U.S. patent application number 17/592700 was filed with the patent office on 2022-05-19 for choline esters.
The applicant listed for this patent is Novartis AG. Invention is credited to Margaret GARNER, William GARNER, David GOODEN, George MINNO.
Application Number | 20220151988 17/592700 |
Document ID | / |
Family ID | |
Filed Date | 2022-05-19 |
United States Patent
Application |
20220151988 |
Kind Code |
A1 |
GARNER; William ; et
al. |
May 19, 2022 |
Choline Esters
Abstract
Compounds, formulations, and methods are provided containing the
choline ester of a reducing agent, especially lipoic acid or
derivatives thereof. The compounds may be administered via a
topical ocular route to treat or prevent oxidative damage.
Inventors: |
GARNER; William; (Eastport,
ME) ; GARNER; Margaret; (Eastport, ME) ;
MINNO; George; (North Andover, MA) ; GOODEN;
David; (Durham, UC) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Novartis AG |
Basel |
|
CH |
|
|
Appl. No.: |
17/592700 |
Filed: |
February 4, 2022 |
Related U.S. Patent Documents
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Application
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Filing Date |
Patent Number |
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16791265 |
Feb 14, 2020 |
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17592700 |
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15837171 |
Dec 11, 2017 |
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16791265 |
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15091389 |
Apr 5, 2016 |
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15837171 |
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14525471 |
Oct 28, 2014 |
9326970 |
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15091389 |
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13851355 |
Mar 27, 2013 |
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14525471 |
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12815526 |
Jun 15, 2010 |
8410162 |
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13851355 |
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61242232 |
Sep 14, 2009 |
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61224930 |
Jul 13, 2009 |
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61187005 |
Jun 15, 2009 |
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International
Class: |
A61K 31/385 20060101
A61K031/385; A61K 9/00 20060101 A61K009/00; A61K 31/21 20060101
A61K031/21; C07D 339/04 20060101 C07D339/04; C07D 343/00 20060101
C07D343/00 |
Claims
1. A compound comprising the choline ester of lipoic acid or a
derivative of lipoic acid.
2. The compound of claim 1, wherein the lipoic acid is alpha lipoic
acid.
3. (canceled)
4. The compound of claim 1, wherein the lipoic acid or derivative
of lipoic acid includes the R enantiomer.
5. A pharmaceutical composition comprising an active agent that is
a reducing agent-choline ester at least one pharmaceutically
acceptable excipient.
6. The pharmaceutical composition of claim 5, wherein the reducing
agent is lipoic acid or a derivative thereof.
7. The pharmaceutical composition of claim 6, wherein the active
agent is lipoic acid choline ester.
8. The pharmaceutical composition of claim 5, wherein the active
agent is present in an amount of about 0.1% to about 10%.
9. (canceled)
10. The pharmaceutical composition of claim 5, wherein the at least
one pharmaceutically acceptable excipient is selected from the
group consisting of a buffer, a tonicity agent, and a viscosity
agent.
11. The pharmaceutical composition of claim 10, wherein the buffer
is a phosphate buffer.
12. The pharmaceutical composition of claim 10, wherein the
viscosity agent is a cellulosic agent.
13. The pharmaceutical composition of claim 5, comprising a
biochemical energy source which is alanine.
14. (canceled)
15. (canceled)
16. (canceled)
17. (canceled)
18. (canceled)
19. A pharmaceutical composition comprising: about 0.25% to about
10% of a reducing agent-choline ester, optionally, about 0.05% to
about 1.0% of a biochemical energy source, about 0.25% to about 1%
buffer, about 0.2% to about 0.6% tonicity agent, and about 0.1% to
about 0.4% viscosity agent.
20. (canceled)
21. (canceled)
22. A method of preventing or treating oxidation damage to cells
comprising administering a pharmaceutical composition of claim
5.
23-26. (canceled)
27. A method for preparing a choline ester comprising: providing a
reducing agent; and reacting the reducing agent with a halogenated
choline to yield a choline ester.
28. The method of claim 27, wherein the reducing agent is lipoic
acid.
29. The method of claim 27, wherein the halogenated choline is
bromocholine bromide.
30. The method of claim 27, wherein the reacting step further
includes a base.
31. (canceled)
32. The method of claim 27 wherein the method is conducted in a
solvent selected from acetone and dimethyl formamide.
33. The method of claim 30, wherein the base is selected from
K.sub.2CO.sub.3, Cs.sub.2CO.sub.3, KF, NaHCO.sub.3 and
KH.sub.2PO.sub.4.
34. The method of claim 30 wherein the base is present in an amount
of about 1 to about 5 equivalents relative to the reducing agent.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit of U.S. Provisional
Application Nos. 61/187,005 filed Jun. 15, 2009, 61/224,930 filed
Jul. 13, 2009, and 61/242,232 filed Sep. 14, 2009, each of which is
incorporated by reference in its entirety.
BACKGROUND OF THE INVENTION
[0002] As we age, our lenses undergo physiological changes that
make it more difficult to focus on near objects. That is why nearly
everyone requires reading glasses, even as early as age 35-40. The
ability of the eye to change focal power, also known as
accommodative amplitude, decreases significantly with age. The
accommodative amplitude is 20 diopters in children and young
adults, but it decreases to 10 diopters by age 25 and to .ltoreq.1
diopter by age 60. The age-related inability to focus on near
objects is called presbyopia. All of us will develop presbyopia and
will use corrective lenses unless a new treatment is found.
[0003] Both presbyopia and cataract are age-related and may share
common etiologies such as lens growth, oxidative stress, and/or
disulfide bond formation.
[0004] There is a need for compositions, formulations and methods
for combating presbyopia and/or cataract, particularly compositions
and methods that minimize toxicity to surrounding healthy
tissues.
BRIEF SUMMARY OF THE INVENTION
[0005] In one embodiment, a compound is provided that is the
choline ester of lipoic acid or a derivative of lipoic acid. In one
embodiment, the lipoic acid is alpha lipoic acid. In another
embodiment, the derivative of lipoic acid is:
6,8-dimercaptooctanoic acid; dihydrolipoate;
5-(1,2-thiaselenolan-5-yl)pentanoic acid; or
5-(1,2-thiaselenolan-3-yl)pentanoic acid. The lipoic acid or
derivative of lipoic acid can include the R enantiomer.
[0006] In another embodiment, a pharmaceutical composition is
provided comprising an active agent that is a reducing
agent-choline ester at least one pharmaceutically acceptable
excipient. In one embodiment, the reducing agent is lipoic acid or
a derivative thereof, e.g., lipoic acid choline ester. The active
agent can be present in an amount of about 0.1% to about 10%, more
specifically about 0.5% to about 10%.
[0007] In one embodiment, the pharmaceutical composition includes a
buffer, a tonicity agent, and/or a viscosity agent. In one
embodiment, the buffer is a phosphate buffer. In another
embodiment, the viscosity agent is a cellulosic agent.
[0008] In one embodiment, the pharmaceutical composition includes a
biochemical energy source, e.g. pyruvate or alanine.
[0009] In one embodiment, the pharmaceutical composition has a pH
of about 4 to about 7.5. In another embodiment, the pharmaceutical
composition has a pH of about 5 to about 6.
[0010] In one embodiment, the pharmaceutical composition is
suitable for topical ocular delivery, e.g., an eye drop.
[0011] In one embodiment, a pharmaceutical composition is provided
that contains: [0012] about 0.25% to about 10% of a reducing
agent-choline ester, [0013] optionally, about 0.05% to about 1.0%
of a biochemical energy source, [0014] about 0.25% to about 1%
buffer, [0015] about 0.2% to about 0.6% tonicity agent, and [0016]
about 0.1% to about 0.4% viscosity agent.
[0017] In another embodiment, a pharmaceutical composition is
provided that contains: [0018] 5% lipoic acid choline ester, [0019]
0.1% ethyl pyruvate, [0020] 0.269% sodium phosphate monobasic
monohydrate, [0021] 0.433% sodium phosphate dibasic anhydrous,
[0022] 0.5% sodium chloride, and [0023] 0.2% hydroxypropyl
methylcellulose.
[0024] In another embodiment, a pharmaceutical composition is
provided that contains: [0025] 5.0% lipoic acid choline ester,
[0026] 0.5% alanine, [0027] 0.269% sodium phosphate monobasic
monohydrate, [0028] 0.433% sodium phosphate dibasic anhydrous,
[0029] 0.5% sodium chloride, and [0030] 0.2% hydroxypropyl
methylcellulose.
[0031] In yet another embodiment, a method of preventing or
treating oxidation damage to cells is provided by administering the
pharmaceutical composition. The method can optionally include
administering a biochemical energy source.
[0032] In one embodiment, the cells are in vivo. In another
embodiment, the cells are ocular cells.
[0033] In one embodiment, administering is administering by topical
ocular delivery.
[0034] In another embodiment, a method is provided for a one-step
synthesis comprising reacting a reducing agent (e.g., lipoic acid)
with a halogenated choline (e.g., bromocholine bromide) to yield a
choline ester.
[0035] In another embodiment, a small portion of the
DHLA-thiolactone can react with low pK lysine protein residues to
form a post-translational acylation product, denoted as
Nepsilon-lipoyl group.
BRIEF DESCRIPTION OF THE DRAWINGS
[0036] FIG. 1 depicts the accommodative amplitude in diopters (D)
of an untreated human lens as a function of age in years. Borja, D
et al. 2008. Optical Power of the Isolated Human Crystalline Lens.
Invest Ophthalmol Vis Sci 49(6):2541-8. Borja et al. calculated the
maximum possible accommodative amplitude of each measured lens
power data point (n=65). As shown, there is good agreement between
the age-dependent loss of accommodation and the maximum amplitude
of accommodation calculated from the isolated lens power.
[0037] FIG. 2 shows a trend graph of the shear modulus versus
position in the lens and age. Weeber, H A et al. 2007. Stiffness
gradient in the crystalline lens. Graefes Arch Clin Exp Ophthalmol
245(9):1357-66. The line at the bottom is the 20-year-old lens; the
line at the top is the 70-year-old lens. The modulus increases with
age for all positions in the lens. Measurements were taken up to
4.0 mm from the lens centre. The lines are extrapolated to a radius
of 4.5 mm (lens diameter 9.0 mm).
[0038] FIG. 3 depicts the average opacity (opacimetry) of an
untreated human lens as a function of age in years. Bonomi, L et
al. 1990. Evaluation of the 701 interzcag lens opacity meter.
Graefe's Arch Clin Exp Ophthalmol 228(5):447-9. Lens opacity was
measured in 73 healthy subjects between 10 and 76 years of age
without slit-lamp evidence of cataract and with a visual acuity of
20/20. These subjects were classified into ten age groups. This
study was carried out using the Interzeag Opacity Meter according
to the procedure described by Flammer and Bebies (Flammer J, Bebie
H. 1987. Lens Opacity Meter a new instrument to quantify lens
opacity. Ophthalmologica 195(2):69-72) and following the
suggestions of the operating manual for the instrument.
[0039] FIG. 4 depicts a scatter plot of the change in .DELTA.D
(micrometers) in the absence (control) and presence of lipoic acid
in lens organ culture experiments. The symbol .dagger-dbl.
designates significantly larger changes in .DELTA.D when compared
to controls. Statistical values are highly significant at
p<0.0001 by unpaired t-test and by Kruskal Wallis test, which
compared medians of each data set. The relative change in Young's
modulus (E) can be calculated as the cubic value derived from the
.DELTA.D of the control divided by the .DELTA.D of the experimental
or E fractional change=(.DELTA.D con/.DELTA.Dexp){circumflex over (
)}3.
[0040] FIG. 5 depicts a scattergram of the percent of the total
protein SH groups in disulfide bonds. Free SH groups were alkylated
with 4-acetamido-4'-maleimidylstilbene-2,2'-sulfonic acid (c, 1
.mu.M, 5 .mu.M, 9.6 .mu.M, 50 .mu.M, 96 .mu.M) or
7-diethylamino-3-(4'maleimidylphenyl)-4-methyl coumarin (500 .mu.M,
and 500 .mu.M c). Following removal of the first alkylating agent,
the S--S bonds were reduced and alkylated with
fluorescein-5-maleimide. Absorption spectra were used to calculated
total protein (A280 nm), free protein SH (A322 or A384), and
protein SS (A490) using the appropriate extinction coefficients.
The symbol .dagger-dbl. indicates statistically significant
difference of mean with mean of control (c, p.ltoreq.0.05). The
symbol ** indicates means of 500 .mu.M lipoic acid and the 500
.mu.M control were significantly different from each other
(p=0.027).
DETAILED DESCRIPTION OF THE INVENTION
[0041] Compounds, formulations, and methods are provided that can
prevent, reduce, reverse, and/or slow the rate of lens growth,
oxidative damage, and/or disulfide bond formation. These compounds,
formulations, and methods may thus effectively prevent or treat
presbyopia and/or cataract.
[0042] The compounds, formulations, and methods described herein
employ an active agent that is the choline ester of a reducing
agent.
Reducing Agents
[0043] The reducing agent is capable of reducing disulfide bonds,
particularly disulfide bond formation in lens membranes and
membrane associated proteins. Accordingly, particularly preferred
reducing agents are capable of entering into the lens epithelial
cells.
[0044] In one embodiment, the reducing agent enters the lens
epithelial cells using a naturally occurring transport mechanism.
For example, lipoic acid enters lens cells via specific plasma
membrane symporters and antiporters. In one embodiment, the
reducing agent is a derivative of lipoic acid that while not
structurally identical to lipoic acid, nevertheless maintains the
capability of utilizing the naturally occurring transport mechanism
for lipoic acid.
[0045] In one embodiment, the reducing agent is lipoic acid or a
derivative thereof. In some embodiments, the reducing agent is
alpha lipoic acid or a derivative thereof. In one embodiment, the
reducing agent is lipoic acid per sc
(5-(1,2-dithiolan-3-yl)pentanoic acid), e.g., alpha lipoic
acid.
[0046] In another embodiment, the reducing agent is a lipoic acid
derivative. Lipoic acid derivatives include, but are not limited
to, 6,8-dimercaptooctanoic acid (dihydrolipoic acid) and
dihydrolipoate. Lipoic acid derivatives also include
seleno-substituted lipoic acid derivatives including, but not
limited to, 5-(1,2-thiaselenolan-5-yl)pentanoic acid and
5-(1,2-thiaselenolan-3-yl)pentanoic acid.
[0047] In another embodiment, the reducing agent can be any of the
reducing agents described in co-pending U.S. patent application
Ser. Nos. 11/946,659, 12/267,260, or 12/390,928.
Choline Esters
[0048] The reducing agent as described above may be provided as a
choline ester. Without being bound by theory, it is believed that
the choline ester may improve the agent's solubility in
pharmaceutical formulations. It may also improve corneal
permeability.
[0049] In one embodiment, the active agent is the choline ester of
lipoic acid. e.g., alpha lipoic acid, or a lipoic acid derivative.
In one embodiment, the active agent is lipoic acid choline ester.
In another embodiment, the active agent is alpha lipoic acid
choline ester.
##STR00001##
The structure may include a counterion, wherein the counterion is
any pharmaceutically acceptable counterion capable of forming a
salt. In yet another embodiment, the active agent is the choline
ester of a lipoic acid derivative.
[0050] Any of the reducing agents can be prepared as a
pharmaceutically acceptable salt. The term "pharmaceutically
acceptable salt" includes salts of the active compounds that are
prepared with relatively nontoxic acids or bases, depending on the
particular substituents found on the compounds described herein.
When compounds of the present invention contain relatively acidic
functionalities, base addition salts can be obtained by contacting
the neutral form of such compounds with a sufficient amount of the
desired base, either neat or in a suitable inert solvent. Examples
of pharmaceutically acceptable base addition salts include sodium,
potassium, calcium, ammonium, organic amino, or magnesium salt, or
a similar salt. When compounds of the present invention contain
relatively basic functionalities, acid addition salts can be
obtained by contacting the neutral form of such compounds with a
sufficient amount of the desired acid, either neat or in a suitable
inert solvent. Examples of pharmaceutically acceptable acid
addition salts include those derived from inorganic acids like
hydrochloric, hydrobromic, nitric, carbonic, monohydrogencarbonic,
phosphoric, monohydrogenphosphoric, dihydrogenphosphoric, sulfuric,
monohydrogensulfuric, hydriodic, or phosphorous acids and the like,
as well as the salts derived from relatively nontoxic organic acids
like acetic, propionic, isobutyric, maleic, malonic, benzoic,
succinic, suberic, fumaric, lactic, mandelic, phthalic,
benzenesulfonic, p-tolylsulfonic, citric, tartaric, oxalic,
methanesulfonic, and the like. Also included are salts of amino
acids such as arginate and the like, and salts of organic acids
like glucuronic or galactunoric acids and the like (see, for
example, Berge et al., "Pharmaceutical Salts", Journal of
Pharmaceutical Science, 1977, 66, 1-19). Certain specific compounds
of the present invention contain both basic and acidic
functionalities that allow the compounds to be converted into
either base or acid addition salts.
[0051] Thus, the compounds of the present invention may exist as
salts, such as with pharmaceutically acceptable acids. The present
invention includes such salts. Examples of such salts include, but
are limited to, hydrochlorides, hydrobromides, sulfates,
methanesulfonates, nitrates, maleates, acetates, citrates,
fumarates, tartrates (e.g., (+)-tartrates, (-)-tartrates, or
mixtures thereof including racemic mixtures), succinates,
benzoates, and salts with amino acids such as glutamic acid. These
salts may be prepared by methods known to those skilled in the
art.
[0052] The neutral forms of the compounds are preferably
regenerated by contacting the salt with a base or acid and
isolating the parent compound in the conventional manner. The
parent form of the compound differs from the various salt forms in
certain physical properties, such as solubility in polar
solvents.
[0053] In one embodiment, the counterion ion is the
1,3-dihydroxy-2-(hydroxymethyl)propan-2-aminium cation (i.e., a
tromethamine salt).
Pharmaceutical Formulations
[0054] The active agent can be combined with one or more
pharmaceutically acceptable excipients to form a pharmaceutical
composition. In the pharmaceutical compositions herein, the active
agent may be present as the choline ester.
[0055] The active agent can be administered as a racemate or as an
enantiomer. Lipoic acid and its derivatives are preferably
administered to include the R form. Synthetic methods to yield a
racemate may be less expensive than stereo-specific processes
including isolation/purification steps. On the other hand,
administering a single enantiomer can lower the therapeutically
effective amount, thus decreasing any toxicity effects of the
active agent.
[0056] As the agents described herein may have therapeutic uses as
described in further detail below, it is preferable to select an
active agent with low toxicity. Additional acceptable lipoic acid
derivatives can be selected by in vitro toxicology testing.
[0057] The amount of the active agent (e.g., the reducing
agent-choline ester) in the pharmaceutical formulation can be
selected based on the condition of the subject to be treated,
including the subject's age, gender, as well as vision and lens
status. Exemplary amounts of the active agent can be about 0.25% to
about 10%, about 0.5% to about 10%, about 1% to about 8%, about 3%
to about 7%, about 2% to about 5%, about 5% to about 7%, or about
5%. In another embodiment, the amount of active agent is less than
about 0.1% (100 mg) or up to about 10% (10000 mg).
[0058] In one embodiment, the pharmaceutical composition is
formulated for ocular use. Ocular formulations include, but are not
limited to, liquid formulations (e.g., solutions, suspensions) for
topical administration as well as formulation for injection or
ocular insert administration. Preferably, the ocular formulation is
formulated for topical administration such as an eye drop, swab,
ointment, gel, or mist (e.g, an aerosol or spray). In one
embodiment, the formulation is an eye drop. For ocular
formulations, the pharmaceutically acceptable excipients are
selected to be compatible with, and suitable for, ocular use. Such
excipients are well known in the art. In one embodiment, excipients
may be selected to improve the solubility of the agent.
[0059] Exemplary excipients include, but are not limited to,
buffers, tonicity agents, viscosity agents, preservatives,
emulsifiers, salts, lubricants, polymers, solvents, and other known
excipients for ocular pharmaceutical formulations. Appropriate
amounts can be determined by one of ordinary skill in the art, but
non-limiting exemplary amounts (in % by weight) are also provided
below.
[0060] In one embodiment, the pharmaceutical composition includes
one or more buffers to adjust or maintain the pH of the
formulation. In one embodiment, the pH is near physiological pH (pH
of tears is about 7). Thus, the pH of the formulation can be about
6 to about 8, about 6.5 to about 7.5, about 6.8 to about 7.2, about
7.1 to about 7.5, or about 7. In another embodiment, the pH is
about 5.5. Thus, the pH of the formulation can be about 4 to about
7, about 4.5 to about 6, about 4.5 to about 5.5, about 5.5 to about
6.5, about 5 to about 6, about 5.25 to about 5.75, or about 5.5.
Exemplary buffers include, but are not limited to, phosphate
buffers (e.g., sodium phosphate monobasic monohydrate, sodium
phosphate dibasic anhydrous), borate buffers, and HBSS (Hank's
Balanced Salt Solution). In one embodiment, the buffer is a
phosphate buffer. In another embodiment, the buffer is sodium
phosphate monobasic monohydrate and/or sodium phosphate dibasic
anhydrous. The buffer amount (amount of either total buffer or a
single buffer excipient) can be 0.1% to about 1.0%, about 0.2% to
about 0.6%, about 0.05% to about 0.5%, about 0.25% to about 0.45%,
or about 0.25%, about 0.43%, or about 0.7%. In one embodiment, the
buffer is about 0.05% to about 0.5% (e.g., about 0.27%) sodium
phosphate monobasic monohydrate and about 0.2% to about 0.6% (e.g.,
about 0.43%) sodium phosphate dibasic anhydrous.
[0061] In one embodiment, the pharmaceutical composition includes
one or more tonicity agents. Although the formulation may be
hypertonic or hypotonic, isotonic formulations are preferred
(260-320 mOsm). Exemplary tonicity agents include, but are not
limited to, sodium chloride. The tonicity agent amount can be about
0.1% to about 5%, about 0.1% to about 2%, about 0.1% to about 1%,
about 0.25% to about 0.75%, about 0.2% to about 0.6%, or about
0.5%. In one embodiment, the tonicity agent is about 0.2% to about
0.6% (e.g., about 0.5%) sodium chloride.
[0062] In one embodiment the pharmaceutical composition includes
one or more viscosity agents to increase the viscosity of the
formulation. Exemplary viscosity agents include, but are not
limited to, cellulosic agents (e.g., hydroxypropyl
methylcellulose), polycarbophil, polyvinyl alcohol. In one
embodiment, the viscosity agent is a cellulosic agent, e.g.,
hydroxypropyl methylcellulose. The viscosity agent amount can be
about 0.1% to about 5%, about 0.1% to about 2%, about 0.1% to about
1%, about 0.1% to about 0.4%, or about 0.2%. In one embodiment, the
viscosity agent is about 0.1% to about 0.4% (e.g., about 0.2%)
hydroxypropyl methylcellulose.
[0063] In one embodiment, the pharmaceutical composition includes
one or more preservatives to minimize microbial contamination or
enhance shelf life. Exemplary preservatives include, but are not
limited to, benzalkonium chloride (BAK), cetrimonium,
chlorobutanol, edetate disodium (EDTA), polyquaternium-1
(Polyquad.RTM.), polyhexamethylene biguanide (PHMB), stabilized
oxychloro complex (PURITE.RTM.), sodium perborate, and SofZia.RTM..
The preservative amount may be, e.g., less than about 0.02%, about
0.004% or less, or about 0.005% to about 0.01%.
[0064] In one embodiment, the pharmaceutical composition includes
one or more stabilizers. Exemplary stabilizers include, but are not
limited to amino acids such as alanine. The stabilizer amount can
be about 0.1% to about 5%, about 0.1% to about 2%, about 0.1% to
about 1%, about 0.25% to about 0.75%, about 0.2% to about 0.6%, or
about 0.5%. In one embodiment, the stabilizer is about 0.2% to
about 0.6% (e.g., about 0.5%) alanine.
[0065] In one embodiment, the pharmaceutical composition includes
one or more emulsifiers. Exemplary emulsifiers include, but are not
limited to, Polysorbate 80.
[0066] The compounds described herein can be used in combination
with one another, with other active agents known to be useful in
ocular disease, or with adjunctive agents that may not be effective
alone, but may contribute to the efficacy of the active agent. For
example, adjunctive agents might include one or more amino acids or
choline (separate from the lipoic acid compound) to enhance the
efficacy of the active agent. The combinations can be advantageous,
e.g., in reducing metabolic degradation.
[0067] The term "co-administer" means to administer more than one
active agent, such that the duration of physiological effect of one
active agent overlaps with the physiological effect of a second
active agent. In some embodiments, co-administration includes
administering one active agent within 0.5, 1, 2, 4, 6, 8, 10, 12,
16, 20, or 24 hours of a second active agent. Co-administration
includes administering two active agents simultaneously,
approximately simultaneously (e.g., within about 1, 5, 10, 15, 20,
or 30 minutes of each other), or sequentially in any order. In some
embodiments, co-administration can be accomplished by
co-formulation, i.e., preparing a single pharmaceutical composition
including both active agents. In other embodiments, the active
agents can be formulated separately. In another embodiment, the
active and/or adjunctive agents may be linked or conjugated to one
another.
[0068] Without being bound by theory, it is believed that the
administration of an active agent, e.g., lipoic acid or a
derivative thereof, and an adjunctive agent such as choline, can be
particularly advantageous in a conjugated form. The conjugate
compound be applied to the cornea, and penetration is achieved due
to the bi-phasic (water and lipid soluble) nature of the conjugate
compound. As the conjugate goes through the cornea, naturally
present esterases (enzymes) separate lipoic acid from choline. The
lipoic acid (now a pro-drug) in the aqueous bathes the lens and
enters the lens epithelial cells (due to low molecular weight and
size), and there is reduced by any one of several oxido-reductases
(enzymes such as thioredoxin and thioltransferase) to form
dihydrolipoic acid. Dihydrolipoic acid now has two extra hydrogen
atoms to donate to a disulfide complex (e.g., protein disulfide
PSSP), separating the two sulfur atoms into sulfhydril molecules
(e.g., protein cysteine residues PSH with free SH groups) thus
breaking the inter-cytosol protein cross-links. Breaking these
cross-link is what reduces the lens stiffness. Once donation of the
hydrogen atoms to the sulfur atom, the dihydrolipoic acid becomes
lipoic acid and is available for recycling in the cell to become
dihydrolipoic acid or converted to a natural degraded by product
thiolactone and excreted.
[0069] In one embodiment, a reducing agent, such as one of the
compounds described herein, is co-administered with a biochemical
energy source. A biochemical energy source facilitates reduction by
participating as an intermediate of energy metabolic pathways,
particularly the glucose metabolic pathway. Exemplary intermediates
of this pathway are depicted by, e.g., Zwingmann, C. et al. 2001.
13C Isotopomer Analysis of Glucose and Alanine Metabolism Reveals
Cytosolic Pyruvate Compartmentation as Part of Energy Metabolism in
Astrocytes. GLIA 34:200-212. Exemplary biochemical energy sources
include, e.g., glucose or a portion thereof (e.g.,
glucose-6-phosphate (G6P)), pyruvate (e.g., ethyl pyruvate), NADPH,
lactate or derivative thereof. G6P may be favored over glucose
since a formulation including glucose may further benefit from the
addition of preservatives. In one embodiment, the biochemical
energy source is an intermediate in a cytosolic metabolic pathway.
Exemplary cytosolic pathway intermediates include, e.g., glucose,
pyruvate, lactate, alanine, glutamate, and 2-oxoglutarate. In
another embodiment, the biochemical energy source is an
intermediate in a mitochondrial metabolic pathway. Exemplary
mitochondrial pathway intermediates include, e.g., pyruvate,
TCA-cycle intermediates, 2-oxoglutarate, glutamate, and glutamine.
In one embodiment, the biochemical energy source is pyruvate
compound (e.g., ethyl pyruvate). In another embodiment, the
biochemical energy source is alaninc. The amount of a biochemical
energy source can be, e.g., about 0.05% to about 1.0%. In one
embodiment, the energy source is 0.1% ethyl pyruvate.
[0070] In one embodiment, the agent is co-administered with
glucose-6-phosphate (G6P), NADPH, or glucose. In one embodiment,
the agent is activated by an endogenous chemical energy, e.g.,
endogenous glucose. For example, endogenous glucose can activate
lipoic acid or a derivative thereof to dihydrolipoic acid (DHLA) or
a corresponding derivative thereof.
[0071] In one embodiment, the pharmaceutical formulation includes a
reducing agent-choline ester as the active agent and one or more
pharmaceutical excipients selected from the group consisting of
buffers, tonicity agents, and viscosity agents.
[0072] The pharmaceutical formulation may be packaged for
administration by any means known in the art including, but not
limited to, individual dose units or multi-dose units, e.g.,
dropper bottles. Multi-dose units may include, for example, about 1
mL to about 100 mL, about 1 mL to about 50 mL, about 1 mL to about
10 mL, about 2 mL to about 7 mL, or about 5 mL. An individual dose
may be, e.g., 1-10 drops, 1-5 drops, or 2-3 drops, wherein each
drop is about 5 to about 50 .mu.l, about 10 to about 30 .mu.l, or
about 20 .mu.l. Depending on the active agent concentration and the
condition of the patient, doses may be administered, for example,
1-4, preferably 1-2 times per day.
Methods of Synthesis
[0073] Although choline esters may be prepared via a multi-step
process as depicted in Example 3, in one embodiment, a one-step
method of synthesis for the choline esters is provided. The method
comprises: providing a reducing agent as described above, reacting
the reducing agent with a halogenated choline to yield a choline
ester of the reducing agent. In one embodiment, the halogenated
choline is bromocholine bromide as follows:
##STR00002##
[0074] In some embodiments, the reaction is conducted in a solvent,
such as acetone or dimethyl formamide (DMF).
[0075] In one embodiment, the reaction mixture further includes a
base. Exemplary bases include, but are not limited to,
K.sub.2CO.sub.3, Cs.sub.2CO.sub.3, KF, NaHCO.sub.3, and
KH.sub.2PO.sub.4. The base can be present in an amount of about 1
to about 5 equivalents relative to the reducing agent. In some
embodiments, the base amount is about 1 eq.
Methods of Treating Oxidation Damage
[0076] The agents described herein can be employed in a method
including the step of providing a reducing agent-choline ester
active agent to a cell, either in vitro or in vivo.
[0077] The active agents described herein can be employed in a
method for treating or preventing oxidation damage to cells. Such a
method includes the step of administering a pharmaceutical
composition comprising a reducing agent-choline ester active agent
to a cell, either in vitro or in vivo.
[0078] As stated above, the agents can be delivered to cells in
vitro or in vivo. In one embodiment, the cells are in vivo. In
either case, the cells can be ocular cells, e.g., lens cells. In
one embodiment, the agent is delivered to a lens, either in vitro
or in vivo. In one embodiment, the compounds described herein can
be used in a method for treating ocular disease. Exemplary ocular
diseases include, but are not limited to: presbyopia, cataract,
macular degeneration (including age-related macular degeneration),
retinopathies (including diabetic retinopathy), glaucoma, and
ocular inflammations. In one embodiment, the ocular disease to be
treated is cataract. In another embodiment, the ocular disease to
be treated is treat presbyopia. Because oxidative damage has been
implicated in other disorders including cancer, the agents may
prove useful for administration to any type of cell exhibiting or
prone to oxidative damage.
[0079] The methods preferably utilize a therapeutically effective
amount of the active agent. The term "therapeutically effective
amount" means an amount that is capable of preventing, reducing,
reversing, and/or slowing the rate of oxidative damage. For ocular
applications, a therapeutically effective amount may be determined
by measuring clinical outcomes including, but not limited to, the
elasticity, stiffness, viscosity, density, or opacity of a
lens.
[0080] Lens elasticity decreases with age, and is a primary
diagnostic and causative factor for presbyopia. Lens elasticity can
be measured as accommodative amplitude in diopters (D). FIG. 1
depicts the average elasticity in diopters of an untreated human
lens as a function of age in years. The lower the value of D, the
less elastic the lens. In one embodiment, the agents described
herein (in the active form) can decrease and/or maintain D at a
value that is greater than the D value exhibited by an untreated
lens of about the same age. In other words, the agents can keep
accommodative amplitude "above the line" (the solid line mean
accommodative amplitude) depicted in FIG. 1. In one embodiment, D
is increased and/or maintained at a value about 2, 5, 7, 10, 15,
25, 50, 100, 150, or 200 percent above the line. However, as
individual lenses may differ with respect to average values,
another embodiment provides any increase in elasticity, maintenance
of elasticity, or reduction in the rate of decline of elasticity
(i.e., reduction in the rate of decrease in diopters) for an
individual lens compared to the elasticity of the same lens before
treatment. In another embodiment, the methods provide an objective
increase in elasticity of at least about 0.1, 0.2, 0.5, 1, 1.2,
1.5, 1.8, 2, 2.5, 3, or 5 diopters.
[0081] Lens elasticity can also be measured by the unit of
elasticity E. The higher the value of E, the less elastic the lens.
FIG. 2 depicts the average elasticity (E) of an untreated human
lens as a function of age in years. In one embodiment, the agents
described herein (in the active form) can decrease and/or maintain
E at a value that is less than the E value exhibited by an
untreated lens of about the same age. In other words, the agents
can keep lens elasticity "below the line" depicted in FIG. 2. In
one embodiment, E is decreased and/or maintained at a value about
2, 5, 7, 10, 15, 25, 50, 100, 150, or 200 percent below the line.
However, as individual lenses may differ with respect to average
values, another embodiment provides any increase inelasticity,
maintenance of elasticity, or reduction in the rate of decline of
elasticity (i.e., reduction in the rate of increase in E value) for
an individual lens compared to the elasticity of the same lens
before treatment.
[0082] Therapeutic efficacy can also be measured in terms of lens
opacity. Lens opacity increases with age and is a primary
diagnostic and causative factor for cataract. FIG. 3 depicts the
average opacity of an untreated human lens as a function of age in
years. In one embodiment, the agents described herein (in the
active form) can decrease and/or maintain opacity at a value that
is less than the opacity value exhibited by an untreated lens of
about the same age. In other words, the agents can keep lens
opacity "below the line" depicted in FIG. 3. In one embodiment,
lens elasticity is decreased and/or maintained at a value about 2,
5, 7, 10, 15, 25, 50, 100, 150, or 200 percent below the line.
However, as individual lenses may differ with respect to average
values, another embodiment provides any decrease, maintenance, or
reduction in the rate of increase of opacity for an individual lens
compared to the opacity of the same lens before treatment.
[0083] Some agents described herein exist naturally in the
untreated eye. Lipoic acid, for example, occurs naturally in eye
tissue. In general, a therapeutically effective amount of the
exogenously administered agent is often at least about 1 or 2
orders of magnitude larger than the natural level of the compound.
In one embodiment, the bioavailable to the lens dose amount of
lipoic acid or a derivative thereof is about 5 .mu.M to about 250
.mu.M or about 10 .mu.M to about 700 .mu.M. The dose amount will
depend on the route of administration as well as the age and
condition of the patient. Similarly, the frequency of dosing will
depend on similar factors as can be determined by one of ordinary
skill in the art.
[0084] Efficacy has been demonstrated in vitro for specific
exemplary dosing. (See Example 2) FIG. 2 shows that the
inelasticity increases by a factor of nearly 20 during the critical
period from age 40 to 55 years. From current data, a 10 .mu.M dose
can decrease the inelasticity over 95% within a millimeter volume
element (voxel). Extrapolation of these results to a volume element
in the human lens suggests that using this treatment dose on a 55
year old person with a 10 kPA lens starting modulus value (see FIG.
2) could be reduced after treatment to a value of about 0.5 kPA
(which then corresponds to a value typically seen with a 40 yr old
person). FIG. 1 permits a conversion of these modulus values to
optical amplitude: accommodative amplitude is normally reduced to
almost 0 above 55 years, while a person at 40-45 years still
exhibits around 4-5 diopters of accommodation.
[0085] This method includes the description of a topical ocular
formulation that will be used to administer one to two drops of the
active agent(s) to the cornea. The formulation will be devised such
to provide sufficient active agent and effect treatment to the
lens. The mechanism of treatment employs using the intrinsic
cellular energy to reduce the active agent lipoate-[S--S] (actually
a pro-drug) to dihydrolipoate [DHLA-(SH).sub.2] (the reduced active
agent). DHLA is then used to reduce protein disulfide bonds and
alter the lens material properties of the lens to restore
accommodative amplitude. The activation of the active agent lipoate
to DHLA is enzymatically formed with endogenous intracellular
oxido-reductase, including such enzymes as thioredoxin, lipoamaide
dehydrogenase, and glutathione reductase. These enzymes use
endogenous NADPH to affect the redox couple and recycle lipoate to
the reduced form: DHLA. DHLA to can however undergo additional
metabolism within the lens to produce a number of other products,
including 7-(2-mercaptoethyl)thiepan-2-one (henceforth referred to
as "DHLA-thiolactone"). A small portion of the DHLA-thiolactone can
react with low pK lysine protein residues to form a
post-translational acylation product, denoted as Nepsilon-lipoyl
group. This later post-translation product is normally localized in
the mitochondrial system and is important with the pyruvate
dehydrogenase-acetyltransferase activity. Any excess
DHLA-thiolactone is released into the aqueous along with DHLA
itself and other byproducts. At 15 minutes to 2 hours after topical
dosing, the amount of DHLA-thiolactone measured in the aqueous
ranges from 10 micro molar levels to 700 micro molar levels.
[0086] The methods include preventative methods that can be
performed on patients of any age. The methods also include
therapeutic methods that can be performed on patients of any age,
particularly patients that are 20, 25, 30, 35, 40, 45, 50, 52, 55,
57, 60, 70, 75, or 80 years of age or older.
[0087] Any numerical values recited herein include all values from
the lower value to the upper value in increments of any measurable
degree of precision. For example, if the value of a variable such
as age, amount, time, percent increase/decrease and the like is 1
to 90, specifically from 20 to 80, and more specifically from 30 to
70, it is intended that values such as 15 to 85, 22 to 68, 43 to
51, 30.3 to 32, etc., are expressly enumerated in this
specification. In other words, all possible combinations of
numerical values between the lowest value and the highest value
enumerated are to be considered to be expressly stated in this
application in a similar manner.
EXAMPLES
Example 1: In Vitro Toxicology Studies
[0088] Cell viability was determined using human umbilical vein
endothelial cells (HUVEC, first passage). Cells were treated with
the active agent in doses ranging from 0.1 .mu.M to 100 .mu.M. The
number of live and dead cells was determined using the
MultiTox-Fluor assay (Promega) or Live/Dead.RTM. assay
(Invitrogen). Logistic plots were used to determine the compound's
LD.sub.50 value. Lipoic acid was not cytotoxic in the concentration
range.
Example 2: In Vitro Efficacy Studies
[0089] Increase in Elasticity: Pairs of mouse lenses were incubated
in medium 200 supplemented with an antibiotic, an antimycotic, in
the presence or absence of lipoic acid (concentrations ranging from
0.5 .mu.M to 500 .mu.M) for 8-15 hours. Each lens was removed from
medium, weighed, and photographed on a micrometer scale. A
coverslip of known weight (0.17899.+-.0.00200 g) was placed on the
lens, and the lens was photographed again on the micrometer scale.
The diameter of each lens with and without the coverslip was
determined from the photographs. The change in lens diameter
produced by the force (coverslip) was computed
.DELTA.D=(D.sub.withcoverslip-D.sub.withoutcoverslip). The results
(FIG. 4, .dagger-dbl.) indicate that lipoic acid at concentrations
.gtoreq.9.6 .mu.M caused a statistically significant increase in
.DELTA.D, p<0.0001.
[0090] Decrease in disulfide bonds: Lipoic acid at concentrations
.gtoreq.9.6 .mu.M caused a statistically significant decrease in
protein disulfides in the mouse lenses where there was a
significant increase in .DELTA.D (FIG. 4). Mouse lenses were
homogenized in a denaturing buffer containing a fluorescent
alkylating agent to modify the free SH groups. After removing the
alkylating agent homogenates were reduced and alkylated with a
different fluorescent alkylating agent. Absorption spectra of the
modified proteins were used to calculate free protein SH and
protein SS groups. The results are shown in FIG. 5.
Example 3: Syntheses of Lipoic Acid Choline Ester
[0091] Lipoic acid choline ester was prepared according to the
following synthetic route. Choline salts of alternative reducing
agents can be similarly prepared by making the appropriate reagents
substitutions. Also, one of ordinary skill in the art would
recognize that these syntheses are provided as guidance and that
reagents, conditions, amounts, temperatures, and the like may be
modified without departing from the general synthetic pathway.
[0092] Step 1
##STR00003##
(R)-2-(dimethylamino)ethyl 5-(1,2-dithiolan-3-yl)pentanoate
[0093] A solution of DCC (11 g, 53 mmol) in anhydrous
CH.sub.2Cl.sub.2 (20 mL) was added with stirring over 10-20 minutes
to a cold (0.degree. C.) solution of R-lipoic acid (10.0 g, 48.5
mmol), N,N-dimethylethanolamine (14.5 mL, 145 mmol, 3 eq.), and
DMAP (600 mg, 4.9 mmol) in anhydrous CH.sub.2Cl.sub.2 (50 mL).
Following complete addition, the cold bath was removed. After 18
hours at room temperature, all volatiles were removed under reduced
pressure, and the resulting residue was purified by flash column
chromatography (SiO.sub.2, 2% MeOH in CH.sub.2Cl.sub.2) providing
the desired product as a clear yellow oil (10.6 g, 79%). All data
consistent with values reported in the literature. (See Courvoisier
C. et al. 2006. Synthesis and effects of 3-methylthiopropanoyl
thiolesters of lipoic acid, methional metabolite mimics. Bioorganic
Chemistry 34(1):49-58.)
[0094] Step 2:
##STR00004##
(R)-2-(5-(1,2-dithiolan-3-yl)pentanoyloxy)-N,N,N-(trimethyl)ethylammonium
Iodide
[0095] Methyl iodide (0.55 mL, 9.0 mmol) was added to a solution of
the amine (2.5 g, 9.0 mmol) in anhydrous CH.sub.2Cl.sub.2 (20 mL).
The reaction mixture was stirred overnight and slowly poured into
diethyl ether (250 mL) with vigorous stirring. The choline salt was
isolated by filtration as a free-flowing pale, yellow sold (3.7 g,
98%).
Example 4: One-Step Synthetic Route
##STR00005##
[0096] Example 5: Eye Drop Formulation of Lipoic Acid Choline
Ester
[0097] The following eye drop formulation was prepared using lipoic
acid choline ester as the active agent.
TABLE-US-00001 Formula A Concentration Ingredient % by weight
Purpose Lipoic acid choline ester 5.0 Active agent Ethyl pyruvate
0.1 Energy source Sodium phosphate 0.269 Buffer monobasic
monohydrate, USP Sodium phosphate 0.433 Buffer dibasic anhydrous,
USP Sodium chloride 0.5 Tonicity agent Hydroxypropylmethylcellulose
0.2 Viscosity agent (HPMC), USP De-ionized, pyrogen free water to
100 mL Solvent
TABLE-US-00002 Formula B Concentration Ingredient % by wieght
Purpose Lipoic acid choline ester 5.0 Active agent Alanine 0.5
Stabilizer Sodium phosphate 0.269 Buffer monobasic monohydrate, USP
Sodium phosphate 0.433 Buffer dibasic anhydrous, USP Sodium
chloride 0.5 Tonicity agent Hydroxypropylmethylcellulose 0.2
Viscosity agent (HPMC), USP De-ionized, pyrogen free water to 100
mL Solvent
[0098] The eye drop formulation has a pH of 7.0.
[0099] The pharmaceutical formulation may be diluted to 100 ml
filtered water (e.g., Millex syringe filter (0.45 micron 33 mm).
The pharmaceutical composition may be packaged for multi-dose
administration, e.g., 2-7 mL (e.g., 5 mL) eyedropper bottle with
screw lid dropper.
[0100] The examples given above are merely illustrative and are not
meant to be an exhaustive list of all possible embodiments,
applications, or modifications of the invention. Thus, various
modifications and variations of the described methods and systems
of the invention will be apparent to those skilled in the art
without departing from the scope and spirit of the invention.
Although the invention has been described in connection with
specific embodiments, it should be understood that the invention as
claimed should not be unduly limited to such specific embodiments.
Indeed, various modifications of the described modes of carrying
out the invention which are obvious to those skilled in the
chemical arts or in the relevant fields are intended to be within
the scope of the appended claims.
[0101] The disclosures of all references and publications cited
above are expressly incorporated by reference in their entireties
to the same extent as if each were incorporated by reference
individually.
* * * * *