U.S. patent application number 11/254400 was filed with the patent office on 2006-02-23 for drug delivery system for hydrophobic drugs.
Invention is credited to Ronald Erwin Boch, Iman Karmadi, Dev Mitra Ranji Singh.
Application Number | 20060039965 11/254400 |
Document ID | / |
Family ID | 25264324 |
Filed Date | 2006-02-23 |
United States Patent
Application |
20060039965 |
Kind Code |
A1 |
Boch; Ronald Erwin ; et
al. |
February 23, 2006 |
Drug delivery system for hydrophobic drugs
Abstract
Compositions comprising microaggregates containing hydrophobic
drugs, as well as methods for their production, are described. Such
microaggregates may include micelle structures or combinations
thereof with liposomes, and constitute an effective delivery
vehicle for a hydrophobic agent. Methods for microaggregate
production include the use of preferred lipid compounds and
processing conditions favo the production of small aggregates for
improved filter sterilization.
Inventors: |
Boch; Ronald Erwin; (North
Vancouver, CA) ; Singh; Dev Mitra Ranji; (Surrey,
CA) ; Karmadi; Iman; (Vancouver, CA) |
Correspondence
Address: |
MORRISON & FOERSTER LLP
12531 HIGH BLUFF DRIVE
SUITE 100
SAN DIEGO
CA
92130-2040
US
|
Family ID: |
25264324 |
Appl. No.: |
11/254400 |
Filed: |
October 20, 2005 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
09833406 |
Apr 11, 2001 |
6984395 |
|
|
11254400 |
Oct 20, 2005 |
|
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|
Current U.S.
Class: |
424/450 ;
514/185; 514/410 |
Current CPC
Class: |
A61P 31/00 20180101;
A61P 29/00 20180101; A61P 27/02 20180101; A61P 19/02 20180101; Y10S
514/937 20130101; A61P 35/00 20180101; A61P 9/00 20180101; A61P
9/10 20180101; A61K 9/19 20130101; A61K 9/127 20130101; A61K
41/0071 20130101; A61P 31/12 20180101; A61K 9/1075 20130101; A61K
41/0057 20130101; A61P 35/02 20180101; A61K 41/0038 20130101 |
Class at
Publication: |
424/450 ;
514/185; 514/410 |
International
Class: |
A61K 9/127 20060101
A61K009/127; A61K 31/555 20060101 A61K031/555; A61K 31/409 20060101
A61K031/409 |
Claims
1. A composition comprising aggregates, said aggregates comprising
saturated and unsaturated phospholipids and one or more
photosensitizer, and wherein the aggregates have an average
diameter of below about 100 nm.
2. The composition of claim 1 wherein said aggregates comprise
micelles.
3. The composition of claim 2 wherein the composition further
comprises liposomes.
4. The composition of claim 1 wherein the one or more
photosensitizers is a green porphyrin.
5. The composition of claim 4 wherein the green porphyrin is a
hydro-monobenzo-porphyrin photosensitiser.
6. The composition of claim 1 wherein said one or more
photosensitizer is BPD-MA, A-EA6, B-EA6 or a combination
thereof.
7. The composition of claim 1 wherein one or more of said saturated
and unsaturated phospholipids comprise a negatively charged
headgroup.
8. The composition of claim 7 wherein said phospholipids comprise
DOPG and DMPC.
9. The composition of claim 8 wherein the ratio of DOPG:DMPC is
40:60.
10. The composition of claim 9 wherein said aggregates further
comprise at least one antioxidant.
11. The composition of claim 10 wherein said at least one
antioxidant is BHT and/or AP.
12. The composition of claim 1 wherein the ratio of
phospholipids:photosensitizer is 8:1.
13. The composition of claim 1 wherein said one or more
photosensitizer is one or more hydro-monobenzo-porphyrin
photosensitizer.
14. The composition of claim 13 wherein said photosensitizer is
BPD-MA, A-EA6 or B-EA6 or combinations thereof.
15. The composition of claim 13 wherein the one or more
photosensitizers is A-EA6 or B-EA6 or a combination thereof.
16. The composition of claim 1 wherein the average diameter is
below about 50 nm.
17. The composition of claim 1 wherein the average diameter is
below about 30 nm.
18. The composition of claim 1 wherein the average diameter is
below about 20 nm.
19. The composition of claim 1 wherein the unsaturated phospholipid
is from a non-animal source.
20. The composition of claim 1 wherein the phospholipid is an egg
phospholipid.
21. The composition of claim 1 wherein the phospholipid is not egg
phosphatidylglycerol.
22. A composition comprising aggregates, said aggregates comprising
saturated and unsaturated phospholipids, at least one of which
comprises a negatively charged head group; and one or more
photosensitizer; wherein the aggregates have an average diameter of
less than 100 nm.
23. A composition of aggregates comprising micelles, said micelles
comprising saturated and unsaturated phospholipids and one or more
photosensitizers, wherein either a) said phospholipids are capable
of forming a lipid bilayer and do not comprise egg phospholipid or
egg phosphatidylglycerol; or b) said phospholipids are capable of
forming a lipid bilayer and said one or more photosensitizer is not
a combination of approximately equal amounts of BPD-MA.sub.C and
BPD-MA.sub.D.
24. A method for making a composition comprising micelles, said
micelles comprising one or more hydrophobic agent and a mixture of
saturated and unsaturated phospholipids, wherein said method
comprises the steps of: producing a mixture of an organic solvent,
a hydrophobic-agent and phospholipids capable of forming a lipid
bilayer to form an "intermediate complex"; removing said solvent to
produce a "presome" material; hydrating said "presome" material
with an aqueous solvent; and processing said hydrated material to
produce micelle comprising aggregates.
25. The method of claim 24 wherein either a) said phospholipids are
capable of forming a lipid bilayer and do not comprise egg
phospholipid or egg phosphatidylglycerol; or b) said phospholipids
are capable of forming a lipid bilayer and said one or more
hydrophobic agent is not a combination of approximately equal
amounts of BPD-MA.sub.C and BPD-MA.sub.D.
26. The method of claim 25 wherein said one or more hydrophobic
agent is a photosensitizer but is not a combination of
approximately equal parts of BPD-MA.sub.C and BPD MA.sub.D.
27. The method of claim 26 wherein said one or more
photosensitizers is BPD-MA, A-EA6, B-EA6, or a combination
thereof.
28. The method of claim 27 wherein said one or more
photosensitizers is BPD-MA.
29. The method of claim 27 wherein said one or more
photosensitizers is A-EA6, B-EA6, or a combination thereof.
30. The method of claim 24 wherein said "intermediate complex" and
said aqueous solvent are low salt.
31. The method of claim 30 wherein said hydrating and processing
steps occur at a temperature of less than about 30.degree. C.
32. The method of claim 24 wherein said steps comprise a) supplying
at a constant speed an organic solvent solution of a mixture of
phospholipids capable of forming a lipid bilayer to a tubular
heater heated externally, b) evaporating the organic solvent in the
heater to prepare a mixture substantially of solids and over heated
organic solvent vapor, c) introducing this mixture at a high speed
of over 0.1 times the sound of speed into the vacuum chamber of not
more than 300 mm Hg to volatize the organic solvent instantaneously
and dry the solids, whereby lipid powder is obtained, and d)
dispersing the resulting lipid powder into a low salt aqueous
solvent at a temperature of less than about 30.degree. C.
33. The method of claim 31 wherein said processing step is by high
energy manipulation.
34. The method of claim 33 wherein said high energy manipulation is
selected from the group consisting of microfluidization,
sonication, high speed shearing, extrusion, sonication and
homogenization.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority from U.S. application Ser.
No. 09/833,406 filed Apr. 11, 2001. The contents of this document
are incorporated herein by reference.
FIELD OF THE INVENTION
[0002] The invention relates to water soluble microaggregates of
water insoluble, poorly soluble or otherwise hydrophobic agents and
phospholipids or lipids which may be used pharmaceutically,
agriculturally or industrially. These microaggregate compositions
may be used to deliver hydrophobic drugs as a pharmaceutical
formulation, hydrophobic compounds related to plant growth as an
agricultural product, and hydrophobic reagents as an industrial
material. Moreover, the microaggregates of the invention comprise
combinations of natural and/or synthetic phospholipids which permit
aggregation with the hydrophobic agents to result in micelles,
liposomes, and mixtures thereof. Particular combinations of
hydrophobic agents and phospholipids or lipids produce
microaggregates that are effective delivery vehicles of said
compounds.
[0003] Additionally, the invention relates to processes for the
production of said microaggregates as delivery systems. These
processes include microfluidization (liquid jet milling), high
shear mixing, and sonication. Particular processes, involving the
use of specific combinations of hydrophobic agents and
phospholipids or lipids, permit the large scale preparation of
effective delivery vehicles for hydrophobic agents.
DESCRIPTION OF THE RELATED ART
[0004] The existence of a wide array of active hydrophobic or
otherwise water insoluble agents is known in the art. Similarly
there is awareness of the need to deliver such active agents to
water based or otherwise aqueous environments. As such, multiple
systems have been development as delivery vehicles for such agents.
These include the use of organic solvents, aqueous/detergent
mixtures, aqueous/organic solvent mixtures (such as co-solvents),
emulsions, liposomes, and micelles. Each of these systems, however,
have limitations arising from considerations such as the degree of
water insolubility and the environment into which delivery is
desired.
[0005] An example of hydrophobic agents in liposomes is taught by
Farmer et al., U.S. Pat. No. 4,776,991, which discloses the
large-scale encapsulation of hemoglobin. Kappas et al., U.S. Pat.
No. 5,010,073, discloses the preparation of liposomes containing a
metalloporphyrin with egg phosphatidyl choline ("EPC") being used
as the lipid. Schneider et al., U.S. Pat. No. 5,270,053, discloses
liposome formulations said to be free of solid particles and larger
lipid aggregates. Parikh et al., U.S. Pat. No. 5,922,355, disclose
microparticles comprising insoluble substances. Lasic (Nature, Vol.
355, pp. 379-380, (1992)) describes the use of mixed micelles
comprising a drug agent and biological lipids.
[0006] Similarly, micelles have also been used to deliver
medications to patients, (Brodin et al., Acta Pharm. Suec. 19
267-284 (1982)) and micelles have been used as drug carriers and
for targeted drug delivery, (Supersaxo et al., Pharm. Res.
8:1286-1291 (1991)), including cancer medications, (Fung et al.,
Biomater. Artif. Cells. Artif. Organs 16: 439 et. seq. (1988); and
Yokoyama et al., Cancer Res. 51: 3229-3236 (1991)).
[0007] Hydrophobic agents of great interest include the
polypyrrolic macrocycle based photosensitizing compounds and, in
particular green porphyrins such as BPD-MA (benzoporphyrin
derivative monoacid ring A, also know by its generic name,
verteporfin). These compounds have been known for some time to be
useful, when combined with light, for the treatment and diagnosis
of a variety of conditions, including tumors, angiogenesis and
neovasculature, restenosis and atherosclerotic plaques, and
rheumatoid arthritis. The porphyrins have a natural tendency to
"localize" in malignant or proliferating tissue, where they absorb
light at certain wavelengths when irradiated. The absorbed light
may result in a cytotoxic effect in the cells, and neighboring
cells, into which the porphyrins have localized. (See, e.g.,
Diamond et al., Lancet, 2:1175-77 (1972); Dougherty et al., "The
Science of Photo Medicine", 625-38 (Regan et al. eds. 1982); and
Dougherty et al., "Cancer: Principles and Practice of Oncology",
1836-44 (DeVita Jr. et al. eds. 1982)). It has been postulated that
the cytotoxic effect of porphyrins is due to the formation of
singlet oxygen when exposed to light (Weishaupt et al., Cancer
Research, 36:2326-29 (1976)).
[0008] Accordingly, preparations containing the porphyrins are
useful in the diagnosis and the detection of important cells and
tissue (see, e.g. "Porphyrin Photosensitization", Plenum Press
(Kessel et al. eds. 1983)), such as those related to tumors,
growing vasculature, arterial blockage and autoimmunity. Similar
photosensitizers have been used in the detection and treatment of
atherosclerotic plaques, as disclosed in U.S. Pat. Nos. 4,512,762
and 4,577,636. In addition to systemic use for the diagnosis and
treatment of various conditions, the porphyrins can be used in a
variety of other therapeutic applications. Porphyrin compounds have
been used topically to treat various skin diseases, as disclosed in
U.S. Pat. No. 4,753,958.
[0009] A number of porphyrin photosensitizer preparations have been
disclosed for therapeutic applications. A photosensitizer
preparation widely used during the early days of photodynamic
therapy both for detection and treatment was a crude derivative of
hematoporphyrin, also called hematoporphyrin derivative ("HPD") or
Lipson derivative, prepared as described by Lipson et al., J. Natl.
Cancer Inst., 26:1-8 (1961). A purified form of the active
component(s) of HPD was prepared by Dougherty and co-workers by
adjustment of the pH to cause aggregation, followed by recovery of
the aggregate, as disclosed in U.S. Pat. Nos. 4,649,151; 4,866,168;
4,889,129; and 4,932,934. A purified form of this product is being
used clinically under the trademark Photofrin.RTM. (Axcan
Pharmaceuticals), which is porfimer sodium.
[0010] Of particular interest is a group of modified porphyrins,
known as "green porphyrins" (Gp), having one or more light
absorption maxima between about 670-780 nm. These Gp compounds have
been shown to confer cytotoxicity against target cells at
concentrations lower than those required for hematoporphyrin or
HPD. Gp compounds can be obtained using Diels-Alder reactions of
protoporphyrin with various acetylene derivatives under the
appropriate conditions. Preferred forms of Gp are the
hydro-monobenzoporphyrin derivatives ("BPD's") as well as BPD-MA,
EA6 and B3 in particular. The preparation and use of the Gp and BPD
compounds are disclosed in U.S. Pat. Nos. 4,920,143, 4,883,790 and
5,095,030, hereby incorporated by reference into the disclosure of
the present application. The preparation and uses of EA6 and B3 are
disclosed in U.S. Pat. Nos. 6,153,639 and 5,990,149 respectively,
also hereby incorporated by reference.
[0011] Many desirable hydro-monobenzoporphyrin photosensitizers,
such as BPD-MA, are not only insoluble in water at physiological
pH's, but are also insoluble in (1) pharmaceutically acceptable
aqueous-organic co-solvents, (2) aqueous polymeric solutions, and
(3) surfactant/micellar solutions. It has recently been shown that
the encapsulation of certain drugs in liposomes, prior to
administration, has a marked effect on the pharmacokinetics, tissue
distribution, metabolism and efficacy of the therapeutic agent. In
an effort to increase the tumor selectivity of porphyrin
photosensitizers, porphyrin compounds have been incorporated into
unilamellar liposomes, resulting in a larger accumulation and a
more prolonged retention of the photosensitizer by both cultured
malignant cells and in experimental tumors in vivo. Jori et al.,
Br. J. Cancer, 48:307-309 (1983); Cozzani et al., In Porphyrins in
Tumor Phototherapy, 177-183, Plenum Press (Andreoni et al. eds.
1984). This more efficient targeting of tumor tissues by
liposome-associated porphyrins may be due in part to the specific
delivery of phospholipid vesicles to serum lipoproteins, which have
been shown to interact preferentially with hyperproliferative
tissue, such as tumors, through receptor-mediated endocytosis. In
this manner, the selectivity of porphyrin uptake by tumors has been
increased, as compared with photosensitizers dissolved in aqueous
solution. See Zhou et al., Photochemistry and Photobiology,
48:487-92 (1988).
[0012] Accordingly, hematoporphyrin and hematoporphyrin dimethyl
esters have been formulated in unilamellar vesicles of dipalmitoyl
phosphatidyl choline (DPPC) and liposomes of dimyristoyl (DMPC) and
distearoyl phosphatidyl choline (DSPC). Zhou et al., supra;
Ricchelli, New Directions in Photodynamic Therapy, 847:101-106
(1987); Milanesi, Int. J. Radiat. Biol., 55:59-69 (1989).
Similarly, HP, porfimer sodium, and tetrabenzoporphyrins have been
formulated in liposomes composed of egg phosphatidyl choline (EPC).
Johnson et al., Proc. Photodynamic Therapy: Mechanisms II, Proc.
SPIE-Int. Soc. Opt. Eng., 1203:266-80 (1990). Additionally, BPD-MA
can be "solubilized" at a concentration of about 2.0 mg/ml in
aqueous solution using an appropriate mixture of phospholipids to
form encapsulating liposomes. Such "solubilized" liposome
compositions are suitable for parenteral administration.
[0013] Further, freeze-dried pharmaceutical formulations comprising
a porphyrin photosensitizer, a disaccharide or polysaccharide, and
one or more phospholipids (such as EPG and DMPC) have been made.
These formulations form liposomes containing an effective amount of
porphyrin photosensitizer upon reconstitution with a suitable
aqueous vehicle and are described in Desai et al., U.S. Pat. No.
6,074,666, which is incorporated by reference. Methods for the
large-scale production of DMPC/EPG liposomes containing a
photosensitizer are disclosed in U.S. Pat. No. 5,707,608, which is
incorporated by reference as if fully set forth.
[0014] It has been a challenge to find suitable pharmaceutical
formulations for hydrophobic polypyrrolic macrocyle based
photosensitizers that can be filter sterilized and freeze dried,
and can also be rapidly reconstituted in an aqueous medium prior to
administration, while retaining a small particle size after
rehydration. Photosensitive compunds such as verteporfin (BPD-MA)
and QLT 0074 (EA6) must be lyophilized for storage, because they
are labile in an aqueous environment.
SUMMARY OF THE INVENTION
[0015] The present invention provides a phospholipid composition
into which hydrophobic photosensitizers may be incorporated that
could be processed into a stable liposome product small enough to
be sterile filtered, lyophilized for storage, and would rapidly
dissolve in an aqueous medium for administration, while maintaining
the small particle size. It was initially believed that the
phospholipids of choice would contain only saturated lipids,
because saturated lipids are more stable, eliminating the need for
anti-oxidants in pharamaceutical preparation. The initial attempts
for a composition using saturated phospholipids failed.
Surprisingly, it was found that the presence of at least some
unsaturated lipid in the composition was essential for a stable,
robust product that would survive the lyophilization process
intact. Additionally, it was found that the presence of at least
some phospholipids having negatively charged polar headgroups
contributed to the stability of the composition.
[0016] Another totally unexpected finding was that bilayer forming
phospholipids comprising a proportion of unsaturated charged lipids
were capable of assuming a micellular structure (with or without
the incorporation of a hydrophobic molecule) if the material was
subjected to a high energy process, such as microfluidization. The
production of micelles from bilayer forming lipids is believed to
be completely novel, and would not have been predicted from the
literature on bilayer forming lipids.
[0017] The present invention relates to microaggregates of lipids
and hydrophobic agents. In particular, the microaggregates are
produced by combining phospholipids and active hydrophobic
compounds. Such compositions may be used in any therapeutic,
agricultural or industrial setting, and as such, they are delivery
vehicles for the active hydrophobic agents. Preferably, the
microaggregates comprise micelles and/or small liposomes containing
a therapeutically acceptable amount of a hydro-monobenzoporphyrin
photosensitizer. The lipids used for microaggregate production
comprise unsaturated lipids, and may be stabilized by the presence
of antioxidants. Preferably, the microaggregates comprise a mixture
of saturated and unsaturated lipids. Preferably, the
microaggregates comprise phospholipids having a headgroup that is
negatively charged over the pH range of 5-7. Alternatively, the
microaggregates may comprise both micelles and liposomes produced
from, or containing, the same combination of phospholipids.
[0018] The present invention also relates to methods of producing
microaggregates comprising lipids and hydrophobic agents. It has
been discovered that with appropriate selection of lipids, salt
conditions, temperature, and size reduction process,
microaggregates comprising differing amounts of liposomes and
micelles can be produced. Appropriately selected combinations of
lipids, low salt conditions, and a high energy process such as
microfluidization can result in the production predominantly
micelle comprising microaggregate compositions.
[0019] The microaggregates of the invention provide nearly 100%
incorporation of a hydrophobic agent such as a
hydro-monobenzoporphyrin photosensitizer, which can be expensive
and usually requires a complicated synthetic procedure to produce.
Thus, there is little reworking necessary and very little waste of
the photosensitizer. In addition, due to their small particle size,
the present microaggregates exhibit the improved filterability
important in producing large quantities of
photosensitizer-containing delivery vehicles. Further, the
microaggregates retain their small size following lyophilization
and reconstituion with an aqueous medium for pharmaceutical
delivery. Such photosensitizing microaggregate compositions are
useful in mediating the destruction of unwanted cells or tissues or
other undesirable materials, or to detect their presence through
fluorescence, upon appropriate irradiation. Particularly preferred
hydro-monobenzoporphyrin photosensitizers used in the practice of
this invention include those having one or more light absorption
maxima in the range of 670-780 nm.
BRIEF DESCRIPTION OF THE DRAWINGS
[0020] Other objects, features, and advantages are evident from the
following descriptions of the various embodiments and the
accompanying drawings, in which:
[0021] FIG. 1 is a graphic representation of .sup.31P-NMR of
liposomes and micelles in the presence of Mn.sup.2+.
DETAILED DESCRIPTION OF THE INVENTION
[0022] The invention relates to water soluble microaggregates (MA)
of hydrophobic agents and phospholipids or lipids. Water soluble
microaggregates are those which are miscible in water or other
aqueous solutions. Microaggregates refer to submicron size
aggregates of regular or irregular, and spherical or non-spherical
shape. For aggregates of roughly spherical shape, the approximate
diameters are less than one micrometer. For significantly
non-spherical aggregates, the approximate diameter of the aggregate
when rotating is less than one micrometer. Aggregates refer to
compositions comprising any aggregated complex of constituent
molecules. Hydrophobic agents refer to those which are poorly
soluble (less than 5 mg/ml water) or insoluble in water or other
aqueous solutions.
[0023] Hydrophobic agents for formulation into the MA of the
invention include any that may be used pharmaceutically,
agriculturally or industrially. These include biologically active,
or otherwise useful, molecules, pharmaceuticals, imaging agents,
and manufacturing reagents as well as precursors and prodrugs of
such substances. Preferred hydrophobic agents are those with
biological activity or other utility in humans and other living
organisms. These include agents that are therapeutics in medicine,
ingredients in cosmetics, and pesticides and herbicides in
agriculture. Examples of such agents include agonists and
antagonists, analgesic and anti-inflammatory agents, anesthetics,
antiadrenergic and antarrhythmics, antibiotics, anticholinergic and
cholinomimetic agents, anticonvulsant agents, antidepressants,
anti-epileptics, antifungal and antiviral agents, antihypertensive
agents, antimuscarinic and muscarinic agents, antineoplastic
agents, antipsychotic agents, anxiolytics, hormones, hypnotics and
sedatives, immunosuppressive and immunoactive agents, neuroleptic
agents, neuron blocking agents, and nutrients. Particularly
preferred agents include porphyrin photosensitizers such as "green
porphyrins" such as BPD-MA, EA6 and B3. Generally, any polypyrrolic
macrocyclic photosensitive compound that is hydrophobic can be used
in the invention.
[0024] Examples of these and other photosensitizers for use in the
present invention include, but are not limited to, angelicins, some
biological macromolecules such as lipofuscin; photosystem II
reaction centers; and D1-D2-cyt b-559 photosystem II reaction
centers, chalcogenapyrillium dyes, chlorins, chlorophylls,
coumarins, cyanines, ceratin DNA and related compounds such as
adenosine; cytosine; 2'-deoxyguanosine-5'-monophosphate;
deoxyribonucleic acid; guanine; 4-thiouridine; 2'-thymidine
5'-monophosphate; thymidylyl(3'-5')-2'-deoxyadenosine;
thymidylyl(3'-5')-2'-deoxyguanosine; thymine; and uracil, certain
drugs such as adriamycin; afloqualone; amodiaquine dihydrochloride;
chloroquine diphosphate; chlorpromazine hydrochloride; daunomycin;
daunomycinone; 5-iminodaunomycin; doxycycline; furosemide;
gilvocarcin M; gilvocarcin V; hydroxychloroquine sulfate;
lumidoxycycline; mefloquine hydrochloride; mequitazine; merbromin
(mercurochrome); primaquine diphosphate; quinacrine
dihydrochloride; quinine sulfate; and tetracycline hydrochloride,
certain flavins and related compounds such as alloxazine; flavin
mononucleotide; 3-hydroxyflavone; limichrome; limiflavin;
6-methylalloxazine; 7-methylalloxazine; 8-methylalloxazine;
9-methylalloxazine; 1-methyl limichrome; methyl-2-methoxybenzoate;
5-nitrosalicyclic acid; proflavine; and riboflavin, fullerenes,
metalloporphyrins, metallophthalocyanines, methylene blue
derivatives, naphthalimides, naphthalocyanines, certain natural
compounds such as
bis(4-hydroxy-3-methoxyphenyl)-1,6-heptadiene-3,5-dione;
4-(4-hydroxy-3-methoxyphenyl)-3-buten-2-one; N-formylkynurenine;
kynurenic acid; kynurenine; 3-hydroxykynurenine;
DL-3-hydroxykynurenine; sanguinarine; berberine; carmane; and
5,7,9(11), 22-ergostatetraene-3.beta.-ol, nile blue derivatives,
NSAIDs (nonsteroidal anti-inflammatory drugs), perylenequinones,
phenols, pheophorbides, pheophytins, photosensitizer dimers and
conjugates, phthalocyanines, porphycenes, porphyrins, psoralens,
purpurins, quinones, retinoids, rhodamines, thiophenes, verdins,
vitamins and xanthene dyes (Redmond and Gamlin, Photochem.
Photobiol., 70(4!:391-475 (1999)).
[0025] Exemplary angelicins include 3-aceto-angelicin; angelicin;
3,4'-dimethyl angelicin; 4,4'-dimethyl angelicin; 4,5'-dimethyl
angelicin; 6,4'-dimethyl angelicin; 6,4-dimethyl angelicin;
4,4',5'-trimethyl angelicin; 4,4',5'-trimethyl-1'-thioangelicin;
4,6,4'-trimethyl-1'-thioangelicin; 4,6,4'-trimethyl angelicin;
4,6,5'-trimethyl-1'-thioangelicin; 6,4,4'-trimethyl angelicin;
6,4',5'-trimethyl angelicin;
4,6,4',5'-tetramethyl-1'-thioangelicin; and 4,6,4',5'-tetramethyl
angelicin.
[0026] Exemplary chalcogenapyrillium dyes include pyrilium
perchlorate, 4,4'-(1,3-propenyl)-bis[2,6-di(1,1-dimethylethyl)]-;
pyrilium perchlorate,
2,6-bis(1,1-dimethyl-ethyl)-4-[1-[2,6-bis(1,1-dimethyl-ethyl)selenopyran--
4-ylidene]-3-propenyl-; pyrilium hexofluoro phosphate,
2,6-bis-(1,1-dimethyl-ethyl)-selenopyran-4-ylidene]-3-propenyl-;
pyrilium hexofluoro phosphate,
2,6-bis(1,1-dimethyl-ethyl)-selenopyran-4-ylidene]-3-propenyl-;
pyrilium perchlorate,
2,6-bis(1,1-dimethyl-ethyl)-4-[1-[2,6-bis(1,1-dimethyl-ethyl)telluropyran-
-4-ylidene]-3-propenyl-; pyrilium hexofluoro phosphate,
2,6-bis(1,1-dimethyl-ethyl)-4-[1-[2,6-bis(1,1-dimethyl-ethyl)telluropyran-
-4-ylidene]-3-propenyl-; pyrilium perchlorate,
2,6-bis(1,1-dimethyl-ethyl)-4-[1-[2,6-bis(1,1-dimethyl-ethyl)thiapyran-4--
ylidene]-3-propenyl]-; selenopyrilium hexofluoro phosphate,
2,6-bis(1,1-dimethyl-ethyl)-4-[1-[2,6-bis(1,1-dimethyl-ethyl)selenopyran--
4-ylidene]-3-propenyl]-; selenopyrilium,
2,6-bis(1,1-dimethylethyl)-4-[1-[2,6-bis(1,1-dimethylethyl)selenopyran-4--
ylidene]-3-propenyl]-; selenopyrilium percheorate,
2,6-bis(1,1-dimethyl-ethyl)-4-[1-[2,6-bis(1,1-dimethyl-ethyl).sub.4-[1-[2-
,6-bis(1,1-dimethyl-ethyl)telluropyran-4-ylidene]-3-propenyl]-;
selenopyrilium hexofluoro phosphate,
2,6-bis(1,1-dimethyl-ethyl)-4-[1-[2,6-bis(1,1-dimethyl-ethyl)telluropyran-
-4-ylidene]-3-propenyl]-; selenopyrilium hexofluoro phosphate,
2,6-bis(1,1-dimethyl-ethyl)-4-[2-[2,6-bis(1,1-dimethyl-ethyl)selenopyran--
4-ylidene]-4-(2-butenyl)]-; selenopyrilium hexofluoro phosphate,
2,6-bis(1,1-dimethyl-ethyl)-4-[2-[2,6-bis(1,1-dimethyl-ethyl)selenopyran--
4-ylidene]-4-(2-pentenyl)]-; telluropyrilium tetrafluoroborate,
2,6-bis(1,1-dimethylethyl)-4-[1-[2,6-bis(1,1-dimethyl-ethyl)-telluropyran-
-4-ylidene]-3-propenyl]-; telluropyrilium hexofluoro phosphate,
2,6-bis(1,1-dimethyl-ethyl)-4-[1-[2,6-bis(1,1-dimethyl-ethyl)telluropyran-
-4-ylidene]-3-propenyl]-; telluropyrilium hexofluoro phosphate,
2,6-bis(1,1-dimethyl-ethyl)-4-[1-[2,6-bis(1,1-dimethyl-ethyl)telluropyran-
-4-ylidene]ethyl-; telluropyrilium hexofluoro phosphate,
2,6-bis(1,1-dimethyl-ethyl)-4-[1-[2,6-bis(1,1-dimethyl-ethyl)-telluropyra-
n-4-ylidene]methyl-; thiopyrilium hexofluoro phosphate,
2,6-bis(1,1-dimethyl-ethyl)-4-[1-[2,6-bis(1,1-dimethyl-ethyl)thiopyran-4--
ylidene]-3-propenyl]-; thiopyrilium hexofluoro phosphate,
2,6-bis(1,1-dimethyl-ethyl).sub.4-[1-[2,6-bis(1,1-dimethyl-ethyl)selenopy-
ran-4-ylidene]-3-propenyl]-; and thiopyrilium hexofluoro phosphate,
2,6-bis(1,1-dimethyl-ethyl)-4-[1-[2,6-bis(1,1-dimethyl-ethyl)telluropyran-
-4-ylidene]-3-propenyl]-.
[0027] Exemplary chlorins dyes include 5-azachlorin dimethyl ester
derivative; 5,10,15,20-tetrakis-(m-hydroxyphenyl) bacteriochlorin;
benzoporphyrin derivative monoacid ring A; benzoporphyrin
derivative monoacid ring-A; porphine-2,18-dipropanoic acid,
7-[2-dimethyl-amino)-2-oxoethyl]-8-ethylidene-7,8-dihydro-3,7,12,17-tetra-
methyl, dimethylester; porphine-2,18-dipropanoic acid,
7-[2-dimethyl-amino)-2-oxoethyl]-8-ethylidene-8-ethyl-7,8-dihydro-3,7,12,-
17-tetramethyl, dimethylester Z; porphine-2,18-dipropanoic acid,
7-[2-dimethyl-amino)-2-oxoethyl]-8-ethylidene-8-ethyl-7,8-dihydro-3,7,12,-
17-tetramethyl, dimethylester Z ECHL; porphine-2,18-dipropanoic
acid,
7-[2-dimethyl-amino)-2-oxoethyl]-8-ethylidene-8-n-heptyl-7,8-dihydro-3,7,-
12,17-tetramethyl, dimethylester Z; tin (II)
porphine-2,18-dipropanoic acid,
7-[2-(dimethylamino-2-oxoethyl]-8-ethylidene-8-n-heptyl-7,8-dihydro-
-3,7,12,17-tetramethyl, dimethylester Z; chlorin e.sub.6; chlorin
e.sub.6 dimethyl ester; chlorin e.sub.6 k.sub.3; chlorin e.sub.6
monomethyl ester; chlorin e.sub.6 Na.sub.3; chlorin p.sub.6;
chlorin p.sub.6-trimethylester; chlorin derivative zinc (II)
porphine-2,18-dipropanoic acid,
7-[2-(dimethylamino)-2-oxoethyl]-8-ethylidene-8-n-heptyl-7,8-dihydro-3,7,-
12,17-tetramethyl, dimethylester Z;
13.sup.1-deoxy-20-formyl-vic-dihydroxy-bacteriochlorin
di-tert-butyl aspartate;
13.sup.1-deoxy-20-formyl-4-keto-bacteriochlorin di-tert-butyl
aspartate; di-L-aspartyl chlorin e.sub.6; mesochlorin;
5,10,15,20-tetrakis-(m-hydroxyphenyl) chlorin;
meta-(tetrahydroxyphenyl)chlorin;
methyl-131-deoxy-20-formyl-4-keto-bacteriochlorin; mono-L-aspartyl
chlorin e.sub.6; photoprotoporphyrin IX dimethyl ester;
phycocyanobilin dimethyl ester; protochlorophyllide a; tin (IV)
chlorin e.sub.6; tin chlorin e.sub.6; tin L-aspartyl chlorin
e.sub.6; tin octaethyl-benzochlorin; tin (IV) chlorin; zinc chlorin
e.sub.6; and zinc L-aspartyl chlorin e.sub.6
[0028] Exemplary chlorophylls dyes include chlorophyll a;
chlorophyll b; oil soluble chlorophyll; bacteriochlorophyll a;
bacteriochlorophyll b; bacteriochlorophyll c; bacteriochlorophyll
d; protochlorophyll; protochlorophyll a; amphiphilic chlorophyll
derivative 1; and amphiphilic chlorophyll derivative 2.
[0029] Exemplary coumarins include 3-benzoyl-7-methoxycoumarin;
7-diethylamino-3-thenoylcoumarin; 5,7-dimethoxy-3-(1-naphthoyl)
coumarin; 6-methylcoumarin; 2H-selenolo[3,2-g][1]benzopyran-2-one;
2H-selenolo[3,2-g][1]benzothiopyran-2-one;
7H-selenolo[3,2-g][1]benzoseleno-pyran-7-one;
7H-selenopyrano[3,2-f][1]benzofuran-7-one;
7H-selenopyrano[3,2-f][1]benzo-thiophene-7-one;
2H-thienol[3,2-g][1]benzopyran-2-one;
7H-thienol[3,2-g][1]benzothiopyran-7-one;
7H-thiopyrano[3,2-f][1]benzofuran-7-one; coal tar mixture; khellin;
RG 708; RG277; and visnagin.
[0030] Exemplary cyanines include benzoselenazole dye; benzoxazole
dye; 1,1'-diethyloxacarbocyanine; 1,1'-diethyloxadicarbocyanine;
1,1'-diethylthiacarbocyanine; 3,3'-dialkylthiacarbocyanines
(n=2-18); 3,3'-diethylthiacarbocyanine iodide;
3,3'-dihexylselenacarbocyanine; kryptocyanine; MC540 benzoxazole
derivative; MC540 quinoline derivative; merocyanine 540; and
meso-ethyl, 3,3'-dihexylselenacarbocyanine.
[0031] Exemplary fullerenes include C.sub.60; C.sub.70; C.sub.76;
dihydro-fullerene;
1,9-(4-hydroxy-cyclohexano)-buckminster-fullerene;
[1-methyl-succinate-4-methyl-cyclohexadiene-2,3]-buckminster-fullerene;
and tetrahydro fullerene.
[0032] Exemplary metalloporphyrins include cadmium (II)
chlorotexaphyrin nitrate; cadmium (II) meso-diphenyl
tetrabenzoporphyrin; cadmium
meso-tetra-(4-N-methylpyridyl)-porphine; cadmium (II) texaphyrin;
cadmium (II) texaphyrin nitrate; cobalt
meso-tetra-(4-N-methylpyridyl)-porphine; cobalt (II)
meso(4-sulfonatophenyl)-porphine; copper hematoporphyrin; copper
meso-tetra-(4-N-methylpyridyl)-porphine; copper (II)
meso(4-sulfonatophenyl)-porphine; Europium (III) dimethyltexaphyrin
dihydroxide; gallium tetraphenylporphyrin; iron
meso-tetra(4-N-methylpyridyl)-porphine; lutetium (III)
tetra(N-methyl-3-pyridyl)-porphyrin chloride; magnesium (II)
meso-diphenyl tetrabenzoporphyrin; magnesium tetrabenzoporphyrin;
magnesium tetraphenylporphyrin; magnesium (II)
meso(4-sulfonatophenyl)-porphine; magnesium (II) texaphyrin
hydroxide metalloporphyrin; magnesium
meso-tetra-(4-N-methylpyridyl)-porphine; manganese
meso-tetra-(4-N-methylpyridyl)-porphine; nickel
meso-tetra(4-N-methylpyridyl)-porphine; nickel (II)
meso-tetra(4-sulfonatophenyl)-porphine; palladium (II)
meso-tetra-(4-N-methylpyridyl)-porphine; palladium
meso-tetra-(4-N-methylpyridyl)-porphine; palladium
tetraphenylporphyrin; palladium (II)
meso(4-sulfonatophenyl)-porphine; platinum (II)
meso(4-sulfonatophenyl)-porphine; samarium (II) dimethyltexaphyrin
dihydroxide; silver (II) meso(4-sulfonatophenyl)-porphine; tin (IV)
protoporphyrin; tin meso-tetra-(4-N-methylpyridyl)-porphine; tin
meso-tetra(4-sulfonatophenyl)-porphine; tin (IV)
tetrakis(4-sulfonatophenyl) porphyrin dichloride; zinc (II)
15-aza-3,7,12,18-tetramethyl-porphyrinato-13,17-diyl-dipropionic
acid-dimethylester; zinc (II) chlorotexaphyrin chloride; zinc
coproporphyrin III; zinc (II)
2,11,20,30-tetra-(1,1-dimethyl-ethyl)tetranaphtho(2,3-b:2',3'-g:2''3''-l:-
2'''3'''-q)porphyrazine; zinc (II)
2-(3-pyridyloxy)benzo[b]-10,19,28-tri(1,1-dimethylethyl)trinaphtho[2',3'--
g:2''3''l::2''',3'''-q] porphyrazine; zinc (II)
2,18-bis-(3-pyridyloxy)dibenzo[b,l]-10,26-di(1,1-dimethyl-ethyl)dinaphtho-
[2',3'-g:2''',3'''-q]porphyrazine; zinc (II)
2,9-bis-(3-pyridyloxy)dibenzo[b,g]-17,26-di(1,1-dimethyl-ethyl)dinaphtho[-
2'',3''-l:2''',3'''-q]porphyrazine; zinc (II)
2,9,16-tris-(3-pyridyloxy)
tribenzo[b,g,l]-24=(1,1-dimethyl-ethyl)naphtho[2''',3'''-q]porphyrazine;
zinc (II) 2,3-bis-(3-pyridyloxy)
benzo[b]-10,19,28-tri(1.1-dimethyl-ethyl)trinaphtho[2',3'-g:2'',3''l:2'''-
,3'''-q]porphyrazine; zinc (II) 2,3,18,19-tetrakis-(3-pyridyloxy)
dibenzo[b,l]-10,26-di(1,1-dimethyl-ethyl)trinaphtho[2',3'-g:2''',3'''-q]p-
orphyrazine; zinc (II) 2,3,9,10-tetrakis-(3-pyridyloxy)
dibenzo[b,g]-17,26-di(1,1-dimethyl-ethyl)dinaphtho[2'',3''-l:2''',3'''-q]-
porphyrazine; zinc (II)
2,3,9,10,16,17-hexakis-(3-pyridyloxy)tribenzo[b,g,l]-24-(1,1-dimethyl-eth-
yl)naphtho[2''',3'''-q]porphyrazine; zinc (II)
2-(3-N-methyl)pyridyloxy)benzo[b]-10,19,28-tri(1,1-dimethyl-ethyl)trinaph-
tho[2',3'-g:2'',3''l:2''',3'''-q]porphyrazine monoiodide; zinc (II)
2,18-bis-(3-(N-methyl)pyridyloxy)dibenzo[b,l]-10,26-di(1,1-dimethylethyl)-
dinaphtho[2',3'-g:2''',3'''-q]porphyrazine diiodide; zinc (II)
2,9-bis-(3-(N-methyl)pyridyloxy)dibenzo[b,g]-17,26-di(1,1-dimethylethyl)d-
inaphtho[2'',3''-l:2''',3'''-q]porphyrazine diiodide; zinc (II)
2,9,16-tris-(3-(N-methyl-pyridyloxy)tribenzo[b,g,l]-24-(1,1-dimethylethyl-
)naphtho[2''',3'''-q]porphyrazine triiodide; zinc (II)
2,3-bis-(3-(N-methyl)pyridyloxy)benzo[b]-10,19,28-tri(1,1-dimethylethyl)t-
rinaphtho[2',3'-g:2''',3'''-l:2''',3'''-q]porphyrazine diiodide;
zinc (II)
2,3,18,19-tetrakis-(3-(N-methyl)pyridyloxy)dibenzo[b,l]-10,26-di(1,1-dime-
thyl)dinaphtho[2',3'-g:2''',3'''-q]porphyrazine tetraiodide; zinc
(II)
2,3,9,10-tetrakis-(3-(N-methyl)pyridyloxy)dibenzo[g,g]-17,26-di(1,1-dimet-
hylethyl)dinaphtho[2'',3''-l:2''',3'''-q]porphyrazine tetraiodide;
zinc (II)
2,3,9,10,16,17-hexakis-(3-(N-methyl)pyridyloxy)tribenzo[b,g,l]-24-(1-
,1-dimethylethyl)naphtho[2''',3'''-q]porphyrazine hexaiodide; zinc
(II) meso-diphenyl tetrabenzoporphyrin; zinc (II) meso-triphenyl
tetrabenzoporphyrin; zinc (II)
meso-tetrakis(2,6-dichloro-3-sulfonatophenyl) porphyrin; zinc (II)
meso-tetra-(4-N-methylpyridyl)-porphine; zinc (II)
5,10,15,20-meso-tetra(4-octyl-phenylpropynyl)-porphine; zinc
porphyrin c; zinc protoporphyrin; zinc protoporphyrin IX; zinc (II)
meso-triphenyl-tetrabenzoporphyrin; zinc tetrabenzoporphyrin; zinc
(II) tetrabenzoporphyrin; zinc tetranaphthaloporphyrin; zinc
tetraphenylporphyrin; zinc (II) 5,10,15,20-tetraphenylporphyrin;
zinc (II) meso (4-sulfonatophenyl)-porphine; and zinc (II)
texaphyrin chloride.
[0033] Exemplary metallophthalocyanines include aluminum
mono-(6-carboxy-pentyl-amino-sulfonyl)-trisulfo-phthalocyanine;
aluminum
di-(6-carboxy-pentyl-amino-sulfonyl)-trisulfophthalocyanine;
aluminum (III) octa-n-butoxy phthalocyanine; aluminum
phthalocyanine; aluminum (III) phthalocyanine disulfonate; aluminum
phthalocyanine disulfonate; aluminum phthalocyanine disulfonate
(cis isomer); aluminum phthalocyanine disulfonate (clinical prep.);
aluminum phthalocyanine phthalimido-methyl sulfonate; aluminum
phthalocyanine sulfonate; aluminum phthalocyanine trisulfonate;
aluminum (III) phthalocyanine trisulfonate; aluminum (III)
phthalocyanine tetrasulfonate; aluminum phthalocyanine
tetrasulfonate; chloroaluminum phthalocyanine; chloroaluminum
phthalocyanine sulfonate; chloroaluminum phthalocyanine
disulfonate; chloroaluminum phthalocyanine tetrasulfonate;
chloroaluminum-t-butyl-phthalocyanine; cobalt phthalocyanine
sulfonate; copper phthalocyanine sulfonate; copper (II)
tetra-carboxy-phthalocyanine; copper (II)-phthalocyanine; copper
t-butyl-phthalocyanine; copper phthalocyanine sulfonate; copper
(II)
tetrakis-[methylene-thio[(dimethyl-amino)methylidyne]]phthalocyanine
tetrachloride; dichlorosilicon phthalocyanine; gallium (III)
octa-n-butoxy phthalocyanine; gallium (II) phthalocyanine
disulfonate; gallium phthalocyanine disulfonate; gallium
phthalocyanine tetrasulfonate-chloride; gallium (II) phthalocyanine
tetrasulfonate; gallium phthalocyanine trisulfonate-chloride;
gallium (II) phthalocyanine trisulfonate; GaPcS.sub.1tBu.sub.3;
GaPcS.sub.2tBu.sub.2; GaPcS.sub.3tBu.sub.1; germanium (IV)
octa-n-butoxy phthalocyanine; germanium phthalocyanine derivative;
silicon phthalocyanine derivative; germanium (IV) phthalocyanine
octakis-alkoxy-derivatives; iron phthalocyanine sulfonate; lead
(II) 2,3,9,10,16,17,23,24-octakis(3,6-dioxaheptyloxy)
phthalocyanine; magnesium t-butyl-phthalocyanine; nickel (II)
2,3,9,10,16,17,23,24-octakis(3,6-dioxaheptyloxy) phthalocyanine;
palladium (II) octa-n-butoxy phthalocyanine; palladium (II)
tetra(t-butyl)-phthalocyanine; (diol)
(t-butyl).sub.3-phthalocyanato palladium(II); ruthenium(II)
dipotassium[bis(triphenyl-phosphine-monosulphonate) phthalocyanine;
silicon phthalocyanine bis(tri-n-hexyl-siloxy)-; silicon
phthalocyanine bis(tri-phenyl-siloxy)-;
HOSiPcOSi(CH.sub.3).sub.2(CH.sub.2).sub.3N(CH.sub.3).sub.2;
HOSiPcOSi(CH.sub.3).sub.2(CH.sub.2).sub.3N(CH.sub.2CH.sub.3).sub.2;
SiPc[OSi(CH.sub.3).sub.2(CH.sub.2).sub.3N(CH.sub.3).sub.2].sub.2;
SiPc[OSi(CH.sub.3).sub.2(CH.sub.2).sub.3N(CH.sub.2CH.sub.3)(CH.sub.2).sub-
.2N(CH.sub.3).sub.2].sub.2; tin (IV) octa-n-butoxy phthalocyanine;
vanadium phthalocyanine sulfonate; zinc (II) octa-n-butoxy
phthalocyanine; zinc (II)
2,3,9,10,16,17,23,24-octakis(2-ethoxy-ethoxy) phthalocyanine; zinc
(II) 2,3,9,10,16,17,23,24-octakis(3,6-dioxaheptyloxy)
phthalocyanine; zinc (II)
1,4,8,11,15,18,22,25-octa-n-butoxy-phthalocyanine;
zn(II)-phthalocyanine-octabutoxy; zn(II)-phthalocyanine; zinc
phthalocyanine; zinc (II) phthalocyanine; zinc phthalocyanine and
perdeuterated zinc phthalocyanine; zinc (II) phthalocyanine
disulfonate; zinc phthalocyanine disulfonate; zinc phthalocyanine
sulfonate; zinc phthalocyanine tetrabromo-; zinc (II)
phthalocyanine tetra-t-butyl-; zinc (II) phthalocyanine
tetra-(t-butyl)-; zinc phthalocyanine tetracarboxy-; zinc
phthalocyanine tetrachloro-; zinc phthalocyanine tetrahydroxyl;
zinc phthalocyanine tetraiodo-; zinc ((I)
tetrakis-(1,1-dimethyl-2-phthalimido)ethyl phthalocyanine; zinc
(II) tetrakis-(1,1-dimethyl-2-amino)-ethyl-phthalocyanine; zinc
(II) phthalocyanine tetrakis(1,1-dimethyl-2-trimethyl
ammonium)ethyl tetraiodide; zinc phthalocyanine tetrasulphonate;
zinc phthalocyanine tetrasulfonate; zinc (II) phthalocyanine
tetrasulfonate; zinc (II) phthalocyanine trisulfonate; zinc
phthalocyanine trisulfonate; zinc (II)
(t-butyl).sub.3-phthalocyanine diol; zinc
tetradibenzobarreleno-octabutoxy-phthalocyanine; zinc (II)
2,9,16,23,-tetrakis-(3-(N-methyl)pyridyloxy)phthalocyanine
tetraiodide; and zinc (II)
2,3,9,10,16,17,23,24-octakis-(3-(N-methyl)pyridyloxy)phthalocyanine
complex octaiodide; and zinc (II)
2,3,9,10,16,17,23,24-octakis-(3-pyridyloxy)phthalocyanine.
[0034] Exemplary methylene blue derivatives include 1-methyl
methylene blue; 1,9-dimethyl methylene blue; methylene blue;
methylene blue (16 .mu.M); methylene blue (14 .mu.M); methylene
violet; bromomethylene violet; 4-iodomethylene violet;
1,9-dimethyl-3-dimethyl-amino-7-diethyl-amino-phenothiazine; and
1,9-dimethyl-3-diethylamino-7-dibutyl-amino-phenothiazine.
[0035] Exemplary naphthalimides blue derivatives include
N,N-bis-(hydroperoxy-2-methoxyethyl)-1,4,5,8-naphthaldiimide;
N-(hydroperoxy-2-methoxyethyl)-1,8-naphthalimide;
1,8-naphthalimide;
N,N-bis(2,2-dimethoxyethyl)-1,4,5,8-naphthaldiimide; and
N,N'-bis(2,2-dimethylpropyl)-1,4,5,8-naphthaldiimide.
[0036] Exemplary naphthalocyanines include aluminum
t-butyl-chloronaphthalocyanine; silicon
bis(dimethyloctadecylsiloxy) 2,3-naphthalocyanine; silicon
bis(dimethyloctadecylsiloxy) naphthalocyanine; silicon
bis(dimethylthexylsiloxy) 2,3-naphthalocyanine; silicon
bis(dimethylthexylsiloxy) naphthalocyanine; silicon
bis(t-butyldimethylsiloxy) 2,3-naphthalocyanine; silicon
bis(tert-butyldimethylsiloxy) naphthalocyanine; silicon
bis(tri-n-hexylsiloxy) 2,3-naphthalocyanine; silicon
bis(tri-n-hexylsiloxy) naphthalocyanine; silicon naphthalocyanine;
t-butylnaphthalocyanine; zinc (II) naphthalocyanine; zinc (II)
tetraacetylamidonaphthalocyanine; zinc (II)
tetraminonaphthalocyanine; zinc (II)
tetrabenzamidonaphthalocyanine; zinc (II)
tetrahexylamidonaphthalocyanine; zinc (II)
tetramethoxy-benzamidonaphthalocyanine; zinc (II)
tetramethoxynaphthalocyanine; zinc naphthalocyanine tetrasulfonate;
and zinc (II) tetradodecylamidonaphthalocyanine.
[0037] Exemplary nile blue derivatives include
benzo[a]phenothiazinium, 5-amino-9-diethylamino-;
benzo[a]phenothiazinium, 5-amino-9-diethylamino-6-iodo-;
benzo[a]phenothiazinium, 5-benzylamino-9-diethylamino-;
benzo[a]phenoxazinium, 5-amino-6,8-dibromo-9-ethylamino-;
benzo[a]phenoxazinium, 5-amino-6,8-diiodo-9-ethylamino-;
benzo[a]phenoxazinium, 5-amino-6-bromo-9-diethylamino-;
benzo[a]phenoxazinium, 5-amino-9-diethylamino-(nile blue A);
benzo[a]phenoxazinium, 5-amino-9-diethylamino-2,6-diiodo-;
benzo[a]phenoxazinium, 5-amino-9-diethylamino-2,-iodo;
benzo[a]phenoxazinium, 5-amino-9-diethylamino-6-iodo-;
benzo[a]phenoxazinium, 5-benzylamino-9-diethylamino-(nile blue 2B);
5-ethylamino-9-diethylamino-benzo[a]phenoselenazinium chloride;
5-ethylamino-9-diethyl-aminobenzo[a]phenothiazinium chloride; and
5-ethylamino-9-diethyl-aminobenzo[a]phenoxazinium chloride.
[0038] Exemplary NSAIDs (nonsteroidal anti-inflammatory drugs)
include benoxaprofen; carprofen; carprofen dechlorinated
(2-(2-carbazolyl) propionic acid); carprofen (3-chlorocarbazole);
chlorobenoxaprofen; 2,4-dichlorobenoxaprofen; cinoxacin;
ciprofloxacin; decarboxy-ketoprofen; decarboxy-suprofen;
decarboxy-benoxaprofen; decarboxy-tiaprofenic acid; enoxacin;
fleroxacin; fleroxacin-N-oxide; flumequine; indoprofen; ketoprofen;
lomelfloxacin; 2-methyl-4-oxo-2H-1,2-benzothiazine-1,1-dioxide;
N-demethyl fleroxacin; nabumetone; nalidixic acid; naproxen;
norfloxacin; ofloxacin; pefloxacin; pipemidic acid; piroxicam;
suprofen; and tiaprofenic acid.
[0039] Exemplary perylenequinones include hypericins such as
hypericin; hypericin monobasic sodium salt; di-aluminum hypericin;
di-copper hypericin; gadolinium hypericin; terbium hypericin,
hypocrellins such as acetoxy hypocrellin A; acetoxy hypocrellin B;
acetoxy iso-hypocrellin A; acetoxy iso-hypocrellin B;
3,10-bis[2-(2-aminoethylamino)ethanol]hypocrellin B;
3,10-bis[2-(2-aminoethoxy)ethanol]hypocrellin B;
3,10-bis[4-(2-aminoethyl)morpholine]hypocrellin B; n-butylaminated
hypocrellin B; 3,10-bis(butylamine) hypocrellin B;
4,9-bis(butylamine) hypocrellin B; carboxylic acid hypocrellin B;
cystamine-hypocrellin B; 5-chloro hypocrellin A or 8-chloro
hypocrellin A; 5-chloro hypocrellin B or 8-chloro hypocrellin B;
8-chloro hypocrellin B; 8-chloro hypocrellin A or 5-chloro
hypocrellin A; 8-chloro hypocrellin B or 5-chloro hypocrellin B;
deacetylated aldehyde hypocrellin B; deacetylated hypocrellin B;
deacetylated hypocrellin A; deacylated, aldehyde hypocrellin B;
demethylated hypocrellin B; 5,8-dibromo hypocrellin A; 5,8-dibromo
hypocrellin B; 5,8-dibromo iso-hypocrellin B;
5,8-dibromo[1,12-CBr.dbd.CMeCBr(COMe)] hypocrellin B;
5,8-dibromo[1,12-CHBrC(.dbd.CH.sub.2)CBr(COMe)] hypocrellin B;
5,8-dibromo[1-CH.sub.2COMe, 12-COCOCH.sub.2Br--] hypocrellin B;
5,8-dichloro hypocrellin A; 5,8-dichloro hypocrellin B;
5,8-dichlorodeacytylated hypocrellin B; 5,8-diiodo hypocrellin A;
5,8-diiodo hypocrellin B; 5,8
diiodo[1,12-CH.dbd.CMeCH(COCH.sub.2I.sub.2)--] hypocrellin B;
5,8-diiodo[1,12-CH.sub.2C(CH.sub.2I).dbd.C(COMe)-] hypocrellin B;
2-(N,N-diethylamino) ethylaminated hypocrellin B;
3,10-bis[2-(N,N-diethylamino)-ethylamine]hypocrellin B;
4,9-bis[2-(N,N-diethyl-amino)-ethylamine] iso-hypocrellin B;
dihydro-1,4-thiazine carboxylic acid hypocrellin B;
dihydro-1,4-thiazine hypocrellin B; 2-(N,N-dimethylamino)
propylamine hypocrellin B;
dimethyl-1,3,5,8,10,12-hexamethoxy-4,9-perylenequinone-6,7-diacetate;
dimethyl-5,8-dihydroxy-1,3,10,13-tetramethoxy-4,9-perylenequinone-6,7-dia-
cetate; 2,11-dione hypocrellin A; ethanolamine hypocrellin B;
ethanolamine iso-hypocrellin B; ethylenediamine hypocrellin B;
11-hydroxy hypocrellin B or 2-hydroxy hypocrellin B; hypocrellin A;
hypocrellin B; 5-iodo[1,12-CH.sub.2C(CH.sub.2I)=C(COMe)-]
hypocrellin B; 8-iodo[1,12-CH.sub.2C(CH.sub.2I).dbd.C(COMe)-]
hypocrellin B; 9-methylamino iso-hypocrellin B;
3,10-bis[2-(N,N-methylamino)propylamine]hypocrellin B;
4,9-bis(methylamine iso-hypocrellin B; 14-methylamine
iso-hypocrellin B; 4-methylamine iso-hypocrellin B; methoxy
hypocrellin A; methoxy hypocrellin B; methoxy iso-hypocrellin A;
methoxy iso-hypocrellin B; methylamine hypocrellin B; 2-morpholino
ethylaminated hypocrellin B; pentaacetoxy hypocrellin A; PQP
derivative; tetraacetoxy hypocrellin B; 5,8,15-tribromo hypocrellin
B; calphostin C, Cercosporins such as acetoxy cercosporin; acetoxy
iso-cercosporin; aminocercosporin; cercosporin;
cercosporin+iso-cercosporin (1/1 molar); diaminocercosporin;
dimethylcercosporin; 5,8-dithiophenol cercosporin; iso-cercosporin;
methoxycercosporin; methoxy iso-cercosporin; methylcercosporin;
noranhydrocercosporin; elsinochrome A; elsinochrome B; phleichrome;
and rubellin A.
[0040] Exemplary phenols include 2-benzylphenol;
2,2'-dihydroxybiphenyl; 2,5-dihydroxybiphenyl; 2-hydroxybiphenyl;
2-methoxybiphenyl; and 4-hydroxybiphenyl.
[0041] Exemplary pheophorbides include pheophorbide a; methyl
13.sup.1-deoxy-20-formyl-7,8-vic-dihydro-bacterio-meso-pheophorbide
a; methyl-2-(1-dodecyloxyethyl)-2-devinyl-pyropheophorbide a;
methyl-2-(1-heptyl-oxyethyl)-2-devinyl-pyropheophorbide a;
methyl-2-(1-hexyl-oxyethyl)-2-devinyl-pyropheophorbide a;
methyl-2-(1-methoxy-ethyl)-2-devinyl-pyropheophorbide a;
methyl-2-(1-pentyl-oxyethyl)-2-devinyl-pyropheophorbide a;
magnesium methyl bacteriopheophorbide d;
methyl-bacteriopheophorbide d; and pheophorbide.
[0042] Exemplary pheophytins include bacteriopheophytin a;
bacteriopheophytin b; bacteriopheophytin c; bacteriopheophytin d;
10-hydroxy pheophytin a; pheophytin; pheophytin a; and
protopheophytin.
[0043] Exemplary photosensitizer dimers and conjugates include
aluminum
mono-(6-carboxy-pentyl-amino-sulfonyl)-trisulfophthalocyanine
bovine serum albumin conjugate; dihematoporphyrin ether (ester);
dihematoporphyrin ether; dihematoporphyrin ether (ester)-chlorin;
hematoporphyrin-chlorin ester; hematoporphyrin-low density
lipoprotein conjugate; hematoporphyrin-high density lipoprotein
conjugate; porphine-2,7,18-tripropanoic acid,
13,13'-(1,3-propanediyl)bis[3,8,12,17-tetramethyl]-;
porphine-2,7,18-tripropanoic acid,
13,13'-(1,11-undecanediyl)bis[3,8,12,17-tetramethyl]-;
porphine-2,7,18-tripropanoic acid,
13,13'-(1,6-hexanediyl)bis[3,8,12,17-tetramethyl]-; SnCe.sub.6-MAb
conjugate 1.7:1; SnCe.sub.6-MAb conjugate 1.7:1; SnCe.sub.6-MAb
conjugate 6.8: 1; SnCe.sub.6-MAb conjugate 11.2: 1; SnCe.sub.6-MAb
conjugate 18.9:1; SnCe.sub.6-dextran conjugate 0.9:1;
SnCe.sub.6-dextran conjugate 3.5:1; SnCe.sub.6-dextran conjugate
5.5:1; SnCe.sub.6-dextran conjugate 9.9:1;
.alpha.-terthienyl-bovine serum albumin conjugate (12:1);
.alpha.-terthienyl-bovine serum albumin conjugate (4:1); and
tetraphenylporphine linked to 7-chloroquinoline.
[0044] Exemplary phthalocyanines include (diol)
(t-butyl).sub.3-phthalocyanine; (t-butyl).sub.4-phthalocyanine;
cis-octabutoxy-dibenzo-dinaphtho-porphyrazine;
trans-octabutoxy-dibenzo-dinaphtho-porphyrazine;
2,3,9,10,16,17,23,24-octakis2-ethoxyethoxy) phthalocyanine;
2,3,9,10,16,17,23,24-octakis(3,6-dioxaheptyloxy) phthalocyanine;
octa-n-butoxy phthalocyanine; phthalocyanine; phthalocyanine
sulfonate; phthalocyanine tetrasulphonate; phthalocyanine
tetrasulfonate; t-butyl-phthalocyanine; tetra-t-butyl
phthalocyanine; and
tetradibenzobarreleno-octabutoxy-phthalocyanine.
[0045] Exemplary porphycenes include
2,3-(2.sup.3-carboxy-2.sup.4-methoxycarbonyl
benzo)-7,12,17-tris(2-methoxyethyl) porphycene;
2-(2-hydroxyethyl)-7,12,17-tri(2-methoxyethyl) porphycene;
2-(2-hydroxyethyl)-7,12,17-tri-n-propyl-porphycene;
2-(2-methoxyethyl)-7,12,17-tri-n-propyl-porphycene;
2,7,12,17-tetrakis(2-methoxyethyl) porphycene;
2,7,12,17-tetrakis(2-methoxyethyl)-9-hydroxy-porphycene;
2,7,12,17-tetrakis(2-methoxyethyl)-9-methoxy-porphycene;
2,7,12,17-tetrakis(2-methoxyethyl)-9-n-hexyloxy-porphycene;
2,7,12,17-tetrakis(2-methoxyethyl)-9-acetoxy-porphycene;
2,7,12,17-tetrakis(2-methoxyethyl)-9-caproyloxy-porphycene;
2,7,12,17-tetrakis(2-methoxyethyl)-9-pelargonyloxy-porphycene;
2,7,12,17-tetrakis(2-methoxyethyl)-9-stearoyloxy-porphycene;
2,7,12,17-tetrakis(2-methoxyethyl)-9-(N-t-butoxycarbonylglycinoxy)
porphycene;
2,7,12,17-tetrakis(2-methoxyethyl)-9-[4-((.beta.-apo-7-carotenyl)benzoylo-
xy]-porphycene; 2,7,12,17-tetrakis(2
methoxyethyl)-9-amino-porphycene;
2,7,12,17-tetrakis(2-methoxyethyl)-9-acetamido-porphycene;
2,7,12,17-tetrakis(2-methoxyethyl)-9-glutaramido-porphycene;
2,7,12,17-tetrakis(2-methoxyethyl)-9-(methyl-glutaramido)-porphycene;
2,7,12,17-tetrakis(2-methoxyethyl)-9-(glutarimido)-porphycene;
2,7,12,17-tetrakis(2-methoxyethyl)-3-(N,N-dimethylaminomethyl)-porphycene-
;
2,7,12,17-tetrakis(2-methoxyethyl)-3-(N,N-dimethylaminomethyl)-porphycen-
e hydrochloride; 2,7,12,17-tetrakis(2-ethoxyethyl)-porphycene;
2,7,12,17-tetra-n-propyl-porphycene;
2,7,12,17-tetra-n-propyl-9-hydroxy-porphycene;
2,7,12,17-tetra-n-propyl-9-methoxy-porphycene;
2,7,12,17-tetra-n-propyl-9-acetoxy porphycene;
2,7,12,17-tetra-n-propyl-9-(t-butyl glutaroxy)-porphycene;
2,7,12,17-tetra-n-propyl-9-(N-t-butoxycarbonylglycinoxy)
porphycene;
2,7,12,17-tetra-n-propyl-9-(4-N-t-butoxy-carbonyl-butyroxy)-porphycene;
2,7,12,17-tetra-n-propyl-9-amino-porphycene;
2,7,12,17-tetra-n-propyl-9-acetamido-porphycene;
2,7,12,17-tetra-n-propyl-9-glutaramido-porphycene;
2,7,12,17-tetra-n-propyl-9-(methyl glutaramido)-porphycene;
2,7,12,17-tetra-n-propyl-3-(N,N-dimethylaminomethyl) porphycene;
2,7,12,17-tetra-n-propyl-9, 10-benzo porphycene;
2,7,12,17-tetra-n-propyl-9-p-benzoyl carboxy-porphycene;
2,7,12,17-tetra-n-propyl-porphycene; 2,7,12,17-tetra-t-butyl-3,6;
13,16-dibenzo-porphycene;
2,7-bis(2-hydroxyethyl)-12,17-di-n-propyl-porphycene;
2,7-bis(2-methoxyethyl)-12,17-di-n-propyl-porphycene; and
porphycene.
[0046] Exemplary porphyrins include 5-azaprotoporphyrin
dimethylester; bis-porphyrin; coproporphyrin III; coproporphyrin
III tetramethylester; deuteroporphyrin; deuteroporphyrin IX
dimethylester; diformyldeuteroporphyrin IX dimethylester;
dodecaphenylporphyrin; hematoporphyrin; hematoporphyrin (8 .mu.M);
hematoporphyrin (400 .mu.M); hematoporphyrin (3 .mu.M);
hematoporphyrin (18 .mu.M); hematoporphyrin (30 .mu.M);
hematoporphyrin (67 .mu.M); hematoporphyrin (150 .mu.M);
hematoporphyrin IX; hematoporphyrin monomer; hematoporphyrin dimer;
hematoporphyrin derivative; hematoporphyrin derivative (6 .mu.M);
hematoporphyrin derivative (200 .mu.M); hematoporphyrin derivative
A (20 .mu.M); hematoporphyrin IX dihydrochloride; hematoporphyrin
dihydrochloride; hematoporphyrin IX dimethylester; haematoporphyrin
IX dimethylester; mesoporphyrin dimethylester; mesoporphyrin IX
dimethylester; monoformyl-monovinyl-deuteroporphyrin IX
dimethylester; monohydroxyethylvinyl deuteroporphyrin;
5,10,15,20-tetra(o-hydroxyphenyl) porphyrin;
5,10,15,20-tetra(m-hydroxyphenyl) porphyrin;
5,10,15,20-tetrakis-(m-hydroxyphenyl) porphyrin;
5,10,15,20-tetra(p-hydroxyphenyl) porphyrin; 5,10,15,20-tetrakis
(3-methoxyphenyl) porphyrin; 5,10,15,20-tetrakis
(3,4-dimethoxyphenyl) porphyrin; 5,10,15,20-tetrakis
(3,5-dimethoxyphenyl) porphyrin; 5,10,15,20-tetrakis
(3,4,5-trimethoxyphenyl) porphyrin;
2,3,7,8,12,13,17,18-octaethyl-5,10,15,20-tetraphenylporphyrin;
Photofrin.RTM.; Photofrin.RTM. II; porphyrin c; protoporphyrin;
protoporphyrin IX; protoporphyrin dimethylester; protoporphyrin IX
dimethylester; protoporphyrin propylaminoethylformamide iodide;
protoporphyrin N,N-dimethylaminopropylformamide; protoporphyrin
propylaminopropylformamide iodide; protoporphyrin butylformamide;
protoporphyrin N,N-dimethylamino-formamide; protoporphyrin
formamide; sapphyrin 13,12,13,22-tetraethyl-2,7,18,23 tetramethyl
sapphyrin-8,17-dipropanol; sapphyrin 2
3,12,13,22-tetraethyl-2,7,18,23 tetramethyl
sapphyrin-8-monoglycoside; sapphyrin 3;
meso-tetra-(4-N-carboxyphenyl)-porphine;
tetra-(3-methoxyphenyl)-porphine;
tetra-(3-methoxy-2,4-difluorophenyl)-porphine;
5,10,15,20-tetrakis(4-N-methylpyridyl) porphine;
meso-tetra-(4-N-methylpyridyl)-porphine tetrachloride;
meso-tetra(4-N-methylpyridyl)-porphine;
meso-tetra-(3-N-methylpyridyl)-porphine;
meso-tetra-(2-N-methylpyridyl)-porphine;
tetra(4-N,N,N-trimethylanilinium) porphine;
meso-tetra-(4-N,N,N''-trimethylamino-phenyl) porphine
tetrachloride; tetranaphthaloporphyrin;
5,10,15,20-tetraphenylporphyrin; tetraphenylporphyrin;
meso-tetra-(4-N-sulfonatophenyl)-porphine; tetraphenylporphine
tetrasulfonate; meso-tetra(4-sulfonatophenyl)porphine;
tetra(4-sulfonatophenyl)porphine; tetraphenylporphyrin sulfonate;
meso-tetra(4-sulfonatophenyl)porphine; tetrakis
(4-sulfonatophenyl)porphyrin;
meso-tetra(4-sulfonatophenyl)porphine;
meso(4-sulfonatophenyl)porphine;
meso-tetra(4-sulfonatophenyl)porphine;
tetrakis(4-sulfonatophenyl)porphyrin;
meso-tetra(4-N-trimethylanilinium)-porphine; uroporphyrin;
uroporphyrin I (17 .mu.M); uroporphyrin IX; and uroporphyrin I (18
.mu.M).
[0047] Exemplary psoralens include psoralen; 5-methoxypsoralen;
8-methoxypsoralen; 5,8-dimethoxypsoralen; 3-carbethoxypsoralen;
3-carbethoxy-pseudopsoralen; 8-hydroxypsoralen; pseudopsoralen;
4,5',8-trimethylpsoralen; allopsoralen; 3-aceto-allopsoralen;
4,7-dimethyl-allopsoralen; 4,7,4'-trimethyl-allopsoralen;
4,7,5'-trimethyl-allopsoralen; isopseudopsoralen;
3-acetoisopseudopsoralen; 4,5'-dimethyl-isopseudopsoralen;
5',7-dimethyl-isopseudopsoralen; pseudoisopsoralen;
3-acetopseudoisopsoralen; 3/4',5'-trimethyl-aza-psoralen;
4,4',8-trimethyl-5'-amino-methylpsoralen;
4,4',8-trimethyl-phthalamyl-psoralen;
4,5',8-trimethyl-4'-aminomethyl psoralen;
4,5',8-trimethyl-bromopsoralen; 5-nitro-8-methoxy-psoralen;
5'-acetyl-4,8-dimethyl-psoralen; 5'-aceto-8-methyl-psoralen; and
5'-aceto-4,8-dimethyl-psoralen Exemplary purpurins include
octaethylpurpurin; octaethylpurpurin zinc; oxidized
octaethylpurpurin; reduced octaethylpurpurin; reduced
octaethylpurpurin tin; purpurin 18; purpurin-18; purpurin-18-methyl
ester; purpurin; tin ethyl etiopurpurin I; Zn(II) aetio-purpurin
ethyl ester; and zinc etiopurpurin.
[0048] Exemplary quinones include 1-amino-4,5-dimethoxy
anthraquinone; 1,5-diamino-4,8-dimethoxy anthraquinone;
1,8-diamino-4,5-dimethoxy anthraquinone; 2,5-diamino-1,8-dihydroxy
anthraquinone; 2,7-diamino-1,8-dihydroxy anthraquinone;
4,5-diamino-1,8-dihydroxy anthraquinone; mono-methylated 4,5- or
2,7-diamino-1,8-dihydroxy anthraquinone; anthralin (keto form);
anthralin; anthralin anion; 1,8-dihydroxy anthraquinone;
1,8-dihydroxy anthraquinone (Chrysazin); 1,2-dihydroxy
anthraquinone; 1,2-dihydroxy anthraquinone (Alizarin);
1,4-dihydroxy anthraquinone (Quinizarin); 2,6-dihydroxy
anthraquinone; 2,6-dihydroxy anthraquinone (Anthraflavin);
1-hydroxy anthraquinone (Erythroxy-anthraquinone);
2-hydroxy-anthraquinone; 1,2,5,8-tetra-hydroxy anthraquinone
(Quinalizarin); 3-methyl-1,6,8-trihydroxy anthraquinone (Emodin);
anthraquinone; anthraquinone-2-sulfonic acid; benzoquinone;
tetramethyl benzoquinone; hydroquinone; chlorohydroquinone;
resorcinol; and 4-chlororesorcinol.
[0049] Exemplary retinoids include all-trans retinal; C.sub.17
aldehyde; C.sub.22 aldehyde; 11-cis retinal; 13-cis retinal;
retinal; and retinal palmitate.
[0050] Exemplary rhodamines include 4,5-dibromo-rhodamine methyl
ester; 4,5-dibromo-rhodamine n-butyl ester; rhodamine 101 methyl
ester; rhodamine 123; rhodamine 6G; rhodamine 6G hexyl ester;
tetrabromo-rhodamine 123; and tetramethyl-rhodamine ethyl
ester.
[0051] Exemplary thiophenes include terthiophenes such as
2,2':5',2''-terthiophene; 2,2':5',2''-terthiophene-5-carboxamide;
2,2':5',2''-terthiophene-5-carboxylic acid;
2,2':5',2''-terthiophene-5-L-serine ethyl ester;
2,2':5',2''-terthiophene-5-N-isopropynyl-formamide;
5-acetoxymethyl-2,2':5',2''-terthiophene;
5-benzyl-2,2':5',2''-terthiophene-sulphide;
5-benzyl-2,2':5',2''-terthiophene-sulfoxide;
5-benzyl-2,2':5',2''-terthiophene-sulphone;
5-bromo-2,2':5',2''-terthiophene;
5-(butynyl-3'''-hydroxy)-2,2':5',2''-terthiophene;
5-carboxyl-5''-trimethylsilyl-2,2':5',2''-terthiophene;
5-cyano-2,2':5',2''-terthiophene;
5,5''-dibromo-2,2':5',2''-terthiophene;
5-(1''',1'''-dibromoethenyl)-2,2':5',2''-terthiophene;
5,5''-dicyano-2,2':5',2''-terthiophene;
5,5''-diformyl-2,2':5',2''-terthiophene;
5-difluoromethyl-2,2':5',2''-terthiophene;
5,5''-diiodo-2,2':5',2''-terthiophene;
3,3''-dimethyl-2,2':5',2''-terthiophene;
5,5''-dimethyl-2,2':5',2''-terthiophene;
5-(3''',3'''-dimethylacryloyloxymethyl)-2,2':5',2''-terthiophene;
5,5''-di-(t-butyl)-2,2':5',2''-terthiophene;
5,5''-dithiomethyl-2,2':5',2''-terthiophene;
3'-ethoxy-2,2':5',2''-terthiophene; ethyl
2,2':5',2''-terthiophene-5-carboxylic acid;
5-formyl-2,2':5',2''-terthiophene;
5-hydroxyethyl-2,2':5',2''-terthiophene;
5-hydroxymethyl-2,2':5',2''-terthiophene;
5-iodo-2,2':5',2''-terthiophene;
5-methoxy-2,2':5',2''-terthiophene;
3'-methoxy-2,2':5',2''-terthiophene;
5-methyl-2,2':5',2''-terthiophene;
5-(3'''-methyl-2'''-butenyl)-2,2':5',2''-terthiophene; methyl
2,2':5',2''-terthiophene-5-[3'''-acrylate]; methyl
2,2':5',2''-terthiophene-5-(3'''-propionate);
N-allyl-2,2':5',2''-terthiophene-5-sulphonamide;
N-benzyl-2,2':5',2''-terthiophene-5-sulphonamide;
N-butyl-2,2':5',2''-terthiophene-5-sulphonamide;
N,N-diethyl-2,2':5',2''-terthiophene-5-sulphonamide;
3,3',4',3''-tetramethyl-2,2':5',2''-terthiophene;
5-t-butyl-5''-trimethylsilyl-2,2':5',2''-terthiophene;
3'-thiomethyl-2,2':5',2''-terthiophene;
5-thiomethyl-2,2':5',2''-terthiophene;
5-trimethylsilyl-2,2':5',2''-terthiophene, bithiophenes such as
2,2'-bithiophene; 5-cyano-2,2'-bithiophene;
5-formyl-2,2'-bithiophene; 5-phenyl-2,2'-bithiophene;
5-(propynyl)-2,2'-bithiophene; 5-(hexynyl)-2,2'-bithiophene;
5-(octynyl)-2,2'-bithiophene;
5-(butynyl-4''-hydroxy)-2,2'-bithiophene;
5-(pentynyl-5''-hydroxy)-2,2'-bithiophene;
5-(3'',4''-dihydroxybutynyl)-2,2'-bithiophene derivative;
5-(ethoxybutynyl)-2,2'-bithiophene derivative, and miscianeous
thiophenes such as 2,5-diphenylthiophene; 2,5-di(2-thienyl)furan;
pyridine, 2,6-bis(2-thienyl)-; pyridine, 2,6-bis(thienyl)-;
thiophene, 2-(1-naphthalenyl)-; thiophene, 2-(2-naphthalenyl)-;
thiophene, 2,2'-(1,2-phenylene)bis-; thiophene,
2,2'-(1,3-phenylene)bis-; thiophene, 2,2'-(1,4-phenylene)bis-;
2,2':5',2'':5'',2'''-quaterthiophene; .alpha.-quaterthienyl;
.alpha.-tetrathiophene; .alpha.-pentathiophene;
.alpha.-hexathiophene; and .alpha.-heptathiophene.
[0052] Exemplary verdins include copro (II) verdin trimethyl ester;
deuteroverdin methyl ester; mesoverdin methyl ester; and zinc
methylpyroverdin.
[0053] Exemplary vitamins include ergosterol (provitamin D2);
hexamethyl-Co a Co
b-dicyano-7-de(carboxymethyl)-7,8-didehydro-cobyrinate
(Pyrocobester); pyrocobester; and vitamin D3.
[0054] Exemplary xanthene dyes include Eosin B (4',5'-dibromo,
2',7'-dinitro-fluorescein, dianion); eosin Y; eosin Y
(2',4',5',7'-tetrabromo-fluorescein, dianion); eosin
(2',4',5',7'-tetrabromo-fluorescein, dianion); eosin
(2',4',5',7'-tetrabromo-fluorescein, dianion) methyl ester; eosin
(2',4',5',7'-tetrabromo-fluorescein, monoanion) p-isopropylbenzyl
ester; eosin derivative (2',7'-dibromo-fluorescein, dianion); eosin
derivative (4',5'-dibromo-fluorescein, dianion); eosin derivative
(2',7'-dichloro-fluorescein, dianion); eosin derivative
(4',5'-dichloro-fluorescein, dianion); eosin derivative
(2',7'-diiodo-fluorescein, dianion); eosin derivative
(4',5'-diiodo-fluorescein, dianion); eosin derivative
(tribromo-fluorescein, dianion); eosin derivative
(2',4',5',7'-tetrachloro-fluorescein, dianion); eosin; eosin
dicetylpyridinium chloride ion pair; erythrosin B
(2',4',5',7'-tetraiodo-fluorescein, dianion); erythrosin;
erythrosin dianion; erythrosin B; fluorescein; fluorescein dianion;
phloxin B (2',4',5',7'-tetrabromo-3,4,5,6-tetrachloro-fluorescein,
dianion); phloxin B (tetrachloro-tetrabromo-fluorescein); phloxine
B; rose bengal
(3,4,5,6-tetrachloro-2',4',5',7'-tetraiodofluorescein, dianion);
rose bengal; rose bengal dianion; rose bengal O-methyl-methylester;
rose bengal 6'-O-acetyl ethyl ester; rose bengal benzyl ester
diphenyl-diiodonium salt; rose bengal benzyl ester triethylammonium
salt; rose bengal benzyl ester, 2,4,6,-triphenylpyrilium salt; rose
bengal benzyl ester, benzyltriphenyl-phosphonium salt; rose bengal
benzyl ester, benzyltriphenyl phosphonium salt; rose bengal benzyl
ester, diphenyl-iodonium salt; rose bengal benzyl ester,
diphenyl-methylsulfonium salt; rose bengal benzyl ester,
diphenyl-methyl-sulfonium salt; rose bengal benzyl ester,
triethyl-ammonium salt; rose bengal benzyl ester, triphenyl
pyrilium; rose bengal bis (triethyl-ammonium) salt)
(3,4,5,6-tetrachloro-2',4',5',7'-tetraiodofluorescein, bis
(triethyl-ammonium salt); rose bengal bis (triethyl-ammonium) salt;
rose bengal bis(benzyl-triphenyl-phosphonium) salt
(3,4,5,6-tetrachloro-2',4',5',7'-tetraiodofluorescein,
bis(benzyl-triphenyl-phosphonium) salt); rose bengal
bis(diphenyl-iodonium) salt
(3,4,5,6-tetrachloro-2',4',5',7'-tetraiodofluorescein,
bis(diphenyl-iodonium) salt); rose bengal di-cetyl-pyridinium
chloride ion pair; rose bengal ethyl ester triethyl ammonium salt;
rose bengal ethyl ester triethyl ammonium salt; rose bengal ethyl
ester; rose bengal methyl ester; rose bengal octyl ester
tri-n-butyl-ammonium salt RB; rose bengal, 6'-O-acetyl-, and ethyl
ester.
[0055] Particularly preferred photosensitizers are the green
porphyrins, such as BPD-DA, -DB, -MA, and -MB, and in particular
BPD-MA, EA6, and B3. These compounds are porphyrin derivatives
obtained by reacting a porphyrin nucleus with an alkyne in a
Diels-Alder type reaction to obtain a monohydrobenzoporphyrin, and
they are described in detail in the issued U.S. Pat. No. 5,171,749,
which is hereby incorporated in its entirety by reference. Of
course, combinations of photosensitizers may also be used. It is
preferred that the absorption spectrum of the photosensitizer be in
the visible range, typically between 350 nm and 1200 nm, more
preferably between 400-900 nm, and even more preferably between
600-900 nm.
[0056] BPD-MA is described, for example, in U.S. Pat. No.
5,171,749; EA6 and B3 are described in U.S. Ser. Nos. 09/088,524
and 08/918,840, respectively, all of which are incorporated herein
by reference. Preferred green porphyrins have the basic structure:
##STR1## where R.sup.4 is vinyl or 1-hydroxyethyl and R.sup.1,
R.sup.2, and R.sup.3 are H or alkyl or substituted alkyl.
[0057] BPD-MA has the structure shown in formula 1 wherein R.sup.1
and R.sup.2 are methyl, R.sup.4 is vinyl and one of R.sup.3 is H
and the other is methyl. EA6 is of formula 2 wherein R.sup.1 and
R.sup.2 are methyl and both R.sup.3 are 2-hydroxyethyl (i.e., the
ethylene glycol esters). B3 is of formula 2 wherein R.sup.1 is
methyl, R.sup.2 is H, and both R.sup.3 are methyl. In both EA6 and
B3, R.sup.4 is also vinyl.
[0058] The representations of BPD-MAC and BPD-MAD, which are the
components of Verteporfin, as well as illustrations of A and B ring
forms of EA6 and B3, are as follows: ##STR2## ##STR3##
[0059] Related compounds of formulas 3 and 4 are also useful; in
general, R.sup.4 will be vinyl or 1-hydroxyethyl and R.sup.1,
R.sup.2, and R.sup.3 are H or alkyl or substituted alkyl.
[0060] Microaggregates
[0061] The MA of the invention results in the production of
phospholipid containing micelles, liposomes, and mixtures thereof.
Phospholipids suitable for use in the invention may be any
naturally occurring or synthetic phospholipid, whether saturated or
unsaturated. They include, but not limited to, the following:
phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine,
phosphatidylinositol, phosphatidylglycerol, phosphatidic acid,
lysophospholipids, egg or soybean phospholipid or combinations
thereof. The phospholipids may be in any form, including salted or
desalted, hydrogenated or partially hydrogenated, or natural,
semisynthetic (modified) or synthetic. In preferred embodiments of
the invention, the phospholipids used are those capable of forming
liposomes, but also able to result in the production of micelles if
a high energy processing step is used for size reduction of
multilammelar liposomes.
[0062] Even more preferred are unsaturated phosphatidylglycerols or
phosphatidylcholines with charged head groups. Such preferred
embodiments include the use of negatively charged mono- or
polyunsaturated phosphatidylglycerols and phosphatidylcholines such
as egg phosphatidylglycerol (EPG),
palmitoyloleoylphosphatidylglycerol (POPG),
dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylcholine
(DPPC), or combinations thereof. The unsaturated fatty acid chain
is preferably on the same phospholipid molecule as the charged
headgroup, but alternatively, the desired combination of
unsaturation and charge could be attained by using a charged
saturated molecule such as DMPG together with an unsaturated
phospholipid molecule. It will generally be preferable to limit the
amount of the unsaturated phospholipid (in other words, not to make
the whole composition from unsaturated phospholipids) because of
the greater stability of saturated phospholipids. Preferably, the
ratio of unsaturated charged phospholipid to the saturated
phospholipid is at least about 1:99, and more preferably the ratio
is at least about 3:97, and even more preferably in the range of
about 10:90 or more. Most preferably, the ratio is in the range of
about 40:60 to about 50:50, but may exceed 50:50.
[0063] The number of unsaturations (double bonds) in the fatty acid
chain can range from about 1-6, but is more preferably about 1 to
3, and most preferably about 1 or about 2.
[0064] Without being bound by theory, and with respect to the
preferential use of unsaturated lipids in the MA of the invention,
it is believed that saturated acyl chains may not be sufficiently
flexible during lyophilization of (removing water from) the MA.
Thus in the case of liposomes, where water is removed from the core
entrapped volume (for which an analogy of making raisins from
grapes is applicable), unsaturated acyl chains permit more
curvature in the lipid membrane and may introduce the necessary
flexibility to allow shrinkage during drying. As such, the micelle
containing MA of the invention are less susceptible to these
effects since they likely lack an inner water core (or
alternatively have a significantly smaller one). This may explain
the robustness of micelle containing MA during lyophilization. The
flexibility of unsaturated lipids may be a likely cause of small
stable micelle structure formation during microfluidization. The
presence of unsaturated lipids also lowers the phase transition
temperature (liquid to gel transition) of the formulation to below
room temperature, and induces a less pronounced transition. The
amount of unsaturated lipid determines the degree to which the
phase transition temperature is decreased. It is also believed that
the presence of a charged headgroup on a phospholipid (for example,
on phosphotidylglycerol) stabilizes small liposomes and micelles
because the repulsive charge prevents fusion into larger liposomal
structures.
[0065] All MA of the invention may comprise, consist of or consist
essentially of any one or more phospholipids in combination with a
hydrophobic agent. Preferably, the phospholipids used in the MA of
the invention are either synthetic or derived from non-animal
sources. More preferably, the phospholipids used in the MA of the
invention include DOPG (1,2 dioleoylphosphatidylglycerol), which is
a doubly unsaturated lipid of plant origin.
[0066] Phosphatidyl glycerols (PGs) may also be present in the MA
of the invention. Examples of such PGs include dimyristoyl
phosphatidyl glycerol (DMPG), DLPG and the like. The incorporation
of such PGs may be used to contribute to the stabilization of
micelles. Other types of suitable lipids that may be included are
phosphatidyl ethanolamines (PEs), phosphatidic acids (PAs),
phosphatidyl serines, and phosphatidyl inositols.
[0067] A range of total lipid to hydrophobic agent ratios may be
use in the practice of the invention. The ratio depends on the
hydrophobic agent being used, but will assure the presence of a
sufficient number of lipid molecules to form stable MA. Appropriate
total lipid:hydrophobic agent ratios may be from about 7:1 and
higher, although lower ratios also do not exhibit adverse effects.
A preferred range is from about 7:1 to 10:1. Of course all
intermediate ratios within this range, such as about 8:1 and about
9:1, are within the scope of the invention. Additionally within the
scope of the invention are the sub-intermediate ratios within the
range, such as from about 7.1:1 to 7.9: 1, about 8.1:1 to 8.9: 1,
and about 9.1:1 to 9.9:1, are within the scope of the invention.
When the number of lipid molecules is not sufficient to form a
stable complex, the lipophilic phase of the MA may become saturated
with hydrophobic agent molecules. Then, any slight change in the
process conditions can force some of the previously encapsulated
hydrophobic agent to leak out onto the surface of the MA, or even
out into the aqueous phase.
[0068] If the concentration of hydrophobic agent is high enough, it
can actually precipitate out from the aqueous layer and promote
aggregation of the MA. The more unencapsulated hydrophobic agent
present, the higher the degree of aggregation. The more
aggregation, the larger the mean aggregate size will be, and the MA
will no longer be of a sufficiently small size for efficient use in
steps such as filter sterilization. Thus slight increases in the
lipid content can increase significantly the filterability of the
liposome composition by increasing the ability to form and maintain
small aggregates. This is particularly advantageous when working
with significant volumes of 500 ml, a liter, five liters, 40
liters, or more, as opposed to smaller batches of about 100-500 ml
or less.
[0069] When larger volumes of MA are being made, a higher molar
ratio of phospholipid provides more assurance of reliable aseptic
filterability by providing smaller aggregates. Moreover, the
substantial potency losses that are common in scale-up batches, due
at least in part to filterability problems, can thus be avoided.
Another means of increasing filterability is by preparation of
micelle containing MA since micelles are smaller than liposomes in
general. Such micelle containing MA are more readily filter
sterilized with a 0.22 micron filter and a preferred embodiment of
the invention. Additional advantages in MA containing the smaller
micelles is reduced loss of the active hydrophobic agent via large
aggregates lost during filtering or other processes; and the
stability of smaller aggregates after reconstitution. Thus a
preferred embodiment of the invention is where the hydrophobic
agent is present in amounts, or in ratios, that favor micelle
formation.
[0070] When a combination of phospholipids is used in the MA of the
invention, a range of relative lipid ratios may be used in
combination with the total lipid:hydrophobic agent ratios described
above. Appropriate lipid ratios for combinations of two
phospholipids range from about 50:50 to about 97:1. Of course all
intermediate ratios within this range, such as about 70:30, about
80:20 and about 90:10, are within the scope of the invention. As
indicated by the use of the 99:1 ratio, sub-intermediate ratios
within the range, such as from about 71:29 to 79:21, about 81:19 to
89:11, and about 91:9 to 97:3, are within the scope of the
invention. Examples of combinations of two phospholipids where such
ratios may be used include DMPC:DMPG, DMPC:EPG, DMPC:POPG and
DMPC:DOPG. An additional example is DMPC: EPG, preferably at a
ratio of about 5:3 respectively. With this combination, even higher
hydrophobic agent:lipid ratios, such as 1:10, 1:15, or 1:20,
respectively, may be used.
[0071] A particularly preferred embodiment of the MA of the
invention comprises hydrophobic agents in an 8:1 total
phospholipid:hydrophobic agent ratio with a 60:40 lipid ratio of a
DMPC:DOPC combination containing antioxidants BHT and AP. In
particular, hydrophobic agents such as EA6 and/or BPD-MA may be
used in such MA. Also preferred are MA compositions comprising EA6
in small liposomes comprising lipids and other components as
described herein.
[0072] Antioxidants
[0073] In preferred embodiments comprising the use of unsaturated
phospholipids, the invention encompasses the use of antioxidants to
prevent oxidation of the phospholipids. Auto-oxidation of
unsaturated acyl chains has been known to be a problem for
long-term storage of liposome formulations. Failure to prevent
oxidative breakdown of unsaturated phospholipids results in
subcomponents such as lyso lipids and fatty acids, which may be
undesirable in some MA compositions. As such, antioxidants suitable
for inclusion in phospholipid containing microaggregates to improve
long-term storage are known in the art. Examples of such
antioxidants include butylated hydroxytoluene (BHT),
alpha-tocopherol, and ascorbyl palmitate (AP) as well as pH
buffering agents such as phosphates and glycine. Preferably, BHT is
present at about 0.01-0.02% by weight and AP at about 0.1-0.2% by
weight.
[0074] BHT is hydrophobic and would be expected to remain in the
lipophilic environments of the MA of the invention. BHT has the
ability to prevent chain propagation during auto-oxidation by
accepting radicals formed during the oxidative breakdown of lipids.
Ascorbic acid has the capacity to act as an antioxidant and to act
with other antioxidants such as alpha-tocopherol. It has been shown
that the BHT/ascorbic acid system allows for BHT regeneration,
following its conversion to a phenoxyl radical after free radical
scavenging from oxidized lipids, thereby resulting in the
appearance of ascorbyl radicals. This latter factor justifies the
relative weight ration of AP to BHT described above. AP was used in
place of ascorbic acid because the hydrophobic nature of the former
would be expected to concentrate the antioxidant within lipophilic
environments.
[0075] Another anti-oxidation considerations is the filling of
container headspaces with nitrogen gas and the sealing of such
containers. Additionally, and because metal ions can catalyze
oxidative processes, the use of high quality drug, excipients, and
containers, the judicious cleaning of manufacturing equipment, and
the appropriate use of metal ion chelators are preferred.
[0076] Cryoprotective Agents and Isotonic Agents
[0077] In a preferred embodiment of the invention, the MA are
stabilized by lyophilization. An advantage to the micelle
containing MA of the invention is the fact that micelles may be
more readily lyophilized in comparison to liposomes due to the
absence of a water core. Lyophilization of liposomes require the
passage of water across at least one lipid bilayer, resulting in
increased processing times and expense. The absence of a water core
also permits micelles to have a greater concentration of
phospholipid per unit volume. Thus a larger amount of hydrophobic
agent can be solubilized by the phospholipid per unit volume of
micelle. This permits the final micelle MA delivery vehicle to have
a higher drug density per unit volume than other delivery vehicles,
such as liposomes alone.
[0078] MA of the invention may contain a cryoprotectant for
stabilizing the MA during lyophilization. Alternatively, the
physical structures of the MA can be preserved by the presence of
sufficient water after lyophilization. This is may be accomplished
by appropriate control of the degree of lyophilization. Since there
is no entrapped volume in micelles, the micelle containing MA of
the invention facilitates greater control over water soluble
components, like solvent or salt, to be removed in the preparation
of delivery vehicles requiring such removal.
[0079] Any cryoprotective agent known to be useful in the art of
preparing freeze-dried formulations, such as di- or polysaccharides
or other bulking agents such as lysine, may be used in the claimed
invention. Further, isotonic agents typically added to maintain
isomolarity with body fluids may be used. In preferred embodiments,
a di-saccharide or polysaccharide is used and functions both as a
cryoprotective agent and as an isotonic agent. In an especially
preferred embodiment, the disaccharide or polysaccharide is
selected from among the group consisting of lactose, trehalose,
maltose, maltotriose, palatinose, lactulose or sucrose, with
lactose or trehalose being preferred. Effective sugars such as
trehalose and lactose are capable of hydrogen bonding to the
phospholipidhead group in place of water. It has also been
hypothesized that effective sugars also act a as a spacing matrix
to decrease the opposition of phospholipids on the exterior of
adjacent MA such as liposomes.
[0080] When the process of hydrating a lipid film is prolonged,
larger liposomes tend to be formed, and hydrophobic agents may even
precipitate. The addition of a disaccharide or polysaccharide
provides the largest surface area for depositing a thin film of MA
and virtually instantaneous subsequent hydration. This thin film
provides for faster hydration so that, when the MA are initially
formed by adding the aqueous phase (hydrated), the MA are of a
smaller and more uniform particle size. This provides significant
advantages in terms of manufacturing ease.
[0081] However, it is also possible that, when a saccharide is
present in the composition of the invention, it is added after dry
lipid film formation, as a part of the aqueous solution used in
hydration. In a particularly preferred embodiment, a saccharide is
added to the dry lipid film of the invention during hydration.
[0082] Disaccharides or polysaccharides are preferred to
monosaccharides for this purpose. To keep the osmotic pressure of
the MA compositions of the invention similar to that of blood, no
more than 4-5% monosaccharides should be added. In contrast, about
9-10% of a disaccharide can be used without generating an
unacceptable osmotic pressure. The higher amount of disaccharide
provides for a larger surface area, which results in smaller
particle sizes being formed during hydration of the lipid film.
[0083] Also, when present, the disaccharide or polysaccharide is
formulated in a preferred ratio of about 10-20 saccharide to
0.5-6.0 total phospholipids, respectively, even more preferably at
a ratio from about 10 to 1.5-4.0. In one embodiment, a preferred
but not limiting formulation is lactose or trehalose and total
phospholipids in a ratio of about 10 to 0.94-1.88 to about
0.65-1.30, respectively.
[0084] The presence of the disaccharide or polysaccharide in the
composition not only tends to yield MA having extremely small and
narrow aggregate size ranges, but also provides MA compositions in
which the hydrophobic agents, such as a hydro-monobenzoporphyrin
photosensitizer, may be stably incorporated in an efficient manner,
i.e., with an encapsulation efficiency approaching 80-100%.
Moreover, MA made with a saccharide typically exhibit improved
physical and chemical stability, such that they can retain an
incorporated hydrophobic agent, such as hydro-monobenzoporphyrin
photosensitizer, without leakage upon prolonged storage, either as
a reconstituted aqueous suspension or as a cryodesiccated
powder.
[0085] Freeze-Drying
[0086] Once formulated, the MA of the invention may be freeze-dried
for long-term storage if desired. For example, BPD-MA, a preferred
hydro-monobenzoporphyrin photosensitizer, has maintained its
potency in a cryodesiccated MA composition for a period of at least
nine months at room temperature, and a shelf life of at least two
years has been projected. If the composition is freeze-dried, it
may be packed in vials for subsequent reconstitution with a
suitable aqueous solution, such as sterile water or sterile water
containing a saccharide and/or other suitable excipients, just
prior to use. For example, reconstitution may be by simply adding
water for injection just prior to administration.
[0087] Various lyophilization techniques are known in the art. For
example, MA containing vials of the invention may be first frozen
to -45.degree. C. and then held there for a period of up to about
90 minutes. This may be followed by a high vacuum primary drying
cycle wherein the temperature is increased slowly to up to about
10.degree. C. for a period usually on the order of about 50 hours.
This may be followed by a 20.degree. C. secondary drying cycle of
up to about 24 hours. Once the lyophilizer pressure stabilizes at
about 55-65 mTorr (73-87 microbar), the cycle is terminated.
Thereafter, the vials may be sealed after overlaying with nitrogen
gas. A general rule for freeze-drying is that a solid, brittle,
non-collapsed, and homogenous cake is preferred for successful
re-hydration.
[0088] Additionally, the use of lyophilization may prevent
hydrolysis of hydrophobic agents susceptible to such reactions. For
example, the photosensitizer BPD-MA may be hydrolyzed to
BPD-DA.
[0089] Size
[0090] In one aspect of the invention, the MA are of a sufficiently
small and narrow size that the aseptic filtration of the
composition through a 0.22 micron hydrophilic filter can be
accomplished efficiently and with large volumes of 500 ml to a
liter or more without significant clogging of the filter. As such
micelle and small liposome containing MA are a preferred embodiment
of the invention. Moreover, and given their smaller size, the MA of
the invention may mainly, or predominantly, contain hydrophobic
agent bearing micelles. The MA of the invention may contain greater
than about 50%, greater than about 60%, greater than about 75%,
greater than about 80%, greater than about 90%, and greater than
about 95% micelles. Even more preferably, the MA of the invention
may contain greater than about 97%, about 98%, or about 99%
micelles. Most preferably in desired circumstances, the MA of the
invention consist only of micelles. Alternatively, the MA of the
invention may in some circumstances (when an extrusion process is
used for size reduction of multilammelar liposomes, rather than a
high energy process such as microfluidization) contain up to 100%
liposomes.
[0091] Micelles refer to microaggregates with the hydrophobic
(lipophilic) "tail" portion of the phospholipids generally oriented
toward the interior of the micelle. Preferably, micelles have the
"tail" portion generally oriented toward the center of the micelle.
Micelles do not have a bilayer structure and so are not considered
vesicles or liposomes. The micelles of the invention have average
diameters of less than about 30 nm (nanometers). Preferably, they
have average diameters of less than about 20 nm.
[0092] Liposomes refer to microaggregates comprising at least one
phospholipid bilayer, composed of two lipid monolayers having a
hydrophobic "tail" region and a hydrophilic "head" region. The
structure of the membrane bilayer is such that the hydrophobic
(nonpolar) "tails" of the lipid monolayers orient themselves
towards the center of the bilayer, while the hydrophilic "heads"
orient themselves toward the aqueous phase. They generally comprise
completely closed, lipid bilayer membranes that contain an
entrapped aqueous volume. Given the bilayer structure, a
significant portion (up to about half) of the phospholipids will
have their hydrophobic (lipophilic) portion generally oriented away
from the center of the liposome. Liposomes include unilamellar
vesicles having a single membrane bilayer or multilamellar vesicles
having multiple membrane bilayers, each bilayer being separated
from the next by an aqueous layer. The average diameters of
liposomes are larger than that of micelles.
[0093] In liposomes, a hydrophobic agent can be entrapped in the
aqueous phase of the liposome or be associated with the "tail"
portion of phospholipids in the lipid bilayer. In micelles, a
hydrophobic agent is left to associate only with the "tail" portion
of phospholipids in the core of the micelle. Additionally, both
micelles and liposomes may be used to help "target" a hydrophobic
drug to an active site or to solubilize hydrophobic drugs for
parenteral administration.
[0094] One aspect of the present invention uses this ability to
form micelles and liposomes by the same mixture of hydrophobic
agent and phospholipids. This would result in MA that have a
bimodal distribution in their diameters, indicating the presence of
both micelles and liposomes. In another aspect of the invention,
the micelles and liposomes are form under conditions that favor one
type of microaggregate over the other in the same mixture.
Conditions that favor micelle formation include the presence of low
salt in the mixture as well as the use of low salt aqueous solution
for hydrating the dried mixture. "Low salt" refers to conditions
containing less than about 0.1 N free cations or anions.
Preferably, it refers to less than about 0.01 N free ions. More
preferably it refers to less than about 0.001 N free ions.
[0095] Preferred MA of the invention have an average aggregate size
diameter of well below about 300 nm, more preferably below from
about 200 nm. Most preferably, the MA of the invention have an
average aggregate size diameter below about 100 nm, and sometimes,
depending on the conditions chosen, in the range of 10-50 nm. The
size of the microaggregates made comprising QLT 0074, DOPG and DMPC
(see Example 1 below) have been sized using three different methods
(using a NICOMP 370 Submicron Particle Sizer, by freeze fracture
analysis and by size exclusion HPLC). Freeze fracture analysis
showed a mixture of micelles (7-15 nm in diameter), and relatively
few liposomes (between 6- and 270 nm diameter). Size exclusion HPLC
indicated mean particle size of 28 nm when tested in four different
media (PBS, 0.9% sodium chloride, 9.2% lactose and 5% dextrose)
with a range or 25-35 nm.
[0096] As discussed herein, the invention controls four major
parameters that can affect the ease of aggregate size reduction to
an unexpected degree. As a result, the filterability, particularly
with standard aseptic filtration, is significantly improved in the
MA of the invention. These parameters are (1) the production of
micelles and small liposomes by use of low salt conditions; (2)
suitable molar ratio of hydro-monobenzoporphyrin photosensitizer to
total phospholipids; (3) temperature during the hydration step; and
(4) temperature during the homogenization or size reduction step.
The latter two parameters are discussed below.
[0097] Filterability can be tested by passing a MA composition
through a Microfluidizer.TM. three times and withdrawing a sample
with a syringe. The syringe is connected to a 0.22 micron
hydrophilic filter and then placed in a syringe pump. The constant
rate of piston movement is set at 10 ml/min, and filtrate is
collected until the filter becomes blocked by large aggregates. The
volume of the filtrate is then measured and recorded in terms of
ml/cm.sup.2 or g/cm.sup.2, with a square centimeter being the
effective filtration area. Thus, filterability for the purposes of
the invention is defined as the maximum volume or weight of MA
composition that can be filtered through a 0.22 micron filter.
[0098] The MA of the invention may be used as a delivery vehicle
for the constituent hydrophobic agent to target any cell or tissue
for which contact with the agent is desired. In preferred
embodiments of the invention, the agent is a photosensitizer to be
delivered prior to light irradiation as part of photodynamic
therapy (PDT). Particularly preferred MA of the invention comprise
a hydro-monobenzoporphyrin photosensitizer, including BPD-MA and
EA6, for use in photodynamic therapy (PDT) or diagnosis.
[0099] The MA of the invention also preferably comprises micelles
which are readily, and significantly, destabilized in the presence
of proteins, salts, charged elements, and/or polymers. Such MA are
well suited as a pharmaceutical formulation to deliver hydrophobic
drugs to fluids such as blood, which contains proteins, salts,
charged elements and polymers. Given the ability to destabilize
after delivery to target conditions, the MA of the invention can
rapidly deliver hydrophobic agents to targets such as the
bloodstream, where the drugs may be picked up or transferred to
blood components for further transport and/or targeting based on
the components' specificities. As such, the MA can be considered
"fast breaking" in that the MA is stable in vitro but, when
administered in vivo, the hydrophobic drug (such as a
photosensitizer) is rapidly released into the bloodstream where it
associates with blood components such as serum lipoproteins.
Another beneficial effect of this transfer is reduced depositing of
hydrophobic agents in various organs, especially the liver. As
such, the pharmokinetics of delivering the hydrophobic agent with
such micelles are altered compared to the use of other delivery
vehicles or systems, such as those that do not release the agent
rapidly or those that do not transfer the agent to blood
components.
[0100] Preparation
[0101] Methods for the production of the MA of the invention
comprise, consist of, and/or consisting essentially of the
combination of hydrophobic agents and phospholipids and subjecting
them to conditions capable of forming micelles, small liposomes or
combinations thereof. Preferably, the methods comprise the use of
phospholipids capable of forming lipid bilayers and result in the
production of stable micelles and/or small liposomes. The resultant
MA, especially those comprising or consisting of micelles of the
invention, do not contain detergents normally used for micelle
production. The absence of detergents can markedly reduce toxicity
known to result in hemolysis and kidney damage. To favor micelle
formation, the MA of the invention are formulated under low salt
conditions because, as noted above, the micelles of the invention
are destabilized by salt.
[0102] Generally, the MA of the invention are produced by
dissolving the desired MA constituent component molecules (such as
desired phospholipids, hydrophobic agent, and optionally
antioxidants and cryoprotectants) into a solvent to form an
"intermediate complex". Preferred solvents are organic or otherwise
non-aqueous. Suitable organic solvents include any volatile organic
solvent, such as diethyl ether, acetone, methylene chloride,
chloroform, piperidine, piperidine-water mixtures, methanol,
tert-butanol, dimethyl sulfoxide, N-methyl-2-pyrrolidone, and
mixtures thereof. Preferably, the organic solvent is
water-immiscible, such as methylene chloride, but water
immiscibility is not required. In any event, the solvent chosen
should not only be able to dissolve all of the components of the
lipid film, but should also not react with, or otherwise
deleteriously affect, these components to any significant
degree.
[0103] The organic solvent is then removed from the resulting
solution to form a dry lipid film by any known laboratory technique
that is not significantly deleterious to the dry lipid film and the
hydrophobic agent. Such techniques include any that remove the
solvent via its gaseous phase, including evaporation or vacuum. In
one embodiment, the solvent is removed by placing the solution
under a vacuum until the organic solvent is evaporated. The solid
residue is the dry lipid film of the invention, which contains
aggregates of the MA components, considered the "presome". The
thickness of the lipid film is not critical, but usually varies
from about 30 to about 45 mg/cm.sup.2, depending upon the amount of
solid residual and the surface area of the vessel which contains
it. In another embodiment of the invention, the solvent is removed
as part the "presome" process of Nanba et al. (U.S. Pat. No.
5,096,629, which is hereby incorporated by reference as if fully
set forth), which heats the "intermediate complex" and subjects it
to dryness via an instantaneous vacuum drying system such as the
CRUX 8B.TM. (Orient Chemical Ind., Ltd., Japan) to produce a lipid
powder containing aggregates of the MA components.
[0104] Once formed, the film or powder may be stored for an
extended period of time, preferably not more than 4 to 21 days,
prior to hydration. Storage may be under an appropriate gas, such
as argon. While the temperature during a lipid film or powder
storage period is also not an important factor, it is preferably
below room temperature, most preferably in the range from about -20
to about 4.degree. C. One advantage to the Nanba et al. "presome"
process is the reduction of batch to batch variability seen with
thin film, which arises due to the use of multiple batches in
evaporation vessels.
[0105] The dry lipid film or powder may be hydrated with an aqueous
solution, preferably containing a disaccharide or polysaccharide if
not previously present. This will result in the formation of large
multilammelar liposomes that can be further processed by extrusion
or a high energy process, such as microfluidization to form the
desired particle size. Examples of useful aqueous solutions used
during the hydration step include sterile water, or a dilute
solution of lactose. In one embodiment of the invention, the
solution is physiologically isotonic, such as 9.2% lactose, which
permits bolus injections. Preferably the aqueous solution is
sterile. Most preferably for the production of micelles and the
stabilization of small liposomes, the solution is low salt. It is
believed that the presence of salts neutralizes the negative
repulsive charges that prevent the aggregation or fusion of these
small particles into larger liposomes.
[0106] The volume of aqueous solution used during hydration can
vary greatly, but should not be so great as about 98% nor so small
as about 3040%. A typical range of useful volumes would be from
about 50 or 60% to about 95%, preferably about 75% to about 95%,
more preferably about 80% to about 90%, and most preferably about
85% to 90%. Of course all subranges from about 30% to about 98% are
included as part of the invention.
[0107] The physical manipulation of material during hydration may
be conducted by a variety of means, including mixing and rotating
on a rotary evaporator, manual swirling of vessels, and the use of
standard laboratory stirrer or shaker means (including stir bars
with stir plates, high shear mixers, paddles and combinations
thereof). Preferred in the practice of the invention are high
agitation methods, such as the use of high-shear mixing or
egg-shaped stir bars.
[0108] Upon hydration, coarse aggregates are formed that
incorporate a therapeutically effective amount of the hydrophobic
agent. The "therapeutically effective amount" can vary widely,
depending on the tissue to be treated and whether the hydrophobic
agent is coupled to a target-specific ligand, such as an antibody
or an immunologically active fragment. Typically, the
therapeutically effective amount is such to produce a dose of
hydrophobic agent within a range of from about 0.1 to about 20
mg/kg, preferably from about 0.15-2.0 mg/kg and, even more
preferably, from about 0.25 to about 0.75 mg/kg. Preferably, the
w/v concentration of the hydrophobic agent in the "intermediate
complex" ranges from about 0.1 to about 8.0-10.0 g/L, when the
mixture becomes such a thick gel that it is not possible to handle
or administer to a subject by the usual means. Most preferably, the
concentration is about 2.0 to 2.5 g/L.
[0109] It should be noted that if the agent is a photosensitizer,
the various parameters used for selective photodynamic therapy are
interrelated. Therefore, the therapeutically effective amount
should also be adjusted with respect to other parameters, for
example, fluence, irradiance, duration of the light used in
photodynamic therapy, and the time interval between administration
of the photosensitizing agent and the therapeutic irradiation.
Generally, all of these parameters are adjusted to produce
significant damage to tissue deemed undesirable, such as
neovascular or tumor tissue, without significant damage to the
surrounding tissue, or to enable the observation of such
undesirable tissue without significant damage to the surrounding
tissue.
[0110] The hydration step should take place at a temperature that
does not exceed the glass transition temperature of the
phospholipid and hydrophobic agent aggregates formed. For
photosensitizers of the invention, this temperature is about
30.degree. C. Preferably the temperature is at room temperature or
lower, such as from 10-25, or even more preferred from
15-20.degree. C. or 17-22.degree. C. An especially preferred
temperature is about 21.degree. C. The glass transition temperature
of the phospholipid and hydrophobic agent aggregates can be
measured by using a differential scanning microcalorimeter. Madden
et al. ("Spontaneous vesiculation of large multilamellar vesicles
composed of saturated phosphatidylcholine and phosphatidylglycerol
mixtures." Biochemistry, Vol. 27, pp. 8724-8730, (1988)) describe
the effects of temperature and ionic strength on vesicle
formation.
[0111] The use of unsaturated charged lipids as encompassed by the
invention may effectively lower the phase transition temperature Tc
(liquid to gel transition) of the formulation to below room
temperature and induce a less pronounced transition. The amount of
unsaturated lipid determines the degree of Tc lowering.
[0112] The particle sizes of the coarse aggregates first formed
during hydration are then homogenized to a more uniform size and/or
reduced to a smaller size range of about less than about 50 to
about 300 nm, depending on the method of size reduction used.
Preferably, this homogenization and/or reduction is also conducted
at a temperature below the glass transition temperature of the
hydrophobic agent-phospholipid complex formed in the hydration
step. For photosensitizers of the invention, such temperature does
not exceed about 30.degree. C., and is preferably below room
temperature of about 25.degree. C. It has been found that the
homogenization temperature with photosensitizers is preferably at
room temperature or lower, e.g., 15-20.degree. C. At higher
homogenization temperatures, such as about 32-42.degree. C., the
relative filterability of the MA composition may improve initially
due to increased fluidity as expected, but then, unexpectedly,
tends to decrease with continuing agitation due to increasing
particle size.
[0113] Various high-speed agitation or high energy system
manipulation processes may be used during the homogenization step.
Examples of such processes include microfluidization (liquid jet
milling), high shear mixing, and sonication. While effective,
sonication is not ideal for use in large scale production of MA.
Processing through the aforementioned high energy system results in
the production of small particles, usually a mixture of small
liposomes and micelles. Extrusion, is another method of size
reduction. Extrusion results in the production of small liposomes
(as small as 50 to 100 nm), but micelles have not been observed by
the inventors in production by this procedure. Extrusion involves
the forcing of hydrated material, under pressure and at
temperatures known to make liposome formulations fluid, through
membrane filters of defined pore sizes. While adequate for
laboratory scale batches of material, extrusion may not be ideal
for large scale processes since 1) the pores become clogged even at
high pressures of greater than 1000 psi, 2) the surface area of the
filter membrane and extruder volume are limitations, and 3)
multiple discontinuous passes through the extruder increases the
likelihood of differences between batches.
[0114] Devices for the above described processes include a
Microfluidizer.TM. (such as a Microfluidics.TM. Model 110F); a
sonicator; a high-shear mixer; a homogenizer; a standard laboratory
shaker or stirrer, or any other agitation device. Of course
modifications in such processes to suit the particular hydrophobic
agent of interest and formation of the desired MA are within the
scope of the invention. In one preferred embodiment of the
invention, these processes are used for the production of MA
containing mainly micelles.
[0115] Such processes may be used to produce MA various ratios of
micelles, liposomes and combinations thereof. In embodiments where
both micelles and liposomes are produced, they may be separated by
the bimodal size distribution seen in combinations of the two. This
arises from the significantly smaller size of micelles in
comparison to liposomes. The analysis of MA size may be performed
by methods including electron microscopy, to exclude large
aggregates as liposomes, and use of a particle sizer, which may be
used in combination with fitting routines for uni- and bimodal
distributions. Another method is by use of manganese chloride
(Mn.sup.2+) mediated nuclear magnetic resonance (.sup.31P-NMR),
where .sup.31Phosphorus labeled headgroups of lipids on the inner
layer of a liposome lipid bilayer are not quenched by Mn.sup.2+
because Mn.sup.2+ cannot readily cross the bilayer to enter the
entrapped volume. Thus liposomes will produce a residual NMR signal
of about 30-40% for large and small liposomes after adding
Mn.sup.2+. All .sup.31P-labeled headgroups of lipids of a micelle,
however, are on the surface and thus fully exposed to Mn.sup.2+
quenching. Thus micelles produce no remaining NMR signal due to
quenching after adding Mn.sup.2+ (see FIG. 1).
[0116] In a preferred embodiment, a high-pressure device such as a
Microfluidizer.TM. is used for agitation. Some models of
microfluidization systems are continuous and batch size scalable
processors. Microfluidization uses high pressure streams of
hydrated material that collide at ultra-high velocities in
precisely defined microchannels. In the interaction chamber, two
streams of fluid at a high speed collide with each other at a
90.degree. angle. The combined forces of shear, impact and
cavitation result in the production of liposomes and micelles. In
microfluidization, a large amount of heat is generated during the
short period of time during which the fluid passes through a high
pressure interaction chamber. As the microfluidization temperature
increases, the fluidity of the membrane also increases, which
initially makes particle size reduction easier, as expected. For
example, filterability can increase by as much as four times with
the initial few passes through a Microfluidizer.TM. device. The
increase in the fluidity of the bilayer membrane promotes particle
size reduction, which makes filtration of the final composition
easier. In the initial several passes, this increased fluidity
mechanism advantageously dominates the process.
[0117] However, as the number of passes and the temperature both
increase, more of the hydrophobic agent molecules are apparently
squeezed out in cases involving liposomes, increasing the tendency
of the liposomes to aggregate into larger particles. At the point
at which the aggregation of vesicles begins to dominate the
process, the sizes cannot be reduced any further.
[0118] For this reason, in the methods of the invention, the
homogenization temperature is cooled down to and maintained at a
temperature no greater than room temperature after the composition
passes through the zone of maximum agitation, e.g., the interaction
chamber of a Microfluidizer.TM. device. An appropriate cooling
system can easily be provided for any standard agitation device in
which homogenization is to take place, e.g., a Microfluidizer.TM.,
such as by circulating cold water into an appropriate cooling
jacket around the mixing chamber or other zone of maximum
turbulence. While the pressure used in such high pressure devices
is not critical, pressures from about 10,000 to about 16,000 psi
are not uncommon.
[0119] Maintaining the hydration temperature and the
homogenizing/reducing step at a temperature below 30.degree. C.
would not have been expected to produce smaller particle sizes. In
fact, the invention is contrary to the conventional wisdom that
small particle sizes are achieved by increasing rather than
decreasing these temperatures. See, e.g., M. Lee et al., "Size
Distribution of Liposomes by Flow Field-Flow Fractionation", J.
Pharm. & Biomed. Analysis 11: 10, 911-20 (1993), equation (6)
showing particle diameter "d" as inversely related to temperature
"T", and FIG. 6b therein showing liposome preparation I (prepared
at about 70.degree. C.) having smaller particle sizes than
preparation II (prepared at about 23.degree. C.).
[0120] As a last step, the MA compositions of the inventions are
preferably aseptically filtered through a filter having an
extremely small pore size, i.e., 0.22 micron. While other
sterilization methods, such as heating and X-ray irradiation are
known, in the art, the use of such methods may result in
irreversible structural changes in lipids and hydrophobic agents
such as many photosensitizers. A wide variety of filtration systems
are known in the art, including Durapore TP cartridges, Millipak
100, Millidisk 40S, and millidisk MCGL. Filter pressures used
during sterile filtration can vary widely, depending on the volume
of the composition, the density, the temperature, the type of
filter, the filter pore size, and the size of the MA. However, as a
guide, a typical set of filtration conditions would be as follows:
filtration pressure of 15-25 psi; filtration load of 0.8 to 1.5
ml/cm.sup.2; and filtration temperature of about 25.degree. C.
Preferably, the hydrophilic Millidisk 40S is used at a load of
approximately 1 ml/cm.sup.2.
[0121] A typical general procedure for producing
hydro-monobenzoporphyrin photosensitizer containing MA of the
invention is described below with additional exemplary detail:
[0122] (1) Sterile filtration of methylene chloride as organic
solvent through a hydrophobic, 0.22 micron filter.
[0123] (2) Addition of DMPC:EPG:BPD-MA at a ratio of 4.7:3.25:1 and
excipients to the filtered organic solvent, dissolving both the
excipients and the photosensitizer to form the "intermediate
complex".
[0124] (3) Filtration of the resulting solution through a 0.22
micron hydrophobic filter.
[0125] (4) Transfer of the filtrate to a rotary evaporator
apparatus, such as that commercially available under the name
Rotoevaporator.
[0126] (5) Removal of the organic solvent to form a dry lipid
film.
[0127] (6) Analysis of the lipid film to determine the level of
organic solvent concentration; optionally continuing removal until
the level of organic solvent is below 0.01%, [0128] (7) Preparation
of a 10% lactose solution. If the MA formulation is to be injected,
this solution should be injectable.
[0129] (8) Filtration of the lactose solution through a 0.22 micron
hydrophilic filter.
[0130] (9) Hydration of the lipid film with the filtered 10%
lactose solution to form coarse aggregates.
[0131] (10) Reduction of the particle sizes of the coarse
aggregates by passing them through a Microfluidizer.TM., optionally
at 9000 psi (pounds per square inch) for about 5 discrete passes to
produce micelles.
[0132] (11) Determination of the reduced aggregated size
distribution of MA.
[0133] (12) Aseptic filtration of the MA composition through a 0.22
micron hydrophilic filter. (Optionally, the solution may first be
pre-filtered with a 5.0 micron or smaller pre-filter.)
[0134] (13) Analysis of photosensitizer potency.
[0135] (14) Filling of vials with the MA composition.
[0136] (15) Freeze-drying.
[0137] The above may be adapted for the selective production of
micelles by conducting all appropriate steps under low salt
conditions to favor subsequent micelle production after hydration.
As such, salt based bulking agents must not be used. In such
applications, the resulting micelles are on the order of about 15
nm in diameter, which is at the lower limit for feasible liposome
sizes. The micelle structure was confirmed by use of
.sup.31P-NMR.
[0138] An alternative general procedure for producing
hydro-monobenzoporphyrin photosensitizer containing MA of the
invention by use of a "presome" process of Nanba et al. (see U.S.
Pat. No. 5,096,629) is described below with additional exemplary
detail:
[0139] (1) Sterile filtration of methylene chloride as organic
solvent through a hydrophobic, 0.22 micron filter.
[0140] (2) Addition of DMPC:DOPG at a ratio of 60:40 with a total
lipid:EA6 at a ratio of 8:1 and antioxidants BHT and AP to the
filtered organic solvent, dissolving both the excipients and the
photosensitizer to form the "intermediate complex".
[0141] (3) Filtration of the resulting solution through a 0.22
micron hydrophobic filter.
[0142] (4) Transfer of the filtrate to liquid tank followed by
feeding to a tubular heater heated externally.
[0143] (5) Removal of the organic solvent by sending the heated
mixture into a vacuum chamber of no more than 300 mm Hg at a speed
over 0.1 times the speed of sound to instantaneously dry the
mixture to form lipid powder.
[0144] (6) Analysis of the lipid powder to determine the level of
organic solvent concentration; optionally continuing removal until
the level of organic solvent is below 0.01%,
[0145] (7) Preparation of a 10% lactose solution. If the MA
formulation is to be injected, this solution should be
injectable.
[0146] (8) Filtration of the lactose solution through a 0.22 micron
hydrophilic filter.
[0147] (9) Hydration of the lipid powder with the filtered 10%
lactose solution to form coarse aggregates.
[0148] (10) Dispersion of the coarse aggregates by stirring them at
high rpm at a temperature below the glass transition temperature of
the photosensitizer and phospholipid containing aggregates.
[0149] (11) Determination of the reduced aggregated size
distribution of MA.
[0150] (12) Aseptic filtration of the MA composition through a 0.22
micron hydrophilic filter. (Optionally, the solution may first be
pre-filtered with a 5.0 micron or smaller pre-filter.)
[0151] (13) Analysis of photosensitizer potency.
[0152] (14) Filling of vials with the MA composition.
[0153] (15) Freeze-drying.
[0154] One means of conducting the above instantaneous drying is by
use of a vacuum drying system such as the CRUX 8B.TM. product of
Orient Chemical Ind., Ltd., Japan. Moreover, the above dispersion
step may be at speeds of about 10,000 rpm, or ranging from 8000 to
15,000 rpm. Such a "presome" process may also be adapted for the
selective production of micelles by conducting all appropriate
steps under low salt conditions to favor subsequent micelle
production after hydration. As such, salt based bulking agents must
not be used.
[0155] As described above, the practice of the methods of the
invention for MA production may be conducted with a variety of
phospholipids and processes. The invention includes the
observation, beyond the use of low salt conditions, that the use of
charged, unsaturated phospholipids, such as EPG and DOPG, as well
as high energy processing (such as microfluidization and
sonication), appears to favor the formation of micelles in
otherwise liposome forming combinations of phospholipids and
hydrophobic agents. The use of unsaturated phospholipids provides a
number of desirable characteristics. These include the ability to
conduct MA production steps at room temperature and to produce
smaller MA when used in combination with saturated lipids.
[0156] Administration and Use
[0157] The use of the hydrophobic agents incorporated in the MA of
the invention may be for any appropriate pharmaceutical,
agricultural or industrial application. With incorporated
photosensitizers, the MA may be used for any condition or in any
method for which the photosensitizers are appropriate in
combination with exposure to light or other electromagnetic
radiation. These include, but are not limited to, the diagnosis or
treatment of cancer, the reduction of activated leukocytes, the
treatment of ocular disorders, the treatment and prevention of
neovasculature and angiogenesis, the destruction of viruses and
cells infected thereby, the treatment of atherosclerotic plaques,
the treatment of restenosis, and others. In addition, many
photosensitizers may be photoactivated by appropriate excitation
wavelengths to fluoresce visibly. This fluorescence can then be
used to localize a tumor or other target tissue. By incorporating
hydrophobic agents in the MA of the invention, more efficient
packaging, delivery and hence administration of the agents can be
obtained.
[0158] Generally speaking, the MA of the invention may be applied
in any manner identical or analogous to the administration of
micelles and liposomes. The concentration of the hydrophobic agent
in the MA of the invention depends upon the nature of the agent as
well as the nature of the administration desired. This dependency
also exists in application of hydro-monobenzoporphyrin
photosensitizers via MA.
[0159] The MA compositions and formulations of the invention may be
administered parenterally or by injection. Injection may be
intravenous, subcutaneous, intramuscular, intrathecal, or even
intraperitoneal. However, the MA may also be administered by
aerosol intranasally or intrapulmonarally, or topically.
Formulations designed for timed release are also with the scope of
the invention.
[0160] The quantity of hydrophobic agent MA formulation to be
administered depends on the choice of active agents, the conditions
to be treated, the mode of administration, the individual subject,
as well as the skill, experience and judgement of the practitioner.
Generally speaking, however, dosages in the range of 0.05-10 mg/kg
may be appropriate. The foregoing range is, of course, merely
suggestive, as the number of variables in regard to an individual
treatment regime is large. Therefore, considerable excursions from
these recommended values are expected.
[0161] For example, and with the use of photosensitizers as a
diagnostic in localizing tumor tissue or in localizing
atherosclerotic plaques, the MA compositions of the invention are
administered systemically in the same general manner as is known
with respect to photodynamic therapy. The waiting period to allow
the drugs to clear from tissues to which they do not accumulate is
approximately the same, for example, from about 30 minutes to about
10 hours. After the compositions of the invention have been
permitted to localize, the location of the target tissue is
determined by detecting the presence of the photosensitizer.
[0162] In diagnosis, the photosensitizers incorporated into MA may
be used along with, or may be labeled with, a radioisotope or other
detecting means. If this is the case, the detection means depends
on the nature of the label. Scintigraphic labels such as technetium
or indium can be detected using ex vivo scanners. Specific
fluorescent labels can also be used but, like detection based on
fluorescence of the photosensitizers themselves, these labels may
require prior irradiation.
[0163] For activation of the photosensitizer applied by the MA of
the invention, any suitable absorption wavelength is used. This can
be supplied using the various methods known to the art for
mediating cytotoxicity or fluorescence emission, such as visible
radiation, including incandescent or fluorescent light sources or
photodiodes such as light emitting diodes. Laser light can also be
used for in situ delivery of light to a localized photosensitizer.
In a typical protocol, for example, several hours prior to
irradiation, approximately 0.5-1.5 mg/kg of green porphyrin
photosensitizer containing MA is injected intravenously and then
excited by an appropriate wavelength.
[0164] The following example is presented to describe the preferred
embodiments, utilities and attributes of the present invention, but
they not meant to limit the invention. The invention is not to be
limited to the particular photosensitizer used in the Example.
EXAMPLE 1
Production of QLT0074 for Injection
[0165] Five hundred mL methylene chloride was added to 0.001 g
butylated hydroxytoluene, 0.01 g ascorbyl palmitate, 3.2 g dioleoyl
phosphatidyl glycerol and 4.8 g dimyristoyl phosphatidyl choline in
a pressure unit and mixed using an overhead stirrer until a clear
solution was obtained. Once the solution was clear, 1 g QLT0074
crystals was slowly added under reduced light and mixed using an
overhead stirrer until the crystals dissolved completely. The
solution was then filtered through a 0.22 .mu.m filter, and
tranfered to a round bottom flask. The flask was on a rotary
evaporator and the methylene chloride was removed under reduced
pressure, with continued drying after the distillation stopped. The
vacuum was broken and the flask was attached to a vacuum manifold
for further drying. Five hundred mL of sterile filtered 9.2% w/v
lactose monohydrate in water for injection was added to the
QLT0074/lipid thin film and agitated at room temperature for at
least 1 h to dissolve and produce multilammelar vesicles. A Model
M-1105 microfluidizer was flushed with water then some lactose
solution, and then the QLT0074/lipid solution until green solution
appeared in the discharge. The following parameters were used: air
pressure, 120 psi; operating pressure, 10,030 psi; inlet air
pressure gauge reading, 62 psi. The cooling coil reservoir was
filled with crushed ice and water to maintain a product temperature
in a range of 16-20.degree. C. The QLT/0074 lipid material was
processed 5 times through the microfluidizer. The resulting
material was then passed through 0.22 .mu.m filters, and aliquoted
into labelled lyophilization vials, with 1 ml per aliquot. The
material was lyophilized using a BCCA lyophilizer, Labconco, serial
# 215369. The lyophilized samples were stored in the dark at
2-8.degree. C.
[0166] All references cited herein, including patents, patent
applications, and publications, are hereby incorporated by
reference in their entireties, whether previously specifically
incorporated or not. As used herein, the terms "a", "an", and "any"
are each intended to include both the singular and plural
forms.
[0167] Having now fully described this invention, it will be
appreciated by those skilled in the art that the same can be
performed within a wide range of equivalent parameters,
concentrations, and conditions without departing from the spirit
and scope of the invention and without undue experimentation.
[0168] While this invention has been described in connection with
specific embodiments thereof, it will be understood that it is
capable of further modifications. This application is intended to
cover any variations, uses, or adaptations of the invention
following, in general, the principles of the invention and
including such departures from the present disclosure as come
within known or customary practice within the art to which the
invention pertains and as may be applied to the essential features
hereinbefore set forth as follows in the scope of the appended
claims.
* * * * *