U.S. patent application number 14/528077 was filed with the patent office on 2015-04-30 for methods of preparing ophthalmic formulations and uses of same.
The applicant listed for this patent is Cognoptix, Inc.. Invention is credited to Haresh Bhagat, Gerald D. Cagle, Paul D. Hartung, Francis X. Smith.
Application Number | 20150118163 14/528077 |
Document ID | / |
Family ID | 51901002 |
Filed Date | 2015-04-30 |
United States Patent
Application |
20150118163 |
Kind Code |
A1 |
Bhagat; Haresh ; et
al. |
April 30, 2015 |
Methods of Preparing Ophthalmic Formulations and Uses of Same
Abstract
The present invention relates to a process for preparing an
ointment comprising subjecting a mixture of a fluorescent molecular
rotor compound, mineral oil, and grinding media to ball milling
agitation and combining this mixture with a hydrophobic vehicle for
an ointment. The invention also relates to an ophthalmic
formulation comprising a pharmaceutically acceptable carrier and a
compound of the following structural Formula (I), or a
pharmaceutically acceptable salt thereof: ##STR00001## Values and
preferred values of the variables in structural Formula (I) are
described herein.
Inventors: |
Bhagat; Haresh; (Fort Worth,
TX) ; Cagle; Gerald D.; (Forth Worth, TX) ;
Smith; Francis X.; (Salem, NH) ; Hartung; Paul
D.; (Acton, MA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Cognoptix, Inc. |
Acton |
MA |
US |
|
|
Family ID: |
51901002 |
Appl. No.: |
14/528077 |
Filed: |
October 30, 2014 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
61898131 |
Oct 31, 2013 |
|
|
|
Current U.S.
Class: |
424/9.6 |
Current CPC
Class: |
A61P 27/02 20180101;
A61K 49/0021 20130101; A61P 25/28 20180101; A61K 9/0048 20130101;
A61P 25/00 20180101; A61K 47/06 20130101; A61P 25/16 20180101 |
Class at
Publication: |
424/9.6 |
International
Class: |
A61K 49/00 20060101
A61K049/00 |
Claims
1. A process for preparing an ophthalmic ointment, comprising: (a)
combining a fluorescent molecular rotor compound with mineral oil
as a levigating agent, thereby obtaining a first mixture; (b)
subjecting the first mixture to ball milling agitation with a
grinding media, thereby obtaining a second mixture; and (c)
combining the second mixture with a hydrophobic vehicle, thereby
obtaining an ointment.
2. The process of claim 1, wherein the second mixture comprises
particles of the fluorescent molecular rotor compound, the
particles having a size distribution characterized by a d(0.95)
particle size of less than about 20 microns.
3. The process of claim 1, wherein the grinding media for ball
milling agitation comprises beads with an average size of at least
3 mm.
4. The process of claim 1, wherein the ratio of the grinding media
volume to the ball milling container volume ranges from about 0.05
to about 0.35.
5. The process of claim 1, wherein the grinding media is a ceramic
selected from the group consisting of magnesia stabilized zirconia,
zirconium silicate, zirconia stabilized alumina, alumina-zirconia
composite, burundum, and yttrium stabilized zirconia.
6. The process of claim 5, wherein the grinding media is yttrium
stabilized zirconia.
7. The process of claim 1, wherein the second mixture is agitated
at about 50% to about 70% of critical mill speed.
8. The process of claim 1, wherein the hydrophobic vehicle is
selected from one of more of: a petrolatum, a mineral oil, a
polyglycol, a silicone oil, a fluorocarbon, a lanolin, a gelled
mineral oil, and a surfactant.
9. The process of claim 8, wherein the hydrophobic vehicle is
selected from one or more of a petrolatum and a mineral oil.
10. The process of claim 1, further comprising irradiating the
fluorescent molecular rotor compound and the mineral oil before
forming the first mixture.
11. The process of claim 1, further comprising irradiating the
first mixture.
12. The process of claim 1, wherein the fluorescent molecular rotor
compound has the following structural Formula (I), or a
pharmaceutically acceptable salt thereof: ##STR00029## wherein:
A.sup.1 is an optionally substituted C6-C 18 arylene, an optionally
substituted C5-C 18 heteroarylene, or is represented by the
following structural formula: ##STR00030## R.sup.1 and R.sup.2 are
each independently hydrogen, optionally substituted C1-C12 alkyl,
an optionally substituted C1-C12 heteroalkyl, optionally
substituted C3-C12 cycloalkyl, or R.sup.1 and R.sup.2 taken
together with the nitrogen atom to which they are attached form an
optionally substituted 3 to 12 membered heterocycloalkyl; R.sup.3
and R.sup.4 are each independently hydrogen, methyl, or ethyl;
R.sup.5 is --OH, optionally substituted --O(C1-C6 alkyl),
--NR.sup.6R.sup.7 or is represented by the following structural
formula: ##STR00031## R.sup.6 and Ware each independently,
hydrogen, methyl, ethyl or R.sup.6 and R.sup.7 taken together with
the nitrogen atom to which they are attached form a 5 to 7 membered
heterocycloalkyl containing one to three ring heteroatoms
independently selected from N, O, and S; wherein: y is an integer
from 1 to 10; R.sup.8, for each occurrence independently, is
hydrogen, --OH, or --CH.sub.2OH; R.sup.9 is hydrogen,
--NR.sup.10R.sup.11, --C(O)R.sup.12, optionally substituted C1-C6
alkyl, optionally substituted C1-C6 heteroalkyl; R.sup.10,
R.sup.11, and R.sup.12 are each independently hydrogen or C1-C6
alkyl.
13. The process of claim 12, wherein A.sup.1 is selected from the
group consisting of optionally substituted phenyl, optionally
substituted naphthyl, an optionally substituted (E)-stilbene, or an
optionally substituted (Z)-stilbene.
14. The process of claim 12, wherein A.sup.1 is optionally
substituted naphthyl.
15. The process of claim 12, wherein R.sup.1 and R.sup.2 taken
together with the nitrogen atom to which they are attached form an
optionally substituted 3 to 12 membered heterocycloalkyl.
16. The process of claim 12, wherein R.sup.5 is ##STR00032##
17. The process of claim 12, wherein R.sup.5 is ##STR00033## y is
3; and R.sup.9 is methyl.
18. The process of claim 12, wherein the fluorescent molecular
rotor compound has the following structural Formula (II) or Formula
(III), or a pharmaceutically acceptable salt thereof: ##STR00034##
wherein: R.sup.13, R.sup.14 and R.sup.15 are each independently
hydrogen, --OH, or optionally substituted --O(C1-C6 alkyl).
19. The process of claim 1, wherein the fluorescent molecular rotor
compound is selected from the group consisting of: ##STR00035##
##STR00036##
20. A process for preparing an ophthalmic ointment, comprising: (a)
combining a fluorescent molecular rotor compound of the following
structural formula: ##STR00037## with mineral oil as a levigating
agent, thereby obtaining a first mixture; (b) subjecting the first
mixture to ball milling agitation, thereby obtaining a second
mixture; and (c) combining the second mixture with a hydrophobic
vehicle, thereby obtaining an ointment.
21. An ophthalmic ointment comprising an effective amount of a
compound of the following structural Formula (I), or a
pharmaceutically acceptable salt thereof: ##STR00038## wherein:
A.sup.1 is an optionally substituted C6-C 18 arylene, an optionally
substituted C5-C18 heteroarylene, or is represented by the
following structural formula: ##STR00039## R.sup.1 and R.sup.2 are
each independently hydrogen, optionally substituted C1-C12 alkyl,
an optionally substituted C1-C12 heteroalkyl, optionally
substituted C3-C12 cycloalkyl, or R.sup.1 and R.sup.2 taken
together with the nitrogen atom to which they are attached form an
optionally substituted 3 to 12 membered heterocycloalkyl; R.sup.3
and R.sup.4 are each independently hydrogen, methyl, or ethyl;
R.sup.5 is --OH, optionally substituted --O(C1-C6 alkyl),
--NR.sup.6R.sup.7 or is represented by the following structural
formula: ##STR00040## R.sup.6 and Ware each independently,
hydrogen, methyl, ethyl or R.sup.6 and R.sup.7 taken together with
the nitrogen atom to which they are attached form a 5 to 7 membered
heterocycloalkyl containing one to three ring heteroatoms
independently selected from N, O, and S; wherein: y is an integer
from 1 to 10; R.sup.8, for each occurrence independently, is
hydrogen, --OH, or --CH.sub.2OH; R.sup.9 is hydrogen,
--NR.sup.10R.sup.11, --C(O)R.sup.12, optionally substituted C1-C6
alkyl, optionally substituted C1-C6 heteroalkyl; R.sup.10R),
R.sup.11 and R12 are each independently hydrogen or C1-C6 alkyl;
and a pharmaceutically acceptable carrier.
22. An ophthalmic ointment comprising an effective amount of a
compound of the following structural Formula (II): ##STR00041##
wherein: R.sup.1 and R.sup.2 are each independently hydrogen,
optionally substituted C1-C12 alkyl, an optionally substituted
C1-C12 heteroalkyl, optionally substituted C3-C12 cycloalkyl, or
R.sup.1 and R.sup.2 taken together with the nitrogen atom to which
they are attached form an optionally substituted 3 to 12 membered
heterocycloalkyl; R.sup.3 and R.sup.4 are each independently
hydrogen, methyl, or ethyl; R.sup.5 is --OH, optionally substituted
--O(C1-C6 alkyl), --NR.sup.6R.sup.7, or is represented by the
following structural formula: ##STR00042## R.sup.6 and R.sup.7 are
each independently, hydrogen, methyl, ethyl, or R.sup.6 and R.sup.7
taken together with the nitrogen atom to which they are attached
form a 5 to 7 membered heterocycloalkyl containing one to three
ring heteroatoms independently selected from N, O, and S; R.sup.10,
R.sup.11 and R.sup.12 are each independently hydrogen or C1-C6
alkyl; R.sup.13 is hydrogen, --OH, or optionally substituted
--O(C1-C6 alkyl); wherein: y is an integer from 1 to 10; R.sup.8,
for each occurrence independently, is hydrogen, --OH, or
--CH.sub.2OH; R.sup.9 is hydrogen, --NR.sup.10R.sup.11,
--C(O)R.sup.12, optionally substituted C1-C6 alkyl, optionally
substituted C1-C6 heteroalkyl; and a pharmaceutically acceptable
carrier.
23. An ophthalmic ointment obtained by the process according to
claim 1.
24. A method of diagnosing a neurodegenerative disease in a
subject, comprising: administering to the subject an effective
amount of the ophthalmic ointment of claim 21.
25. The method of claim 24, wherein the neurodegenerative disorder
is an amyloidogenic disorder.
26. The method of claim 25, wherein the amyloidogenic disorder is
selected from Alzheimer's Disease (AD), Familial AD, Sporadic AD,
Creutzfeld-Jakob disease, variant Creutzfeld-Jakob disease,
spongiform encephalopathies, Prion diseases (including scrapie,
bovine spongiform encephalopathy, and other veterinary
prionopathies), Parkinson's disease, Huntington's disease (and
trinucleotide repeat diseases), amyotrophic lateral sclerosis,
Down's Syndrome (Trisomy 21), Pick's Disease (Frontotemporal
Dementia), Lewy Body Disease, neurodegeneration with brain iron
accumulation (Hallervorden-Spatz Disease), synucleinopathies
(including Parkinson's disease, multiple system atrophy, dementia
with Lewy Bodies, and others), neuronal intranuclear inclusion
disease, tauopathies (including progressive supranuclear palsy,
corticobasal degeneration, hereditary frontotemporal dementia (with
or without Parkinsonism), and Guam amyotrophic lateral
sclerosis/parkinsonism dementia complex.
27. A method of diagnosing a neurodegenerative disease in a
subject, comprising: administering to the subject an effective
amount of the ophthalmic ointment of claim 22.
28. The method of claim 27, wherein the neurodegenerative disorder
is an amyloidogenic disorder.
29. The method of claim 28, wherein the amyloidogenic disorder is
selected from Alzheimer's Disease (AD), Familial AD, Sporadic AD,
Creutzfeld-Jakob disease, variant Creutzfeld-Jakob disease,
spongiform encephalopathies, Prion diseases (including scrapie,
bovine spongiform encephalopathy, and other veterinary
prionopathies), Parkinson's disease, Huntington's disease (and
trinucleotide repeat diseases), amyotrophic lateral sclerosis,
Down's Syndrome (Trisomy 21), Pick's Disease (Frontotemporal
Dementia), Lewy Body Disease, neurodegeneration with brain iron
accumulation (Hallervorden-Spatz Disease), synucleinopathies
(including Parkinson's disease, multiple system atrophy, dementia
with Lewy Bodies, and others), neuronal intranuclear inclusion
disease, tauopathies (including progressive supranuclear palsy,
corticobasal degeneration, hereditary frontotemporal dementia (with
or without Parkinsonism), and Guam amyotrophic lateral
sclerosis/parkinsonism dementia complex.
30. The method of claim 23, comprising: determining the amount of
amyloid aggregate in the supranuclear region and/or cortical lens
region of the eye.
Description
RELATED APPLICATION
[0001] This application claims the benefit of U.S. Provisional
Application No. 61/898,131, filed on Oct. 31, 2013. The entire
teachings of the above application are incorporated herein by
reference.
BACKGROUND OF THE INVENTION
[0002] Early diagnosis and detection of diseases and disease risk
generally improves disease prognosis and expands treatment options.
Recently, it has been discovered that analyzing the lenses of the
eyes can yield indications of various types of diseases. For
example, neurodegenerative disease such as an amyloidogenic
disorders have recently been linked to the presence or increase in
the amount of aggregate in the supranuclear region and/or cortical
lens region of the eye. An example of an amyloidogenic disorder is
Alzheimer's disease (AD), which is characterized by a progressive
loss of cognitive abilities including memory loss, personality
changes, and impaired reasoning. Innovative formulations and
processes for preparing formulations such as ophthalmic
formulations and diagnostic ointments for diagnosis and detection
of neurodegenerative diseases will help alleviate the burden of
these health issues.
SUMMARY OF THE INVENTION
[0003] It has now been discovered that an ointment comprising a
fluorescent molecular rotor compound possesses advantageous
properties (e.g., uniformity of particle content) when it is
manufactured by methods described and claimed herein. For example,
it has been found that by using the method described and claimed
herein an ointment is obtained that is suitable for use in the eye
of a subject, in particular for diagnostic purposes. It has been
found that the method is particularly advantageous in the case of
fluorescent molecular rotor compounds. By applying the method, a
fluorescent molecular rotor compound, which is provided in
particulate form, is transformed to a mixture of particles with
uniform particle size distribution without significant loss of
compound material. In example embodiments, a process for preparing
an ophthalmic formulation comprising a fluorescent molecular rotor
compound provides a formulation useful to diagnose and to assess
disease risk for amyloidogenic disorders. In some embodiments, the
ophthalmic formulation used to diagnose and assess disease risk for
amyloidogenic disorders is an ointment. For example, ointments of
the present invention can be manufactured by a method comprising:
[0004] (a) combining a fluorescent molecular rotor compound with
mineral oil as a levigating agent, thereby obtaining a first
mixture; [0005] (b) subjecting the first mixture to ball milling
agitation with a grinding media, thereby obtaining a second
mixture; and [0006] (c) combining the second mixture with a
hydrophobic vehicle, thereby obtaining an ointment.
[0007] In one embodiment, the fluorescent molecular rotor compound
is represented by structural Formula (I), or a pharmaceutically
acceptable salt thereof:
##STR00002## [0008] wherein: [0009] A.sup.1 is an optionally
substituted C6-C18 arylene, an optionally substituted C5-C18
heteroarylene, or is represented by the following structural
formula:
[0009] ##STR00003## [0010] R.sup.1 and R.sup.2 are each
independently hydrogen, optionally substituted C1-C12 alkyl, an
optionally substituted C1-C12 heteroalkyl, optionally substituted
C3-C12 cycloalkyl, or R.sup.1 and R.sup.2 taken together with the
nitrogen atom to which they are attached form an optionally
substituted 3 to 12 membered heterocycloalkyl; [0011] R.sup.3 and
R.sup.4 are each independently hydrogen, methyl, or ethyl; [0012]
R.sup.5 is --OH, optionally substituted --O(C1-C6 alkyl),
--NR.sup.6R.sup.7 or is represented by the following structural
formula:
[0012] ##STR00004## [0013] R.sup.6 and R.sup.7 are each
independently, hydrogen, methyl, ethyl or R.sup.6 and R.sup.7 taken
together with the nitrogen atom to which they are attached form a 5
to 7 membered heterocycloalkyl containing one to three ring
heteroatoms independently selected from N, O, and S; [0014]
wherein: [0015] y is an integer from 1 to 10; [0016] R.sup.8, for
each occurrence independently, is hydrogen, --OH, or --CH.sub.2OH;
[0017] R.sup.9 is hydrogen, --NR.sup.10R.sup.11, --C(O)R.sup.12,
optionally substituted C1-C6 alkyl, optionally substituted C1-C6
heteroalkyl; [0018] R.sup.10, R.sup.11 and R.sup.12 are each
independently hydrogen or C1-C6 alkyl.
[0019] In some embodiments, the present invention relates to a
process for preparing an ointment, preferably an ophthalmic
ointment, comprising: [0020] a) combining a fluorescent molecular
rotor compound of the following structural formula:
[0020] ##STR00005## [0021] with mineral oil as a levigating agent,
thereby obtaining a first mixture; [0022] b) subjecting the first
mixture to ball milling agitation, thereby obtaining a second
mixture; and [0023] c) combining the second mixture with a
hydrophobic vehicle, thereby obtaining an ointment.
[0024] In some embodiments, the present invention relates to an
ophthalmic formulation comprising a fluorescent molecular rotor
compound represented by structural Formula (I), structural Formula
(II), structural Formula (III), or a pharmaceutically acceptable
salt thereof and a pharmaceutically acceptable carrier.
[0025] In certain embodiments, a process for preparing an
ophthalmic ointment comprising a fluorescent molecular rotor
compound and mineral oil subjected to ball milling agitation
provides a formulation with enhanced uniformity of content compared
to formulations prepared by other processes. Enhanced uniformity of
content can include uniformity of particle size and uniformity of
dispersion of the fluorescent molecular rotor compound particles.
For example, particles of the fluorescent molecular rotor compound
having a size distribution characterized by a d(0.95) particle size
of less than about 10 microns. For example, particle dispersion and
particle size uniformity can be enhanced when the process comprises
grinding media or grinding beads with an average particle size of
at least 3 mm for ball milling agitation.
[0026] Ophthalmic formulations and ointments prepared by processes
of the invention involving ball milling agitation can provide more
stable and uniform formulations, thus facilitating administration
of the formulation to the eye and allowing for more precise dosing
of the active compound. In addition, the uniform particle size
distribution renders it feasible to provide formulations that are
potentially less irritating to, e.g., the eye than formulations
prepared using other processes. In some embodiments, the
formulations are useful in eye tests for diagnosing amyloidogenic
disorders. In a preferred embodiment, the formulations are useful
for aiding in the diagnosis of, and to assess disease risk, for
amyloidogenic disorders in an eye test.
DETAILED DESCRIPTION OF THE INVENTION
[0027] A description of example embodiments of the invention
follows.
Glossary
[0028] "Alkyl" used alone or as part of a larger moiety such as
"haloalkyl" or "alkoxyalkyl" refers to a straight or branched,
saturated aliphatic group having the specified number of carbons,
typically having 1 to 12 carbon atoms. More particularly, the
aliphatic group may have 1 to 8, 1 to 6, or 1 to 4 carbon atoms.
Alkyl is exemplified by groups such as methyl, ethyl, n-propyl,
isopropyl, n-butyl, isobutyl, tert-butyl, n-hexyl, and the
like.
[0029] "Heteroalkyl" refers to a 1 to 12 membered straight or
branched, saturated aliphatic group having one to three heteroatoms
independently selected from nitrogen, oxygen and sulfur. Examples
of heteroalkyl groups include ethylenediamine, hexylamine, and the
like.
[0030] "Cycloalkyl" refers to a 3 to 12 membered fully saturated or
partially unsaturated monocyclic ring system. Examples of
cycloalkyl groups include cyclohexane, cyclopentane, cycloundecane,
and the like.
[0031] "Heterocycloalkyl" refers to a 3 to 7 membered fully
saturated or partially unsaturated monocyclic ring system,
containing one to three ring heteroatoms independently selected
from nitrogen, oxygen and sulfur. Examples of optionally
substituted heterocycloalkyl groups include piperidine,
(C.sub.1-C.sub.4)alkylpiperidine, morpholine, piperazine,
succinimide, and the like.
[0032] "Halogen" and "halo" refer to fluoro, chloro, bromo or
iodo.
[0033] "Cyano" refers to the group --CN.
[0034] "Carbonyl" refers to a divalent --C(O)-- group.
[0035] "Aryl" refers to an aromatic carbocyclic group of from 6 to
18 carbon atoms having a single ring or multiple condensed rings.
The term "aryl" also includes aromatic carbocycle(s) fused to
cycloalkyl or heterocycloalkyl groups. Examples of aryl groups
include phenyl, benzo[d][1,3]dioxole, naphthyl, phenantrenyl, and
the like.
[0036] "Arylene" refers to a disubstituted aryl group as defined
above.
[0037] "Heteroaryl" refers to a 5 to 18 membered monocyclic,
bicyclic or tricyclic heteroaromatic ring system, containing one to
four ring heteroatoms independently selected from nitrogen, oxygen
and sulfur. The term "heteroaryl" also includes heteroaromatic
ring(s) fused to cycloalkyl or heterocycloalkyl groups. Particular
examples of heteroaryl groups include optionally substituted
pyridyl, pyrrolyl, pyrimidinyl, furyl, thienyl, imidazolyl,
oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, pyrazolyl,
1,2,3-triazolyl, 1,2,4-triazolyl, 1,2,3-oxadiazolyl,
1,2,4-oxadiazolyl, 1,2,5-oxadiazolyl, 1,3,4-oxadiazolyl,
1,3,4-triazinyl, 1,2,3-triazinyl, benzofuryl,
[2,3-dihydro]benzofuryl, isobenzofuryl, benzothienyl,
benzotriazolyl, isobenzothienyl, indolyl, isoindolyl, 3H-indolyl,
benzimidazolyl, imidazo[1,2-a]pyridyl, benzothiazolyl,
benzoxa-zolyl, quinolizinyl, quinazolinyl, pthalazinyl,
quinoxalinyl, cinnolinyl, napthyridinyl, pyrido[3,4-b]pyridyl,
pyrido[3,2-b]pyridyl, pyrido[4,3-b]pyridyl, quinolyl, isoquinolyl,
tetrazolyl, 1,2,3,4-tetrahydroquinolyl,
1,2,3,4-tetrahydroisoquinolyl, purinyl, pteridinyl, carbazolyl,
xanthenyl, benzoquinolyl, and the like.
[0038] "Heteroarylene" refers to a disubstituted heteroaryl group
as defined above.
[0039] "Stilbene" refers to a diarylethene and is represented by
the following structural formula:
##STR00006##
In the above structural formula, the use of a wavy line ""
indicates that the substituents R.sup.3 and R.sup.4 can be either
in the E or in the Z configuation.
[0040] "Amino" means --NH.sub.2. "Alkylamine" and "dialkylamine"
mean --NHR and --NR.sub.2, respectively, wherein R is an alkyl
group.
[0041] Suitable substituents for "alkyl", "cycloalkyl", "arylene",
etc., are those which will form a stable compound of the invention.
Examples of suitable substituents are those selected from the group
consisting of hydrogen, halogen, --CN, --OH, --NH.sub.2,
(C.sub.1-C.sub.4)alkyl, (C.sub.1-C.sub.4)haloalkyl,
(C.sub.3-C.sub.7)cycloalkyl, (5-7 membered)heterocycloalkyl,
(C.sub.1-C.sub.4)alkyl(5-7 membered)heterocycloalkyl, (5-7
membered)heterocycloalkyl(C.sub.1-C.sub.4)alkyl,
--NH(C.sub.1-C.sub.6)alkyl, --N((C.sub.1-C.sub.6)alkyl).sub.2,
(C.sub.1-C.sub.6)alkoxyalkyl, (C.sub.1-C.sub.6)alkoxycarbonyl,
--CONH.sub.2, --OCONH.sub.2, --CONH(C.sub.1-C.sub.4)alkyl,
--OCONH(C.sub.1-C.sub.4)alkyl, --CON((C.sub.1-C.sub.4)alkyl).sub.2,
--CO(C.sub.1-C.sub.4)alkyl, --OCO(C.sub.1-C.sub.4)alkyl,
--C(O)O(C.sub.1-C.sub.4)alkyl, --OC(O)O(C.sub.1-C.sub.4)alkyl,
--C(O)H or --CO.sub.2H. For example, suitable substituents can be
selected from the group consisting of hydrogen, methyl, --OMe,
--N((C.sub.1-C.sub.6)alkyl).sub.2, piperidine, morpholine,
piperazine, 1-methylpiperazine, and 2-morpholinoethanamine.
[0042] Pharmaceutically acceptable salts of the compounds disclosed
herein are included in the present invention. For example, an acid
salt of a compound containing an amine or other basic group can be
obtained by reacting the compound with a suitable organic or
inorganic acid, resulting in pharmaceutically acceptable anionic
salt forms. Examples of anionic salts include the acetate,
benzenesulfonate, benzoate, bicarbonate, bitartrate, bromide,
calcium edetate, camsylate, carbonate, chloride, citrate,
dihydrochloride, edetate, edisylate, estolate, esylate, fumarate,
glyceptate, gluconate, glutamate, glycollylarsanilate,
hexylresorcinate, hydrobromide, hydrochloride, hydroxynaphthoate,
iodide, isethionate, lactate, lactobionate, malate, maleate,
mandelate, mesylate, methylsulfate, mucate, napsylate, nitrate,
pamoate, pantothenate, phosphate/diphospate, polygalacturonate,
salicylate, stearate, subacetate, succinate, sulfate, tannate,
tartrate, teoclate, tosylate, triethiodide, and trifluoroacetate
salts.
[0043] Salts of the compounds containing an acidic functional group
can be prepared by reacting with a suitable base. Such a
pharmaceutically acceptable salt can be made with a base which
affords a pharmaceutically acceptable cation, which includes alkali
metal salts (especially sodium and potassium), alkaline earth metal
salts (especially calcium and magnesium), aluminum salts and
ammonium salts, as well as salts made from physiologically
acceptable organic bases such as trimethylamine, triethylamine,
morpholine, pyridine, piperidine, picoline, dicyclohexylamine,
N,N'-dibenzylethylenediamine, 2-hydroxyethylamine,
bis-(2-hydroxyethyl)amine, tri-(2-hydroxyethyl)amine, procaine,
dibenzylpiperidine, dehydroabietylamine,
N,N'-bisdehydroabietylamine, glucamine, N-methylglucamine,
collidine, quinine, quinoline, and basic amino acids such as lysine
and arginine.
[0044] A "fluorescent molecular rotor compound" is a compound with
the ability to form an excited state through the rotation of one
portion of the compound structure with respect to the rest of the
molecule. The emission of the fluorescent molecular rotor compound
depends, for example, on the polarity and the viscosity of the
microenvironment surrounding the compound. The fluorescent
molecular rotor compound fluoresces after photon absorption when
the rotation is constrained, for example, by dye aggegration,
protein binding, and solvent interactions. Emission is blocked in
microenvironments where fluorescent molecular rotor compound
rotates freely.
[0045] The fluorescent molecular rotor compounds of the invention
may possess one or more chiral centers or double bonds and so exist
in a number of stereoisomeric forms. All stereoisomers, racemates,
diastereomers, tautomers, geometric isomers, individual isomers and
mixtures thereof are included in the scope of the present
invention. Racemic compounds may either be separated using
preparative HPLC and a column with a chiral stationary phase or
resolved to yield individual enantiomers utilizing methods known to
those skilled in the art. In addition, chiral intermediate
compounds may be resolved and used to prepare chiral compounds of
the invention.
[0046] Fluorescent molecular rotor compounds suitable for use in
the present invention include, but are not limited to the
fluorescent molecular rotor compound categories and specific
fluorescent molecular rotor compounds listed below and the
fluorescent molecular rotor compounds represented by Formula (I),
Formula (II), and Formula (III).
[0047] As used herein, the phrases "ball milling" or "ball milling
agitation" refer to a process of grinding that uses a container
partially filled with material to be ground combined with a
grinding media that rotates around an axis to reduce particle size
and, if needed, to disperse the particles in a suitable vehicle.
The ball milling process of the invention preferably results in
approximately spherical or ellipsoidal particles.
[0048] As used herein, a "mill speed" refers to the speed of
rotating a container as described in the above paragraph in a ball
milling process. The mill speed at which all grinding media (or
grinding beads) are forced to the internal surface of a ball
milling container is a "critical mill speed," and no desired
grinding occurs at or above critical mill speed. In some
embodiments, the ball milling agitation occurs at a mill speed
expressed as some percentage of critical mill speed, for example at
greater than 30% of critical mill speed. In another embodiment, the
ball milling agitation occurs at about 50% to about 70% of critical
mill speed.
[0049] As used herein, a "grinding media" or "grinding beads"
refers to material, preferably an inert material, used in a balling
milling process. Several parameters determine the performance of a
given material in a ball milling process. Typically, a material is
selected with a crushing strength and a wear resistance, which is
preferably greater than that of the container used in the milling
process. In addition, a suitable grinding material has a higher
density than the material which is to subjected to the grinding
process. For example, the grinding media consists of materials
including ceramics, glass, steel, and agate. For example, the
ceramic grinding material is selected from magnesia stabilized
zirconia, zirconium silicate, zirconia stabilized alumina,
alumina-zirconia composite, burundum, and yttrium stabilized
zirconia. In some embodiments, the grinding media can have a
spherical, a bead, a rod, or a natural shape. In a preferred
embodiment, the grinding media comprises ceramic beads, preferably
yttrium stabilized zirconia beads. In a preferred embodiment, the
grinding media comprises beads with an average diameter in the
range from 0.1 to 25 mm, preferably in the range from 0.5 to 15 mm,
more preferably in the range from 2 mm to 10 mm, even more
preferably in the range from 3 to 8 mm, most preferably in the
range from 4 to 6 mm. In another embodiment, the grinding media
comprises beads having a diameter of 5 mm. Preferably, the grinding
media comprises ceramic beads having a diameter of 5 mm. In some
embodiments, larger bead sizes are preferable over smaller bead
sizes. In particular, it may be preferred in such embodiments to
use bead sizes of more than 2 mm in diameter, preferably from 4 to
10 mm in diameter. Such larger bead sizes may in some embodiments
allow to obtain smaller particle sizes, which may again be
preferable to ensure homogenous particle size distribution.
[0050] As used herein, "size distribution" of particles refers to
the number of particles in a first mixture or in a second mixture
as defined above that fall into each of various size ranges given
as a percentage of the total number of all sizes in the sample of
interest. An important characteristic of the size distribution of
particles is the d(0.95), which is the size, in microns, below
which 95% by volume of the particles are found. Thus, a d(0.95) of
less than 10 microns (.mu.m) means that 95 percent by volume of the
particles in a composition have a diameter less than 10 microns. In
a preferred embodiment of the invention, at least 90%, the d(0.9),
preferably at least 95%, the d(0.95), even more preferably at least
99%, the d(0.99) of the particles in a second mixture or in an
ophthalmic ointment, respectively, have a diameter of less than 10
.mu.m, wherein less than 10%, preferably less than 5%, more
preferably less than 2% of the particles have a diameter of less
than 5 .mu.m.
[0051] As used herein, "median particle diameter" refers to a
particle diameter, with respect to which 50% of the particles have
a smaller diameter and 50% of the particles have a larger diameter
in the sample of interest. For example, a d(0.5) of less than 10
.mu.m means that 50% by volume of the particles in a composition
have a diameter less than 10 .mu.m, which corresponds to a median
particle diameter of less than 10 .mu.m. The median particle
diameter represents the particle diameter in the middle of all
sizes of particles in the sample of interest. In one embodiment,
the median particle diameter is less than 10 .mu.m, preferably less
than 9 .mu.m, more preferably less than 8 .mu.m, even more
preferably less than 7 .mu.m and most preferably less than 6 .mu.m.
Preferably, the median particle diameter is in the range from 2 to
10 .mu.m, more preferably in the range from 3 to 8 .mu.m and even
more preferably from 4 to 7 .mu.m. Most preferably, the median
particle diameter is between 4 and 6 .mu.m. For such preferable
median particle sizes, the size distribution of particles also has
certain values, preferably the d(0.90), more preferably the d(0.95)
value of less than 10 .mu.m.
[0052] As used herein, a "hydrophobic vehicle" includes any vehicle
or mixture of vehicles that are insoluble or have very limited
solubility in water. The hydrophobic vehicle, for example, is
suitable for administration to the eye. In particular, the
hydrophobic vehicle is tolerated by the ocular tissue. A
hydrophobic vehicle is also a non-aqueous medium. For example, the
hydrophobic vehicle is selected from one or more of a petrolatum, a
mineral oil, a polyglycol, a silicone oil, a fluorocarbon, a
lanolin, a gelled mineral oil, and a surfactant.
[0053] A "petrolatum" is a semi-solid mixture of saturated
hydrocarbons obtained from petroleum. For example, a petrolatum
includes petroleum jelly, white petrolatum, white petroleum, yellow
petrolatum, red petrolatum, soft paraffin, paraffin jelly, and
mineral jelly.
[0054] A "mineral oil" is a mixture of C15 to C40 alkanes from a
non-vegetable (mineral) source. For example, a mineral oil is
preferably a paraffinic oil, more preferably a naphthenic oil or
even more preferably an aromatic oil. For example, a mineral oil
includes nujol, light mineral oil, heavy mineral oil, paraffin oil,
and liquid petrolatum. In some embodiments, light mineral oil has a
density of about 0.83 to about 0.86 g/mL at 25.degree. C. In some
embodiments, heavy mineral oil has a density of about 0.875 to
about 0.905 g/mL at 25.degree. C.
[0055] A "polyglycol" is a polymer of ethylene, propylene or
butylenes oxides used as a synthetic lubricant base. For example,
polyglycols include polyethylene glycols (PEGs, for example with a
molecular weight of about 400 to about 4000 g/mol),
methoxypolyethylene glycols (MPEGs), polypropylene glycols (PPGs),
polybutylene glycols (PBGs). PEGs also include for example PEG
derivatives such as polyethylene glycol 40 stearate (PEG-40
stearate).
[0056] A "silicone oil" is a liquid polymerized siloxane with
organic side chains. For example, a polymer that contains units of
the following structural formula:
##STR00007##
with viscosity in the range of about 5 centistokes to about 100,000
centistokes at 25.degree. C. is also referred to as dimethicone and
polydimethylsiloxane (PDMS). For example, a silicone oil includes
methicone (a monomethylated linear siloxane polymer) and
derivatives of linear siloxane polymers of dimethicone and
methicone.
[0057] A "fluorocarbon" is a molecule consisting of carbon atoms,
fluorine atoms, and at least one atom that is not fluorine such as
hydrogen. In certain embodiments, a fluorocarbon is not a
perfluorocarbon, i.e., a fluorocarbon having every hyrdrogen
replaced with a fluorine atom. For example, a fluorocarbon includes
1,1,1,2-tetrafluoroethane (HFA-134a or HCFC-134a or
C.sub.2H.sub.2F.sub.4), 1-chloro-2,2,2-trifluoroethane (HFC-133a or
C.sub.2H.sub.2F.sub.3Cl), 1,1,1,3,3,3-hexafluoropropane (HFA-236fa
or C.sub.3H.sub.2F.sub.6), and 1,1,1,2,3,3,3-heptafluoropropane
(HFC-227ea or C.sub.3HF.sub.7).
[0058] A "lanolin" refers to a waxy substance secreted by
wool-bearing animals and encompasses lanolin derivatives. For
example, lanolin includes lanolin, lanolin wax, lanolin alcohol,
PEG-75 lanolin, acetylated lanolin, hydroxylated lanolin, isopropyl
lanolate, and acetylated lanolin alcohol.
[0059] A "gelled mineral oil" comprises mineral oil and
hydrogenated copolymer to form a gel. Example hydrogenated
copolymers include hydrogenated butylene/ethylene/stryrene
copolymer and hydrogenated ethylene/propylene/styrene copolymer.
Example gelled mineral oils include Plastibase 50 W.TM. and
ViscUp.RTM. 160 (Lonza, Basel, Switzerland).
[0060] As used herein, the term "surfactant" refers to a
surface-active agent or a substance that tends to reduce the
surface tension of the liquid in which it is dissolved. Suitable
surfactants include polysorbates, poloxamers, Triton, sodium
dodecyl sulfate, sodium lauryl sulfate, and betaines. For example,
surfactants include polyoxyethylene (20) sorbitan monolaurate
(Tween.RTM. 20, e.g. from Sigma-Aldrich), polyoxyethylene (20)
sorbitan monopalmitate (Tween.RTM. 40), polyoxyethylene (20)
sorbitan monooleate (Tween.RTM. 80), poloxamer 188,
polyoxyethylene-polyoxypropylene block copolymer (Pluronic.RTM.
F-68, e.g. from Sigma-Aldrich), polyethyleneglycol
660-12-hydroxystearate (Solutol.RTM. HS 15, BASF), cocamidopropyl
betaine, linoleyl betaine, myristyl betaine, cetyl betaine,
polyethoxylated castor oil (Cremophor.RTM., now Kolliphor BASF),
and lecithin.
[0061] The ointments and ophthalmic formulations can be
administered to the subject in conjunction with a fluorescent
molecular rotor compound and an "pharmaceutically acceptable
carrier" as part of a pharmaceutical composition. Pharmaceutically
acceptable carriers may contain inert ingredients which do not
interact with the compound. Standard pharmaceutical formulation
techniques can be employed, such as those described in Remington's
Pharmaceutical Sciences, Mack Publishing Company, Easton, Pa.
Pharmaceutically acceptable carriers for the ophthalmic
formulations include, for example, petrolatum, a mineral oil, a
polyglycol, a silicone oil, a fluorocarbon, a lanolin, a gelled
mineral oil, and a surfactant.
[0062] A "preservative" is a compound which can be added to the
formulation to reduce bacterial activity or undesirable chemical
changes in the formulations. Examples of preservatives include
benzyl alcohol, ethanol, methanol, isopropanol, butyl paraben,
ethyl paraben, methyl paraben, propyl paraben, cathechol,
2-chlorophenol, m-cresol, phenol, resorcinol, xylitol,
2,6-dimethylcyclohexanol, 2-methyl-2,4-pentadiol,
polyvinylpyrrolidone, benzethonium chloride, merthiolate
(thimersosal), benzoic acid, benzalkonium chloride, chlorobutanol,
sodium benzoate, sodium propionate, and cetylpyridinium
chloride.
[0063] As used herein, an "effective amount" refers to an amount of
a fluorescent molecular rotor compound sufficient to diagnose or to
assess risk of the target disorder. Examples of effective amounts
typically range from about 0.0001 mg/kg of body weight to about 500
mg/kg of body weight. A person of ordinary skill will be able to
determine an effective amount based on body weight and the nature
of the fluorescent molecular rotor compound.
[0064] As used herein, the term "subject" refers to a mammal,
preferably a human, but can also mean an animal in need of
veterinary diagnosis or risk assessment, e.g., companion animals
(e.g., dogs, cats, and the like), farm animals (e.g., cows, sheep,
pigs, horses, and the like) and laboratory animals (e.g., rats,
mice, guinea pigs, and the like).
[0065] It has now been discovered that an ointment comprising a
fluorescent molecular rotor compound possesses advantageous
properties (e.g., uniformity of particle content) when it is
manufactured by methods described and claimed herein, wherein these
properties render the ointment particularly suitable for
administration to ocular tissue, preferably for administration to
the lens. For example, ointments of the present invention can be
manufactured by a method comprising: [0066] (a) combining a
fluorescent molecular rotor compound with mineral oil as a
levigating agent, thereby obtaining a first mixture; [0067] (b)
subjecting the first mixture to ball milling agitation with a
grinding media, thereby obtaining a second mixture; and [0068] (c)
combining the second mixture with a hydrophobic vehicle, thereby
obtaining an ointment.
[0069] In some embodiments, the process includes a second mixture
comprising particles of the fluorescent molecular rotor compound,
the particles having a size distribution characterized by a d(0.9)
to a d(0.99) of less than 10 microns. For example, the particles
can have a size distribution characterized by a d(0.95) particle
size from about 1 to about 25 microns. Example embodiments have a
size distribution characterized by a d(0.95) particle size from
about 5 to about 20 microns. For example, the particles can have a
size distribution characterized by a d(0.95) particle size of less
than about 10 microns.
[0070] In one embodiment, the median particle size diameter is less
than 10 .mu.m, preferably less than 9 .mu.m, more preferably less
than 8 .mu.m, even more preferably less than 7 .mu.m and most
preferably less than 6 .mu.m. Preferably, the median particle
diameter in the range from 2 to 10 .mu.m, more preferably in the
range from 3 to 8 .mu.m and even more preferably from 4 to 7 .mu.m.
Most preferably, the median particle diameter is between 4 and 6
.mu.m.
[0071] Particle size of the fluorescent molecular rotor compound
can be determined by laser light diffraction, transmission electron
microscopy, scanning electron microscopy, light microscopy, X-ray
diffractometry and light scattering methods or Coulter counter
analysis (see, for example, "Characterization of Bulk Solids" D.
McGlinchey, Ed., Blackwell Publishing, 2005). For example, the
particle size of a fluorescent molecular rotor compound can be
determined using a laser light diffraction analyzer for particle
size analysis manufactured by Malvern instruments, model
Mastersizer 2000 with wet sampling system manufactured by Malvern,
model number Hydro 2000S. The d(0.95) for all samples can be
calculated using the software provided by Malvern instruments for
the model Mastersizer 2000.
[0072] For example, the grinding media can comprise beads with an
average size or diameter of 0.03, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5,
0.65, 0.8, 1, 1.25, 1.5, 1.75, 2, 2.3, 2.7, 3, 5, 10, 15, 20, and
25 mm. In some embodiments, the process includes grinding media for
ball milling agitation comprises particles with an average size of
at least 3 mm.
[0073] In some embodiments, the grinding media is a ceramic
selected from the group consisting of magnesia stabilized zirconia,
zirconium silicate, zirconia stabilized alumina, alumina-zirconia
composite, burundum, and yttrium stabilized zirconia. Preferably,
the grinding media comprises magnesia stabilized zirconia, more
preferably zirconium silicate, even more preferably zirconia
stabilized alumina, even more preferably alumina-zirconia composite
and most preferably burundum. In some embodiments the grinding
media is yttrium stabilized zirconia such as YTZ.RTM. grinding
media (Tosoh Corp., Tokyo, Japan).
[0074] In some embodiments, the process includes a ratio of the
grinding media volume to the weight of mineral oil and fluorescent
molecular rotor compound ranging from about 0.05 to about 0.7. For
example, 7 mL of YTZ.RTM. grinding media can be mixed with about 10
g of mineral oil and about 0.5 g of fluorescent molecular rotor
compound for a ratio of about 0.67. In some embodiments the ratio
of grinding media volume to mineral oil and fluorescent molecular
rotor compound is about 0.26.
[0075] In one embodiment, the hydrophobic vehicle is selected from
one or more of a petrolatum, a mineral oil, a polyglycol, a
silicone oil, a fluorocarbon, a lanolin, a gelled mineral oil, and
a surfactant. In some embodiments, the hydrophobic vehicle is
selected from one or more of a petrolatum and a mineral oil. For
example, the hydrophobic vehicle can be a combination of heavy
mineral oil and white petroleum. Table 1 below outlines example
vehicle formulations that can be used in the ointments of the
invention. Column 1 denotes a formulation name while columns 2 and
3 outline the amount of components (mg) to be used in every gram of
the formulation. For example, the 80/20 vehicle comprises 80% white
petroleum (800 mg of white petroleum per 1 g of ointment) and 20%
heavy mineral oil (200 mg of white petroleum per 1 g of ointment).
In some embodiments, the ophthalmic ointment comprises Compound 11
at 0.5% (w/w or 5 mg of Compound 11 per g of ointment), 20% heavy
mineral oil (199 mg of white petroleum per 1 g of ointment), and
80% white petroleum (796 mg of white petroleum per 1 g of
ointment).
TABLE-US-00001 TABLE 1 Example ointment preparations of the
invention. Component Conc. Formulation Component (mg/g) 80/20
Vehicle Heavy mineral oil, USP 200 White Petroleum, USP 800 75/25
Vehicle Heavy mineral oil, USP 250 White Petroleum, USP 750 70/30
Vehicle Heavy mineral oil, USP 300 White Petroleum, USP 700 80/20
Ointment with 0.5% Compound 11 5 (w/w) Compound 11 Heavy mineral
oil, USP 199 White Petroleum, USP 796
[0076] In some embodiments, the ointment can be filled into bottles
with a controlled dropper tip, bottles with a natural stream tip, 1
mL syringes, and tubes as containers to dispense the ointment.
[0077] The ointment or ophthalmic formulation can be sterilized by
methods known in the art before administration to subjects. For
example, the ointment can be sterilized by electron beam
irradiation. In some embodiments, the electron beam exposure can be
in the range of 14 kGy to 21 kGy. In preferred embodiments, the
components of the ointment are sterilized before manufacture and
preparation of the ointment by heat or electron beam irradiation.
The process for preparing an ointment can also include
sterilization by irradiating the fluorescent molecular rotor
compound and the mineral oil before forming the first mixture to
undergo ball milling agitation. In some embodiments, the first
mixture can also be sterilized by electron beam irradiation after
combining the fluorescent molecular rotor compound and the mineral
oil but before ball milling agitation. The percent area of the
fluorescent molecular rotor compound in the irradiated samples can
be compared to a non-irradiated sample as determined by the
concentration analysis described in Example 3. Changes in the
relative area of the related substance peak compared to the area of
the fluorescent molecular rotor compound before and after
irradiation can be compared to evaluate degradation of the
fluorescent molecular rotor compound due to formulation and
ointment irradiation.
[0078] For example, processes to prepare ointments of the invention
include an ointment that can be used for diagnostic purposes to
detect and diagnose neurodegenerative diseases. The processes of
the invention can be used to prepare an ointment that can be used
for diagnostic purposes by contacting an ocular tissue of a
subject, e.g., a human subject, with the fluorescent molecular
rotor compound which binds to an amyloid protein or pre-amyloid
protein aggregate. The fluorescent molecular rotor compound
preferentially binds to amyloid proteins compared to other
.beta.-pleated sheet containing proteins. Neurodegenerative
diseases such as amyloidogenic disorders have recently been linked
to the presence or increase in the amount of aggregate in the
supranuclear region and/or cortical lens region of the eye. In a
preferred embodiment of the invention, the ointment is used in
diagnosis or for aiding in a diagnostic method. Such a method may
involve determining the amount of amyloid aggregate in the
supranuclear region and/or cortical lens region of the eye.
Preferably, the ointment, for example, is used in diagnosis of
amyloidogenic disorders such as Alzheimer's Disease (AD), Familial
AD, Sporadic AD, Creutzfeld-Jakob disease, variant Creutzfeld-Jakob
disease, spongiform encephalopathies, Prion diseases (including
scrapie, bovine spongiform encephalopathy, and other veterinary
prionopathies), Parkinson's disease, Huntington's disease (and
trinucleotide repeat diseases), amyotrophic lateral sclerosis,
Down's Syndrome (Trisomy 21), Pick's Disease (Frontotemporal
Dementia), Lewy Body Disease, neurodegeneration with brain iron
accumulation (Hallervorden-Spatz Disease), synucleinopathies
(including Parkinson's disease, multiple system atrophy, dementia
with Lewy Bodies, and others), neuronal intranuclear inclusion
disease, tauopathies (including progressive supranuclear palsy,
corticobasal degeneration, hereditary frontotemporal dementia (with
or without Parkinsonism), and Guam amyotrophic lateral
sclerosis/parkinsonism dementia complex. These disorders may occur
alone or in various combinations. Aggregate analysis is also useful
to detect Transmissible Spongiform Encephalopathies (TSEs), which
are prion-mediated diseases characterized by fatal spongiform
neurodegeneration of the brain and are associated with severe and
fatal neurological signs and symptoms. TSE prionopathies include
Creutzfeld-Jacob Disease (CJD); new variant, Creutzfeld-Jacob
Disease (nv-CJD); Gertsmann-Straussler-Scheinker syndrome; fatal
familial insomnia; Kuru; Alpers Syndrome; Bovine Spongiform
Encephalopathy (BSE); scrapie; and chronic wasting disease
(CWD).
[0079] For example, the ointments and ophthalmic formulations can
utilize amyloid-binding bioavailable lipophilic fluorescent
molecular rotor compounds to detect amyloid peptides in the eye.
Examples of fluorescent molecular rotor compounds that have been
used to analyze brain tissue (but not eye tissue) include X-34 and
{(trans,
trans)-1-bromo-2,5-bis-(3-hydroxycarbonyl-4-hyrdoxy)styrlbenzene
(BSB)} (Styren et al., 2000, J. Histochem., 48:1223-1232; Link et
al., 2001, Neurobiol. Aging, 22:217-226; and Skovronsky et al.,
2000, Proc. Natl., Acad. Sci. U.S.A., 97(13):7609-7614). These
fluorescent molecular rotor compounds emit light in the blue-green
range, thus the level of fluorescence, which is diagnostically
relevant, exceeds the amount of human lens autofluorescence in the
blue-green range. For example, other useful fluorescent molecular
rotor compounds include Me-X04
(1,4-bis(4'-hydroxystyryl)-2-methoxybenzene), Chrysamine or
Chrysamine derivative compounds such as {(trans,
trans)-1-bromo-2,5-bis-(3-hydroxycarbonyl-4-hyrdoxy)styrlbenzene
(BSB)}. Such compounds are described in Mathis et al., Curr. Pharm.
Des., 10(13):1469-93 (2004); U.S. Pat. Nos. 6,417,178; 6,168,776;
6,133,259; and 6,114,175, each of which is hereby incorporated by
reference in its entirety. Nonspecific amyloidphilic fluorescent
molecular rotor compounds such as thioflavin T, thioflavin S or
Congo red dye may also be used. For example, the following
structural formulas may be fluorescent molecular rotor
compounds:
##STR00008##
[0080] In one embodiment, the fluorescent molecular rotor compound
is represented by structural Formula (I), or a pharmaceutically
acceptable salt thereof:
##STR00009## [0081] wherein: [0082] A.sup.1 is an optionally
substituted C6-C18 arylene, an optionally substituted C5-C18
heteroarylene, or is represented by the following structural
formula:
[0082] ##STR00010## [0083] R.sup.1 and R.sup.2 are each
independently hydrogen, optionally substituted C1-C12 alkyl, an
optionally substituted C1-C12 heteroalkyl, optionally substituted
C3-C12 cycloalkyl, or R.sup.1 and R.sup.2 taken together with the
nitrogen atom to which they are attached form an optionally
substituted 3 to 12 membered heterocycloalkyl; [0084] R.sup.3 and
R.sup.4 are each independently hydrogen, methyl, or ethyl; [0085]
R.sup.5 is --OH, optionally substituted --O(C1-C6 alkyl),
--NR.sup.6R.sup.7, or is represented by the following structural
formula:
[0085] ##STR00011## [0086] R.sup.6 and Ware each independently,
hydrogen, methyl, ethyl, or R.sup.6 and R.sup.7 taken together with
the nitrogen atom to which they are attached form a 5 to 7 membered
heterocycloalkyl containing one to three ring heteroatoms
independently selected from N, O, and S; [0087] wherein: [0088] y
is an integer from 1 to 10; [0089] R.sup.8, for each occurrence
independently, is hydrogen, --OH, or --CH.sub.2OH; [0090] R.sup.9
is hydrogen, --NR.sup.10R.sup.11, --C(O)R.sup.12, optionally
substituted C1-C6 alkyl, or optionally substituted C1-C6
heteroalkyl; [0091] R.sup.10, R.sup.11 and R.sup.12 are each
independently hydrogen or C1-C6 alkyl.
[0092] In some embodiments, A.sup.1 is selected from the group
consisting of an optionally substituted phenyl, an optionally
substituted naphthyl, an optionally substituted (E)-stilbene, or an
optionally substituted (Z)-stilbene. In another embodiment, A.sup.1
is an optionally substituted naphthyl. Values and preferred values
of the remainder of the variables are as defined above and below
with respect to Formula (I).
[0093] In a preferred embodiment, the fluorescent molecular rotor
compound, which is used in a process for preparing an opththalmic
ointment is a compound according to structural Formula (II). The
compound of Formula (II) is a compound of Formula (I), wherein
A.sup.1 is represented by the following structural formula:
##STR00012##
and is represented by the following structural Formula (II), or a
pharmaceutically acceptable salt thereof:
##STR00013## [0094] wherein: [0095] R.sup.13 is hydrogen, --OH, or
optionally substituted --O(C1-C6 alkyl). Values and preferred
values of the remainder of the variables are as defined above and
below with respect to Formula (I).
[0096] In a preferred embodiment, the fluorescent molecular rotor
compound, which is used in a process for preparing an opththalmic
ointment is a compound according to structural Formula (III). The
compound of Formula (III) is a compound of Formula (I), wherein
A.sup.1 is represented by the following structural formula:
##STR00014## [0097] and is represented by the following structural
Formula (III), or a pharmaceutically acceptable salt thereof:
[0097] ##STR00015## [0098] wherein: [0099] R.sup.14 and R.sup.15
are each independently hydrogen, --OH, or optionally substituted
--O(C1-C6 alkyl). Values and preferred values of the remainder of
the variables are as defined above and below with respect to
Formula (I).
[0100] In some embodiments, R.sup.1 and R.sup.2 are both optionally
substituted C1-C12 alkyl. In other embodiments, R.sup.1 and R.sup.2
are both selected from the group consisting of methyl, ethyl,
propyl, and butyl. Values and preferred values of the remainder of
the variables are as defined above and below with respect to
Formula (I).
[0101] In some embodiments, R.sup.1 and R.sup.2 taken together with
the nitrogen atom to which they are attached form an optionally
substituted 3 to 12 membered heterocycloalkyl. In another
embodiment, R.sup.1 and R.sup.2 taken together with the nitrogen
atom to which they are attached form heterocycloalkyl selected from
the group consisting of piperidine, morpholine, piperazine, and
1-methylpiperazine. Values and preferred values of the remainder of
the variables are as defined above and below with respect to
Formula (I), Formula (II), or Formula (III).
[0102] In some embodiments, R.sup.5 is
##STR00016##
Values and preferred values of the remainder of the variables are
as defined above and below with respect to Formula (I), Formula
(II), or Formula (III).
[0103] In some embodiments, R.sup.5 is
##STR00017## [0104] y is 1; [0105] R.sup.8 is --CH.sub.2OH; and
[0106] R.sup.9 is --OH. Values and preferred values of the
remainder of the variables are as defined above and below with
respect to Formula (I), Formula (II), or Formula (III).
[0107] In some embodiments, R.sup.5 is
##STR00018## [0108] y is 3; and [0109] R.sup.9 is methyl. Values
and preferred values of the remainder of the variables are as
defined above and below with respect to Formula (I), Formula (II),
or Formula (III).
[0110] In some embodiments, R.sup.5 is
##STR00019## [0111] y is 4; and [0112] R.sup.9 is methyl. Values
and preferred values of the remainder of the variables are as
defined above and below with respect to Formula (I), Formula (II),
or Formula (III).
[0113] In some embodiments, A.sup.1 is selected from the group
consisting of an optionally substituted phenyl, an optionally
substituted naphthyl, an optionally substituted (E)-stilbene, or an
optionally substituted (Z)-stilbene; R.sup.1 and R.sup.2 are both
optionally substituted C1-C12 alkyl; and R.sup.5 is
##STR00020##
Values and preferred values of the remainder of the variables are
as defined above and below with respect to Formula (I).
[0114] In some embodiments, A.sup.1 is selected from the group
consisting of an optionally substituted phenyl, an optionally
substituted naphthyl, an optionally substituted (E)-stilbene, or an
optionally substituted (Z)-stilbene; R.sup.1 and R.sup.2 taken
together with the nitrogen atom to which they are attached form an
optionally substituted 3 to 12 membered heterocycloalkyl; and
R.sup.5 is
##STR00021##
Values and preferred values of the remainder of the variables are
as defined above and below with respect to Formula (I).
[0115] In some embodiments, A.sup.1 is an optionally substituted
phenyl; R.sup.1 and R.sup.2 are both optionally substituted C1-C12
alkyl; and R.sup.5 is
##STR00022##
Values and preferred values of the remainder of the variables are
as defined above and below with respect to Formula (I).
[0116] In some embodiments, A.sup.1 is an optionally substituted
phenyl; R.sup.1 and R.sup.2 taken together with the nitrogen atom
to which they are attached form an optionally substituted 3 to 12
membered heterocycloalkyl; and R.sup.5 is
##STR00023##
Values and preferred values of the remainder of the variables are
as defined above and below with respect to Formula (I).
[0117] In some embodiments, A.sup.1 is an optionally substituted
naphthyl; R.sup.1 and R.sup.2 are both optionally substituted
C1-C12 alkyl; and R.sup.5 is
##STR00024##
Values and preferred values of the remainder of the variables are
as defined above and below with respect to Formula (I).
[0118] In some embodiments, A.sup.1 is an optionally substituted
naphthyl; R.sup.1 and R.sup.2 taken together with the nitrogen atom
to which they are attached form an optionally substituted 3 to 12
membered heterocycloalkyl; and R.sup.5 is
##STR00025##
Values and preferred values of the remainder of the variables are
as defined above and below with respect to Formula (I).
[0119] In some embodiments, the fluorescent molecular rotor
compound is selected from the group consisting of:
##STR00026## ##STR00027##
[0120] In some embodiments, the present invention relates to an
ophthalmic formulation comprising an effective amount of a compound
of the following structural Formula (I), structural Formula (II),
or structural Formula (III), or a pharmaceutically acceptable salt
thereof:
##STR00028## [0121] and a pharmaceutically acceptable carrier.
[0122] The fluorescent molecular rotor compounds of structural
Formula (I) can be synthesized by any methods known to those of
skill in the art. For example, suitable fluorescent molecular rotor
compounds can be synthesized by the methods described in PCT
Publication WO 2011/072257, which is hereby incorporated by
reference in its entirety.
EXEMPLIFICATION
Example 1
Optimization of Ball Milling Agitation Conditions for Mixtures of
Fluorescent Molecular Rotor Compounds in Mineral Oil
[0123] Experiments were conducted to test whether the particle size
of the grinding media used for ball milling agitation affected the
fluorescent molecular rotor compound particle size. Mixtures of
Compound 11 HCl salt in heavy mineral oil were subjected to ball
milling agitation with grinding media YTZ.RTM. beads with an
average diameter of 0.5 mm, 2 mm, and 5 mm.
[0124] Grinding media consisting of 5.0703 g of YTZ.RTM. beads with
an average diameter of 0.5 mm (a yttrium stabilized zirconia
ceramic grinding media, Tosoh Corp., Tokyo, Japan), 0.5043 g of
Compound 11 HCI salt, and 9.9775 g of heavy mineral oil (Spectrum
Chemicals) were added to a 20 mL amber glass bottle. The bottle
containing the mixture was placed on a US Stoneware Ball Mill (SN
CZ-92040) at a speed setting of 25, which corresponds to about 50
rpm, for ball milling agitation for one day. Visual inspection
showed large chunks of Compound 11 salt remaining after 1 day. The
appearance did not change significantly upon continued ball milling
agitation for an additional 3 days (4 days total). Since increasing
the amount of time of ball milling agitation did not affect
Compound 11 particle size, an additional 20.73 g of YTZ.RTM.
grinding media was added to the mixture. The mixture was then
allowed to undergo ball milling agitation for one more day.
Afterwards, most of the large chunks of Compound 11 salt were
gone.
[0125] Further experiments were performed using larger size
YTZ.RTM. grinding media with average diameters of 5 mm and 2 mm
(yttrium stabilized zirconia ceramic grinding media, Tosoh Corp.,
Tokyo, Japan) to evaluate whether increasing the particle size of
the grinding media could decrease the time of ball milling
agitation. A mixture of 10.22 g of YTZ.RTM. beads with an average
diameter of 5 mm (Tosoh Corp., Tokyo, Japan), 0.5048 g of Compound
11 HCI salt and 10.02 g of heavy mineral oil (Spectrum Chemicals)
were added to a 20 mL amber glass bottle before ball milling
agitation using a US Stoneware Ball Mill for three days. A mixture
of 5.09 g of YTZ.RTM. beads with an average diameter of 2 mm (Tosoh
Corp., Tokyo, Japan), 0.5013 g of Compound 11 HCl salt and 9.94 g
of heavy mineral oil (Spectrum Chemicals) were added to a 20 mL
amber glass bottle before ball milling agitation using a US
Stoneware Ball Mill for three days. Visual inspection showed that
the process using 5 mm compared to 2 mm YTZ.RTM. beads as grinding
media produced a more homogeneous mixture after 3 days of ball
milling agitation. Processes using grinding media with larger
average bead diameter (5 mm compared to 2 mm and 0.5 mm) increased
the uniformity of the mixture of heavy mineral oil and Compound 11
particles, which was desirable for first mixtures used to prepare
final ointments of the invention. Also, grinding media with smaller
average bead diameter can create practical problems for handling
such as recovering material from the surface of the beads.
Example 2
Microscopic Examination of Mixture after Ball Milling Agitation to
Evaluate Uniformity
[0126] Microscopic examination of mixtures after ball milling can
be used to assess uniformity and to estimate the particle size of
the fluorescent molecular rotor compounds. Microscopic examination
of the mixtures containing Compound 11 were performed using a
compound optical microscope with a calibrated eyepiece micrometer,
or reticle. For example, the eyepiece micrometer, reticle, can be
aligned and calibrated with the stage micrometer by aligning the
zero line of the reticle with the zero line of the stage
micrometer, and the ratios between the micrometers or scales can be
used to determine sizes and distances. The microscopic analysis
showed predominately fine particles using the 0.5 mm grinding media
beads for the ball milling process after ball milling agitation for
4 days, but the measured concentration for this mixture was
inconsistent with the expected concentration. Microscopic
examination of the mixture of Compound 11 HCl salt in heavy mineral
oil subjected to ball milling agitation with YTZ.RTM. beads with an
average diameter of 5 mm was more homogenous than the process using
YTZ.RTM. beads with an average diameter of 2 mm (Tosoh Corp.,
Tokyo, Japan), which showed a range of sizes of Compound 11
particles. Microscopic examination showed a uniform mixture of
particles approximately 5 microns in diameter, which was within the
target range for fluorescent molecular rotor compound particle
size, with the process using the 5 mm grinding media beads.
Example 3
HPLC Analysis of Concentration of Fluorescent Molecular Rotor
Compounds in Mixtures
[0127] The following HPLC method was used to analyze whether the
concentration of fluorescent molecular rotor compounds in mixtures
of mineral oil and Compound 11 was consistent with the expected
concentration of the mixture. The HPLC analysis was also used to
evaluate degradation of Compound 11 in the mixture. The HPLC
instrument used was a Waters 2695 Separation Module, or equivalent
containing a pump capable of delivering a gradient flow rate of 1.0
mL/min or equivalent and a Waters 2487 Multiwavelength Detector, or
dual wavelength detector capable of detection at 215 nm and 320 nm,
or equivalent. The column used was a Waters Deltapak C18 column, 5
.mu.m, 3.9.times.150 mm, (Waters Corp., Milford, Mass., Catalog
No.: WAT011793). The flow rate was 1.0 mL/min with detection at a
wavelength of 215 nm and a column temperature of 25.degree. C. The
injection volume used for the concentration sample evaluation was
20 .mu.L and for the related substances sample evaluation was 50
.mu.L. The mobile phases were A: 0.1% trifluoroacetic acid in
water, C: methanol, and D: ethanol, and the gradient program shown
in Table 2 was used for the HPLC method.
TABLE-US-00002 TABLE 2 Example mobile phase gradient program. Time
(min) % A % C % D 0 50 50 0 10 50 50 0 12 0 100 0 14 0 0 100 18 0 0
100 20 0 100 0 22 50 50 0 32 50 50 0
[0128] Table 3 shows the sample components found by HPLC analysis
with the approximate retention time (RT) and relative retention
time (RRT), which is calculated as a ratio relative to the
retention time of Compound 11 (8.5 min). In a mixture sample
compared to a standard sample, increased relative peak area of the
related substance peak (RT of 3.8 min, Table 3) compared to the
peak area of Compound 11 (RT of 8.5 min, Table 3) would indicate
degradation of Compound 11. The concentration in the mixture sample
of Compound 11 was determined by analyzing the HPLC peak with a
retention time of 8.5 min (Table 3) compared to a standard sample
of Compound 11.
TABLE-US-00003 TABLE 3 Example retention times (RT) and percentages
of components detected. Component Approximate RT % Detected Related
Substance 3.8 min 0.23% Compound 11 8.5 min 99.77% Unknown 9.2 min
N/A
[0129] The HPLC analysis showed no significant degradation of
Compound 11 based on changes in the related substance peak (RT of
3.8 min, Table 3), which corresponds to a peak that appears after
forced degradation, after ball milling agitation. However, the
concentration of the mixture of Compound 11 and heavy mineral oil
after ball milling agitation with YTZ.RTM. grinding media beads of
an average diameter of 0.5 mm was determined by HPLC (RT of 8.5
min, Table 3) to be 4.75 mg/g as compared to an expected
concentration of 48.12 mg/g (504.3 mg/10.48 g, mineral oil and
Compound 11). The concentration discrepancy indicated that Compound
11 was not uniformly dispersed in the mixture and that ball milling
agitation with 0.5 mm beads provided unacceptable mixtures. The
concentration as measured by HPLC of the mixture of Compound 11 and
heavy mineral oil after ball milling agitation with beads of an
average diameter of 5 mm was 44.9 mg/g as compared to an expected
concentration of 47.98 mg/g (504.8 mg/10.52 g, mineral oil and
Compound 11), which was within the acceptable range. The process of
the invention using ball milling agitation with YTZ.RTM. 5 mm beads
provided a mixture with an acceptable concentration of Compound 11
without degradation of Compound 11 as analyzed by HPLC.
Example 4
Preparation of Ointment Formulations with Fluorescent Molecular
Rotor Compounds
[0130] Based on optimized ratios of vehicle components for ointment
consistency, ointments can be prepared using the claimed process. A
mixture of 10.22 g of YTZ.RTM. beads with an average diameter of 5
mm (Tosoh Corp., Tokyo, Japan), 0.5048 g of Compound 11 HCI salt
and 10.02 g of heavy mineral oil (Spectrum Chemicals) is added to a
20 mL amber glass bottle. The mixture is subjected to ball milling
agitation using a US Stoneware Ball Mill for three days. After
removing the grinding media beads, the mixture is added to a
mixture containing about 10 g of heavy mineral oil and about 79.5 g
of white petroleum. The ointment has a calculated concentration
of:
504.8 mg/(10.02 g+0.5048 g+10 g+80 g)=504.8 mg/100.5248 g or 5.02
mg/g of Compound 11 in the final ointment or 0.5% (w/w) Compound
11.
[0131] The teachings of all patents, published applications and
references cited herein are incorporated by reference in their
entirety.
[0132] While this invention has been particularly shown and
described with references to example embodiments thereof, it will
be understood by those skilled in the art that various changes in
form and details may be made therein without departing from the
scope of the invention encompassed by the appended claims.
* * * * *