NZ713007B2 - Methods of inhibiting cataracts and presbyopia - Google Patents
Methods of inhibiting cataracts and presbyopia Download PDFInfo
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- NZ713007B2 NZ713007B2 NZ713007A NZ71300714A NZ713007B2 NZ 713007 B2 NZ713007 B2 NZ 713007B2 NZ 713007 A NZ713007 A NZ 713007A NZ 71300714 A NZ71300714 A NZ 71300714A NZ 713007 B2 NZ713007 B2 NZ 713007B2
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Abstract
Described herein are methods of inhibiting or reversing the progression of cataract formation or presbyopia in an eye by administering a ?-crystallin charge masking agent of the formulae shown. Both presbyopia and cataracts are caused by aggregation of the soluble crystalline lens proteins called the crystallins. e crystallins.
Description
METHODS OF INHIBITING CATARACTS AND PRESBYOPIA
FIELD OF THE DISCLOSURE
The present disclosure relates to methods of inhibiting or reversing the
progression of age related changes in the crystalline lens of an eye.
BACKGROUND
The crystalline lens of the eye is a transparent structure that is suspended
immediately behind the iris, which brings rays of light to a focus on the retina. The lens
contains both soluble and insoluble proteins; together they constitute 35 percent of the wet
weight of the lens. In a young, healthy lens, the soluble proteins, commonly referred to as
crystallins, constitute 90 percent of the lens proteins. During the aging process, the lens
crystallins form insoluble aggregates, which, at least in part, account for the decreased
deformability of the lens nucleus, which characterizes presbyopia, the loss of the eye’s ability
to change focus to see near objects. The formation of insoluble aggregates of lens crystallins
in presbyopia is believed to be an early stage in the formation of age-related cataracts.
Cataracts are defined by cloudiness or opacification in the crystalline lens of
the eye. As an individual ages, cataracts form as the crystallins present in the lens are
converted into aggregates, resulting in increased lens opacity. Specifically, there is a
progressive decrease in the concentration of the soluble chaperone, α-crystallin, in human
lens nuclei with age, as it becomes incorporated into high molecular weight aggregates and
insoluble protein. The presence of aggregates compromises the health and function of the
lens and left untreated, cataracts can lead to substantial vision loss or even blindness.
Presently, the most common treatment for cataracts is surgery.
Crystallins are structural proteins most highly expressed in the lens fiber cells
of the vertebrate eye. The crystallins are divided into two subfamilies: the α-crystallins (αA
and αB) which are members of the small heat shock protein superfamily, also functioning as
structural proteins and molecular chaperones; and the evolutionarily-linked superfamily of β-
and γ-crystallins which function primarily as structural proteins in the lens, and contribute to
the transparency and refractive properties of lens structure. In addition to their role in
cataract development, αA-crystallin and αB-crystallin have been implicated in
neurodegenerative diseases, like Alexander’s disease, Creutzfeldt-Jacob disease, Alzheimer’s
disease and Parkinson’s disease.
U.S. Patent Application 2008/0227700 describes deaggregation of proteins
using peptides having chaperone activities as a therapeutic treatment. Specifically, αB
peptides were used to deaggregate pH-induced aggregates of β-crystallin as measured by light
scattering. Provision of a continuous supply of alpha crystallins into the lens is a challenge.
What is needed are alternative methods suitable for the deaggregation of crystallins for the
inhibition and/or reversal of cataracts and presbyopia.
SUMMARY
In one aspect, a method of inhibiting or reversing the progression of cataract
formation or presbyopia in an eye comprises contacting the eye with an effective cataract or
presbyopia-inhibiting amount of an ophthalmic composition comprising at least one γ-
crystallin charge masking agent, wherein the charge masking agent is not a polypeptide.
In another aspect, an ophthalmic composition comprises a bifunctional
molecule containing a leaving group covalently linked to a molecular bristle.
In another aspect, a method of inhibiting or reversing the progression of age
related degeneration of a crystalline lens in an eye comprises contacting the eye with an
effective degeneration-inhibiting amount of an ophthalmic composition comprising at least
one γ-crystallin charge masking agent, wherein the γ-crystallin charge masking agent is not a
polypeptide.
In yet another aspect, a method of treating a disease relating to protein folding
in a patient in need thereof comprises administering a therapeutically effective amount of a
bifunctional molecule containing a leaving group covalently linked to a molecular bristle.
In a further aspect, use of an ophthalmic composition comprising at least one
γ-crystallin charge masking agent in the manufacture of a medicament for inhibiting or
reversing the progression of cataract formation, presbyopia, or age related degeneration of a
crystalline lens in an eye, wherein the γ-crystallin charge masking agent is a bifunctional
molecule containing a reactive group covalently linked to a molecular bristle,
wherein the reactive group is NH , a succinimide, a carboxylic acid, isocyanate,
isothiocyanate, sulfonyl chloride, aldehyde, carbodiimide, acyl azide, anhydride,
fluorobenzene, carbonate, N-hydroxysuccinimide ester, imidoester, epoxide and fluorophenyl
ester, and
wherein the molecular bristle is a polyethylene glycol, an alkoxy-polyethylene glycol,
or an alkoxypolyethylene glycol having 4 to 200 oxyethylene, alkoxyethylene or
aryloxyethylene groups; poly(2-hydroxypropyl)methacrylamide (HPMA); poly(2-
hydroxyethyl)methacrylate (HEMA);a ply(2-oxaziline), poly(m-phosphocholine, poly lysine,
or poly glutamic acid, the molecular bristle having a molecular weight of 150 to 8000.
In a further aspect, an ophthalmic composition comprising a bifunctional
molecule containing a reactive group covalently linked to a molecular bristle,
wherein the reactive group is NH , a succinimide, a carboxylic acid, isocyanate,
isothiocyanate, sulfonyl chloride, aldehyde, carbodiimide, acyl azide, anhydride,
fluorobenzene, carbonate, N-hydroxysuccinimide ester, imidoester, epoxide and fluorophenyl
ester,and
wherein the molecular bristle is a polyethylene glycol, an alkoxy-polyethylene glycol,
or an alkoxypolyethylene glycol having 4 to 200 oxyethylene, alkoxyethylene or
aryloxyethylene groups; poly(2-hydroxypropyl)methacrylamide (HPMA); poly(2-
hydroxyethyl)methacrylate (HEMA);a ply(2-oxaziline), poly(m-phosphocholine, poly lysine,
or poly glutamic acid, the molecular bristle having a molecular weight of 150 to 8000.
In a further aspect, use of a bifunctional molecule containing a leaving group
covalently linked to a molecular bristle in the manufacture of a medicament for treating a
diseases relating to protein folding in a patient in need thereof, a bifunctional molecule
containing a leaving group covalently linked to a molecular bristle,
wherein the leaving group is a succinimide functional group, a carboxylic acid
functional group, isocyanate, isothiocyanate, sulfonyl chloride, aldehyde, carbodiimide, acyl
azide, anhydride, fluorobenzene, carbonate, N-hydroxysuccinimide ester, imidoester, epoxide
and fluorophenyl ester,and
wherein the molecular bristle is a polyethylene glycol, an alkoxy-polyethylene glycol,
or an alkoxypolyethylene glycol having 4 to 200 oxyethylene, alkoxyethylene or
aryloxyethylene groups; poly(2-hydroxypropyl)methacrylamide (HPMA); poly(2-
hydroxyethyl)methacrylate (HEMA);a ply(2-oxaziline), poly(m-phosphocholine, poly lysine,
or poly glutamic acid, the molecular bristle having a molecular weight of 150 to 8000.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 shows MALDI-TOF data for modification of ɣ-crystallin with
PEG24.
Figure 2 shows the NICF of ɣ-crystallin modified with PEG24.
Figure 3 shows the distribution function of ɣ-crystallin modified with PEG24.
Figure 4 shows the graph of Γ versus q for ɣ-crystallin modified with PEG24.
Figure 5 shows MALDI-TOF data for modification of ɣ-crystallin with PEG4.
Figure 6 shows the NICF of ɣ-crystallin modified with PEG4.
Figure 7 shows the distribution function of ɣ-crystallin modified with PEG4.
Figure 8 shows the graph of Γ versus q for ɣ-crystallin modified with PEG4.
Figure 9 shows MALDI-TOF data for modification of ɣ-crystallin with
CAPEG.
Figure 10 shows the NICF of ɣ-crystallin modified with CAPEG4.
Figure 11 shows the distribution function of ɣ-crystallin modified with
CAPEG4.
Figure 12 shows the graph of Γ versus q for ɣ-crystallin modified with
CAPEG4.
Figure 13 shows the distribution function of ɣ-crystallin modified with
MMPEG.
Figure 14 shows the distribution function of ɣ-crystallin modified with Biotin.
Figure 15 shows the distribution function of ɣ-crystallin modified with Biotin-
PEG.
Figure 16 shows the distribution function of ɣ-crystallin modified with sulfo-
N-hydroxysuccinimide acetate.
Figure 17 shows embodiments of charge masking groups. In each structure, R
is the molecular bristle.
Figure 18 shows embodiments of molecular bristles.
Figure 19 shows a schematic of the measurement of trans-epithelial transport.
Figure 20 shows the CAPEG4 that was transported to the bottom of the cell in
the tran-epithelial transport experiment.
Figure 21 shows the pH of Opisol® versus the mg/ml of added CAPEG4.
The above-described and other features will be appreciated and understood by
those skilled in the art from the following detailed description, drawings, and appended
claims.
DETAILED DESCRIPTION
Disclosed herein are methods of disaggregating/preventing formation of a γ-
crystallin aggregate comprising contacting the γ-crystallin aggregate with a composition
comprising a γ-crystallin charge masking agent in an amount sufficient to disaggregate and/or
prevent formation of the γ-crystallin aggregate. One of ordinary skill in the art would
recognize that while the molecules disclosed herein are described as a γ-crystallin charge
masking agents they may also disaggregate/prevent protein aggregation of β-crystallin as
well. Further disclosed are methods of inhibiting or reversing the progression of cataract
formation in an eye which comprises contacting the eye with an effective cataract-inhibiting
amount of an ophthalmic composition comprising a γ-crystallin charge masking agent. Also
disclosed are methods of inhibiting or reversing the progression of presbyopia in an eye
which comprises contacting the eye with an effective presbyopia -inhibiting amount of an
ophthalmic composition comprising a γ-crystallin charge masking agent. In specific
embodiments, the γ-crystallin charge masking agent is not a polypeptide.
The inventors herein have employed techniques such as dynamic light
scattering to study the aggregates formed by γ-crystallins in solution. Both the β and γ-
crystallins are highly stable structural proteins comprising four Greek-key motifs in two
domains. While the β-crystallins form dimers as well as hetero- and homo-oligomers, the γ-
crystallins are monomers in the eye. Further, while the β-crystallins exhibit a repulsive force
in solution, the γ-crystallins exhibit an attractive interaction attributed to nonspecific protein
or water interactions. It has also been hypothesized that thiol modifications cause aggregates
of γ-crystallin to form in solution.
The human γ-crystallin family contains five members, the γA-D crystallins
and γ-S crystalline. The γA-D crystallins are expressed early in development and are
primarily found in the lens core; γC and γD-crystallin are most prevalent. Unfolding and
refolding of γ-D crystalline in vitro has been shown to lead to increased protein aggregation
due to the lack of stability of the refolded protein. γS-crystallin has been shown to be a key
protein in the suppression of aggregation of other crystalline proteins, leading to a clear lens.
Without being held to theory, it is believed that the aggregation of γ-crystallin
is both an electrostatic and hydrophobic phenomenon, with the electrostatic forces
dominating. Adding the heat shock proteins αA- and αB-crystallin disrupts γ-crystallin
aggregation. A γ-crystallin charge masking agent that can disrupt electrostatic interactions
can substitute for the chaperone activity of α-crystallin and prevent/reduce γ-crystallin
aggregate size.
Treatment with γ-crystallin charge masking agents can be used to treat
diseases and/or conditions resulting from aggregation of γ-crystallins such as cataracts and
presbyopia. As used herein, a cataract is an opacity of the crystalline lens of the eye caused
by altered protein interactions in the lens. Protein interactions include misfolding of proteins
as well as protein-protein interactions such as aggregation. Presbyopia is the impairment of
vision due to advancing years or old age. Symptoms of presbyopia include decreased
focusing ability for near objects, eyestrain, difficulty reading fine print, fatigue while reading
or looking at an illuminated screen, difficulty seeing clearly up close, less contrast when
reading print, need for brighter and more direct light for reading, needing to hold reading
material further away in order to see it clearly, and headaches, especially headaches when
using near vision. Individuals suffering from presbyopia may have normal vision, but the
ability to focus on near objects is at least partially lost over time, and those individuals come
to need glasses for tasks requiring near vision, such as reading. Presbyopia affects almost all
individuals over the age of 40 to a greater or lesser degree.
In the method of inhibiting the progression of cataract formation in an eye, the
eye may already contain one or more developing or fully developed cataracts before it is
contacted with the γ-crystallin charge masking agent. Accordingly, the method can be used
to inhibit the formation of further cataracts in the eye, or to inhibit the formation of mature
cataracts from the developing cataracts already present in the eye. Alternatively, the eye may
be free of any developing or fully developed cataracts before it is contacted with the γ-
crystallin charge masking agent.
In the method of reversing the progression of cataract formation in an eye, at
least partial to full reversal of cataracts in the eye is achieved by contacting the eye with a γ-
crystallin charge masking agent as disclosed herein.
Similarly, in the method of inhibiting the progression of presbyopia in an eye,
the individual may already be experiencing one or more symptoms of presbyopia before the
eye is contacted with the γ-crystallin charge masking agent. Accordingly, the method can be
used to reduce the progression of the symptom(s) experienced, or to inhibit the formation of
additional symptoms of presbyopia. Alternatively, the eye may be free of any symptoms of
presbyopia before it is contacted with the γ-crystallin charge masking agent.
In the method of reversing the progression of presbyopia in an eye, at least
partial to full reversal of the symptoms of presbyopia in the eye is achieved by contacting the
eye with a γ-crystallin charge masking agent as disclosed herein.
As used herein, γ-crystallin charge masking agent is a molecule suitable to
interfere with γ-crystallin electrostatic protein-protein interactions which lead to γ-crystallin
aggregation. In one embodiment, the masking agent is not a polypeptide. γ-crystallin charge
masking agents prevent γ-crystallin aggregates from forming and/or reduce the size of pre-
formed aggregates.
In one embodiment, the γ-crystallin charge masking agent is a high
concentration salt solution, having a salt concentration over 400 mM. The term “salt” as used
herein, is intended to include an organic or inorganic salt, including but not limited to one or
more of NaCl, KCl, ammonium halides such as NH Cl, alkaline earth metal halides such as
CaCl , sodium acetate, potassium acetate, ammonium acetate, sodium citrate, potassium
citrate, ammonium citrate, sodium sulphate, potassium sulphate, ammonium sulphate,
calcium acetate or mixtures thereof. Additional organic salts include alkylammonium salts
such as ethylammonium nitrate, sodium citrate, sodium formate, sodium ascorbate,
magnesium gluconate, sodium gluconate, trimethamine hydrochloride, sodium succinate, and
combinations thereof. Without being held to theory, it is believed that the identity of the ion,
e.g., Li+, Na+ and K+, can affect the ability of the γ-crystallin charge masking agent to
prevent γ-crystallin aggregation.
It was unexpectedly shown herein that salt, e.g. KCl, concentrations of less
than 300 nM did not provide a reduction in the size of γ-crystallin aggregates. However, at
KCl concentrations to 400 to 1000 mM, aggregate formation was effectively inhibited.
In another embodiment, the γ-crystallin charge masking agent is a bifunctional
molecule containing a leaving group covalently linked to a molecular bristle. The
bifunctional molecule interacts with charges on the γ-crystallin molecules, such as positively
charged lysine and arginine residues and negatively charged glutamate and aspartate residues.
The molecular bristle is a hydrophilic, water-soluble species that provides distance between
the γ-crystallin molecules, preventing aggregation. Without being held to theory, it is
believed that the bifunctional molecule reacts and effectively puts the molecular bristle onto
the protein, and the leaving group is expelled during the reaction. The covalently attached
molecular bristle prevents aggregation of the γ-crystallin molecules. Without being held to
theory, it is believed that the bifunctional molecules described herein may also act as β-
crystallin interaction inhibitors.
Exemplary leaving groups (also called reactive groups) include succinimide
and carboxylic acid functional groups, specifically N-hydroxysuccinimide and COOH. In
some embodiments, the leaving group is biocompatible. Other examples of leaving groups
include isocyanate, isothiocyanate, sulfonyl chloride, aldehyde, carbodiimide, acyl azide,
anhydride, fluorobenzene, carbonate, N-hydroxysuccinimide ester, imidoester, epoxide, and
fluorophenyl ester. Figure 17 shows embodiments of leaving groups wherein the R group is
the molecular bristle. When the bifunctional molecule contains COOH, water leaves when it
reacts with a protein’s amine group. When the bifunctional molecule contains N-
hydroxysuccinimide water is not released. In an NH reaction, water leaves when NH reacts
with a COOH group on the protein.
Exemplary molecular bristles include linear or branched polyethylene glycols
having 4 or more oxyethylene groups, such as 4 to 200 oxyethylene groups. Also included
are modified polyethylene glycols such as alkoxy- and aryloxy polyethylene glycols having 4
to 200, specifically 4 to 24 oxyethylene, alkloxy ethylene or aryloxy ethylene groups.
Alternative molecular bristles include poly(2-hydroxypropyl)methacrylamide (HPMA),
poly(2-hydroxyethyl)methacrylate (HEMA), poly(2-oxazilines), poly(m-phosphocholine),
poly lysine, and poly glutamic acid. Figure 18 shows embodiments of molecular bristles. In
one embodiment, the molecular bristle has a number average molecular weight of 150 to
8000.
In a specific embodiment, the bifunctional γ-crystallin charge masking agent
In one embodiment, the bifunctional γ-crystallin charge masking agents
described herein are also useful in the treatment of diseases relating to protein folding such as
Alzheimer’s disease, Parkinson’s disease, and Huntington’s disease. In a specific
embodiment, the bifunctional γ-crystallin charge masking agents are administered as oral
compositions.
In one embodiment, the γ-crystallin charge masking agent is not a polypeptide.
“Polypeptide”, “peptide” and “protein” are used interchangeably herein to refer to a polymer
of amino acid residues. The terms apply to amino acid polymers in which one or more amino
acid residue is an artificial chemical mimetic of a corresponding naturally occurring amino
acid, as well as to naturally occurring amino acid polymers and non-naturally occurring
amino acid polymers.
An advantage of the γ-crystallin charge masking agents described herein is
that they are expected to be effective in the presence of post-translational modifications of γ-
crystallins, including, for example, transamidation, oxidation, modified and dysfunctional
connexins, and high concentrations of inorganic and organic ions such as Ca .
The γ-crystallin charge masking agents are contacted with the eye to inhibit
the progression of cataracts and/or reduce existing cataracts, or to inhibit and/or reduce the
symptoms of presbyopia. As used herein, the term “contacting the eye” encompasses
methods of directly applying the γ-crystallin charge masking agent to the eye. In the above-
described method, suitable means known to those of ordinary skill in the art may be used to
contact the eye with the compound. Examples of such methods include, but are not limited
to, the compound being injected into the eye, being dropped or sprayed into the eye, applied
in the form of an ophthalmic device, applied by iontophoresis, or otherwise topically applied
to the eye.
As used herein, the term “effective cataract-inhibiting amount” means an
amount which will inhibit the progression or formation of cataracts in an eye or inhibit the
progression or formation of mature cataracts from developing cataracts already present in the
eye. The effective cataract-inhibiting amount of the γ-crystallin charge masking agent will
depend on various factors known to those of ordinary skill in the art. Such factors include,
but are not limited to, the size of the eye, the number and progression of any fully developed
or developing cataracts already present in the eye, and the mode of administration. The
effective cataract-inhibiting amount will also depend on whether the pharmaceutical
composition is to be administered a single time, or whether the pharmaceutical composition is
to be administered periodically, over a period of time. The period of time may be any
number of days, weeks, months, or years. In one embodiment, the effective cataract-
inhibiting amount of the γ-crystallin charge masking agent, specifically the bifunctional
molecules described herein, is about 0.001 g to about 0.1 g. Specifically, the effective
cataract-inhibiting amount is about 0.01 g to about 0.05 g.
As used herein, the term “effective presbyopia -inhibiting amount” means an
amount which will reduce a symptom of presbyopia in an eye or inhibit the progression of
additional symptoms of presbyopia in the eye. The effective presbyopia -inhibiting amount
of the γ-crystallin charge masking agent will depend on various factors known to those of
ordinary skill in the art. Such factors include, but are not limited to, the size of the eye, the
number and type of symptoms already present in the individual, and the mode of
administration. The effective cataract-inhibiting amount will also depend on whether the
pharmaceutical composition is to be administered a single time, or whether the
pharmaceutical composition is to be administered periodically, over a period of time. The
period of time may be any number of days, weeks, months, or years. In one embodiment, the
effective presbyopia -inhibiting amount of the γ-crystallin charge masking agent, specifically
the bifunctional molecules described herein, is about 0.001 g to about 0.1 g. Specifically, the
effective presbyopia -inhibiting amount is about 0.01 g to about 0.05 g.
As used herein the term “ophthalmic composition” refers to a
pharmaceutically acceptable formulation, delivery device, mechanism or system suitable for
administration to the eye. The term “ophthalmic compositions” includes but are not limited
to solutions, suspensions, gels, ointments, sprays, depot devices or any other type of
formulation, device or mechanism suitable for short term or long term delivery of β -
crystallin electrostatic interaction inhibitors to the eye. In contrast to oral formulations, for
example, ophthalmic compositions exhibit specific technical characteristics associated with
their application to the eyes, including the use of pharmaceutically acceptable ophthalmic
vehicles that avoid inducing various reactions such as, for example, irritation of the
conjunctiva and cornea, closure of the eyelids, secretion of tears and painful reactions.
Specific ophthalmic compositions are advantageously in the form of ophthalmic solutions or
suspensions (i.e., eye drops), ophthalmic ointments, or ophthalmic gels containing β –
crystallin electrostatic interaction inhibitors. Depending upon the particular form selected,
the compositions may contain various additives such as buffering agents, isotonizing agents,
solubilizers, preservatives, viscosity-increasing agents, chelating agents, antioxidizing agents,
antibiotics, sugars, and pH regulators.
Examples of preservatives include, but are not limited to chlorobutanol,
sodium dehydroacetate, benzalkonium chloride, pyridinium chlorides, phenethyl alcohols,
parahydroxybenzoic acid esters, benzethonium chloride, hydrophilic dihalogenated
copolymers of ethylene oxide and dimethyl ethylene-imine, mixtures thereof, and the like.
The viscosity-increasing agents may be selected, for example, from methylcellulose,
hydroxyethylcellulose, carboxymethylcellulose, hydroxypropylmethylcellulose, polyvinyl
alcohol, carboxymethylcellulose, chondroitin sulfate, and salts thereof. Suitable solubilizers
include, but are not limited to, polyoxyethylene hydrogenated castor oil, polyethylene glycol,
polysorbate 80, and polyoxyethylene monostearate. Typical chelating agents include, but are
not limited to, sodium edetate, citric acid, salts of diethylenetriamine pentaacetic acid,
diethylenetriamine pentamethylenephosphonic acid, and stabilizing agents such as sodium
edetate and sodium hydrogen sulfite.
Exemplary buffers include, but are not limited to borate buffers, phosphate
buffers, carbonate buffers, acetate buffers and the like. The concentration of buffer in the
ophthalmic compositions may vary from about 1 mM to about 150 mM or more, depending
on the particular buffer chosen.
As used herein, the term “vehicle” is intended to include a carrier, diluent or
excipient suitable for ophthalmic use. “Excipient” refers to an ingredient that provides one or
more of bulk, imparts satisfactory processing characteristics, helps control the dissolution
rate, and otherwise gives additional desirable characteristics to the compositions. In
particular, the excipients are selected such that the ophthalmic composition does not trigger a
secretion of tears that will entrain the active ingredient. Acceptable excipients are well
known to a person skilled in the art, who will know how to select them depending on the
desired formulation.
In one embodiment, the γ-crystallin charge masking agent is administered in
the form of an ophthalmic device, such as a contact lens or a punctal plug. Suitable
ophthalmic devices included biocompatible devices with a corrective, cosmetic or therapeutic
quality.
In one embodiment, the γ-crystallin charge masking agent may be adhered to,
incorporated into or associated with a contact lens, optionally as a controlled-release
composition. The contact lens may be produced using the known materials, for example
hydrogels, silicone hydrogels, silicone elastomers and gas permeable materials such as
polymethylmethacrylate (PMMA), methacrylic acid ester polymers, copolymers of
oligosiloxanylalkyl(meth)acrylate monomers/methacrylic acid and the like. Specific
examples of materials for water-containing soft ophthalmic lenses include those described in
U.S. Pat. No. 5,817,726, 2-hydroxyethyl methacrylate polymers as described in U.S. Pat. No.
,905,125, ophthalmic lens materials as described in European Patent Application No.
781,777, the hydrogel lens which is coated with a lipid layer in advance as described in U.S.
Pat. No. 5,942,558; all incorporated herein for their teachings regarding contact lenses.
Generally used contact lens such as hard or rigid cornea-type lens, and gel, hydrogel or soft-
type lens which are produced from the above known materials may be used.
It is common in the contact lens industry to characterize contact lenses into
two major categories; conventional and silicone hydrogels. The conventional based
hydrogels started as poly(hydroxyethyl methacrylate) (poly HEMA) and evolved to
polyHEMA copolymers with other hydrophilic moieties such as n-vinyl pyrrolidone (nVP),
acrylamide, dimethyl acrylamide, and methacrylated phosphorylcholines. Polyvinyl alcohol
lenses may also be employed.
The silicone hydrogels (SiH) typically consist of copolymers of methacrylated
or meth(acrylamide) silicone monomers, prepolymers or macromers with typical
conventional hydrogel monomers. Examples of silicone monomers include “Tris”, alkyl
terminated, methacrylated polydimethylsiloxane (PDMS), and block copolymers of silicone
and hydrophilic monomers. ABA triblock copolymers are common where the A group is a
hydrophilic block and the B group is the silicone monomer block. In addition to the
methacrylates, other reactive groups include vinyl, acrylamide, or any other reactive group
capable of chain reaction polymerization. Crosslinking and polymerization can also be
achieved via step-growth polymerization using monomers with bi-functionality. An example
is the reaction of a hydroxyl group with a carboxylic acid group in two amino acids or from
terepthalic acid and ethylene glycol.
Plasma based coating methods are commonly used on silicone hydrogels
including plasma oxidation and plasma coatings.
A sustained-release γ-crystallin charge masking agent composition may be
produced, for example, by incorporating in, associating with or adhering to the contact lens
the γ-crystallin charge masking agent composition according to the known methods for
producing the contact lenses with sustained-release drugs as described in U.S. Pat. Nos.
,658,592; 6,027,745; WO2003/003073; US0079197, incorporated herein for their
teachings regarding contact lenses and sustained release. Specifically, the contact lens may
be produced by adhering the γ-crystallin charge masking agent to a part of a finely-divided or
gel sustained-releasing agent such as polyvinyl pyrrolidone, sodium hyaluronate and the like.
In addition, sustained release may be produced by forming a γ-crystallin charge masking
agent composition reservoir such as by producing a contact lens from a member which forms
a front surface of the lens and a member which forms a rear surface of the lens.
In one embodiment the charge masking agent may be inserted into the aqueous
or vitreous as an injection with controlled release.
In one embodiment, the γ-crystallin charge masking agent is administered in a
punctal plug. As used herein, the term punctal plug refers to a device of a size and shape
suitable for insertion into the inferior or superior lacrimal canaliculus of the eye through,
respectively, the inferior or superior lacrimal punctum.
In one embodiment, the γ-crystallin charge masking agent is administered by
iontophoresis. Iontophoresis is a technique using a small electric charge to deliver a
medicine or other chemical through the skin.
In one embodiment, the ophthalmic composition is administered using
ultrasound enhancement.
The invention is further illustrated by the following non-limiting examples.
Examples
Example 1: Cloning of γ-Crystallin
The ɣD- and ɣS-crystallin DNA sequences are in pQe1 plasmids that were
provided by the King Labs at Massachusetts Institute of Technology (Cambridge, MA). ɣ-
crystallin protein sequences contain a 6x N-terminal histidine tag (his tag) for purification
purposes. The plasmids were transformed into a cloning competent cell line to create
additional plasmid DNA. Plasmids were subsequently transformed into an expression
competent cell line for protein synthesis (TAM 1 E. coli cells (Active Motif. Carlsbad, CA)).
ɣ-crystallin plasmid DNA was chemically transformed into M15pRep E. coli
cells for protein synthesis. 1 L cultures were grown for protein purification. ɣ-crystallin
protein was purified by Ni affinity chromatography. The N-terminal His tag contained on the
ɣ-crystallin proteins preferentially binds to the Ni column. The bound protein can be eluted
with an imidazole gradient which competitively binds to the Ni, releasing the purified
protein. Purity was confirmed by SDS PAGE gel electrophoresis and fast protein liquid
chromatography (FPLC).
Example 2: Effect of pH and Salt on Purified ɣD- and ɣS- crystallin
Because pH and salt have an effect on the aggregation of β-crystallin, the
effect of pH and salt on γ-crystallin was also studied. Particle sizes were measured using
dynamic light scattering (DLS).
Experimental dynamic light scattering (DLS) was measured using an ALV
goniometer instrument which had an ALV-5000/E correlator equipped with 288 channels
(ALV, Langen Germany) and a 2W argon laser (Coherent Inc., Santa Clara, CA), with a
working power of approximately 40 mW. Scattering intensity was measured at angles
between 30° and 90° at 5° intervals, corresponding to a scattering wave vector (q) range
6 7 -1
between 8.41x10 and 2.30x10 m . The scattering wave vector is defined as q = 4πn sin
(θ/2) / λ, where θ is the scattering angle, and λ= 514.5, the wavelength of the argon laser in
vacuum, and n is 1.33, the refractive index of water. The temperature of the sample was held
at constant temperature of 4 to 37 ± 0.1°C by a circulating water bath.
The correlation function was analyzed using CONTIN analysis to calculate lag
times for the correlation functions at measured angles. The peaks represent the diffusive
mode of the proteins. The delay times (τ) were converted to Γ and graphed versus q . The
slope of the fitted line is the diffusion coefficient (D). The lines are fitted such that the
intercept is zero because a non-zero value of q=0 is unphysical. The Γ versus q plots were
linear with r values of 0.95 or greater.
DLS measurements were performed on both modified and unmodified ɣD-
and ɣS-crystallin proteins at 150mM NaCl, 20mM Na HPO /NaH PO buffer pH 6.8 at a
2 4 2 4
concentration of 0.5 mg/mL. All solutions were filtered with 0.22 µm hydrophobic PVDF
membranes (Fisher Scientific) into 10 mm diameter borosilicate glass tubes and sealed.
Solutions were allowed to equilibrate for thirty minutes prior to measuring light scattering.
Individual solutions of α-A, α-B, γD- and γ-S crystallin proteins were
investigated to understand the size scale and how the proteins behave in dilute solution.
(Table 1) Temperature had little effect on protein size in solution as measured by DLS.
Table 1: DLS measurements performed on both modified and unmodified γD- and γ-S -
crystallin proteins
αA αB ɣD ɣS
Temp R R R R R R
h h hf hs hf hs
(°C) (nm) (nm) (nm) (nm) (nm) (nm)
4º 12 12 3 116 3 100
22º 12 13 2.8 104 2.7 102
37º 13 13 2.7 109 2.6 95
Considering that the molecular weight of αA-crystallin is 19.9 kDa and α-B
crystalline is 20.16 kDa, the size seen in the DLS experiment is consistent with α-crystallin’s
known assembly into 300-1200 kDa species in solution as well as in the human eye.
The 3 nm R for the γ-crystallins represents a single protein species which
correlates well with the molecular weights of 20.6 kDa and 20.9 kDa for monomeric γD- and
γ-S crystallin, respectively. Both species also show aggregates with R values of 110 and 100
nm for γD- and γS- crystallins, respectively. Aggregation of γ-crystallin has previously been
attributed to the attractive interactions between γ-crystallins. The lack of specific protein-
protein interactions in the γ-crystallins allows them to form large aggregates in solution.
γ-crystallins were subjected to a variety of experimental conditions, including
a range of pHs (5,6,7,8,9,10,11), concentrations of KCl (100mM, 150 mM, 300 mM, 500
mM, 1000 mM) and temperature (4.5ºC, 22ºC, 37ºC). The pH and salt concentration were
adjusted via overnight dialysis at 4ºC and their final concentration adjusted to 1.0 g/L.
Table 2: Effect of pH and salt on γD- and γ-S crystallin
ɣD ɣS
Rh Rh
Variable (nm) Rh (nm) (nm) Rh (nm)
100 mM NaCl 2.8 108 2.7 104
150 mM NaCl 2.8 104 2.7 102
300 mM NaCl 2.6 91 2.9 100
400 mM NaCl 2.7 - 2.6 -
500 mM NaCl 2.8 - 2.5 -
1000 mM NaCl 2.5 - 2.6 -
pH 5 3.9 100 4.8 141
pH 6 2.7 118 2.5 110
pH 8 2.9 102 3.0 96
pH 9 3.5 97 2.9 106
pH 10 3.2 - 3.4 90
pH 11 3.1 - 3.1 -
As can be seen in Table 2, varying pH above 10 removed the aggregates from
γD- and γS-crystallin solutions. Salt concentrations over 300 mm KCl did reduce γ-crystallin
aggregates to individual protein particles. These results indicate that the large aggregates of
γ-crystallin can be disrupted or prevented by interfering with the electrostatic interactions
between the γ-crystallins.
It had been hypothesized that disulfide bonds might mediate the observed γ-
crystallin aggregates. The DLS of the γ-crystallin proteins was also measured in 5 mM DTT,
and in the presence of 1.0 g/L α-crystallin. As shown in Table 3, DTT had no effect on the
size of the γ-crystallin aggregates. Thus, this hypothesis was incorrect.
Further, the synthesized α-crystallin proteins were mixed with γ-crystallin
proteins to determine if the chaperoning ability of α-crystallin disrupts the γ-crystallin
aggregates. The α-A and α-B crystallins were each individually mixed with γD- or γS-
crystallin in a molar ratio of 3:1, respectively, mimicking the ratio found in the human eye
lens. All solutions were allowed to equilibrate for an hour at 4ºC. Upon incubation with α-A
or α-B crystallin, the large γ-crystallin aggregate of several hundred nanometers disappeared
and the individual γ-crystallin and α-crystallin macromolecules were seen. (Table 3) These
data support previous work demonstrating that the α-crystallins suppress nonspecific protein
aggregation thus preventing the aggregation of γ-crystallin proteins.
Table 3: Effect of DTT and chaperones on γD- and γ-S crystallin
ɣD ɣS
Rh Rh Rh Rh
f s f s
Additive (nm) (nm) (nm) (nm)
---- 2.8 104 2.7 102
mM
DTT 2.6 108 3 102
αA 2.7 16 2.8 18
αB 3.2 17 3 19
Without being held to theory, it is hypothesized that the increase in size of α-
crystallins (from 63-68 nm to 75nm) is due to the α-crystallin interaction with denatured or
misfolded γ-crystallin. It is clear that the addition of α-crystallin prevents or disrupts the
large γ-crystallin species from forming in solution and by disrupting electrostatic charges
between the γ-crystallins. The α-crystallins are able to disrupt the large aggregates of γ-
crystallin that appear at high concentrations of γ-crystallin. Thus, in the absence of α-
crystallin, γ-crystallin will form large soluble aggregates through electrostatic forces that can
be interrupted at high pH and high salt concentrations.
Materials and Methods for Characterization of Aggregates
Chemical modification of ɣD- and ɣS- crystallin was undertaken to modify the
aggregation behavior of these proteins. The methods used to characterize the modified ɣD-
and ɣS- crystalline are Matrix Assisted Laser Desorption Ionization – Time of Flight
(MALDI-TOF), circular dichroism, and dynamic light scattering.
Matrix Assisted Laser Desorption Ionization – Time of Flight
Approximately 2mg of regular or modified ɣ-crystallin protein was dialyzed
overnight into 5mM tris (hydroxymethyl) aminomethane hydrochloride (Tris HCl) pH 7. The
solution was lyophilized (freeze dried) overnight to obtain a dry crystallin protein powder.
Mass spectrometry data were obtained on an Omniflex MALDI-TOF mass
spectrometer (Bruker Daltonics, Inc., Billerica MA) equipped with a 337nm nitrogen laser.
Samples (2mg/mL) were mixed (1:1) with a matrix consisting of 0.1% trifluoroacetic acid
(TFA), 50% acetonitrile and 3,5-dimethoxyhydroxycinnamic acid. 1μL of solution was
subsequently deposited on a stainless steel target. The instrument was used in linear mode
for data acquisition.
Circular Dichroism
Crystallin samples were dialyzed overnight into 10mM Na HPO /NaH PO
2 4 2 4
buffer pH 6.8 and measured at a concentration of 0.5mg/mL. CD spectra were measured on a
Jasco J715 spectropolarimeter at 22°C using a quartz cell of 1mm path length. After allowing
the sample to equilibrate for five minutes, spectra were obtained in the range of 250 to 195
Dynamic Light Scattering
Experimental dynamic light scattering (DLS) was measured using an ALV
goniometer instrument which had an ALV-5000/E correlator equipped with 288 channels
(ALV, Langen Germany) and a 2W argon laser (Coherent Inc., Santa Clara, CA), with a
working power of approximately 40 mW. Scattering intensity was measured at angles
between 30° and 90° at 5° intervals, corresponding to a scattering wave vector (q) range
6 7 -1
between 8.41x10 and 2.30x10 m . The scattering wave vector is defined as q = 4πn sin
(θ/2) / λ, where θ is the scattering angle, and λ= 514.5, the wavelength of the argon laser in
vacuum, and n is 1.33, the refractive index of water. The temperature of the sample was held
at constant temperature of 4 to 37 ± 0.1°C by a circulating water bath.
The correlation function was analyzed using CONTIN analysis to calculate lag
times for the correlation functions at measured angles. The peaks represent the diffusive
mode of the proteins. The delay times (τ) were converted to Γ and graphed versus q . The
slope of the fitted line is the diffusion coefficient (D). The lines are fitted such that the
intercept is zero because a non-zero value of q=0 is unphysical. The Γ versus q plots were
linear with r values of 0.95 or greater.
DLS measurements were performed on both modified and unmodified ɣD-
and ɣS-crystallin proteins at 150mM NaCl, 20mM Na HPO /NaH PO buffer pH 6.8 at a
2 4 2 4
concentration of 0.5 mg/mL. All solutions were filtered with 0.22 µm hydrophobic PVDF
membranes (Fisher Scientific) into 10 mm diameter borosilicate glass tubes and sealed.
Solutions were allowed to equilibrate for thirty minutes prior to measuring light scattering.
Example 3- Chemical Modification of ɣD- and ɣS- crystalline with PEG24
The first modification of ɣ-crystallin was done with NHSPEG24. Amino
acids containing primary amines, lysine and arginine, can perform a nucleophilic substitution
on N-hydroxy succinimide (NHS) functionalized poly(ethylene glycol) (PEG). NHS is an
activated ester which accelerates the Sn2 reaction mechanism because it is a good leaving
group. The nucleophilic substitution produces a protein modified with PEG or a PEGylated
ɣ-crystallin protein. PEGylation was chosen because modification of proteins with PEG has
been shown to increase solubility and not affect the three dimensional structure or properties.
In particular, PEG24 was chosen because of its reasonable molecular weight (1100.39) added
and spacer arm length (8.82nm).
NHSPEG24
Modification of ɣ-crystallin with PEG24 was successful as demonstrated by
the large increase in ɣ-crystallin molecular weight observed in MALDI-TOF. The maximum
relative intensity for ɣD-crystallin was at 24,148 m/z or 2 PEG24 units while for ɣS-crystallin
the peak occurred at 26,711 m/z which corresponds to 4 PEG24 units. (Figure 1) The excess
reactant and reaction conditions were sufficient as there was no unmodified ɣ-crystallin
present in solution. The higher resolution MALDI-TOF data of ɣD-crystallin shows an
additional two distinct peaks at 23,056 m/z and 25,206 m/z corresponding to 1 and 3 PEG24
modifications.
CD spectroscopy demonstrated that both crystallin proteins appear to keep
their native state despite modification. ɣD-crystallin showed increased peaks and depths
which could be attributed to a slight difference in protein concentration. The good fit with
experimental data is expected as it has been previously demonstrated that PEGylation does
not interfere with a protein’s secondary structure. (data not shown)
DLS was performed on ɣD- and ɣS-crystallin modified with PEG24 at 22°C
and 37°C. The NICF provided a distribution function with a single set of peaks which has an
angular dependency that can be seen in the graph of Γ versus q . (Figures 2-4) The PEG24
modified ɣ-crystallin protein had a small size distribution with no additional mode at longer
relaxation times which would indicate aggregate formation. For ɣD-crystallin, the R
calculated from the diffusion coefficient was 3.1nm at 22° and 37°C, while ɣS-crystallin had
an R of 3.2nm at 22° and 37°C. The R values correlated well with the 24.1 kDa and
26.7kDa molecular weights of ɣD- and ɣS-crystallin, respectively, measured by MALDI-
TOF.
PEGylation of ɣ-crystallin by NHSPEG24 effectively prevented any
aggregation events in dilute solutions as evident in DLS. PEG is a hydrophilic polymer and
has been shown to increase the solubility of proteins in solution. Without being held to
theory, it is believed that if the aggregation phenomena are a result of solubility issues
associated with hydrophobic interactions or electrostatics, then the increased solubility
associated with PEGylation prevents the ɣ-crystallin proteins from aggregation.
Along the same line of thought, without being held to theory, it is believed that
the overall surface charge of the proteins has been altered by the reaction with PEG. At a
protein’s isoelectric point, there is a decrease in solubility which results from the charge
neutrality associated with the isoelectric point. Amino acids containing primary amines were
the target sites for this type of modification. The primary amine of lysine and arginine can
have a positive charge associated with it depending on the protein makeup and solution
conditions. By having NHSPEG react onto the lysine and arginine groups the potentially
charged sites were occupied by the hydrophilic PEG, thereby changing the protein surface
charge. If the aggregation event is a result of electrostatic interactions or a result of the
proteins proximity to the isoelectric point then this rational would explain why the aggregate
is absent from solution. Modification of ɣ-crystallin protein with PEG via other reaction
mechanisms will be used to examine this possibility.
Without being held to theory, a final explanation for the lack of aggregates in
solution is a spacer issue. In the field of hard spherical colloids it has been established that
the addition of spacer molecules can reduce aggregation. Adding the PEG24 moiety to ɣ-
crystallin provides a hydrophilic spacer molecule on the surface of the protein. The spacing
between proteins provided by PEG could be all that is needed to prevent aggregation of ɣ-
crystallin protein.
Example 4- Chemical Modification of ɣD- and ɣS- crystalline with PEG4
ɣ -crystallin proteins were modified with PEG4 to investigate the effect of
spacer arm length on ɣ-crystallin protein aggregation. The reaction was again performed
with NHS functionalized PEG (NHSPEG4) to keep the method of modification the same.
PEG4 has a molecular weight added of 219.33 g/mol and spacer arm of 1.6 nm, both of
which are smaller than PEG24.
NHSPEG4
MALDI-TOF data showed an increase in the overall ɣ-crystallin molecular
weight indicating modification with PEG4. (Figure 5) The highest relative intensity seen for
ɣD-crystallin occurred at 22,820 m/z, while for ɣS-crystallin this occurred at 23,329 m/z
which correspond to 4 and 5 PEG4 units, respectively, being added to either protein. It
should be noted that there is a Gaussian distribution around the relative intensity peak
suggesting that there are proteins which contain both a greater and lesser degree of
modification. MALDI-TOF data also showed that no unmodified crystallin protein is
present.
CD spectroscopy again showed that PEGylation did not significantly affect the
secondary structures of ɣ and α-crystallin proteins. (data not shown) Excellent agreement
was seen between the native and PEG4 tailored ɣ-crystallin proteins.
The NICF of ɣ-crystallin protein modified with PEG4 had a monoexponetial
decay which indicates a single size scale present in solution. (Figure 6) Similar to ɣ-
crystallin modified with PEG24, no aggregate was present in solution. The distribution
function (Figure 7) again showed a single set of peaks that demonstrate linear angular
dependence in the Γ versus q graph (Figure 8). The Rh of ɣD-crystallin measured by DLS
was 2.8 and 2.9 nm at 22°C and 37°C respectively. The R size correlates well with the
modified protein weight of nearly 22.8 kDa. A slight increase of the protein monomeric size
can also been observed in DLS as the R of the modified ɣD-crystallin protein is slightly
larger than its unmodified predecessor. The PEG4 modified ɣS-crystallin protein had an R
of 2.9 at 22°C and 37°C. The overall size of the modified protein did increase in comparison
to unmodified ɣS-crystallin and the R is consistent with a molecular weight of
approximately 23.3 kDa.
ɣ-crystallin protein modified with NHSPEG4 and NHSPEG24 showed no
aggregation at 22° and 37°C. Modification with PEG4 generally resulted in four to five low
molecular weight additions while in the case of PEG24 two to three groups were added per ɣ-
crystallin protein. The higher number of modifications made per protein with PEG4 provided
no significant benefit in preventing aggregation. Similarly, adding a greater total weight with
PEG24 to ɣ-crystallin provided no advantage to preventing aggregation.
The ɣ-crystallin proteins modified with PEG24 had a slightly larger R in
comparison to the PEG4 modification. As both modifications resulted in no aggregate it was
concluded that spacer arm length does not significantly contribute to the prevention of
aggregation. It is predicted that larger PEG chains would produce similar results.
Example 5- Chemical Modification of ɣD- and ɣS- crystalline with CAPEG
Modification by CAPEG4 was performed to investigate an alternative reaction
mechanism for the PEGylation of ɣ-crystallin. At physiological pH (6.8), the primary amine
of CAPEG4 is slightly more positive and capable of reacting with the negatively charged
amino acids, aspartic acid and glutamic acid. Alternatively, positively charged amino acids
are still capable of reacting with the carboxylic acid of CAPEG4 (CA). CAPEG4 was
selected for similar reasons as NHSPEG4, those being a small added molecular weight added
per unit (265.3 g/mol) and a short spacer arm (1.81 nm).
CAPEG4
The MALDI-TOF showed a considerable increase in ɣ-crystallin molecular
weight which is the result of modification by CAPEG4. (Figure 9) There was no unmodified
ɣ-crystallin in either solution which suggests that reaction conditions were sufficient for
protein modification. The relative intensity peak at 23,273 m/z for ɣD-crystallin represents 5
CAPEG units having been added to the protein. A higher resolution MALDI-TOF spectrum
for ɣS-crystallin showed several distinct mass peaks to include 22,867 m/z or 2 CAPEG4
units, a relative intensity maximum at 23,089 m/z or 3 CAPEG4 units, and 23,591 m/z or 5
CAPEG4 units.
The CD spectroscopy of CAPEG4 modification of ɣ-crystallin showed a
similar trend to modifications made with PEG4 and PEG24. (Data not shown) ɣS-crystallin
+ CAPEG4 showed excellent correlation with the unmodified ɣS-crystallin protein. The
modified ɣD-crystallin again showed a slight variation at 220nm but overall the curve is of a
similar shape as the native ɣD-crystallin.
Modifying ɣ-crystallin with CAPEG also prevented aggregation of proteins.
The NICF can be described by a monoexponetial function which produces a distribution
function that has a linear Γ vs q dependence. (Figure 10-12) The modified ɣ-crystallin
protein was seen in its monomeric form at both 22° and 37°C where the R was temperature
independent. For ɣD-crystallin, the diffusion coefficient provided a calculated R of 2.8nm,
while for ɣS-crystallin this value was 2.9m. The R values correlate well with the 23kDa and
23.5kDa modified molecular weights of ɣD- and ɣS-crystallin, respectively, in addition to
being similar in size to ɣ-crystallin modified with PEG4.
PEGylation with CAPEG at physiological pH (6.8) targeted acidic amino
acids. Similar to the reactions done with NHSPEG, the CAPEG modification should alter the
overall surface charge of the protein. By reacting with negatively charaged amino acids the
hydrophilic PEG chain occupies the potentially charged amino acid. Having a similar size
and number of modifications (four to five) as NHSPEG4, ɣ-crystallin tailored with CAPEG4
offers a very similar modification. A potential advantage to using CAPEG4 over NHSPEG is
that there is no NHS leaving group in the reaction.
Without being held to theory, the CAPEG modification again suggests that the
protein’s surface charge is a key factor to the formation of ɣ-crystallin aggregates. As a PEG
moiety is being added to the protein it is also possible that the hydrophobic nature of the
surface is altered, preventing aggregation. The crucial information gained from this
experiment is that modification through acidic or basic amino acids can prevent aggregation.
Example 6- Chemical Modification of ɣD- and ɣS- crystalline with MMPEG
Maleimide functionalized PEG is yet another reaction mechanism by which
the ɣ-crystallin proteins may be PEGylated. Thiol groups are capable of Michael addition
over the double bond of the maleimide functional group. Both ɣ-crystallin proteins contain
multiple cysteine amino acid residues which contain a thiol end group. PEGylation of ɣ-
crystallin with MMPEG24 (MM) thus occurs by cysteine amino acids. Modification via this
reaction mechanism is unique to previous PEGylation experiments because the overall
protein charge should not be affected. In particular, MMPEG24 was chosen because the high
molecular weight added per unit (1239.44 g/mol) and long spacer arm (9.53 nm) will be
comparable with PEG24.
N NH
MMPEG24
The MALDI-TOF spectra of ɣ-crystallin modified with MMPEG24 showed
three distinct peaks can be observed with minimal to no unmodified ɣ-crystallin. (data not
shown) Four ɣD-crystallin peaks were observed at 23187, 24426, 25666 m/z corresponding
to 1, 2, and 3 modifications. In the case of ɣS-crystallin peaks were observed at 23565,
24426, and 25666 m/z corresponding to 1, 2, and 3 modifications. The highest relative
intensity for both crystallin proteins occurred at 2 modifications.
Excellent agreement is again seen between the native and PEGylated ɣ-
crystallin proteins. It is known that in some instances the thiol groups of cysteine are
involved in intramolecular disulfide bonding which give structural integrity providing the
secondary structure. As both MM modified ɣD- and ɣS-crystallin spectra show a secondary
structure similar to native ɣ-crystallin state it was concluded that the modification did not
alter the proteins structure. (data not shown)
PEGylating ɣ-crystallin through its cysteine residues did not prevent
aggregation. The NICF curves (data not shown) have an angular dependence which resulted
in the distribution function having two distinct sets of peaks (Figure 13). The Γ versus q
graph showed two slopes with a linear angular dependence indicating the presence of a slow
and fast diffusion coefficient. (data not shown) At 37°C, the slow mode resulted in an R of
80nm for ɣD-crystallin and 85nm for ɣS-crystallin. The large size indicateed that the protein
was aggregating in solution. The fast mode corresponded to monomeric ɣ-crystallin protein,
with an R of 2.8nm and 3.0 nm for ɣD- and ɣS-crystallin at 37°C.
The fact that PEGylation via maleimide functionality did not prevent
aggregation provides critical information regarding the formation of ɣ-crystallin aggregates.
Due to MMPPEG24 and NHSPEG24 both resulting in a similar number of modifications the
surface coverage and spacer groups around the ɣ-crystallin proteins must also be similar. The
aggregate observed with MMPEG24 modification demonstrates that prevention of
aggregation cannot be not entirely dependent on hydrophilic spacer molecules.
A key insight into the aggregation mechanism is gained in realizing that
PEGylating the protein via uncharged amino acids does not prevent aggregation. The key
difference between MMPEG and NHSPEG or CAPEG is that PEGylation via maleimide
reaction does not target charged amino acids of the protein. Without being held to theory,
this suggests that surface charge and electrostatics play a major role in the formation of
aggregates and must be targeted when disrupting aggregation.
Example 7- Chemical Modification of ɣD- and ɣS- crystalline with BIOTIN
Biotin is a molecule commonly used for protein modification due to its high
binding affinity for avidin. Proteins can be tagged with biotin, undergo reactions in vivo or in
vitro, then be separated or purified from the bulk using an avidin column. The NHSBiotin
molecule was chosen because it reacts through the same mechanism as PEG4 and PEG24
however the added molecule will no longer be hydrophilic but rather contain a highly charged
end group. The molecular weight added per unit is 226.38 g/mole with a spacer arm of 1.38
NHSBiotin
The ɣ-crystallin proteins were successfully modified with biotin as shown in
the MALDI-TOF. (data not shown) ɣD-crystallin’s peak modification was eight units
(23758 m/z) while ɣS-crystallin’s peak occurrence was four units (23325 m/z). Both systems
show no unmodified ɣ-crystallin indicating reaction conditions were sufficient for thorough
modification.
The CD spectra showed a sizeable shift upwards as compared with the native
structure of the protein. (data not shown) A shallower well was observed around 220nm
which goes against the trend of good correlation between modification and secondary
structure. The change in spectra could be an indication that the secondary structure of the
proteins has been perturbed by the modification. Despite the shift in spectra, the overall
shape of the curve is similar and there was no indication of a disordered or random coil.
Aggregation of ɣ-crystallin protein was observed for ɣ-crystallin modified
with biotin. The NICF showed a slow and a fast mode which resulted in the distribution
function having two sets of peaks (Figure 14). Angular dependence of the distribution
function can be seen in the Γ versus q graph, where a qualitative difference was seen
between the slow and fast modes. At 22°C, the R of the fast and slow modes were 2.8nm
and 150nm for ɣD-crystallin, while for ɣS-crystallin these values were 2.6nm and 180nm. A
slow and fast mode was also observed at 37°C with the R values being similar to those at
22°C.
The aggregation of ɣ-crystallin modified with NHSBiotin shows that the
functionality of the modifying molecule (i.e., the molecular bristle) is important.
Mechanistically, biotin modified ɣ-crystallin in the same mannor as NHSPEG. Only
modifying charged amino acids of ɣ-crystallin is thus not sufficient in preventing
aggregation. A comparable degree of modification occurred with biotin and PEG4 meaning a
similar shift of the protein’s isoelectric point. Due to aggregation occuring in the case of ɣ-
crystallin modified with biotin it is not sufficient to purely shift the isoelectric point of the ɣ-
crystallin protein to prevent aggregation. DLS measurements of the biotinylated system also
further support the notion that aggregation cannot be prevented merely by adding spacer
molecules onto the surface of ɣ-crystallin.
The stark contrast in chemical properties between PEG and biotin is the reason
one prevents aggregation and the other does not. PEG is a flexible hydrophilic molecule
whereas the biotin functionaly is capable of hydrogen bonding and stabilizing a negative
charge. The properties of PEG thus help the solubility of proteins whereas biotin would
contribute to the hydrophobic and electrostatic nature of the aggregates.
Example 8- Chemical Modification of ɣD- and ɣS- crystalline with BIOTIN-PEG
Modifying ɣ-crystallin proteins with biotin resulted in aggregation so it was
proposed to use NHSBiotinPEG which incorporates a hydrophilic spacer between the protein
and biotin functionality. It was proposed that the hydrophilic nature of PEG would be the
key factor in the prevention of ɣ-crystallin aggregation. The NHS functionalization is used to
keep the reaction mechanism constant. The molecular weight added per unit was 825.64
g/mol and the spacer arm was 5.6 nm.
NHSBiotinPEG12
An appreciable shift in molecular weight was observed in MALDI-TOF
indicating modification of the ɣ-crystallin proteins with BiotinPEG12. (data not shown) ɣD-
crystallin had three distinct peaks observable at 22760, 23582, and 24404 m/z corresponding
to one, two, and three modifications. The resolution of ɣS-crystallin provided MALDI-TOF
peaks at 23001, 23816, 24669, 25470, and 26122 m/z corresponding to one-five
modifications. The MALDI-TOF spectra also showed no unmodified ɣ-crystallin
demonstrating sufficient reaction conditions.
The modified ɣ-crystallin protein’s secondary structure was in good agreement
with that of the native ɣ-crystallin as seen in the CD spectra. (data not shown) The well
observed with CD spectra for ɣ-crystallin modified with just biotin was no longer evident.
The PEG portion of the modification most likely allowed for increased solubility in addition
to providing a spacer between the protein and biotin functionality.
Despite the incorporation of PEG on the biotin functionality there was still
aggregate present in solution as can be seen in the slow and fast mode of the NICF. (datya
not shown) The distribution function (Figire 15) contained two sets of peaks that
demonstrate an angular dependence. The Γ versus q graph clearly showed two distinct linear
fits, the slope of which provide a slow and fast diffusive mode. (data not shown) The fast
diffusion coefficient corresponds to the monomeric ɣ-crystallin where ɣD-crystallin had an
R of 2.7nm at 22°C, while ɣS-crystallin had the value was 2.6nm. The slow diffusion
coefficient represented the large aggregate present in solution with an Rh of 105nm for ɣD-
crystallin and 115nm for ɣS-crystallin at 22°C. Aggregate of a similar R was also observed
at 37°C.
The use of PEG as a spacer between the protein and biotin functionality did
not aid in preventing aggregation. Without being held to theory and with reference to the
discussion earlier concerning biotin shifting the isoelectric point of ɣ-crystallin, these
experimental results also suggest hydrophobics to dominate over electrostatics in aggregates
forming. The long spacer arm of BiotinPEG also did not prevent aggregation giving
additional support to the trend of spacer molecule length not being a crucial factor in deturing
aggregation. It should be noted that the ɣ-crystallin tailored with BiotinPEG has a slow mode
or aggregate R which was smaller than the biotin modification suggesting that PEG does aid
in curbbing aggregation.
Example 9- Chemical Modification of ɣD- and ɣS- crystalline with Sulfo-N-
hydroxysuccinimide acetate (SA)
Due to previous modifications with large molecular weights and spacer arms
still leading to aggregation, a minimally invasive modification was studied. The final
modification was done with sulfo-N-hydroxysuccinimide acetate, which incorporated a
minimal molecular weight (43 g/mol) and spacer arm (acetate molecule) per unit added. The
reaction mechanism is similar to that of PEG4 so by reacting through the charged amino acid
groups the isoelectric point of ɣ-crystallin should be affected.
A subtle shift in the weight of the ɣ-crystallin proteins was observed in
MALDI-TOF. (data not shown) The peak relative intensity for ɣD-crystallin occurred at
22209 m/z and for ɣS-crystallin at 22562 m/z corresponding to six and seven acetate groups,
respectively. The peaks showed no unmodified protein, indicating sufficient reaction
conditions. A Gaussian distribution around the peak relative intensity indicated that some
crystallin proteins have a higher and a lower degree of modification.
A CD spectrum for both modified ɣ-crystallin proteins demonstrated a smaller
well around 220nm as compared to the native ɣ-crystallin. (data not shown) The change in
CD spectra was also observed with the biotin modification. There is reason to believe that
the secondary structure might be slightly affected by the modification although there are no
indications of a random or disordered state.
Looking at the NICF it is evident that modification of ɣ-crystallin with acetate
groups did not prevent aggregation. (data not shown) The distribution function is shown in
Figure 16. The angular dependence of the slow and fast diffusive modes can be seen in the Γ
versus q graph. (data not shown) For ɣD-crystallin at 37°C, the fast and slow diffusion
coefficients correspond to an R of 2.6nm and 85nm. The R values for ɣS-crystallin at 37°C
were 2.5nm and 90nm. The fast mode R is an appropriate size for the modified ɣ-crystallin
proteins whose molecular weight is approximately 22kDa. R values obtained from
measurements made at room temperature showed similar results.
The SA modification was performed in an attempt to shift the protein’s
isoelectric point with a minimal spacer arm. Despite an aggregate being present in solution,
the aggregate size was smaller in comparison to unmodified ɣ-crystallin. Through a high
number of modifications per protein it is possible that a substantial shift in isoelectric point
did reduce the size of the aggregate. Withour being held to theory, as aggregation is still
present in solution it was concluded that the ɣ-crystallin protein’s proximity to the isoelectric
point is not solely responsible for the aggregation phenomena.
The results of Examples 3-9 are summarized in the following table:
Table 4: Summary of results with bifunctional charge masking agents
ɣS ɣD
R (nm) R (nm) (nm) (nm)
PEG4 22ºC 2.9 2.8
PEG4 37ºC 2.9 2.9
PEG24 22ºC 3.2 3.1
PEG24 37ºC 3.2 3.1
CAPEG4
22ºC 2.8 2.9
CAPEG4
37ºC 3 2.8
MMPEG24
22ºC 3.1 100 2.9 85
MMPEG24
37ºC 3 85 2.8 80
Biotin 22ºC 2.6 180 2.8 150
Biotin 37ºC 2.8 210 2.7 170
BiotinPEG
22ºC 2.6 115 2.7 105
BiotinPEG
37ºC 2.8 110 2.6 100
SA 22ºC 2.5 92.5 2.5 82.5
SA 37ºC 2.5 90 2.6 85
The results shown herein show that electrostatics are an important component
of γ-crystallin aggregation. The electrostatic aggregation can be effectively interrupted using
high salt concentrations or bifuncional charge masking agents. A increased understanding of
the mechanism of γ-crystallin aggregation has provided a new class of agents that are
particularly useful in the treatment of cataracts and presbyopia.
Methods: Harvesting and characterization of human cadaver lenses
Within about 24 hours of death, the eyeball is harvested, sliced and the
vitrteous is removed. The lens is excised and placed in an incubation medium called
Optisol®. Optisol® is a corneal storage medium conatining chondroitin sulfate, dextran 40,
optisol base powder, sodium bicarbonate, gentamycin, amino acids, sodium pyruvate, L-
glutamine, 2-mercaptoethanol and purified water. OD is the right eye and OS is the left eye.
CAPEG4
Lens opacities are classified according to the LOCS III system. LOCS III
measurements are taken with a slit lamp miroscope. The LOCS III contains an expanded set
of standards selected from the Longitudinal Study of Cataract slide library at the Center for
Clinical Cataract Research, Boston, Mass. It includes six slit lamp images for grading
nuclear color (NC) and nuclear opalescence (NO), five retroillumination images for grading
cortical cataract (C), and five retroillumination images for grading posterior subcapsular (P)
cataract. Cataract severity is graded on a decimal scale, with the standards have regularly
spaced intervals on the scale.
Example 10: Testing of CAPEG4 in human cadaver lenses- transport across the epithelium
The transport of the CAPEG4 across an epithelium construct was studied. The
epithelium is the outer layer of the cornea and transport across the epithelium has proven to
be challenging. The goal was to deomnstrate the CAPEG4 construct could be used in a eye
drop formulation for transport through the front of the eye.
Specifically, the CAPEG4 construct in solution was added to the top of an
apparatus containing culture medium and an epithelium construct. (Figure 19). The
epithelium construct consists of a layer(s) of human corneal epithelial cells. Aliquots were
taken at the top and the bottom of the cultre medium from 15 to 120 minutes. Figure 20
shows the CAPEG4 that was transported to the bottom of the cell.
Figure 21 shows the pH of Opisol® versus the mg/ml of added CAPEG4. The
data is also presented in tables 5 and 6. Overall, it is demonstrated that the reduction in
corticle and posterior subcapsular cataracts is due to the CAPEG4 and not a pH effect of the
medium.because here was no reduction in corticle cataracts by simply changing (lowering)
the pH of the Optisol® media with citric acid to the same pH of Optisol® containing the
CaPEG amine. Reduction in opacity required the presence of the active agent.
Table 5: 1 mg/ml CAPEG4
Time Top Bottom
Min mcg/well Average mcg/well Ave rage % (stock)
427 449 4 13 2.4
472 22
448 446 26 31 5.8
445 37
454 421 8 9 1.7
388 11
459 443 9 8 1.5
428 8
467 463 17 31 5.7
459 45
453 481 21 25 4.7
510 30
1000
543 543
mcg/mL
Time 0 446 450 4 3 0.6
harvest: 453 3
Table 5: 16 mg/mL
CAPEG4
Time Top Bottom
Min mcg/well Ave rage mcg/well Ave rage % (stock)
5842 6375 58 60 0.7
6908 62
6099 6312 93 99 1.2
6526 105
6450 6972 160 149 1.8
7494 139
45 6313 6335 677 548 6.7
6356 418
6122 6457 525 525 6.4
6792 na
16000
8186
mcg/mL 8186
Blank
insret at 7857 1837 1837 23
min 7857
Example 11: Testing of CAPEG4 with human cadaver lenses- reduction of cataracts in
human cadaver lenses
In all cases, the control is Optisol® medium. The remaining data points are
for isolated human cadaver lenses treated with CAPEG4 solutions with Optisol®.
Table 6 shows the results for lenses incubated with control medium or 10
mg/mL CAPEG4 versus time. In table 6, under the heading cortical, there is a LOCS III
grade of 1.0, 1.0 and 0.9 for a lens incubated with 10 mg/ml CAPEG4. The LOCS grades for
the control are 1.0, 1.2 and 0.9. At 10 mg/mL there was no significant change in the
experimental versus control samples.
Table 6- LOCS III Grading of cataracts incubated with Optisol® or 10 mg/ml CAPEG4
versus time
[CaPEG] Time Cortical NO NC PS
(mg/mL) (HR)
(OD) 0 1.0 5.0 5.0 0.9
Contol (OS)
0 1.2 5.0 5.0 0.9
Optisol Only
24 1.0 5.0 5.0 0.9
Control
24 1.2 5.0 5.0 0.9
Optisol Only
72 0.9 5.0 5.0 0.9
Control 72 0.9 5.0 5.0 1.0
Table 7 shows the results for lenses incubated with control medium or 50
mg/mL CAPEG4 versus time. In table 7, under the heading cortical, there is a LOCS III
grade of 1.0 for all samples and a Nuclear opalescence (NO) grade of 2.0 for all samples.
There were no significant differences between the controls and 50 mg/mL incubation up tp 72
hours for cortical cataracts or for NO. Nuclear color (NC) increased slightly with time for
both the control and upon incubation in the 50 mg/mL CAPEG4 solution. There was a slight
improvement in posterior subcapsular cataraact from 1.1 to 0.9 at o and 24 hours,
respectively.
Table 7- LOCS III Grading of cataracts incubated with Optisol® or 50 mg/ml CAPEG4
versus time
[CaPEG] Time Cortical NO NC PS
(mg/mL) (HR)
50 (OD) 0 1.0 2.0 1.7 1.1
Contol (OS) 2.0
0 1.0 1.8 0.9
Optisol Only
50 24 1.0 2.0 1.7 0.9
Control 2.0 0.9
24 1.0 1.8
Optisol Only
50 72 1.0 2.0 1.9 0.9
Control 72 1.0 2.0 2.0 0.9
Table 8 shows the results for lenses incubated with control medium or 100
mg/mL CAPEG4 versus time. There is a decrease in opalescence (NO from 1.8 to 1.2) at 0
and 24 hours, respectively for CAPEG4 incubation and minor improvements in cortical and
nuclear color up to 72 hours.
Table 8- LOCS III Grading of cataracts incubated with Optisol® or 100 mg/ml CAPEG4
versus time
[CaPEG] Time Cortical NO NC PS
(mg/mL) (HR)
100 (OD) 0 3.0 1.8 1.3 0.9
Contol (OS)
0 0.9 1.9 1.5 0.9
Optisol Only
100 24 2.9 1.2 1.1 0.9
Control 0.9
24 0.9 1.8 2.0
Optisol Only
100 72 2.7 1.2 1.1 0.9
Control 72 0.9 1.9 2.0 0.9
Table 9 is a repeat of the experiment in Table 8 with different eyes. Under PS
(Posterior subcapsular cataracts) the sample incubated at 100 mg/ml CAPEG4 had values of
1.8, 1.5, and 1.0 at 0, 24 and 72 hours, respectively. There was also a reduction in cortical
cataracts from 3.0 to 2.6 to 2.0 at 0, 24, and 72 hours, respectively.
Table 9- LOCS III Grading of cataracts incubated with Optisol® or 100 mg/ml CAPEG4
versus time
[CaPEG] Time Cortical NO NC PS
(mg/mL) (HR)
100 (OD) 0 3.0 3.0 3.0 1.8
Contol (OS) 3.0 3.0
0 1.0 1.5
Optisol Only
100 24 2.6 3.0 3.0 1.5
Control 3.0 3.0
24 1.0 1.5
Optisol Only
100 72 2.0 3.0 3.2 1.0
Control 72 1.0 3.0 3.1 1.5
Table 10 shows the results for lenses incubated with control medium or 200
mg/mL CAPEG4 versus time. Cortical cataracts were reduced from 2.5 to 2.2 to 2.0 at 0, 24,
and 72 hours, respectively. The control also exhibit a slight reduction in cortical cataracts
from 3.3 to 3.0 at 24 and 72 hours, respectively.
Table 10- LOCS III Grading of cataracts incubated with Optisol® or 200 mg/ml CAPEG4
versus time
[CaPEG] Time Cortical NO NC PS
(mg/mL) (HR)
200 (OD) 0 2.5 2.4 2.4 0
Contol (OS)
0 3.3 2.5 2.5 0
Optisol Only
200 24 2.2 2.4 2.4 0
Control
24 3.3 2.5 2.5 0
Optisol Only
200 72 2.0 2.4 2.4 0
Control 72 3.0 2.5 2.5 0
Table 11 shows the results for lenses incubated with control medium or 200
mg/mL CAPEG4 versus time. The only decrease in cataract was observed in cortical
cataracts for the CAPEG4 solution going from 1.3 to 1.0 to 0.9 at 0, 24, and 72 hours,
respectively.
Table 11- LOCS III Grading of cataracts incubated with Optisol® or 200 mg/ml CAPEG4 (in
Optisol®) versus time
[CaPEG] Time Cortical NO NC PS
(mg/mL) (HR)
200 (OD) 0 1.3 3.2 2.8 0.9
Contol (OS)
0 1.4 3.2 3.0 0.9
Optisol Only
200 24 1.0 3.2 2.8 0.9
Control
24 1.4 3.2 3.0 0.9
Optisol Only
200 72 0.9 3.2 2.8 0.9
Control 72 1.4 3.2 3.0 0.9
Table 12 shows the results of changing the pH of Optisol® with no added
CAPEG4. The pH was lowered via the addition of citric acid. There was no effect of
changing the pH of the Optisol® on the cataracts.
Table 12: Efect of pH of Optisol® on cataracts
pH Time Cortical NO NC PS
(HR)
6.470 (OD) 0 2.0 5.0 5.2 0.9
6.085 (OD) 0 2.7 2.3 2.0 0.9
6.470 24 2.0 5.0 5.2 0.9
6.075 24 2.7 2.3 2.0 0.9
6.470 72 2.0 5.0 5.2 0.9
6.075 72 2.7 2.3 2.0 0.9
For cadaver lens studies, the different starting point for LOCSIII grade is due
to the varying starting condition in the patients. As the concentration of the CAPEG4
solutions in Optisol increased, wrinkling of the lens capsule was observed and subjectively
exhibited some loss of lens volume.
The terms “a” and “an” do not denote a limitation of quantity, but rather
denote the presence of at least one of the referenced item. The term “or” means “and/or”.
The terms “comprising”, “having”, “including”, and “containing” are to be construed as
open-ended terms (i.e., meaning “including, but not limited to”). All ranges disclosed herein
are inclusive and combinable.
Embodiments are described herein, including the best modes known to the
inventors. Variations of such embodiments will become apparent to those of ordinary skill in
the art upon reading the foregoing description. The skilled artisan is expected to employ such
variations as appropriate, and the disclosed methods are expected to be practiced otherwise
than as specifically described herein. Accordingly, all modifications and equivalents of the
subject matter recited in the claims appended hereto are included to the extent permitted by
applicable law. Moreover, any combination of the above-described elements in all possible
variations thereof is encompassed unless otherwise indicated herein or otherwise clearly
contradicted by context.
Claims (6)
1. The use of an ophthalmic composition in the manufacture of a medicament for contacting the eye and inhibiting or reversing the progression of cataract formation, presbyopia, or an age related degeneration of a crystalline lens in an eye, said composition comprising
2. The use of an ophthalmic composition as claimed in claim 1, wherein the ophthalmic composition is in the form of a solution, suspension, gel, ointment, spray, depot device, as an eye drop or comprised within an ophthalmic device.
3. The use of an ophthalmic composition as claimed in claim 2, wherein the ophthalmic device is a contact lens or a punctal plug.
4. The use of an ophthalmic composition according to any one of claims 1 to 3, wherein said ophthalmic composition is formulated for administration by injection, iontophoresis or ultrasound enhancement.
5. An ophthalmic composition comprised within a contact lens or a punctal plug; said composition comprising 5-24 .
6. An ophthalmic composition comprising a γ-crystallin charge masking agent in an amount of from 0.001g to 0.1g per dosage form selected from 5-24. together with one or more of buffering agents, isotonizing agents, solubilizers, preservatives, viscosity-increasing agents, chelating agents, antioxidizing agents, antibiotics, sugars, or pH regulators, in the form of a solution, suspension, gel, ointment, spray, depot device, or as an eye drop.
Applications Claiming Priority (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US201361782860P | 2013-03-14 | 2013-03-14 | |
US61/782,860 | 2013-03-14 | ||
PCT/US2014/027852 WO2014152818A1 (en) | 2013-03-14 | 2014-03-14 | Methods of inhibiting cataracts and presbyopia |
Publications (2)
Publication Number | Publication Date |
---|---|
NZ713007A NZ713007A (en) | 2020-11-27 |
NZ713007B2 true NZ713007B2 (en) | 2021-03-02 |
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