U.S. patent application number 11/917869 was filed with the patent office on 2009-05-21 for lkktet and/or lkktnt peptide compositions and methods for treating or preventing tissue deterioration, injury or damage.
This patent application is currently assigned to REGENERX BIOPHARMACEUTICALS, INC.. Invention is credited to David Crockford, Jack Finkelstein Jr., Allan L. Goldstein, Gabriel Sosne.
Application Number | 20090131313 11/917869 |
Document ID | / |
Family ID | 37570786 |
Filed Date | 2009-05-21 |
United States Patent
Application |
20090131313 |
Kind Code |
A1 |
Sosne; Gabriel ; et
al. |
May 21, 2009 |
LKKTET AND/OR LKKTNT PEPTIDE COMPOSITIONS AND METHODS FOR TREATING
OR PREVENTING TISSUE DETERIORATION, INJURY OR DAMAGE
Abstract
A pharmaceutical, ophthalmic or cosmetic combination or method
utilizes a peptide agent include amino acid sequence LKKTET or
LKKTNT, a conservative variant thereof, or a stimulating agent that
stimulates production of an LKKTET or LKKTNT peptide, or a
conservative variant thereof, and further utilizes a quaternary
ammonium salt, wherein the agent and the salt can be administered
to a subject separately or together.
Inventors: |
Sosne; Gabriel; (Oak Park,
MI) ; Crockford; David; (Newburyport, MA) ;
Goldstein; Allan L.; (Washington, DC) ; Finkelstein
Jr.; Jack; (Chevy Chase, MD) |
Correspondence
Address: |
ROTHWELL, FIGG, ERNST & MANBECK, P.C.
1425 K STREET, N.W., SUITE 800
WASHINGTON
DC
20005
US
|
Assignee: |
REGENERX BIOPHARMACEUTICALS,
INC.
Bethesda
MD
|
Family ID: |
37570786 |
Appl. No.: |
11/917869 |
Filed: |
June 19, 2006 |
PCT Filed: |
June 19, 2006 |
PCT NO: |
PCT/US06/23758 |
371 Date: |
January 30, 2009 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60691261 |
Jun 17, 2005 |
|
|
|
60776947 |
Feb 28, 2006 |
|
|
|
Current U.S.
Class: |
514/1.1 |
Current CPC
Class: |
A61P 27/14 20180101;
A61K 9/10 20130101; A61P 31/04 20180101; A61P 43/00 20180101; A61K
9/0048 20130101; A61P 27/04 20180101; A61P 29/00 20180101; A61K
9/14 20130101; A61P 31/12 20180101; A61K 38/2292 20130101; A61K
9/08 20130101; A61P 27/06 20180101; A61K 38/08 20130101; A61P 31/00
20180101; A61K 47/186 20130101; A61P 31/10 20180101; A61P 27/02
20180101 |
Class at
Publication: |
514/12 ;
514/17 |
International
Class: |
A61K 38/16 20060101
A61K038/16; A61K 38/08 20060101 A61K038/08 |
Claims
1. A composition for administration to skin tissue of a subject,
comprising a peptide agent comprising amino acid sequence LKKTET a
conservative variant thereof, LKKTNT or a conservative variant
thereof, KLKKTET, LKKTETQ, Thymosin .beta.4 (T.beta.4), a T.beta.4
isoform, analogue or derivative, T.beta.4 sulfoxide, an N-terminal
variant of T.beta.4, a C-terminal variant of T.beta.4,
T.beta.4.sup.ala, T.beta.9, T.beta.10, T.beta.11, T.beta.12,
T.beta.13, T.beta.14, T.beta.15 gelsolin, vitamin D binding protein
(DBP), profilin, cofilin, depactin, Dnasel, vilin, fragmin,
severin, capping protein, .beta.-actinin or acumentin, or a
stimulating agent that stimulates production in said tissue of said
peptide agent, the composition further comprising a quaternary
ammonium salt and a topical carrier for application to skin tissue
of said subject.
2. The composition of claim 1 wherein said peptide agent comprises
amino acid sequence LKKTET, amino acid sequence LKKTETQ, T.beta.4,
an N-terminal variant of T.beta.4, or an isoform of T.beta.4.
3. The composition of claim 1 wherein said quaternary ammonium salt
comprises benzalkonium chloride.
4. The composition of claim 1 wherein said peptide agent is at a
concentration of about 0.001-1,000 mg/ml and said quaternary
ammonium salt is present in said composition within a range of
about 0.001-1% by weight.
5. The composition of claim 1 wherein said composition is in a form
of a solution, gel, cream, paste, lotion, spray, suspension,
dispersion, salve, hydrogel, ointment or foam formulation.
6. The composition of claim 5 wherein said composition is a
cosmetic formulation.
7. A pharmaceutical or cosmetic combination comprising a peptide
agent comprising amino acid sequence LKKTET or a conservative
variant thereof, LKKTNT or a conservative variant thereof, KLKKTET,
LKKTETQ, Thymosin .beta.4 (T.beta.4), a T.beta.4 isoform, analogue
or derivative, T.beta.4 sulfoxide, an N-terminal variant of
T.beta.4, a C-terminal variant of T.beta.4, T.beta.4.sup.ala,
T.beta.9, T.beta.10, T.beta.11, T.beta.12, T.beta.13, T.beta.14,
T.beta.15, gelsolin, vitamin D binding protein (DBP), profilin,
cofilin, depactin, Dnasel, vilin, fragmin, severin, capping
protein, .beta.-actinin or acumentin, or a stimulating agent that
stimulates production of said peptide agent, the combination
further comprising a quaternary ammonium salt, wherein said peptide
agent and said salt can be administered to a subject separately or
together.
8. The combination of claim 7 wherein said peptide agent comprises
amino acid sequence LKKTET, amino acid sequence LKKTETQ, T.beta.4,
an N-terminal variant of T.beta.4, or an isoform of T.beta.4.
9. The combination of claim 7 wherein said quaternary ammonium salt
is benzalkonium chloride.
10. The combination of claim 7 comprising a pharmaceutical,
ophthalmic or cosmetic composition comprising said peptide agent at
a concentration of about 0.001-1,000 mg/ml, and wherein said
quaternary ammonium salt is present in said composition at a about
0.0001-1% by weight.
11. The combination of claim 7 comprising an ophthalmic composition
further comprising an opthalmically acceptable carrier.
12. The combination of claim 11 wherein said composition comprises
an eye drop composition.
13. A method of treatment for treating, preventing, inhibiting or
reducing tissue deterioration, injury or damage resulting from
administration of a quaternary ammonium salt to a subject,
comprising administering said quaternary ammonium salt to said
subject, and administering to said subject a peptide agent
comprising amino acid sequence LKKTET or a conservative variant
thereof, LKKTNT or a conservative variant thereof, KLKKTET,
LKKTETQ, Thymosin .beta.4 (T.beta.4), a T.beta.4 isoform, analogue
or derivative, T.beta.4 sulfoxide, an N-terminal variant of
T.beta.4, a C-terminal variant of T.beta.4, T.beta.4.sup.ala,
T.beta.9, T.beta.10, T.beta.11, T.beta.12, T.beta.13, T.beta.14,
T.beta.15, gelsolin, vitamin D binding protein (DBP), profilin,
cofilin, depactin, Dnasel, vilin, fragmin, severin, capping
protein, .beta.-actinin or acumentin or a stimulating agent that
stimulates in said tissue production of said peptide agent.
14. The method of claim 13 wherein said agent is administered to
said subject prior to, concurrently with or after said
administration of said quaternary ammonium salt.
15. The method of claim 14 wherein said agent is administered
concurrently with said salt.
16. The method of claim 15 wherein said agent and said salt are
administered together as a composition.
17. The method of claim 16 wherein said composition further
comprises an ophthalmically acceptable carrier.
18. The method of claim 17 wherein said composition comprises an
eye drop composition.
19. The method of claim 16 wherein said composition further
comprises a cosmetically acceptable carrier.
20. The method of claim 19 wherein said composition is in a form of
a solution, gel, cream, paste, lotion, spray, suspension,
dispersion, salve, hydrogel, ointment or foam formulation.
21. The method of claim 13 wherein said peptide agent comprises
amino acid sequence KLKKTET, amino acid sequence LKKTETQ, T.beta.4,
an N-terminal variant of T.beta.4, a C-terminal variant of
T.beta.4, or an isoform of T.beta.4.
22. The method of claim 13 wherein said quaternary amino salt
comprises benzalkonium chloride.
23. The method of claim 16 wherein said peptide agent is included
in said composition at a concentration within a range of about
0.001-1,000 mg/ml, and said quaternary ammonium salt is present in
said composition within a range of about 0.0001-1% by weight.
24. The composition of claim 1 wherein said peptide agent comprises
T.beta.4.
25. The combination of claim 7 wherein said peptide agent comprises
T.beta.4.
26. The method of claim 13 wherein said peptide agent comprises
T.beta.4.
Description
CROSS-REFERENCE TO RELATED APPLICATION
[0001] This application claims the benefit of U.S. Provisional
Application No. 60/691,261, filed Jun. 17, 2005 and U.S.
Provisional Application No. 60/776,947, filed Feb. 28, 2006.
BACKGROUND OF THE INVENTION
[0002] 1. Field of the Invention
[0003] The present invention relates to the field of LKKTET and/or
LKKTNT compositions and methods.
[0004] 2. Description of the Background Art
[0005] Thymosin beta 4 initially was identified as a protein that
is up-regulated during endothelial cell migration and
differentiation in vitro. Thymosin beta 4 was originally isolated
from the thymus and is a 43 amino acid, 4.9 kDa ubiquitous
polypeptide identified in a variety of tissues. Several roles have
been ascribed to this protein including a role in a endothelial
cell differentiation and migration, T cell differentiation, actin
sequestration, vascularization and wound healing.
[0006] Many T.beta.4 isoforms have been identified and have about
70%, or about 75%, or about 80% or more homology to the known amino
acid sequence of T.beta.4. Such isoforms include, for example,
T.beta.4.sup.ala, T.beta.9, T.beta.10, T.beta.11, T.beta.12,
T.beta.13, T.beta.14 and T.beta.15. Similar to T.beta.4, the
T.beta.10 and T.beta.15 isoforms have been shown to sequester
actin. T.beta.4, T.beta.10 and T.beta.15, as well as these other
isoforms share an amino acid sequence, LKKTET or LKKTNT, that
appears to be involved in mediating actin sequestration or binding.
Although not wishing to be bound by any particular theory, the
activity of peptide agents as described herein may be due, at least
in part, to the anti-inflammatory activity of such agents. T.beta.4
also can modulate actin polymerization (e.g. .beta.-thymosins
appear to depolymerize F-actin by sequestering free G-actin).
T.beta.4's ability to modulate actin polymerization may be due to
its ability to bind to or sequester actin via the LKKTET or LKKTNT
sequence.
[0007] There remains a need in the art for LKKTET and/or LKKTNT
compositions and methods.
SUMMARY OF THE INVENTION
[0008] According to one embodiment, a composition for
administration to skin tissue of a subject, comprises a peptide
agent comprising amino acid sequence LKKTET or LKKTNT, a
conservative variant thereof, or a stimulating agent that
stimulates production in said tissue of an LKKTET or LKKTNT
peptide, or a conservative variant thereof, the composition further
comprising a quaternary ammonium salt and a topical carrier for
application to skin tissue of said subject.
[0009] According to another embodiment, a pharmaceutical,
ophthalmic or cosmetic combination comprises a peptide agent
comprising amino acid sequence LKKTET or LKKTNT, a conservative
variant thereof, or a stimulating agent that stimulates production
of an LKKTET or LKKTNT peptide, or a conservative variant thereof,
the combination further comprising a quaternary ammonium salt,
wherein said agent and said salt can be administered to a subject
separately or together.
[0010] According to a further embodiment, a method of treatment for
treating, preventing, inhibiting or reducing tissue deterioration,
injury or damage resulting from administration of a quaternary
ammonium salt to a subject, comprises administering said quaternary
ammonium salt to said subject, and administering to said subject a
peptide agent comprising amino acid sequence LKKTET or LKKTNT, a
conservative variant thereof, or a stimulating agent that
stimulates in said tissue production of an LKKTET or LKKTNT
peptide, or a conservative variant thereof.
DETAILED DESCRIPTION OF THE INVENTION
[0011] Many compositions that are administered to subjects for
various purposes contain one or more ingredients that may cause
injury to the subject.
[0012] For example, preservatives such as benzalkonium chloride
(BAK), which are present in many ophthalmic and cosmetic products,
may cause surface irritation and/or injury when the product is
administered. Symptoms may include redness and discomfort.
[0013] There is a significant and growing problem with ocular
surface irritation and apoptosis with chronic use of eye drops
containing quaternary ammonium salts such as BAK. Patients often
develop symptoms such as redness and general eye discomfort. A
peptide agent in accordance with the present invention, such as
thymosin beta 4 (T.beta.4), can be utilized to treat or prevent
damage or injury to eye tissue when administered to a subject in
combination with contacting the eye tissue with a quaternary
ammonium salt such as BAK.
[0014] Examples of quaternary ammonium salts to which the present
invention may be applicable include BAK, cetrimide, benzoxonium
chloride, and the like.
[0015] According to one embodiment, the peptide agent is
administered together with the quaternary ammonium salt such as BAK
as part of the same formulation, e.g., as an additive to a
formulation containing BAK or other quaternary ammonium salt.
Quaternary ammonium salts such as BAK are ingredients commonly used
as preservatives in many ocular solutions, such as glaucoma eye
drops.
[0016] In alternative embodiments, the peptide agent can be
administered before administration of the quaternary ammonium salt
such as BAK, and/or during administration of the quaternary
ammonium salt such as BAK, and/or after administration of the
quaternary ammonium salt such as BAK.
[0017] According to one embodiment the invention is particularly
useful in preventing damage to corneal epithelial eye tissue,
including prevention of apoptosis of cells of such eye tissue. For
example, the invention may be utilized as an adjuvant for
anti-glaucoma drops or other eye drops.
[0018] In accordance with one aspect, an ophthalmically acceptable
composition comprises a peptide agent comprising amino acid
sequence LKKTET or LKKTNT, or a conservative variant thereof.
[0019] In accordance with another aspect, a method of treating eye
tissue comprises topically administering to said eye tissue an
ophthalmically acceptable composition comprising a peptide agent
comprising amino acid sequence LKKTET or LKKTNT, or a conservative
variant thereof.
[0020] In one embodiment, the invention provides a method of
treatment by contacting eye tissue with an effective amount of a
composition which contains a peptide agent as described herein.
Examples of direct administration include, for example, contacting
the tissue, by direct application with a solution, lotion, salve,
gel, cream, paste, spray, suspension, dispersion, hydrogel,
ointment, oil or foam comprising a peptide agent as described
herein.
[0021] Without being found to any specific theory,
actin-sequestering peptides such as thymosin beta 4 (T.beta.4 or
TB4) and other agents including actin-sequestering peptides or
peptide fragments containing amino acid sequence LKKTET or LKKTNT
or conservative variants thereof, promote healthy eye tissue.
[0022] Thymosin beta 4 was initially identified as a protein that
is up-regulated during endothelial cell migration and
differentiation in vitro. Thymosin beta 4 was originally isolated
from the thymus and is a 43 amino acid, 4.9 kDa ubiquitous
polypeptide identified in a variety of tissues. Several roles have
been ascribed to this protein including a role in a endothelial
cell differentiation and migration, T cell differentiation, actin
sequestration, vascularization and wound healing.
[0023] According to one embodiment the invention is preferably
applicable to Thymosin .beta.4, and/or T.beta.4 isoforms, analogues
or derivatives, including KLKKTET, LKKTETQ, oxidized T.beta.4,
T.beta.4 sulfoxide, N-terminal variants of T.beta.4 and C-terminal
variants of T.beta.4.
[0024] According to one embodiment, compositions which may be used
in accordance with the present invention include peptide agents
such as Thymosin .beta.4 (T.beta.4), and/or T.beta.4 isoforms,
analogues or derivatives, including oxidized forms of T.beta.4
including T.beta.4 sulfoxide, N-terminal variants of T.beta.4, and
C-terminal variants of T.beta.4, and polypeptides or peptide
fragments comprising or consisting essentially of the amino acid
sequence LKKTET and conservative variants thereof. International
Application Serial No. PCT/US99/17282, incorporated herein by
reference, discloses isoforms of T.beta.4 which may be useful in
accordance with the present invention as well as amino acid
sequence LKKTET or LKKTNT and conservative variants thereof, which
may be utilized with the present invention. International
Application Serial No. PCT/GB99/00833 (WO 99/49883), incorporated
herein by reference, discloses oxidized Thymosin .beta.4 which may
be utilized in accordance with the present invention. Although the
present invention is described primarily hereinafter with respect
to T.beta.4 and T.beta.4 isoforms, it is to be understood that the
following description is intended to be equally applicable to amino
acid sequence LKKTET or LKKTNT, peptides and fragments comprising
or consisting essentially of LKKTET or LKKTNT, conservative
variants thereof and/or T.beta.4 isoforms, analogues or
derivatives, including oxidized T.beta.4, N-terminal variants of
T.beta.4, and C-terminal variants of T.beta.4.
[0025] According to one embodiment a composition in accordance with
the present invention can be administered daily, every other day,
every other week, every other month, etc., with a single
application or multiple applications per day of administration,
such as applications 2, 3, 4 or more times per day of
administration.
[0026] Many T.beta.4 isoforms have been identified and have about
70%, or about 75%, or about 80% or more homology to the known amino
acid sequence of T.beta.4. Such isoforms include, for example,
T.beta.4.sup.ala, T.beta.9, T.beta.10, T.beta.11, T.beta.12,
T.beta.13, T.beta.14 and T.beta.15. Similar to T.beta.4, the
T.beta.10 and T.beta.15 isoforms have been shown to sequester
actin. T.beta.4, T.beta.10 and T.beta.15, as well as these other
isoforms share an amino acid sequence, LKKTET or LKKTNT, that
appears to be involved in mediating actin sequestration or binding.
T.beta.4 has anti-inflammatory activity, and also can modulate
actin polymerization (e.g. .beta.-thymosins appear to depolymerize
F-actin by sequestering free G-actin). T.beta.4's ability to
modulate actin polymerization may be due to its ability to bind to
or sequester actin via the LKKTET or LKKTNT sequence. Thus, as with
T.beta.4, other proteins which are anti-inflammatory and/or bind or
sequester actin, or modulate actin polymerization, including
T.beta.4 isoforms having the amino acid sequence LKKTET, are likely
to be effective, alone or in a combination with T.beta.4, as set
forth herein.
[0027] Thus, it is specifically contemplated that known T.beta.4
isoforms, such as T.beta.4.sup.ala, T.beta.9, T.beta.10, T.beta.11,
T.beta.12, T.beta.13, T.beta.14 and T.beta.15, as well as T.beta.4
isoforms not yet identified, will be useful in the methods of the
invention. As such T.beta.4 isoforms are useful in the methods of
the invention, including the methods practiced in a subject. The
invention therefore further provides compositions comprising
T.beta.4, as well as T.beta.4 isoforms T.beta.4.sup.ala, T.beta.9,
T.beta.10, T.beta.11, T.beta.12, T.beta.13, T.beta.14 and
T.beta.15, and an ophthalmically acceptable carrier.
[0028] In addition, other agents or proteins having anti
inflammatory activity and/or actin sequestering or binding
capability, or that can mobilize actin or modulate actin
polymerization, as demonstrated in an appropriate sequestering,
binding, mobilization or polymerization assay, or identified by the
presence of an amino acid sequence that mediates actin binding,
such as LKKTET or LKKTNT, for example, can similarly be employed in
the methods of the invention. Such proteins may include gelsolin,
vitamin D binding protein (DBP), profilin, cofilin, depactin,
Dnasel, vilin, fragmin, severin, capping protein, .beta.-actinin
and acumentin, for example. As such methods include those practiced
in a subject, the invention further provides pharmaceutical
compositions comprising gelsolin, vitamin D binding protein (DBP),
profilin, cofilin, depactin, Dnasel, vilin, fragmin, severin,
capping protein, .beta.-actinin and acumentin as set forth herein.
Thus, the invention includes the use of an polypeptide comprising
the amino acid sequence LKKTET or LKKTNT and conservative variants
thereof.
[0029] As used herein, the term "conservative variant" or
grammatical variations thereof denotes the replacement of an amino
acid residue by another, biologically similar residue. Examples of
conservative variations include the replacement of a hydrophobic
residue such as isoleucine, valine, leucine or methionine for
another, the replacement of a polar residue for another, such as
the substitution of arginine for lysine, glutamic for aspartic
acids, or glutamine for asparagine, and the like.
[0030] According to one embodiment, a composition in accordance
with the present invention is an eye drop formulation.
[0031] According to one embodiment, a composition for use in the
invention contains a peptide agent as described herein at a
concentration within a range of from about 0.001-1000 micrograms
per ml (mcg/ml), more preferably about 0.1-100 mcg/ml, most
preferably about 1-10 mcg/ml. In particularly preferred
embodiments, the peptide agent is T.beta.4.
[0032] According to one embodiment the invention is useful for
treating or preventing damage or injury to eye tissue resulting
from contacting eye tissue with a quaternary ammonium salt such as
BAK at concentrations within a range of about 0.0001-1% by weight,
preferably within a range of about 0.001-0.1% by weight, and more
preferably within a range of about 0.002-0.05% by weight. In one
embodiment, the quaternary ammonium salt is at a concentration of
about 0.005-0.02% by weight.
[0033] According to one embodiment the invention also includes a
pharmaceutical composition comprising a peptide agent as described
herein in an ophthalmically acceptable carrier. Such carriers
include, e.g., those listed herein.
[0034] According to one embodiment the actual dosage or reagent,
formulation or composition that provides treatment may depend on
many factors, including the size and health of a subject. However,
persons of ordinary skill in the art can use teachings describing
the methods and techniques for determining clinical dosages as
disclosed in PCT/US99/17282, supra, and the references cited
therein, to determine the appropriate dosage to use.
[0035] According to one embodiment the methods and compositions
using or containing a peptide agent as described herein may be
formulated into compositions by admixture with ophthalmically
acceptable non-toxic excipients or carriers.
[0036] According to one embodiment the topical formulation
containing the active compound can also contain a physiologically
compatible vehicle, as those skilled in the ophthalmic art can
select using conventional criteria. The vehicles can be selected
from the known ophthalmic vehicles which include, but are not
limited to, saline solution, water polyethers such as polyethylene
glycol, polyvinyls such as polyvinyl alcohol and povidone,
cellulose derivatives such as methylcellulose and hydroxypropyl
methylcellulose, petroleum derivatives such as mineral oil and
white petrolatum, animal fats such as lanolin, polymers of acrylic
acid such as carboxypolymethylene gel, vegetable fats such as
peanut oil and polysaccharides such as dextrans, and
glycosaminoglycans such as sodium hyaluronate and salts such as
sodium chloride and potassium chloride.
[0037] According to one embodiment an ophthalmic composition is
advantageously applied topically to the eye, especially in the form
of a solution, a suspension, an ointment, gel or foam.
[0038] According to one embodiment there are used for a
corresponding ophthalmic composition customary pharmaceutically
acceptable excipients and additives known to the person skilled in
the art, for example those of the type mentioned below, especially
carriers, stabilizers, solubilizers, tonicity enhancing agents,
buffer substances, preservatives, thickeners, complexing agents and
other excipients. Examples of such additives and excipients can be
found in U.S. Pat. Nos. 5,134,124 and 4,906,613. Such compositions
are prepared in a manner known per se, for example by mixing the
active ingredient with the corresponding excipients and/or
additives to form corresponding ophthalmic compositions. The active
ingredient is preferably administered in the form of eye drops, the
active ingredient being conventionally dissolved, for example, in a
carrier. The solution is, where appropriate, adjusted and/or
buffered to the desired pH and, where appropriate, a stabilizer, a
solubilizer or a tonicity enhancing agent is added. Where
appropriate, preservatives and/or other excipients are added to an
ophthalmic composition.
[0039] Carriers used in accordance with the present invention are
typically suitable for topical or general administration, and are
for example water, mixtures of water and water-miscible solvents,
such as C.sub.1-C.sub.7-alkanols, vegetable oils or mineral oils
comprising from 0.5 to 5% by weight hydroxyethylcellulose, ethyl
oleate, carboxymethylcellulose, polyvinyl-pyrrolidone and other
non-toxic water-soluble polymers for ophthalmic uses, such as, for
example, cellulose derivatives, such as methylcellulose, alkali
metal salts of carboxymethylcellulose, hydroxymethylcellulose,
hydroxyethylcellulose, methylhydroxypropylcellulose and
hydroxypropylcellulose, acrylates or methacrylates, such as salts
of polyacrylic acid or ethyl acrylate, polyacrylamides, natural
products, such as gelatin, alginates, pectins, tragacanth, karaya
gum, xanthan gum, carrageenin, agar and acacia, starch derivatives,
such as starch acetate and hydroxypropyl starch, and also other
synthetic products, such as polyvinyl alcohol,
polyvinylpyrrolidone, polyvinyl methyl ether, polyethylene oxide,
preferably cross-linked polyacrylic acid, such as neutral Carbopol,
or mixtures of those polymers. Preferred carriers are water,
cellulose derivatives, such as methylcellulose, alkali metal salts
of carboxymethylcellulose, hydroxymethylcellulose,
hydroxyethylcellulose, methylhydroxypropylcellulose and
hydroxypropylcellulose, neutral Carbopol, or mixtures thereof.
[0040] According to one embodiment the solubilizers used for an
ophthalmic composition of the present invention are, for example,
tyloxapol, fatty acid glycerol poly-lower alkylene glycol esters,
fatty acid poly-lower alkylene glycol esters, polyethylene glycols,
glycerol ethers or mixtures of those compounds. The amount added is
typically sufficient to solubilize the active ingredient. For
example, the concentration of the solubilizer is from 0.1 to 5000
times the concentration of the active ingredient. Lower alkylene
means linear or branched alkylene with up to and including 7
C-atoms. Examples are methylene, ethylene, 1,3-propylene,
1,2-propylene, 1,5-pentylene, 2,5-hexylene or 1,7-heptylene. Lower
alkylene is preferably linear or branched alkylene with up to and
including 4 C-atoms.
[0041] Examples of buffer substances are acetate, ascorbate,
borate, hydrogen carbonate/carbonate, citrate, gluconate, lactate,
phosphate, propionate and TRIS (tromethamine) buffers. Tromethamine
and borate buffer are preferred buffers. The amount of buffer
substance added is, for example, that necessary to ensure and
maintain a physiologically tolerable pH range. The pH range is
typically in the range of from 5 to 9, preferably from 6 to 8.2 and
more preferably from 6.8 to 8.1.
[0042] Tonicity enhancing agents are, for example, ionic compounds,
such as alkali metal or alkaline earth metal halides, such as, for
example, CaCl.sub.2, KBr, KCl, LiCl, NaBr, NaCl, or boric acid.
Non-ionic tonicity enhancing agents are, for example, urea,
glycerol, sorbitol, mannitol, propylene glycol, or dextrose. For
example, sufficient tonicity enhancing agent is added to impart to
the ready-for-use ophthalmic composition an osmolality of
approximately from 50 to 1000 mOsmol, preferred from 100 to 400
mOsmol, more preferred from 200 to 400 mOsmol and even more
preferred from 280 to 350 mOsmol.
[0043] Examples of preservatives are quaternary ammonium salts,
such as cetrimide, benzalkonium chloride or benzoxonium chloride,
alkyl-mercury salts of thiosalicylic acid, such as, for example,
thimerosal, phenylmercuric nitrate, phenylmercuric acetate or
phenylmercuric borate, parabens, such as, for example,
methylparaben or propylparaben, alcohols, such as, for example,
chlorobutanol, benzyl alcohol or phenyl ethanol, guanidine
derivatives, such as, for example, chlorohexidine or
polyhexamethylene biguanide, or sorbic acid. Preferred
preservatives are cetrimide, benzalkonium chloride, benzoxonium
chloride and parabens. Where appropriate, a sufficient amount of
preservative is added to the ophthalmic composition to ensure
protection against secondary contaminations during use caused by
bacteria and fungi.
[0044] According to one embodiment the ophthalmic compositions may
comprise further non-toxic excipients, such as, for example,
emulsifiers, wetting agents or fillers, such as, for example, the
polyethylene glycols designated 200, 300, 400 and 600, or Carbowax
designated 1000, 1500, 4000, 6000 and 10,000. Other excipients that
may be used if desired are listed below but they are not intended
to limit in any way the scope of the possible excipients. They are
especially complexing agents, such as disodium-EDTA or EDTA,
antioxidants, such as ascorbic acid, acetylcysteine, cysteine,
sodium hydrogen sulfite, butyl-hydroxyanisole,
butyl-hydroxy-toluene or .alpha.-tocopherol acetate; stabilizers,
such as a cyclodextrin, thiourea, thiosorbitol, sodium dioctyl
sulfosuccinate or monothioglycerol; or other excipients, such as,
for example, lauric acid sorbitol ester, triethanol amine oleate or
palmitic acid ester. Preferred exipients are complexing agents,
such as disodium-EDTA and stabilizers, such as a cyclodextrin. The
amount and type of excipient added is in accordance with the
particular requirements and is generally in the range of from
approximately 0.0001 to approximately 90% by weight. A cyclodextrin
is composed of several glucose units which have three free hydroxy
groups per glucose. The amount of a cyclodextrin used in accordance
with one embodiment may preferably range from 0.01-20% by weight,
more preferably from 0.1-15% by weight and even more preferably
from 1-10% by weight.
[0045] According to one embodiment the present invention relates
also to an ophthalmic composition, which comprises a
therapeutically effective amount of a peptide agent as described
herein a carrier, a solubilizer and another therapeutically
effective pharmaceutical agent which may be, for example, an
antibiotic, an antiallergic, an anesthetic, another antiphlogistic,
a corticosteroide, an agent suitable for lowering intra-ocular
pressure, or another drug.
pH (Hydrogen Ion Concentration)
[0046] According to one embodiment, the pH of the inventive
formulations should be as close to that of the tear film as
possible. The physiologic pH of tears is approximately 7.4.+-.0.2.
Thus, from a comfort, tolerability and safety perspective, this
would be the optimal pH of ophthalmic preparations.
[0047] Stimulation of tear secretion and eye blinking causes the pH
to decrease in value. When the eyelid remains open for extended
periods of time the tear-film is alkalized by equilibrium with the
partial pressure of CO.sub.2 in the surrounding air and a pH value
of greater than 9 is attained. Both decreases and increases in pH
occur without adverse consequences. Thus, there is some latitude in
the pH range when formulating inventive formulations around pH of
approximately 7.4.
[0048] Also, when a formulation is administered to the eye, it
stimulates the flow of tears. Tear fluid is capable of quickly
diluting and buffering small volumes of added substances,
suggesting the eye can tolerate a fairly wide pH range offered by
certain formulations.
[0049] Consequently, ophthalmic formulations may be within a range
of from about pH 3.5 to 11.5. However, ophthalmic formulations may
display pH ranges somewhat more narrowly from 3.5 to 9, preferably
from 4.5 to 8 and most preferably from pH 5.5 to 7.8. The most
preferred pH range is advantageous from the perspective solubility,
chemical stability and therapeutic activity of the inventive
compositions and a useful and relatively narrow range to prevent
corneal damage.
Buffer Systems
[0050] According to one embodiment buffer systems are composed of a
weak acid or base and its conjugate salt. The buffering capacity of
the components in the system acts in such a way that, despite the
addition of an acid or base and exposure to external influences of
temperature, pressure, volume, redox potential, body fluids and
tears, the pH will remain essentially constant. Although buffer
capacity should be large enough to resist changes in the product pH
(i.e., pH drift) for a reasonable shelf-life (i.e., under storage
conditions), the buffer capacity of inventive ophthalmic
formulations should be low enough to allow rapid readjustment of
the product to physiologic pH upon administration to the eye.
According to one embodiment buffer capacities for ophthalmic
products should be within the range of 0.05 to 1.0. Preferred and
most preferred buffer capacities range from 0.02 to 0.2 and 0.01 to
0.1, respectively for certain inventive compositions. Buffer
capacity is determined by the following formula:
*.beta.=.DELTA.B/.DELTA.pH
where .beta. is buffer capacity, .DELTA.B is the gram equivalent of
strong acid/base to change pH of 1 liter of buffer solution and
.DELTA.pH is the change caused by the addition of strong
acid/base.
[0051] According to one embodiment appropriate buffer systems may
be sodium salts of the following acids: acetic; ascorbic; boric;
carbonic; phosphoric; citric; gluconic; lactic; and propionic.
Calcium salts of carbonic or propionic acid form appropriate buffer
systems as do potassium salts of phosphoric acid. Tris buffer
(tromethamine) is used intravenously as an alkalizer for the
correction of metabolic acidosis and is one of the preferred
buffers for use in this Invention. Other preferred buffers are
acetate, phosphate, citrate and borate. In certain instances a
buffer system involving proton donor and proton acceptor groups of
the amino acid residues of proteins may be preferable to the
acid-base or an amine-base buffer.
[0052] The specific amount of a buffer substance used will vary and
depends upon the amount that is deemed necessary to maintain a
pH-environment suitable for the stability of inventive composition
and to ensure and maintain a physiologically tolerable pH
range.
Tonicity Agents
[0053] Of those listed herein the objective is to adjust the
tonicity of the inventive ophthalmic compositions to that of
natural tears to approximate physiological tonicity (e.g., 0.9%
saline). For example, sodium chloride, potassium chlorides, calcium
chloride, dextrose and/or mannitol may be added to the inventive
peptide agent formulation. The amount of tonicity agent will vary,
depending on the particular agent to be added. In general, certain
inventive compositions will have a tonicity agent in an amount
sufficient to cause the final composition to have an ophthalmically
acceptable osmolality preferably 150-450 mOsm and most preferably
250-350 mOsm.
[0054] The preferred tonicity agents are sodium salts and potassium
salts, particularly sodium and potassium chloride. The most
preferred tonicity agent is sodium chloride.
Lubricants/Demulcents/Viscosity Enhancers
[0055] According to one embodiment compounds may be included which
sooth the eye, reduce surface tension and improve wettability
(contact) of an otherwise hydrophobic epithelial corneal surface,
approximate the consistency of tears. Such compounds may also
enhance the viscosity of the inventive compositions, allowing an
inventive formulation to remain in the eye longer thus giving the
peptide agent more time to exert its therapeutic activity or
undergo absorption to reach the desired target.
[0056] Suitable viscosity enhancers in ophthalmic formulations and
their concentration ranges used in certain inventive compositions
include but are not limited to: (a) Monomeric polyols, such as
tyloxapol (0.1-1%), glycerol (0.2-1%), propylene glycol (0.2 to
1%), ethylene glycol (0.2-1%); (b) Polymeric polyols, such as
polyethylene glycol (e.g., PEG 300, PEG 400)(0.2-1%); (c) Cellulose
derivatives (polymers of the cellulose family), such as
hydroxyethylcellulose (0.2-2.5%), hypromellose (0.2 to 2.5%),
hydroxypropylmethyl cellulose (0.2-2.5%), methycellulose
(0.2-2.5%), carboxymethylcellulose sodium (0.2 to 2.5%),
hydroxylpropylcellulose (0.2-2.5%); (d) Dextrans, such as dextran
70 (0.1% when used with another polymeric demulcent agent); (e)
Water-soluble proteins such as gelatin (0.01%); (f) Vinyl polymers
such as polyvinyl alcohol (0.1-4%), polyvinyl pyrollidine (0.1-4%);
(g) Other polyols, such as polysorbate 80 (0.2-1%), povidone
(0.1-2%); (h) Carbomers, such as carbomer 934P, carbomer 941,
carbomer 940, and carbomer 974P, and (i)
Polysaccharides/Glycosaminoglycans, such as hyaluronan (hyaluronic
acid/hyaluronate) (0.1-3%), chondroitin sulfate (0.1-3%).
[0057] More than one viscosity enhancer may be added to an
inventive composition to increase the viscosity of the carrier
(vehicle). A preferred enhancer in the carrier of an inventive
peptide agent formulation is carboxymethycellulose.
Viscosity
[0058] Viscosity describes a material's internal resistance to flow
or change in form, when a stress is applied. The viscosity of a
material (solution, semi-viscous gel, suspension, oleaginous
ointments and ointment gels (viscous gels) is given in poise units.
The unit, centipoise ("cp" or the plural "cps") is equal to 0.01
poise and is most often used in pharmaceutical applications.
Compounds used to enhance viscosity are available in various grades
such as 15 cps, 100 cps, etc., etc. The grade number refers to the
viscosity which results when a fixed percentage aqueous solution of
the enhancer is made. Generally, solutions are 1% or 2%; however,
they can be as high as 4% with certain enhancers. Viscosity is
measured at 20.degree. or 25.degree. C.
[0059] A suitable viscosity in an ophthalmic solution is between 25
and 50 centipoises (cps). The actual concentration of an enhancer
required to produce that desired viscosity will depend on the grade
of the enhancer. For example, if methycellulose 25 cps is used, a
1% solution will create a viscosity of 25 cps. If methycellulose
4000 cps is used, an 0.25% solution provides the desired viscosity.
Standard references give tables of viscosities produced by
percentage solutions and grades of ingredients.
[0060] According to one embodiment inventive formulations will
exhibit a viscosity of >1 to 100,000 centipoises (cps) or
greater. Inventive ointment compositions (oleaginous or viscous
gels) may have viscosity grades that are greater than 100,000 cps.
This is because ophthalmic ointments are intended to be thick when
standing to prevent them from flowing away from the intended area
of use. Following application and over time, temperatures within
the conjunctival sac, or on the surface of the eye, where these
ointments are deposited, will cause these ointments to "melt" and
begin to flow.
[0061] The preferred viscosity ranges of various inventive
formulation types are found in the table below:
TABLE-US-00001 Formulation Type Viscosity Range (cps) Aqueous
Solution >1 to <1000 Suspension >1 to <1000 Gel >3
to 40,000 Ointment, gel >20,000 to 100,000 Ointment, oleaginous
>20,000 to 100,000
Reducing Agents/Antioxidants/Oxygen-Sequestering Agents
[0062] Certain inventive compositions have the potential to be
degraded by oxidation. Consequently, steps during the manufacture,
control and packaging of an inventive composition may include
protecting inventive compositions, susceptible to oxidation, by (1)
displacing oxygen with nitrogen or a dense inert gas such as argon,
(2) adding a reducing agent to minimize oxidative effects, (3) the
introduction of a decoy molecule.
[0063] Common antioxidant (reducing) agents which may be used in
ophthalmic formulations up to a concentration of 0.1% or more are
sodium sulfite, sodium thiosulfite, sodium bisulfite, sodium
metabisulfite, and thiourea. Sulfites can cause allergic-type
reactions in certain people; consequently, patients receiving this
type of antioxidant should be questioned about this potential
reaction before being treated with an inventive composition
containing the antioxidant. Other useful antioxidants compatible
with the inventive compositions are ascorbic acid, EDTA/disodium
edetate, acetic acid, citric acid, glutathione and acetylcysteine.
These agents may also be regarded as stabilizers.
[0064] A decoy molecule or an oxygen sequestering protective agent
may be added as stabilizers to an inventive formulation to minimize
oxidative effects on the inventive formulation. The molecular decoy
must have at least the same capability of being oxidized as the
inventive formulation. One such decoy, for an inventive composition
containing methionine is the amino acid, methionine, itself. Free
methionine added to an inventive composition containing the amino
acid methionine would compete for oxygen in the process of being
oxidized to methionyl sulfoxide. A free oxygen-consuming agent is
one that prevents other oxygen-reactive amino acids in the
inventive composition/preptide from being oxidized. For the
purposes of certain inventive compositions but not limited to such,
a free oxygen-consuming agent is methionine.
Ophthalmic Ointments/Oleaginous Emollient Bases
[0065] Ophthalmic ointments tend to keep an active agent in contact
with the eye longer than suspensions and certainly solutions. Most
ointments, tend to blur vision, as they are not removed easily by
the tear fluid. Thus, ointments are generally used at night as
adjunctive therapy to eye drops used during the day.
[0066] Oleaginous ointment bases of inventive compositions are
mixtures of mineral oil, petrolatum and lanolin all have a melting
point close to body temperature. In the case of the inventive
compounds, the compositions may include mineral oil, petrolatum or
lanolin. According to one embodiment preferred compositions would
include a combination of petrolatum, mineral oil and lanolin. The
most preferred composition is an ointment combination containing
white petrolatum, mineral oil and lanolin (anhydrous).
[0067] The peptide agent comprising amino acid sequence LKKTET or
LKKTNT or a conservative variant thereof is dissolved in a small
amount of purified water or 0.9% saline to affect dissolution. This
aqueous solution is incorporated into anhydrous lanolin and the
then "liquid" lanolin (up to 10%) is mixed with the remaining
ointment/oleaginous emollient base ingredients, mineral oil (up to
30%) and white petrolatum (up to 60%).
[0068] Ophthalmic ointment tubes are typically small holding
approximately 1-5 grams of ointment, preferably 3.5 grams, and
fitted with narrow gauge tips which permit the extrusion of narrow
bands of ointment measured in inches or fractions thereof for
dosing purposes.
Preservatives
[0069] Sterility is an absolute requirement of all ophthalmic
formulations. Contaminated formulations may result in eye
infections that could ultimately cause blindness, especially if the
P. aeruginosa microbe is involved. Therefore, ophthalmic
formulations as described herein must be prepared using techniques,
unique for solutions, gels, suspensions and ointments of the
inventive compositions that assure sterility. Sterile formulations
must be packaged in sterile containers. Most topical ophthalmic
products are typically packaged in multidose form. As such
preservatives are required to prevent microbial contamination of an
otherwise sterile product during use. Suitable preservatives
include: Quaternary ammonium compounds (salts), such as
benzalkonium chloride (0.001 to 0.02%), benzethonium chloride,
cetalkonium chloride, cetrimide, benzododecinium bromide and
benzoxonium chloride; Alkyl-mercury salts of thiosalicylic acid,
such as thimerosal (0.001 to 0.005%); Parabens, such as
methylparaben and propylparaben; Chelating agents, such as disodium
edetate, sodium gluconate, sodium propionate; Other agents, such as
chlorobutanol, boric acid, sorbic acid, phenylethanol (0.25%);
Purite.RTM. chlorine dioxide; Polyquad.RTM. polyquatemium-1
(0.001%); and Aldox.RTM. myristamidopropyl diethylamine (0.005%);
or other agents known to those skilled in the art.
[0070] Such preservatives are typically employed at a level of from
0.001% to 1.0% (w/v) to ensure protection against secondary
microbial contaminations during use caused by bacteria, mold, and
fungi.
[0071] Maximum concentrations of the following selected
preservatives, presently approved for use in ophthalmic
formulations, are shown in the table below:
TABLE-US-00002 Agent Maximum Concentration % Benzalkonium chloride
(BAK) 0.01 Benzethonium chloride 0.01 Clorobutanol 0.5
Phenylmercuric acetate 0.004 Phenylmercuric nitrate 0.004
Thimerosal (thiomersal) 0.01 Methyparaben 0.2 Propylparaben 0.04
Source: FDA Advisory Panel on OTC Opthalmic Drug Products, Final
report. December 1979
[0072] Selection of the appropriate preservative is based upon its
anti-microbial effectiveness with the chosen inventive composition.
Preferred preservatives for use in the inventive formulations are
the combination of methylparaben (0.080% -0.1%) and propylparaben
(0.016% -0.024%), benzalkonium chloride (BAK) (0.005% -0.02%, where
0.01% w/v is most preferred), a combination of BAK and EDTA
(0.01-0.5%), which when used together have synergistic effects.
[0073] Unit dose compositions of the present invention will be
sterile but unpreserved. Such compositions for the most part will
not contain preservatives. Consequently, these compositions cannot
be re-used and once-opened they must be discarded.
Bulking/Stabilizing Agents
[0074] Bulking/stabilizing agent(s) may be advantageous to maintain
the hydration state of a lubricant, emollient or vehicle enhancer
comprising an inventive composition during long-term storage.
Associations appear to occur within or among polymer chains of
these substances which after time favor the reduction of hydration
state of these chains. These associations may be in the form of
hydrogen bonds within and among the polymer chains which can
manifest as a change in viscosity and texture of an ophthalmic
formulation/composition in the present invention. Lyophilization
bulking agents, which are principally sugars, may also be
considered stress protectants, protecting compounds during the
lyophilization cycle. Agents which greatly decelerate or eliminate
this reduced state of hydration are a class of stabilizing or
hydration-enhancing agents, the polyols at concentrations of 0.2 to
5% by weight. Representatives of such polyols are mannitol,
sorbitol, glycerol, sucrose, related sugars, and the like. A most
preferred stabilizing agent is the hydroscopic mannitol at
concentrations ranging from 0.2% to 5% by weight.
[0075] Additionally, 50 mM amino acid stabilizers such as alanine
(Ala), lysine (Lys), glycine (Gly) and glutamic acid (Glu) have
been incorporated into the formulated peptide agent containing
sequence LKKTET or LKKTNT or a conservative variant thereof to
improve recovery from reconstituted aqueous solutions following
lyophilization. The preferred amino acid stabilizers are arginine
and glycine, while the most preferred 50 mM amino acid is
glycine.
Inventive Peptide Administration
Exemplary Topical Delivery (For Surface-Acting Effects)
[0076] Peptide agents comprising amino acid sequences LKKTET or
LKKTNT or a conservative variant thereof are administered to the
surface of the eye for local effects to treat, for example: [0077]
1. Corneal epithelial wounds caused by but not limited to chemical
burns, recurrent corneal erosions, epithelial debridement during
surgery, corneal resurfacing procedures, Laser-assisted In Situ
Keratomileusis (LASIK); [0078] 2. Corneal epithelial thinning
caused by quaternary ammonium salts, such as BAK and the like;
[0079] 3. Ocular inflammation (alone or in combination with
corticosteroids) to treat, for example, conjunctivitis,
blepharitis, keratitis, uveitis, scleritis, retinitis, optic
neuritis, and temporal arteritis; [0080] 4. Microbial infection
(alone or in combination with antibacterial, antifungal, or
antiviral agents or in combination with both antimicrobials and
anti-inflammatory agents); [0081] 5. Dry eye syndrome
(xerophthalmia); [0082] 6. Red eye [alone or in combination with
ocular decongestants (adrenergic vasoconstrictors of the
conjunctiva), such as ephedrine, naphazoline, phenylephirine,
tetrahydrozoline and antihistamines, such as pheniramine maleate)
to whiten the eye]; [0083] 7. Elevated intraocular pressure (IOP)
and Glaucoma; and [0084] 8. Inflammatory or irritative conditions
after traumatic injury or surgery or in various eye irritation
disorders
[0085] Topical inventive peptide agents are formulated as
solutions, suspensions, gels and ointments. Inventive peptide agent
formulations may be administered directly or indirectly by collagen
sponges, inserts or the like. Every ophthalmic product, including
topical ophthalmics, should be sterile in its final container to
prevent microbial contamination of the eye. Preservatives are added
to the formulation when packaged in a multidose container for more
than one use to maintain sterility once the container has been
opened. Ophthalmic formulations require that the pH, buffer
capacity, viscosity and tonicity of the formulation are carefully
controlled. Preferred pH ranges, buffers, viscosities and
tonicities have been described herein.
Exemplary Formulation: Topical Solution for Eye Drops
[0086] Each milliliter of a topically-applied inventive peptide
formulation contains the following peptide agent comprising amino
acid sequence LKKTET or LKKTNT or a conservative variant as shown
below:
[0087] Peptide at a concentration with a range of from about 0.001
to 1,000 mg/ml, preferably from about 0.01 mg/ml to 600 mg/ml, more
preferably peptide at a concentration of about 0.1 mg/ml to 60
mg/ml, ost preferably peptide at a concentration of about 1 mg/ml
to 6 mg/ml.
[0088] Preferred Carrier/Vehicle: 20 mM Sodium citrate; 50 mM
glycine; 3% sucrose; NaOH or HCl to adjust pH; Purified water,
USP.
Exemplary Formulation: Topical Suspension for Eye Drops
[0089] With regard to ophthalmic suspensions containing a peptide
agent comprising amino acid sequence LKKTET or LKKTNT, particles
must be less than 10 microns in size to minimize irritation to the
eye. There may be a tendency of the solid undissolved particles to
adhere to the conjunctiva. As drug is absorbed, these particles
will dissolve to replenish the absorbed drug. This reservoir or
depot effect increases the contact time and duration of action of a
suspension compared to a solution.
[0090] Each milliliter of a topically-applied inventive peptide
formulation contains the following peptide agent comprising amino
acid sequence LKKTET or LKKTNT or a conservative variant as shown
below:
[0091] Peptide at a concentration with a range of from about 0.001
to 1,000 mg/ml, preferably from about 0.01 mg/ml to 600 mg/ml, more
preferably peptide at a concentration of about 0.1 mg/ml to 60
mg/ml, most preferably peptide at a concentration of about 1 mg/ml
to 6 mg/ml.
[0092] Preferred Carrier/Vehicle: Peptide-encapsulation in
poly(lactide-co-glycolide) PLGA microspheres; 20 mM Sodium citrate;
50 mM glycine; 3% sucrose; NaOH or HCl to adjust pH; Purified
water, USP.
Exemplary Formulation: Topical Gel for Eye Drops
[0093] Each milliliter of a topically-applied inventive peptide
formulation contains the following peptide agent comprising amino
acid sequence LKKTET or LKKTNT or a conservative variant as shown
below:
[0094] Peptide at a concentration of from about 0.001 to 1,000
mg/ml, preferably from about 0.01 mg/ml to 600 mg/ml, more
preferably peptide at a concentration of about 0.1 mg/ml to 60
mg/ml, most preferably peptide at concentration of about 1 mg/ml to
6 mg/ml.
[0095] Preferred Carrier/Vehicle: carboxymethylcellulose sodium
(0.5 to 1%); dibasic sodium phosphate; sodium chloride; propylene
glycol; methylparaben; propylparaben; NaOH/HCl to adjust pH;
Purified water, USP.
Exemplary Formulation: Topical Ointments
[0096] Each milliliter of a topically-applied inventive peptide
formulation contains the following peptide agent comprising amino
acid sequence LKKTET or LKKTNT or a conservative variant as shown
below:
[0097] Peptide at a concentration of from about 0.001 to 1,000
mg/ml, preferably from about 0.01 mg/ml to 600 mg/ml, more
preferably peptide at concentration of about 0.1 mg/ml to 60 mg/ml,
most preferably peptide at concentration of about 1 mg/ml to 6
mg/ml.
[0098] Preferred Carrier/Vehicle (1): carboxymethycellulose sodium
(2.5%); dibasic sodium phosphate; propylene glycol; methylparaben;
propylparaben; sodium chloride; NaOH/HCl to adjust pH; purified
water.
[0099] Preferred Carrier/Vehicle (2): "liquid" lanolin (10%);
mineral oil (30%), and white petrolatum (60%).
Exemplary Topical Delivery for Steroid-Sparing Effects
[0100] Corticosteroids inhibit the inflammatory response to a
variety of inciting agents. [0101] Dexamethasone ophthalmic
suspension (0.1%); Dexamethasone ophthalmic ointment (0.05%); and
Dexamethasone Sodium Phosphate Ophthalmic Solution (0.1%) [0102]
Fluorometholone Ophthalmic Ointment (0.1%); Fluorometholone
Ophthalmic Suspensions (0.25-1%); and Fluorometholone Acetate
Ophthalmic Suspension (0.1%) [0103] Lotoprednol etabonate (0.5%)
[0104] Medrysone Ophthalmic Suspension (1%) [0105] Prednisolone
Acetate Ophthalmic Suspensions (0.12-1%) and Prednisolone Sodium
Phosphate Opthmalmic Solutions (0.125-1%) [0106] Rimexolone
Ophthalmic Suspension (1%)
[0107] These agents, however, can elevate intraocular pressure
(IOP) and, in susceptible individuals, can induce glaucoma with
damage to the optic nerve, defects in visual acuity and fields of
vision, and posterior subcapsular cataract formation. Cataract
formation is a complication more likely to occur with high-dose,
longterm use. Some corticosterioids, such as fluorometholone
acetate, medrysone, and loteprednol cause less elevation of IOP
than others. Prolonged use may also suppress the host immune
response and thus aid in the establishment of secondary ocular
infections from fungi and viruses liberated from ocular tissue.
Topical corticosteroids are known to delay or slow wound
healing.
[0108] Administration of topically-applied eye drops or ointments
containing an inventive peptide agent comprising amino acid
sequence LKKTET or LKKTNT or a conservative variant to inhibit an
inflammatory response to an inciting agent has the potential to be
steroid-sparing.
Exemplary Intraocular Drug Delivery--Conjunctival/Sclera
Instillation
[0109] The topical conjunctival route of entry plays an important
role in the penetration of drugs into the anterior segment.
Furthermore, topically applied drugs have been shown to have access
to the sclera from the conjuctiva. The potential for transport or
diffusion through the sclera lies in the large and accessible
surface area of this tissue, with its high degree of hydration,
hypocellularity, and permeability that do not decline significantly
with age. As such, it is conceivable that inventive compositions
could find their way to the posterior segment by this noninvasive
route of administration. Data suggest that the sclera is readily
permeable to even large molecular weight compounds (.about.150 kD),
much larger than a peptide agent comprising amino acid sequence
LKKTET or LKKTNT or a conservative variant thereof. The recent
finding that topically-applied nepafenac inhibited choroidal and
retinal neovascularization by decreasing the production of VEGF,
and a large molecular weight peptide like insulin (5.8 kD) can
accumulate in the retina and optic nerve after topical
administration indicates that topically applied inventive
compositions, all of which having molecular weights of <150 kD,
could not only reach the posterior segment through conjunctival
penetration, but that they can also be therapeutic. Topical
solutions, suspensions, gels or ointments of peptide agents
comprising amino acid sequences LKKTET or LKKTNT or conservative
variants described above are suitable formulations for topical
conjuctival and scleral application.
[0110] Additionally, subconjuctival administration by injection of
inventive compositions of the peptide agent comprising amino acid
sequence LKKTET or LKKTNT or a conservative variant thereof is
useful in delivering anti-inflammatory and anti-microbial regimens,
sensitive to the inventive composition, to treat serious ocular
inflammation and ocular infections, such as uveitis and
endophthalmitis, and glaucoma.
[0111] Preferred Injectable Formulation: Each ml of a peptide agent
comprising amino acid sequence LKKTET or LKKTNT or a conservative
variant contains:
[0112] Peptide at a concentration of from about 0.001 to 1,000
mg/ml, preferably from about 0.01 mg/ml to 600 mg/ml, more
preferably peptide at a concentration of about 0.1 mg/ml to 60
mg/ml, most preferably peptide at a concentration of about 1 mg/ml
to 6 mg/ml.
[0113] Preferred Carrier/Vehicle: 20 mM Sodium citrate; 50 mM
glycine; 3% sucrose; NaOH or HCl to adjust pH; Water for Injection,
USP.
Exemplary Intraocular Drug Delivery--Transcorneal Instillation
[0114] Topically applied drugs do penetrate into the intraocular
environment through the hydrophobic cornea; however, transcorneal
transport is not the most effective process as it is estimated that
only one- to three-tenths of a dose penetrates into the eye while
most of the drug remains confined to the superficial epithelium
layer. Passive diffusion of inventive peptide compositions across
the cornea is largely influenced by their solubility, molecular
weight and degree of ionization. Having a net negative charge and a
relatively high molecular weight, an inventive peptide agent,
formulated as a topically-applied drug will find it difficult to
penetrate the intact cornea. This is supported by the fact that
pores, localized between epithelial cells in the cornea allow
paracellular permeation of only small molecules of about
500-molecular weight or less. However, in cases where the intact
corneal epithelium has been disrupted by erosion, for example, or
exposed to a substance or penetrating agent that opens tight
junctions between epithelial cells, inventive compositions will
pass through the cornea into the intraocular space more
efficiently.
[0115] Topical solutions, gels or ointments of peptide agents
comprising amino acid sequences LKKTET or LKKTNT or conservative
variants described above are suitable formulations for transcorneal
instillation.
Exemplary Intraocular Drug Delivery--Periocular Injection
[0116] Periocular injection formulations of inventive peptide
agents are used in cases where ocular inflammation is not
responding to a topical eye drops alone and in inflammatory
conditions such as anterior uveitis, posterior uveitis,
endophthalmitis, and optic neuritis. The peptide agent is injected
just below the conjunctiva or in the space below Tenon's capsule.
Here, more absorption will occur and consequently, more drug will
be available to the desired site. Periocular injection is additive
to topical therapy but lacks the convenience and is not
well-tolerated to be considered a first line treatment.
[0117] Preferred Injectable Formulation: Each ml of the peptide
agent comprising amino acid sequence LKKTET or LKKTNT or a
conservative variant contains:
[0118] Peptide at a concentration of from about 0.001 to 1,000
mg/ml, preferably from about 0.01 mg/ml to 600 mg/ml, more
preferably peptide at a concentration of about 0.1 mg/ml to 60
mg/ml, most preferably peptide at a concentration of about 1 mg/ml
to 6 mg/ml.
[0119] Preferred Carrier/Vehicle: 20 mM Sodium citrate; 50 mM
glycine; 3% sucrose; NaOH or HCl to adjust pH; Water for Injection,
USP.
Exemplary Intraocular Drug Delivery--IntraVitreal/IntraAqueous
Administration
[0120] As an alternative to transcorneal, transconjuctival, and
transscleral transport, delivery of inventive peptide agents to the
intraocular tissues can be achieved by injection into the vitreous
or aqueous cavitiy. The vitreous is made of a hydrogel (water,
hyaluronic acid and collagen), which fills the cavity between the
retina and the lens, while the aqueous is a watery fluid which
fills the cavity between the lens and the iris. Intravitreal or
intra-aqueous injections of an inventive peptide agent, formulated
as a solution allows for immediate exposure of intraocular tissues
to the peptide agent. To achieve a continuous intraocular presence
of an inventive agent, which may be quickly eliminated from the
vitreous, would require repeated injections that increase the risk
of endophthalmitis, damage to lens, retinal detachment and may be
poorly tolerated. To obviate this obstacle, the inventive peptide
agent would be encapsulated within phospholipid membranes, i.e.,
liposomes, biodegradable microspheres, nanoparticles, or
biodegradable lactone based polymers that includes polyesters made
by polycondensation of L-lactide, glycolide, caprolactone,
dioxanone, cyclic carbonates and their derivatives. Polylactide and
polyglycolide, known also as poly(lactic-acid) PLA and
poly(glycolic-acid) PGA, respectively, and particularly their
co-polymers poly(lactide-co-glycolide) PLGA are the most
investigated biodegradable polymers, which also can also be applied
as carriers for the inventive peptide agents. Additionally,
peptide-polymer conjugates such as the covalent linkage of the
peptide agent comprising LKKTET or LKKTNT or a conservative variant
thereof with synthetic and natural polymers such as polyethylene
glycol (PEG) and dextran, including the cyclodextrans, allow for an
improve pharmacokinetic profile, resulting in a decrease in
inventive peptide clearance.
[0121] Intravitreal or intra-aqueous administration of an inventive
peptide agent may be indicated in treatment of ocular inflammation,
ocular infection (bacterial, fungal or viral) and the glaucomatous
eye by controlling F-actin architecture in outflow pathway cells.
(Read AT et al., Exp Eye Res, 2006 Jun:82(6):974-85).
[0122] A Preferred Injectable Formulation: Each ml of the peptide
agent comprising amino acid sequence LKKTET or LKKTNT or a
conservative variant contains:
[0123] Peptide at a concentration of from about 0.001 to 1,000
mg/ml, preferably from about 0.01 mg/ml to 600 mg/ml, more
preferably peptide at a concentration of about 0.1 mg/ml to 60
mg/ml, most preferably peptide at a concentration of about 1 mg/ml
to 6 mg/ml.
[0124] Preferred Carrier/Vehicle (1): 20 mM Sodium citrate; 50 mM
glycine; 3% sucrose; NaOH or HCl to adjust pH; Water for Injection,
USP.
[0125] Preferred Carrier/Vehicle (2): Peptide encapsulation in PLGA
microspheres; 20 mM Sodium citrate; 50 mM glycine; 3% sucrose; NaOH
or HCl to adjust pH; Water for Injection, USP.
Exemplary Formulation Dosing
Topical Solutions and Suspensions:
[0126] One drop per administration and at least five minutes
between administrations is recommended. Immediately after
instilling a drop on the eye, place pressure on the lacrimal sac
for one or two minutes to reduce the rate of drug loss through this
pathway. Injectable Dosing: Use 27-30 gauge needle, 0.5 inch
length.
[0127] A composition according to one embodiment may be in
lyophilized form, or in a form capable of being lyophilized,
comprising a peptide agent comprising amino acid sequence LKKTET or
LKKTNT, a conservative variant thereof, or a stimulating agent that
stimulates production of an LKKTET or LKKTNT peptide, or a
conservative variant thereof, the composition further comprising at
least one amino acid stabilizing agent. The composition may
comprise a peptide agent comprising amino acid sequence LKKTET or
LKKTNT, a conservative variant thereof, or a stimulating agent that
stimulates production of an LKKTET or LKKTNT peptide, or a
conservative variant thereof, and at least one of a lyophilization
bulking agent or an amino acid stabilizing agent, said composition
being in lyophilized form. The composition may further comprise at
least one of an acidic or basic pH adjusting agent capable of
adjusting pH of the composition to a desired physiologically
acceptable pH level in an aqueous medium, and a buffer to
substantially maintain said desired pH in said aqueous medium. The
amino acid stabilizing agent may comprise at least one of alanine,
lysine, glycine or glutamic acid. The amino acid stabilizing agent
may comprise at least one 50 mM amino acid stabilizer. The amino
acid stabilizing agent may comprise 50 mM glycine. The composition
may further comprise a bulking agent comprising at least one of
carbohydrates, sugar alcohols, mono-, di-, and poly-saccharides,
polyols, mannitol, sorbitol, glycerol, sucrose or dextrose. The pH
adjusting agent may include at least one of NaOH or HCl. The buffer
may include at least one of a sodium salt of at least one of
acetic, ascorbic, boric, carbonic, phosphoric, citric, gluconic,
lactic or propionic acids, a calcium salt of carbonic or propionic
acids, a potassium salt of phosphoric acid, Tris buffer, acetate,
phosphate, citrate or borate buffers. The buffer may be sodium
citrate. The buffer may have a buffer capacity of about 0.05-1.0.
The buffer may have a buffer capacity of about 0.02-0.2, or about
0.01-0.1. The desired pH level may be within a range of about
3.5-11.5, about 3.5-9, about 4.5-8, or about 5.0-7.8. The desired
pH level may be about 5.5. The peptide agent may comprise amino
acid sequence KLKKTET, amino acid sequence LKKTETQ, T.beta.4, an
N-terminal variant of T.beta.4, a C-terminal variant of T.beta.4,
or an isoform of T.beta.4. The composition may further include an
aqueous medium, wherein said peptide agent is present in said
aqueous medium at a concentration within a range of about
0.001-1,000 mg/ml. The composition may further include at least one
steroid.
[0128] A composition according to another embodiment is for
administration to skin tissue of a subject, and comprises a peptide
agent comprising amino acid sequence LKKTET or LKKTNT, a
conservative variant thereof, or a stimulating agent that
stimulates production of an LKKTET or LKKTNT peptide, or a
conservative variant thereof, in said tissue, the composition
further comprising a quaternary ammonium salt and a topical carrier
for application to skin tissue of said subject. The peptide agent
may comprise amino acid sequence LKKTET, amino acid sequence
LKKTETQ, T.beta.4, an N-terminal variant of T.beta.4, or an isoform
of T.beta.4. The quaternary ammonium salt may comprise benzalkonium
chloride. The peptide agent may be at a concentration of about
0.001-1,000 mg/ml and said quaternary ammonium salt may be present
in said composition at about 0.001-1% by weight. The composition
may be in a form of a solution, gel, cream, paste, lotion, spray,
suspension, dispersion, salve, hydrogel, ointment or foam
formulation. The composition may be a cosmetic formulation.
[0129] A pharmaceutical or cosmetic combination according to
another embodiment comprises a peptide agent comprising amino acid
sequence LKKTET or LKKTNT, a conservative variant thereof, or a
stimulating agent that stimulates production of an LKKTET or LKKTNT
peptide, or a conservative variant thereof, the combination further
comprising a quaternary ammonium salt, wherein said agent and said
salt can be administered to a subject separately or together. The
peptide agent may comprise amino acid sequence LKKTET, amino acid
sequence LKKTETQ, T.beta.4, an N-terminal variant T.beta.4, or an
isoform of T.beta.4. The quaternary ammonium salt may be
benzalkonium chloride. The combination may comprise a
pharmaceutical, ophthalmic or cosmetic composition comprising said
peptide agent at a concentration of about 0.001-1,000 mg/ml, and
wherein said quaternary ammonium salt may be present in said
composition at about 0.0001-1% by weight. The combination may
comprise an ophthalmic composition further comprising an
opthalmically acceptable carrier. The composition may comprise an
eye drop composition.
[0130] According to a further embodiment, method of treatment for
treating, preventing, inhibiting or reducing tissue deterioration,
injury or damage resulting from administration of a quaternary
ammonium salt to a subject, comprises administering said quaternary
ammonium salt to said subject, and administering to said subject a
peptide agent comprising amino acid sequence LKKTET or LKKTNT, a
conservative variant thereof, or a stimulating agent that
stimulates production of an LKKTET or LKKTNT peptide, or a
conservative variant thereof in said tissue. The agent may be
administered to said subject prior to, concurrently with or after
said administration of said quaternary ammonium salt. The agent and
said salt may be administered together as a composition. The
composition may further comprise an opthalmically acceptable
carrier. The composition may comprise an eye drop composition. The
composition may comprise a cosmetically acceptable carrier. The
composition may be in a form of a solution, gel, cream, past,
lotion, spray, suspension, dispersion, salve, hydrogel, ointment or
foam formulation. The peptide agent may comprise amino acid
sequence KLKKTET, amino acid sequence LKKTETQ, T.beta.4, an
N-terminal variant of T.beta.4, a C-terminal variant of T.beta.4,
or an isoform of T.beta.4. The quaternary ammonium salt may
comprise benzalkonium chloride. The peptid agent may be included in
said composition at a concentration within a range of about
0.001-1,000 mg/ml, and said quaternary ammonium salt may be present
in said composition within a range of about 0.0001-1% by
weight.
[0131] A composition according to still a further embodiment
comprises a peptide agent comprising amino acid sequence LKKTET or
LKKTNT, a conservative variant thereof, or a stimulating agent that
stimulates production of an LKKTET or LKKTNT peptide, or a
conservative variant thereof, and an opthalmically acceptable
carrier, an antimicrobially effective preservative, a tonicity
agent for providing said composition with an opthalmically
acceptable tonicity, a comfort enhancing agent, at least one of an
acidic or basic pH adjusting agent capable of adjusting pH of the
composition to a desired ophthalmically acceptable pH level, and a
buffer to substantially maintain said desired pH level. The
composition may further comprise at least one of an antioxidant or
an oxygen sequestering agent. The antioxidant or oxygen
sequestering agent may comprise at least one of sodium sulfite,
sodium thiosulfite, sodium bisulfite, sodium metabisulfite,
thiourea, ascorbic acid, EDTA/disodium edentate, acetic acid,
citric acid, glutathione, acetylcysteine or methionine. The
antioxidant or oxygen sequestering agent may be at a concentration
in said composition within a range of about 0.0001-1.0% by weight.
The antimicrobially effective preservative may comprise at least
one of a quaternary ammonium compound, benzalkonium chloride,
benzethonium chloride, cetalkonium chloride, cetrimide,
benzododecinium bromide, benzoxonium chloride, an alkyl-mercury
salt of thiosalicylic acid, thimerosal, phenylmercuric nitrate,
phenylmercuric acetate, phenylmercuric borate, a paraben,
methylparaben, propylparaben, a chelating agent, disodium edentate,
sodium gluconate, sodium propionate, an alcohol, chlorobutanol,
benzyl alcohol, phenyl ethanol, a guanidine derivative,
chlorohexidine, polyhexamethylene biguanide, sorbic acid, boric
acid, chlorine dioxide, polyquatemium or myristamidopropyl
diethylamine. The preservative may be present in said composition
at a concentration within a range of about 0.0001-5.0% (w/v). The
composition may further include at least one opthalmically
acceptable stabilizing agent. The stabilizing agent may comprise at
least one of a polyol, mannitol, sorbitol, glycerol, sucrose, an
amino acid stabilizer, alanine (Ala), lysine (Lys), glycine (Gly)
or glutamic acid (Glu). The stabilizing agent may be present in
said composition at a concentration within a range of about
0.01-10% by weight. The tonicity agent may comprise at least one of
an ionic compound, alkali metal halide, alkaline earth metal
halide, CaCl2, KBr, KCl, LiCl, NaBr, NaCl, boric acid, a non-ionic
compound, urea, glycerol, sorbitol, mannitol, propylene glycol, or
dextrose. The composition may have an osmolality within a range of
about 50 to 1000 mOsmol. The adjusting agent may include at least
one of NaOH or HCL. The buffer may include at least one of a sodium
salt of acetic acid, a sodium salt of ascorbic acid, a sodium salt
of boric acid, a sodium salt of carbonic acid, a sodium salt of
phosphoric acid, a sodium salt of citric acid, a sodium salt of
gluconic acid, a sodium salt of lactic acid, a sodium salt of
propionic acid, a calcium salt of carbonic acid, a calcium salt of
propionic acid, a potassium salt of phosphoric acid, Tris buffer,
an acetate buffer, a phosphate buffer, a citrate buffer or a borate
buffer. The desired pH level may be within a range of about
3.5-11.5. The buffer may have a buffer capacity of about 0.05-1.0.
The peptide agent may comprise amino acid sequence KLKKTET, amino
acid sequence LKKTETQ, T.beta.4, an N-terminal variant of T.beta.4,
a C-terminal variant of T.beta.4, or an isoform of T.beta.4. The
composition may further comprise an aqueous medium, wherein said
peptide agent is present in said aqueous medium at a concentration
within a range of about 0.001-1,000 mg/ml. The composition may be
in a form of a solution, gel, cream, paste, lotion, spray,
suspension, dispersion, salve, hydrogel, ointment or foam
formulation. The composition may comprise an eye drop composition.
The peptide agent may be present in said composition encapsulated
with at least one of phospholipid membranes, liposomes,
microspheres, nanoparticles or biodegradable polymers, or as a
peptide-polymer conjugate.
EXAMPLE
[0132] Thymosin beta 4 (T.beta..sub.4), a 43 amino acid molecule,
promotes ocular wound healing, decreases ocular inflammation, and
has anti-apoptotic effects on corneal epithelium. In this study,
the effect of T.beta..sub.4 on the survival of cultured human
corneal epithelial cells exposed to benzalkonium chloride (BAK) was
measured.
[0133] Human corneal epithelial cells at approximately 80%
confluence were treated with 0%, 0.001%, 0.01%, or 0.1% BAK for 15
minutes. After 3 and 24 hours of recovery in culture medium, cell
proliferation was measured using a calorimetric BrdU incorporation
assay. Apoptosis was measured using a calorimetric annexin-based
cell death assay. Studies were repeated in the presence of 1 mcg/ml
T.beta..sub.4, an in vitro dosage demonstrated effective in several
published studies. To further assess the ability of Tb.sub.4 to
prevent apoptosis, corneal epithelial cells were treated with 0.01%
BAK.+-.T.beta..sub.4 over a 5 day time course.
[0134] At all BAK concentrations used, corneal epithelial cell
proliferation was inhibited, and apoptosis was increased, compared
to control at 3 and 24 hours recovery time. At the 3 and 24 hour
time points, T.beta..sub.4 did not abrogate the deleterious effects
of BAK; cell proliferation was not promoted by Tb.sub.4 and
apoptosis was not inhibited. However, at longer times in culture (2
to 5 days), T.beta..sub.4 treatment significantly inhibited the
BAK-initiated epithelial cell apoptosis. In addition,
T.beta..sub.4-treated cells demonstrated decreased apoptosis
compared to those cultured in medium alone for 5 days.
[0135] BAK, a preservative used in many commercially available
ocular solutions, induces corneal epithelial cell apoptosis in
culture, suggesting that long-term exposure is deleterious to
corneal health. The study reported here indicates that
T.beta..sub.4 is able to overcome the deleterious pro-apoptotic
effects of BAK. Since many BAK-containing eye drops are typically
used for extended periods of time, T.beta..sub.4 is indicated to be
a useful additive to solutions containing this preservative.
* * * * *