U.S. patent application number 11/524718 was filed with the patent office on 2007-08-16 for ethylene glycol esters as photoactive agents.
This patent application is currently assigned to QLT Inc.. Invention is credited to Ronald E. Boch, David Dolphin, David W.C. Hunt, Ashok Jain, Julia G. Levy, Anna M. Richter, Ethan D. Sternberg, Andrew Norman Tovey, Elizabeth M. Waterfield.
Application Number | 20070191329 11/524718 |
Document ID | / |
Family ID | 32512178 |
Filed Date | 2007-08-16 |
United States Patent
Application |
20070191329 |
Kind Code |
A1 |
Sternberg; Ethan D. ; et
al. |
August 16, 2007 |
Ethylene glycol esters as photoactive agents
Abstract
New compounds useful in photodynamic therapy are of the formula
##STR1## and their 1,4-diene isomers and the metallated and/or
labeled and/or conjugated forms thereof wherein each R.sup.1 is
independently alkyl(1-6C); each n is independently an integer of
0-6; and R.sup.2 is vinyl or a derivative form thereof.
Inventors: |
Sternberg; Ethan D.;
(Vancouver, CA) ; Dolphin; David; (Vancouver,
CA) ; Levy; Julia G.; (Vancouver, CA) ;
Richter; Anna M.; (Vancouver, CA) ; Hunt; David
W.C.; (Surrey, CA) ; Jain; Ashok; (Vancouver,
CA) ; Waterfield; Elizabeth M.; (Vancouver, CA)
; Boch; Ronald E.; (North Vancouver, CA) ; Tovey;
Andrew Norman; (Vancouver, CA) |
Correspondence
Address: |
MORRISON & FOERSTER LLP
12531 HIGH BLUFF DRIVE
SUITE 100
SAN DIEGO
CA
92130-2040
US
|
Assignee: |
QLT Inc.
Vancouver
CA
|
Family ID: |
32512178 |
Appl. No.: |
11/524718 |
Filed: |
September 20, 2006 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10825270 |
Apr 14, 2004 |
7122569 |
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|
11524718 |
Sep 20, 2006 |
|
|
|
09588206 |
Jun 6, 2000 |
6756396 |
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|
10825270 |
Apr 14, 2004 |
|
|
|
09313106 |
May 17, 1999 |
6153639 |
|
|
09588206 |
Jun 6, 2000 |
|
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|
09088524 |
Jun 1, 1998 |
5929105 |
|
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09313106 |
May 17, 1999 |
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08852494 |
May 7, 1997 |
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09088524 |
Jun 1, 1998 |
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Current U.S.
Class: |
514/185 ;
514/410; 540/145 |
Current CPC
Class: |
Y10S 514/825 20130101;
G16H 20/30 20180101; C07D 487/22 20130101; A61K 41/0071 20130101;
Y10S 514/885 20130101; A61P 37/02 20180101; G16H 70/20
20180101 |
Class at
Publication: |
514/185 ;
514/410; 540/145 |
International
Class: |
A61K 31/555 20060101
A61K031/555; C07D 487/22 20060101 C07D487/22; A61K 31/409 20060101
A61K031/409 |
Claims
1. A compound of the formula ##STR5## and their 1,4-diene isomers
and the metallated and/or labeled and/or conjugated forms thereof
wherein each R.sup.1 is independently alkyl (1-6C); each n is
independently an integer of 0-6; and R.sup.2 is vinyl or a
derivative form thereof.
2. The compound of claim 1 wherein R.sup.2 is vinyl, --CHOR',
--CHO, --COOR', --CH(OR')CH.sub.3, --CH(OR')CH.sub.2OR',
--CH(SR')CH.sub.3, --CH(NR').sub.2CH.sub.3, --CH(CN)CH.sub.3,
--CH(COOR')CH.sub.3, --CH(OOCR')CH.sub.3, --CH(NR'COR')CH.sub.3,
--CH(CONR.sub.12)CH.sub.3, --CH(halo)CH.sub.3, or
--CH(halo)CH.sub.2(halo) wherein R' is H, or a hydrocarbon radical
(1-6C) optionally substituted with a heteroatom substitutent.
3. The compound of claim 1 wherein R.sup.2 is an organic group of
less than 12C resulting from direct or indirect derivatization of a
vinyl substitutent.
4. The compound of claim 1 wherein R.sup.2 is a group containing
1-3 tetrapyrrole-type nuclei.
5. The compound of claim 1 which is in a metallated form.
6. The compound of claim 1 which is in conjugated form.
7. The compound of claim 1 which is labeled.
8. The compound of claim 1 which does not contain a metal ion.
9. The compound of claim 1 wherein R.sup.2 is vinyl.
10. The compound of claim 1 wherein each R.sup.1 is methyl.
11. The compound of claim 1 wherein both n are 2.
12. The compound of claim 11 wherein R.sup.2 is vinyl and both
R.sup.1 are methyl.=
13. The compound of claim 1 which is of formulas 1-4.
14. The compound of claim 13 wherein R.sup.2 is vinyl, --CHOR',
--CHO, --COOR', --CH(OR')CH.sub.3, --CH(OR')CH.sub.2OR',
--CH(SR')CH.sub.3, --CH(NR').sub.2CH.sub.3, --CH(CN)CH.sub.3,
--CH(COOR')CH.sub.3, --CH(OOCR')CH.sub.3, --CH(NR'COR')CH.sub.3,
--CH(CONR'.sub.2)CH.sub.3, --CH(halo)CH.sub.3, or
--CH(halo)CH.sub.2(halo) wherein R' is H, or a hydrocarbon radical
(1-6C) optionally substituted with a heteroatom substitutent.
15. The compound of claim 13 wherein R.sup.2 is an organic group of
less than 12C resulting from direct or indirect derivatization of a
vinyl substitutent.
16. The compound of claim 13 wherein R.sup.2 is vinyl.
17. The compound of claim 13 wherein each R.sup.1 is methyl.
18. The compound of claim 13 wherein both n are 2.
19. The compound of claim 18 wherein R.sup.2 is vinyl and both
R.sup.1 are methyl.
20. The compound of claim 12 which is of the formula ##STR6## and
the metallated and/or labeled and/or conjugated forms thereof.
21. The compound of claim 20 which is in a metallated form.
22. The compound of claim 20 which is in conjugated form.
23. The compound of claim 20 which is labeled.
24. The compound of claim 20 which does not contain a metal
ion.
25. A pharmaceutical composition which comprises the compound of
claim 1 in admixture with at least one pharmaceutically acceptable
excipient.
26. A pharmaceutical composition which comprises the compound of
claim 20 in admixture with at least one pharmaceutically acceptable
excipient.
27. An improved method to conduct photodynamic therapy or diagnosis
wherein said method comprises administering a photoactive compound
to a subject in need of said therapy or diagnosis wherein the
improvement comprises use of the compound of claim 1 as the
photoactive agent.
28. An improved method to conduct photodynamic therapy or diagnosis
wherein said method comprises administering a photoactive compound
to a subject in need of said therapy or diagnosis wherein the
improvement comprises use of the compound of claim 20 as the
photoactive agent.
29. An improved method to conduct photodynamic therapy or diagnosis
wherein said method comprises administering a photoactive compound
to a subject in need of said therapy or diagnosis wherein the
improvement comprises use of the compound of claim 21 as the
photoactive agent.
30. An improved method to conduct photodynamic therapy or diagnosis
wherein said method comprises administering a photoactive compound
to a subject in need of said therapy or diagnosis wherein the
improvement comprises use of the compound of claim 22 as the
photoactive agent.
31. An improved method to conduct photodynamic therapy or diagnosis
wherein said method comprises administering a photoactive compound
to a subject in need of said therapy or diagnosis wherein the
improvement comprises use of the compound of claim 23 as the
photoactive agent.
32. An improved method to conduct photodynamic therapy or diagnosis
wherein said method comprises administering a photoactive compound
to a subject in need of said therapy or diagnosis wherein the
improvement comprises use of the compound of claim 24 as the
photoactive agent.
Description
[0001] This application is a continuation of U.S. Ser. No.
10/825,270 filed Apr. 14, 2004, which is a continuation of U.S.
Ser. No. 09/588,206 filed Jun. 6, 2000 and now U.S. Pat. No.
6,76,396, which is a continuation of Ser. No. 09/313,106 filed May
17, 1999 and now U.S. Pat. No. 6,153,639, which is a continuation
of U.S. Ser. No. 09/088,524 filed Jun. 1, 1998 and now U.S. Pat.
No. 5,929,105, which is a continuation-in-part of U.S. Ser. No.
08/852,494 filed 7 May 1997 and now abandoned, the contents of
which are incorporated herein by reference.
TECHNICAL FIELD
[0002] The invention relates to compounds useful in photodynamic
therapy (PDT) and related applications. In particular, it concerns
ethylene glycol esters of monohydrobenzoporphyrins.
BACKGROUND ART
[0003] Photodynamic therapy (PDT) generally involves the
administration of compounds that are capable of absorbing light,
typically in the visible range, but also in the near ultraviolet,
followed by irradiation of locations in the subject for which a
toxic or inhibitory effect is desired. PDT was initially developed
using hematoporphyrin and related compounds in the treatment of
tumors, as it appeared that these compounds would "home" to
locations containing rapidly dividing cells. The tumor could then
be irradiated with light absorbed by the hematoporphyrin and
destruction of the surrounding tissue resulted. PDT has since been
shown to be useful for treatment of atherosclerotic plaques,
restenosis, infections in the blood stream, rheumatoid arthritis,
psoriasis and in the treatment of ocular conditions not necessarily
limited to tumors.
[0004] U.S. Pat. No. 5,171,749 and patents issuing on related
applications, U.S. Pat. Nos. 5,283,255; 5,399,583; 4,883,790;
4,920,143; and 5,095,030; all of which are incorporated herein by
reference, describe and claim a class of photoactive compounds
useful in PDT designated the monohydrobenzoporphyrins, or "BPDs."
This class is obtained by Diels-Alder reaction of a mono- or
disubstituted alkyne with protoporphyrin-IX and the resultant
compounds can further be isomerized, reduced, and/or derivatized to
obtain a large class of BPDs. As disclosed in these patents, a
particularly useful subclass of this group results from hydrolysis
or partial hydrolysis of the ester groups of the 2-carboxyethyl
side-chains on rings C and D. Esterification as protection of these
groups during the Diels-Alder reaction results in initial products
which contain 2-carbalkoxyethyl groups. It was found that facile
hydrolysis of these esters could readily be conducted, leaving any
carbalkoxy groups associated with the Diels-Alder product obtained
from a dicarbalkoxyalkyne virtually completely unhydrolyzed. This
resulted in four species of compounds, BPD-MA, BPD-MB, BPD-DA and
BPD-DB as depicted in FIG. 1; this figure taken from U.S. Pat. No.
5,171,749. In this depiction, R.sup.1 and R.sup.2 are carbalkoxy
groups, typically carbomethoxy or carboethoxy, and R is alkyl
(1-6C).
[0005] BPD-MA was found to have particularly useful properties for
PDT and is currently in clinical development. However, there
remains a need for additional specific forms of photoactive agents
which expand the repertoire of photoactive compounds for the
variety of indications to which PDT is applied, as noted above. The
present invention provides compounds in which rings C and D contain
ethylene glycol esters of the carboxyalkyl substitutents. These
compounds have pharmacokinetic properties which are advantageous in
certain instances where PDT is employed.
DISCLOSURE OF THE INVENTION
[0006] The compounds of the invention are useful new additions to
the repertoire of compounds that find application in photodynamic
therapy and related methodologies that employ photoactive
compounds. The presence of ethylene glycol esters in these
molecules provides them with characteristics that permit expansion
of the scope of conditions under which such photoactive compounds
are employed and fine tuning of the treatment.
[0007] Thus, in one aspect, the invention is directed to compounds
of the formula ##STR2##
[0008] and the metallated and/or labeled and or conjugated forms
thereof
[0009] wherein R.sup.1 is alkyl (1-6C), preferably methyl, n is an
integer of 0-6, preferably 2, and R.sup.2 is vinyl or a derivative
thereof, preferably vinyl.
[0010] The invention also is directed to compounds of the formula
##STR3##
[0011] and the metallated and/or labeled and or conjugated forms
thereof wherein R.sup.1, n, and R.sup.2 are defined as described
above. These analogs are derived from protoporphyrin III and
protoporphyrin XIII respectively, in a manner similar to that in
which the compounds of formulas 1 and 2 are derived from
protoporphyrin IX. The invention also includes isomers of the
various forms of formulas 1-4 which result from the unrearranged
Diels-Alder condensation products (i.e., the 1,4-diene) as
described in U.S. Pat. No. 4,883,790, incorporated herein by
reference. These structures are also set forth in FIG. 14.
[0012] In other aspects, the invention related to methods of
diagnosis and treatment using the compounds of formula 1, 2, 3 or 4
or their 1,4-diene isomers, as shown in FIG. 14, or mixtures
thereof.
BRIEF DESCRIPTION OF THE DRAWINGS
[0013] FIG. 1 shows the compounds of the prior art, BPD-MA, BPD-MB,
BPD-DA and BPD-DB.
[0014] FIG. 2 shows the kinetics of uptake of B-EA6 by L1210
cells.
[0015] FIG. 3 shows the kinetics of release of B-EA6 by L1210
cells.
[0016] FIG. 4 shows a graphic depiction of the pharmacokinetics of
B-EA6 in vivo.
[0017] FIG. 5 shows a comparison of the kinetics of uptake of B-EA6
by normal splenocytes and L1210 cells.
[0018] FIG. 6 shows the time course of PDT using B-EA6 in mice as
compared to mice treated with BPD-MA and BPD-MB.
[0019] FIG. 7 shows the effect of B-EA6 on microvasculature in
mice.
[0020] FIG. 8 shows a comparison of the spectra in plasma of BPD-MA
and B-EA6.
[0021] FIGS. 9A and 9B show the cytotoxic effect of photodynamic
treatment using A-EA6 in comparison with BPD-MA in L1210 cells and
in dendritic cells.
[0022] FIG. 10 shows the comparative effects of A-EA6 and BPD-MA in
decreasing the surface expression of MHC I receptors.
[0023] FIG. 11 shows the effect of photodynamic therapy using A-EA6
and BPD-MA on stress and mitogenic pathway kinases in HL60
cells.
[0024] FIG. 12 shows the comparative effect of PDT using A-EA6 and
BPD-MA on caspase activation in HL60 cells.
[0025] FIG. 13 shows the comparative effect of PDT using A-EA6 and
BPD-MA on DNA fragmentation in HL60 cells.
[0026] FIG. 14 shows the structures of the unrearranged Diels-Alder
products that are precursors to the compounds of formulas 1-4.
MODES OF CARRYING OUT THE INVENTION
[0027] The compounds of the invention are related to those
disclosed in the BPD patents cited above, but differ in that they
contain esters of ethylene glycol in the substitutents on rings C
and D. These compounds can be prepared by simple hydrolysis of the
carbalkoxyalkyl or carbalkoxyl substitutents and reesterification
of the resulting carboxyl groups in the C and D-rings of the
benzoporphyrins, or can be obtained directly by
transesterification.
[0028] It will be noted that compounds 1 and 2 and their 1,4-diene
isomers are individual species of the genus, described in the
above-referenced U.S. patents, obtained through a process which
comprises a Diels-Alder reaction with protoporphyrin IX. Compounds
3 and 4 and their 1,4-diene isomers are prepared in a completely
analogous manner but using protoporphyrin III or protoporphyrin
XIII as substrates for the Diels-Alder reaction. Since
protoporphyrin IX is not symmetric with respect to the A and B
rings, two possible products result depending on whether the
Diels-Alder addition occurs in the A or B ring. On the other hand,
protoporphyrins III and XIII are symmetric with respect to these
rings, and therefore only one product results in each case
regardless of the site of addition.
[0029] In the compounds of the invention, R.sup.2 is preferably
vinyl, but may also be a derivative thereof. The vinyl group in
ring A or B is readily derivatized to other embodiments of R.sup.2
by addition or oxidation. The addition or oxidation products can be
further substituted if the added substitutents are functional as
leaving groups, for example, --Br may be substituted by --OH,
--OR'', --NH.sub.2, --NHR'', or --NR.sub.2'', etc., where R'' is a
hydrocarbon radical. For instance, one of the added substitutents
may be hydrogen and the other halo, hydroxy, lower alkoxy, amino,
or an amide, sulfhydryl or an organosulfide or an additional
hydrogen. The compounds of the invention include various groups as
R.sup.2 including substitutents which provide additional porphyrin
or porphyrin-related ring systems.
[0030] Thus, R.sup.2 may be vinyl, --CHOR', --CHO, --COOR',
--CH(OR')CH.sub.3, --CH(OR')CH.sub.2OR', --CH(SR')CH.sub.3,
--CH(NR').sub.2CH.sub.3, --CH(CN)CH.sub.3, --CH(COOR')CH.sub.3,
--CH(OOCR')CH.sub.3, --CH(NR'COR')CH.sub.3,
--CH(CONR'.sub.2)CH.sub.3, --CH(halo)CH.sub.3, or
--CH(halo)CH.sub.2(halo) wherein R' is H, or a hydrocarbon radical
(1-6C) optionally substituted with a heteroatom substitutent or
wherein R.sup.2 is an organic group of less than 12C resulting from
direct or indirect derivatization of the vinyl group, or wherein
R.sup.2 is a group containing 1-3 tetrapyrrole-type nuclei.
[0031] As used herein, the term "alkyl" refers to a saturated
straight or branched chain hydrocarbon which may, if it contains a
sufficient number of carbon atoms, be cyclic or contain a cyclic
portion. Typical examples are methyl, ethyl, t-butyl, cyclohexyl,
and the like.
[0032] A "hydrocarbon radical" refers to a monovalent substitutent
containing only carbon and hydrogen which may be straight or
branched chain, saturated or unsaturated, aromatic or nonaromatic
or both, and cyclic or noncyclic. Thus, a hydrocarbon radical of
1-10C could include cyclopentylethyl, 2-pentenyl, 3-butynyl,
2,4-dimethylhexyl, and the like.
[0033] In some embodiments of the invention, the hydrocarbon
radical may be substituted with a heteroatom-containing
substitutent. Such substitutents include --OR, --NR.sub.2, --SR,
--COOR, --CONR.sub.2, --OOCR, --NRCOR, --SOR, --SO.sub.2R,
--SO.sub.3R, halo, --CN, and the like, wherein R is H or alkyl
(1-6C). Cyclic amines include pyridyl, pyrimidyl, thiazolyl,
quinolyl, and so forth. Thus, they may include single ring or fused
ring systems and may contain additional heteroatoms.
[0034] It will be noted that the compounds of the invention contain
at least one chiral center and thus may exist in various
stereoisomeric forms. If desired, such stereoisomers, including
enantiomers, may be separated using techniques standard in the art;
however, racemic mixtures or mixtures containing more than one
diastereomer may also be used. The compounds as indicated in
formulas 1-4 and in FIG. 14, therefore, are representative of the
individual optical isomers, enantiomers or diasteriomers as the
case may be, as well as mixtures of these individual chiral
isomers.
[0035] If desired, the compounds of the invention can be prepared
in metallated forms by treating the tetrapyrrole-type nucleus with
an appropriate ion such as magnesium ion, zinc ion, stannous ion
and the like, to obtain a metal complex. The metal ion may also be
a radiolabel. Generally, the metal ion is inserted using the
appropriate salts under conditions standard in the art. For
example, zinc ion can be introduced by treating the compound with
zinc acetate in 1:1 methylene chloride:methanol.
[0036] The compounds may also contain label, including
radioisotopes, chromophores, and fluorescent labels. Radioisotope
labeling is generally useful when the compounds are to be followed
in vivo or used to label specific moieties. Useful cationic
moieties that are radioisotopes include technetium, gallium and
indium. In addition, radioisotopes of heteroatoms, such as
.sup.131I or .sup.32P, in the molecule itself, or inclusion of
.sup.14C may be used to label the molecule.
[0037] As further described in the BPD-related patents set forth
above, the compounds of the invention may be coupled, if desired,
to a targeting agent which will direct the molecule to a specific
tissue or organ. Such targeting agents include antibodies,
receptors, receptor-ligands and the like. Linkage of the targeting
agent to the compound is conducted using standard techniques. By a
"conjugated form" is meant a compound of formulas 1-4 coupled to a
targeting agent, as above described.
[0038] Preferred embodiments of the compounds of formulas 1-4 and
their 1,4-diene isomers include those wherein both n equal 2, or
those wherein both R.sup.1 are ethyl or methyl, preferably methyl,
and those wherein R.sup.2 is vinyl. Particularly preferred are
compounds of the formula ##STR4##
[0039] Both A-EA6 and B-EA6 are effective photosensitizers; it
appears that A-EA6 is the easier to formulate.
[0040] The various forms of the compounds of the invention can be
used in the photodynamic therapy techniques generally known in the
art. As set forth in the Background section above, photodynamic
therapy can be conducted using a plethora of protocols and for a
variety of indications. In addition, compounds of this type exhibit
pharmacological activity in the absence of light in some instances.
Standard pharmaceutical compositions, including liposomal
compositions as preferred, are used as desired in such
applications.
[0041] The following examples are intended to illustrate but not to
limit the invention. While the Examples illustrate and demonstrate
the surprising pharmacokinetic properties of two members of the
species of the invention, A-EA6 and B-EA6, it is expected that the
remaining compounds described by formulas 1-4 and their 1,4-diene
isomers will have similar variations in these properties. Hence,
the small class of compounds contained in the present invention
offers valuable additions to the repertoire of photodynamic agents
useful in treating the various conditions to which this therapy has
been directed.
EXAMPLE 1
Preparation of Two Forms of EA6
A. To prepare B-EA6, the starting material is BPD-DB as the
dimethyl ester--i.e., BPD-DB as shown in FIG. 1 wherein R.sup.1 and
R.sup.2 are both COOMe an R'' is vinyl.
[0042] To 2.0 g (2.7 mM) BPD-DB in 50 mL ethylene glycol and 100 mL
dichloromethane was added 1.0 mL sulfuric acid. The reaction was
stirred for 18 hr. at room temperature. Then the reaction was added
to a stirring mixture of 100 mL 5% aqueous ammonium acetate and 100
mL dichloromethane. The organic layer was isolated and then washed
twice with 50 mL water. The solvent was removed by rotary
evaporation. The dark green residue was then chromatographed on 75
g alumina (deactivated with 5% water) and eluted with a gradient of
0.5%-5.0% methanol in dichloromethane. The solvent from the
fractions containing product was then removed by rotary
evaporation. The residue was dried in vacuo overnight to provide
2.02 g (89%) of the analytically pure green sold title
compound.
[0043] B. In a manner similar to that set forth in paragraph A, but
substituting BPD-DA for BPD-DB, the isomeric form, A-EA6 was
prepared.
EXAMPLE 2
Comparison of Uptake and Release of B-EA6 and BPD-MA by L1210
Cells
[0044] BPD-MA or B-EA6 were incubated at 3 .mu.g/ml in the presence
of 10% fetal bovine serum with 10.sup.7/mL of L 1210 cells, a
murine leukemia cell line. Intracellular content of the
photosensitizers was measured by fluorescence of cell lysates at
various times. The maximum concentration reached was 145.9
ng/10.sup.6 cells for B-EA6 and 149.5 ng/10.sup.6 cells for BPD-MA.
The time course of uptake is shown in FIG. 2 as a percentage of
cell content at 60 min by which time uptake had reached a maximum
in both cases. As shown, B-EA6 is taken up more rapidly and reaches
805 of its maximum concentration after only 5 min and reached its
maximum uptake within 15 min.
[0045] The kinetics of release of these drugs from L1210 cells was
measured by preloading the cells at 3 .mu.g/ml for 1 hr and then
placing the cells in drug-free medium containing 10% fetal bovine
serum. Remaining intracellular drug content was measured at various
time points by lysing the cells and measuring fluorescence. As
shown in FIG. 3 (again as a percent of starting intracellular
content), BPD-MA and B-EA6 showed different kinetics of release.
Initial release of B-EA6 was much more rapid, but release was more
complete in the case of BPD-MA.
[0046] It was unexpected that the in vitro pharmacokinetics of
B-EA6 were more rapid than those of BPD-MA. While the higher
retention of B-EA6 could be attributed to its increased size as
compared to BPD-MA, the faster transfer through the cellular
membrane was unexpected.
EXAMPLE 3
Comparison of In Vivo Pharmacokinetics
[0047] Either BPD-MA or B-EA6 was administered by intravenous
injection into DBA/2 mice at a dose of 4 mg/kg using 3 mice per
time point. The drug content of plasma, skin, liver and kidney was
determined by fluorescence in the tissue extracts. FIG. 4 shows the
results plotted as a percentage of the concentration in the
relevant tissue 15 min postinjection. As seen in FIG. 4, neither
BPD-MA nor B-EA6 accumulated in plasma, liver or kidney; however,
BPD-MA accumulated in skin within the first 3 hr; B-EA6 does
not.
[0048] The more rapid accumulation of B-EA6 as compared to BPD-MA,
as here confirmed in vivo by more rapid clearance from all tissues,
constitutes an advantage. The treatment with light can be carried
out soon after injection of the photosensitizer and due to the
rapid clearance, no prolonged skin or eye photosensitivity will be
exhibited. Thus, the subjects treated can resume normal lives
without special precautions such as avoiding bright light and
wearing dark eyeglasses.
[0049] The half-life of B-EA6 and BPD-MA in various tissues was
then computed in the time-frame 15 min-3 hr and the results are
shown in Table 1: TABLE-US-00001 TABLE 1 Tissue Half-Lives of B-EA6
and BPD-MA T1/2* (15 min-3 hours) Tissue B-EA6 BPD-MA Liver 0.6 2.4
Spleen 0.8 10.9 Kidney 0.8 5.6 Skin 1.9 .sup. 0** Muscle 11.1
ND.dagger. Plasma 0.6 2.0 *shown in hours **BPD-MA concentration in
the skin increased for up to 3 hr .dagger.ND = not determined
[0050] The half-life of BPD-MA in this time-frame could not be
computed in skin since its concentration increased during the 3 hr
period. As shown in Table 1, generally, B-EA6 has a much shorter
half-life than BPD-MA in most tissues. The lack of accumulation of
B-EA6 in normal skin as compared to BPD-MA was unexpected, and
indicates more rapid clearance than that of BPD-MA. As set forth
above, this is advantageous as skin photosensitivity is the only
recognized side effect of photodynamic therapy utilizing
photosensitizers.
[0051] The pharmacokinetics were also determined using an in vivo
mouse tumor model. Groups of 10 DBA/2 mice containing M1
rhabdomyosarcoma tumors were injected intravenously with a
liposomal formulation of BPD-MA at various dosages of 0.75-1.5
mg/kg. The tumors were irradiated with 690 nm laser light at 50 or
150 J/cm.sup.2 at various times after injection. The results, as
shown in Table 2, were determined in terms of the percentage of
mice in each group that were tumor-free on day 7 after injection.
TABLE-US-00002 TABLE 2 Results of Bioassay PDT Conditions Drug**
Time Light*** Percent Tumor Dose post IV dose Free on Day 7*
(mg/kg) (min) (J/cm.sup.2) BPD-MA B-EA6 0.75 15 50 (4/5) 50% 30 50
70% 0% 1.0 15 50 100% 90% 30 50 90% 0% 1.5 180 150 70% 0% *tumor
model = MI tumor in DBA/2 mice - each PDT condition was tested in
10 animals **the drugs were liposomally formulated and injected
intravenously ***690 nm laser light.
[0052] As shown in Table 2, BPD-MA treated mice showed substantial
survival rates when postinjection times ranged from 15-180 min. On
the other hand, B-EA6 treated mice showed no response at 30 min or
180 min; however, significant responses were obtained when
irradiation was supplied after only 15 min.
[0053] These data demonstrate that PDT using B-EA6 will be
effective in early treatment with light. The lack of effect of
later times postinjection indicates, again, rapid clearance of
B-EA6 which is advantageous for the reasons set forth above.
EXAMPLE 4
Determination of LD.sub.50 with and without Serum
[0054] Either B-EA6 or BPD-MA was incubated for 1 hr with L1210
cells at a range of concentrations and exposed to 9 J/cm.sup.2
broad spectrum light. This determination was made in the absence of
serum and in the presence of 10% serum. The results are shown in
Table 3. TABLE-US-00003 TABLE 3 LD.sub.50 Values No serum 10% serum
BPD-MA 3.7 ng/ml 54.0 ng/ml B-EA6 4.7 ng/ml 19.7 ng/ml
[0055] As shown, BPD-MA and B-EA6 have comparable LD.sub.50 values
in the absence of serum; however, in the presence of serum, B-EA6
shows a substantially better retention of effectiveness.
[0056] In most instances, the presence of serum greatly reduces the
photoactivity of agents used in PDT, such as BPD-MA. Surprisingly,
B-EA6 shows more affinity for cell membranes than for plasma
components and is thus very slightly affected by the presence of
serum in the cellular environment. Thus, in vivo, its activity may
be higher than that of BPD-MA and other compounds of this
family.
EXAMPLE 5
In Vitro Efficacy of B-EA6
[0057] The ability of B-EA6 to exert a cytotoxic effect on L1210
cells in vitro was further tested by incubating the cells with
B-EA6 at various concentrations for 1 hr in the absence of serum.
After excess drug was removed, the cells were exposed to 9
J/cm.sup.2 broad spectrum light (380-750 nm) and cell survival was
determined by the MTT assay (Mosmann, T. et al. J Immunol Meth
(1983) 65:55-63). The percentage of killed cells was calculated in
reference to survival of cells exposed to light only. At a
concentration of approximately 7 ng/ml, 80% of the cells were
killed; at 15 ng/ml, almost 100% of the cells did not survive. As
stated above, the LD.sub.50 for B-EA6 is approximately 4.7
ng/ml.
[0058] The somewhat lower effect of B-EA6 as compared to BPD-MA in
vitro makes even more unexpected the comparatively higher activity
of B-EA6 as compared to BPD-MA in vivo in the presence of serum as
demonstrated in Example 4.
EXAMPLE 6
Selectivity of B-EA6 for Tumor Cells
[0059] The ability of L1210 cells to accumulate B-EA6 was compared
to the ability of splenocytes to do so. B-EA6 at 3 .mu.g/ml was
incubated with each cell type and the cell content of B-EA6 was
determined by fluorescence in cell lysates. FIG. 5 shows a
comparison of uptake for the two cell types in ng/10.sup.6 cells.
As shown, L1210 cells were able to take up approximately 140
ng/10.sup.6 cells reaching this value after approximately 20 min.
Splenocytes, on the other hand, accumulated less than 20
ng/10.sup.6 cells after an hour of incubation.
[0060] DBA/2 mice bearing M1 (rhabdomyosarcoma) tumor, grown
subcutaneously in their flanks, were used as a model to show that
B-EA6 demonstrated selectivity for tumors. Mice were administered
0.75 mg/kg of B-EA6 in a liposomal formulation intravenously. After
15 min, a 1 cm area which included a 5 mm diameter tumor was
exposed to 50 J/cm.sup.2 of 70 mW light of 690 nm wavelength from
an argon-pumped dye laser. The exposure effectively eliminated the
tumor, but did not affect the surrounding normal skin. Thus, B-EA6
demonstrates tumor specificity.
EXAMPLE 7
Immunomodulation by B-EA6
[0061] Balb/C mice (5-8 mice per group) were tested using the
delayed skin photosensitivity assay also called the contact
hypersensitivity (CHS) assay. The mice were painted in the flank
with the sensitizing agent dinitrofluorobenzene (DNFB) and 5 days
later, one ear is challenged with DNFB, while the other serves as a
control. The swelling is an indicator of immune response. Mice were
injected intravenously with 1 mg/kg liposomal B-EA6 and either
irradiated with 15 J/cm.sup.2 light over the whole body or exposed
to ambient light. The ability of this treatment to prevent the
immune response as demonstrated by inhibition of ear swelling was
determined. The results showed that administering B-EA6 combined
with either after irradiation with 15 J/cm.sup.2 whole body light
or with ambient light decreased swelling in the test ear as
compared to untreated mice. The swelling in both cases was only
approximately 60% of the that shown in mice without treatment.
[0062] In an additional assay to determine immunomodulation, murine
peritoneal macrophages were isolated, purified and activated by
recombinant interferon-.gamma. (100 U/ml). The activated cells were
incubated for 1 hr at 37.degree. C. with B-EA6 at a range of
concentrations and then exposed to 690 nm LED light at 5
J/cm.sup.2. Expression levels of MHC I, MHC II, CD54, CD80 and CD86
were determined 24 hr later using FITC conjugated antibodies and a
cell sorter. The results are shown in Table 4 for B-EA6 at 0.5
ng/ml in comparison to similar experiments using BPD-MA at 2.5
ng/ml. TABLE-US-00004 TABLE 4 Effect of Low-Dose PDT with B-EA6 on
Expression Levels of Cell Surface Antigens by Murine Peritoneal
Macrophages MHC MHC CD54 CD80 CD86 Compound Class I Class II
(ICAM-1) (B7-1) (B7-2) BPD-MA 99.1 .+-. 79.3 .+-. 105.4 .+-. 3.0%
93.5% 99.2% (2.5 ng/ml) 4.3% 10.1% BPD-B-EA6 100.4% 71.8% 106.9%
102.3% 92.2% (0.5 ng/ml)
[0063] The results in the table are given as a percent of
expression as compared to cells treated with light only. As shown,
BPD-MA and B-EA6 were both able to reduce expression of MHC II, but
not the remaining surface markers. Thus, although B-EA6 has
advantageous pharmacokinetics, it retains the immunomodulatory
activity of BPD-MA and other compounds of this group.
EXAMPLE 8
Effect of B-EA6 in an Arthritis Model
[0064] MRL-Ipr mice spontaneously develop arthritis; this was
enhanced by intradermal injection of Freund's Adjuvant. Various
numbers of MRL-Ipr mice were treated with PDT on days 0, 10, and 20
after injection of the adjuvant. PDT consisted of 0.5 mg/kg
liposomal B-EA6 injected intravenously followed by exposure of the
ventral part of the mice to red (560-900 nm) light at 80 J/cm.sup.2
at 1 hr post-B-EA6 injection. The mice were observed and symptoms
scored every 5 days for 30 days. The results are shown in FIG. 6 in
comparison to mice similarly treated with BPD-MA and BPD-MB. As
shown in FIG. 6, whether measured by the incidence of clinical
symptoms (i.e., the percentage of mice exhibiting these symptoms)
or by the change in bimaleolar ankle width in millimeters, B-EA6
(shown as solid circles) was effective in preventing the sequellae
of adjuvant injection.
[0065] Again, the retention of immunomodulatory activity of B-EA6
is demonstrated.
EXAMPLE 9
Effect of B-EA6 on Microvasculature
[0066] The mouse cremaster muscle model was used. B-EA6 was
administered intravenously at 2 mg/kg and starting at 5 and 15 min
postinjection, surgically exposed arterioles and venules were
irradiated with light at an intensity of 25 J/cm.sup.2 per 5 min
beginning at 5 min and 15 min after injection of the B-EA6. The
vessels were measured as red blood column diameter as a percentage
of controls.
[0067] The results are shown in FIG. 7. While transient vessel
closure could be obtained when irradiation was started at 5 min,
permanent closure was obtained when radiation was started after 15
min.
[0068] The enhanced capacity of B-EA6 to constrict or occlude
vasculature, as demonstrated in this Example, in combination with
more rapid pharmacokinetics, make B-EA6 particularly advantageous
in treating neovascular diseases in the eye.
EXAMPLE 10
Absorption Spectrum of B-EA6
[0069] BPD-MA and B-EA6 have similar absorption spectra in plasma
before and after 4-hr exposure to fluorescent (380-750 nm) light. A
comparison of these spectra is shown in FIG. 8. The similarity of
the spectrum of B-EA6 to the spectrum of BPD-MA is advantageous
since the use of BPD-MA as a therapeutic agent useful in PDT is
well developed. The similarity in their spectra indicates that the
same light sources can be used for B-EA6 as are successful in
treatment with BPD-MA.
EXAMPLE 11
In Vitro Cytotoxicity of A-EA6
[0070] In a manner similar to that set forth in Example 5, the
cytotoxicity of A-EA6 in vitro on two different cell lines was
tested and compared with BPD-MA. Either L1210 cells or the
dendritic cell line D2SC/1 was incubated for one hour at 37.degree.
C. with either A-EA6 or BPD-MA. After removal of excess drug, the
cells were exposed to 690 nm light at 5 J/cm.sup.2 light. Cell
survival was determined 18-24 hours later using the MTT
colorimetric assay described in Example 5. Percent cells killed was
calculated by reference to cells exposed to light only. As shown in
FIG. 9A, A-EA6 showed comparable cytotoxicity to BPD-MA with
respect to L1210 cells in the absence of serum but was markedly
more toxic in the presence of serum than BPD-MA. The open circles
represent A-EA6 plus serum; the closed circles represent BPD-MA
plus serum; open squares represent A-EA6 in the absence of serum;
and closed squares represent BPD-MA in the absence of serum.
[0071] As shown in FIG. 9B, in dendritic cells where BPD-MA has an
LD.sub.50 of 6 ng/ml and A-EA6 has an LD.sub.50 of 2.7 ng/ml, A-EA6
was toxic at lower concentrations than BPD-MA in the presence of 5%
fetal calf serum. In FIG. 9B, closed circles represent BPD-MA and
open squares represent A-EA6.
[0072] In a similar determination, but measuring MHC I receptors
rather than cytotoxicity, A-EA6 was effective in decreasing
expression of these receptors at lower concentrations. In this
determination, dendritic cells were incubated for 1 hour at a drug
concentration less than its LD.sub.50; 2.5 ng/ml and 5 ng/ml for
BPD-MA and 1 ng/ml and 2.5 ng/ml for A-EA6. The cells were treated
with 690 nm light at 5 J/cm.sup.2 and then labeled with the
appropriate antibody 3 hours post-treatment and assessed by flow
cytometry. The results were measured as the percent of the mean
channel fluorescence intensity for light-treated control cells.
These results are shown in FIG. 10; BPD-MA gave an 18% and a 29%
reduction, respectively, at 2.5 ng/ml and 5 ng/ml; A-EA6 lowered
the channel fluorescence by approximately 25% at both 1 ng/ml and
2.5 ng/ml concentrations.
EXAMPLE 12
Effect of A-EA6 on Intracellular Signaling
[0073] The conditions of the study set forth in Example 11 were
repeated using HL-60 cells as the target and comparing the effects
of A-EA6 and BPD-MA on cytotoxicity, on the mitogenic pathway
kinase p70 S6K, and on the stress pathway kinases c-jun and HSP27.
The results are shown in FIG. 11. At sublethal concentrations,
A-EA6 showed stronger activation of the stress pathway kinases and
stronger inhibition of the mitogenic pathway kinases.
[0074] The effect on caspase activation in HL-60 cells was also
measured. A-EA6 showed a stronger activation of caspases than did
BPD-MA. This effect is desirable as it is associated with
apoptosis. Using apoptosis to remove unwanted cells causes the
least effect on surrounding normal cells and tissues. The
comparison of A-EA6 with BPD-MA is shown in FIG. 12.
[0075] FIG. 13 shows a similar comparison when percent DNA
fragmentation was measured in HL-60 cells. Again, A-EA6 was
effective at lower concentrations than BPD-MA.
EXAMPLE 13
In Vivo Photodynamic Therapy Using A-EA6
[0076] In a protocol similar to that set forth in Example 3, either
A-EA6 or BPD-MA was injected intravenously into mice harboring M1
tumors at a dose of 1 mg/kg. This was followed by whole body
irradiation with 50 J/cm.sup.2 of 690 nm laser light at various
times after administration of the drug. The number of tumor-free
animals on day 7 was determined and the results are shown in Table
5. TABLE-US-00005 TABLE 5 Irradiation Day 7 tumor-free
Photosensitizer time (post i.v.) animals BPD-MA 15 min 10/10 30 min
9/10 A-EA6 15 min 2/2 30 min 6/6
[0077] These results show A-EA6 is at least as effective as BPD-MA
in this assay.
EXAMPLE 14
Immunomodulatory Activity
[0078] Flanks of control and test mice were painted with the
antigen DMFB and their ears were challenged 5 days later by pasting
with the same compound. Test animals were treated with whole-body
PDT using BPD-MA or A-EA6, by injecting the photosensitizer
intravenously and then exposing the animals to red LED light at 15
J/cm.sup.2. The percent suppression of ear swelling was calculated
in comparison to controls. The results are shown in Table 6 and
indicate that A-EA6 had a stronger immunomodulatory effect in this
assay than did BPD-MA. TABLE-US-00006 TABLE 6 Photosensitizer Dose
(mg/kg) Percent suppression BPD-MA 1.0 49% A-EA6 1.0 68% A-EA6 0.3
59%
* * * * *