U.S. patent application number 12/189695 was filed with the patent office on 2009-02-26 for modulators of retinol-retinol binding protein (rbp)-transthyretin (ttr) complex formation.
This patent application is currently assigned to SIRION THERAPEUTICS, INC.. Invention is credited to Yun Han, Jay Lichter, Nathan L. Mata, Kenneth Widder.
Application Number | 20090054532 12/189695 |
Document ID | / |
Family ID | 36337095 |
Filed Date | 2009-02-26 |
United States Patent
Application |
20090054532 |
Kind Code |
A1 |
Mata; Nathan L. ; et
al. |
February 26, 2009 |
Modulators of Retinol-Retinol Binding Protein (RBP)-Transthyretin
(TTR) Complex Formation
Abstract
Described herein are methods and compositions for the detection
of transthyretin (TTR), retinol binding protein (RBP) and retinol
complex formation. The methods and compositions described herein
also provide for the screening of modulators of retinol-RBP-TTR
complex formation. Furthermore, the methods and compositions
provide for therapeutic agents for the treatment and/or prevention
of age-related macular degeneration and/or dystrophies.
Inventors: |
Mata; Nathan L.; (San Diego,
CA) ; Han; Yun; (San Diego, CA) ; Widder;
Kenneth; (Rancho Santa Fe, CA) ; Lichter; Jay;
(Rancho Santa Fe, CA) |
Correspondence
Address: |
WILSON SONSINI GOODRICH & ROSATI
650 PAGE MILL ROAD
PALO ALTO
CA
94304-1050
US
|
Assignee: |
SIRION THERAPEUTICS, INC.
San Diego
CA
|
Family ID: |
36337095 |
Appl. No.: |
12/189695 |
Filed: |
August 11, 2008 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11267395 |
Nov 4, 2005 |
7432307 |
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12189695 |
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60625532 |
Nov 4, 2004 |
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60629695 |
Nov 19, 2004 |
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60660904 |
Mar 11, 2005 |
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60672405 |
Apr 18, 2005 |
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Current U.S.
Class: |
514/725 ;
436/164 |
Current CPC
Class: |
A61P 27/02 20180101;
G01N 2500/02 20130101; Y10S 514/912 20130101; G01N 33/542 20130101;
A61P 21/04 20180101; G01N 2800/164 20130101; A61K 31/215 20130101;
A61K 2300/00 20130101; A61P 17/00 20180101; A61K 2300/00 20130101;
A61K 2300/00 20130101; A61K 31/203 20130101; A61K 49/0041 20130101;
A61K 49/0052 20130101; A61P 27/00 20180101; A61K 31/203 20130101;
A61K 2300/00 20130101; A61K 49/0056 20130101; A61K 31/16 20130101;
A61K 31/16 20130101; A61K 31/215 20130101; A61K 41/0061 20130101;
G01N 33/6893 20130101; A61K 41/0061 20130101 |
Class at
Publication: |
514/725 ;
436/164 |
International
Class: |
A61K 31/07 20060101
A61K031/07; G01N 21/00 20060101 G01N021/00; A61P 27/00 20060101
A61P027/00 |
Claims
1. (canceled)
2. (canceled)
3. (canceled)
4. A method of identifying a therapeutic agent for the treatment of
macular degenerations or dystrophies comprising: a. incubating an
assay mixture comprising at least one candidate therapeutic agent
for the treatment of macular degenerations or dystrophies, labeled
TTR, RBP and retinol under conditions sufficient to permit
formation of a retinol-RBP-labeled TTR complex, wherein the TTR
label is a fluorophore: and b. measuring the emission spectra of
the retinol-RBP-labeled TTR complex by fluorescence resonance
energy transfer (FRET); wherein a change in the emission spectra of
the retinol-RBP-labeled TTR complex after incubation of the
candidate therapeutic agent indicates modulation of the
retinol-RBP-labeled TTR complex by the candidate therapeutic
agent.
5. (canceled)
6. The method of claim 4, wherein the fluorophore is an acceptor
fluorescence moiety.
7. (canceled)
8. The method of claim 4, wherein the fluorophore absorbs at
between 380 nm and 480 nm and emits at between 520 nm and 600
nm.
9. The method of claim 4, wherein the TTR is labeled with a
fluorophore chosen from the group consisting of:
N-((2-(iodoacetoxy)ethyl)-N-methyl)amino-7-nitrobenz-2-oxa-1,3-diazole,
4-dihexadecylamino-7-nitrobenz-2-oxa-1,3-diazole,
6-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)hexanoic acid,
succinimidyl
6-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)hexanoate, lucifer
yellow iodoacetamide, N-(5-aminopentyl)-4-amino-3,6-
disulfo-1,8-naphthalimide,
N,N'-dimethyl-N-(iodoacetyl)-N'-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)ethyle-
nediamine,
1-(2-maleimidylethyl)-4-(5-(4-methoxyphenyl)oxazol-2-yl)pyridin-
ium methanesulfonate,
1-(2,3-epoxypropyl)-4-(5-(4-methoxyphenyl)oxazol-2-yl)pyridinium
trifluoromethanesulfonate,
1-(3-(succinimidyloxycarbonyl)benzyl)-4-(5-(4-methoxyphenyl)oxazol-2-yl)
pyridinium bromide, 3-(4-carboxybenzoyl)quinoline-2-carboxaldehyde,
3-(2-furoyl)quinoline-2-carboxaldehyde and ##STR00003##
10. The method of claim 4, wherein the dye-TTR is labeled with
##STR00004##
11. The method of claim 6, wherein the excitation wavelength is
between 275 nm and 295 nm and the emission wavelength is measured
at between 330 nm and 650 nm.
12. The method of claim 6, wherein the excitation wavelength is
between 315 and 345 nm, and the emission wavelength is measured at
between 525 and 600 nm.
13. The method of claim 4, wherein the candidate therapeutic agent
is a small molecule.
14. The method of claim 4, wherein the candidate therapeutic agent
is a retinyl derivative.
15. The method of claim 14, wherein the retinyl derivative is
N-(4-hydroxyphenyl)retinamide, N-(4-methoxyphenyl)retinamide, or
ethylretinamide
16. (canceled)
17. (canceled)
18. (canceled)
19. (canceled)
20. (canceled)
21. (canceled)
22. (canceled)
23. (canceled)
24. (canceled)
25. (canceled)
26. (canceled)
27. The method of claim 4, wherein the emission spectra of the
retinol-RBP-labeled TTR complex is decreased after incubation of
the candidate therapeutic agent.
28. A method of identifying a therapeutic agent for the treatment
of macular degenerations or dystrophies comprising: a. incubating
an assay mixture comprising at least one candidate therapeutic
agent for the treatment of macular degenerations or dystrophies,
labeled TTR, RBP and retinol under conditions sufficient to permit
formation of a retinol-RBP-labeled TTR complex, wherein the TTR
label is a fluorophore with an absorbance spectra between 380 and
480 nm and an emission spectra between 520 and 600 nm; and b.
measuring the emission spectra of the retinol-RBP-labeled TTR
complex; wherein a change in the emission spectra of the
retinol-RBP-labeled TTR complex after incubation of the candidate
therapeutic agent indicates modulation of the retinol-RBP-labeled
TTR complex by the candidate therapeutic agent.
29. The method of claim 28, wherein the TTR is labeled with a
fluorophore chosen from the group consisting of:
N-((2-(iodoacetoxy)ethyl)-N-methyl)amino-7-nitrobenz-2-oxa-1,3-diazole,
4-dihexadecylamino-7-nitrobenz-2-oxa-1,3-diazole,
6-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)hexanoic acid,
succinimidyl
6-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)hexanoate, lucifer
yellow iodoacetamide,
N-(5-aminopentyl)-4-amino-3,6-disulfo-1,8-naphthalimide,
N,N'-dimethyl-N-(iodoacetyl)-N'-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)ethyle-
nediamine,
1-(2-maleimidylethyl)-4-(5-(4-methoxyphenyl)oxazol-2-yl)pyridin-
ium methanesulfonate,
1-(2,3-epoxypropyl)-4-(5-(4-methoxyphenyl)oxazol-2-yl)pyridinium
trifluoromethanesulfonate,
1-(3-(succinimidyloxycarbonyl)benzyl)-4-(5-(4-methoxyphenyl)oxazol)
pyridinium bromide, 3-(4-carboxybenzoyl)quinoline-2-carboxaldehyde,
3-(2-furoyl)quinoline-2-carboxaldehyde and ##STR00005##
30. The method of claim 29, wherein the emission spectra of the
retinol-RBP-labeled TTR complex is detected by fluorescence
resonance energy transfer (FRET).
31. The method of claim 29, wherein the emission spectra of the
retinol-RBP-labeled TTR complex is decreased after incubation of
the candidate therapeutic agent.
32. The method of claim 4, wherein the candidate therapeutic agent
is a small molecule.
33. The method of claim 28, wherein the candidate therapeutic agent
is a retinyl derivative.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This Application claims the benefit of U.S. Provisional
Application serial number 60/625,532 filed Nov. 4, 2004, U.S.
Provisional Application Ser. No. 60/629,695, filed on Nov. 19,
2004, U.S. Provisional Application Ser. No. 60/660,904, filed on
Mar. 11, 2005, U.S. Provisional Application Ser. No. 60/672,405,
filed on Apr. 18, 2005, the disclosures of all of which are hereby
incorporated by reference in their entirety.
FIELD OF THE INVENTION
[0002] The methods and compositions described herein are directed
to the treatment of ophthalmic conditions.
BACKGROUND OF THE INVENTION
[0003] Retinoids are essential for maintenance of normal growth,
development, immunity, reproduction, vision and other physiological
processes. Conversely, abnormal production or processing of
retinoids correlates with the manifestation of disease processes,
including macular degeneration.
[0004] Macular degeneration is a group of eye diseases that is the
leading cause of blindness for those aged 55 and older in the
United States, affecting more than 10 million Americans. Some
studies predict a six-fold increase in the number of new macular
degeneration over the next decade, taking o the characteristics of
an epidemic. Age-related macular degeneration or dystrophy, a
particularly debilitating disease, leads to gradual loss of vision
and eventually severe damage to the central vision.
[0005] To date, there is no effective cure for macular
degeneration. Therefore, there is an urgent need to provide for
assays that screen for therapeutic compositions to treat these
diseases.
SUMMARY OF THE INVENTION
[0006] Described herein are methods and compositions for detecting
agents which modulate the formation of a retinol-retinol binding
protein (RBP)-transthyretin (TTR) complex. Also described herein
are methods and compositions for detecting, quantitating, and/or
monitoring the retinol-RBP-TTR complex. Also presented herein are
treatment methods for ophthalmic conditions, including the wet and
dry forms of the macular degenerations and dystrophies and
geographic atrophy, comprising administration of a compound that
modulates me formation of a tetinol-retinol binding protein
(RBP)-transthyretin (TTR) complex.
[0007] In one embodiment, the methods and compositions described
herein provide for the detecting and/or quantitating of
retinol-RBP-TTR complex formation in a sample comprising RBP,
retinol and TTR comprising measuring the emission spectra of the
complex, wherein at least some of the TTR further comprises a
label. In one embodiment, the TTR is labeled with a fluorescence
moiety. In another embodiment, the fluorescence moiety is an
acceptor fluorescence moiety. Alternatively, the complex may be
detected by fluorescence resonance energy transfer.
[0008] In further embodimments, the fluorescence moiety described
herein absorbs at between 330 rim and 480 nm and emits at between
520 nm and 600 nm. In yet another embodiment, the fluorescence
moiety may be chosen from the group consisting of:
N-((2-(iodoacetoxy)ethyl)-N-methyl)amino-7-nitrobenz-2oxa-1,3-diazole,
4-dihexadecylamino-7-nitrobenz-2-oxa-1,3-diazole,
6-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)hexanoic acid,
succinimidyl
6-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)hexanpate, lucifer
yellow iodoacetamide,
N-(5-aminopentyl)-4-amino-3,6-disulfo-1,8-naphthalimide,
N,N'-dimethyl-N-(iodoacetyl)-N'-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)ethyle-
nediamine,
1-(2-maleimidylethyl)-4-(5-(4-methoxyphenyl)oxazol-2-yl)pyridin-
ium
[0009] methanesulfonate,
1-(2,3-epoxypropyl)-4-(5-(4-methoxyphenyl)oxazol-2-yl)pyridinium
trifluoromethanesulfonate,
1-(3-(succinimidyloxycarbonyl)benzyl)-4-(5-94-methoxyphenyl)oxazol-2-yl)p-
yridinium bromide, 3-(4-carboxybenzoyl)quinoline-2-carboxaldehyde,
3-(2-furoyl)quinoline-2-carboxaldehyde or an ALEXA FLUOR.RTM.
(fluorescent chemicals and biomolecule labeling kit) dye. In some
embodiments, the sample may be illuminated so as to excite at least
some of the amino acid groups of RBP.
[0010] Alternatively, the labeled TTR-RBP-retinol complex described
herein may be excited at a wavelength of between 275 nm and 295 nm
and the emission wavelength measured at between 330 nm and 650 nm.
In other embodiments, the excitation wavelength for measuring the
emission spectra may be between 315 and 345 nm, and the emission
wavelength may be measured at between 525 and 600 nm.
[0011] In further embodiments, the labeled TTR may be immobilized
on a solid support. Alternatively, the RBP may be immobilized on a
solid support. The solid support described herein may comprise a
nanoparticle. In other embodiments, the sample comprising the
labeled TTR, RBP and retinol maybe incubated in a microtiter plate
or a microarray. In another embodiment, the sample described herein
further comprises an inhibitor of retinol-RBP-TTR complex
formation. Furthermore, in one embodiment the inhibitor is a
retinyl derivative.
[0012] The methods and compositions described herein also provide
for detecting and/or quantitating retinol-RBP-TTR complex formation
in a sample, the sample comprising RBP, retinol and TTR attached to
a fluorescence moiety, comprising incubating the sample under
conditions sufficient to permit formation of a retinol-RBP-TTR
complex and measuring the emission spectra of the complex. In
another embodiment, the complex is detected by fluorescence
resonance energy transfer, wherein the fluorescence moiety may
absorb at between 380 nm and 480 nm and emit at between 520 nm and
600 nm. In other embodiments, the method described herein may
provide an excitation wavelength for measuring the emission spectra
at between 275 nm and 295 nm and the emission wavelength may be
measured at between 330 nm and 650 nm. In yet another embodiment,
the method described herein may provide an excitation wavelength
for measuring the emission spectra at between 315 and 345 nm, and
the emission wavelength may be measured at between 525 and 600
nm.
[0013] In further embodiments, the methods described herein provide
for screening of modulators of retinol-RBP-TTR complex formation in
a sample, the sample comprising at least one candidate modulator,
labeled TTR, RBP and retinol, further comprising incubating the
sample under conditions sufficient to permit formation of a
retinol-RBP-TTR complex and measuring the emission spectra of the
complex, wherein a change in the emission spectra of the complex
after incubation of the candidate modulator indicates modulation of
the retinol-RBP-TTR complex. In some embodiments, the TTR label is
a fluorescence moiety. In other embodiments, the fluorescence
moiety is an acceptor fluorescence moiety.
[0014] In yet another embodiment, the complex is detected by
fluorescence resonance energy transfer. In other embodiments, the
fluorescence moiety absorbs at between 380 nm and 480 nm and emits
at between 520 nm and 600 nm. Alternatively, the fluorescence
moiety may be chosen from the group consisting of:
N-((2-(iodoacetoxy)ethyl)-N-methyl)amino-7-nitrobenz-2-oxa-1,3-diazole,
4-dihexadecylamino-7-nitrobenz-2-oxa-1,3-diazole;
6-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)hexanoic acid,
succinimidyl
6-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)hexanoate, lucifer
yellow iodoacetamide,
N-(5-aminopentyl)-4-amino-3,6-disulfo-1,8-naphthalimide,
N,N'-dimethyl-N-(iodoacetyl)-N'-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)ethyle-
nediamine,
1-(2-maleimidylethyl)-4-(5-(4-methoxyphenyl)oxazol-2-yl)pyridin-
ium methanesulfonate,
1-(2,3-epoxypropyl)-4-(5-(4-methoxyphenyl)oxazol-2-yl)pyridinium
trifluoromethanesulfonate,
1-(3-(succinimidyloxycarbonyl)benzyl)-4-(5-(4-methoxyphenyl)oxazol-2-yl)
pyridinium bromide, 3-(4-carboxybenzoyl)quinoline-2-carboxaldehyde,
3-(2-furoyl)quinoline-2-carboxaldehyde or an ALEXA FLUOR.RTM.
dye.
[0015] In one embodiment, the sample may be illuminated with
sufficient light to excite at least some of the amino acid moieties
of the RBP. In another embodiment, at least some of the RBP of the
sample forms the retinol-RBP-TTR complex in the sample.
[0016] In further embodiments, the excitation wavelength may be set
at between 275 and 295 nm and the emission wavelength measured at
between 330 and 650 nm. Alternatively, the excitation wavelength
may be set at between 315 and 345 nm, and the emission wavelength
measured at between 525 and 600 nm.
[0017] Furthermore, the methods described herein may comprise a
labeled TTR immobilized on a solid support. Alternatively, the RBP
may be immobilized on a solid support. In some embodiments, the
solid support may be a nanoparticle. In other embodiments, the
sample is incubated in a microliter plate or microarray.
[0018] In further embodiments, the methods described herein provide
for screening of modulators of retinol-RBP-TTR complex formation in
a sample, the sample comprising at least one candidate modulator, a
TTR attached to an acceptor fluorescence moiety, RBP and retinol,
the method further comprising incubating the sample, under
conditions sufficient to permit formation of a retinol-RBP-TTR
complex, and measuring the emission spectra of the sample, wherein
a change in the emission spectra of the complex after incubation of
the candidate modulator indicates modulation of the retinol-RBP-TTR
complex.
[0019] In another embodiment, the methods described herein further
provide for the screening of modulators of retinol-RBP-TTR complex
formation in a sample, the sample comprising at least one candidate
modulator, a TTR attached to an acceptor fluorescence moiety, RBP
and retinol, the method further comprising incubating the sample
under conditions; sufficient to permit formation of a
retinol-RBP-TTR complex, and measuring the emission spectra of the
sample by fluorescence resonance energy transfer, wherein a change
in the emission spectra of the complex after incubation of the
candidate modulator indicates modulation of the retinol-RBP-TTR
complex.
[0020] In one embodiment, the methods described herein further
provide for the screening of modulators of retinol-RBP-TTR complex
formation in a sample, the sample comprising at least one candidate
modulator, a TTR attached to an acceptor fluorescence moiety, RBP
and retinol, the method further comprising incubating the sample
under conditions sufficient to permit formation of a
retinol-RBP-TTR complex, adding at least one candidate inhibitor,
and measuring the emission spectra of the sample, wherein a change,
in the emission spectra of the complex after incubation of the
candidate modulator indicates modulation of the retinol-RBP-TTR
complex.
[0021] In further embodiments, the methods described herein provide
for screening of modulators of retinol-RBP-TTR complex formation in
vivo, the method comprising injecting labeled TTR into a subject,
introducing at least one candidate modulator into the subject,
removing a biological sample from the subject, and measuring the
emission spectra of the sample, wherein a change in the emission
spectra of the complex after introduction of the candidate
modulator indicates modulation of the retinol-RBP-TTR complex in
vivo.
[0022] In another embodiment, the methods described herein provide
for screening of modulators of retinol-RBP-TTR complex formation in
vivo, the method comprising injecting TTR attached to an acceptor
fluorescence moiety into a subject, introducing at least one
candidate modulator into the subject, removing a biological sample
from the subject, and measuring the emission spectra of the sample
by fluorescence resonance energy transfer, wherein a change in the
emission spectra of the complex after introduction of the candidate
modulator indicates modulation of the retinol-RBP-TTR complex in
vivo.
[0023] In further embodiments, a kit is provided for screening
modulators of retinol-RBP-TTR complex formation comprising TTR and
a means for labeling at least a portion of the TTR. In some
embodiments, the kit further comprises retinol and RBP. In another
embodiment, the kit provides for a fluorescent labeling means for
labeling TTR. In other embodiments, the fluorescence label in the
kit is an acceptor fluorescence moiety. In some embodiments, the
acceptor fluorescence moiety in the kit is chosen from the group
consisting of:
N-((2-(iodoacetoxy)ethyl)-N-methyl)amino-7-nitrobenz-2-oxa-1,3-diazole,
4-dihexadecylamino-7-nitrobenz-2-oxa-1,3-diazole,
6-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)hexanoic acid,
succinimidyl
6-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)hexanoate, lucifer
yellow iodoacetamide,
N-(5-aminopentyl)-4-amino-3,6-disulfo-1,8-naphthalimide,
N,N'-dimethyl-N-(iodoacetyl)-N'-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)ethyle-
nediamine,
1-(2-malemidylethyl)-4-(5-(4-methoxyphenyl)oxazol-2-yl)pyridini- um
methanesulfonate,
1-(2,3-epoxypropyl)-4-(5-(4-methoxyphenyl)oxazol-2yl)pyridinium
trifluoromethanesulfonate,
1-(3-(succinimidyloxycarbonyl)benzyl)-4-(5-(4-methoxyphenyl)oxazol-2-yl)p-
yridinium bromide, 3-(4-carboxybenzoyl)quinoline-2-carboxaldehyde,
3-(2-furoyl)quinoline-2-carboxaldehyde or ALEXA FLUOR.RTM. dye.
[0024] In another embodiment, the acceptor fluorescence moiety of
the kit absorbs at between 380 nm and 480 nm and emits at between
520 nm and 600 nm. Furthermore, the RBP and/or the labeled TTR of
the kit may be immobilized on a solid support. In one embodiment,
the solid support of the kit may be a nanoparticle.
[0025] In further embodiments, a labeled TTR molecule is provided,
wherein the labeled TTR is attached to a fluorescence moiety. In
some embodiments, the fluorescence moiety attached to the labeled
TTR molecule may be chosen from the group consisting of:
N-((2-(iodoacetoxy)ethyl)-N-methyl)amino-7-nitrobenz-2-oxa-1,3-diazole,
4-dihexadecylamino-7-nitrobenz-2-oxa-1,3-diazole,
6-(N-(7-nitrobenz-2-oxa-1,3-diazol-4yl)amino)hexanoic acid,
succinimidyl
6-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)hexanoate, lucifer
yellow iodoacetamide,
N-(5-aminopentyl)-4-amino-3,6-disulfo-1,8-naphthalimide,
N,N'-dimethyl-N-(iodoacetyl)-N'-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)ethyle-
nediamine,
1-(2-maleimidylethyl)-4-(5-(4-methoxyphenyl)oxazol-2-yl)pyridin-
ium methanesulfonate,
1-(2,3-epoxypropyl)-4-(5-(4-methoxyphenyl)oxazol-2-yl)pyridinium
trifluoromethanesulfonate,
1-(3-(succinimidyloxycarbonyl)-4-(5-(4-methoxyphenyl)oxazol-2-yl)
pyridinium bromide, 3-(4-carboxybenzoyl)quinoline-2-carboxaldehyde,
3-(2-furoyl)quinoline-2-carboxaldehyde or ALEXA FLUOR.RTM. dye. In
other embodiments, the acceptor fluorescence moiety attached to the
labeled TTR molecule may absorb at between 380 nm and 480 nm and
emit at between 520 nm and 600 nm. Furthermore, the labeled TTR may
be attached to a solid support.
[0026] In further embodiments, a composition is provided comprising
labeled TTR, RBP and retinol. In one embodiment, the methods and
compositions further provide for a complex formed by the
composition comprising labeled TTR, RBP and retinol.
[0027] In yet another embodiment, the composition further comprises
at least one candidate therapeutic agent. In some embodiments, the
candidate therapeutic agent is a small molecule, a polypeptide, a
nucleic acid, or an antibody. In other embodiments, the candidate
therapeutic agent is a retinyl derivative.
[0028] In further embodiments, the labeled TTR of the composition
may be attached to a fluorescence moiety. In one embodiment, the
fluorescence moiety may be
N-((2-(iodoacetoxy)ethyl)-N-methyl)amino-7-nitrobenz-2-oxa-1,3-diazole,
4-dihexadecylamino-7-nitrobenz-2-oxa-1,3-diazole,
6-(N-(7-nitrobenz-2-oxa-1,3-diazol-4yl)amino)hexanoic acid,
succinimidyl
6-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)hexanoate, lucifer
yellow iodoacetamide,
N-(5-aminopentyl)-4-amino-3,6-disulfo-1,8-naphthalimide,
N,N'-dimethyl-N-(iodoacetyl)-N'-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)ethyle-
nediamine,
1-(2-maleimidylethyl)-4-(5-(4-methoxyphenyl)oxazol-2-yl)pyridin-
ium methanesulfonate,
1-(2,3-epoxypropyl)-4-(5-(4-methoxyphenyl)oxazol-2-yl)pyridinium
trifluoromethanesulfonate,
1-(3-(succinimidyloxycarbonyl)-4-(5-(4-methoxyphenyl)oxazol-2-yl)
pyridinium bromide, 3-(4-carboxybenzoyl)quinoline-2-carboxaldehyde,
3-(2-furoyl)quinoline-2-carboxaldehyde an or ALEXA FLUOR.RTM. dye.
Alternatively, the fluorescence moiety of the composition may
absorb at between 380 nm and 480 nm and emits at between 520 nm and
600 nm. In one embodiment, the labeled TTR is attached to a solid
support.
[0029] In other embodiments, the methods described herein provide
for identifying a therapeutic agent for the treatment of macular
degenerations (including both the wet forms and dry forms of
age-related macular degeneration) or dystrophies comprising
incubating a sample comprising at least one candidate therapeutic
agent, labeled TTR, RBP and retinol under conditions sufficient to
permit formation of a retinol-RBP-labeled TTR complex, and
measuring the emission spectra of the complex, wherein the
candidate therapeutic agent decreases me emission spectra of the
complex after incubation.
[0030] In some embodiments, the TTR label is a fluorescence moiety.
In other embodiments, the fluorescence moiety is an acceptor
fluorescence moiety. In some embodiments, the complex is detected
by fluorescence resonance energy transfer. In other embodiments,
the fluorescence moiety absorbs at between 380 nm and 480 nm and
emits at between 520 nm and 600 nm. In one embodiment, the
fluorescence moiety may be
N-((2-(iodoacetoxy)ethyl)-N-methyl)amino-7-nitrobenz-2-oxa-1,3-diazole,
4-dihexadecylamino-7-nitrobenz-2-oxa-1,3-diazole,
6-(N-(7-nitrobenz-2-oxa-1,3-diazol-4yl)amino)hexanoic acid,
succinimidyl
6-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)hexanoate, lucifer
yellow iodoacetamide,
N-(5-aminopentyl)-4-amino-3,6-disulfo-1,8-naphthalimide,
N,N'-dimethyl-N-(iodoacetyl)-N'-(7-nitrozenz-2-oxa-1,3-diazol-4-yl)ethyle-
nediamine,
1-(2-maleimidylethyl)-4-(5-(4-methoxyphenyl)oxazol-2-yl)pyridin-
ium methanesulfonate,
1-(2,3-epoxypropyl)-4-(5-(4-methoxyphenyl)oxazol-2-yl)pyridinium
trifluoromethanesulfonate,
1-(3-(succinimidyloxycarbonyl)-4-(5-(4-methoxyphenyl)oxazol-2-yl)
pyridinium bromide, 3-(4-carboxybenzoyl)quinoline-2-carboxaldehyde,
3-(2-furoyl)quinoline-2-carboxaldehyde an or ALEXA FLUOR.RTM. dye.
In one embodiment, the sample described herein is illuminated with
sufficient light to excite at least some of the amino acid moieties
of the RBP. Furthermore, in other embodiments at least some of the
RBP in the sample forms the retinol-RBP-TTR complex.
[0031] In yet another embodiment, the excitation wavelength is
between 275 nm and 295 nm and the emission wavelength is measured
at between 330 nm and 650 nm. Alternatively, the excitation
wavelength is between 315 and 345 nm, and the emission wavelength
measured at between 525 and 600 nm. In some embodiments, the
labeled TTR or the RBP is immobilized on a solid support. The solid
support may also be a nanoparticle. In other embodiments, the
sample is incubated in a microtiter plate or a microarray.
[0032] In further embodiments, the candidate therapeutic agent
described herein is a small molecule, a polypeptide, a nucleic acid
or an antibody. In other embodiments, the candidate therapeutic
agent is a retinyl derivative. Alternatively, the retinyl
derivative is N-(4-hydroxyphenyl)retinamide (also referred to
herein as "HPR" or "fenretinide" or "4-hydroxyphenylretinamide" or
"hydroxyphenyl retinamide"), N-(4-methoxyphenyl)retinamide ("MPR";
the most prevalent metabolite of HPR), or ethylretinamide.
[0033] In further embodiments, the methods described herein provide
for identifying a therapeutic agent for the treatment of macular
degenerations (including both the wet forms and dry forms of
age-related macular degeneration) or dystrophies, wherein the
method comprises incubating a sample comprising at least one
candidate therapeutic agent, a TTR attached to an acceptor
fluorescence moiety, RBP and retinol under conditions sufficient to
permit formation of a TTR-RBP-retinol complex, and detecting the
emission spectra of the sample by fluorescence resonance energy
transfer, wherein the candidate therapeutic agent decreases the
emission spectra of the complex after incubation.
[0034] In another embodiment, the methods described herein provide
a modulator of the formation of a complex comprising retinol, RBP
and TTR, wherein the modulator also modulates formation of a
complex comprising retinol, RBP and labeled TTR. In one embodiment,
the TTR is fluorescently labeled. In some embodiments, the complex
of the modulator is in an in vitro sample. In other embodiments,
the complex is in an in vivo sample. In one embodiment, the
modulator further comprises a retinyl derivative. Alternatively,
the retinyl derivative of the modulator is
N-(4-hydroxyphenyl)retinamide ("HPR"),
N-(4-methoxyphenyl)retinamide ("MPR"), or ethylretinamide.
[0035] In any of the aforementioned screening or detection or
measuring or quantitation methods, strategies, compositions and
kits, the following further embodiments may further be used singly
or in any combination: (a) the sample has not been frozen; (b) the
sample has been frozen for up to about two weeks; (c) the sample
does not contain dimethylsulfoxide; (d) the sample contains up to
about 8% dimethylsulfoxide; (e) the sample is performed using
high-throughput methods; (f) the sample is contained within a well
of a 384-well microtiter plate; (g) the mol % of label to
tetrameric TTR is less than 5%; (h) the mol % of label to
tetrameric TTR is less than 3%;, (i) the mol % of label to
tetrameric TTR is less than 2.5%; (j) the mol % of label to
tetrameric TTR is less than 1.8%; (k) a compound of Formula (I) is
used; (l) fenretinide is used; (m) a metabolite of fenretinide is
used; (n) addition of a modulator of retinol-RBP-TTR formation to
the sample results in a decrease the detected signal; (b) the
detected signal from the retinol-RBP-TTR complex is measured as a
function of the concentration of modulator; (p) the detected signal
from the retinol-RBP-TTR complex is measured using a spectrometer;
(r) the detected signal from the retinol-RBP-TTR complex is
measured using a double-grating emission spectrometer; (s) the
modulator of retinol-RBP-TTR complex formation reduces serum
retinol levels when administered to a human; (t) the modulator of
retinol-RBP-TTR complex formation reduces ocular levels of retinol
when administered to a human; (u) the modulator of retinol-RBP-TTR
complex formation reduces ocular levels of retinoids when
administered to a human; (v) the modulator of retinol-RBP-TTR
complex formation reduces ocular levels of A2E when administered to
a human; or (w) the modulator of retinol-RBP-TTR complex formation
is used to treat age-related macular degeneration (including both
the wet forms and dry forms of age-related macular degeneration) in
a human patient when administered to the human patient. In any of
me aforementioned embodiments, the "TTR" can be labeled with a
fluorophore.
[0036] In another embodiment are treatment methods for ophthalmic
conditions, including macular degenerations (including both the wet
forms and dry forms of age-related macular degeneration) and
dystrophies and geographic atrophy, comprising administration of a
compound that modulates the formation of a retinol-retinol binding
protein (RBP)-transthyretin (TTR) complex. In further embodiments
of the treatment methods, the compound that modulates the formation
of a retinol-retinol binding protein (RBP)-transthyretin (TTR)
complex is an all-trans retinyl derivative, wherein the all-trans
retinyl derivative is administered at least once in an effective
amount and has the structure of Formula (I):
##STR00001## [0037] wherein X.sub.1 is selected from the group
consisting of NR.sub.2, O, S, CHR.sup.2; R.sup.1 is
(CHR.sup.2).sub.x-L.sup.1-R.sup.3, wherein x is, 0,1,2, or 3;
L.sup.1 is a single bond or --C(O)--; R.sup.2 is a moiety selected
from the group consisting of H, (C.sub.1-C.sub.4)alkyl, F,
(C.sub.1-C.sub.4fluoroalkyl, (C.sub.1-C.sub.4)alkoxy, --C(O)OH,
--C(O)--NH.sub.2, --(C.sub.1-C.sub.4)alkylamine,
--C(O)-(C.sub.1-C.sub.4)alkyl, --C(O)-(C.sub.1-C.sub.4)fluoroalkyl,
--C(O)-(C.sub.1-C.sub.4)alkylamine and
--C(O)-(C.sub.1-C.sub.4)alkoxy; and R.sup.3 is H or a moiety,
optionally substituted with 1-3 independently selected
substituents, selected from the group consisting of
(C.sub.2-C.sub.7)alkenyl, (C.sub.2-C.sub.7)alkynyl, aryl,
(C.sub.3-C.sub.7)cycloalkyl, (C.sub.5-C.sub.7)cycloalkenyl, and a
heterocycle, provided that R.sup.3 is not H when both x is 0 and
L.sup.1 is a single bond; or an active metabolite, or a
pharmaceutically acceptable prodrug or solvate thereof.
[0038] In further embodiments (a) X.sup.1 is NR.sup.2, wherein
R.sup.2 is H or (C.sub.1-C.sub.4)alkyl; (b) x is 0; (c) x is 1 and
L1 is --C(O)--; (d) R.sup.3 is an optionally substituted aryl;(e)
R.sup.3 is an optionally substituted heteroaryl; (f) X.sup.1 is NH
and R.sup.3 is an optionally substituted aryl, including yet
further embodiments in which (i) the aryl group has one
substituent, (ii) the aryl group has one substituent selected from
the group consisting of halogen, OH, O(C.sub.1-C.sub.4)alkyl,
NH(C.sub.1-C.sub.4)alkyl, O(C.sub.1-C.sub.4)fluoroalkyl, and
N[(C.sub.1-C.sub.4)alkyl].sub.2, (iii) the aryl group has one
substituent, which is OH, (v) the aryl is a phenyl, or (vi) the
aryl is naphthyl; (g) the compound is
##STR00002##
or an active metabolite, or a pharmaceutically acceptable prodrug
or solvate thereof; (h) the compound is 4-hydroxyphenylretinamide,
or a metabolite, or a pharmaceutically acceptable prodrug or
solvate thereof; (i) the compound is 4-methpxyphenylretinamide, or
(j) 4-oxo fenretinide, or a metabolite, or a pharmaceutically
acceptable prodrug or solvate thereof.
[0039] In further embodiments, the administration of a compound of
Formula (I) is used to treat ophthalmic conditions by lowering the
levels of serum retinol in the body of a patient. In further
embodiments (a) the effective amount of the compound is
systemically administered to the mammal; (b) the effective amount
of the compound is administered orally to the mammal; (c) the
effective amount of the compound is intravenously administered to
the mammal; (d) the effective amount of the compound is
ophthalmically administered to the mammal; (e) the effective amount
of the compound is administered by iontophoresis; or (f) the
effective amount of the compound is administered by injection to
the mammal.
[0040] In further embodiments the mammal is a human, including
embodiments wherein (a) the human is a carrier of the mutant ABCA4
gene for Stargardt Disease or the human has a mutant ELOV4 gene for
Stargardt Disease, or has a genetic variation in complement factor
H associated with age-related macular degeneration, or (b) the
human has an ophthalmic condition or trait selected from the group
consisting of Stargardt Disease, recessive retinitis pigmentosa,
geographic atrophy (of which scotoma is one non-limiting example),
photoreceptor degeneration, dry-form AMD, recessive cone-rod
dystrophy, exudative (or wet-form) age-related macular degeneration
cone-rod dystrophy, arid retinitis pigmentosa. In further
embodiments the mammal is an animal model for retinal
degeneration.
[0041] In further embodiments, are methods comprising multiple
administrations of the effective amount of the compound, including
further embodiments in which (i) the time between multiple
administrations is at least one week; (ii) the time between
multiple administrations is at least one day; and (iii) the
compound is administered to the mammal on a daily basis; or (iv)
the compound is administered to the mammal every 12 hours. In
further Or alternative embodiments, the method comprises a drug
holiday, wherein the administration of the compound is temporarily
suspended or the dose of the compound being administered is
temporarily reduced; at the end of the drug holiday, dosing of the
compound is resumed. The length of the drug holiday can vary from 2
days to 1 year.
[0042] In further embodiments are methods comprising administering
at least one additional agent selected from the group consisting of
an inducer of nitric oxide production, an anti-inflammatory agent,
a physiologically acceptable antioxidant, a physiologically
acceptable mineral a negatively charged phospholipid, a carotenoid,
a statin, an anti-angiogenic drug, a matrix metalloproteinase
inhibitor, 13-cis-retinoic acid (including, derivatives of
13-cis-retinoic acid), 11-cis-retinoic acid (including derivatives
of 11-cis-retinoic acid), 9-cis-retinoic acid (including
derivatives of 9-cis-retinoic acid), and retinylamine derivatives.
In further embodiments:
[0043] (a) the additional agent is an inducer of nitric oxide
production, including embodiments in which the inducer of nitric
oxide production is selected from the group consisting of
citrulline, ornithine, nitrosated L-arginine, nitrosylated
L-arginine, nitrosated N-hydroxy-L-arginine, nitrosylated
N-hydroxy-L-arginine, nitrosated L-homoarginine and nitrosylated
L-homoarginine;
[0044] (b) the additional agent is; an anti-inflammatory agent,
including embodiments in which the anti-inflammatory agent is
selected from the group consisting of a non-steroidal
anti-inflammatory drug, a lipoxygenase inhibitor, prednisone,
dexamethasone, and a cyclooxygenase inhibitor;
[0045] (c) the additional agent is at least one physiologically
acceptable antioxidant, including embodiments in which the
physiologically acceptable antioxidant is selected from the group
consisting of Vitamin C, Vitamin E, betarcarotene, Coenzyme Q, and
4-hydroxy-2,2,6,6-tetramethylpiperadine-N-oxyl, or embodiments in
which (i) the at least one physiologically acceptable antioxidant
is administered with the compound having the structure of Formula
(I), or (ii) at least two physiologically acceptable antioxidants
are administered with the compound having the structure of Formula
(I);
[0046] (d) the additional agent is at least one physiologically
acceptable mineral, including embodiments in which the
physiologically acceptable mineral is selected from the group
consisting of a zinc (II) compound, a Cu(II) compound, and a
selenium (II) compound, or embodiments further comprising
administering to the mammal at least one physiologically acceptable
antioxidant;
[0047] (e) the additional agent is a negatively charged
phospholipid, including embodiments in which the negatively charged
phospholipid is phosphatidylglycerol;
[0048] (f) the additional agent is a carotenoid, including
embodiments in which the carotenoid is selected from the group
consisting of lutein and zeaxarithin;
[0049] (g) the additional agent is a statin including embodiments
in which the statin is selected from the group consisting of
rosuvastatin, pitivastatin, simvastatin, pravastatin, cerivastatin,
mevastatin, velostatin, fluvastatin, compactin, lovastatin,
dalvastatin, fluindostatin, atorvastatin, atorvastatin calcium, and
dihydrocompactin;
[0050] (h) the additional agent is an anti-angiogenic drug,
including embodiments in which the the anti-angiogenic drug is
Rhufab V2, Tryptophanyl-tRNA synthetase, an Anti-VEGF pegylated
aptamer, Squalamine, anecortave acetate, Combretastatin A4 Prodrug,
Macugen.TM., mifepristone, subtenon triamcinolone acetonide,
intravitreal crystalline triamcinolone acetonide, AG3340,
fluocinolone acetonide, and VEGF-Trap;
[0051] (i) the additional agent is a matrix metalloproteinase
inhibitor, including embodiments in which the matrix
metalloproteinase inhibitor is a tissue inhibitors of
metalloproteinases, .alpha..sub.2-macroglobulin, a tetracycline, a
hydroxamate, achelator, a synthetic MMP fragment, a succinyl
mercaptopurine, a phosphonamidate, and a hydroxaminic acid;
[0052] (j) the additional agent is 13-cis-retinoic acid; (including
derivatives of 13-cis-retinoic acid), 11-cis-retinoic acid
(including derivatives ,of 11-cis-retinoic acid), or 9-cis-retinoic
acid (including derivatives of 9-cis-retinoic acid);
[0053] (k) the additional agent is a retinylamine derivative,
including an all-trans-retinylamine derivative, a
13-cis-retinylamine derivative, a 11-cis-retinylamine derivative,
or a 9-cis-retinylamine derivative;
[0054] (l) the additional agent is administered (i) prior to the
administration of the compound having the structure of Formula (I),
(ii) subsequent-to the administration of the compound having the
structure of Formula (I), (iii) simultaneously with the
administration of the compound having the structure of Formula (I),
or (iv) both prior and subsequent to the administration of the
compound having the structure of Formula (I); of
[0055] (m)the additional agent and the compound having the
structure of Formula (I),are administered in the same
pharmaceutical composition.
[0056] In further embodiments are methods comprising administering
extracorporeal rheopheresis to the mammal. In further embodiments
are methods comprising administering to the mammal a therapy
selected from the group consisting of limited retinal
translocation, photodynamic therapy, drusen lasering, macular hole
surgery, macular translocation surgery, Phi-Motion, Proton Beam
Therapy, Retinal Detachment and Vitreous Surgery, Scleral Buckle,
Submacular Surgery, Transpupillary Thermotherapy, Photosystem I
therapy, Microcurrent Stimulation, anti-inflammatory agents, RNA
interference, administration of eye medications such as phospholine
iodide or echothiophate or carbonic anhydrase inhibitors, microchip
implantation, stem cell therapy, gene replacement therapy ribozyme
gene therapy, photoreceptor/retinal cells transplantation, and
acupuncture.
[0057] In further embodiments are methods comprising the use of
laser photocoagulation to remove drusen from the eye of the
mammal.
[0058] In further embodiments are methods comprising administering
to the mammal at least once an effective amount of a second
compound having the structure of Formula (I), wherein the first
compound is different from the second compound.
[0059] In further embodiments, an apparatus capable of detecting
and/or quantitating retinol-RBP-TTR complex formation is provided,
wherein at least a portion of the TTR is fluorescently labeled.
[0060] Other objects, features and advantages of the methods and
compositions described herein will become, apparent from the
following detailed description. It should be understood, however,
that the detailed description and the specific examples, while
indicating specific embodiments, are given byway of illustration
only, since various changes and modifications within the spirit and
scope of the invention will become apparent to those skilled in the
art from this detailed description.
[0061] All publications and patent applications mentioned in this
specification are herein incorporated by reference to the same
extent as if each individual publication or patent application was
specifically and individually indicated, to be incorporated by
reference.
BRIEF DESCRIPTION OF THE DRAWINGS
[0062] The novel features of the methods and compositions disclosed
herein are set forth with particularity in the appended claims. A
better understanding of the features and advantages will be
obtained by reference to the following detailed description that
sets forth illustrative embodiments, in which the principles
disclosed herein are utilized, and the accompanying drawings of
which:
[0063] FIG. 1 illustrates a flowchart of one embodied method;
[0064] FIG: 2 illustrates FRET detection of TTR-RBP-retinol complex
formation with labeled TTR;
[0065] FIG. 3 presents a schematic of one example of the
instrumentation for detecting and/or measuring the presence of
fluorescent compounds in a sample;
[0066] FIG. 4 illustrates results from aromatic amino acid
quenching versus FRET detection of retinol-RBP-TTR complex
formation;
[0067] FIG. 5 illustrates results from aromatic amino acid
quenching versus FRET detection of retinol-RBP-TTR complex
formation.
[0068] FIG. 6 illustrates the relationship of serum HPR levels to
serum retinol levels and ocular levels of retinoids and A2E.
[0069] FIG. 7 illustrates the effect of administering HPR to wild
type mice on (A) serum retinol levels and (B) ocular retinoid
levels.
[0070] FIG. 8 illustrates an example of a FRET spectrum taken of an
RBP-TTR complex in the absence and presence of HPR, wherein the TTR
has been labeled with a fluorescence moiety.
[0071] FIG. 9 illustrates an example of dose dependent inhibition
of retinol-RBP-TTR complex formation by HPR as determined using the
FRET methods described herein.
[0072] FIG. 10 illustrates a comparison of the inhibition of
retinol-RBP-TTR complex formation using HPR, 13-cis-retinoic acid
and all-trans-retinoic acid as determined using the FRET methods
described herein.
[0073] FIG. 11 illustrates a comparison of the inhibition of
retinol-RBP-TTR complex formation using HPR as determined using the
FRET methods described herein and fluorescence anisotropy
methods.
DETAILED DESCRIPTION OF THE INVENTION
[0074] Reference will now be made in detail to embodiments of the
methods and compositions disclosed herein. Examples of the
embodiments are illustrated in the following Examples section.
[0075] Unless defined otherwise, all technical and scientific terms
used herein have the same meaning as is commonly understood by one
of skill in the art to which this invention belongs. All patents
and publications referred to herein are incorporated by
reference.
[0076] One of the methods for detecting agents which modulate the
formation of a retinol-RBP-TTR complex is presented schematically
in FIG. 1. An optional first step is the preparation of the sample.
The sample can comprise purified components of the retinol-RBP-TTR
complex; alternatively the sample can comprise a biological
specimen including, but not limited to, blood, sera, tissue, mucus,
saliva, tears, urine or feces. Further, the methods and
compositions described herein may be used with test and/or
laboratory animals, such as mice, rats, non-human primates, and the
like. Such test and/or laboratory animals may be alive or dead.
Such further preparations include homogenization of the biological
specimens, including dispersal or suspension in another media, and
the like. Further, the samples may include of be derived from
cultured cells or from tissue banks, or from storage centers.
[0077] After optionally preparing the sample, complex formation is
detected, for example, by illumination with light. The methods and
devices described herein are not limited by the type or source of
light, that is, the light may originate, by way of example only,
from a lamp, laser, or light-emitting diode. The light may be
pulsed (in any sequence) or continuous; further the light may be
coherent or non-coherent; further the light may be polarized or
nonpolarized; further the light may pass through filters
(including, but not limited to band-pass filters), blocking (e.g.,
spatial filtering) and/or focusing devices; further the light may
illuminate all or only a portion of the sample. The wavelength
range or ranges used for illuminating the sample depend upon the
fluorescent compound or compounds to be detected, further detail is
provided herein for specific compounds. Preferably, the
wavelength(s) of light used in the illuminating step should excite
the fluorescent compound so as to emit a fluorescence signal that
can be subsequently detected and/or measured. In addition, the
wavelength range used for illumination may also include wavelengths
that are not well absorbed (if at all) by the fluorescence
compounds of interest; such light may be used as a reference signal
or for background subtraction. However, the use of such a reference
or background signal is not required by the methods and devices
described herein. In addition, the absorbance of the illuminating
light may also be measured and used separately or in combination
with the fluorescence signal measured and/or detected in the
detection step. Such an absorbance signal may be diagnostic for a
particular fluorescent compound, as described elsewhere herein. The
key requirement for the illumination step is that the fluorescent
compound or compounds to be detected absorb at least a portion of
the light applied to the sample. The use of a microprocessor may
also, be used to control the illumination step. In any sample,
there may be more than one type of fluorescent compound; if more
than one type of fluorescent compound is presented, then the
illuminating light may be provided so as to be absorbed by only one
type of compound or by multiple types of compounds.
[0078] After illuminating the sample, the fluorescence emitted from
the fluorescent compounds in the samplers detected. Such emitted
fluorescence may be detected by any number of methods, or a
combination of methods. For example, the fluorescence signal may be
detected at only one wavelength, at different wavelengths, at a
range of wavelengths, or over multiple ranges of wavelengths. If a
specific fluorescence is being detected and/or measured, then one
of the methods described herein examines a specific range of
wavelengths that corresponds to the major component
[0079] The information or data acquired from the detection step may
be stored temporarily or permanently in a variety of media,
including by way of example only, film, computer memory or any
other form of archival material. Such record-keeping and/or storage
of information and data is generally associated with laboratory
practices, and/or patient diagnosis and treatment, as well as for
testing the effectiveness of a modulator or therapeutic agent (in
vivo or in vitro). By storing or archiving such information, one of
skill in the art can also create a temporal analysis of the sample.
Furthermore, the archived information, data or images can be
further processed (e.g., magnified, enriched, deconvoluted,
pseudocolored, quantitated) as desired.
[0080] The optional sample preparation, the illumination of the
sample, the detection of fluorescence and the optional storage of
information can be considered one detection cycle. As such, it is
contemplated herein the repetition of this detection cycle on a
sample. Certainly, if the sample is already prepared, it may not be
necessary to re-prepare the sample, especially if the time interval
between detection cycles is short (e.g., less than 10 seconds, less
than one minute, less than 5 minutes or less than one hour or even
less than one day). By way of example only, it may be necessary to
repeat a detection cycle to ensure the accuracy of the
measurements, in which case, the time interval between detection
cycles may be relatively short. If the interval between detection
cycles is longer, it may be necessary to store the sample. In
addition, if the sample is being tested with a therapeutic agent or
a potential modulator (e.g., in the testing and/or design of anew
drug), the interval between detection cycles may be used to provide
further manipulation to the sample. The time between detection
cycles may be less than 10 seconds, less than one minute, more than
5 minutes, more than one hour, or even more than one day. The
duration of time between detection cycles and the number of times
the detection cycle is repeated is within the discretion of one of
skill in the art. In any case, the duration of time between
detection cycles does not have to be uniform and may be a
combination of multiple repeat cycles.
[0081] The information collected from a detection cycle or cycles
may be optionally used for a number of purposes in which the
absorbance and/or fluorescence detected from a sample is used as a
surrogate marker and/or risk factor for the status of a sample.
Non-limiting examples include (a) measuring the effectiveness of a
therapeutic candidate for a relevant ophthalmic disease or
condition (including the retinal and/or macular degenerations
(including both the wet forms and dry forms of age-related macular
degeneration) or dystrophies) in an in vitro sample or an in vivo
sample (including the injection of components of the
retinol-RBP-labeled TTR into a living laboratory animal, including
a rodent or an ABCA4 knockout mouse) by measuring changes in the
amount of fluorescent compound(s) in a sample following
administration of the therapeutic candidate to the sample; and (b)
measuring the effectiveness of a treatment for a relevant
ophthalmic disease or condition (including the retinal and/or
macular degenerations (including both the wet forms and dry forms
of age-related macular degeneration) or dystrophies) in an in vivo
sample (including the injection of components of the
retinol-RBP-labeled TTR into a living laboratory animal, including
a rodent or ah ABCA4 knockout mouse), by measuring changes in the
amount of fluorescent compound(s) in a sample following
administration of a treatment to the laboratory animal.
[0082] As used herein, the term "ABGA4 gene" refers to a gene
encoding the rim protein or RmP. The ABCA4 gene is also known as
the ABCR gene.
[0083] As used herein, the term "anti-oxidant" refers to a
synthetic or natural substance that can prevent, delay or otherwise
inhibit the Oxidation of a compound or biological substance.
[0084] As used herein, the term "deconvoluting" refers to the
process of converting data, information and/or images into (at
least in part) constituent components. For example, a fluorescence
or absorbance spectrum that features a complex wave form can be
mathematically deconvoluted into the separate absorbance or
fluorescence peaks that comprise the complex wave form. Suitable
mathematical procedures and algorithms are well-known in the art,
and suitable software packages for deconvolving data, information
and/or images are commercially available.
[0085] As used herein, the term "disruption of the visual cycle" or
the like refers to any means for modulating the activity, directly
or indirectly, of at least one enzyme involved in the visual
cycle.
[0086] As used herein, the term "dispersing" refers to suspending a
substance in another medium. Dispersing can include steps for
homogenizing, fractionating, breaking up, fluidizing or decreasing
the size of a substance in order to facilitate the suspending
step.
[0087] As used herein, a retinyl derivative refers to compound that
can be produced by reacting one of the various cis or trans retinal
isomers with another compound or series of compounds.
[0088] As used herein, the term "age-related macular degeneration
or dystrophy" or "ARMD" refers to a debilitating disease, which
include wet and dry forms of ARMD. The dry form of ARMD, which
accounts for about 90 percent of all cases, is also known as
atrophic, nonexudative, or drusenoid (age-related) macular
degeneration. With the dry form of ARMD, drusen typically
accumulate in the retinal pigment epithelium (RPE) tissue
beneath/within the Bruch's membrane. Vision loss can then occur
when drusen interfere with the function of photoreceptors in the
macula. The dry form of ARMD results in the gradual loss of vision
over many years. The dry form of ARMD can lead to the wet form of
ARMD. The wet form of ARMD, also known as exudative or neovascular
(age-related) macular degeneration, can progress rapidly and cause
severe damage to central vision. The macular dystrophies include
Stargardt Disease, also known as Stargardt Macular Dystrophy of
Fundus Flavimaculatus, which is the most frequently encountered
juvenile onset form of macular dystrophy.
[0089] As used herein, the term "mammal" refers to all mammals
including humans. Mammals include, by way of example only, humans,
non-human primates, cows, dogs,; cats, goats, sheep pigs, rats,
mice and rabbits.
[0090] As used herein, the term "biological sample" refers to the
eyes, plasma, bloody urine, feces, tissue, mucus, tears or saliva
of a mammal.
[0091] As used herein, the term "effective amount" refers to the
total amount of the therapeutic agent in the pharmaceutical
formulation or method that is sufficient to show a meaningful
subject or patient benefit.
[0092] As used herein, the term "measuring the emission
fluorescence" refers to any means for either (a) detecting the
presence of a fluorescent compound by detecting the presence of its
fluorescence following excitation by some form of illumination, (b)
measuring the amount of a fluorescent compound by measuring the
intensity (absolute or relative) of the fluorescence emitted by the
fluorescent compounds in a sample following excitation by some form
of illumination, and (c) a combination of the above.
[0093] As used herein, the term "emission spectra" refers to a plot
of relative intensity of emitted radiation as a function of
wavelength.
[0094] As used herein, the term "emission wavelength" refers to the
maximal wavelength or wavelength range of emitted radiation upon
excitation by light energy.
[0095] As used herein, the term "excitation wavelength" refers to
the wavelength of an external energy source equivalent to the
photon of energy hv.sub.EX supplied by the external source, such as
an incandescent lamp or a laser, arid absorbed by the fluorophore,
creating an excited electronic singlet state.
[0096] As used herein, the term "fluorescence moiety" refers to a
fluorescent species or substance.
[0097] As used herein, the term "acceptor fluorescence moiety"
refers to a fluorescent species of substance in FRET detection
which accepts a donor electron from a donor fluorescent
species.
[0098] As used herein, the term "fluorescence resonance energy
transfer" or "FRET" refers to the transfer of energy between an
acceptor and donor species, wherein the absorption spectrum of the
acceptor species overlaps the emission spectrum of the donor
species.
[0099] As used herein,, the term "ophthalmic disease or condition"
refers to any disease or condition involving the eye or related
tissues. Non-limiting examples include diseases or conditions
involving degeneration of the retina and/or macula, including the
retinal and/or macular dystrophies and the retinal and/or macular
degenerations.
[0100] As used herein, the term "immobilized" refers to the
covalent or non-covalent attachment of a chemical or biological
species to a support.
[0101] As used herein, the term "primate" refers to the highest
order of mammals; includes man, apes and monkeys.
[0102] As used herein, the term "risk" refers to the probability
that an event will occur.
The Visual Cycle
[0103] The vertebrate retina contains two types of photoreceptor
cells. Rods are specialized for vision under low light conditions.
Cones are less sensitive provide vision at high temporal and
spatial resolutions, and afford color perception. Under daylight
conditions, the rod response is saturated and vision is mediated
entirely by cones. Both cell types contain a structure called the
outer segment, comprising a stack of membranous discs. The
reactions of visual transduction take place on the surfaces of
these discs. The first step in vision is absorption of a photon by
an opsin-pigment molecule, which involves 11-cis to all-trans
isomerization of the retinal chromophore. Before light sensitivity
can be regained, the resulting all-trans-retinal must dissociate
from the opsin apoprotein and isomerize to 11-cis-retinal.
[0104] Further information regarding the anatomical organization of
the vertebrate eye, the visual cycle for regeneration of rhodopsin,
arid the biogenesis of A2E-oxiranes is provided in U.S. Provisional
Pat. App. No. 60/582,293, filed Jun. 23, 2004, U.S. Provisional
Pat. App. No. 60/602,675, filed Aug. 18, 2004 and U.S. Provisional
Pat. App. No. 60/622,213, filed Oct. 25, 2004, the contents of
which are incorporated by reference in their entirety.
Macular or Retinal Degeneration.
[0105] As discussed above,-macular degeneration (also referred to
as retinal degeneration) is a disease of the eye that involves
deterioration of the macula, the central portion of the retina.
Approximately 85% to 90% of the cases of macular degeneration are
the "dry" (atrophic or non-neovascular) type.
[0106] In "dry" macular degeneration, the deterioration of the
retina is associated with the formation of small yellow deposits,
known as drusen, under, the macula. This phenomena leads to a
thinning and drying out of the macula. The location and amount of
thinning in the retinal caused by the drusen directly correlates,
to the amount of central vision loss. Degeneration of the pigmented
layer of the retina and photoreceptors overlying drusen become
atrophic and cause a slow of central vision. This often occurs over
a decade or more.
[0107] Most people who lose vision from age related macular
degeneration have "wet" macular degeneration. In "wet"
(neovascular) macular degeneration, abnormal blood vessels from the
choroidal layer of the eye, known as subretinal neovascularization
grow under the retina and macula. These blood vessels tend to
proliferate with fibrous tissue, and bleed and leak fluid under the
macula, causing the macula to bulge or move and distort the central
vision. Acute vision loss occurs as transudate of hemorrhage a the
retina. Permanent vision loss occurs as the outer retina becomes
atrophic or replaced by fibrous tissues.
Stargardt Disease
[0108] Stargardt Disease is a macular dystrophy that manifests as a
recessive form of macular degeneration with an onset during
childhood. See e.g., Allikmets et al., Science, 277:1805-07 (1997).
Stargardt Disease is characterized clinically by progressive loss
of central vision and progressive atrophy of the RPE overlying the
macula. Mutations in the human ABCA4 gene for RmP are responsible
for Stargardt Disease. Early in the disease course, patients show
delayed dark adaptation but otherwise normal rod function.
Histologically, Stargardt D is associated with deposition of
lipofuscin pigment granules in RPE cells.
[0109] Besides Stargardt Disease, mutations in ABCA4 have been
implicated in recessive retinitis pigmentosa, recessive cone-rod
dystrophy, and non-exudative age-related macular degeneration
(AMD), see e.g., Lewis et al., Am. J. Hum. Genet., 64:422-34
(1999), although the prevalence of ABCA4 mutations in AMD is still
uncertain. See Allikmets, Am. J. Hum. Gen., 67:793-799 (2000).
Similar to Stargardt Disease, these diseases are associated with
delayed rod dark-adaptation. Lipofuscin-deposition in RPE cells is
also seen prominently in AMD, see Kliffen et al, Microsc. Res.
Tech., 36:106-22 (1997) and some cases of retinitis pigmentosa and
cone-rod dystrophy.
[0110] An eye doctor examining a patient at this stage may note the
presence of these drusen, even though most people have no
symptoms.. When drusen have been noted on examination, monitoring
will be needed over time. Many people over the age of 60 will have
some drusen.
Modulation of Retinol-Retinol Binding Protein (RBP)-Transthyretin
(TTR) Binding
[0111] The methods and compositions described herein are useful for
the detection and screening of modulators of retinol binding to
retinol binding protein (RBP), and the transport complex
retinol-RBP-TTR. Vitamin A (all-trans retinol) is a vital cellular
nutrient which cannot be synthesized de novo and therefore must be
obtained from dietary sources. Following digestion, retinol in food
material is transported to the liver bound to, lipid aggregates.
See Bellovino et al., Mol. Aspects Med., 24:411-20 (2003); Once in
the liver, retinol forms a complex with retinol binding protein
(RBP) and is then secreted into the blood circulation. Before the
retinol-RBP holoprotein can be delivered to extra-hepatic target
tissues, such as the eye, it must bind with transthyretin (TTR).
Zanotti and Berni, Vitam. Horm., 69:271-95 (2004). It is this
secondary complex which allows retinol to remain in the circulation
for prolonged periods. Association with TTR facilitates RBP release
from hepatocytes, arid prevents renal filtration of the RBP-retinol
complex. The retinol-RBP-TTR complex is delivered to target tissues
where retinol is taken up arid utilized for various cellular
processes. Delivery of retinol to cells through the circulation by
the RBP-TTR complex is the major pathway through which cells and
tissue acquire retinol.
[0112] Retinol binding protein, or RBP, is a single polypeptide
chain, with a molecular weight of approximately 21 kD. RBP has been
cloned arid sequenced, and its amino acid sequence determined.
Colantuni et al., Nuc. Acids Res., 11:7769-7776 (1983). The
three-dimensional structure of RBP reveals a specialized
hydrophobic pocket designed to bind and protect the fat-soluble
vitamin retinol. Newcorner et al., EMBO J., 3:1451-1454 (1984). In
in vitro experiments, cultured hepatocytes have been shown to
synthesize and secrete RBP. Blaner, W. S., Endocrine Rev.,
10:308-316 (1989). Subsequent experiments have demonstrated that
many cells contain mRNA for RBP, suggesting a widespread
distribution of RBP synthesis throughout the body (Blaner (1989)).
Most of the RBP secreted by the liver contains retinol in a 1:1
molar ratio, and retinol binding to RBP is required for normal RBP
secretion.
[0113] In cells, RBP tightly binds to retinol in the endoplasmic
reticulum, where it is found in high concentrations. Binding of
retinol to RBP initiates a translocation of retinol-RBP from
endoplasmic reticulum to the Golgi complex, followed by secretion
of retinol-RBP from the cells. RBP secreted from hepatocytes also
assists in the transfer of retinol from hepatocytes to stellate
cells, where direct secretion of retinol-RBP into plasma takes
place.
[0114] In plasma, approximately 95% of the plasma RBP is associated
with transthyretin (TTR) in a 1:1 mol/mol ratio, wherein
essentially all of the plasma vitamin A is bound to RBP. TTR is a
well-characterized plasma protein consisting of four identical
subunits with a molecular weight of 54,980. The full
three-dimensional structure, elucidated by X-ray diffraction,
reveals extensive .beta.-sheets arranged tetrahedrally. Blake et
al., J. Mol. Biol., 121:339-356 (1978). A channel runs through the
center of the tetramer in which is located two binding sites for
thyroxine. However, only one thyroxine molecule appears to be bound
normally to TTR due to negative cooperativity. The complexation of
TTR to RBP-retinol is thought to reduce the glomerular filtration
of retinol, thereby increasing me half-life of retinol and RBP in
plasma by about threefold. See e.g., Blomhoff (1994).
[0115] Retinol uptake from its complexed retinol-RBP-TTR form into
cells occurs by binding of RBP to cellular receptors on target
cells. This interaction leads to ehdocytosis of the RBP-receptor
complex and subsequent release of retinol from the complex, or
binding of retinol to cellular retinol binding proteins (CRBP), and
subsequent release of apoRBP by the cells into the plasma. Other
pathways contemplate alternative mechanisms for the entry of
retinol into cells, including uptake of retinol alone into the
cell. See Blomhoff (1994) for review;
[0116] A2E, the major fluorophore of lipofuscin, is formed in
macular or retinal degeneration or dystrophy, including age-related
macular degeneration and Stargardt Disease, due to excess
production of the visual-cycle retinoid, all-trans-retinaldehyde, a
precursor of A2E. Reduction of vitamin A and all-trans
retinaldehyde in the retina, therefore, would be beneficial in
reducing A2E and lipofuscin build-up, and treatment of age-related
macular degeneration. Studies have confirmed that reducing serum
retinol may have a beneficial effect of reducing A2E and lipofuscin
in RPE. For example, animals maintained on a vitamin A deficient
diet have been shown to demonstrate significant reductions in
lipofuscin accumulation. Katz et al., Mech. Ageing Dev., 35:291-305
(1986); Katz et al., Mech. Ageing Dev., 39:81-90(1987); Katz et
al., Biochem. Biophys. Acta, 924:432-41 (1987). Further evidence
that reducing vitamin A levels may be beneficial in the progression
of macular degeneration and dystrophy was shown by Radu and
colleagues, where reduction in ocular vitamin A levels resulted in
reductions in both lipofuscin and A2E. Radu et al., Proc. Natl.
Acad. Sci. USA, 100:4742-7 (2003); Radu et al., Proc. Natl; Acad.
Sci. USA, 101:5928-33 (2004).
[0117] Administration of the retinoic acid analog,
N-4-(hydroxyphenyl)retinamide, has been shown to cause profound
reductions in serum retinol arid RBP. Formelli et al., Cancer Res.
49:6149-52 (1989); Formelli et al., J. Clin Oncol., 11:2036-42
(1993); Torrisi et al., Cancer Epidemiol. Biomarkers Prev.,
3:507-10 (1994). In vitro studies have demonstrated that HPR
interferes with the normal interaction of TTR with RBP, Malpeli et
al., Biochim. Biophys. Acta 1294: 48-54 (1996); Holven et al., Int.
J. Cancer 71:654-9 (1997).
[0118] Modulators (e.g. HPR) mat inhibit delivery of retinol to
cells either through interruption of binding of retinol to RBP of
the RBP-TTR complex, or the increased renal excretion of retinol
and RBP, therefore, would be useful in decreasing serum levels, and
buildup of retinol and its derivatives in target tissues such as
the eye. The methods and compositions described herein provide for
the detection and screening of such modulators, and provides kits
for the screening of retinol binding modulators.
[0119] One embodiment provides for retinol, retinol binding protein
(RBP) and transthyretin (TTR) for the monitoring of retinol-RBP-TTR
complex formation. Retinol, or vitamin A, binds to various binding
proteins, including retinol binding protein, for transport to
target organs such as the eye. Vitamin A is a generic term which
may designate any compound possessing the biological activity,
including binding activity, of retinol. One retinol equivalent (RE)
is the specific biologic activity of 1 .mu.g of all-trans retinol
(3.33 IU) or 6 .mu.g (10 IU) of beta-carotene. Beta-carotene,
retinol and retinal (vitamin A aldehyde) all possess effective and
reliable vitamin A activity.
[0120] Some examples of potential modulators of retinol-RBP-TTR
complex formation include derivatives of vitamin A, such as
tretinoin (all trans-retinoic acid) and isotretinoin
(13-cis-retinoic acid), which are used in the treatment of acne and
certain other skin, disorders. Other derivatives include
fenretinide (N-(4-hydroxyphenyl)retinamide), MPR and
ethylretinamide. In some aspects of the methods and compositions
disclosed herein, it is contemplated that derivatives of retinol,
retinyl derivatives and related retinoids may be used alone, or in
combination with, other derivatives of retinol or related
retinoids.
[0121] Fenretinide (hereinafter referred to as hydroxyphenyl
retinamide) is particularly useful in the compositions and methods
disclosed herein. As will be explained below, fenretinide may be
used as a modulator of retinol, RBP and TTR complex formation. In
some aspects of the methods and compositions described herein,
derivatives of fenretinide maybe used instead of, or in combination
with, fenretinide. As used herein, a "fenretinide derivative"
refers to a compound whose chemical structure comprises a 4-hydroxy
moiety and a retinamide. The most prevalent metabolite of HPR is
MPR, a compound that can also reduce serum retinol levels. As such,
reference herein to HPR or fenretinide includes die metabolite
MPR.
[0122] In some embodiments, derivatives of fenretinide mat may be
used include, but are not limited to, C-glycoside and arylamidc
analogues of N-(4-hydroxyphenyl) retinamide-O-glucuronide,
including but not limited to 4-(retinamido)phenyl-C-glucurohide,
4-(retinamido)phenyl-C-glucoside, 4-(retinamido)phenyl-C-xyloside,
4-(retinamido)benzyl-C-glucuronide,
4-(retinamido)benzyl-C-glucoside, 4-(retinamido)benzyl-C-xyloside;
and retinoyl .beta.-glucuronide analogues such as, for example,
1-(.beta.-D-glucopyranosyl) retinamide and
1-(D-glucopyranosyluronosyl) retinamide, described in U.S. Pat.
Nos. 5,516,792, 5,663,377, 5,599,953, 5,574,177, and Bhatnagar et
al., Biochem. Pharmacol., 41:1471-7 (1991), each incorporated
herein by reference;
[0123] In other embodiments, other vitamin A derivatives may be
used, including those disclosed in U.S. Pat. No. 4,743,400,
incorporated herein by reference. These retinoids include, for
example, all-trans retinoyl chloride, all-trans-4-(methoxyphenyl)
retinamide, 13-cis-4-(hydroxyphenyl) retinamide and
all-trans-4-(ethoxyphenyl) retinamide; U.S. Pat. No. 4,310,546,
incorporated herein by reference, describes
N-(4-acyloxyphenyl)-all-trans retinamides, such as, for example,
N-(4-acetoxyphenyl)-all-trans-retinamide,
N-(4-propionyloxyphenyl)-all-trans-retinamide and
N-(4-n-butyryloxyphenyl-)-all-trans-retinamide, all of which are
contemplated for use in certain embodiments.
[0124] Other vitamin A derivatives or metabolites, such as
N-(1H-tetrazol-5-yl)retinamide, N-ethylretinamide,
13-cis-N-ethylretinamide, N-butylretinamide, etretin (acitretin),
etretinate, tretinoin (all-trans-retinoic acid) or isotretinoin
(13-cis-retinoic acid) maybe contemplated for use in certain
embodiments. See U.S. Provisional Patent Applications Nos.
60/582,293 and 60/602,675; see also Turton et al., Int. J. Exp.
Pathol., 73:551-63 (1992), all herein incorporated by
reference).
[0125] Other potential modulators include, but are not limited to,
non-steroidal antiinflammatory drugs, including, by way of example
only, flufenamic acid, mefenamic acid, meclofenamic acid,
diflunisal, diclofenac, flurbiprofen; fenoprofen, and indomethacin.
Other potential modulators include, but are not limited to,
biaryls, biarylamines, stilbenes, and dibenzofurans. Further
exemplary potential modulators can be found in Purkey, et al.,
Proc. Natl. Acad. Sci, 98:5566-71 (2001), which is hereby
incorporated by reference in its entirety.
[0126] Other potential modulators include small, molecules,
polypeptides, nucleic acids and antibodies. For example, the
methods and compositions described herein may be used to screen
small molecule libraries, nucleic, acid libraries, peptide
libraries or antibody libraries in conjunction with the teachings
disclosed herein. Methods for screening libraries, Such as
combinatorial libraries and other libraries disclosed above, can be
found in U.S. Pat. Nos. 5,591,646; 5,866,341; and 6,343,257, which
are hereby incorporated by reference in its entirety.
[0127] In one embodiment, the methods and compositions disclosed
herein can be applied to the labeling of a retinol-RBP-TTR complex
member. In this approach, labels, such as enzymes, fluorescers,
radiolabels, chemiluminescers, specific binding pairs, such as
avidin and biotin, ligands, and antibodies, e.g. digoxin and
antidigoxin, and the like, where the protein of interest, for
example TTR, may be labeled with the labels indicated above. In one
embodiment, a fluorophore is attached to the TTR. In some
embodiments, the proteins are labeled with a fluorophore with an
absorbance spectra of between 380 and 480 nm, and an emission
spectra of between 520 nm and 600 nm. In another embodiment, the
proteins may be labeled with a fluorophore with an absorbance
spectra between 410 and 490 nm, and an emission spectra of between
530 and 595 nm. One of ordinary skill in the art will recognize
that other fluorophore moieties may be used in conjunction with the
teachings disclosed herein in order to identify modulators of
retinol-RBP-TTR complex formation.
[0128] Representative flurophores that may be used include
N-((2-(iodoacetoxy)ethyl)-N-methyl)amino-7-nitrobenz-2-oxa-1,3-diazole,
4-dihexadecylamino-7-nitrobenz-2-oxa-1,3-diazole,
6-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)hexanoic acid,
succinimidyl
6-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)hexanoate, lucifer
yellow iodoacetamide,
N-(5-aminopentyl)-4-amino-3,6-disulfo-1,8-naphthalimide,
N,N'-dimethyl-N-(iodoacetyl)-N'-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)ethyle-
nediamine,
1-(2-maleimidylethyl)-4-(5-(4-methoxyphenyl)oxazol-2-yl)pyridin-
ium methanesulfonate,
1-(2,3-epoxypropyl)-4-(5-(4-methoxyphenyl)oxazol-2-yl)pyridinium
trifluoromethanesulfonate,
1-(3-succinimidyloxycarbonyl)benzyl)-4-(5-(4-methoxyphenyl)oxazol-2-yl)
pyridinium bromide, and
3-(4-carboxybenzoyl)quinoline-2-carboxaldehyde,
3-(2-furoyl)quinoline-2-carboxaldehyde. In one embodiment, the
proteins are labeled with a dye with minimum interference from
background protein or retinol fluorescence. Such background
interference may come from internal aromatic amino acid residues on
RBP, or from retinol itself. Therefore, a dye that absorbs and
emits at a wavelength distinct from RBP or retinol would be
desirable in the teachings disclosed herein.
[0129] One embodiment provides the use of an ALEXA FLUOR.RTM. dye
(fluorescent chemicals and biomolecule labeling kit, Molecular
Probes, Inc.), with an absorbance spectra between 380 and 480 nm
and an emission spectra between 520 and 600 nm. For example, ALEXA
FLUOR.RTM. 430 absorbs at 430 nm, and emits at approximately 540
nm. One of ordinary skill in the art will appreciate the many
varieties of dyes available to the practitioner to which, together
with the disclosures presented herein, will allow a practitioner to
select a fluorophore dye suitable for the purposes disclosed
herein.
[0130] Proteins (such as TTR) may be labeled with fluorophores
using commercially available kits, including ALEXA FLUOR.RTM. 430
protein labeling kit (Molecular Probes, Inc., Eugene, Oreg.).
Alternatively, proteins may be labeled wherein the fluorophore
labels possess reactive linkers, see e.g. U.S. Pat. No. 6,140,041,
herein incorporated by reference, or wherein the fluorophore labels
covalently attach to the protein of interest. Other labeling means
will become apparent to those of ordinary skill in the art
dependent upon the type of fluorophore chosen.
[0131] The labels disclosed herein may also comprise an acceptor
fluorescence moiety, preferably for use in fluorescence resonance
energy transfer (FRET) detection. FRET detects signals representing
a binding event, in which the fluorescence of a sample is altered
by a change in the distance separating a fluorescence resonance
energy donor moiety from a fluorescence resonance energy acceptor
moiety that is either another fluorophore of a quencher.
Combinations of a fluorophore and an interacting molecule or moiety
are known as "FRET pairs." A transfer of energy between two members
of a FRET-pair requires that the absorption spectrum of the second
member of the pair overlaps the emission spectrum of the first
member of the pair.
[0132] In FRET detection, the first probe comprises a first probe
fluorescent donor or acceptor. The second probe comprises a second
fluorescent donor or acceptor. The first fluorescent donor or
acceptor and the second fluorescent donor of acceptor are selected
to form a donor/acceptor pair comprising a fluorescent donor and a
fluorescent acceptor capable of fluorescence resonance energy
transfer with each other in response to activation of the
fluorescent donor by light of a predetermined wavelength or band of
wavelengths.
[0133] The excitation and emission spectra of a fluorescent moiety
and the moiety to which it is paired determines whether it is a
fluorescent donor or a fluorescent acceptor. The fluorescent dyes
are selected so that the emission spectrum of the donor fluorophore
overlaps the excitation spectrum of the acceptor fluorophore.
Furthermore, in some methods, a donor fluorophore having a high
extinction coefficient and low fluorescence quantum yield is paired
with an acceptor fluorophore that does not strongly emit at the
excitation wavelength of me donor fluorophore. A cyanine donor
(e.g., CYA) and a rhodamine dye (e.g., R110, R6G; TAMRA arid ROX)
are an example of such a pair. Further guidance regarding the
selection of donor and acceptor pairs that can effectively be used
with the methods disclosed herein include: Fluorescence
Spectroscopy (Pesce et al., Eds;) Marcel Dekker, New York, (1971);
White et. al., Fluorescence Analysis; A Practical Approach, Marcel
Dekker, New York, (1970); Berlman, Handbook of Fluorescence Spectra
of Aromatic Molecules, 2nd ed., Academic Press; New York, (1971);
Griffiths, Colour and Constitution of Organic Molecules, Academic
Press, New York, (1976); Indicators; (Bishop, Ed.). Pergamon Press,
Oxford, 19723; and Haugland, Handbook of Fluorescent Probes arid
Research Chemicals, Molecular Probes, Eugene (1992).
[0134] The efficiency of fluorescence resonance energy transfer has
been reported to be proportional to D.times.10.sup.-6, where D is
the distance between the donor and acceptor (Forster, Z.
Naturforsch A. 4:321-327 (1949)). Accordingly, fluorescence
resonance energy transfer typically occurs at distances of between
10-70 .ANG.. Detection is performed by detecting light emitted by
the fluorescent donor, the fluorescent acceptor, or both
fluorescent donor and acceptor (i.e. a ratio). In one embodiment,
the fluorescent donor and fluorescent acceptors are both
fluorophores. The first fluorophore is activated with light of the
appropriate wavelength or band of wavelengths, which is a function
of the particular fluorophore. In an alternative embodiment, the
fluorescent donor is a fluorophore and the fluorescent acceptor is
a quencher and detection is performed by measuring the emission of
the fluorophore.
[0135] In one embodiment, a member of the retinol-RBP-TTR complex
is labeled with a fluorescent acceptor. For example, a fluorescent,
acceptor molecule may be attached to a TTR protein or polypeptide
and subsequently detected through a FRET detection means.
Representative flurophores that may be used include
N-((2-(iodoacetoxy)ethyl)-N-methyl)amino-7-nitrobenz-2-oxa-1,3-diazole,
4-dihexadecylamino-7-nitrobenz-2-oxa-1,3-diazole,
6-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)hexanoic acid,
succinimidyl-6-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)hexanoate,
lucifer yellow iodoacetamide,
N-(5-aminopentyl)-4-amino-3,6-disulfo-1,8-naphthalimide,
N,N-dimethyl-N-(iodoacetyl)-N'-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)ethylen-
ediamine,
1-(2-maleimidylethyl)-4-(5-(4-methoxyphenyl)oxazol-2-yl)pyridini-
um methanesulfonate,
1-(2,3-epoxypropyl)-4-(5-(4-methoxyphenyl)oxazol-2-yl)pyridinium
trifluoromethanesulfonate,
1-(3-(succinimidyloxycarbonyl)benzyl)-4-(5-(4-methoxyphenyl)oxazol-2-yl)
pyridinium bromide, and
3-(4-carboxybenzoyl)quinoline-2-carboxaldehyde,
3-(2-furoyl)quinoline-2-carboxaldehyde. The proteins may be labeled
With an ALEXA FLUOR.RTM. dye, with an absorbance spectra between
380 and 480 nm and an emission spectra between 520 and 600 nm.
[0136] In one embodiment, a retinol-RBP-TTR protein complex,
wherein TTR is labeled with an acceptor fluorophore, may be excited
with light between 275 and 295 nm, and the emission wavelength
measured at between 330 and 650 nm. Although not bound by any
particular theory, it is believed that aromatic amino acid groups
on RBP act as donor fluorophores and donate electrons to retinol,
which acts as both an acceptor and donor fluorophore. The
approximately 340 nm emission from the activated aromatic-amino
acid groups on RBP, therefore, excites retinol, which absorbs at
approximately 325 nm, and in turn, emits at approximately 470 nm
which excites the acceptor fluorophore groups on me labeled TTR,
allowing FRET detection at an emission wavelength characteristic of
the acceptor fluorophore. Aromatic; amino acids absorb light at a
wavelength of approximately 280 .mu.m, and emit at approximately
325-350 nm, depending on the assay environment.
[0137] FIG. 2 is a depiction of FRET detection with labeled TTR.
The absorbance and excitation emission spectra of RBP and retinol
is shown in FIG. 2A, 2B and 2E. Excitation of RBP, through
excitation of aromatic amino acid residues, occurs at 280 nm and
produces emission at 340 nm. Absorbance of TTR labeled with ALEXA
FLUOR.RTM. 430 (FIG. 2C-2E) occurs at 430 nm, with emission at 540
nm. Upon excitation of the labeled retinol-RBP-TTR complex at 280
nm, an emission radiation signal at approximately 540 nm wavelength
will be detected through FRET to the acceptor ALEXA FLUOR.RTM.430
label on TTR (see FIG. 2E).
[0138] In an alternative embodiment, a retinol-RBP-TTR protein
complex, wherein TTR is labeled with an acceptor fluorophore, maybe
excited with light between 315 and 345 nm, and the emission
wavelength measured at between 525 and 600 nm (see FIG. 2).
Although not bound by any particular theory, it is believed that
energy transfer from retinol to the acceptor fluorophore on a
labeled TTR takes place, resulting in an emission wavelength
characteristic of the acceptor fluorophore (see FIG. 2E).
Assay Conditions
[0139] An exemplary embodiment of the methods and compositions
disclosed herein is to detect and/or quantitate retinol-RBP-TTR
complex in a sample, which requires Conditions conducive to
retinol-RBP-TTR complex formation. One embodiment is to incubate
RBP, TTR and retinol in physiological conditions, e.g. in phosphate
buffered saline (PBS) or the equivalent. Alternatively, other
conditions that approximate physiological conditions in vitro may
also be used. Other embodiments include incubation solvents, such
as ethanol or hexane, see, e.g., Ong, D. E., J. Biol. Chem.,
259:1476-1482 (1984), or other buffer formulations which allow
retinol-RBP-TTR complex formation. In further embodiments,
dimethylsulfoxide is added to the sample to promote the solubility
of potential modulators of retinol-RBP-TTR complex formation.
Amounts of dimethylsulfoxide up to at least 8% by volume may be
added without adversely inhibiting (by itself) retinol-RBP-TTR
complex formation. Further, the sample may be fresh or frozen until
needed without adversely inhibiting (by itself) retinol-RBP-TTR
complex formation.
[0140] Purified protein components, including TTR and RBP, may be
used in conjunction with the teachings disclosed herein. Examples
of purification of TTR and RBP are found in Berni et al., Anal.
Biochem., 150:273-277 (1985); Peterson, P. A., J. Biol. Chem.,
246:34-43 (1971); Berni and Lamberti, Comp. Biochem. Physiol., 94B:
79-83 (1989); Ong, D. E., J. Biol. Chem., 259:1476-1482 (1984),
herein incorporated by reference in its entirety. Purification
includes any improvement in the amount of desired protein in a
sample relative to the native sample (for example, if a native
sample contains 1.5% of a desired protein, purification includes
steps that increase the amount of desired protein in a sample
derived from that native sample to more than 1.5%). Alternatively,
the components of the methods and compositions disclosed herein
maybe naturally occurring in a biological sample from a mammal. For
example, labeled TTR may be added to a biological, sample from a
mammal, and potential modulators tested according to the methods
and compositions disclosed herein. The mammal is preferably a
human, however other mammals, such as primates, horse, dog, sheep,
goat, rabbit, mice or rats may also be used. A biological sample
may comprise, but is not limited to, plasma, blood, urine, feces,
tears Or saliva.
[0141] Upon formation of the retinol-RBP-TTR complex, a potential
modulator is added to the reaction mix and disruption of the
complex monitored by monitoring label activity, for example
fluorescence strength. A decrease in fluorescence as compared to a
reaction mixture without added modulator, for example, will
indicate disruption of the complex by the modulator. Conversely, no
change in fluorescence activity will indicate non-disruption of the
retinol-RBP-TTR complex. Addition of the potential modulator may
occur before, during or after complex formation.
[0142] The compounds and compositions disclosed herein can also be
used in assays utilizing a reagent comprised of a retinol-RBP-TTR
complex formation member attached to a solid support. For example,
RBP or labeled TTR may be attached to biotin, and subsequently
bound to a solid support coated or modified with strept/avidin.
Alternatively, RBP of labeled TTR may be derivatized and conjugated
to active groups on the surface of the solid phase support.
Examples of derivatizing protein or peptides for binding assays can
be found in U.S. Pat. Nos. 4,478,946; 5,169,756, herein
incorporated in its entirety by reference. Proteins of polypeptides
may alternatively be adsorbed onto the solid phase. Coupling or
adsorption of the protein or polypeptide may be followed by
non-specific blocking of exposed binding sites on the surface of
the solid phase.
[0143] The solid phase may be a test tube wall or microtiter plate,
or may be a glass or silicon slide, microarray, microchip or other
semi-conductor or microanalysis platform for attaching proteins or
peptides to a solid phase. Alternatively, the solid phase may also
be ahead (nanoparticle, microparticles, magnetic beads or the like)
comprising agarose, polystyrene, latex, semi-conductor materials
and polymethacrylate.
[0144] The assay can be performed either without separation
(homogeneous) or with separation (heterogeneous) of any of the
assay components or products. The present methods and compositions
find particular use in homogeneous assays where the reactions can
be carried out in solution phase. In these assays any dissociation
of a labeled retinol, RBP, or TTR member into free label can reduce
the sensitivity of the assay since binding of, for example,
unlabeled TTR can compete with binding of labeled TTR that in rum
is related to the presence or amount of modulator to be
determined.
[0145] Heterogeneous assays usually involve one or more separation
steps and can be competitive or noncompetitive. A variety of
competitive and non-competitive heterogeneous assay formats are
disclosed in U.S. Pat. No. 5,089,390, incorporated herein by
reference. In a typical competitive heterogeneous assay, a support
having an antigen for analyte bound, thereto is contacted with a
medium containing the sample and analyte conjugated to a detectable
label, such as an enzyme (the "conjugate"), fluorescent moiety or
radiolabel. The analyte in the sample competes with the conjugate
for binding to the antibody. After separating the support and the
medium, the label activity of the support or the medium is
determined by conventional techniques and is related to the amount
of analyte in the sample.
[0146] A typical non-competitive sandwich assay is an assay
disclosed in U.S. Pat. No. 4,486,530, incorporated herein by
reference. In this method, a sandwich complex, for example an
immune complex, is formed in an assay medium. The complex comprises
the analyte, a first antibody, or binding member; that binds to the
analyte and a second antibody, or binding member that binds to the
analyte or a complex of the analyte and the first antibody, or
binding member. Subsequently, the sandwich complex is detected and
is related to the presence and/or amount of analyte in me sample.
The sandwich complex is detected by virtue of the presence in the
complex of a label wherein either or both the first antibody and
the second antibody, or binding members, contain labels or
substituents capable of combining with labels.
[0147] Sandwich assays find use for the most part in the detection
of antigen and receptor analytes. In the assay, the analyte is
bound by two receptor moieties, or antibodies, specific for the
analyte. In one approach a first incubation of unlabeled antibody,
or binding member, coupled to a support, otherwise known as the
insolubilized binding group, is contacted with a medium containing
a sample suspected of containing the analyte. After a wash and
separation step, the support is contacted with a medium containing
the second antibody or binding member, which generally contains a
label, for a second incubation period. The support is again washed
and separated from the medium and either the medium or the support
is examined for the presence of label. The presence and amount of
label is related to the presence or amount of the analyte. For a
more detailed discussion of this approach see U.S. Pat. No. Re
29,16 and U.S. Pat. No. 4,474,878, the relevant disclosures of
which are incorporated herein by reference.
[0148] In a variation of the above sandwich assay, the sample in a
suitable medium is contacted with labeled antibody or binding
member for the analyte and incubated for a period of time. Then,
the medium is contacted with a support to which is bound a second
antibody, or binding member, for the analyte. After an incubation
period, the support is separated from the medium and washed to
remove unbound reagents. The support or the medium is examined for
the presence of the label, which is related to the presence or
amount of analyte. For a more detailed discussion of this approach
see U.S. Pat. No. 4,098,876, the relevant disclosure of which is
incorporated herein by reference.
[0149] In another variation of the above, the sample, the first
antibody (or binding member) bound to a support and the labeled
antibody (or labeled binding member) are combined in a medium and
incubated in a single incubation step. Separation, wash steps and
examination for label are as described above. For a more detailed
discussion of this approach see U.S. Pat. No. 4,244,940, the
relevant disclosure of which is incorporated herein by
reference.
[0150] Homogenous assays require no separation of assay components,
and is particularly useful in conjunction with high-throughput
assays, therefore, one embodiment is to provide a means for a
homogeneous assay for detecting and/or quantitating modulators of
TTR-RBP-retinol complex formation. In particular, the fluorescence
resonance energy transfer assays are amenable to a homogeneous,
assay format. In fluorescence resonance energy transfer, a
fluorescent signal is not detected unless an energy transfer event
between a donor fluorescent moiety and an acceptor fluorescent
moiety occurs. In the absence of a binding event, therefore, there
is a minimal necessity for separating the labeled and unlabeled
components. Similarly, there is a minimal necessity for separation
of the labeled and unlabeled components if no perturbation of the
complex after formation occurs when an agent is added to the
mixture. Therefore, one other aspect is to provide a homogenous
assay format utilizing FRET detection of a disruption of complex
formation of TTR, RBP and retinol.
[0151] The methods and compositions disclosed herein also have
application to all of the above heterogeneous assays, wherein the
disruption of the binding event above is monitored following
addition of the modulator. For example, the insolubilized binding
member, either labeled TTR or RBP, can be formed by combining
avidin bound to a support with a bis-biotin compound on the labeled
TTR or RBP, in accordance with the teachings disclosed herein. This
may be done prior to, during or after the formation of the labeled
TTR-RBP-retinol complex. A potential modulator of TTR-RBP-retinol
complex formation can then be added, and fluorescence monitored to
determine if disruption has occurred. Alternatively, or in
conjunction therewith, the labeled TTR or RBP can also be bound to
the solid substrate, and subsequently the modulator is added
together with the other components of the TTR-RBP-retinol
complex.
In Vivo Detection of Modulator Activity
[0152] In addition to the in vitro methods disclosed above, the
methods and compositions disclosed herein may also be used in
conjunction with in vivo detection and/or quantitation of modulator
activity of the TTR-RBP-retinol complex formation. For example,
labeled TTR may be injected into a subject, wherein a candidate
modulator was added before, during or after the injection of the
labeled TTR. The subject may be a mammal, for example a human;
however other mammals. such as primates, horse, dog, sheep, goat,
rabbit, mice or rats may also be used. A biological sample is then
removed from the subject and the label detected. A biological
sample may comprise, but is not limited to, plasma, blood, urine,
feces, mucus, tissue, tears of saliva.
Measurement of Label
[0153] The labeled reagents disclosed herein may take place using
any of the conventional means known to those of ordinary skill in
the art, depending upon the nature of the label. For example, a
schematic of one example of a device that maybe used with the
fluorescence methods described herein is presented in FIG. 3; the
various mirrors and lenses depicted within this figure are for
illustrative purposes and not to provide a limitation to the design
of the device that may be used with the detection, measurement arid
analytical methods described herein. The light is provided from a
source (as described elsewhere herein) which is subsequently passed
through a double-grating excitation spectrometer, which can
comprise a series of mirrors and lenses. In addition the
double-grating excitation spectrometer may include a microprocessor
and associated software for controlling the action of the mirrors
and lenses; as well as for recording any information regarding the
properties of the light passing through the double-grating
excitation spectrometer. Other methods and designs for
manipulating, controlling and/or measuring the light prior to
contact with the sample may be used, in such a device.
[0154] After passing through the double-grating excitation
spectrometer, the light passes through a sample compartment; in the
case of FIG. 3, the sample, compartment is designed as a T-box
sample compartment module although other designs are considered
well within the scope of the devices described herein. A series of
lenses and mirrors may also be arranged within the sample module.
In addition, the sample module may also reside within the
double-grating spectrometers; i.e., the sample compartment does not
have to exist as a distinct module. The components and properties
of the sample compartment module may also be controlled, monitored
and/or recorded using a microprocessor and associated software, or
by means of an analog device, or more directly by the end-user of
the device. After the source light interacts with the sample, the
resultant light from the sample (via reflection, emission,
transmission, and the like) can be further analyzed. A portion of
the source light may also be used as a reference beam, in which
case the reference beam may not make contact with the sample. In
the example device presented schematically in FIG. 3, the resultant
light (also described herein as the measured light and the received
light) can further pass through a series of mirrors and lenses
within the sample compartment; in addition, a portion of the
resultant light may also be sent to other devices or
instruments.
[0155] In FIG. 3, after passing through the series of optional
mirrors and lenses in the sample compartment, the resultant light
passes through a double-grating emission spectrometer, which may
include a further series of lenses and mirrors. As with the
double-grating excitation spectrometer, the double-grating emission
spectrometer may include a microprocessor and associated software
for controlling the action of the mirrors and lenses, as well as
for recording any information regarding the properties of the light
passing through the double-grating emission spectrometer. Other
methods and designs for manipulating, controlling and/or measuring
the light after contact with the sample may be used in such a
device. In the final stage of the device presented schematically in
FIG. 3, the resultant light interacts with a photo-multiplier tube,
which can be used as part of an instrument for recording the
properties of the resultant light. Methods for recording, storing
and analyzing the properties of the resultant light are described
herein and maybe incorporated into the device presented
schematically in FIG. 3. Such a device may also include a means for
providing a series of measurements, including but not limited to,
various timing devices, choppers, and associated hardware,
microprocessors, data storage devices, and software. In accordance
with the teachings disclosed herein, measurements may take place
only once; or multiple measurements, preferably at least two times,
of the same sample may also be performed. If multiple measurements
are taken, a brief delay in subsequent measurements may take place.
In some embodiments, for example, at least ten seconds is given
before subsequent measurements are taken. In other embodiments,
non-specific background fluorescence is taken into account by
separately measuring a sample, for example, of retinol-RBP-labeled
TTR in the absence of potential modulators.
[0156] Devices suitable for the methods describe herein may include
software for controlling the illumination step, the detecting step,
archiving information, manipulating or deconvolving images, data or
information from the detection step, and the like. .
[0157] Examples of monitoring devices for chemiluminescence,
radiolabels and other labeling compounds can be found in U.S. Pat.
No. 4,618,485; 5,981,202, the relevant disclosures of which are
herein incorporated by reference.
High-Throughput Assays
[0158] The methods and compositions disclosed herein also relate to
a high throughput assay for rapidly screening a plurality of
modulators in conjunction with the methods and compositions
disclosed herein. The assay detects and/or quantitates potential
modulators of a retinol-RBP-labeled TTR complex, wherein the
potential modulators or therapeutic candidates are added prior to,
during or after complex formation. Inhibition of binding by the
modulators or agents, which may include the library compounds
disclosed above, causes a change in the amount of an optically
detectable label that is attached to the TTR protein or
polypeptide, the labeled TTR or RBP being optionally attached to
solid supports. The degree of modulation is determined by measuring
the amounts of label in solution, or in the case of an attached TTR
or RBP, bound to the solid supports. These amounts are compared
with the amount of label that is present in the absence of a
modulator. Measurement may be performed using a high throughput
optical device, including microtiter plate fluorescent readers of
microchip array fluorescent readers. The assay may be homogeneous,
i.e., no separation step is required to remove unbound label, since
the amount of bound label is distinguished by scanning of the
individual cells or solid supports. Alternatively, the assay may be
heterogeneous with the inclusion of washing steps to remove any
free components, e.g. uncomplexed labeled TTR. The method allows
exceptional sensitivity and high throughput to be obtained in
assays using small volumes, and small amounts of test compound.
Kits
[0159] One further embodiment is a kit for performing assays for
screening for modulators using the methods and compositions
disclosed herein. The kit comprises a TTR probe and a means for
labeling the TTR protein or polypeptide. The TTR may be labeled
with a fluorescent molecule, including a fluorescent acceptor
moiety, in accordance with the embodiments disclosed herein.
Alternatively, the kit may comprise RBP and retinol, and the TTR
may be either free or attached (either bound or adsorbed) to a
solid support.
Treatment Methods, Dosages and Combination Therapies.
[0160] There is a wide variety of treatments and therapies patients
may consider for macular or retinal degenerations and dystrophies,
Which include: photodynamic therapy (PDT), low dose radiation
therapy, submacular surgery, RPE transplantation, macular
translocation surgery, laser treatment of drusen, and medications
which can include an effective amount of a retinyl derivative,
including derivatives of all-trans-retinal and 13-cis-retinal.
[0161] Other methods may be used to treat macular degenerations and
dystrophies, retinal degenerations, and geographic atrophy. Thus,
administration of a therapeutically effective amount of a compound
of Formula (I) to a human having a macular degeneration (including
both the wet forms and dry forms of age-related macular
degeneration), macular dystrophy, retinal degeneration and/or
geographic atrophy can be used to treat such conditions. Thus,
fenretinide and its active metabolites maybe so administered. For
such treatment methods, doses, pharmaceutical formulations and
additional experimental details see U.S. Provisional Pat. App. No.
60/582,293, filed Jun. 23, 2004 and U.S. Provisional Pat. App. No.
60/602,675 filed Aug. 18, 2004, U.S. Provisional Application Ser.
No. 60/625,532 filed Nov. 4, 2004, U.S. Provisional Application
Ser. No. 60/629,695, filed on Nov. 19, 2004, U.S. Provisional
Application Ser. No. 60/660,904, filed on Mar. 11, 2005, and U.S.
Provisional Application Ser. No. 60/672,405 filed on Apr. 18, 2005,
the disclosures of which are specifically included in their
entirety.
[0162] Further combinations that may be used to provide benefit to
an individual include the use of genetic testing to determine
whether that individual is a carrier of a mutant gene that is known
to be correlated, with certain ophthalmic conditions. By way of
example only, defects in the human ABCA4 gene are thought to be
associated with four distinct retinal phenotypes including
Stargardt disease, cone-rod dystrophy, age-related macular
degeneration and retinitis pigmentosa. Such patients would be
expected to find therapeutic and/or prophylactic benefit in the
methods described herein.
[0163] In addition to the aforementioned ingredients, the
formulations disclosed herein may further include one or more
optional accessory ingredient(s) utilized in the art of
pharmaceutical formulations, i.e., diluents, buffers, flavoring
agents, colorants, binders, surface active agents, thickeners,
lubricants, suspending agents, preservatives (including
antioxidants) and the like.
EXAMPLES
[0164] The following ingredients, processes and procedures for
practicing the methods disclosed herein correspond to that
described above. The procedures below describe with particularity a
presently preferred embodiment of the process for the detection and
screening of modulators to retinol binding. Any methods, materials,
reagents or excipients which are not particularly described will be
generally known and available those skilled in the assay and
screening arts.
Example 1
Labeling of TTR
[0165] TTR (Sigma Chemical Go.) was dissolved in phosphate buffered
saline (PBS) buffer (pH 7.2), and the protein concentration
adjusted to about 2-3 mg/ml. Prior to labeling, 10% (v/v) of 1M
NaHCO.sub.3 (pH 8.3) was added to raise the sample pH.
[0166] A 4-times molar excess of ALEXA FLUOR.RTM. 430 (Molecular
Probes) stock solution (in DMSO) was added to the protein solution.
The mixture was incubated at room temperature on a Nutator in the
dark. After 30 min, another aliquot of the fluorescence probe was
added at approximately the same quantity as the first aliquot. The
mixture was incubated for another 30 minutes.
[0167] ALEXA FLUOR.RTM. 430 labeled-TTR was separated from the free
probe using Econo-Pac 10 DG desalting column (Bio-Rad). The protein
fraction was concentrated and the residue amount of free probe
further removed using Centricon YM-3 (Millipore) concentration
devices.
[0168] The absorbance of the labeled protein solution at 280 and
434 nm was measured using a spectrofluorimeter. The protein
concentration was calculated according to the following
equation:
T T R ( M ) = [ A 280 - ( A 434 .times. 0.28 ) ] .times. dilution
factor 77600 ##EQU00001##
where 0.28 is the correction factor to account for the probe
absorbance at 280 nm, and 77,600 cm.sup.-1M.sup.-1 is the molar
extinction coefficient of TTR.
[0169] (a) The degree of labeling was then calculated according to
the following equation:
moles probe per mole protein = A 434 .times. dilution factor 16000
.times. protein concentration ( M ) ##EQU00002##
where 16,000 cm.sup.-1M.sup.-1 is the molar extinction coefficient
of Alexa Fluor 430 at 434 nm.
Example 2
Fluorescent Measurement of TTR and RBP Binding
[0170] 3 ml of 1 .mu.M TTR or Alexa Fluor 430-labeled TTR was
prepared in PBS buffer and placed in a 3-ml fluorescent cuvette.
The emission spectrum was measured between 290 nm to 650 nm with
the excitation wavelength at 280 nm, and emission spectrum between
340 nm to 650 nm with the excitation wavelength at 330 nm, using a
Jobin-Yvon Fluorolog 3 spectrofluorimeter. The band pass of the
spectrofluorimeter was set at 1.5 nm.
[0171] Small aliquots of concentrated holo-RBP (retinol bound to
RBP) stock solutions was then sequentially added to the TTR
solution. The final concentrations of RBP were 0.33 .mu.M, 0.67
.mu.M, 1 .mu.M, 1.33 mM, 1.67 .mu.M and 2 .mu.M, respectively.
After each addition, the sample was mixed and incubated at room
temperature for 20 min. The fluorescence spectra was measured as
above. The contribution of RBP to the emission was subtracted from
the measured spectra by using blanks of RBP at appropriate
concentrations.
[0172] Two emission peaks were particularly monitored, TTR protein
emission at 330 nm (excitation at 280 nm), and Alexa Fluor 430
emission at 540 nm (excitation at 330 nm), at different RBP to TTR
ratios (see FIG. 4).
Example 3
Comparison of Aromatic Amino Acid Quenching
[0173] The currently used method for detection of RBP-TTR
interaction relies on quenching of TTR protein (aromatic amino
acid) fluorescence in the present of increasing concentrations of
RBP. Analysis of crude sera is not permissible in this assay due to
the presence of other interfering proteins in sera. Therefore, RBP
must be purified from crude sera prior to analysis of TTR binding.
This is a time consuming process which is not amenable to
high-throughput screening. An example of data obtained using this
technique is provided in FIG. 4A. In FIG. 4A, the fluorescent
emission of the retinol-RBP-unlabeled TTR complex is measured with
excitation at 280 nm. The different curves in the graph represent
increasing concentration of RBP added to the sample mixture. At
higher amounts of RBP, where increasing levels of RBP are complexed
to unlabled TTR, the complexed RBP gradually quenches the aromatic
amino acid fluorescent signal on TTR.
[0174] In order to overcome limitations of me routine binding
assay, a high-throughput assay was developed to examine the
kinetics of RBP-TTR interaction (see FIG. 4B). In this assay,
commercially prepared TTR is modified with a fluorescent probe
which is excited only when a retinoid protein complex is bound to
it,(i.e. retinol-RBP). Thus, non-specific protein and/or unbound
retinoid-protein species present in crude sera will not interfere
with the detection of the RBP-labeled TTR complex. In addition, the
assay is 2 to 3 times more sensitive arid demonstrates a greater
dynamic range and linearity for detection and quantitation. A
direct comparison of the two techniques is provided (FIG. 5). The
assay technique may be adapted to a 96-well plate format to
facilitate screening of large numbers of samples in a
high-throughput format.
Example 4
In-Vivo Analyses of the Relationship of Serum HPR Levels to the
Levels of Serum Retinol, and Ocular Retinoids and A2E
[0175] In in vitro assays, inhibition of LRAT activity results in a
net reduction in the all-trans retinyl ester pool and, therefore, a
reduction in 11-cis retinol. In order to further explore the role
of HPR in the visual cycle, the in vivo effects of HPR in mice have
been examined. Thus, HPR was administered to ABCA4 null mutant mice
(5-20 mg/kg, i.p. in DMSO) for periods of 28 days. Control mice
received only the DMSO vehicle. At the end of the treatment period,
the concentrations of retinol and HPR in serum and retinoid content
in ocular tissues was measured. Profound reductions in serum
retinol as a function of increasing serum HPR was observed. This
effect was associated with commensurate reductions in ocular
retinoids and A2E (a toxic retinoid-based fluorophore). Thus, the
calculated percent reduction for each of the measured retinoids,
and A2E, was nearly identical (see FIG. 6). These results indicate
that HPR has a limited effect on visual cycle proteins (e.g., LRAT)
when administered systemically; instead these results indicate that
reduction of ocular retinoides and A2E resulting from systemic
administration of HPR results from reductions in serum retinol
levels. If systemically-administered HPR inhibits ocular LRAT, then
reduction of ocular retinoids and A2E should exceed the reduction
in serum retinol.
[0176] In order to ensure that the observed effects of HPR in ABCA4
null mice were not due to the genetic mutation, HPR (20 mg/kg, i.p.
in DMSO) was administered to wild type mice for 5 days. Control
mice received only the DMSO vehicle. On the final day of HPR
treatment, the mice were exposed to constant illumination (1000 lux
for 10 min) in order "stimulate" the visual cycle to generate
visual chromophore. Immediately following the illumination period,
the animals were sacrificed and the concentrations of retinoids in
serum and ocular tissue were determined. The data (see FIG. 7)
reveal no significant inhibition in synthesis of either retinyl
esters or visual chromophore. As in the previous study, HPR caused
a significant reduction in serum retinol (.about.55%), ocular
retinol (.about.40%) and ocular retinal (.about.30%). Although HPR
did accumulate within ocular tissues during the treatment period
(.about.5 .mu.M), no effect on LRAT or Rpe65/isomerase activities
was observed.
[0177] Genetic crosses of RBP4.sup.-/- mice with ABGA4.sup.-/- mice
was undertaken to examine the role of RBP in mediation of retinol
levels in serum and ocular tissue. Mice from the first generation
of this cross (i.e., RBP4/ABCA4.sup.+/-) show comparable levels of
RBP-retinol reduction as observed in the HPR study when me
administered dose was 10 mg/kg (.about.50-60% reduction in serum
RBP-retinol). Moreover, the RBP4/ABCA4.sup.+/- mice show
commensurate reductions in ocular retinol (.about.60% reduction).
These findings are consistent with data obtained during
pharmacological modulation of RBP-retinol with HPR and, therefore,
strongly suggest that A2E-based fluorophores will be reduced
proportionately.
[0178] The inhibition of LRAT activity observed during in vitro
analyses has not been observed in mice receiving acute and chronic
doses of HPR. Such differences in in vivo and in vitro observations
may arise from the differential accessibility of HPR to visual
cycle enzymes in vitro versus in vivo. That is, in the in vitro
assays, HPR is pre-incubated with enzyme source material before me
addition of substrate and initiation of the assay. HPR may become
more accessible to visual cycle proteins during the pre-incubation
and assay period and produce the observed results. The situation in
vivo may be very different. As all of the enzymatic proteins of the
visual cycle are hydrophobic in nature, accessibility by small
polar molecules such as HPR maybe significantly hindered.
Example 6
High-throughput Assay for Detection of RBP/TTR Interaction
[0179] Reduction of serum retinol and RBP are correlated with
concomitant reductions in toxic lipofuscin fluorophores. Because
compounds that affect RBP-TTR interaction will directly affect
fluorophore levels in the eye, a high-throughput screen for small
molecules which prevent interaction of RBP with TTR was developed.
This screen employs probe-labeled forms of RBP and TTR which
participate in a unique fluorescence resonance energy transfer
(FRET) event when complexed. Compounds which interfere with RBP-TTR
interaction prevent FRET. Sample spectra taken during the course of
this type of assay are shown in FIG. 8. These data show,
interaction of RBP-TTR (0.5 .mu.M unlabeled RBP+0.5 .mu.M
Alexa430-TTR) in the absence (solid line) and presence (dashed
line) of HPR (1 .mu.M). The sample is incubated at 37.degree. C.
for 30 min and then illuminated with 330 nm light. The emission
spectra are shown in the range of 400-600 nm. HPR binds to RBP and
prevents interaction with TTR, and here this property of HPR is
utilized here to validate the ability of this screen to detect
inhibition of RBP-TTR interaction. The presence of HPR is
associated with significantly reduced retinol and TTR-probe
fluorescence indicating loss of complexation. Additionally, the
design of this assay permits discrimination between compounds which
interact with RBP versus those which interact with TTR. Thus, by
using two distinct excitation energies (280 nm and 330 nm, for
protein and retinoid respectively) and implementing simultaneous
monitoring of the retinol and TTR-probe fluorescence, the "target"
of a presumptive small molecule can be easily determined.
Example 7
Adaptation and Optimization of Assay to 384-Well Plate Format
[0180] In order to facilitate screening of large numbers of test
compounds, the RBP-TTR assay has been adapted to a 384-well plate
format. This transition required re-evaluation of the reagent
concentrations and minimal assay volume for maintaining solubility
and detection sensitivity. Under the 384-well plate format, the
assay can be efficiently performed in a 50 .mu.l volume using 0.5
.mu.M apo-RBP, 0.5 .mu.M TTR, 2-8 .mu.M test compound and 1 .mu.M
retinol. The microplate with minimal fluorescence background, best
optical clarity arid most appropriate well design was determined to
be Coming's model #3711.
[0181] Dimethyl sulfoxide (DMSO) Will be used to deliver test
compounds to the RBP-TTR assay mixture. Further, in order to
determine the kinetic properties of inhibition observed with a
particular compound, the test compounds are evaluated at varied
concentrations. The most convenient method of achieving this
objective in a high-throughput assay is to add increasing volumes
of a fixed compound concentration. This approach results in
increasing concentrations of DMSO in the assay mixture.
Accordingly, studies were carried out to determine the tolerance of
the RBP-TTR assay for increasing concentrations of DMSO. It was
determined that concentrations up to 8% DMSO, v/v had no effect on
RBP-TTR interaction either in the absence or presence of HPR, which
was used as the positive control for RBP-TTR inhibition.
[0182] An important consideration for generation of the
TTR-AlexaFluor 430 protein is the molar amount of AlexaFluor 430
required to effectively label (and in certain circumstances, to
optimally label) the TTR protein without compromising the inherent
affinity of TTR for RBP. This concern has economical implications
as the requirement for increased mol % of AlexaFluor 430 means
increased cost. Studies were conducted to determine the lowest mole
% of AlexaFluor 430 required to effectively label TTR without
affecting binding to RBP. It was determined that 1.7 mol % of
AlexaFluor 430 is required to label 1 mole of tetrameric TTR.
[0183] In order to further facilitate high throughput of the
RBP-TTR assay, the test compounds with be added to master plates at
one time and the plates stored at -20.degree. C. for up to 2 weeks
before adding retinol and conducting the assay. The present assay
is quite stable under these conditions. No loss in sensitivity is
observed after 2 weeks of storage at -20.degree. C.
Example 8
Assay Validation and Comparison to Conventional Techniques
[0184] HPR is an effective inhibitor of RBP-TTR interaction as
Shown by chromatographic and spectrophotometric measurement
techniques (See, e.g., Radu R A, Han Y, Bui T V, Nusinowitz S, Bok
D; Lichter J, Widder K, Travis G H and Mata N L; Reductions in
Serum Vitamin A Arrest Accumulation of Toxic Retinal Fluorophores;
A Potential Therapy for Treatment of Lipofuscin-based Retinal
Diseases, Invest Ophthalmol. Vis Sci., in press (2005)). Thus, HPR
may be used as a positive control to validate the capacity of the
high throughput assay to detect inhibitors of RBP-TTR interaction.
Accordingly, HPR was employed at varied concentrations (from 0-4
.mu.M), using the conditions specified in Example 7, to evaluate
the high throughput assay. As shown in FIG. 9, the high throughput
assay is effective to detect compounds which, like HPR, inhibit
RBP-TTR interaction.
[0185] Physiologically, RBP-retinol must complex with TTR in order
to achieve a high steady-state concentration of RBP-retinol. This
interaction creates a large molecular size complex which resists
glomerular filtration and permits delivery of retinol to
extra-hepatic target tissues. Inhibition of RBP-TTR interaction
results in a reduction of circulating RBP as the relatively small
sized RBP-ligand complex would be lost through glomerular
filtration; The reduction in circulating RBP then causes a
reduction in circulating retinol. This effect has been established
in vivo for HPR by several investigators. This, effect has also
been observed in vivo using all-trans and 13-cis retinoic acids
(See, e.g., Berni R, Clerici M, Malpeli G, Cleris L, Formelli F;
Retinoids: in vitro interaction with retinol-binding; protein and
influence on plasma retinol, FASEB J. (1993) 7:1179-84).
[0186] The mechanism of action underlying this effect can be
explained by the disruption of RBP-TTR interactions. In order to
explore this possibility and to further validate the RBP-TTR
screen, the effects of all-trans retinoic and 13-cis retinoic acid,
using the conditions, the conditions specified for analysis of HPR,
were examined. The data obtained (see FIG. 10) are entirely
consistent with the in vivo data. This finding further validates
the ability of this assay to detect known physiological inhibitors
of RBP-TTR interaction.
Example 9
Comparison of Assay to Conventional Techniques
[0187] Current high throughput methodologies used to detect
complexation between two proteins are limited to those employing
fluorescence techniques. A popular method is fluorescence
anisotropy. This method, which measures change in molecular volume
(or size), has been used successfully in analytical studies to
measure interaction between RBPT-retinol and TTR (see, e.g., van
Jaarsveld P P, et al., J Biol Chem., 248:4698-705 (1973); Kopelman
M, et al., Biochim Biophys Acta. 439:449-60 (1976); Malpeli G, et
al., Biochim Biophys Acta., 1294:48-54 (1996)). In this approach,
the fluorescence emission of retinol is monitored, at 0 and 90
degree angles, in the absence and presence of TTR. Although this
technique is quite sensitive, it is riot quantitative. Thus, the
output value would be the same for all degrees of RBP-TTR binding;
A binding of 100% of the RBP-retinol present in the assay to TTR
could not be distinguished from a binding of only 10%. This
shortcoming of the anisotropy technique is in FIG. 11. Here, the
effect of HPR on RBP-TTR interaction is measured using our routine
FRET (high-throughput) assay and by traditional fluorescence
anisotropy.
[0188] In addition to the technical impasses of fluorescence
anisotropy to screen for
[0189] compounds which affect RBP-TTR interaction, few commercially
available instruments are available with high throughput capability
which offer fluorescence anisotropy with detection in the near UV
range. On the other hand, the FRET assay which can be employed on
any conventional fluorescent microplate reader:
[0190] All of the methods disclosed and claimed herein can be made
and executed without undue experimentation in light of the present
disclosure. It will be apparent to those of skill in the art that
variations may be applied without departing from the concept,
spirit and scope of the invention. More specifically, it will be
apparent that certain agents that both chemically and
physiologically related may be substituted for the agents described
herein while the same or similar results would be achieved. All
such similar substitutes and modifications apparent to those
skilled in the art are deemed to be within the spirit, scope and
concept of the invention as defined by the appended claims.
* * * * *