U.S. patent application number 13/081138 was filed with the patent office on 2011-10-13 for gamma-polyglutamic acid-based ocular irrigating solutions.
This patent application is currently assigned to NATIONAL HEALTH RESEARCH INSTITUTES. Invention is credited to Ko-Hua Chen, Yu-Chun CHEN, Yen-Hsien Lee, Feng-Huei Lin, Wen-Yu Su.
Application Number | 20110251137 13/081138 |
Document ID | / |
Family ID | 43921105 |
Filed Date | 2011-10-13 |
United States Patent
Application |
20110251137 |
Kind Code |
A1 |
CHEN; Yu-Chun ; et
al. |
October 13, 2011 |
GAMMA-POLYGLUTAMIC ACID-BASED OCULAR IRRIGATING SOLUTIONS
Abstract
Ophthalmic irrigating solutions are disclosed. The ophthalmic
irrigating solution comprises: a) .gamma.-polyglutamic acid
(.gamma.-PGA) and/or salt thereof in an amount effective to
increase the viscosity of the irrigating solution; and b) an
ophthalmically acceptable aqueous vehicle for the .gamma.-PGA
and/or salt thereof. Also disclosed is a method of irrigating
ocular tissues of a patient, in which the method comprises
introducing to the ocular tissues of the patient an ophthalmic
irrigating solution comprising .gamma.-PGA) and/or salt thereof in
an amount sufficient to irrigate the ocular tissues of the
patient.
Inventors: |
CHEN; Yu-Chun; (Miaoli
County, TW) ; Su; Wen-Yu; (Miaoli County, TW)
; Lee; Yen-Hsien; (Miaoli County, TW) ; Chen;
Ko-Hua; (Taipei, TW) ; Lin; Feng-Huei; (Miaoli
County, TW) |
Assignee: |
NATIONAL HEALTH RESEARCH
INSTITUTES
Miaoli County
TW
|
Family ID: |
43921105 |
Appl. No.: |
13/081138 |
Filed: |
April 6, 2011 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
61322738 |
Apr 9, 2010 |
|
|
|
Current U.S.
Class: |
514/20.8 |
Current CPC
Class: |
A61K 31/74 20130101;
A61K 31/198 20130101; A61P 27/04 20180101; A61K 31/765 20130101;
A61P 27/02 20180101 |
Class at
Publication: |
514/20.8 |
International
Class: |
A61K 38/02 20060101
A61K038/02; A61P 27/04 20060101 A61P027/04 |
Claims
1. A method of irrigating ocular tissues of a patient, comprising:
introducing to the ocular tissues of the patient an ophthalmic
irrigating solution in an amount sufficient to irrigate the ocular
tissues of the patient, the solution comprising: (a)
.gamma.-polyglutamic acid (.gamma.-PGA) and/or salt thereof in an
amount effective to increase the viscosity of the solution; and (b)
an ophthalmically acceptable aqueous vehicle for the .gamma.-PGA
and/or salt thereof.
2. The method of claim 1, wherein the irrigating solution has a
viscosity of 0.32 to 50, or 0.32 to 30, or 0.32 to 3.93
centipoise.
3. The method of claim 1, wherein the irrigating solution has an
osmolarity of 290 to 320 mOsm per Liter.
4. The method of claim 1, wherein the irrigating solution does not
contain cross-linked polyglutamic acid and has no additional
polyamino acid or polymer.
5. The method of claim 1, wherein the irrigating solution has a
refractive index of 1.330 to 1.344.
6. The method of claim 1, wherein the ophthalmically acceptable
aqueous vehicle comprises a balanced salt solution containing
electrolytes, a buffer and an energy source.
7. An ophthalmic irrigating solution comprising: (a)
.gamma.-polyglutamic acid (.gamma.-PGA) and/or salt thereof in an
amount effective to increase the viscosity of the irrigating
solution; and (b) an ophthalmically acceptable aqueous vehicle for
the .gamma.-PGA and/or salt thereof.
8. The ophthalmic irrigating solution of claim 7, wherein the
irrigating solution has a viscosity of 0.32 to 50, or 0.32 to 30,
or 0.32 to 3.93 centipoise.
9. The ophthalmic irrigating solution of claim 7, wherein the
irrigating solution has an osmolarity of 290 to 320 mOsm per
Liter.
10. The ophthalmic irrigating solution of claim 7, wherein the
irrigating solution has a refractive index of 1.330 to 1.344.
11. The ophthalmic irrigating solution of claim 7, wherein the
irrigating solution does not contain cross-linked polyglutamic acid
and has no additional polyamino acid or polymer.
12. The ophthalmic irrigating solution of claim 7, wherein the
ophthalmically acceptable aqueous vehicle comprises a balanced salt
solution containing electrolytes, a buffer and an energy
source.
13. The ophthalmic irrigating solution of claim 7, wherein the
.gamma.-PGA has a molecular weight of 10,000 to 2,000,000 Daltons,
or 1,000,000 to 2,000,000 Daltons.
14. A pharmaceutical kit comprising: (a) an ophthalmic irrigating
solution according to claim 7; and (b) an a package insert
containing printed instructions for irrigating ocular tissues of a
patient.
15. A method of reducing stress-induced damage to ocular tissues of
a patient during eye surgery, comprising: introducing to the ocular
tissues of the patient during the eye surgery an ophthalmic
irrigating solution in an amount sufficient to irrigate the ocular
tissues of the patient, the irrigating solution comprising: (a)
.gamma.-polyglutamic acid (.gamma.-PGA) and/or salt thereof in an
amount effective to increase the viscosity of the solution; and (b)
an ophthalmically acceptable aqueous vehicle for the .gamma.-PGA
and/or salt thereof.
16. The method of claim 15, wherein the eye surgery includes
surgical vitrectomy, cataract extraction, lens aspiration, anterior
segment reconstruction and phacoemulsification.
17. The method of claim 1, wherein the irrigating solution has a
viscosity of 0.32 to 3.93 centipoise.
18. The ophthalmic irrigating solution of claim 7, wherein the
irrigating solution has a viscosity of 0.32 to 3.93 centipoise.
19. The ophthalmic irrigating solution of claim 7, wherein the
ophthalmically acceptable aqueous vehicle further comprises an
antioxidant.
20. The ophthalmic irrigating solution of claim 8, wherein the
irrigating solution has an osmolarity of 290 to 320 mOsm per Liter.
Description
REFERENCE TO RELATED APPLICATION
[0001] The present application claims the priority to U.S.
Provisional Application Ser. No. 61/322,738, filed Apr. 9, 2010,
which is herein incorporated by reference in its entirety.
FIELD OF THE INVENTION
[0002] The present invention relates generally to ocular solutions,
and more specifically to ocular irrigating solutions.
BACKGROUND OF THE INVENTION
[0003] Irrigating solutions are widely used in intraocular surgical
procedures, such as phacoemulsification, vitrectomy surgery and
glaucoma surgery. Phacoemulsification is a surgery to remove the
lens of the eye that has developed an opacification, which is
referred to as a cataract. Vitrectomy is a surgery to remove some
or all of the vitreous humor from an eye. Glaucoma surgery is
associated with a laser treatment or making a cut in the eye to
reduce the intraocular pressure. In Taiwan, according to a report
of National Health Insurance Department, about 150,000 ocular
surgical procedures were performed annually. The effect of
intraocular surgery is related to the irrigating solution used. An
improper irrigating solution may cause damages to cornea or lens,
resulting in poor vision, blind spots and even loss of vision.
[0004] A desired irrigating solution is supposed to have a
composition that is close to aqueous humor and an osmolarity of
between 290 mOsm and 320 mOsm. The major function of irrigating
solutions is for maintaining endothelium cell integrity, corneal
thickness and retinal tissue. Moreover, an appropriate irrigating
solution should preserve the viability of corneal endothelial cells
during cataract surgery, provide an energy source (i.e., glucose),
maintain appropriate tonicity and electrolyte concentration, and
protect corneal endothelium cells from fluctuation of pH value.
[0005] Balanced Salt Solution (BSS.RTM.) and BSS PLUS.RTM. have
been frequently used for intraocular irrigation. The composition of
BSS PLUS.RTM. is close to that of the aqueous humor. Basically, the
composition of BSS PLUS.RTM. has four parts: 1) adequate buffer
(i.e., bicarbonate), 2) energy source (i.e., glucose). 3) stable pH
value between 7 and 8 (i.e., HEPES). 4) antioxidant agent (i.e.,
glutathione). However, these intraocular irrigating solutions are
not effectively enough to protect corneal (endothelial) cells,
which are most liable to sustain physical damage in ophthalmic
operations. Studies have indicated that a sophisticated intraocular
surgery, such as phacoemulsification, may cause potential
complications. Some possible reasons for causing complications
include the mechanical effects of ultrasound, physical trauma
caused by nonaspirated lens fragments, heat production and even
osmotic irregularities caused by the irrigating solution. All of
these may result in damages to corneal endothelium and even may
have the risk of leading to irreversible bullous keratopathy.
[0006] Therefore, a heretofore unaddressed need exists in the art
to address the aforementioned deficiencies and inadequacies,
especially in connection with development of ocular irrigating
solutions with an improved functionality to protect intraocular
tissues, particularly corneal (endothelial) cells.
SUMMARY OF THE INVENTION
[0007] In one aspect, the invention relates to a method of
irrigating ocular tissues of a patient. The method comprises
introducing to the ocular tissues of the patient an ophthalmic
irrigating solution in an amount sufficient to irrigate the ocular
tissues of the patient, in which the solution comprises: a)
.gamma.-polyglutamic acid (.gamma.-PGA) and/or salt thereof in an
amount effective to increase the viscosity of the solution; and b)
an ophthalmically acceptable aqueous vehicle for the .gamma.-PGA
and/or salt thereof.
[0008] In another aspect, the invention relates to an ophthalmic
irrigating solution comprising: a) .gamma.-polyglutamic acid
(.gamma.-PGA) and/or salt thereof in an amount effective to
increase the viscosity of the irrigating solution; and b) an
ophthalmically acceptable aqueous vehicle for the .gamma.-PGA
and/or salt thereof.
[0009] Further in another aspect, the invention relates to a
pharmaceutical kit comprising: a) an ophthalmic irrigating solution
as aforementioned; and b) a package insert containing printed
instructions for irrigating ocular tissues of a patient.
[0010] Yet in another aspect, the invention relates to a method of
reducing stress-induced damage to ocular tissues of a patient
during eye surgery. The method comprises introducing to the ocular
tissues of the patient during the eye surgery an ophthalmic
irrigating solution in an amount sufficient to irrigate the ocular
tissues of the patient, in which the irrigating solution comprises:
a) .gamma.-polyglutamic acid (.gamma.-PGA) and/or salt thereof in
an amount effective to increase the viscosity of the solution; and
b) an ophthalmically acceptable aqueous vehicle for the .gamma.-PGA
and/or salt thereof.
[0011] These and other aspects will become apparent from the
following description of the preferred embodiment taken in
conjunction with the following drawings, although variations and
modifications therein may be affected without departing from the
spirit and scope of the novel concepts of the disclosure.
[0012] The accompanying drawings illustrate one or more embodiments
of the invention and, together with the written description, serve
to explain the principles of the invention. Wherever possible, the
same reference numbers are used throughout the drawings to refer to
the same or like elements of an embodiment.
BRIEF DESCRIPTION OF THE DRAWINGS
[0013] FIGS. 1A-1C show the effects of .gamma.-PGA and dextrose
concentrations on the osmolarity of the irrigating solution.
[0014] FIG. 2 is a graph showing the effect of .gamma.-PGA on the
viscosity of the irrigating solution.
[0015] FIG. 3 is a graph showing the effect of .gamma.-PGA on the
refractive index of the irrigating solution.
[0016] FIGS. 4A-4B show .gamma.-PGA had no significant effect on
cell proliferation. (A) bovine corneal endothelial cell. (B) human
retinal pigment epithelial cells.
[0017] FIGS. 5A-5B show .gamma.-PGA had no significant cytotoxic
effect on cells. (A) bovine corneal endothelial cells. (B) human
retinal pigment epithelial cells.
[0018] FIGS. 6-7 show a collection of photomicrographs of cells
stained with dyes for simultaneous detection of viable and dead
cells after culture with or without .gamma.-PGA under a fluorescent
microscope. (6A) bovine corneal endothelial cells cultured for one
day. (6B) bovine corneal endothelial cells cultured for 3 days.
(7A) human retinal pigment cultured for one day. (7B) human retinal
pigment epithelial cells cultured for 3 days. The negative control:
Alumina (Al.sub.2O.sub.3) extraction medium; the positive control:
Triton x-100 (0.1%) in DMEM/F12 medium.
[0019] FIG. 8A shows two vein detained needles inserted into an eye
of a rabbit.
[0020] FIG. 8B shows perfusion of irrigation solution by a
peristaltic pump.
[0021] FIG. 9 is a graph showing changes in cornea thickness during
the period of ocular irrigation.
DETAILED DESCRIPTION OF THE INVENTION
Definitions
[0022] The terms used in this specification generally have their
ordinary meanings in the art, within the context of the invention,
and in the specific context where each term is used. Certain terms
that are used to describe the invention are discussed below, or
elsewhere in the specification, to provide additional guidance to
the practitioner regarding the description of the invention. For
convenience, certain terms may be highlighted, for example using
italics and/or quotation marks. The use of highlighting has no
influence on the scope and meaning of a term; the scope and meaning
of a term is the same, in the same context, whether or not it is
highlighted. It will be appreciated that same thing can be said in
more than one way. Consequently, alternative language and synonyms
may be used for any one or more of the terms discussed herein, nor
is any special significance to be placed upon whether or not a term
is elaborated or discussed herein. Synonyms for certain terms are
provided. A recital of one or more synonyms does not exclude the
use of other synonyms. The use of examples anywhere in this
specification including examples of any terms discussed herein is
illustrative only, and in no way limits the scope and meaning of
the invention or of any exemplified term. Likewise, the invention
is not limited to various embodiments given in this
specification.
[0023] Unless otherwise defined, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention pertains. In the
case of conflict, the present document, including definitions will
control.
[0024] As used herein, "around", "about" or "approximately" shall
generally mean within 20 percent, preferably within 10 percent, and
more preferably within 5 percent of a given value or range.
Numerical quantities given herein are approximate, meaning that the
term "around", "about" or "approximately" can be inferred if not
expressly stated.
[0025] As used herein, when a number or a range is recited,
ordinary skill in the art understand it intends to encompass an
appropriate, reasonable range for the particular field related to
the invention.
[0026] By a viscosity of 0.32 to 50 centipoise it meant that all
hundredth, tenth and integer unit amounts within the range are
specifically disclosed as part of the invention. Thus, 0.32, 0.33,
0.34 . . . and 0.7, 0.8, 0.9 and 1, 2, 3, 4 . . . 47, 48, 49 and 50
centipoise unit amounts are included as embodiments of this
invention.
[0027] By an osmolarity of 290 to 320 mOsm it meant that all
integer unit amounts within the range are specifically disclosed as
part of the invention. Thus, 290, 291, 292 . . . and 317, 318, 319
and 320 mOsm unit amounts are included as embodiments of this
invention.
[0028] By a refractive index of 1.330 to 1.344 it meant that all
thousandth unit amounts within the range are specifically disclosed
as part of the invention. Thus, 1.330, 1.331, 1.332 . . . and
1.340, 1.341, 1.342, 1.343 and 1.344 unit amounts are included as
embodiments of this invention.
[0029] By a molecular weight of 10,000 to 2,000,000 Daltons it
meant that all integer unit amounts within the range are
specifically disclosed as part of the invention. Thus, 10,000,
10,001, 10,002 . . . and 1,999,997, 1,999,998, 1,999,999 and
2,000,000 Daltons unit amounts are included as embodiments of this
invention.
[0030] By 0.2.about.1% (w/v) it meant that all tenth and integer
unit amounts within the range are specifically disclosed as part of
the invention. Thus, 0.2, 0.3, 0.4 . . . and 0.7, 0.8, 0.9 and 1%
unit amounts are included as embodiments of the invention.
[0031] As used herein, ".gamma.-PGA" shall generally mean
"gamma-polyglutamic acid and/or salt thereof" or
"gamma-polyglutamate." Glutamic acid has 2 carboxyl groups. One of
them, .gamma.-carboxyl group is linked with .alpha.-amino group and
PGA is formed.
[0032] The terms Gamma-Poly(glutamic acid) and Gamma-Polyglutamic
acid (.gamma.-PGA) are interchangeable.
[0033] As used herein, the term "in an effective amount to increase
the viscosity of the irrigating solution" shall generally mean that
the viscosity of the irrigating solution is increased in the
presence of .gamma.-PGA compared to the viscosity of the irrigating
solution without the addition of .gamma.-PGA.
[0034] Cross-linked polyglutamic acid consists of mesh structure of
tens of millions by molecular weight. Compared to PGA, cross-linked
PGA has higher water absorption capability. The molecular weight of
cross-linked polyglutamic acid is more than 10,000,000.
[0035] As used herein, "an aqueous physiologically acceptable
solution" shall generally mean but not limited to sterile saline or
sterile buffered solution.
[0036] As used herein, an "antioxidant" is a molecule capable of
slowing or preventing the oxidation of other molecules.
Antioxidants include but not limited to glutathione, vitamin C, and
vitamin E.
[0037] Osmosis is the movement of solvent molecules through a
selectively-permeable membrane into a region of higher solute
concentration, aiming to equalize the solute concentrations on the
two sides. Net movement of solvent is from the less-concentrated
(hypotonic) to the more-concentrated (hypertonic) solution, which
tends to reduce the difference in concentrations. This effect can
be countered by increasing the pressure of the hypertonic solution,
with respect to the hypotonic. The osmotic pressure is defined to
be the pressure required to maintain an equilibrium, with no net
movement of solvent. The osmotic pressure depends on the molar
concentration of the solute but not on its identity. Osmosis is
important in biological systems, as many biological membranes are
semipermeable.
[0038] Osmolarity is the measure of solute concentration, defined
as the number of osmoles (Osm) of solute per liter (L) of solution
(osmol/L or Osm/L). The osmolarity of a solution is usually
expressed as Osm/L. Osmolarity measures the number of osmoles of
solute particles per unit volume of solution.
[0039] The invention relates to the discovery of the viscoelastic
material poly-.gamma.-glutamic acid (.gamma.-PGA) as an additional
ingredient in irrigating solutions to reduce injury caused by eye
surgery. Dispersive viscoelastic materials have the positive effect
on protecting intraocular tissues during phacoemulsification and
aspiration (PEA). Viscoelastic materials can reduce the turbulence
within the anterior and posterior chambers of the eye and help
contain the movement of tissue fragments and air bubbles within the
eye. Besides, such kinds of viscoelastic materials can facilitate
the removal of lens fragments and make it easier for a surgeon to
track the fragments with the tip of a surgical hand piece.
[0040] Poly-.gamma.-glutamic acid (.gamma.-PGA), a natural polymer
of the amino acid glutamic acid (GA), is synthesized by several
bacteria (all Gram-positive), one archaea and one eukaryote. The
structure of PGA is shown below:
##STR00001##
[0041] Poly-.gamma.-glutamic acid has a molecular weight ranging
from about 10,000 up to 2 millions. It can be produced to meet the
requirements of different applications. It is well known for its
application in food industry. .gamma.-PGA is a major composition of
mucilage of "natto" (one kind of traditional Japan food), which was
first discovered by Ivnovics as a capsule of Bacillus anthracis in
1937. .gamma.-PGA is a unique polyanionic polymer composed of D
form and/or L form glutamic acid residues connected by
.gamma.-amide bonds (between the .alpha.-amino and
.gamma.-carboxylic groups). It is a hydrophilic, viscous,
biodegradable and non-toxic biomaterial. Due to the unique
properties on ion trapping and high water absorbance, it has been
widely used in various applications, such as metal chelate,
absorbent, cryoprotectant, ageing inhibitor, drug carrier and
humectant. .gamma.-PGA has been widely used as a biomaterial with a
fine swelling ability during the past few years. The
biocompatibility makes it practicable for use in clinical fields
such as bioglue, tissue engineering and drug delivery systems.
[0042] The invention relates to the discovery that .gamma.-PGA has
the ability to adjust the osmolarity and viscosity of an
ophthalmically acceptable irrigating solution. Adequate osmolarity
and slight viscosity of an ophthalmic irrigating solution should be
able to reduce complications and injury of cornea. The present
invention relates to use of .gamma.-PGA as an additive for an
ophthalmically acceptable irrigating solution or a surgical
solution to provide the anterior and posterior chambers of the eye
with protections during surgical procedures that require
irrigation.
[0043] .gamma.-PGA serves as an agent for adjusting the osmolarity
and avoiding edema phenomena of the tissue. The osmolarity of the
ophthalmically acceptable irrigating solution in the presence of
.gamma.-PGA is in the range of 290-320 mOsm. The high molecular
weight (1020 k Daltons) .gamma.-PGA has the ability to adjust the
viscosity of the irrigating solution. An optimal viscosity can
reduce tissue injury caused by phacoemulsification.
[0044] Various types of vehicles for the .gamma.-PGA may be
utilized. However, the vehicle preferably contains electrolytes, a
buffer (e.g., bicarbonate, phosphate or a combination thereof), and
an energy source. These agents help to maintain the normal function
of corneal tissues during the surgical procedure and promote a
rapid recovery of visual acuity subsequent to the surgery. However,
the invention is not limited relative to the types of balanced salt
solutions or other electrolyte/nutrient solutions that may be
utilized to form the irrigating solutions described herein.
[0045] In one aspect, the invention relates to a method of
irrigating ocular tissues of a patient. The method comprises
introducing to the ocular tissues of the patient an ophthalmic
irrigating solution in an amount sufficient to irrigate the ocular
tissues of the patient, in which the solution comprises: a)
.gamma.-polyglutamic acid (.gamma.-PGA) and/or salt thereof in an
amount effective to increase the viscosity of the solution; and b)
an ophthalmically acceptable aqueous vehicle for the .gamma.-PGA
and/or salt thereof.
[0046] In another aspect, the invention relates to an ophthalmic
irrigating solution comprising: a) .gamma.-polyglutamic acid
(.gamma.-PGA) and/or salt thereof in an amount effective to
increase the viscosity of the irrigating solution; and b) an
ophthalmically acceptable aqueous vehicle for the .gamma.-PGA
and/or salt thereof.
[0047] Further in another aspect, the invention relates to a
pharmaceutical kit comprising: a) an ophthalmic irrigating solution
as aforementioned; and b) an a package insert containing printed
instructions for irrigating ocular tissues of a patient.
[0048] In one embodiment of the invention, the irrigating solution
has a viscosity of 0.32 to 50, or 0.32 to 30, or 0.32 to 3.93
centipoise.
[0049] In another embodiment of the invention, the irrigating
solution has an osmolarity of 290 to 320 mOsm per Liter.
[0050] In another embodiment of the invention, the irrigating
solution has a viscosity of 0.32 to 50, or 0.32 to 30, or 0.32 to
3.93 centipoise and an osmolarity of 290 to 320 mOsm per Liter.
[0051] In another embodiment of the invention, the irrigating
solution has a refractive index of 1.330 to 1.344.
[0052] In another embodiment of the invention, the irrigating
solution does not contain cross-linked polyglutamic acid and has no
additional polyamino acid or polymer.
[0053] In another embodiment of the invention, the ophthalmically
acceptable aqueous vehicle comprises a balanced salt solution
containing electrolytes, a buffer and an energy source.
[0054] In another embodiment of the invention, the concentration of
.gamma.-PGA in the ophthalmic irrigating solution ranges from
0.2-1% or 0.2-0.8% (w/v).
[0055] In another embodiment of the invention, the .gamma.-PGA has
a molecular weight of 10,000 to 2,000,000 Daltons, or 1,000,000 to
2,000,000 Daltons.
[0056] Further in another embodiment of the invention, the
ophthalmically acceptable aqueous vehicle further comprises an
antioxidant.
[0057] Yet in another aspect, the invention relates to a method of
reducing stress-induced damage to ocular tissues of a patient
during eye surgery. The method comprises introducing to the ocular
tissues of the patient during the eye surgery an ophthalmic
irrigating solution in an amount sufficient to irrigate the ocular
tissues of the patient, in which the irrigating solution comprises:
a) .gamma.-polyglutamic acid (.gamma.-PGA) and/or salt thereof in
an amount effective to increase the viscosity of the solution; and
b) an ophthalmically acceptable aqueous vehicle for the .gamma.-PGA
and/or salt thereof.
[0058] In one embodiment of the invention, the eye surgery includes
surgical vitrectomy, cataract extraction, lens aspiration, anterior
segment reconstruction and phacoemulsification.
EXAMPLES
[0059] Without intent to limit the scope of the invention,
exemplary instruments, apparatus, methods and their related results
according to the embodiments of the present invention are given
below. Note that titles or subtitles may be used in the examples
for convenience of a reader, which in no way should limit the scope
of the invention. Moreover, certain theories are proposed and
disclosed herein; however, in no way they, whether they are right
or wrong, should limit the scope of the invention so long as the
invention is practiced according to the invention without regard
for any particular theory or scheme of action.
Materials and Methods
Materials and Reagent
[0060] All materials and reagents used were purchased from
Sigma-Aldrich, Inc. (St. Louis, Mo., USA) unless otherwise stated.
Poly-.gamma.-glutamate (molecular weight 1020 k Da) was purchased
from VEDAN Enterprise Corporation. Osmometer standards were
obtained from Advanced Instruments, Inc. (Norwood, Mass., USA).
[0061] Antibiotic, trypsin and fetal bovine serum were obtained
from Invitrogen (Carlsbad, Calif.). Flasks and culture well/dishes
were obtained from Orange Scientific (Braine-l'Alleud, Belgium).
Qick Cell Proliferation Assay Kit was procured from BioVision (CA,
USA) and cytotoxicity assay was purchased from Promega (CytoTox 96
Assay kit, WI, USA). Bovine cornea endothelial cells (bCE cells)
and human retina pigmented epithelium cells (hRPE cells) were
obtained from the National Center for Cell Sciences (Food Industry
Research and Development Institute, Hsinchu, Taiwan).
Methods
Osmolarity Evaluation of the Irrigating Solution
[0062] The osmolarity of the solution was measured in duplicates
using a 3D3 Single-Sample
[0063] Osmometer manufactured by Advanced Instrument, Co., Inc. To
investigate the effect of .gamma.-PGA, irrigating solutions
containing various concentrations of .gamma.-PGA, 0.2%, 0.4%, 0.6%,
0.8%, 1% and control (without .gamma.-PGA) were prepared.
Calibration standards (100 and 1500 mOsm per Liter) were used to
calibrate the performance of the osmometer. The pH value of the
.gamma.-PGA-containing irrigating solution was 7.4.+-.0.1. Sodium
bicarbonate was used as a pH buffer and double distilled water was
used to make irrigating solutions.
Viscosity Evaluation of the Irrigating Solution
[0064] A HAAKE RheoStress 600 (Thermo Fisher Scientific Inc.,
Waltham, Mass., USA) instrument with parallel plate geometry was
used to evaluate the viscosity of the irrigating solution. The
temperature was controlled by control units. Two working
temperatures, room temperature (25.degree. C.) and body temperature
(37.degree. C.), were evaluated. The gap height between the upper
(35 mm in diameter) and bottom stainless steel plates was set at
1.05 mm. Controlled rate rotation ramp mode was used to obtain the
viscosity curve of the irrigating solution. The range of the shear
rate was from 1 s.sup.-1 to 500 s.sup.-1.
Refractive Index Evaluation of the Irrigating Solution
[0065] A DR-A 1 refractometer (ATAGO, Japan) was used to measure
the refractive index (RI). Irrigating solutions containing 0.2,
0.4, 0.6, 0.8 and 1% (w/v) of .gamma.-PGA were prepared at room
temperature and placed carefully on the prism. While looking
through the eyepiece, the control knob was turned until the shadow
line was centered in the crosshairs. The value of refractive index
was taken from the digital screen.
Biocompatibility Studies of .gamma.-PGA
[0066] Different concentrations of .gamma.-PGA were added to
culture medium to evaluate the biocompatibility. Two hundred
microliters of the medium was tested on a monolayer of corneal
endothelium cells and retinal pigment epithelium cells. Cells were
seeded onto 96-well tissue culture plates at a cell density of
5.times.10.sup.3 cells/well, allowed to adhere overnight at
37.degree. C. under 5% carbon dioxide atmosphere. Groups including
a negative control (Al.sub.2O.sub.3 extraction medium), a positive
control (0.1% Triton X-100 contained medium) and experimental
groups (medium with 0.2%, 0.4%, 0.6%, 0.8% and 1% .gamma.-PGA) were
tested in hexaplicate. After incubation at 37.degree. C. for 24 h
and 72 h, the cell viability and cytotoxicity evaluations were
quantitatively assessed using Quick Cell Proliferation Assay Kit II
and CYTOTOX 96.RTM. Non-Radioactive Cytotoxicity Assay.
[0067] For cell viability evaluation, the test medium after 72 h
incubation was discarded and 0.2 ml of water-soluble tetrazolium-8
(WST-8) working solution was transferred into each well. After 2 h
incubation, the WST-8 working solution showed color changes due to
cleavage of tetrazolium salt and formation of formazan by cellular
mitochondrial dehydrogenases. The viability of corneal endothelium
cells and retinal pigment epithelium cells was quantitatively
assessed by spectrophotometer readout at 450 nm. The reference
wavelength was 650 nm.
[0068] For cytotoxicity evaluation, 0.05 ml of the incubation
medium was transferred into 96-well ELSA plates, mixed with 0.05 ml
of the substrate mix and incubated for 30 minutes in the dark. The
tetrazolium salt in the substrate mix reacts with lactate
dehydrogenase (LDH) and gives a red formazan product. LDH released
into the medium was quantitatively assessed by spectrophotometer
readout at 490 nm. Medium (without incubation with cells) was also
evaluated to serve as a culture medium background. All corneal
endothelium cells and retinal pigment epithelium cells were lysed
with lysis solution (1% TRITON X-100) and the OD.sub.490 value was
read. Percent cytotoxicity was expressed as follows:
% Cytotoxicity = Medium O . D . - Blank O . D . Total Lysis O . D .
- Blank O . D . .times. 100 ##EQU00001##
Fluorescence Staining
[0069] Cells cultured in the medium containing .gamma.-PGA at a
concentration of 0.2, 0.4, 0.6, 0.8 and 1% (w/v) for one day and 3
days were respectively stained with a LIVE/DEAD staining kit
(Molecular Probes # L3224, Eugene, Oreg., USA) and photographed by
using NIS Element software.
In Vivo Study of the .gamma.-Poly(Glutamic Acid)-Based Ocular
Irrigation Solution
[0070] Six eyes of three New Zealand white rabbits (2-3 kg) were
used. The surgeries were performed under general anesthesia by
intramuscular injection of ketalar/Chanazine 2% (Ketamine: 22 mg/kg
BW; Xylazine: 4-6 mg/kg BW). Under an operating microscope, two
vein detained needles were carefully inserted through cornea
without touching the lens (FIG. 8A). One of the vein detained
needles was connected to a bottle full of the experimental ocular
irrigating solution, and the other was connected to an empty bottle
(FIG. 8B). The corneal endothelium was perfused with the irrigation
solution at 37.degree. C. by using a peristaltic pump (flow rate of
5 mL/min) (FIG. 8B) for 60 minutes. The right eye was irrigated
with 0.4% (w/v) of .gamma.-poly(glutamic acid)-based ocular
irrigating solution and the left eye irrigated with a normal saline
solution. Corneal thickness was measured at intervals of five to
ten minutes during the course of perfusion.
Corneal Thickness Measurement
[0071] An ultrasonic pachymeter DGH 550 (DGH Technology) with a
hand-held transducer was used to measure central corneal thickness.
The DGH 550 is an ultrasonic pachymeter that uses echo spike
techniques to measure the thickness of the cornea. Cornea thickness
was measured using the Ultrasonic pachymeter at intervals of five
to ten minutes during the course of perfusion.
Statistical Analysis
[0072] Statistical analysis was conducted at least in triplicate,
and the results are reported as mean.+-.standard deviation (SD).
Analysis of variance (ANOVA) was used to evaluate the influence of
.gamma.-PGA on biocompatibility. Differences with p values less
than 0.05 were considered statistically significant.
Results
Osmolarity of the .gamma.-PGA-Containing Irrigating Solution
[0073] The effects of dextrose and .gamma.-PGA on the osmolarity of
the irrigating solution are shown in FIGS. 1A and 1B, respectively.
Table 1 shows the compositions of dextrose-containing (FIG. 1A) and
.gamma.-PGA-containing (FIG. 18) irrigating solutions.
TABLE-US-00001 TABLE 1 Dextrose-containing .gamma.-PGA-containing
Ingredient (mM) irrigating solution irrigating solution NaCl 122
122 KCl 5.08 5.08 CaCl.sub.2 1.05 1.05 MgCl.sub.2 0.98 0.98
NaHCO.sub.3 25.0 25.0 Na.sub.2HPO.sub.4 3.0 3.0 HCl or NaOH Adjust
pH to 7.2~7.4 Adjust pH to 7.2~7.4 Dextrose 0~1 -- r-PGA -- 0~1
(%)
[0074] The osmolarities of the irrigating solutions containing 5 mM
dextrose and 0.2, 0.4, 0.6, 0.8 and 1% .gamma.-PGA were 304, 309,
314, 319 and 325 mOsm, respectively (FIG. 1C). When the .gamma.-PGA
concentration was decreased from 1% to 0.2%, the osmolarity of the
5 mM dextrose-containing irrigating solution decreased from 325 to
304 mOsm. However, the osmolarity of the irrigating solutions
containing 1, 2, 3, 4, 5 mM dextrose without .gamma.-PGA were 295,
296, 296, 298, 300 mOsm, respectively (FIG. 1A). Thus, dextrose had
less effect on the osmolarity of the irrigating solution than
.gamma.-PGA.
Viscosity of the .gamma.-PGA-Containing Irrigating Solution
[0075] FIG. 2 shows .gamma.-PGA increased the viscosity of the
irrigating solution in a concentration-dependent manner and the
presence of 5 mM dextrose had no impact on the viscosity. The
viscosities of the irrigating solutions containing 5 mM dextrose
and 0, 0.2, 0.4, 0.6, 0.8, or 1% .gamma.-PGA at room temperature
(25.degree. C.) were 0.62, 1.15, 1.72, 2.39, 3.11, and 3.93
centipoise (cP), respectively (Table 2). The viscosity of the
irrigation solution decreased when the temperature was increased to
the body temperature (37.degree. C.). The viscosities of the
irrigating solutions containing 5 mM dextrose and 0, 0.2, 0.4, 0.6,
0.8, or 1% .gamma.-PGA at 37.degree. C. were 0.32, 0.75, 1.20,
1.73, 2.33, and 2.96 cP, respectively (Table 3).
TABLE-US-00002 TABLE 2 Viscosity at 25.degree. C. (cP) Dextrose
conc. .gamma.-PGA (%) 0 mM 5 mM 0 0.59 0.62 0.2 1.11 1.15 0.4 1.71
1.72 0.6 2.39 2.39 0.8 3.16 3.11 1.0 3.98 3.93
TABLE-US-00003 TABLE 3 Viscosity at 37.degree. C. (cP) Dextrose
conc. .gamma.-PGA (%) 0 mM 5 mM 0 0.48 0.32 0.2 0.70 0.75 0.4 1.26
1.20 0.6 1.84 1.73 0.8 2.36 2.33 1.0 3.01 2.96
Refractive Index of .gamma.-PGA-Containing Irrigating Solution
[0076] The refractive indexed of the irrigating solutions
containing 0.25, 0.5, 0.75 and 1% (w/v) of .gamma.-PGA were 1.3346,
1.3350, 1.3355 and 1:3360, respectively (FIG. 3). The composition
of the irrigating solution comprises 122 mM NaCl, 5.08 mM KCl, 1.05
mM CaCl.sub.2, 0.98 mM MgCl.sub.2, 25.0 mM NaHCO.sub.3, 3.0 mM
Na.sub.2HPO.sub.4, 0.about.1% (w/v) of .gamma.-PGA, and HCl or NaOH
to adjust pH to 7.2-7.4.
Biocompatibility of .gamma.-PGA
[0077] Cell viability and cytotoxicity were evaluated on bovine
corneal endothelium (bCE) cells and human retinal pigment
epithelium (hRPE) cells cultured in the .gamma.-PGA-containing
medium on day 1 and day 3 by WST-8 and LDH assays (FIGS. 4A-B, and
FIGS. 5A-B). The WST-8 assay was used to measure the number of
viable cells. The OD.sub.450nm of the medium from bCE cells treated
with 0.2%, 0.4%, 0.6%, 0.8%, and 1% (w/v) of .gamma.-PGA were
0.20.+-.0.02, 0.19.+-.0.02, 0.20.+-.0.02, 0.19.+-.0.01 and
0.17.+-.0.04 on day 1; 0.53.+-.0.05, 0.47.+-.0.04, 0.46.+-.0.10,
0.44.+-.0.06 and 0.41.+-.0.03 on day 3, respectively (FIG. 4A). The
OD.sub.450nm of the medium from hRPE cells treated with 0.2%, 0.4%,
0.6%, 0.8%, and 1% .gamma.-PGA were 0.47.+-.0.05, 0.51.+-.0.05,
0.52.+-.0.02, 0.51.+-.0.06 and 0.47.+-.0.07 on day 1, 0.88.+-.0.08,
0.88.+-.0.03, 0.81.+-.0.07, 0.78.+-.0.09 and 0.79.+-.0.10 on day 3,
respectively (FIG. 4B). .gamma.-PGA at a concentration from 0.2 to
1% had no effect on bCE cell and hRPE cell viability.
[0078] Cell death was assayed by quantifying plasma membrane damage
or rupture. The LDH cytotoxicity detection is a colorimetric assay
for dead and plasma membrane-damaged cells. LDH present in the
culture supernatant (due to plasma membrane damage) participates in
a coupled reaction which converts a yellow tetrazolium salt into a
red, formazan-class dye, which is measured by absorbance at 492 nm.
The amount of formazan is directly proportional to the amount of
LDH in the culture medium, which is in turn directly proportional
to the number of dead or damaged cells. The percentages of bCE cell
cytotoxicity in the medium containing 0.2%, 0.4%, 0.6%, 0.8% and 1%
.gamma.-PGA were 7.77.+-.3.5%, 8.90.+-.3.5%, 5.76.+-.2.8%,
10.86.+-.2.8% and 10.65.+-.2.5% on day 1; 16.11.+-.5.8%,
12.11.+-.6.6%, 8.66.+-.4.0%, 4.61.+-.3.7% and 2.76.+-.0.4% on day
3, respectively. For hRPE cells, the percentages of cytotoxicity in
the medium containing 0.2%, 0.4%, 0.6%, 0.8% and 1% .gamma.-PGA
were 2.97.+-.0.6%, 2.74.+-.0.4%, 3.95.+-.1.0%, 2.99.+-.1.0% and
4.78.+-.0.9% on day 1; 2.85.+-.0.3%, 2.77.+-.1.5%, 3.74.+-.1.5%,
8.11.+-.1.9% and 10.37.+-.0.9% on day 3, respectively. There was no
significant difference between .gamma.-PGA-containing medium and
the negative control group (FIGS. 5A-B). The percentage of cell
cytotoxicity represents the number of dead cells divided by the
total cell number and was calculated according to the following
formula:
% Cytotoxicity = Medium O . D . - Blank O . D . Total Lysis O . D .
- Blank O . D . .times. 100. ##EQU00002##
Fluorescence Staining of Cells
[0079] The Live/Dead staining kit utilizes two fluorescent dyes,
calcein-AM and ethidium homodimer (EthD-1). Calcein AM is a widely
used green fluorescent cell marker and is membrane-permeable. Once
inside the cells, Calcein AM (a non-fluorescent molecule) is
hydrolyzed by intracellular esterases into negatively charged green
fluorescent calcein. The fluorescent calcein is retained in the
cytoplasm in live cells. It is an end-point assay for cell
viability. The fluorescent signal is monitored using a 485 nm
excitation wavelength and a 530 nm emission wavelength. The
fluorescence signal generated from the assay is proportional to the
number of living cells in the sample. Dead cells have damaged
membranes. Ethidium homodimer-1 (EthD-1) enters damaged cells and
is fluorescent when bound to nucleic acids. EthD-1 produces a
bright red fluoresce in damaged or dead cells. Nearly all of bCE
and hRPE cells were viable in .gamma.-PGA-containing culture medium
(FIGS. 6A-B and 7A-B).
Effect of .gamma.-PGA-Containing Irrigating Solutions on Corneal
Thickness
[0080] Corneal thickness was measured during intraocular perfusion
with the irrigating solution through two vein detained needles
inserted into the eye of a rabbit (FIG. 8A). The movement of the
irrigating solution was driven by a peristaltic pump (FIG. 8B).
Corneal thickness was increased during the initial 20 minutes of
perfusion. FIG. 9 shows the change in the corneal thickness in the
group irrigated with .gamma.-poly(glutamic acid)-based ocular
solution was significantly less than that in the normal saline
group. The changes in the corneal thickness in the normal
saline-irrigated group and in the 0.4% (w/v) of
.gamma.-poly(glutamic acid)-containing solution-irrigated group
were 47 .mu.m and 32 .mu.m, respectively. The initial increase in
the corneal thickness might due to the injury caused by insertion
of the vein detained needles. Afterwards, the cornea perfused with
.gamma.-poly(glutamic acid)-based ocular irrigation solution
increased its thickness only slightly for the remaining 1
hr-perfusion with the corneal thickness swelling about 36 .mu.m
(FIG. 9). In contrast, the cornea perfused with a normal saline
solution showed a continuous increase in the thickness during the
remaining 60-minute perfusion with the thickness swelling about 58
.mu.m by the end of 1 hr perfusion, which was roughly 1.6-fold
increase in the corneal thickness compared with the group irrigated
with .gamma.-poly(glutamic acid)-based ocular irrigating
solution.
[0081] The foregoing description of the exemplary embodiments of
the invention has been presented only for the purposes of
illustration and description and is not intended to be exhaustive
or to limit the invention to the precise forms disclosed. Many
modifications and variations are possible in light of the above
teaching.
[0082] The embodiments and examples were chosen and described in
order to explain the principles of the invention and their
practical application so as to enable others skilled in the art to
utilize the invention and various embodiments and with various
modifications as are suited to the particular use contemplated.
Alternative embodiments will become apparent to those skilled in
the art to which the present invention pertains without departing
from its spirit and scope. Accordingly, the scope of the present
invention is defined by the appended claims rather than the
foregoing description and the exemplary embodiments described
therein.
[0083] Some references, which may include patents, patent
applications and various publications, are cited and discussed in
the description of this invention. The citation and/or discussion
of such references is provided merely to clarify the description of
the present invention and is not an admission that any such
reference is "prior art" to the invention described herein. All
references cited and discussed in this specification are
incorporated herein by reference in their entireties and to the
same extent as if each reference was individually incorporated by
reference.
* * * * *