use in treating the gum disease gingivitis (caused
by bacteria) and other pathogenic oral bacteria, E. coli O157:H7
and Listeria monocytogenes, buccal mucosa (in rats), multi-drug
resistant malignancies, Helicobacter pylori, and human leukaemic T
cells. Thus, a comparison of fullerene-based PS in accordance with
the invention with TBO provides a measure of efficacy as compared
with that of a well-used and understood PS. This compound was
chosen not only because it is widely used in PDI studies, but also
because it is cationic (like CI1-3) and accordingly should
associate with the negatively charged cell walls of bacterial
pathogens.
[0234] In order to obtain a comparison of the CI1-3
photosensitizers with TBO, PDI experiments were conducted with
CI1-3 compounds and TBO at identical concentrations. In these
studies, the concentration of fullerene derivatives or TBO was 10
.mu.M for all microbes except S aureus where concentration was
reduced to 1 .mu.M. Incubation time was 10 minutes in all cases.
Survival fraction at 0 J/cm.sup.2 gives "dark" toxicity of
fullerene derivatives or TBO. Broad band white light was used for
fullerene derivatives (400-700 nm) and 620-650 nm red light was
used for TBO.
[0235] Referring to FIGS. 12-15, it can be seen that under the same
conditions, all three of the tested cationic compounds, i.e.,
CI1-3, exhibited PDI of the four microorganisms investigated (E.
coli, P. aeuriginosa, S. aureus, and C. albicans) that is far
superior to that of TBO. See also Example 14, infra.
Example 11
Mammalian Cell Toxicity of Fullerenes Relative to TBO
[0236] This Example demonstrates that fullerene-based PS agents in
accordance with the present invention are less toxic at high light
levels than is the commonly used photosensitizer toluidine blue O
(TBO).
[0237] At the fluences used to carry out PDI of pathogens, the
C.sub.1-3 cationic PS exhibited negligible toxicity to L929
mammalian cells (see, for example, FIGS. 9-11). Nevertheless, the
toxicity at high fluences was compared with that of TBO, which is
one of the few PS compounds that also exhibits relatively low
mammalian toxicity.
[0238] Remarkably, as shown in FIG. 16, results of this study show
that CI2 is considerably less toxic to mammalian cells at high
fluences than TBO, making it superior not only in PDI but also in
its selectivity for microbes. See also Example 15, infra.
Example 12
Screening of Derivatized Fullerenes as Antimicrobial PS
[0239] This Example describes further characterization of
embodiments of functionalized (derivatized) fullerenes in
accordance with the present invention with respect to their ability
to mediate photodynamic inhibition (PDI) against gram-negative and
gram-positive bacteria.
[0240] Derivatized fullerenes NI1-3 and CI1-3 were prepared as
described above and used in assays to assess their potential to
mediate PDI against the gram-positive bacterium S. aureus after 10
min incubations with 100 .mu.M concentrations of fullerenes under
conditions of "no wash," or "wash," i.e., with or without
centrifugation to remove the bacteria after the 10 minute
incubation and resuspension in fresh PBS before illumination, as
described above.
[0241] The results of these experiments are shown in FIG. 17A. More
particularly, FIG. 17A shows results of experiments in which S.
aureus (10.sup.8 cells per ml) were incubated as described, then
followed (or not followed) by a wash, i.e., centrifugation and
resuspension as described, and subsequent illumination with 400-700
nm light at an irradiance of 200 mW/cm.sup.2. Aliquots were removed
from the suspension after the various fluences of light (0-120
J/cm.sup.2) had been delivered and the CFU had been determined.
Values shown in FIG. 17 are means of six independent experiments
and bars are SEM. Single asterisks (*) denote p<0.05; double
asterisks (**) denote p<0.01 by two-tailed unpaired t test.
[0242] From the results of "no wash" experiments, it was seen that
compounds CI2 and CI3 were completely dark toxic to S. aureus and
gave zero colonies or >99.9999% killing, regardless of the
amount of light delivered. Compound CI1 showed significant dark
toxicity (99%; e.g., see first point of curve with squares in FIG.
18). Referring again to FIG. 17, by contrast, compounds NI1-3 show
only minor dark toxicity toward S. aureus (i.e., 60%-80%; see FIG.
17A).
[0243] When relatively large fluences of broad-band white light
were delivered to bacterial suspensions still containing the
fullerenes, a fluence-dependent loss of viability of S. aureus
ranging from 2-4 logs of killing was observed, as shown in FIG. 17A
(closed symbols). Compounds NI1-3 displayed significant differences
in effectiveness between members of the series. Their effectiveness
was NI3>NI2>NI1, and the differences in the survival fraction
were significant (p<0.05) at the two highest fluences (80 and
120 J/cm.sup.2) (FIG. 17A).
[0244] In order to test whether the fullerenes actually bound to
the bacterial cells, PDI was compared with and without a wash as
described. As can be seen by comparing curves with open and closed
symbols in FIG. 17A, there was no difference in killing with and
without a wash, indicating that the fullerenes bound to the
bacteria, and could not easily be washed out.
[0245] Compounds NI1-3 were also tested under the same conditions
(100 .mu.M incubation for 10 min without wash) with the
gram-negative bacteria E. coli. Referring to FIG. 17B, it is seen
that there was no dark toxicity and only a very small amount of
light-mediated killing (less than 90%). NH was significantly less
effective than NI2-3 (p<0.05).
Example 13
Cationic Fullerenes Mediate Photodynamic Inactivation of Three
Microbial Classes
[0246] Compound CI1 showed significant dark toxicity toward S.
aureus at 100 .mu.M; therefore the concentration of CI1 was
decreased in the incubation mixture in a step-wise manner to 50,
25, 10, and 1 M. These experiments, carried out with a wash, are
illustrated in FIG. 18. More particularly, FIG. 18 shows PDI of S.
aureus under the specified conditions, followed by a wash and
illumination with white light. Values in the graphs represent means
of six independent experiments and the bars are SEM.
[0247] As is shown in FIG. 18, the dark toxicity decreased as the
concentration was decreased until, at 10 and 1 .mu.M, dark toxicity
was nonexistent. When PDI experiments were carried out after
incubation of S. aureus with these concentrations of CI1, a
fluence-dependent loss of viability was observed in all cases with
comparatively low doses of light (4-8 J/cm.sup.2). Remarkably, the
PDI killing curves were not significantly different among the
different concentrations (compare the slopes of curves in FIG. 18).
The difference between the curves was solely in the survival
fraction at 0 J/cm.sup.2, i.e., the dark toxicity.
[0248] Since an initial screening experiment had suggested that the
bis- and tris-cationic fullerenes CI2 and CI3 would be more potent
than CH (higher dark toxicity), they were tested against S. aureus
at 1 .mu.M with a wash. These results are shown in FIG. 19A.
[0249] More particularly, and for comparison, FIGS. 19A-B and 20A-B
show PDI of various bacteria and yeast tested with cationic
fullerene derivatives CI1-3 as follows--(FIG. 19A): S. aureus
incubated with a 1 .mu.M concentration of CI1-3; (FIGS. 19B, 20A,
B): E. coli, (19B), C. albicans (20A), and P. aeruginosa (20B), all
at 10.sup.8 cells per ml, incubated with CI1-3 at 10 .mu.M
concentrations for 10 min, followed by a wash and illumination with
white light. Values are means of six independent experiments, and
bars are SEM. *p<0.05, **p<0.01, ***p<0.001, by two-tailed
unpaired t test.
[0250] As shown in FIG. 19A, compounds CI2 and CI3 were highly
active, with 2 and 1 J/cm.sup.2 of light being sufficient to kill
4-5 logs, respectively. All three killing curves were significantly
different (p<0.01).
[0251] As it is known that gram-positive bacteria are much more
susceptible to PDI than gram-negative bacteria or fungal species
(Malik, Z., Ladan, H., and Nitzan, Y. (1992). Photodynamic
inactivation of Gram-negative bacteria: problems and possible
solutions. J. Photochem. Photobiol. B 14, 262-266, Nitzan, Y.,
Gutterman, M., Malik, Z., and Ehrenberg, B. (1992). Inactivation of
Gram-negative bacteria by photosensitized porphyrins. Photochem.
Photobiol. 55, 89-96), the cationic fullerenes CI1-3 were tested
against other microorganisms, at a concentration of 10 .mu.M with a
wash. FIG. 19B shows the light-mediated killing of gram-negative E.
coli with the three cationic fullerenes. It can be seen that CI2
and CI3 were highly effective, with 2 J/cm.sup.2 giving 4 and 6
logs of killing, respectively. CI1 was much less potent, requiring
8 J/cm.sup.2 to give 3 logs of killing (p<0.001). There was only
minimal dark toxicity.
[0252] Referring now to FIG. 20A, it is shown that effective
killing was also achieved against the yeast C. albicans, in which
CI3 was slightly more effective than CI2 and both were much better
than CI1 (p<0.001).
[0253] As shown in FIG. 20B, the gram-negative bacterium P.
aeruginosa was more resistant than the other organisms tested. The
maximum light dose delivered was doubled to 16 J/cm.sup.2 in an
effort to increase killing, but this had only a minimal effect. CI2
and CI3 were able to kill 3-5 logs, whereas CH gave 2 logs of
killing of P. aeruginosa.
Example 14
Comparison of PDI Mediated by Photosensitizers CI1-3- and Toluidine
Blue O
[0254] This Example describes experiments to compare PDI of
gram-negative bacteria by the derivatized cationic fullerenes of
the present invention with known photo sensitizer TBO in the
presence of serum.
[0255] In order to obtain an objective measure of how cationic
fullerenes performed as antimicrobial photosensitizers, they were
compared as above with a widely used phenothiazinium dye, i.e.,
TBO. In order to be able to directly compare the PDI-mediated
killing of bacteria with killing of mammalian cells by photodynamic
therapy (PDT), 10% serum was added to the bacterial suspension,
because this is the standard growth condition used for mammalian
fibroblasts. It has previously been shown that the addition of
serum to bacterial PS incubations significantly reduces the
effectiveness of the PS, probably because the PS binds to serum
proteins, thus reducing the effective concentration available to
bind to bacteria (Wilson, M., and Pratten, J. (1995). Lethal
photosensitisation of Staphylococcusaureusin vitro: effect of
growth phase, serum, and pre-irradiation time. Lasers Surg. Med.
16, 272-276, Lambrechts, S. A., Aalders, M. C., Verbraak, F. D.,
Lager-berg, J. W., Dankert, J. B., and Schuitmaker, J. J. (2005).
Effect of albumin on the photodynamic inactivation of
microorganisms by a cationic porphyrin. J. Photochem. Photobiol. B
79, 51-57).
[0256] In these experiments, the PDI of E. coli was tested using
CI1-3 and TBO under the same conditions (i.e., at 10 .mu.M
concentration, for a 10 min incubation in the presence of 10% FBS,
followed with a wash).
[0257] The results of this experiment are shown in FIG. 21A, and
are compared with results from mammalian fibroblasts in FIG. 21B.
More particularly FIG. 21 shows a comparison of PDI of E. coli and
L929 fibroblasts incubated with either CI1-3 or TBO E. coli was
incubated fullerenes CI1-3 or TBO under the conditions described
and illumination with white or red light, respectively (FIG.
21A).
[0258] Referring to FIG. 21A, it is seen that TBO was almost
ineffective in mediating PDI of E. coli under these conditions.
When the CI1-3 mediated killing of E. coli under these conditions
is compared with that shown in FIG. 19B (i.e., with no serum), it
is seen that the effectiveness of CI1 was unchanged, whereas the
killing mediated by CI2 and CI3 was reduced by about 1 log in the
presence of serum (compare FIGS. 19B, 21A).
[0259] Other experiments testing other microorganisms showed that
TBO at the same concentration and fluence as was used for the
fullerenes (i.e., 1 .mu.M for S. aureus and 10 .mu.M for both P.
aeruginosa and C. albicans) produced less than 1 log of killing in
the presence or absence of serum.
[0260] The above studies were performed in order to make
comparisons between the effectiveness and selectivity of the
cationic fullerenes with an established antimicrobial PS, i.e., the
phenothiazinium dye TBO under the same conditions. As discussed,
TBO has been widely used to kill multiple classes of microbes in
vitro after illumination with red light (Matevski, D., Weersink,
R., Tenenbaum, H. C., Wilson, B., Ellen, R. P., and Lepine, G.
(2003). Lethal photosensitization of periodontal pathogens by a
red-filtered Xenon lamp in vitro. J. Periodontal Res. 38, 428-435,
52. Romanova, N. A., Brovko, L. Y., Moore, L., Pometun, E.,
Savitsky, A. P., Ugarova, N. N., and Griffiths, M. W. (2003).
Assessment of photodynamic destruction of Escherichia coli O157:H7
and Listeria monocytogenes by using ATP bioluminescence. Appl.
Environ. Microbiol. 69, 6393-6398, Soukos, N. S., Wilson, M.,
Burns, T., and Speight, P. M. (1996). Photodynamic effects of
toluidine blue on human oral keratinocytes and fibroblasts and
Streptococcussanguisevaluated in vitro. Lasers Surg. Med. 18,
253-259, Usacheva, M. N., Teichert, M. C., and Biel, M. A. (2001).
Comparison of the methylene blue and toluidine blue
photobactericidal efficacy against Gram-positive and Gram-negative
microorganisms. Lasers Surg. Med. 29, 165-173, and Wilson, M.
(2004). Lethal photosensitisation of oral bacteria and its
potential application in the photodynamic therapy of oral
infections. Photochem. Photobiol. Sci. 3, 412-418). It has also
been tested in several animal models of localized infections. Wong
et al. (Wong, T. W., Wang, Y. Y., Sheu, H. M., and Chuang, Y. C.
(2005). Bactericidal effects of toluidine blue-mediated
photodynamic action on Vibriovulnificus. Antimicrob. Agents
Chemother. 49, 895-902) used topical TBO and red light to cure an
otherwise fatal wound infection with Vibrio anguillarum in mice,
Komerik et al. (Komerik, N., Nakanishi, H., MacRobert, A. J.,
Henderson, B., Speight, P., and Wilson, M. (2003). In vivo killing
of Porphyromonasgingivalisby toluidine blue-mediated
photosensitization in an animal model. Antimicrob. Agents
Chemother. 47, 932-940) used TBO and light to treat a rat model of
periodontal infection, and Teichert et al. (Teichert, M. C., Jones,
J. W., Usacheva, M. N., and Biel, M. A. (2002). Treatment of oral
candidiasis with methylene blue-mediated photodynamic therapy in an
immunodeficient murine model. Oral Surg. Oral Med. Oral Pathol.
Oral Radiol. Endod. 93, 155-160) used the closely related
phenothiazinium dye methylene blue combined with light to treat a
mouse model of oral candidiasis.
[0261] In these studies, TBO (under the same conditions as cationic
fullerenes, i.e., 1 or 10 .mu.M, 10 min incubation, and up to 16
J/cm.sup.2 of red light) did not kill more than 90% of any of the
microbial species. Therefore, these studies show that exemplary
cationic fullerenes CI2 and CI3 are many orders of magnitude more
effective as antimicrobial agents than TBO, a widely used
antimicrobial PS.
Example 15
Cationic Fullerenes Selectivity Kill Microbes and Spare Healthy
Cells
[0262] In order to assess the selectivity of light-mediated killing
of microbes over mammalian cells, mouse L929 fibroblasts were
incubated with CI1-3 and with TBO under the same conditions (i.e.,
10 .mu.M concentration for 10 min in 10% FBS with a wash), followed
by delivery of white or red light, respectively, up to 120
J/cm.sup.2.
[0263] Results of this study are shown in FIG. 21B, and contrasted
with the results for E. coli shown in FIG. 21A. Referring to FIG.
21B, CI1-3 did show some dark toxicity (20%-60% killing) and some
additional phototoxicity (20%-30%) toward L929 cells. However, TBO
displayed a different shape of killing curve, with little dark
toxicity but a pronounced light-dependent toxicity, until the limit
of the viability assay was reached at 80 J/cm.sup.2.
[0264] To employ antimicrobial PS to treat localized infections in
animals or patients, it is necessary to address the question of
selectivity of the PS for microbial cells as compared to host
mammalian cells, as was done in the above-described experiments.
This selectivity may be relatively easy to demonstrate because
antimicrobial PDI is often carried out with relatively short
incubation times (minutes) before illumination, whereas mammalian
cells in tissue culture are frequently incubated with PS for
periods of hours (even 24 hr). Hence, if killing is compared
between microbes and mammalian cells after a short incubation time,
it is likely to favor microbial killing.
[0265] Another difficulty in comparisons between killing microbes
and mammalian cells depends upon differences in the viability
assays employed in each case. The CFU assay for microorganisms can
detect 6 logs of killing, whereas the MTT assay for mammalian cell
viability has a maximum detection limit of 2 logs of killing. These
differences in assay methods notwithstanding, it is nevertheless
clear from the data presented herein that the fullerenes show a
greater level of selectivity for microbes over mammalian cells than
is observed for TBO under the same conditions (FIG. 21B).
Example 16
Design of Fullerene-Based Antimicrobial Photosensitizers
[0266] The effectiveness of various photosensitizers (PS)
considered for antimicrobial PDT can be judged on several criteria.
Preferably the PS are able to kill multiple classes of microbes at
relatively low PS concentrations and low fluences of light. PS
should be reasonably nontoxic in the dark and should demonstrate
selectivity for microbial cells over mammalian cells. PS should
ideally have large extinction coefficients in the red part of the
spectrum.
[0267] As disclosed herein, cationic fullerenes fulfill many of
these criteria. As shown in FIG. 1, the fullerenes have broad
absorption in the UV range, with a tail that extends well into the
visible spectrum (to 550 nm in the case of CI1). The UV absorption
decreases as the number of substituents on the fullerene is
increased, and, consequently, the degree of conjugation is
decreased. TBO, however, like many other PS used for PDT and PDI,
has an absorption peak in the red at 635 nm. Many reports show that
PDT in vivo is more effective with red light and near infrared
light, as both the absorption and scattering of light by tissue
decrease as the wavelength increases (Anderson, R. R., and Parrish,
J. A. (1981). The optics of human skin. J. Invest. Dermatol. 77,
13-19). A broad-band pass filter that gives an output of the entire
visible spectrum (400-700 nm) was used to excite the fullerenes
that maximized the absorption by the tail in the visible range. UV
light was not used to excite the fullerenes, as UV light is highly
germicidal and can kill most microorganisms. FIG. 1 shows that the
effective absorption of the delivered wavelength ranges was not
very different between the fullerenes and TBO.
[0268] Screening experiments carried out against S. aureus at a 100
.mu.M concentration show that a C.sub.60 fullerene series
substituted with pyrrolidinium groups (exemplified by compounds
CI1-3) behaves very differently from a C.sub.60 series substituted
with di-serinol groups (exemplified by compounds NI1-3). The
cationic fullerenes give high levels of dark toxicity (except for
CI1), whereas the di-serinol-functionalized C.sub.60 show a typical
loss of colony-forming ability that is light dose-dependent.
However, cationic fullerenes were highly effective PS at lower
concentrations. This finding agrees with reports that PS with one
(or preferably more) cationic groups are efficient antimicrobial PS
(Hamblin, M. R., and Hasan, T. (2004). Photodynamic therapy: a new
antimicrobial approach to infectious disease? Photo-chem.
Photobiol. Sci. 3, 436-150, Minnock, A., Vernon, D. I., Schofield,
J., Griffiths, J., Parish, J. H., and Brown, S. B. (1996).
Photoinactivation of bacteria. Use of a cationic water-soluble zinc
phthalocyanine to photoinactivate both Gram-negative and
Gram-positive bacteria. J. Photochem. Photobiol. B 32, 159-164,
Merchat, M., Bertolini, G., Giacomini, P., Villanueva, A., and
Joni, G. (1996). Meso-substituted cationic porphyrins as efficient
photosensitizers of Gram-positive and Gram-negative bacteria. J.
Photochem. Photobiol. B 32, 153-157, Demidova, T. N., and Hamblin,
M. R. (2004). Photodynamic therapy targeted to pathogens. Int. J.
Immunopathol. Pharmacol. 17, 245-254, Demidova, T. N., and Hamblin,
M. R. (2005). Effect of cell-photo-sensitizer binding and cell
density on microbial photoinactivation. Antimicrob. Agents
Chemother. 49, 2329-2335). Quarternary nitrogen-based groups are
superior to primary, secondary, or tertiary amino groups, as the
positive charge is less dependent on the pH of the surrounding
media, or the pKa of the molecules with which the PS is
interacting.
[0269] Microbial cells possess overall negative charges, and it is
thought that cationic PS bind to these groups on the outer layers
of the cell surface. Gram-positive and fungal cells have relatively
permeable outer layers of peptidoglycan and lipoteichoic acid or
.beta.-glucan, respectively, although the mannan layer of Candida
species can present a permeability barrier. This permeability
allows cationic, and to a lesser extent, noncationic PS to diffuse
inward to the plasma membrane, a site at which the generation of
reactive oxygen species under illumination can damage the membrane
structure, allowing for leakage of essential components and causing
cell death. Our finding that NI1-3 are equally effective against
the gram-positive S. aureus with and without a wash demonstrates
that the neutrally charged fullerenes are indeed able to penetrate
to a sufficient extent into the cell that they can not easily be
washed out.
[0270] By contrast, gram-negative bacteria have a double membrane
structure that presents a barrier to diffusion of many PS. Cationic
compounds such as CI1-3 are able to displace divalent cations
(Ca.sup.2+ and Mg.sup.2+) that play a role in the attachment of
lipopolysaccharide to the outer membrane (Lambrechts, S. A.,
Aalders, M. C., Langeveld-Klerks, D. H., Khayali, Y., and
Lagerberg, J. W. (2004). Effect of monovalent and divalent cations
on the photoinactivation of bacteria with meso-substituted cationic
porphyrins. Photochem. Photobiol. 79, 297-302). Such displacement
is thought to weaken the structure of the outer permeability,
allowing the PS to penetrate further in a process that has been
termed "self-promoted uptake" (Hancock, R. E., and Bell, A. (1988).
Antibiotic uptake into Gram-negative bacteria. Eur. J. Clin.
Microbiol. Infect. Dis. 7, 713-720).
[0271] In this disclosure it has been shown inter alia that bis-
and tris-cationic fullerenes are highly active antimicrobial PS
that mediate the destruction of a broad spectrum of microbial
classes and show better selectivity for microbes over mammalian
cells than TBO, a widely used antimicrobial PS. Accordingly,
functionalized fullerenes, and preferably cationic fullerenes, hold
great promise as effective antimicrobial photosensitizers,
particularly in those situations in which red light activation is
not important for the light to penetrate deep into tissue.
[0272] Those skilled in the art will recognize, or be able to
ascertain using no more than routine experimentation, many
equivalents to the specific embodiments of the invention described
herein. Such equivalents are intended to be encompassed by the
following claims. All references disclosed herein are incorporated
by reference in their entirety.
REFERENCES
[0273] It is believed that a review of the following references
will appreciate understanding of the present invention. Some of
these documents are referred to throughout the present disclosure
by a number, as indicated below. [0274] 1. Kroto, H. W., Heath, J.
R., O'Brien, S. C., Curl, R. F., and Smalley, R. E. (1985). C60:
Buckminsterfullerene. Nature 318, 162-163. [0275] 2. Jensen, A. W.,
Wilson, S. R., and Schuster, D. I. (1996). Biological applications
of fullerenes. Bioorg. Med. Chem. 4, 767-779. [0276] 3. Bosi, S.,
Da Ros, T., Spalluto, G., and Prato, M. (2003). Fullerene
derivatives: an attractive tool for biological applications. Eur.
J. Med. Chem. 38, 913-923. [0277] 4. Dugan, L. L., Lovett, E. G.,
Quick, K. L., Lotharius, J., Lin, T. T., and O'Malley, K. L.
(2001). Fullerene-based antioxidants and neurodegenerative
disorders. Parkinsonism Relat. Disord. 7, 243-246. [0278] 5.
Tagmatarchis, N., and Shinohara, H. (2001). Fullerenes in medicinal
chemistry and their biological applications. Mini Rev. Med. Chem.
1, 339-348. [0279] 6. Brettreich, M., and Hirsch, A. (1998). A
highly water-soluble dendro[60]fullerene. Tetrahedron Lett. 39,
2731-2734. [0280] 7. Da Ros, T., Prato, M., Novello, F., Maggini,
M., and Banfi, E. (1996). Easy access to water-soluble fullerene
derivatives via 1,3-dipolar cycloadditions of azomethine ylides to
C(60). J. Org. Chem. 61, 9070-9072. [0281] 8. Foley, S., Crowley,
C., Smaihi, M., Bonfils, C., Erlanger, B. F., Seta, P., and
Larroque, C. (2002). Cellular localisation of a water-soluble
fullerene derivative. Biochem. Biophys. Res. Commun. 294, 116-119.
[0282] 9. Bosi, S., Da Ros, T., Spalluto, G., Balzarini, J., and
Prato, M. (2003). Synthesis and anti-HIV properties of new
water-soluble bis-functionalized[60]fullerene derivatives. Bioorg.
Med. Chem. Lett. 13, 4437-4440. [0283] 10. Schinazi, R. F.,
Sijbesma, R., Srdanov, G., Hill, C. L., and Wudl, F. (1993).
Synthesis and virucidal activity of a water-soluble,
configurationally stable, derivatized C60 fullerene. Antimicrob.
Agents Chemother. 37, 1707-1710. [0284] 11. Tsao, N., Luh, T. Y.,
Chou, C. K., Wu, J. J., Lin, Y. S., and Lei, H. Y. (2001).
Inhibition of group A streptococcus infection by carboxyfullerene.
Antimicrob. Agents Chemother. 45, 1788-1793. [0285] 12. Dugan, L.
L., Gabrielsen, J. K., Yu, S. P., Lin, T. S., and Choi, D. W.
(1996). Buckminsterfullerenol free radical scavengers reduce
excitotoxic and apoptotic death of cultured cortical neurons.
Neurobiol. Dis. 3, 129-135. [0286] 13. Jin, H., Chen, W. Q., Tang,
X. W., Chiang, L. Y., Yang, C. Y., Schloss, J. V., and Wu, J. Y.
(2000). Polyhydroxylated C(60), fullerenols, as glutamate receptor
antagonists and neuroprotective agents. J. Neurosci. Res. 62,
600-607. [0287] 14. Tsai, M. C., Chen, Y. H., and Chiang, L. Y.
(1997). Polyhydroxylated C60, fullerenol, a novel free-radical
trapper, prevented hydrogen peroxide- and cumene
hydroperoxide-elicited changes in rat hippocampus in-vitro. J.
Pharm. Pharmacol. 49, 438-445. [0288] 15. Mashino, T., Nishikawa,
D., Takahashi, K., Usui, N., Yamori, T., Selci, M., Endo, T., and
Mochizuki, M. (2003). Antibacterial and antiproliferative activity
of cationic fullerene derivatives. Bioorg. Med. Chem. Lett. 13,
4395-4397. [0289] 16. Mashino, T., Usui, N., Okuda, K., Hirota, T.,
and Mochizuki, M. (2003). Respiratory chain inhibition by fullerene
derivatives: hydrogen peroxide production caused by fullerene
derivatives and a respiratory chain system. Bioorg. Med. Chem. 11,
1433-1438. [0290] 17. Mashino, T., Shimotohno, K., Ikegami, N.,
Nishikawa, D., Okuda, K., Takahashi, K., Nakamura, S., and
Mochizuki, M. (2005). Human immunodeficiency virus-reverse
transcriptase inhibition and hepatitis C virus RNA-dependent RNA
polymerase inhibition activities of fullerene derivatives. Bioorg.
Med. Chem. Lett. 15, 1107-1109. [0291] 18. Castano, A. P.,
Demidova, T. N., and Hamblin, M. R. (2004). Mechanisms in
photodynamic therapy: part one--photosensitizers, photochemistry
and cellular localization. Photodiag. Photodyn. Ther. 1, 279-293.
[0292] 19. Yamakoshi, Y., Umezawa, N., Ryu, A., Arakane, K.,
Miyata, N., Goda, Y., Masumizu, T., and Nagano, T. (2003). Active
oxygen species generated from photoexcited fullerene (C60) as
potential medicines: O2-* versus 102. J. Am. Chem. Soc. 125,
12803-12809. [0293] 20. Liu, Y., Zhao, Y. L., Chen, Y., Liang, P.,
and Li, L. (2005). A water-soluble bcyclodextrin derivative
possessing a fullerene tether as an efficient photodriven
DNA-cleavage reagent. Tetrahedron Lett. 46, 2507-2511. [0294] 21.
Kasermann, F., and Kempf, C. (1997). Photodynamic inactivation of
enveloped viruses by buckminsterfullerene. Antiviral Res. 34,
65-70. [0295] 22. Sera, N., Tokiwa, H., and Miyata, N. (1996).
Mutagenicity of the fullerene C60-generated singlet oxygen
dependent formation of lipid peroxides. Carcinogenesis 17,
2163-2169. [0296] 23. Burlaka, A. P., Sidorik, Y. P., Prylutska, S.
V., Matyshevska, O. P., Golub, O. A., Prylutskyy, Y. I., and
Scharff, P. (2004). Catalytic system of the reactive oxygen species
on the C60 fullerene basis. Exp. Oncol. 26, 326-327. [0297] 24.
Tabata, Y., Murakami, Y., and Ikada, Y. (1997). Photodynamic effect
of polyethylene glycol-modified fullerene on tumor. Jpn. J. Cancer
Res. 88, 1108-1116. [0298] 25. Moan, J., and Peng, Q. (2003). An
outline of the hundred-year history of PDT. Anticancer Res. 23,
3591-3600. [0299] 26. Dolmans, D. E., Fukumura, D., and Jain, R. K.
(2003). Photodynamic therapy for cancer. Nat. Rev. Cancer 3,
380-387. [0300] 27. Brown, S. B., and Mellish, K. J. (2001).
Verteporfin: a milestone in opthalmology and photodynamic therapy.
Expert Opin. Pharmacother. 2, 351-361. [0301] 28. Wainwright, M.
(1998). Photodynamic antimicrobial chemotherapy (PACT). J.
Antimicrob. Chemother. 42, 13-28. [0302] 29. Maisch, T., Szeimies,
R. M., Jori, G., and Abels, C. (2004). Antibacterial photodynamic
therapy in dermatology. Photochem. Photobiol. Sci. 3, 907-917.
[0303] 30. Hamblin, M. R., and Hasan, T. (2004). Photodynamic
therapy: a new antimicrobial approach to infectious disease?
Photo-chem. Photobiol. Sci. 3, 436-450. [0304] 31. Minnock, A.,
Vernon, D. I., Schofield, J., Griffiths, J., Parish, J. H., and
Brown, S. B. (1996). Photoinactivation of bacteria. Use of a
cationic water-soluble zinc phthalocyanine to photoinactivate both
Gram-negative and Gram-positive bacteria. J. Photochem. Photobiol.
B 32, 159-164. [0305] 32. Nitzan, Y., Dror, R., Ladan, H., Malik,
Z., Kimel, S., and Gottfried, V. (1995). Structure-activity
relationship of porphines for photoinactivation of bacteria.
Photochem. Photobiol. 62, 342-347. [0306] 33. Merchat, M.,
Bertolini, G., Giacomini, P., Villanueva, A., and Jori, G. (1996).
Meso-substituted cationic porphyrins as efficient photosensitizers
of Gram-positive and Gram-negative bacteria. J. Photochem.
Photobiol. B 32, 153-157. [0307] 34. Minnock, A., Vernon, D. I.,
Schofield, J., Griffiths, J., Parish, J. H., and Brown, S. B.
(2000). Mechanism of uptake of a cationic water-soluble pyridinium
zinc phthalocyanine across the outer membrane of Escherichia coli.
Antimicrob. Agents Chemother. 44, 522-527. [0308] 35. Wharton, T.,
Kini, V. U., Mortis, R. A., and Wilson, L. J. (2001). New
non-ionic, highly water-soluble derivatives of C60 designed for
biological compatibility. Tetrahedron Lett. 42, 5159-5162. [0309]
36. Wharton, T., and Wilson, L. J. (2002). Highly-iodinated
fullerene as a contrast agent for X-ray imaging. Bioorg. Med. Chem.
10, 3545-3554. [0310] 37. Maggini, M., Scorrano, G., and Prato, M.
(1993). Addition of azomethine ylides to C60: synthesis,
characterization, and functionalization of fullerene pyrrolidines.
J. Am. Chem. Soc. 115, 9798-9799. [0311] 38. Cassell, A. M.,
Scrivens, W. A., and Tour, J. M. (1998). Assembly of DNA/fullerene
hybrid materials. Angew. Chem. Int. Ed. Engl. 37, 1528-1530. [0312]
39. Malik, Z., Ladan, H., and Nitzan, Y. (1992). Photodynamic
inactivation of Gram-negative bacteria: problems and possible
solutions. J. Photochem. Photobiol. B 14, 262-266. [0313] 40.
Nitzan, Y., Gutterman, M., Malik, Z., and Ehrenberg, B. (1992).
Inactivation of Gram-negative bacteria by photosensitized
porphyrins. Photochem. Photobiol. 55, 89-96. [0314] 41. Wilson, M.,
and Pratten, J. (1995). Lethal photosensitisation of
Staphylococcusaureusin vitro: effect of growth phase, serum, and
pre-irradiation time. Lasers Surg. Med. 16, 272-276. [0315] 42.
Lambrechts, S. A., Aalders, M. C., Verbraak, F. D., Lager-berg, J.
W., Dankert, J. B., and Schuitmaker, J. J. (2005). Effect of
albumin on the photodynamic inactivation of microorganisms by a
cationic porphyrin. J. Photochem. Photobiol. B 79, 51-57. [0316]
43. Anderson, R. R., and Parrish, J. A. (1981). The optics of human
skin. J. Invest. Dermatol. 77, 13-19. [0317] 44. Demidova, T. N.,
and Hamblin, M. R. (2004). Photodynamic therapy targeted to
pathogens. Int. J. Immunopathol. Pharmacol. 17, 245-254. [0318] 45.
Demidova, T. N., and Hamblin, M. R. (2005). Effect of
cell-photo-sensitizer binding and cell density on microbial
photoinactivation. Antimicrob. Agents Chemother. 49, 2329-2335.
[0319] 46. Lambrechts, S. A., Aalders, M. C., Langeveld-Klerks, D.
H., Khayali, Y., and Lagerberg, J. W. (2004). Effect of monovalent
and divalent cations on the photoinactivation of bacteria with
meso-substituted cationic porphyrins. Photochem. Photobiol. 79,
297-302. [0320] 47. Hancock, R. E., and Bell, A. (1988). Antibiotic
uptake into Gram-negative bacteria. Eur. J. Clin. Microbiol.
Infect. Dis. 7, 713-720. [0321] 48. Mashino, T., Okuda, K., Hirota,
T., Hirobe, M., Nagano, T., and Mochizuki, M. (1999). Inhibition of
E. coli growth by fullerene derivatives and inhibition mechanism.
Bioorg. Med. Chem. Lett. 9, 2959-2962. [0322] 49. Hancock, R. E.,
and Wong, P. G. (1984). Compounds which increase the permeability
of the Pseudomonas aeruginosa outer membrane. Antimicrob. Agents
Chemother. 26, 48-52. [0323] 50. Hancock, R. E. (1986). Intrinsic
antibiotic resistance of Pseudomonas aeruginosa. J. Antimicrob.
Chemother. 18, 653-656. [0324] 51. Matevslci, D., Weersink, R.,
Tenenbaum, H. C., Wilson, B., Ellen, R. P., and Lepine, G. (2003).
Lethal photosensitization of periodontal pathogens by a
red-filtered Xenon lamp in vitro. J. Periodontal Res. 38, 428-435.
[0325] 52. Romanova, N. A., Brovko, L. Y., Moore, L., Pometun, E.,
Savitsky, A. P., Ugarova, N. N., and Griffiths, M. W. (2003).
Assessment of photodynamic destruction of Escherichia coli O157:H7
and Listeria monocytogenes by using ATP bioluminescence. Appl.
Environ. Microbiol. 69, 6393-6398. [0326] 53. Soukos, N. S.,
Wilson, M., Burns, T., and Speight, P. M. (1996). Photodynamic
effects of toluidine blue on human oral keratinocytes and
fibroblasts and Streptococcus sanguis evaluated in vitro. Lasers
Surg. Med. 18, 253-259. [0327] 54. Usacheva, M. N., Teichert, M.
C., and Biel, M. A. (2001). Comparison of the methylene blue and
toluidine blue photobactericidal efficacy against Gram-positive and
Gram-negative microorganisms. Lasers Surg. Med. 29, 165-173. [0328]
55. Wilson, M. (2004). Lethal photosensitisation of oral bacteria
and its potential application in the photodynamic therapy of oral
infections. Photochem. Photobiol. Sci. 3, 412-418. [0329] 56. Wong,
T. W., Wang, Y. Y., Sheu, H. M., and Chuang, Y. C. (2005).
Bactericidal effects of toluidine blue-mediated photodynamic action
on Vibriovulnificus. Antimicrob. Agents Chemother. 49, 895-902.
[0330] 57. Komerik, N., Nakanishi, H., MacRobert, A. J., Henderson,
B., Speight, P., and Wilson, M. (2003). In vivo killing of
Porphyromonasgingivalisby toluidine blue-mediated
photosensitization in an animal model. Antimicrob. Agents
Chemother. 47, 932-940. [0331] 58. Teichert, M. C., Jones, J. W.,
Usacheva, M. N., and Biel, M. A. (2002). Treatment of oral
candidiasis with methylene blue-mediated photodynamic therapy in an
immunodeficient murine model. Oral Surg. Oral Med. Oral Pathol.
Oral Radiol. Endod. 93, 155-160. [0332] 59. Chen, Y. W., Hwang, K.
C., Yen, C. C., and Lai, Y. L. (2004). Fullerene derivatives
protect against oxidative stress in RAW 264.7 cells and
ischemia-reperfused lungs. Am. J. Physiol. Regul. Integr. Comp.
Physiol. 287, R21-R26. [0333] 60. Taylor, A. E. (2004). Fullerene
derivatives protect against oxidative stress in murine macrophage
line cells and ischemia-reper-fused lungs. Am. J. Physiol. Regul.
Integr. Comp. Physiol. 287, R1-R2. [0334] 61. Hamano, T., Okuda,
K., Mashino, T., Hirobe, M., Arakane, K., Ryu, A., Mashiko, S., and
Nagano, T. (1997). Singlet oxygen production from fullerene
derivatives: effect of sequential functionalization of the
fullerene core. Chem. Commun. 21-22. [0335] 62. Jett, B. D.,
Hatter, K. L., Huycke, M. M., and Gilmore, M. S. (1997). Simplified
agar plate method for quantifying viable bacteria. Biotechniques
23, 648-650. [0336] 63. Earle, W. R., Schilling, E. L., Stark, T.
H., Straus, N. P., Brown, M. F., and Shelton, E. (1943). Production
of malignancy in vitro. IV. The mouse fibroblast cultures and
changes seen in the living cells. J. Natl. Cancer Inst. 4, 165-212.
[0337] 64. Merlin, J. L., Azzi, S., Lignon, D., Ramacci, C.,
Zeghari, N., and Guillemin, F. (1992). MTT assays allow quick and
reliable measurement of the response of human tumour cells to
photodynamic therapy. Eur. J. Cancer 28A, 1452-1458.
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