U.S. patent application number 10/225341 was filed with the patent office on 2003-06-19 for method for treating meibomian gland disease.
Invention is credited to Lokeshwar, Balakrishna L., Pflugfelder, Stephen C., Selzer, Marie.
Application Number | 20030114426 10/225341 |
Document ID | / |
Family ID | 22187748 |
Filed Date | 2003-06-19 |
United States Patent
Application |
20030114426 |
Kind Code |
A1 |
Pflugfelder, Stephen C. ; et
al. |
June 19, 2003 |
Method for treating meibomian gland disease
Abstract
A method for treating a patient having meibomian gland disease,
ocular irritation associated with delayed tear clearance, or
recurrent corneal epithelial erosion, is disclosed. Preferably, the
method concerns treatment of a patient with topical tetracycline, a
derivative or analogue of tetracycline, or a chemically modified
tetracycline (CMT). Oral administration of a CMT is also disclosed
as part of the method for treating meibomian gland disease, ocular
irritation associated with delayed tear clearance, or recurrent
corneal epithelial erosion.
Inventors: |
Pflugfelder, Stephen C.;
(Miami, FL) ; Lokeshwar, Balakrishna L.; (Miami,
FL) ; Selzer, Marie; (Fort Lauderdale, FL) |
Correspondence
Address: |
VENABLE, BAETJER, HOWARD AND CIVILETTI, LLP
P.O. BOX 34385
WASHINGTON
DC
20043-9998
US
|
Family ID: |
22187748 |
Appl. No.: |
10/225341 |
Filed: |
August 22, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10225341 |
Aug 22, 2002 |
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09307182 |
May 7, 1999 |
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6455583 |
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60084873 |
May 8, 1998 |
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Current U.S.
Class: |
514/152 |
Current CPC
Class: |
Y10S 514/912 20130101;
A61P 31/00 20180101; A61P 27/02 20180101; A61P 27/00 20180101; A61K
31/65 20130101; A61P 33/00 20180101; A61P 7/02 20180101 |
Class at
Publication: |
514/152 |
International
Class: |
A61K 031/65 |
Claims
What is claimed is:
1. A method of treating a patient having meibomian gland disease,
aqueous tear deficiency, delayed tear clearance or recurrent
corneal epithelial erosion, said method comprising: topically
administering an effective amount of tetracycline to an eye of the
patient.
2. The method of claim 1, wherein said tetracycline is an
antimicrobial tetracycline.
3. The method of claim 2, wherein said antimicrobial tetracycline
is oxytetracycline.
4. The method of claim 2, wherein said antimicrobial tetracycline
is doxycycline.
5. The method of claim 1, wherein said tetracycline is a
non-antimicrobial tetracycline.
6. The method of claim 5, wherein said non-antimicrobial is a
member selected from the group consisting of
4-dedimethylaminotetracycline, 4-dedimethylamino-5-oxytetracycline,
4-dedimethylamino-7-chlorotetracycli- ne,
4-hydroxy-4-dedimethylaminotetracycline,
4-dedimethylamino-12a-deoxyte- tracycline,
4-dedimethylamino-11-hydroxy-12a-deoxytetracycline,
4-dedimethylamino-7-dimethylaminotetracycline,
6-dimethyl-6-deoxy-4-dedim- ethylaminotetracycline,
6-o-deoxy-5-hydroxy-4-dedimethylaminotetracycline,
11a-chlortetracycline, 12a-deoxytetracycline and 2-nitrilo analogs
of tetracycline.
7. The method of claim 1, wherein said treatment increases tear
clearance in the eye of the patient.
8. The method of claim 1, wherein said treatment comprises
inhibiting a member selected from the group consisting of matrix
metalloproteinase activity in tear fluid, synthesis and activation
of interleukin-1.beta., conversion of precursor interleukin-1.beta.
to mature interleukin-1.beta., ocular surface inflammation and
reactive oxygen species in tear fluid and ocular surface
epithelium.
9. The method of claim 8, wherein said matrix metalloproteinase
comprises matrix metalloproteinase-9.
10. The method of claim 1, wherein said treatment comprises
increasing production of interleukin-1 receptor antagonist by
corneal epithelium.
11. The method of claim 1, wherein said treatment comprises
reducing interleukin-1-.alpha. concentration in tear fluid.
12. A method of treating a patient having meibomian gland disease,
aqueous tear deficiency, delayed tear clearance or recurrent
corneal epithelial erosion, said method comprising: orally
administering an effective amount of tetracycline to an eye of the
patient.
13. The method of claim 12, wherein said tetracycline is an
antimicrobial tetracycline.
14. The method of claim 13, wherein said antimicrobial tetracycline
is oxytetracycline.
15. The method of claim 13, wherein said antimicrobial tetracycline
is doxycycline.
16. The method of claim 12, wherein said tetracycline is a
non-antimicrobial tetracycline.
17. The method of claim 16, wherein said non-antimicrobial is a
member selected from the group consisting of
4-dedimethylaminotetracycline, 4-dedimethylamino-5-oxytetracycline,
4-dedimethylamino-7-chlorotetracycli- ne,
4-hydroxy-4-dedimethylaminotetracycline,
4-dedimethylamino-12a-deoxyte- tracycline,
4-dedimethylamino-11-hydroxy-12a-deoxytetracycline,
4-dedimethylamino-7-dimethylaminotetracycline,
6-dimethyl-6-deoxy-4-dedim- ethylaminotetracycline,
6-o-deoxy-5-hydroxy-4-dedimethylaminotetracycline,
11a-chlortetracycline, 12a-deoxytetracycline and 2-nitrilo analogs
of tetracycline.
18. The method of claim 12, wherein said treatment increases tear
clearance in the eye of the patient.
19. The method of claim 12, wherein said treatment comprises
inhibiting a member selected from the group consisting of matrix
metalloproteinase activity in tear fluid, synthesis and activation
of interleukin-1.beta., conversion of precursor interleukin-1.beta.
to mature interleukin-1.beta., ocular surface inflammation and
reactive oxygen species in tear fluid and ocular surface
epithelium.
20. The method of claim 19, wherein said matrix metalloproteinase
comprises matrix metalloproteinase-9.
21. The method of claim 12, wherein said treatment comprises
increasing production of an interleukin-1 receptor antagonist by
corneal epithelium.
22. The method of claim 12, wherein said treatment comprises
reducing interleukin-1-.alpha. concentration in tear fluid.
23. A composition for treating a patient having meibomian gland
disease, said composition comprising an antimicrobial tetracycline
or a non-antimicrobial tetracycline as an active ingredient in a
balanced salt solution.
24. The composition of claim 23, wherein said active ingredient has
a final concentration of between about 0.001% to about 1.0%.
25. The composition of claim 23, wherein said active ingredient has
a final concentration of about 0.025%.
26. The composition of claim 23, wherein said active ingredient is
doxycycline.
27. The composition of claim 23, wherein said antimicrobial
tetracycline comprises a member selected from the group consisting
of a topical ointment, a gel and a sustained-release preparation.
Description
BACKGROUND OF THE INVENTION
[0001] Meibomian gland disease is the most common tear film and
ocular surface disorder causing eye irritation. The incidence of
the disease increases with age, and occurs in approximately 50% of
patients with the skin disease, rosacea. A conservative estimate of
the number of patients affected with this condition is 10 million
in the United States alone. It has been reported that 15% of
patients with ocular rosacea develop recurrent corneal epithelial
erosions, a potentially sight-threatening problem.
[0002] Common complaints of patients suffering from meibomian gland
disease include blurred or filmy vision, burning or foreign body
sensations in the eye, photophobia, and pain severe enough to
awaken the person from sleep. Although patients with this condition
usually have normal production of aqueous tears by their lacrimal
glands, their meibomian glands can atrophy and this is frequently
accompanied by metaplasia of the ductal epithelium of these glands.
Anterior erosion of the mucocutaneous junction of the eyelid is
often noted, as well as eyelid and conjunctival infection, eyelid
margin irregularity, corneal epithelial changes, and corneal
vascularization.
[0003] The mechanisms responsible for the eyelid and ocular surface
changes and irritation symptoms experienced by patients with
meibomian gland disease were heretofore unknown. Therefore,
previous treatments of meibomian gland disease were directed only
to treatment of presumed infection of the eyelids or meibomian
glands, or had particular disadvantages that made such treatments
of little use for long periods of time. For example, patients with
meibomian gland disease have been symptomatically treated with
artificial tears, but these agents provide limited, if any,
improvement. Topically applied steroids to the eyelids or ocular
surface are effective as short-term pulse therapies. However,
steroids are not good long-term solutions because of the potential
side-effects e.g., cataract and glaucoma. Meibomian gland disease
is currently not curable or reversible; therefore, patients with
this condition must be treated for life.
[0004] Orally administered tetracyclines and tetracycline analogues
(e.g., doxycycline and minocycline) having antibiotic activity are
commonly and effectively used for prophylactic or therapeutic
treatment of meibomian gland disease. The mechanism by which
tetracyclines work in treating meibomian gland disease is not
known, but some relief of symptoms has been reported. However, one
disadvantage for using antimicrobially active tetracyclines or
tetracycline analogues orally in the treatment of meibomian gland
disease is that a high percentage of patients are unable to
tolerate oral tetracyclines for extended periods of time. The
intolerance to tetracyclines can manifest itself in
gastrointestinal problems, e.g., epigastric pain, nausea, vomiting,
and diarrhea, or other problems related to taking long-term oral
antibiotics, such as mucosal candidiasis. At the present time there
are no available long-term treatments of meibomian gland
disease.
[0005] Recently, tetracycline and tetracycline analogues have been
reported to have antimicrobial-inflammatory effects (e.g.,
reduction of IL-1 and nitric oxide production) and to inhibit
synthesis and activation of MMPs. Tetracyclines have been reported
to be effective in treating diseases where chronic inflammation and
tissue destruction due to increased collagenase activity have been
implicated. These diseases include rheumatoid arthritis, sterile
corneal ulceration, and periodontitis.
[0006] Certain modifications of the chemical structure of
tetracycline result in a tetracycline analogue which lacks
antimicrobial activity. These non-antimicrobial, chemically
modified tetracyclines (CMTs) retain their anti-inflammatory and
anti-collagenolytic activities. Topical administration of
antimicrobial tetracyclines or non-antimicrobial CMTs, or oral
administration of non-antimicrobial CMTs represent a major advance
in the therapy of patients having meibomian gland disease because
both treatment modalities eliminate systemic toxicity and would
allow lifetime administration of these therapeutic agents for this
chronic disease.
BRIEF SUMMARY OF THE INVENTION
[0007] The subject invention concerns a class of agents and methods
of using those agents for treatment of a patient having meibomian
gland disease, including relief of symptoms or conditions
associated with the disease, such as ocular irritation, delayed
tear clearance, or recurrent corneal epithelial erosion. More
specifically, the subject invention concerns agents and methods for
decreasing ocular irritation, and surface inflammation, improving
tear clearance, reducing tear IL-1.alpha. concentration, or
inhibiting IL-1.alpha.-mediated matrix metalloproteinase activity
which is increased in patients with meibomian gland disease or
delayed tear clearance. The subject invention can thus be useful
for reducing eye irritation, improving tear clearance, reducing
IL-1.alpha. concentration in the tear fluid, or inhibiting MMP
activity in the tear fluid of patients with delayed tear clearance
and increased tear fluid IL-1.alpha..
[0008] In a preferred embodiment, the subject invention concerns
use of an effective amount of a topically administered
antimicrobial tetracycline or tetracycline analogue, or a
non-antimicrobial analogue of tetracycline. Non-antimicrobial
tetracycline analogues are commonly referred to and accepted in the
scientific literature as "chemically modified tetracyclines"
(CMTs). These compounds, or compositions comprising those
compounds, can be used for treating meibomian gland disease, ocular
irritation associated with delayed tear clearance, or recurrent
corneal epithelial erosion. Alternatively, CMTs can be administered
orally for treating meibomian gland disease, ocular irritation
associated with delayed tear clearance, or recurrent corneal
epithelial erosion.
BRIEF DESCRIPTION OF THE DRAWINGS
[0009] FIG. 1 shows IL-1.alpha. concentrations in tear fluid for
rosacea patients, ideal controls, and age-matched control subject
groups.
[0010] FIG. 2 shows gelatinase activity plotted against IL-1.alpha.
concentrations for normal control (NL) patients and meibomian gland
disease (MGD) patients.
[0011] FIG. 3 shows fluorescein tear concentration plotted against
IL-1.alpha. concentration for normal control (NL) patients and
meibomian gland disease (MGD) patients.
[0012] FIG. 4 shows fluorescein tear concentration plotted against
gelatinase activity normal control (NL) patients and meibomian
gland disease (MGD) patients.
[0013] FIG. 5 shows the concentration (pg/ml) of mature IL-1.alpha.
in supernatants of human corneal epithelial cell cultures.
Media=negative control; LPS=cultures treated with
lipopolysaccharide for 24 h; LPS MP=cultures treated with LPS and
the corticosteroid methylprednisolone for 24 h; LPS Doxy=cultures
treated with LPS and doxycycline (5 .mu.g/ml) for 24 h.
[0014] FIG. 6 shows the ratio of the mature to precursor forms of
IL-1.alpha. in supernatants of human corneal epithelial cell
cultures. Media=negative control; LPS=cultures treated with
lipopolysaccharide for 24 h; LPS MP=cultures treated with LPS and
the corticosteroid methylprednisolone for 24 h; LPS Doxy=cultures
treated with LPS and doxycycline (5 .mu.g/ml) for 24 h.
[0015] FIG. 7 shows the concentration (pg/ml) of IL-1RA in
supernatants of human corneal epithelial cell cultures.
Media=negative control; LPS=cultures treated with
lipopolysaccharide for 24 h; Doxy=cultures treated with doxycycline
(5 .mu.g/ml) for 24 h; CMT 3=cultures treated with chemically
modified tetracycline CMT 3 (1 .mu.g/ml) for 24 hr;
Doxy+LPS=cultures treated with LPS and doxycycline (5 .mu.g/ml) for
24 h; CMT 3+LPS=cultures treated with LPS and chemically modified
tetracycline CMT 3 (1 .mu.g/ml) for 24 hr.
[0016] FIG. 8 is a gelatin zymogram of human corneal epithelial
cell supernatant: doxycycline treatment and MMP-3 activation. Lane
Legend: (1) 92 kDa purified pro-MMP-9 standard (0.1 ng); (2)
Corneal epithelial supernatant+50 ,.mu.g/mL doxycycline; (3)
Supernatant+50 .mu.g/mL doxycycline+1.5 ng/.mu.L MMP-3; (4)
Supernatant+100 .mu.g/mL doxycycline+1.5 ng/.mu.L MMP-3; (5)
Supernatant from cell culture pre-treated with doxycycline; (6)
Supernatant from cell culture pre-treated with doxycycline+1.5
ng/.mu.L MMP-3.
DETAILED DESCRIPTION OF THE INVENTION
[0017] This invention concerns, in a preferred embodiment, the use
of topically applied antimicrobial tetracyclines, including
tetracycline or a tetracycline analogue, or topically or orally
administered chemically-modified tetracyclines (CMTs) for treatment
of a patient having meibomian gland disease. According to the
subject invention, treatment of a patient having meibomian gland
disease includes reducing or reversing irritation symptoms, delayed
tear clearance, recurrent corneal epithelial erosion or aqueous
tear deficiency. The CMTs lack antimicrobial activity. For purposes
of this invention, tetracycline and its analogues having
antimicrobial activity are termed "antimicrobial tetracyclines" and
the tetracycline analogues lacking antimicrobial activity are
termed "CMTs".
[0018] We recently discovered that patients with meibomian gland
disease have significantly delayed tear clearance, rapid tear
break-up time (often instantaneous), and tear film instability. In
addition, it was found that meibomian gland disease patients have
significantly greater concentrations of the pro-inflammatory
cytokine, interleukin I alpha (IL-1.alpha.) in their tear
fluid.
[0019] IL-1.alpha. has been reported to have multiple biological
effects, including increasing expression of adhesion molecules by
vascular endothelial and epithelial cells that facilitate binding
of inflammatory cells, inducing expression of chemotactic cytokines
such as interleukin 8, stimulating production of prostaglandins
that increase vascular permeability and pain, stimulating
production of nitric oxide that causes vasodilation and redness,
and stimulation of production of matrix-degrading enzymes, termed
matrix metalloproteinases (MMPs), by several different cell types,
including mucosal epithelium.
[0020] We found that the activity of one member of the MMP family,
MMP-9 (also termed gelatinase B), is markedly increased in the tear
fluid of patients with meibomian gland disease associated with
rosacea, as compared to normal asymptomatic patients. In addition,
activity of gelatinase B appears to be inversely correlated with
tear clearance.
[0021] These data indicate that antimicrobial tetracyclines and
tetracycline analogues and CMTs possess biological activities
capable of neutralizing the inflammatory and matrix degrading
factors that have been found to be strongly correlated with the
development of ocular rosacea in our studies.
[0022] Advantageously, topically applied tetracyclines or CMTs can
have greater efficacy because of the higher concentrations that can
be achieved at the disease site. Because of their lack of
antimicrobial-bacterial effect and greater therapeutic activity,
CMTs can have fewer systemic or other side effects than
tetracyclines, whether administered orally or topically.
[0023] Topical administration of a tetracycline analogue in an
ointment (e.g., oxytetracycline) or in solution (e.g., doxycycline
eye drops), to patients having meibomian gland disease associated
with rosacea resulted in complete resolution of symptoms in 73% (8
of 11) of the patients treated with the tetracycline analogue
ointment and more than 90% (10 of 11) of the patients treated with
the tetracycline analogue solution.
[0024] Several patients who had previously used oral tetracycline
stated that improvement in their symptoms was better with topically
applied oxytetracycline than orally administered tetracycline.
Three patients had moderate but not complete relief of symptoms.
Three other patients experienced burning from the medication, one
of which ceased treatment.
[0025] The tetracycline utilized in the present invention may be
any of the readily available, pharmaceutically acceptable
tetracyclines known in the medical art. Included in this group of
tetracyclines are those such as chlortetracycline, which is
marketed under the tradename Acronize, Aureocina, Aureomycin,
Biomitsin, Biomycin, and Chrysomykine; Demeclyeycline marketed as
Ledermycin, Detravis, Meciclin, and Mexocine; Doxycyline marketed
as Vibramycin, Vibramycin Hyclace, Liomycin Vibradox, Panamycin,
Titradox, Hydramycin, Tecacin; Lymecycline which is marketed as
Armyl, Mucomycin, Tetramyl, Tetralysal; Methacycline which is
marketed as Adriamicina, Cyclobiotic, Germicilclin, Globociclina,
Megamycine, Pindex, Londomycin; Optimycin, Rondomycin; Minocycline
which is marketed as Minocin, Klinomycin, Vectrin; Oxytetracycline
which is marketed as Biostat, Oxacycline, Oxatets, Oxydon,
Oxymycin, Oxytan, Oxytetracid, Ryomycin, Stezazin, Tetraject,
Terramycin, Tetramel, Tetran, Dendarcin, Dendarcin;
Rolitetracycline marketed as Bristacin, Reverin, Superciclin,
Syntetrex, Syntetrin, Synotodecin, Tetraverin, Transcycline,
Velacicline, Velacycline; and Tetracycline marketed as Achromycin,
Ambramycin, Cyclomycin, Polycycline, Tetrabon, and Tetracyn.
[0026] The active salts which are formed through protonation of the
dimethylamino group on carbon atom 4, exist as crystalline
compounds and are very stable in water. However, these amphoteric
antibiotics will crystallize out of aqueous solutions of their
salts unless stabilized by an excess of acid. The hydrochloride
salts are used most commonly for oral administration. Water soluble
salts may be obtained also from bases such as sodium or potassium
hydroxides but are not stable in aqueous solution, they are also
formed with divalent and polyvalent metals.
[0027] The tetracyclines or CMTs used according to the present
invention are preferably orally administered at a dosage level from
about 10% to about 100%, and preferably about 20% to about 80% of
the normal antibiotic therapeutic dose of the particular
tetracycline compound being employed. By normal antibiotic
therapeutic dose is meant the dosage of the particular tetracycline
compound which is commonly used and recommended for the treatment
of bacterial infection. Alternatively, sub-antimicrobial dose means
a dose having no significant antimicrobial effect in vitro or in
vivo. More than 100% of the normal antibiotic therapeutic dose can
be utilized in the method of the present invention. The normal
antibiotic therapeutic dose of the tetracycline is, for the most
part, well studied and well documented.
[0028] Tetracyclines and chemically modified tetracyclines for
topical administration can be added to buffered and electrolyte
balanced aqueous solutions, buffered and electrolyte balanced
aqueous solutions with a lubricating polymer, mineral oil or
petrolatum-based ointment, other oils, liposomes, cylcodextrins,
sustained release polymers or gels in a range from 0.001 to 3%
(weight per volume or weight per weight). These preparations can be
administered to the preocular tear film or onto the eyelid skin or
lid margin 1 to 6 times per day for a period up to the lifetime of
the patient. For example, an eye drop solution comprising
doxycycline as an active ingredient was prepared by dissolving
pharmaceutical grade doxycycline hydrochloride powder in an
electrolyte-balanced salt solution (BSS.TM., Alcon, Ft. Worth,
Tex.) to a final concentration of 0.025%.
[0029] In a preferred embodiment, the compounds useful according to
the subject invention are tetracyclines which have been chemically
modified so as to substantially reduce or eliminate antimicrobial
properties and increase their antimicrobial-inflammatory activity.
Methods for reducing antimicrobial properties of a tetracycline are
disclosed in The Chemistry of the Tetracyclines, Ch. 6, Mitscher,
Ed., at p. 211. As pointed out by Mitscher, modification at
positions 1, 2, 3, 4, 10, and 12a can lead to loss of antimicrobial
activity. Such chemically modified tetracyclines (CMTs) are
included in the preferred embodiment of the present invention,
since they can be used without disturbing the normal microbial
flora of the treated mammal as would happen with extended exposure
to antimicrobial tetracyclines.
[0030] Preferable CMTs used according to the subject invention
include those lacking a dimethylamino side chain at position 4. For
example, 4-dedimethylamino-tetracycline,
4-dedimethylamino-5-oxytetracycline,
4-dedimethylamino-7-chlortetracycline,
4-hydroxy-4-dedimethylaminotetracy- cline,
4-dedimethylamino-12a-deoxytetracycline,
4-dedimethylamino-11-hyclr- oxy-12a-deoxytetracycline,
4-dedimethylamino-7-dimethylaminotetracycline,
6-dimethyl-6-deoxy-4-dedimethylaminotetracycline,
6-o-deoxy-5-hydroxy-4-d- edimethylaminotetracycline,
11a-chlortetracycline, 12a-deoxytetracycline, and the 2-nitrilo
analogs of tetracycline.
[0031] The amount of tetracycline or CMT administered to
effectively treat meibomian gland disease, ocular irritation
associated with delayed tear clearance, or recurrent corneal
epithelial erosion is an amount that significantly improves tear
clearance, reduces IL-1.alpha. concentration in the tear fluids, or
IL-1.alpha. mediated production of MMPs, or MMP activity. The
maximal dosage for humans is the highest dosage that does not cause
clinically important side effects. For the purpose of the present
invention, side effects include clinically important disruption of
the normal flora as well as toxic effects to the eye surface.
[0032] The dosage of tetracyclines administered in the present
invention is also additionally dependent upon the age and weight of
the person being treated, the mode of administration, and the type
and severity of the inflammatory or matrix-degrading disease being
treated. For illustrative purposes, a suitable amount of the
antimicrobial tetracycline, doxycycline, is 0.1-4.0 mg/kg/day. In
the case of a non-antimicrobial tetracycline, for example, the dose
for 4-dedimethylaminotetracycline can be 0.1-30 mg/kg/day. However,
in either case, the preferred method of treatment includes
tetracycline or CMT compositions administered topically to the eye
in suitable pharmaceutical carriers. The pharmaceutical carrier may
be in the form of a solution, suspension, ointment, gel, or other
form suitable for topical administration of the tetracycline or
topical or oral administration of CMT to the affected mammal. For
oral administration, the CMTs utilized according to the subject
invention can be formulated in the form of tablets, capsules,
elixirs, or the like. Additionally, the tetracyclines or CMTs of
the present invention can be reasonably incorporated into a polymer
carrier delivery system for use topically, subconjunctivally, or
orally. In addition, other means of administration are
contemplated, such as by injection, either intramuscularly or
intravenously.
[0033] The following Examples describe in detail compositions and
methods illustrative of the present invention. It will be apparent
to those skilled in the art that many modifications, both of
materials and methods, may be practiced without departing from the
purpose and intent of this disclosure.
EXAMPLE 1
Identification of Increased IL-1.alpha. Concentrations in Meibomian
Gland Disease Patients
[0034] In one study, tear fluid concentrations of
Interleukin-1-alpha (IL-1.alpha.), Tumor Necrosis Factor-.alpha.
(TNF-.alpha.), and Epidermal Growth Factor (EGF) in patients having
ocular rosacea were compared with those concentrations in normal
patients (controls).
[0035] Fourteen (14) patients with severe meibomian gland disease,
facial rosacea, and symptoms of ocular irritation were examined for
ocular surface disease, tear production and tear clearance rate
(TCR). For meibomian gland disease assessment, meibomian glands
were examined by slit lamp biomicroscopy and graded for the
presence of orifice metaplasia, expressibility of meibum, and
meibomian gland acinar dropout, as previously described and known
from the scientific literature. Twelve (12) controls,
frequency-matched for age, and fifteen (15) ideal normals were
assessed using the same parameters.
[0036] Minimally stimulated tear samples (20 .mu.l) were drawn from
each subject and analyzed using a sandwich ELISA to detect
IL-1.alpha. and TNF-.alpha. and EGF. Sandwich ELISA assays for EGF,
IL-1.alpha. and TNF-.alpha. were performed using commercial kits
(R&D Systems, Minneapolis, Minn.). Prior to each analysis tears
were extracted in a masked fashion by the laboratory technician
from the saturated rods by centrifuging at 3,500 g for 5 minutes
within the pipette tip.
[0037] The samples from both eyes of each patient were pooled. The
rods and pipette tips were carefully removed and the tears
aspirated (2.mu.l for EGF assay and 4 .mu.l for IL-1.alpha. and
TNF-.alpha. assays) and diluted in ELISA buffer (supplied by
manufacturer) to a final volume of 200 .mu.l. The tear samples were
transferred to wells of microliter plate and the ELISA performed
according to the instructions provided by the manufacturer.
Cytokine concentrations were determined from the standard curve as
described previously in the literature. The results of the cytokine
concentration assays for each subject group are shown in Table 1
below.
1TABLE 1 Inflammatory Cytokines in Tears Tumor Necrosis Subject
Group Factor-.alpha. (pg/ml) Interleukin-1.alpha. (pg/ml) Rosacea
patients <10 pg/ml 45.4 .+-. 4.6 Age-matched controls <10
pg/ml 22.6 .+-. 5.0 (p = 0.003) Ideal controls <10 pg/ml 17.1
.+-. 3.4 (p < 0.001) P values listed for each control group are
derived from comparison of the mean value of that group with the
mean value of the same variable in the rosacea group.
[0038] Tear IL-1 .alpha. concentration was significantly higher in
rosacea patients than in age-matched (p=0.003) and ideal normal
controls (p<0.001) (FIG. 1). TNF-.alpha. was not detected in
patients or controls, indicating levels of less than 10 pg/ml. EGF
was not significantly higher in rosacea patients than in
age-matched controls. Tear Clearance LN(TCR) was significantly
lower in rosacea patients than in both age-matched (p=0.048) and
ideal controls (p=0.002). Schirmer 1 scores were statistically
lower in rosacea patients than ideal controls (p=0.013), but not
age-matched controls. IL-1.alpha. was correlated inversely with
tear clearance LN(TCR) (r=0.58 p<0.0001) and Schirmer 1 (r=0.39,
p=0.012).
[0039] It was concluded that concentrations of IL-1.alpha. are
present in normal tears but are elevated in ocular rosacea, whereas
TNF-.alpha. is not. The reduced tear clearance LN(TCR), its inverse
correlation with IL-1.alpha. and the absence of TNF-.alpha. the
tears of these patients, suggests that the increased concentration
of IL-1.alpha. observed, may be largely due to failure of clearance
of cytokine normally produced at the ocular surface.
[0040] In summary, there is a differential increase in the level of
the inflammatory cytokine IL-1.alpha. in the tear fluid in patients
having ocular rosacea. Much of this elevation can be caused by
reduced tear clearance, which can encourage tear stagnation and
perpetuate ocular surface inflammation by stimulating production of
inflammatory molecules.
EXAMPLE 2
Correlation of Gelatinise Activity with IL-1.alpha. Concentration
and Tear Clearance
[0041] Tear fluorescein clearance was correlated with IL-1.alpha.
concentration and 92 kD gelatinase (MMP 9) activity in the tears of
patients. Thirteen patients with ocular rosacea (including 1
patient with recurrent epithelial erosion, 2 with recurrent
peripheral corneal infiltrates and vascularization and 2 patients
with epithelial basement membrane dystrophy) and 13 normal subjects
with normal aqueous tear production and no irritation symptoms were
evaluated. Tear fluorescein clearance was evaluated by measuring
fluorescence in tear fluid collected from the inferior meniscus 15
minutes after instillation of 5 ml of 2% Na-fluorescein with a
Cytofluor II. IL-1.alpha. was measured by ELISA using an R&D
Systems kit. Gelatinase activity was evaluated by gelatin
zymography, comparing tear activity to purified 92 kD gelatinase
(MMP 9).
[0042] Compared to normal controls, patients with ocular rosacea
had greater delay of fluorescein clearance (p<0.001), higher
tear IL-1.alpha. concentration (p<0.001), and greater 92 kD
gelatinase activity (p<0.001). Tear IL-1.alpha. concentration
was correlated with gelatinase activity (rho=0.58, p<0.002)
(FIG. 2); delayed tear clearance was correlated with elevated tear
IL-1.alpha. concentration (rho=0.84, p<0.001) (FIG. 3) and
increased gelatinase activity (rho=0.84, p<0.001)(FIG. 4).
EXAMPLE 3
Reduced Tear Clearance in Ocular Irritation
[0043] Reduced tear clearance is commonly found in most patients
with ocular irritation irrespective of the patient's tear
production. Forty (40) abnormal patients presenting with a chief
complaint of ocular irritation and forty (40) asymptomatic controls
of similar age distribution were used to correlate and compare a
new method of measuring tear fluorescein clearance and the Schirmer
1 test with the severity of ocular irritation symptoms, presence of
meibomian gland disease, corneal fluorescein staining scores, and
corneal and conjunctival sensitivity. All subjects completed a
symptom questionnaire, a baseline ocular examination, fluorescein
clearance test (FCT) and Schirmer test.
[0044] Methods. The fluorescein clearance test (FCT) was performed
by measuring the fluorescein concentration in minimally-stimulated
tear samples collected from the inferior tear meniscus 15 minutes
after instillation of 5 .mu.l of 2% sodium fluorescein with a
Cytofluor II fluorophotometer. Severity of ocular irritation was
assessed with a symptom questionnaire.
[0045] Schirmer 1 test, biomicroscopic meibomian gland evaluation,
corneal fluorescein staining score and corneal and conjunctival
sensation scores with the Cachet-Bonnet anesthesiometer were
assessed in all subjects.
[0046] Results. Irritation symptoms correlated with higher log tear
fluorescein concentration (symptomatic 3.08+/-0.62 units/.mu.l,
normal control 1.89+/-0.7 units/.mu.l, p<0.005) and lower
Schirmer 1 test scores (symptomatic 12.6 mm, normal control 22.3
mm, p<0.005). The fluorescein clearance test showed greater
predictive value for identifying ocular irritation than the
Schirmer 1 test.
EXAMPLE 4
Doxycycline Therapy Improves Tear Clearance and Reduces IL-1
Concentration in the Tear Fluid
[0047] Six patients were treated with oral doxycycline 50 mg orally
twice a day for 3 weeks. An improvement in irritation symptoms was
observed in 5 of 6 patients. Additionally, decreased conjunctival
hyperemia and corneal fluorescein staining was observed in all
patients. An improvement in fluorescein clearance was noted in 5 of
6 patients and reduction of tear fluid IL-1 concentration was also
observed in 5 of 6 patients. These findings indicate that
tetracyclines reduce two of the significant risk factors for ocular
irritation in patients with meibomian gland disease.
EXAMPLE 5
Resolution of Symptoms of Meibomian Gland Disease by Topical
Administration of Oxytetracycline
[0048] Eleven (11) patients with ocular irritation symptoms due to
meibomian gland disease associated with rosacea and delayed tear
clearance were treated with a commercially available
oxytetracycline ointment (Terak, Akorn Pharmaceuticals) as a
topical administration to the ocular surface or the eyelids.
Seventy-three percent (73%) of these patients had complete
resolution of irritation symptoms. See Table 2 below.
2TABLE 2 Patients with Ocular Rosacea Treated with Oxytetracycline
Ointment Patient's Clinical Number Initials BPEI # Date Treated
Response 1 LP 410606 Jan. 2, 1997 E 2 HB 393796 Jan. 13, 1997 E 3
HP 214814 Dec. 23, 1996 E 4 BP 358105 Dec. 2, 1996 G mild burning 5
JS 547453 Jan. 10, 1997 G burned and stopped 6 FC 225688 Jan. 7,
1997 E 7 JI 422804 Jan. 13, 1997 E 8 RR 401297 Jan. 13, 1997 E 9 CA
88261 Jan. 27, 1997 E 10 DL 531404 Jan. 21, 1997 G Mild burning 11
ES 554804 Dec. 16, 1996 E E = Excellent response, complete or
almost complete relief of symptoms. G = Good response, symptoms
moderately, but not completed relieved
[0049] Several patients who had previously used oral tetracycline
stated that improvement in their symptoms was better with topically
applied oxytetracycline than orally administered tetracycline.
Three patients had moderate but not complete relief of symptoms.
Three other patients experienced burning from the medication, one
of which ceased treatment.
EXAMPLE 6
Resolution of Ocular Irritation by Administration of Topical
Doxycycline Solution
[0050] Moderate to complete relief of ocular irritation symptoms
was also observed in 10 of 11 patients experiencing ocular
irritation due to meibomian gland disease and delayed tear
clearance that were treated with topical administration of a 0.025%
aqueous solution of doxycycline to their eyes 3 to 4 times per day
for 2 weeks. See Table 3. The solution was prepared as described
herein. A reduction of corneal fluorescein staining was observed in
several of these patients.
3TABLE 3 Patients Treated With Topical Doxycycline No. BPEI #
DIAGNOSIS RESPONSE 1 585447 MGD, DTC complete 2 460606 MGD, DTC
moderate, better than oral 3 537137 MGD, DTC Complete, better than
oral 4 606807 MGD, DTC complete 5 590395 MGD, DTC complete 6 593046
MGD, DTC moderate 7 503909 MGD, DTC Complete, better than oral 8
558105 MGD, DTC Moderate, not as good as oral 9 405797 MGD, DTC no
effect 10 576264 MGD, DTC moderate 11 562422 MGD, DTC complete MGD
= Meibomian gland disease DTC = delayed tear clearance
[0051] It should be understood that the examples and embodiments
described herein are for illustrative purposes only and that
various modifications or changes in light thereof will be suggested
to persons skilled in the art and are to be included within the
spirit and purview of this application and the scope of the
appended claims.
EXAMPLE 7
Doxycycline Inhibits the Conversion of Precursor IL-1 (.beta. to
its Mature Form in Cultured Human Corneal Epithelium
[0052] The effect of Doxycycline was evaluated on the
concentrations of precursor and mature IL-1.beta. in the
supernatants of human corneal epithelial cell cultures that were
stimulated with lipopolysaccharide (LPS). Doxycycline significantly
decreased the level of the mature biologically active form of
IL-1.beta. compared to LPS-stimulated cultures that were not
stimulated with doxycycline (FIG. 5). Furthermore, doxycycline
markedly decreased the ratio of mature to precursor IL-1.beta.
(FIG. 6). A decrease in the level of IL-1.beta. mRNA in
doxycycline-treated cultures was also observed. These findings
demonstrate that doxycycline inhibits that synthesis and activation
of the inflammatory cytokine IL-1.beta..
EXAMPLE 8
Doxycycline Increases Release of the Anti-inflammatory Form of
IL-1, IL-1RA.
[0053] e-1 receptor antagonist (IL-1RA) is a molecule produced by
epithelial and inflammatory cells that binds to the type 1 IL-1
receptor but does not initiate signal transduction.
[0054] IL-1RA has an anti-inflammatory effect by competing with the
pro-inflammatory forms of IL-1 (IL-1.alpha. and IL-1.beta. for the
type 1 receptor. The addition of doxycycline or the chemically
modified tetracycline CMT-3 to human corneal epithelial cell
cultures significantly increased the release of IL-1RA in to the
media compared to untreated cultures. Furthermore, the
concentration of IL-1RA in corneal epithelial cultures stimulated
with LPS and treated with either doxycycline or CMT-3 was greater
than cultures exposed to LPS alone (FIG. 7). These findings
indicate tetracyclines are anti-inflammatory due to their ability
to stimulate IL-1RA release by the human corneal epithelium.
EXAMPLE 9
Doxycycline Decreases MMP-9 Activity in Human Corneal Epithelia
Cultures
[0055] Exposure of human corneal epithelial cultures to doxycycline
(5.multidot.g/ml) for 24 hours decreased the level of pro-MMP9
activity in the supernatant by 70% compared to untreated cultures
(FIG. 8). MMP-9 is the metalloproteinase responsible for initiating
sterile corneal ulceration and it is also capable of cleaving
precursor IL-1.beta. into its mature form.
[0056] All cited references are hereby incorporated herein by
reference.
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